FIZIOLOGIA
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CO-CHIEF EDITORSASSOCIATE EDITORSEXECUTIVE EDITORSFRANCISC SCHNEIDERCARMEN PANAITESCUCARMEN TATUFLORINA BOJINMIHAI NECHIFORSORIN RIGAGABRIELA TANASIEDACIANA NISTORCALIN MUNTEANEDITORIAL BOARD
ARDELEAN AURELBĂDĂRĂU ANCA
BENEDEK GYORGYBENGA GHEORGHECOJOCARU MANOLEGĂLUȘCAN ATENAIANCAU MARIAMIHALAŞ GEORGETA
MUNTEAN DANINAMUREŞAN ADRIANANESTIANU VALERIUOPREA TUDORPANAITESCU CARMEN(Arad)(Bucureşti)(Szeged)(Cluj)(Bucureşti)(Timișoara)(Craiova)(Timişoara)(Timişoara)(Cluj)(Craiova)(New Mexico)(Timişoara)PĂUNESCU VIRGILPETROIU ANAPODARIU ANGELA CODRUTA
RÂCZ OLIVERRIGA DANSABĂU MARIUS
SAULEA I. AURELSIMIONESCU MAIASWYNGHEDAUW BERNARDTANGUAY M. ROBERTTATU ROMULUS FABIANVLAD AURELIANVOICU VICTORZĂGREAN LEON

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Fiziologia - Physiology • 2023.2(105)CUPRINSSECVENȚIEREA DE GENERAȚIE URMĂTOARE (NGS) CA INSTRUMENT IMPORTANT PENTRU ABORDAREATERAPEUTICĂ ÎN MELANOM…………………………….…….…………………………….…….…………………………….………….5
Șerban Daniela, Bojin Florina, Gavriliuc Oana, Paunescu Virgil
DESCIFRAREA UNEI REȚELE COMPLICATE: FACTORII IMUNOLOGICI ÎN INFERTILITATE ȘI IMPACTUL LOR ASUPRA
SĂNĂTĂȚII REPRODUCTIVE…………………………………………...................…………………………………………..................11
Grijincu Manuela, Zbarcea Lauriana, Buzan Roxana, Anghel Simona, Ivan Alexandra, Telea Ada, Bojin Florina
INFECȚIA
ȘI
COLONIZAREABACTERIANĂLA
FEMEILEINSARCINATEȘILANOU-NASCUȚI ……………………………………………………………………………….……………………………………………….19
Izabella Petre, Rabia Tasdemir, Ion Petre, Laurentiu Cezar Tomescu, Cristian Furau, Ana Draghici, Anca Bordianu
ANEVRISM INTRACRANIAN DEZVALUIT PRIN DEFICIT NEUROLOGIC - O EXPLORARE A LITERATURII ȘI O
PROVOCARE DIAGNOSTICĂ……….….……….………….………….………….………….………….………….………….………….28
Emilia Burada, Madalina Aldea, Denisa Pirscoveanu, Burdusel Daiana, Raluca Elena Sandu, Roxana Surugiu, Carmen Valeria
Albu
EXPLORAREA FACTORILOR DE RISC ȘI PROGNOSTIC ÎN ACCIDENTUL VASCULAR CEREBRAL ISCHEMIC…………...39
Elena Anca Pinosanu, Burdusel Daiana, Roxana Surugiu, Madalina Aldea, Raluca Elena Sandu
VEZICULELE EXTRACELULARE ÎN ACCIDENTUL VASCULAR CEREBRAL ISCHEMIC: DE LA BIOMARKERI LAPERSPECTIVE TERAPEUTICE……………………………………………………………………………………………………………..48
Roxana Surugiu, Burdusel Daiana, Emilia Burada, Amelia Dumitrescu-Genunche, Raluca Elena Sandu
EXPLORAND DIVERSITATEA: MODELE MURINE PENTRU INDUCEREA SEPSULUI IN CERCETAREA PRECLINICA……55
Alexandra Daniela Rotaru Zavaleanu, Mihai Ruscu, Venera Dinescu, Ramona Vasile, Sorin Dinescu
ADRESABILITATEA PACIENȚILOR CU AFECȚIUNI PSIHIATRICE ÎN CONTEXTUL PANDEMIEI COVID-19
Madalina Aldea, Victor Gheorman, Alexandra Daniela Rotaru Zavaleanu, Daiana Burdusel, Roxana Surugiu, Daniela-Gabriela
Glavan…………………………………………………………………………………………………………………………………………..59
UZUL NOCIV DE ALCOOL, DATE DEMOGRAFICE, PREVALENTA SI COMPLICATII……………………………………………..68
Madalina Aldea, Raluca-Elena Sandu, Daniela-Gabriela Glavan, Roxana Surugiu, Tudor-Adrian Bălseanu
EVALUAREA POSIBILELOR MODIFICĂRI PSIHOMETRICE INDUSE DE PRACTICAREA RUGĂCIUNII ÎN TRADIȚIA
CREȘTINĂ ORTODOXĂ…………………………………………………………………………………………………………………………………..75
Adrian Sorin Mihalache, Oana-Maria Vicu, Delia Oana Popa, Leon Zagrean

3

CONTENTS
NEXT GENERATION SEQUENCING (NGS) AS IMPORTANT TOOL FOR THERAPEUTIC APPROACH IN
MELANOMA....................................................... ...................................................... ......................................................................5Șerban Daniela, Bojin Florina, Gavriliuc Oana, Paunescu Virgil
DECIPHERING THE INTRICATE NETWORK: IMMUNOLOGIC FACTORS IN INFERTILITY AND THEIR IMPACT ON
REPRODUCTIVE HEALTH…………………………………………………………………………………………………………………..11
Grijincu Manuela, Zbarcea Lauriana, Buzan Roxana, Anghel Simona, Ivan Alexandra, Telea Ada, Bojin Florina
ENDOMETRIOSIS - A CAUSE OF INFERTILITY?…………………………………………... ………………………………………….19
Izabella Petre, Rabia Tasdemir, Ion Petre, Laurentiu Cezar Tomescu, Cristian Furau, Ana Draghici, Anca Bordianu
INTRACRANIAL ANEURYSM UNVEILING AS NEUROLOGICAL DEFICITS: A LITERATURE REVIEW AND DIAGNOSTIC
CHALLENGE……………………………………………………………..………………........................................................................28
Emilia Burada, Madalina Aldea, Denisa Pirscoveanu, Burdusel Daiana, Raluca Elena Sandu, Roxana Surugiu, Carmen Valeria
Albu
EXPLORING
PREDICTORSANDOUTCOMESINISCHEMICSTROKE………………………………………………………………………………………………………………………………………..39
Elena Anca Pinosanu, Burdusel Daiana, Roxana Surugiu, Madalina Aldea, Raluca Elena Sandu
EXTRACELLULAR VESICLES IN ISCHEMIC STROKE: POTENTIAL BIOMARKERS AND THERAPEUTIC AVENUES……...48
Roxana Surugiu, Burdusel Daiana, Emilia Burada, Amelia Dumitrescu-Genunche, Raluca Elena Sandu
EXPLORING DIVERSITY: MURINE MODELS FOR INDUCING SEPSIS IN PRECLINICAL RESEARCH……...........................55
Alexandra Daniela Rotaru Zavaleanu, Mihai Ruscu, Venera Dinescu, Ramona Vasile, Sorin Dinescu
A PSYCHIATRY PERSPECTIVE OF COVID-19 DISEASE IN PATIENT HOSPITAL ADMISSION………………………………….59
Madalina Aldea, Victor Gheorman, Alexandra Daniela Rotaru Zavaleanu, Daiana Burdusel, Roxana Surugiu, Daniela-Gabriela
Glavan
ALCOHOL MISUSE – DEMOGRAPHY, PREVALENCE AND COMPLICATIONS……………………………………………………68
Madalina Aldea, Raluca-Elena Sandu, Daniela-Gabriela Glavan, Roxana Surugiu, Tudor-Adrian Bălseanu
EVALUATION OF POSSIBLE PSYCHOMETRIC CHANGES INDUCED BY THE PRACTICE OF PRAYER IN THE ORTHODOXCHRISTIAN TRADITION……………………………………………………………………………………………………………………..75
Adrian Sorin Mihalache, Oana-Maria Vicu, Delia Oana Popa, Leon Zagrean

Fiziologia - Physiology • 2023.2(105)NEXT GENERATION SEQUENCING (NGS) AS IMPORTANT TOOL FOR
THERAPEUTIC APPROACH IN MELANOMAȘERBAN DANIELA1, BOJIN FLORINA2,3, GAVRILIUC OANA2,3, PAUNESCU VIRGIL2,3
1Clinical Emergency City Hospital Timisoara

2“Victor Babes” University of Medicine and Pharmacy Timisoara; Department of Functional Sciences, Immunology;

Immuno-Physiology and Biotechnologies Center (CIFBIOTEH)
3Clinical Emergency County Hospital “Pius Brinzeu” Timisoara – OncoGen

ABSTRACT
Sequencing technology proposes new insights into neoantigens and mutation-targeted therapies derived from transcriptional
patterns. Combined with classic diagnostic methods, immune profile and microenvironmental assessment, can provide in-depth
information that can reframe our understanding of human cancer biology. In order to know tumor biology, it is necessary to obtain
information about the genetic changes in the DNA of the tumor cells. We performed an extensive genomic characterization by
next-generation sequencing (NGS) of three melanoma cell lines with the aim of analyzing the genetic changes occurring inmelanoma and their clinical and therapeutic relevance. We identified known melanoma driver mutations, such as BRAF V600E,
NRAS Q61R, and additionally found other mutated genes that appear in melanoma pathogenesis and some of them targeted bynovel therapies. These cell lines are used to improve the genetic understanding of melanoma and to study the functionalsignificance of individual mutational changes, but turning cancer genomic data into knowledge remains a challenge. For
establishing the appropriate therapeutic approach in melanoma, the personalized treatment should be envisioned, based on bothnext generation sequencing, surface markers expression, as well as microenvironment assessment.
Key words: NGS, melanoma, BRAF, targeted therapy

INTRODUCTION

from which cutaneous melanoma develops. UVradiation ( UVA and also UVB) is one of the maincauses of DNA damage having harmful effects onskin cells. Intermittent and intense exposure to sun,as well as exposure to UV rays from artificial sources,
has been associated to an increased risk ofmelanoma development [2].
The different clinical subtypes of melanoma havespecific genetic alterations and are classified asfollows based on sun-induced damage: non-CSD(non-chronic sun-induced damage): melanomas onskin without chronic sun-induced damage; CSD(chronic sun-induced damage): melanomas on skinwith chronic sun-induced damage; and acral:melanomas on the palms, soles, or sub-ungual sites.
Outside of the skin, melanocytes exist as well andcan give rise to non-cutaneous melanomas on
mucosal membranes, the uveal tract of the eye, or
leptomeninges. Different genetic profiles important fortherapeutic decisions have been identified in different

The application of next-generation sequencing(NGS) has increased over the past ten years,
facilitating the identification of cancer driver genes
and opening new opportunities for cancer genomic
research, including melanoma. The skin cancer
associated with the highest mortality is cutaneous
melanoma. It has a somatic (sporadic) or germline(familial) origin. Almost 90% of melanoma cases are
represented by the somatic form [1]. The main
germline mutations found in the familial form are the
CDKN2A and CDK4 genes. The most frequent
genetic alterations occurring in melanoma are related
to the BRAF, NRAS, KIT and NF1 genes. According
to the presence of these mutations, melanoma isclassified into four genomic subtypes: BRAF-mutant,
NRAS-mutant, NF1-loss and triple wild-type [1].
Cutaneous melanocytes are located in the basal
layer of the epidermis and they represent the cells
5melanoma subtypes [3].
Non-CSD subtype
was described to have the highest amount BRAF
mutations (56%) compared to CSD, acral, andmucosal subtypes (6%, 21%, and 3%, respectively).
In contrast, the incidence of KIT aberrations was 28%,
36%, and 39% in CSD, acral, and mucosal subtypes,
respectively. NRAS mutations were described in 5%
to 20% of the subtypes [3].
Considering the different subtypes of melanoma
and their different genetic profiles, sequencing
techniques used have included: targeted sequencing(focusing on specific regions of the genome),
whole-exome sequencing: WES (the entire coding
region of the genome), and whole-genome
sequencing: WGS (the entire coding and non-coding
regions of the genome) [1,4].
Identifying the presence of targetable mutations is an
important step in melanoma management because
effective targeted therapies are available. With thedecreasing cost of next-generation sequencing (NGS),
precision medicine can be clinically practiced basedon the genetic profile of the patient's melanoma that isused for diagnosis and personalized treatment [4,5].
MATERIALS AND METHODS

We used 3 melanoma cell lines for morphological,
immunophenotypic characterization and NGS
analysis of single nucleotide variation (SNV).
The cells lines were purchased from ATCC,
cultured and expanded according to producer
specified conditions, at 37°C, in 5% CO2 in air
atmosphere: A375 (CRL-1619; in Dulbecco’s Modified
Eagle’s Medium, 10% FBS), A2058 (CRL-11147; inDulbecco’s Modified Eagle’s Medium, 10% FBS) and
SK-MEL-2 (HTB-68, in Eagle’s Minimum Essential
Medium, 10% FBS).
Cells were submitted to immunophenotypic
analysis using FACSVerse 3 lasers capableflowcytometer and a 12 markers panel including:CD90, CD105, HLA-DR, PD1, PD-L1, MICA/B,
CD146, CD29, Fas, FasL, CD56 and EGFR.
The typical NGS workflow comprises different
steps, from nucleic acids extraction to variant
annotation. The process generally begins withconverting nucleic acids (RNA or DNA) from biologicalsamples to a biomaterial compatible with thesequencing system intended for the study. NGS
experiment can be divided into the following four main
steps: (1) DNA purification from tumor cells (2)preparation of libraries, (3) gene sequencing, and (4)gene analysis.
NGS - ThermoFisher Scientific kits and reagents/ IonTorrent platform: DNA purification-GeneJET
Genomic DNA Purification Kit; Library Preparation-Ion
Ampliseq Library Preparation on the Ion Chef System+ Ampliseq Cancer Hotspot Panel v2 Chef-Ready Kit(49 genes, 207 hotspots); Automated template
preparation, chip loading, and sequencing-Ion 510
and Ion 520 and Ion 530 Kit Chef + Ion GeneStudio
S5 System; Analysis - Ion Reporter and Oncomine
Reporter software.
RESULTS

Currently, cultured melanoma cell models arewidely used to study the mechanisms involved intumor cell progression and to develop personalized
therapies. In the present study, we performed an
extensive
genomic
characterizationbynext-generation sequencing (NGS) of threemelanoma cell lines. We also performed amorphological and immunophenotypic analysis. We
used the following melanoma cell lines: A375, A2058
and SK-MEL-2. A375 is derived from a skin primarymelanoma of a 54-year-old female. A2058 has been
isolated from a lymph node metastasis of a
43-year-old male patient. SK-MEL-2 has been
isolated from a metastatic site on the thigh of a
60-year-old male.
The A375 cell line expressed increased levels of
the following markers on the cell surface: CD105,
HLA-DR, PD-L1, CD146, CD29, Fas, CD56, EGFR
(Figure 1). High expression of CD105, CD29 andCD146 are associated with a high risk of metastasis.
CD146 is correlated with melanoma progression
being highly expressed on metastatic melanoma
cells.

Figure 1. Expression of surface markers of the A375 cellline.

Also, the A2058 cell line expressed high amountsof PD-L1, CD146, CD29, Fas, CD56, EGFR as wellas PD-1. This cell line has not expressed CD105 andHLA-DR (Figure 2).

Fiziologia - Physiology • 2023.2(105)Figure 4. NGS analysis for A375 cell line.

Figure 2. Expression of surface markers of the A2058 cellline.

For A2058 we identified the following mutations:
BRAF V600E, PTEN L112Q, TP53 V274F and
SMARCB1.
The identification of certain mutations may
contraindicate the choice of treatment, as for example
in this cell line with a PTEN mutation, treatment withdabrafenib leads to a shorter median progression-freesurvival (PFS) [2], outlining the importance of NGS
analysis in guiding the therapeutic decision (Figures 5,
6).

The SK-MEL-2 had a completely different
expression of the markers on the cell surface with agood level of expression of PD-L1, CD146, CD29,
CD56, EGFR (Figure 3).

Figure 3. The expression of surface markers for SK-MEL-2 cellline.

All three cell lines have good expression of
PD-L1, used in clinical practice to evaluate melanoma
before checkpoint inhibitors therapy [3].
The E-cadherin is found on normal melanocytes
and is important for attachment to keratinocytes.
Down-regulation of E-cadherin is involved inmelanoma progression, reflecting the ability ofmelanocytes to invade the dermis. All three cell lines
did not express E-cadherin.
In order to obtain the genetic profile of thesemelanoma cell lines, we performed next-generation
sequencing. We analyzed the data using Oncomine
Reporter software, with which we identified relevantbiomarkers currently targeted by approved therapies
by: FDA, NCCN, EMA and ESMO. The report also
contains information about the clinical trials targetingthe mutations identified in the sample.
The NGS analysis revealed for A375 cell line thepresence of BRAF V600E mutation and CDKN2A E61
and E69 mutations (Figure 4).

Figure 5. Mutations identified by NGS analysis for A2058
cell line.

Figure 6. Relevant biomarkers identified by NGS analysis
for A2058 cell line.

7

The SK-MEL-2 cell line presented mutations in
NRAS Q61R, TP53 G245S, and KIT M541L (Figure
7).

Figure 7. NGS analysis for SK-MEL-2 cell line.

DISCUSSIONS

The most common mutation was BRAF V600E
which appeared in two samples (A375 and A2058), ina mutually exclusive manner with the NRAS Q61R
mutation, which we found in one cell line (SK-MEL-2).
For melanoma cell lines A375 and A2058, apart of
BRAF V600E we identified other mutations which are
not targeted yet by current approved therapies:
CDKN2A E61 and E69, PTEN L112Q, TP53 V274F,
and SMARCB1. In SK-MEL-2 cell line we found theassociation between KIT mutation, frequently found inmucosal melanoma and NRAS mutation, found in acralmelanoma.
Our study has several limitations. Unfortunately,
we were unable to find out from the manufacturer ofthe cell lines the location of the primary tumors in thecase of A2058 and SK-MEL-2 cell lines. We also
lacked clinical data about tumors, such as exposure to
the sun and the existence of other cases of melanoma
in the family. It would be interesting to know thelocation of the primary tumor, its NGS analysis, thecomparison with the one from the metastasis, but also
if the patient underwent therapy because over thecourse of the disease, the genome of a cancer
changes, especially in response to the selection
pressure of treatment.
Tumors contain a multitude of mutations, few ofwhich are essential for maintaining a malignant state.
Advances in DNA sequencing technologies, including
next-generation sequencing (NGS) facilitate theidentification of potential targetable changes. In
cutaneous melanoma, mutations in the signaling

components BRAF, NRAS and NF1 commonly lead toactivation of mitogen-associated protein kinase(MAPK) pathway. The MAPK pathway can be targetedwith FDA approved small molecule inhibitors: BRAF
inhibitor + MEK inhibitor (dabrafenib + trametinib,
binimetinib + encorafenib).
BRAF is the most common genetic alteration in
cutaneous melanoma, being present in 40–60% ofcases and representing one of the main therapeutictargets. BRAF mutations mainly affect the 600th
codon. Replacement of valine with glutamic acid in the600th codon (V600E) is the most common mutation,
occurring in over 80% of cases. V600K is found in 15%
of melanomas and V600R/M/D/G are described in lessthan 5% [3]. Currently, there is an association ofclinical features of the patient with the BRAFV600E
mutation, such as: younger age, skin sites with
non-chronic sun-induced damage and superficial
melanoma subtype. BRAFV600K mutation has beencorrelated with chronic sun-induced damage and older
age [2,3].
In patients with BRAF V600-mutated melanoma,
selective BRAF inhibitors have shown remarkabletherapeutic activity. Unfortunately, within a few monthsof starting BRAFi +/ MEKi treatment, nearly all patientswith BRAF-mutated metastatic melanoma will develop
resistance to therapy and experience tumor
recurrence, outlining the need for more clinicalresearch to discover the processes that lead to thedevelopment of treatment resistance. As the diseaseprogresses, the genome of a tumor evolves, especiallyin response to the selection pressure of treatment. In
order to find presumably de novo mutations and themechanisms that led to resistance to therapy, a preand post-treatment NGS analysis may facilitate theiridentification [6,7].
The presence of NRAS mutations has been
described in 10-25% of cutaneous melanomas, innodular subtypes and melanomas developing in skinsites with chronic sun induced damage, raising thequestion that they are a result of UV-related
mutagenesis. In contrast to BRAF, NRAS is not atherapeutic target. BRAF and NRAS mutations areusually mutually exclusive, nevertheless there havealso been descriptions of their coexistence [2].
Neurofibromin 1 (NF1) is a negative regulator ofRas and has frequently undergone loss of function inmalignancy and thus considered as a tumor
suppressor. Loss of NF1 has been found to besignificantly increased in cutaneous melanoma without
either BRAF or NRAS mutations and has been
Fiziologia - Physiology • 2023.2(105)associated with increased activation of the MAPK
pathway [8].
The melanoma subtypes with the most C-KIT
mutations are mucosal and acral melanomas. KIT is atransmembrane receptor tyrosine kinase that is widely
expressed in a range of normal cell types and is alsoessential for the regulation of normal melanocyte
differentiation, growth and migration. Mucosal
melanoma is a rare and aggressive subtype ofmelanoma with a less favorable outcome, and KITmutations are more prevalent in this tumor. In mucosal
melanoma there is a lack of understanding andidentification of oncogenic drivers, and NGS analysis
could help to reveal them. Tumors such as
gastrointestinal stromal tumors (GISTs) andcutaneous melanoma with KIT exon 11 or exon 13
mutations have a better response to KIT targeted
therapy suggesting that specific changes may be moresensitive to KIT inhibition, outlining the importance of
NGS analysis. Imatinib, nilotinib, and dasatinib are KIT
inhibitors that have demonstrated variable clinicalefficacy in the treatment of KIT mutated mucosal
melanoma, but further research is needed [8,9].
Neoantigens can develop from somatic mutationsof tumor DNA, representing antigens derived frommutations that can be recognized and attacked bycells of the immune system. The probability of
neoantigen formation increases with the number ofsomatic mutations a tumor contains and tumor
mutational burden (TMB) might serve as a helpful
indicator of tumor neoantigen load. The highest
amounts of TMB have been found in melanoma,according to numerous studies that have been able tomap and describe TMB variants in different types ofcancer [9,10].

resistance is highly common, so further research is
required to find new targeted treatments and additional
mutations causing resistance to therapy will need to be
identified.
Cultured cell models of melanoma are often used
to study response to treatment and mechanisms
underlying therapy resistance. Due to the genetic
diversity of melanoma cell lines, it would be useful tohave access to broad panels of well-characterized celllines, and with the help of NGS analysis we can define
this database.
Given that significant advances have already led
to the development of a variety of analysis tools for
NGS results, it seems likely that NGS will soon beavailable for large-scale uses, for diagnosis andpersonalized treatment.
REFERENCES
1.

2.
3.

4.

5.

CONCLUSIONS

6.

The molecular pathways of melanoma are crucial
to be revealed, because understanding themechanisms of operation and the significance ofgenetic changes will lead to new targeted therapies for
personalized treatment with better chances of cure.
The management of metastatic melanoma has been
greatly improved by targeting the MAPK signaling
pathway, with BRAF mutations being the mostcommon and important alteration that can be targeted.
For patients with metastatic melanoma who have a
BRAF mutation, the combination of BRAF/MEK
inhibition is the first line of treatment. However, drug

7.

8.
9.

9

Muñoz-Barrera A, Rubio-Rodríguez LA, Díaz-de UseraA,
JáspezD,
Lorenzo-Salazar
JM,
González-Montelongo R, et al. From Samples toGermline and Somatic Sequence Variation: A Focus on
Next-Generation Sequencing in Melanoma Research.
Vol. 12, Life. MDPI; 2022.
Teixido C, Castillo P, Martinez-Vila C, Arance A, Alos L.
Molecular markers and targets in melanoma. Vol. 10,
Cells. MDPI; 2021.
NCCN Clinical Practice Guidelines in Oncology (NCCN
Guidelines ® ) NCCN Evidence Blocks TM Melanoma:
Cutaneous [Internet]. 2023. Available from:www.nccn.org/patents
Morton C, Sarker D, Ross P. Next-generation
sequencing and molecular therapy. Clinical Medicine,
Journal of the Royal College of Physicians of London.
2023 Jan 1;23(1):65–9.
Garman B, Anastopoulos IN, Krepler C, Brafford P,
Sproesser K, Jiang Y, et al. Genetic and Genomic
Characterization of 462 Melanoma Patient-Derived
Xenografts, Tumor Biopsies, and Cell Lines. Cell Rep.
2017 Nov 14;21(7):1936–52.
Santamaria-Barria JA, Matsuba C, Khader A, Scholar
AJ, Garland-Kledzik M, Fischer TD, et al. Age-related
next-generation sequencing mutational analysis in
1196 melanomas. J Surg Oncol. 2023 Jun
1;127(7):1187–95.
Griewank KG, Schilling B. Next-Generation Sequencing
to Guide Treatment of Advanced Melanoma. Vol. 18,
American Journal of Clinical Dermatology. Springer
International Publishing; 2017. p. 303–10.
Nassar KW, Tan AC. The mutational landscape ofmucosal melanoma. Vol. 61, Seminars in Cancer
Biology. Academic Press; 2020. p. 139–48.
Riviere P, Goodman AM, Okamura R, Barkauskas DA,
Whitchurch TJ, Lee S, et al. High tumor mutationalburden correlates with longer survival inimmunotherapy-naïve patients with diverse cancers.
Mol Cancer Ther. 2020 Oct 1;19(10):2139–45.
10. Chan TA, Yarchoan M, Jaffee E, Swanton C, Quezada
SA, Stenzinger A, et al. Development of tumor mutation

burden as an immunotherapy biomarker: Utility for the
oncology clinic. Vol. 30, Annals of Oncology. Oxford
University Press; 2019. p. 44–56.

SECVENȚIEREA DE GENERAȚIE URMĂTOARE (NGS) CA INSTRUMENT IMPORTANT
PENTRU ABORDAREA TERAPEUTICĂ ÎN MELANOMREZUMAT

Tehnologia de secvențiere propune noi perspective asupra neoantigenelor și a terapiilor țintite. Metodele clasice dediagnostic împreună cu evaluarea profilul imun și micromediului tumoral, pot oferi informații noi pentru o mai bunăînțelegere a biologiei tumorilor maligne. Pentru o cunoaștere aprofundată a biologiei tumorale, este necesară obținerea
de informații referitoare la modificările genetice din ADN-ul celulelor tumorale. În studiul de față, am efectuat ocaracterizare genomică extinsă prin secvențierea de generație următoare (NGS) a trei linii celulare de melanom, cu
scopul de a analiza modificările genetice prezente în aceste linii celulare și relevanța clinică și terapeutică a acestora. Am
identificat mutații cunoscute implicate în patogeneza melanomului, precum BRAF V600E, NRAS Q61R dar și mutații
noi pentru care terapii țintite se află în dezvoltare. Aceste linii celulare sunt folosite pentru a studia semnificația
funcțională a modificărilor genetice individuale. Pentru stabilirea abordării terapeutice adecvate în melanom, trebuie avut
în vedere tratamentul personalizat, bazat atât pe secvențierea de generație următoare, expresia markerilor de suprafață,cât și pe evaluarea micromediului tumoral.
Cuvinte cheie: NGS, melanom, mutația BRAF, terapii țintite

Fiziologia - Physiology • 2023.2(105)DECIPHERINGTHEINTRICATE
NETWORK:IMMUNOLOGIC FACTORS IN INFERTILITY AND THEIRIMPACT ON REPRODUCTIVE HEALTHGRIJINCU MANUELA1, ZBARCEA LAURIANA1, BUZAN ROXANA1, ANGHEL SIMONA1,2, IVAN
ALEXANDRA1,2, TELEA ADA1, BOJIN FLORINA1,2
1Clinical Emergency County Hospital "Pius Brinzeu" Timisoara, Center for Gene and Cellular Therapies in the Treatment of Cancer
Timisoara-OncoGen, No 156 Liviu Rebreanu, 300723 Timisoara, Romania
2Department of Functional Sciences, Immuno-Physiology and Biotechnologies Center, “Victor Babes” University of Medicine and Pharmacy,
No 2 Eftimie Murgu Square, 300041 Timisoara, Romania

Received July 16th 2023. Accepted September 20th 2023. Address for correspondence: Zbarcea Lauriana, PhD student,
Clinical Emergency County Hospital "Pius Brinzeu" Timisoara, Center for Gene and Cellular Therapies in the Treatment ofCancer Timisoara-OncoGen, No 156 Liviu Rebreanu, 300723 Timisoara, Romania; e-mail lauriana_zbarcea@yahoo.com
ABSTRACTThis paper investigates the intricate interplay of fetal human leukocyte antigen (HLA) and maternal killer cell immunoglobulin-like receptor(KIR) interactions, focusing on their implications for pregnancy outcomes. The study explores the impact of HLA mismatches, particularly in thecontext of the HY immunity system, on the maternal immune response. The presence of HLA antibodies is assessed, with consideration given
to the correlation with total immunoglobulin levels, aiming to discern associations with potential pregnancy complications. Complement activity
is examined, adding a layer of understanding to the dynamics of fetal HLA and maternal KIR interactions.
Furthermore, the paper delves into the delicate balance between Th1 and Th2 helper T cells, crucial components of the immune system,and their potential influence on maternal-fetal immune tolerance. Natural Killer Cell Cytotoxic Activity (NKa) is investigated, providing insights
into the cytotoxicity of these immune cells and their role in the context of maternal-fetal interactions. The findings presented contribute to anuanced comprehension of the immunological factors shaping pregnancy outcomes and may inform future research and clinical approaches
aimed at optimizing maternal and fetal health.
Key words: immunological factors, infertility, reproductive health

INTRODUCTION
Recurrent implantation failure (RIF) is characterized
by the inability to achieve pregnancy after three or moreembryo transfer cycles involving high-quality blastocysts.
RIF can stem from various factors, including altered uterine,
male, or embryo-related factors. Disruptions in endometrial
receptivity, gene expression in multiple pathways,
immunologic disturbances in peripheral blood and/or theendometrium, and epigenetic changes are linked to RIF.
Immunologically, women with RIF often exhibit an altered
Th1/Th2 ratio and changes in NK cell and macrophage
numbers. However, the diversity of immune dysfunction
among women with RIF suggests a heterogeneouscondition with varied immune responses, emphasizing theneed for personalized therapies based on individual
immune statuses. Tacrolimus is commonly recommended
for those with a high Th1/Th2 ratio, while intravenous IgG issuggested for those with elevated NK cell numbers or HLA
mismatch. Women with a hyperactivated immune status

may receive progesterone support, prednisolone, vitamin E,
and intralipid treatment to mitigate inflammation andoxidative stress. Conversely, those with a hypo-active
immune status might benefit from endometrial scratching
and intrauterine hCG administration. Standardized tests for
immune status evaluation and well-powered randomized
controlled trials are essential to determine the efficacy of
personalized therapies for women experiencing RIF.
In this paper we present a set of tests that should be
performed in case of RIF, related to the immune status ofboth partners.
1.

Parental histocompatibility

Fetal HLA and maternal KIR interactions
The mother's uterine natural killer cells recognize theunique HLAs of a developing embryo through receptors
known as killer immunoglobulin-like receptors (KIRs).
11

Some maternal KIRs exhibit better responsiveness toembryos expressing HLA-C2, influencing the flow ofoxygen and nutrition through the placenta via spiral artery
remodeling. Furthermore, if the embryo possesses agreater (or in some cases, an equal) number of HLA-C2
alleles compared to the mother, it may pose a potential risk.
This examination assesses the patient's HLA-C2 content,
the anticipated embryo's HLA-C2 content, and whether thematernal KIRs form a compatible match with HLA-C2.
Significant compatibility issues arise between thepatient's KIR genes and the fetal HLA-C alleles, potentiallyleading to a compromised immune tolerance toward theembryo's HLAs and hindered spiral artery remodeling. The
patient and their healthcare provider may consider
investigating the advantages of immune-modulating
treatments to enhance immune tolerance [1-6].
Clinical studies have revealed that the combination ofmaternal KIR-AA and fetal HLA-C2, as opposed to fetal
HLA-C1, is associated with an elevated risk of recurrent
losses [4], preeclampsia [3], and low birth weight [5].
Notably, the frequency of maternal KIR AA is higher inpregnancies that result in loss or are affected by
preeclampsia, especially when the fetus has more C2
genes than the mother or when fetal C2 is inherited
paternally [4]. Additionally, a study comparing HLA-C andKIR haplotypes in couples with three or more spontaneous
miscarriages (RPL) versus those with no fertility issues
demonstrated a significant association. The absence ofactivating KIR in the affected woman (KIR AA genotype)and an increased HLA-C2 group frequency were observedin couples experiencing recurrent pregnancy loss [6].
HLA mismatches
For a healthy pregnancy, a mother's immune system
must establish tolerance to the embryo, acknowledging its
genetic uniqueness. Yet, if the embryo inherits paternal
HLAs closely resembling the maternal ones, a robust
tolerance may not develop. This test assesses the extent of
mismatches between maternal and paternal HLA Class II
alleles to gauge the potential for immune tolerance
development.
If there are fewer than 4 mismatches, there is amoderate risk that the similarity of HLA Class II alleles
could hinder the maternal immune system from developing
tolerance toward the embryo. The patient and theirhealthcare provider may consider investigating
immune-modulating treatments to enhance immune
tolerance.
Human Leukocyte Antigen (HLA) genes, commonly
known as the major histocompatibility complex (MHC)
genes, constitute a group of genes located on chromosome
6 and play a crucial role in presenting antigens to T cells to
initiate immune responses. Typically, this immune reaction
results in the elimination of cells displaying "non-self"
peptides. There are two classes of HLA molecules: class I,

which includes HLA-A, -B, -C, and class II, which includes
HLA-DR, -DQ, and -DP. HLA molecules are pivotal in organtransplantation and are implicated in various diseases,
including autoimmune disorders [7-8].
In organ transplantation, a mismatch in HLA between
a donor and recipient may lead to graft rejection, often due
to antibody formation [9]. Conversely, a certain degree ofdifference in HLA alleles between the mother and father[10], inherited by the embryo and defined as a mismatch, isessential for actively establishing immune tolerance of theembryo [11]. Consequently, couples with significant
matching of HLA alleles may be more susceptible toinfertility, repeated implantation failure, and recurrent
pregnancy loss [12].
HY immunity
Embryos possessing a Y chromosome express
proteins known as HY antigens (male-specific minor
histocompatibility antigens) on their cells. On occasion,
when a mother gives birth to a baby with a Y chromosome,her immune system may generate an immune response
against these HY antigens, potentially affecting subsequentpregnancies. The mother's HLAs play a crucial role intriggering this immune response, with certain HLA alleles
increasing the likelihood of such a reaction. This test
examines whether the patient carries these higher-risk
HY-restricting alleles, which could elevate the risk,
especially if they have previously given birth to a boy. (Anallele refers to an alternative version of a gene at a specificchromosome location.
In the initial pregnancy carrying a male fetus, thematernal immune system may be triggered by allogenic
fetal cells containing male-specific minor histocompatibility
inherited antigens (HYrHLA allele) encoded by genes
situated on the Y chromosome [13]. In certain women, thisactivation can result in an immediate immune response,
prompting the production of HY antibodies by B cells.
These antibodies may persist in the maternal serum for an
extended period, potentially contributing to subsequent
recurrent miscarriages (regardless of the embryo's gender)
and the delivery of male infants with lower birth weights[14-15].
Although found in 30% of women, anti-HY antibodies
have been associated with secondary recurrent
miscarriage in subsequent pregnancies, along with othercomplications like stillbirth, placental abruption, or fetal
growth retardation. These occurrences are outcomes of an
inflammatory environment. In a comprehensive cohort
study involving women with unexplained secondary
recurrent pregnancy loss (RPL), findings indicated thatthese patients faced a higher likelihood of miscarriage intheir subsequent pregnancies when their firstborn was aboy compared to a girl (46% versus 24%, respectively)[16].
For those patients with a firstborn son, the presence ofFiziologia - Physiology • 2023.2(105)H-Y restricting HLA class II alleles (HLADRB115, HLADQB105:01/02, and HLA–DRB3*03:01) has beencorrelated with a diminished chance of live birth and alower male/female ratio among subsequent births(indicating an increased loss rate of male embryos) [17].
Patients with no copies of HY-restricting alleles have asubsequent live birth rate of 73%, which decreases to 58%
with one allele copy and further to 49% with two allele
copies.

Complement activity
The complement system, an integral part of the innate
immune system, acts as a potent initiator of inflammation
upon activation. Within this system, the proteins C3 and C4
play pivotal roles, and their activation has been linked topregnancy complications, particularly pre-eclampsia.
Complement is a system of serum proteins that forms a
crucial effector arm of the innate immune system,
associated with macrophage activation and inflammation.
There are three distinct pathways through which thecomplement system is activated: the classical pathway
activated by antigen-antibody complexes, the alternative
pathway activated by microbial surfaces, and the lectin
pathways activated by microbial surfaces. These pathways
converge, leading to the generation of anaphylatoxins (C3a,C4a, and C5a) and the membrane attack complex MAC(C5b,6,7,8, and 9), which are potent inflammatory
molecules. The complement and coagulation pathways
closely interact, with C5a inducing procoagulant activity
and reducing fibrinolysis. Additionally, C3a and C5a
activate endothelial cells and platelets, resulting inincreased levels of adhesion factors and procoagulant
activity.
Numerous studies have delved into complement
activation in the circulation of women with preeclampsia,
revealing heightened complement activity compared topregnancies in healthy control subjects [19-21]. Similarly,
clinical investigations support the significance ofcomplement activity, evident in lower levels of C3 and C4complement levels associated with obstetricalcomplications in patients with antiphospholipid syndrome[22-25] or Systemic Lupus Erythematosus [26]. Notably, acorrelation has been established between elevated levelsof complement activation fragments (Bb and C3a) andpreterm birth [27-28]. Intriguingly, certain proteins involvedin complement activation, such as factor B and H, havebeen identified as useful biomarkers for predicting preterm
birth as early as 15 weeks into pregnancy [29].
The profound effects of complement activation on theplacenta, leading to inflammation and placental injury,
stand out as major contributors to fetal loss in APS, in
addition to triggering pre-eclampsia.

HLA antibodies
Occasionally, an individual's immune systemgenerates antibodies against HLAs. These antibodies mayeither be directed against "self" HLAs (autoantibodies),
target "non-self" HLAs from prior full-term pregnancies or
blood transfusions, or specifically focus on the HLAs of apartner that the embryo will inherit. While HLA antibodies
are common and typically not problematic, partner-specific
HLA-C antibodies (a subset of Class I antibodies) canpresent a significant risk and are associated with early
miscarriages and secondary infertility. This test evaluates
the presence, quantity, and type of HLA antibodies in apatient, if any.
Pregnancy is characterized by profound alterations in
the maternal immune system, facilitating the implantationand growth of the semi-allogeneic fetus within the uterus. B
lymphocytes (B cells) play a crucial role in the maternal
immune adaptation to fetal implantation among immune
cells.
Certain subsets of B cells can generate antibodies
targeting components of the embryo/fetus determined bypaternal genetics, notably paternally derived HLA
molecules. Anti-HLA antibodies are present inapproximately one third of healthy and successful
pregnancies [18]. However, the presence ofpartner-specific anti-HLA antibodies, particularly thosecapable of fixing complement, can be detrimental to themaintenance of pregnancy, potentially leading to
miscarriage or subsequent complications such as
preeclampsia, intrauterine growth restriction, or stillbirth.
2.

Inflammation

Total immunoglobulin level
Whether stemming from an autoimmune condition,
infection, allergies, or another origin, various inflammatory
markers have been linked to difficulties in reproduction. In
the course of a healthy pregnancy, the body needs totransition to a relatively anti-inflammatory state to sustain
the pregnancy. The following tests represent somewell-established markers of inflammation in reproductive
immunology. The outcomes of these tests can be utilized toassess whether interventions to alleviate inflammation
might enhance a patient's likelihood of reproductive
success.

Th1/Th2 helper T cells ratio
Certain immune cells, such as T helper cells, generate
cytokines that instruct the immune system to intensify its
activity (pro-inflammatory) or to ease off (anti-inflammatory).
Th1 and Th2 cells, specific types of T helper cells, generate
pro-inflammatory compounds, and the balance between
Th1 and Th2 can play a role in gauging the extent ofinflammation in a patient.
CD4+ T cells, CD8+ T cells, NKT cells, and NK cells
constitute four major effector cell types within the immune
13

system. These cells can react to foreign antigens, including
those derived from the paternal side present inconceptuses, and can initiate either immunogenic or
tolerogenic responses. Upon antigen priming by
antigen-presenting cells (APCs), naïve CD4+ T cells candifferentiate into various lineages, such as Th1, Th2, Th17,
and Treg cells (regulatory T cells), depending on the
involved APCs and the soluble molecules secreted during
priming. These lineages are distinguished by uniquecytokine expression profiles, with flow cytometry identifying
cells expressing specific cytokines (IFNγ for Th1, IL-4 for
Th2, IL-17 for Th17, and IL-10 for Treg). Analogous
lineages exist for CD8+ T cells, NKT cells, and NK cells,
and their profiles can be similarly characterized. The
presence of TNFα-positive cells serves as a generalmarker of cellular activation.
The relative balances of these lineages within eachcell type can help characterize underlying immune
conditions. For instance, certain autoimmune conditions,like rheumatoid arthritis, exhibit Th1 dominance, whileothers, such as systemic lupus erythematosus, show Th2
dominance. Conditions like endometriosis, PCOS, andatopy are also characterized by distinct intracellular
cytokine profiles. These profiles can manifest many years
before the full clinical manifestation and diagnosis of anunderlying autoimmune or inflammatory condition.
Combined with other genetic and cellular data, they can be
employed to characterize preclinical or asymptomaticconditions influencing the immune response to foreign
antigens. Changes in intracellular cytokine profiles can also
be used to monitor the maternal immune response during
pregnancy, evaluate the effectiveness of immune
treatments, and identify whether any pregnancy failures
have an immunological basis.
Women with a history of recurrent implantation failureor recurrent miscarriage exhibit altered intracellular
cytokine profiles in peripheral blood. These alterations
encompass an increased ratio of Th1 to Th2 cells, elevatedlevels of TNFα-positive cells, reduced levels of
IL-10-positive cells, and heightened levels of Th17 cells[30-31]. The typical (non-pathological) response topregnancy is characterized by a tolerogenic reaction topaternal antigens within the conceptus. This tolerogenic
response involves specific modifications to the intracellular
cytokine profile of various immune cell types. Suchtolerogenic responses include a shift towards Th2
dominance, indicated by a decrease in the Th1/Th2 ratio,
and an elevation in levels of IL-10-positive cells [32-33].
Pathological immune responses to a conceptus
indicate a failure to establish proper immunologicaltolerance to paternally derived antigens. Instead, an
immunogenic response is triggered, potentially leading tocellular and/or humoral (antibody-mediated) reactions.
Such pathological immune responses to a conceptus canmanifest in various clinical outcomes, including

implantation failure, spontaneous abortion, preeclampsia,
intrauterine growth restriction (IUGR), and stillbirth. The
defective development of immunological tolerance during
pregnancy is characterized in peripheral blood, marked bya failure to shift towards Th2 dominance, a failure toincrease IL-10-positive cells, and an elevation in levels of
IL-17-positive cells [34-35].
Natural Killer Cell Cytotoxic Activity (NKa)Natural killer (NK) cells, often labeled as "killers" dueto their cytotoxic capabilities, possess a broader range offunctions beyond cell elimination. These cells contribute tovarious activities, such as promoting the healthy
development of the placenta in the uterus. This test
assesses the extent to which the patient's NK cells are
inclined toward cytotoxic activity. Elevated NK cytotoxic
activity has been linked to recurrent pregnancy loss.
Natural Killer (NK) cells represent a subset ofcytotoxic innate lymphocytes [36]. Elevated natural killer
cell activity has been observed in individuals with
autoimmune conditions like Graves' disease andHashimoto's thyroiditis when compared to a healthy control
group [37]. NK cells may play a direct role in these
diseases, either through their potential autoreactivity or byinteracting with dendritic cells, macrophages, or T
lymphocytes, thus contributing to excessive inflammationor promoting adaptive autoimmune responses [38]. During
early pregnancy, NK cells dominate the lymphocyte
population in the decidua [39]. While endometrial NK cells
exhibit distinct phenotypic features from circulating NK
cells—possessing weak cytotoxicity and immunoregulatory
activities [40] - it is speculated that peripheral blood NK
cells maintain a strong correlation with NK cell populations
found in the decidua [41].
Natural Killer (NK) cell activity has been linked to thepathogenesis of recurrent pregnancy loss [42-43], even incases where the embryo exhibits normal chromosomal
characteristics [44]. A clinical investigation has
demonstrated that women with elevated NK cell activity
before conception face a substantially higher risk ofpregnancy loss (3.5-fold higher risk) compared to thosewith normal levels [42]. Recent studies further emphasize arobust association between heightened NK cell activity andrecurrent spontaneous abortion [45-47], suggesting that NK
cell activity could serve as a predictive biomarker.
Intravenous immunoglobulin (IVIG) has been shown to
reduce NK cell activity in women experiencing recurrent
pregnancy losses [48], potentially enhancing the overall
pregnancy outcome. However, the significance ofpre-conceptional NK activity as a predictive indicatorremains debated, as one study found no correlation
between NK activity and the risk of subsequent loss [49].
It's worth noting that this study may be biased as it
exclusively considers clinical losses as miscarriages,
excluding all chemical losses.

Fiziologia - Physiology • 2023.2(105)and other complications during pregnancy. APS iscategorized as primary when it occurs without anunderlying disease and secondary when associated withan underlying pathology, such as systemic lupus
erythematosus (SLE).
Among various antiphospholipid antibodies, anti-beta
2 glycoprotein I (anti-β2-GP1) antibodies are particularly
instrumental in supporting the diagnosis of APS [50].
Numerous studies indicate that anti-β2-GP1 antibodies are
more specific for APS compared to anti-cardiolipin
antibodies [50-52]. All three isotypes of anti-β2-GP1 (IgG,
IgM, and IgA) have been linked to thrombosis [53-55]. The
presence of either or both β2-GP1 IgG and IgM antibodies
serves as an independent risk factor for thrombosis andpregnancy-related complications [52].
APS stands out as the most prevalent acquired risk
factor for recurrent pregnancy loss, exerting its influence bydisrupting placental function and compromisingmaternal-fetal blood exchange. This heightened risk
extends to various pregnancy complications, including
stillbirth, intrauterine death, pre-eclampsia (PE), prematurebirth, and fetal growth restriction.
Among women experiencing recurrent pregnancy loss
(RPL), 26.4% of cases were associated with the presenceof antiphospholipid antibodies [55], while pregnancy
complications were identified in up to 20% of pregnancies
affected by APS [56]. A comprehensive meta-analysis
involving over 200,000 participants revealed a 2.5-foldincrease in the risk of spontaneous abortion in women with
APS [57]. Additionally, another meta-analysis found thatmoderate to high levels of anti-cardiolipin antibodies (aCL)
were linked to an elevated risk of PE [58]. In instanceswhere anti-phospholipid antibodies are both high andpersistent, the risk escalates for preterm birth and fetal
growth restriction [59-60]. Notably, heightened titers of aCL
and anti-β2GPI antibodies were associated with a 3- to
5-fold increased likelihood of stillbirth [61].
Collectively, these studies underscore the importanceof detecting, closely monitoring, and providing appropriate
care to pregnant women with APS to facilitate successful
pregnancies.

Regulatory T cells
Specialized immune cells known as regulatory T cells(Treg cells) play a crucial role in suppressing inflammationand preventing the rejection of the embryo by the uterus.
Insufficient Treg levels in the uterus have been associatedwith infertility, recurrent miscarriage, and pregnancy
complications. This test evaluates the patient's circulating
Treg cell levels, offering insights into the potential
recruitment of Treg cells to the uterus during pregnancy.
3.

Autoimmunity

Antinuclear antibodies (ANAs)
The likelihood of experiencing adverse pregnancy
outcomes is elevated in individuals with autoimmune
diseases, making expectant mothers with conditions suchas antiphospholipid syndrome, lupus, rheumatoid arthritis,and other autoimmune disorders classified as high-risk
pregnancies. Nevertheless, a significant number ofindividuals go undiagnosed for autoimmune conditions.
These tests aid in assessing a patient's susceptibility todifferent autoimmune conditions, enabling tailoredtreatment approaches for each specific condition.
Within a cell, the nucleus houses essential DNA andvarious proteins crucial for maintaining cellular function.
Occasionally, an individual's immune system may initiate
an attack on nucleus-associated proteins, producing
antinuclear antibodies (ANAs). Elevated concentrations of
ANAs could suggest the presence of an autoimmune
disease, such as lupus. These tests evaluate the existenceof various ANAs: ANA Ds-DNA, Anti-Jo1 Ab, ANA RNP,ANA SM, ANA SCL, Antichromatin, ANA CENT.
Antiphospholipid antibodies (APAs)
Phospholipids play a vital role in the composition ofhuman cell membranes. On occasion, an individual's
immune system may initiate an attack on its own
phospholipids, producing antiphospholipid antibodies(APAs). As phospholipids are present in blood cells, thepresence of APAs can contribute to issues such as bloodclots, miscarriages, or complications during pregnancy.
These tests are designed to evaluate the existence ofvarious APAs: anticardiolipin (IgG, IgM, IgA), anti-beta2
glycoprotein I (IgG, IgM, IgA), anti-phosphatidylserine (IgG,
IgM, IgA), lupus anticoagulant dPT, PTT-LA, Thrombin
time.
Antiphospholipid antibody syndrome (APS) is an
autoimmune disorder characterized by the production of
antiphospholipid antibodies by the immune system,
targeting its own components. The existence of these
antibodies can instigate a thrombophilic disorder, resulting
in excessive clotting and contributing to conditions such as
venous thromboembolism, stroke, multiple miscarriages,

CCP antibodies and rheumatoid factor
Antibodies such as cyclic citrullinated protein (CCP)
and rheumatoid factor serve as indicators for rheumatoid
arthritis and certain other autoimmune conditions. These
tests evaluate the presence of specific antibodies, including
anti-CCP antibodies IgG/IgA, and levels of rheumatoid
factor.
CONCLUSION
Progress in reproductive medicine has markedly
improved the efficacy of fertility treatments. Despite theseadvancements, certain women encounter recurrent
15

implantation failure (RIF) following in-vitro fertilization (IVF)or recurrent pregnancy loss (RPL). Imbalances in the
immune system and the inability to establish immune
tolerance to the fetus have been identified as potentiallymodifiable factors contributing to idiopathic RIF and RPL.
Consequently, there is a growing trend in the use of
immunomodulatory agents as a therapeutic approach toenhance the likelihood of a successful pregnancy in thesecases. Advancements in immunological diagnostics are
crucial to align with the evolving clinical requirements inthis emerging field, providing clinicians with the necessary
guidance to make optimal and safe therapeutic decisions.

13.
14.

15.

References
16.
1.

Moffett-King A. Natural killer cells and pregnancy. Nat
Rev Immunol., 2002; 2(9):656-63.
2. Chazara O, Xiong S, Moffett A. Maternal KIR and fetal
HLA-C: a fine balance. J Leukoc Biol. 2011;
90(4):703-16.
3. Hiby SE,Walker JJ, O’Shaughnessy KM, Redman CW,
Carrington M, Trowsdale J, et al. Combinations ofmaternal KIR and fetal HLA-C genes influence the riskof preeclampsia and reproductive success. J Exp Med.,
2004; 200(8):957-65.
4. Hiby SE, Apps R, Sharkey AM, Farrell LE, Gardner L,
Mulder A, Claas FH, Walker JJ, Redman CW, Morgan
L, Tower C, Regan L, Moore GE, Carrington M, Moffett
A. Maternal activating KIRs protect against humanreproductive failure mediated by fetal HLA-C2. J Clin
Invest. 2010; 120(11): 4102-10.
5. Hiby SE, Apps R, Chazara O, Farrell LE, Magnus P,
Trogstad L, Gjessing HK, Carrington M, Moffett A.
Maternal KIR in combination with paternal HLA-C2
regulate human birth weight. J Immunol. 2014;
192(11):5069-73.
6. Hiby SE, Regan L, Lo W, Farrell L, Carrington M,
Moffett A. Association of maternal killer-cell
immunoglobulin-like receptors and parental HLA-C
genotypes with recurrent miscarriage. Hum Reprod.
2008; 23(4): 972-6.
7. Doherty PC,Zinkernagel RM. A biological role for the
major histocompatibility antigens. Lancet 1975;
1(7922):1406-9.
8. Complete sequence and gene map of a human major
histocompatibility complex. The MHC sequencing
consortium. Nature, 1999; 401(6756):921-3.
9. Jucaud V. The Immunogenicity of HLA Class II
Mismatches: The Predicted Presentation of Nonself
Allo-HLA-Derived Peptide by the HLA-DR Phenotypeof the Recipient Is Associated with the Formation of
DSA. J Immunol Res. 2017; 2017: 2748614.
10. Ober C. Studies of HLA, fertility and mate choice in ahuman isolate. Hum Reprod Update. 1999; 5(2):103-7.
11. Papúchová H, Meissner TB, Li Q, Strominger JL,
Tilburgs T. The Dual Role of HLA-C in Tolerance and
Immunity at the Maternal-Fetal Interface. Front
Immunol. 2019; 10: 2730.
12. Ober C, Hyslop T, Elias S, Weitkamp LR, Hauck WW.
Human leukocyte antigen matching and fetal loss:

17.

18.

19.

20.

21.

22.

23.

24.

25.

results of a 10-year prospective study. Hum Reprod.
1998; 13(1):33-8.
Christiansen OB, Steffensen R, Nielsen HS. Anti-HY
responses in pregnancy disorders. Am J Reprod
Immunol. 2011; 66 Suppl 1: 93-100.
Kolte AM, Steffensen R, Christiansen OB, Nielsen HS.
Maternal HY-restricting HLA class II alleles areassociated with poor long-term outcome in recurrent
pregnancy loss after a boy. Am J Reprod Immunol.
2016; 76(5): 400-405.
Nielsen HS, Steffensen R, Varming K, Van Halteren
AG, Spierings E, Ryder LP, Goulmy E, Christiansen
OB. Association of HY-restricting HLA class II alleles
with pregnancy outcome in patients with recurrent
miscarriage subsequent to a firstborn boy. Hum Mol
Genet. 2009; 18(9): 1684-91.
Christiansen OB, Steffensen R, Nielsen HS. The
impact of anti-HY responses on outcome in current
and subsequent pregnancies of patients with recurrent
pregnancy losses. J Reprod Immunol. 2010; 85(1):9-14.
Nielsen HS, Steffensen R, Varming K, Van Halteren
AG, Spierings E, Ryder LP, Goulmy E, Christiansen
OB. Association of HY-restricting HLA class II alleles
with pregnancy outcome in patients with recurrent
miscarriage subsequent to a firstborn boy. Hum Mol
Genet. 2009; 18(9): 1684-91.
Regan L, Braude PR, Hill DP. A prospective study ofthe incidence, time of appearance and significance ofanti-paternal lymphocytotoxic antibodies in humanpregnancy. Human Reproduction, 1991; 6(2): 294-298.
Ye Y, Kong Y, Zhang Y. Complement split products
C3a/C5a and receptors: are they regulated bycirculating angiotensin II type 1 receptor autoantibody
in severe preeclampsia? Gynecol Obstet Invest. 2016;
81(1): 28-33.
Boij R, Svensson J, Nilsson-Ekdahl K, Sandholm K,
Lindahl TL, Palonek E, et al. Biomarkers of coagulation,
inflammation, and angiogenesis are independently
associated with preeclampsia. Am J Reprod Immunol.
2012; 68: 258-70.
Derzsy Z, Prohászka Z, Rigó J, Füst G, Molvarec A.
Activation of the complement system in normal
pregnancy and preeclampsia. Mol Immunol. 2010; 47:
1500-6.
Reggia R, Ziglioli T, Andreoli L, Bellisai F, Iuliano A,
Gerosa M, et al. Primary anti-phospholipid syndrome:
any role for serum complement levels in predicting
pregnancy complications? Rheumatology. 2012; 51:
2186-90.
Oku K, Atsumi T, Bohgaki M, Amengual O, Kataoka H,
Horita T, et al. Complement activation in patients withprimary antiphospholipid syndrome. Ann Rheum Dis.
2009; 68: 1030-5.
Breen KA, Seed P, Parmar K, Moore GW, Stuart-Smith
SE, Hunt BJ. Complement activation in patients withisolated antiphospholipid antibodies or primary
antiphospholipid syndrome. Thromb Haemost. 2012;
107: 423-9.
De Carolis S, Botta A, Santucci S, Salvi S, Moresi S,
Di Pasquo E, et al. Complementemia and obstetricFiziologia - Physiology • 2023.2(105)

26.

27.

28.

29.

30.
31.
32.
33.
34.
35.
36.
37.

38.
39.

40.

41.

outcome in pregnancy with antiphospholipid syndrome.
Lupus. 2012; 21: 776-8.
Hazeltine M, Rauch J, Danoff D, Esdaile JM,
Tannenbaum H. Antiphospholipid antibodies insystemic lupus erythematosus: evidence of an
association
withpositive
Coombs’andhypocomplementemia. J Rheumatol. 1988; 15: 80-6.
Lynch AM, Gibbs RS, Murphy JR et al. Complement
activation fragment bb in early pregnancy andspontaneous preterm birth. Am J Obstet Gynecol 2008;
199: 354.e1-354.e8
Lynch AM, Gibbs RS, Murphy JR et al. Early elevationsof the complement activation fragment C3a andadverse pregnancy outcomes. Obstet Gynecol. 2011;
117: 75-83.
Lynch AM, Wagner BD, Deterding RR et al. The
relationship of circulating proteins in early pregnancy
with preterm birth. Am J Obstet Gynecol 2016; 214:
517.e1–517.e8.
Yockey LJ, Iwasaki A. Interferons and Proinflammatory
Cytokines in Pregnancy and Fetal Development.
Immunity. 2018; 49(3): 397-412.
Raghupathy R, Kalinka J. Cytokine imbalance inpregnancy complications and its modulation. Front
Biosci. 2008; 13: 985-94.
Saito S, Nakashima A, Shima T, Ito M. Th1/Th2/Th17
and regulatory T-cell paradigm in pregnancy. Am J
Reprod Immunol. 2010; 63(6): 601-10.
Ali S, Majid S, Niamat Ali M, Taing S. Evaluation of T
cell cytokines and their role in recurrent miscarriage.
Int Immunopharmacol. 2020; 82: 106347.
Sykes L, MacIntyre DA, Yap XJ, Teoh TG, Bennett PR.
The Th1:Th2 dichotomy of pregnancy and pretermlabour. Mediators Inflamm. 2012; 2012:967629.
Raghupathy R. Pregnancy: success and failure within
the Th1/Th2/Th3 paradigm. Semin Immunol. 2001;
13(4): 219-27.
Moffett A, Shreeve N. First do no harm: Uterine naturalkiller (NK) cells in assisted reproduction. Hum Reprod.,2015; 30(7): 1519-1525.
Hidaka Y, Amino N, Iwatani Y, Kaneda T, Nasu M,
Mitsuda N, Tanizawa O, Miyai K. Increase in peripheral
natural killer cell activity in patients with autoimmune
thyroid disease. Autoimmunity. 1992; 11(4): 239-46.
Fogel LA, Yokoyama WM, French AR. Natural killer
cells in human autoimmune disorders. Arthritis Res
Ther. 2013; 15(4): 216.
Ntrivalas EI, Kwak-Kim JYH, Gilman-Sachs A,
Chung-Bang H, Ng SC, Beaman KD et al. Status of
peripheral blood natural killer cells in women withrecurrent spontaneous abortions and infertility of
unknown aetiology. Hum Reprod., 2001; 16(5):855-861.
Koopman LA, Kopcow HD, Rybalov B, Boyson JE,
Orange JS, Schatz F, Strominger JL. Human decidual
natural killer cells are a unique NK cell subset withimmunomodulatory potential. J Exp Med., 2003; 198(8):1201-1212.
Male V, Hughes T, McClory S, Colucci F, Caligiuri MA,
Moffett A. Immature NK cells, capable of producing
IL-22, are present in human uterine mucosa. J
Immunol., 2010; 185(7): 3913-3918.

42. Aoki K, Kajiura S, Matsumoto Y, et al. Preconceptional
natural-killer cell activity as a predictor of miscarriage.
Lancet (London, England). 1995; 345: 1340-1342.
43. Higuchi K, Aoki K, Kimbara T, Hosoi N, Yamamoto T,
Okada H. Suppression of natural killer cell activity by
monocytes following immunotherapy for recurrent
spontaneous aborters. Am J Reprod Immunol., 1995;
33(3): 221-227.
44. Kwak-Kim J, Gilman-Sachs A. Clinical implication ofnatural killer cells and reproduction. Am J Reprod
Immunol, 2008; 59(5): 388-400.
45. Perricone C, De Carolis C, Giacomelli R, et al. Highlevels of NK cells in the peripheral blood of patientsaffected with anti-phospholipid syndrome and recurrent
spontaneous abortion: a potential new hypothesis.
Rheumatology(Oxford,England).
2007;
46:1574-1578.
46. Seshadri S, Sunkara SK. Natural killer cells in female
infertility and recurrent miscarriage: a systematic
review and meta-analysis. Hum Reprod Update. 2014;
20:429-438.
47. Miyaji M, Deguchi M, Tanimura K, Sasagawa Y,
Morizane M, Ebina Y, Yamada H. Clinical factors
associated with pregnancy outcome in women withrecurrent pregnancy loss. Gynecol Endocrinol. 2019;23: 1-6.
48. Ahmadi M, Ghaebi M, Abdolmohammadi-Vahid S,
Abbaspour-Aghdam S, Hamdi K, Abdollahi-Fard S,
Danaii S, Mosapour P, Koushaeian L, Dolati S,
Rikhtegar R, Oskouei FD, Aghebati-Maleki L, Nouri M,
Yousefi M. NK cell frequency and cytotoxicity incorrelation to pregnancy outcome and response to
IVIG therapy among women with recurrent pregnancy
loss. J Cell Physiol. 2019; 234(6): 9428-9437.
49. Katano K, Suzuki S, Ozaki Y, Suzumori N, Kitaori T,
Sugiura-Ogasawara M. Peripheral natural killer cell
activity as a predictor of recurrent pregnancy loss: alarge cohort study. Fertil Steril. 2013; 100(6): 1629-34.
50. Harris EN, Pierangeli SS, Gharavi AE. Diagnosis of the
antiphospholipid syndrome: A proposal for use oflaboratory tests. Lupus. 1998; 7(Suppl 2): S144-S148.
51. Carreras LO, Forastiero RR, Martinuzzo ME. Whichare the best biological markers of the antiphospholipid
syndrome? J Autoimmun. 2000; 15(2): 163-172.
52. Levine JS, Branch DW, Rauch J. The antiphospholipid
syndrome. N Engl J Med. 2002; 346(10): 752-763.
53. Reddel SW, Krilis SA. Testing for and clinicalsignificance of anticardiolipin antibodies. Clin Diagn
Lab Immunol. 1999; 6(6): 775-782.
54. Brey RL, Abbott RD, Curb JD, et al. Beta(2)-Glycoprotein 1-dependent anticardiolipin antibodies
and risk of ischemic stroke and myocardial infarction:
The Honolulu Heart Program. Stroke. 2001; 32(8):1701-1706.
55. Greco TP, Amos MD, Conti-Kelly AM, Naranjo JD, Ijdo
JW. Testing for the antiphospholipid syndrome:
Importance of IgA anti-beta 2-glycoprotein I. Lupus.
2000; 9(1): 33-41.
56. Rai RS, Regan L, Clifford K, et al. Antiphospholipid
antibodies and beta 2-glycoprotein-I in 500 womenwith recurrent miscarriage: results of a comprehensive
17

screening approach. Hum Reprod 1995; 10(8):2001-2005.
57. Cervera R, Serrano R, Pons-Estel GJ, et al.
Euro-Phospholipid Project Group (European Forum on
Antiphospholipid Antibodies). Morbidity and mortality inthe antiphospholipid syndrome during a 10-year period:a multicentre prospective study of 1000 patients. Ann
Rheum Dis 2015; 74(6): 1011-1018.
58. Liu L, Sun D. Pregnancy outcomes in patients withprimary antiphospholipid syndrome: A systematic
review and meta-analysis. Medicine (Baltimore). 2019;
98(20): e15733.
59. Do Prado AD, Piovesan DM, Staub HL, Horta BL.
Association of anticardiolipin antibodies withpreeclampsia: a systematic review and meta-analysis.
Obstet Gynecol 2010; 116(6): 1433-1443.
60. Clark EAS, Silver RM, Branch DW. Do
antiphospholipid antibodies cause preeclampsia and
HELLP syndrome? Curr Rheumatol Rep 2007; 9(3):219-225 54.
61. Yamada H, Atsumi T, Kobashi G, et al.
Antiphospholipid antibodies increase the risk ofpregnancyinduced hypertension and adverse
pregnancy outcomes. J Reprod Immunol 2009; 79(2):188-195.
62. Silver RM, Parker CB, Reddy UM, et al.
Antiphospholipid antibodies in stillbirth. Obstet Gynecol
2013; 122(3): 641-657.

DESCIFRAREA UNEI REȚELE COMPLICATE: FACTORII IMUNOLOGICI ÎN INFERTILITATE ȘI
IMPACTUL LOR ASUPRA SĂNĂTĂȚII REPRODUCTIVEREZUMAT
Această lucrare cercetează intricata interacțiune dintre antigenul leucocitar uman fetal (HLA) și receptorii asemănători imunoglobulinei(KIR) de pe celulele NK materne, concentrându-se asupra implicațiilor lor asupra rezultatelor sarcinii. Studiul investighează impactul
nepotrivirilor HLA, în special în contextul sistemului imunitar HY, asupra răspunsului imun matern. Se analizează prezența anticorpilor HLA,
având în vedere corelarea cu nivelurile totale de imunoglobuline, în încercarea de a identifica eventualele asocieri cu complicațiile posibile ale
sarcinii. Activitatea complementului este examinată pentru a adăuga o înțelegere mai profundă a dinamicii interacțiunilor dintre HLA-ul fetal și
KIR-ul matern.
De asemenea, lucrarea investighează echilibrul subtil dintre celulele T helper Th1 și Th2, componente esențiale ale sistemului imunitar, și
influența lor potențială asupra toleranței imune materno-fetale. Se analizează activitatea citotoxică a celulelor ucigașe naturale (NKa), oferind
perspective asupra citotoxicității acestor celule imune și a rolului lor în contextul interacțiunilor materno-fetale. Descoperirile prezentate
contribuie la o înțelegere complexă a factorilor imunologici care influențează rezultatele sarcinii și pot ghida cercetările și abordările cliniceviitoare menite să optimizeze sănătatea atât a mamei, cât și a fătului.
Cuvinte cheie: factori imunologici, infertilitate, sănătate reproductivă

Titlul proiectului: ROHU-339, „Romanian-Hungarian cross-border project with a wider focus on diagnostics related toinfertility, healthy pregnancy and newborn care”, acronim HEALTH-PREGN-RO-HU
Editorul materialului: Spitalul Clinic Județean de Urgență „Pius Brînzeu” Timișoara
Data publicării: decembrie 2023
Acest proiect este cofinanțat de Uniunea Europeană prin Fondul European de Dezvoltare Regională în cadrul Programului Interreg
V-A România-Ungaria.
Conţinutul acestui material nu reprezintă în mod necesar poziţia oficială a Uniunii Europene.

Fiziologia - Physiology • 2023.2(105)ENDOMETRIOSIS - A CAUSE OF INFERTILITY?
IZABELLA PETRE1,2*, RABIA TASDEMIR2, ION PETRE3, LAURENTIU CEZAR TOMESCU4,CRISTIAN FURAU5, ANA DRAGHICI6, ANCA BORDIANU7
1 Department XII, Discipline of Obstetrics and Gynaecology III, “Victor Babeș” University of Medicine of and Pharmacy,Timisoara, Romania
2 Emergency County Hospital Timisoara
3 Department III, Medical Informatics and Biostatistics Discipline, Timisoara, “Victor Babeș” University of Medicine and Pharmacy, Timisoara,
Romania
4 Ovidius University of Constanta,Faculty of Medicine, Romania
5 Department of Obstetrics and Gynecalogy, “Vasile Goldiş” Western University of Arad, Romania
6 Emergency Hospital for Children Louis Ţurcanu, Timisoara, Romania
7 Departament of Plastic Surgery and Reconstructive Microsurgery Bagdasar-Arseni, Emergency Hospital Bucharest, University of Medicine andPharmacy “Carol Davila“, Bucharest, Romania*Correspondence to: petre.izabella@umft.ro

ABSTRACT
Endometriosis, marked by endometrial tissue outside the uterus, is a an important factor in infertility due to tubal and ovarian dysfunction. The
lack of consensus on detection methods leads to delayed diagnoses, impacting therapeutic strategies. Around half a million patients in
Romania suffer from this condition. This study aimed to correlate endometriosis with infertility. The study included 45 diagnosed patients
endometriosi were aged 30 to 40 years.
The study explored correlations between endometriosis and infertility in 45 diagnosed patients, noting a peak occurrence at age 40. Early
menarche (11-14 years) and high nulliparity percentages aligned with previous findings linking younger menarche and infertility inendometriosis. Various correlations between pregnancies, lesion size, and serum Ca-125 were found, indicating a potential link between larger
lesions and higher Ca-125 values, suggesting contributions of these factors to infertility. The study's emphasis on the multifactorial nature of thedisease and its small sample size underscore the need for further investigation.
The thesis choice stems from the lack of consensus on endometriosis etiopathogenesis, diagnosis, and management, compounded by thenormalization of symptoms due to societal stigmatization of menstrual discussions. Clinical and paraclinical links between endometriosis andinfertility were identified, highlights the multifactorial nature of infertility and calling for a comprehensive approach to diagnosis andmanagement. Further research is crucial to unravel the complex interplay between genetic and environmental factors in endometriosis.
In summary, endometriosis poses challenges to fertility through debated mechanisms, delayed diagnoses, and societal stigmatization. The
study highlights correlations and emphasizes the multifactorial nature of infertility, stressing the need for tailored management and further
research.
Key words: endometriosis, early menarche, lesion size, Ca-125, nulliparity, infertility

INTRODUCTION
Endometriosis,
anincompletely
elucidated
inflammatory disease, is the presence of endometrial tissue
outside the uterus. It is associated with subfertility andchronic pelvic pain. The links between this disease andinfertility are a much-debated topic in today's literature.
With this study, we aimed both to establish an answer tothe question "To what extent can endometriosis beconsidered a cause of infertility?" and to establish somepredictors of infertility in endometriosis patients. This could
prove useful in early diagnosis and more efficient andpersonalised management of the disease.
There are several ways in which endometriosis affects
fertility. These include tubal dysfunction, ovariandysfunction, adverse influences of peritoneal fluid on
fertility-related phenomena, mechanical obstructions in the

tubal lumen, changes in tubal-ovarian anatomical
relationships.
In addition, there is still no consensus on effective
minimally invasive methods for detecting endometriosis,
leading to delayed diagnosis.
Both the early diagnosis of endometriosis and thepredictors of infertility associated with it are necessary forthe development of the most effective therapeutic strategy
tailored to the patient's needs. A set of well-defined
investigations would contribute significantly to thisapproach.
It occurs in about 10-15% of women in the
reproductive period and in about 20-50 to 70% of womenwith infertility, according to some studies (1,2,3).
The highest prevalence was recorded between 25 and35 years (4). Reproductive women younger than 35 years
19

of age have twice the risk of infertility if they haveendometriosis than those without the condition (5). In
addition, many cases are asymptomatic for a long period,
with prevalence ranging from 6% to 43% (6), so the true
prevalence becomes difficult to estimate. Unfortunately, formany women there is also a delay in the diagnosis ofendometriosis, leading to unnecessary suffering andreduced quality of life (7).
In Romania it is estimated that there are half a million
patients with endometriosis. 70% of patients with chronic
pelvic pain and 50% of infertile patients have the dsease,
but an accurate diagnosis is difficult to make. Numerous
studies show a 7–10-year delay in diagnosing women indeveloping countries. 47% of patients see more than 5
doctors before the diagnosis of endometriosis isestablished (8).
MATERIAL AND METHOD
The aim of the study was to look for a correlation
between endometriosis and infertility and to detect thepossible existence of predictive factors for these twoentities.
We included in the study all patients who wereadmitted between 01.01.2019 - 31.12.2019 in the
Obstetrics and Gynecology I and II wards of theEmergency Clinical County Hospital "Pius Brȋnzeu"
Timisoara. The main inclusion criterion was the diagnosisof endometriosis. Patient data were collected fromobservation sheets provided by the clinic archive. Data
related to anamnestic, clinical and paraclinical examination,
drug treatment, surgical procedures, discharge diagnoses
were reviewed. Microsoft Excel and SPSS were used forthe database and statistical analysis.
A total of 45 patients diagnosed with endometriosis
were registered. In the first stage, we described the
database using tables and graphical representations, aswell as descriptive statistics specific to the variables
studied.

Most of them are aged between 30 and 40 years(48.89%). We also found that the number of patientsdiagnosed with endometriosis increases with age up to 40
years and then decreases again. The mean age of patientswith endometriosis included in our study was 33 years. The
youngest patient was 17 years old and the oldest 49 years
old. According to Medscape, this trend is similar to otherstudies, and it is generally accepted that endometriosis is adisease of women of reproductive age, with mostdiagnoses being made between the ages of 25 and 35 (9).
RESULTS AND DISCUSSIONS
Many women with endometriosis have an increased
risk of infertility, i.e. their ability to conceive spontaneously
is affected. The percentage of women able to do so varies
according to the severity of the disease, i.e. the moreadvanced the disease, the lower the chance of
spontaneous conception. There are numerous mechanisms
potentially involved in the development of infertility inwomen with endometriosis, e.g. mechanical changes in the
reproductive
organs,chronic
inflammation,
pathophysiological mechanisms affecting the ovum,
especially in ovarian endometriosis (10).
Regarding the age of menarche onset, in our study
most of the patients had menarche at 13 (24.44%) - 14
years (37.78%), but it can be seen that there is a tendencyof menarche onset at younger ages (11-14 years), because
after the age of 14 years, the percentages decreasesignificantly. The lowest age of menarche onset was 10
years and the highest 17 years. (Figure 2). We observed
also that the number of patients diagnosed withendometriosis increases with age until 40 years and thendecreases again. The mean age of endometriosis patients
included in our study was 33 years. The youngest patientwas 17 years old and the oldest 49 years old. According to
Medscape, this trend is similar to other studies, and it isgenerally accepted that endometriosis is a disease of
women of reproductive age, with most diagnoses being
made between the ages of 25 and 35 (11).

Fig. 2. Histogram of the distribution of patients according to thetime of menarche onset
Fig 1. Histogram of patient distribution by age group

Fiziologia - Physiology • 2023.2(105)Multiple studies mention a trend similar to our study,i.e., the occurrence of menarche at a younger age in
people suffering from endometriosis, some evenconsidering this as a risk factor for the development ofendometriosis, being explained by the theory of retrograde
menstruation (12,13).

Percentage of total
patients

No. of patients with nopregnancies

26

57,78%

No. of patients with at least
one pregnancy

1942,22%

No. of patients with at least
one birth

18

40,00%

Approximately 60% of the patients had no previous
pregnancies, which may be partly explained by their
younger age (Table 1). However, endometriosis may be acause of infertility, which could explain this high percentageof women who had not had a spontaneous conception at
the time of the study. The most pregnancies recorded in
one patient were 3. Again, the literature shows anincreased infertility rate in women with diagnosed
endometriosis. In a large cohort study, the risk of infertility
was doubled in women under 35 diagnosed withendometriosis compared to those without endometriosis(14).
The endometriotic lesion locations were diverse in ourgroup of patients: ovary, uterus, pelvis, parietal - at thelevel of post-caesarean section scar and other locations
perecum omentum, rectum, intestinal loops, vagina.
However, by far the most common location was the ovary(73.33%). In terms of which ovary is more commonly
affected, our study found a relatively similar incidence
between the right and left ovary, with one third of cases
having bilateral involvement. According to Medscape, themost common locations of endometriosis are the ovaries,
with some studies showing a frequency of up to 96.4% ofendometriosis located in the ovary (15).

No. ofpatients

Percentage of
total patients

Ovary

33

73,33%

Uterus5

11,11%

8,89%

Post-Cesarean section scar

9

20,00%

Other sites

2

4,44%

Table III. Distribution of patients according to serum Ca125 value
Serum Ca125 levelNo. ofpatients

Percentage of
total patients<46 U/mL

16

35,56%≥46 U/mL

8

17,78%

Unknown

21

46,67%The neutrophil/lymphocyte ratio was within normal
parameters for the majority of patients (86.67%),
suggesting a low degree of body stress. (Table 3),
However, the results are not statistically conclusive in ourstudy, and a number of studies in the literature prove theoppositesituation,inwhich
increased
neutrophil/lymphocyte ratio is strongly correlated withendometriosis, especially in combination with increased
Ca-125 value (19,20).
Table IV. Distribution of patients according toneutrophil/lymphocyte ratio

Table II. Distribution of patients according to the location of theendometriotic lesion
Lesion site

4

Quite a large percentage of patients had no Ca-125
value mentioned in the observation sheet (46.67%). Of theremainder, 35.56% had a serum Ca-125 value within
normal limits, almost double those with Ca-125 greaterthan or equal to 46 U/mL. The mean Ca-125 value was
39.37 U/mL, the highest value being 102.8 U/mL and thelowest 8.6 U/mL (Table 3). Similar to our study, anotherstudy detected a link between increased serum Ca-125
level and endometriosis (16). However, there are no
relevant data in the literature at the acute time point
recommending the use of Ca-125 as a screening method
for endometriosis, nor as a disease severity parameter(17,18).

Table I. Distribution of patients by number of pregnancies andbirthsNo. ofpatients

Pelvis21

Neutrophile/lymphocyte ratio No. ofpatients

Percentage of
total patients

Between 1 and 6

39

86,67%

Between 6 and 9

2

4,44%

Between 9 and 18

0

0,00%

≥18

1

2,22%

Unknown

index

3

Next,
we
calculated
descriptive
statistics
corresponding to the numerical variables of the study.
These are: central tendency parameters (mean, median,mode), dispersion parameters (standard deviation,
variance, standard error, minimum, maximum, amplitude),degree of skewness and degree of flattening of the
distribution. All these calculations are reported in the tables
below (see Table 5-6).
Table V. Descriptive statistics corresponding to the numericalvariables of the study
VolumeHighest lesion ofAge
Menarchemeasurement biggest(years)(age)(cm)
lesion(ml)
Average

33,37777
5,382571
78

35,6177

Standard
error

1,216174
0,384963
2

13,85112 0,222542

Median33

511,375

14

Mode

24

4

6,24

14

Standard
deviation

8,158344
2,277472
55

80,76519 1,459304

Variant

66,55858
5,186878596523,016 2,129568

Symmetry
index-0,46854
0,862616
99

19,40859 0,847647

Flattening
index

0,158539 0,892024

4,202307 0,073556

Amplitude

32

10,07

435,2545 7

Minimum

17

1,93

1,545544 10

Maximum

49

12

436,8

13,32558

17

Table VI. Descriptive statistics corresponding to the numericalvariables of the study (continued)
No. ofNo. of Ca-125 Neutrophilepregnancies births
level/lymphocyte(UI/mL)
ratio
Average

1,02272727

0,613636 39,37417 3,479481

Standard
error

0,18244474

0,113636 5,217265 0,489377

Median

0,5

0

Mode

0

0

Standard
deviation

1,21020153

0,753778 25,55927 3,171524

Variant

1,46458774

0,568182 653,2765 10,05857

Symmetry
index-1,1494377

0,988515 0,299851 23,77434

Flattening

0,69697561

1,129548 0,967657 4,420711

32,95

2,727454
4,923077

Amplitude

3

3

94,2

20,16963

Minimum

00

8,60,905637

Maximum

3

3

102,8

21,07527

We also wanted to examine the extent to which certain
variables in the study are influenced by each other. To
achieve this, we performed correlation and regression
analysis. We tested whether there is a possible association
between the number of loads, lesion size, lesion volume,
Ca-125, and neutrophil/lymphocyte ratio, respectively. We
also examined the degree of association between lesion
size, i.e. lesion volume and age, serum Ca-125 andneutrophil/lymphocyte ratio. We used Pearson's coefficientfor the degree of association of the variables, and todemonstrate statistical significance we set as level ofsignificance. The results obtained are illustrated in thefollowing tables and figures (see Table 7-16, Figure 3-5).
Table VII. Correlation between number of tasks and highest
lesion measurePregnanLesioncies no.
size
Pearson1-.068
correlation
PregnanSig.
cies no.
.697(2-tailed)N
35
35
Pearson-.068
1
correlation
Lesion
Sig.
size
.697
(2-tailed)N
35
35

In this case we obtained a weak but statisticallyinsignificant negative association (r = -0.68, p = 0.697).
Table VIII. Correlation between number of loads and lesion
volumePregnanLesicies no.
onvolumePregnanPearson1.223
cies no.
correlation
Sig.
.205
(2-tailed)N
34
34
LesionvolumePearsoncorrelationSig.
(2-tailed)
N

.223

1

.205
35

34

In the given situation, we obtained a positive, statisticallyinsignificant association (r = 0.223, p = 0.205).
Table IX. Correlation between number of loads and Ca-125 value

Fiziologia - Physiology • 2023.2(105)Pregnancies no.
Pregnancies no.

Ca-125value

PearsoncorrelationSig.
(2-tailed)
N

1

Lesi
on
volume.249

size

Ca-125value.252

PearsoncorrelationSig.
(2-tailed)
N

23

23

-.249

1

23

Table X. Correlation between number of loads andneutrophil/lymphocyte ratio value
Pregnan
Neutrophils/
cies no.
lymphocytes ratio
1
-.030
.853
41
-.030

41
1

32
1

.986
2332

40

In this case, we obtained an extremely weak, positive,
statistically insignificant association (r = 0.003, p = 0.988).

.853
41

32
-.003

Table XIII. Correlation between highest lesion measurement andage
AgLesionesize
AgPearson1.003
ecorrelation
Sig. (2-tailed)
.988
N
35
35
Le
Pearson.00
1
sion
correlation
3
size
Sig. (2-tailed)
.98
8
N
35
35

Here we obtained a statistically insignificant negative
association (r = -0.249, p = 0.252).

Pregnanc
Pearsonies no.
correlationSig.
(2-tailed)N
Neutrophi
Pearsonls/
correlation
lymphocytes
Sig.
ratio
(2-tailed)N.986

In this situation we obtained an extremely weak,
positive, statistically insignificant association (r = 0.003, p =0.986).

.252
23

correlationSig.
(2-tailed)N
PearsoncorrelationSig.
(2-tailed)N

Table XIV. Correlation between lesion volume and Ca-125
LesionCa-1
volume25 value
LesionPearson1.156
volumecorrelation
Sig. (2-tailed)
.511
N
20
20
Ca-125Pearson
-.156
1
value
correlation
Sig. (2-tailed)
.511
N
20
20

41

In this case, we obtained a very weak negative
association, not statistically significant (r = -0.030, p =0.853).
Table XI. Correlation between highest lesion measurement andCa-125
LesionCa-1
size
25 value
LesionPearson1.365
size
correlation
Sig. (2-tailed)
.104
N
21
21
Ca-125Pearson
-.365
1
value
correlation
Sig. (2-tailed)
.104
N
21
21

Here we obtained a weak, positive, statisticallyinsignificant association (r = 0.156, p = 0.511).
Table XV. Correlation between lesion volume andneutrophil/lymphocyte ratio

LesionvolumeHere we obtained a positive, statistically insignificant
correlation (r = 0.365, p = 0.104).

LesionvolumeTable XII. Correlation between highest lesion measurement andneutrophil/lymphocyte ratio
LesionLesisizeonvolumeLesionPearson1.003

Neutrophils/
23

PearsoncorrelationSig.
(2-tailed)N
Pearsoncorrelation

1

Neutrophils/lymphocytes
ratio
-.118
.527

31
-.11
8

31
1

lymphocytes
ratio

Sig.
(2-tailed)
N

.527
31

31

In this case we obtained a weak, negative, statisticallyinsignificant correlation (r = -0.118, p = 0.527).
Table XVI. Correlation between lesion volume and age
LesionAgevolumeLesionPearson1.034
volumecorrelation
Sig. (2-tailed)
.849
N
34
34
Age
Pearson-.034
1
correlation
Sig. (2-tailed)
.849
N
34
34

In this situation we obtained a very weak, positive,
statistically insignificant correlation (r = 0.034, p = 0.8849).
The highest association was present between number ofloads and lesion volume, number of loads and serum
Ca-125 and the highest lesion measurement and serum
Ca-125.

Figure 5. Degree of association between lesion size and serum
Ca-125 value

In the correlation and regression analysis we observeda low degree of association between the variables studied,which may be attributed to the small number of samples inthe study group, especially given the incompletely
elucidated multifactorial nature of the disease. There are a
few associations worth mentioning. The positive
association between the number of pregnancies and lesion
volume (r = 0.22, p = 0.2) can be explained by the
association of these two variables independently withpatient age. The negative association between number ofpregnancies and Ca-125 value (r = 0.24, p = 0.2) isinterpreted as an increase in Ca-125 is correlated with alow number of pregnancies in a patient (Figure 6).

Fig 3. Degree of association between number of loads and lesion
volumeserum Ca-125

Fig. 6. Degree of association between number of loads andserum Ca-125

Fig. 4. Degree of association between number of loads andserum Ca-125

Last but not least, the strongest positive association in
our study was between lesion size and Ca-125 value (r =0.36, p = 0.1) - the larger the lesion size, the higher the
Ca-125 value.
The strongest association was present between
number of loads and lesion volume, number of loads andserum Ca-125 value and largest lesion size and serum
Ca-125. These three measurements will be represented byscatterplots to show their distribution (see Figure 7).

Fiziologia - Physiology • 2023.2(105)

link between infertility and endometriosis exists, the view
today is that infertility in endometriosis is multifactorial.
In conclusion, in recent years, endometriosis is seen
as a multifactorial disease, with both genetic andenvironmental risk factors, and its contribution to subfertility
status is not yet fully elucidated, but it can no longer beseen as the sole causative factor. A comprehensive
approach to the diagnosis and management of this diseaseis therefore needed.

Fig.7. Degree of association between number of loads
and lesion volume

REFERENCES
1. Kumar V., Abbas A.K., Aster J.C.: Robbins Basic
Pathology,10th edition, Elsevier,2018 (cap. 19, pg. 721).
2. Parasar P, Ozcan P, Terry KL. Endometriosis: Epidemiology,
Diagnosis and Clinical Management. Curr Obstet Gynecol
Rep. 2017 Mar;6(1):34-41. doi: 10.1007/s13669-017-0187-1.
Epub 2017 Jan 27. PMID: 29276652; PMCID: PMC5737931.
3. Tanbo T, Fedorcsak P. Endometriosis-associated infertility:
aspects of pathophysiological mechanisms and treatment
options.
Acta
ObstetGynecol
Scand.
2017
Jun;96(6):659-667. doi: 10.1111/aogs.13082. Epub 2017 Jan
30. PMID: 27998009.
4. Filip L, Duică F, Prădatu A, Crețoiu D, Suciu N, Crețoiu SM,
Predescu DV, Varlas VN, Voinea SC. Endometriosis
Associated Infertility: A Critical Review and Analysis on
Etiopathogenesis and Therapeutic Approaches. Medicina(Kaunas).
2020
Sep
9;56(9):460.
doi:
10.3390/medicina56090460. PMID: 32916976; PMCID:
PMC7559069
5. Tanbo T, Fedorcsak P. Endometriosis-associated infertility:
aspects of pathophysiological mechanisms and treatment
options.
Acta
ObstetGynecol
Scand.
2017
Jun;96(6):659-667. doi: 10.1111/aogs.13082. Epub 2017 Jan
30. PMID: 27998009.
6. Malvezzi, H., Marengo, E.B., Podgaec, S. et al.
Endometriosis: current challenges in modeling a multifactorialdisease of unknown etiology. J Transl Med 18, 311 (2020).
https://doi.org/10.1186/s12967-020-02471-0
7. Broi MGD, Ferriani RA, Navarro PA. Ethiopathogenic
mechanisms of endometriosis-related infertility. JBRA Assist
Reprod.
2019Aug22;23(3):273-280.
doi:
10.5935/1518-0557.20190029. PMID: 31091056; PMCID:
PMC6724396.
8. Dumitru C, Mladin NC, Craina M, Petre I, Moleriu LC, Bardan
R, Bonte DC, Ungureanu E, Stefaniga SA, Susan R, Pop E,
Suciu N. Can the CA-125 and Neutrophil-to-lymphocyte Ratio
Values be Considered as a Diagnostic Value in Ovarian
Endometriosis?.
Rev
Chem.[internet].
2018
Dec;69(12):3561-3564.
Availablefrom:https://doi.org/10.37358/RC.18.12.6792
9. Davila G, Kapoor D, Alderman E, et al. Endometriosis; May
2021
article;https://emedicine.medscape.com/article/271899-overview#a5.
10. Hirsch M, Begum MR, Paniz É, Barker C, Davis CJ, Duffy J.
Diagnosis and management of endometriosis: a systematic
review of international and national guidelines. BJOG. 2018
Apr;125(5):556-564. doi: 10.1111/1471-0528.14838. Epub
2017 Nov 27. PMID: 28755422
11. Davila G, Kapoor D, Alderman E, et al. Endometriosis; May
2021
article;The lesion size was generally small, but larger inpatients without previous pregnancies compared to thosewith previous pregnancies. This result is consistent with the
hypothesis that ovarian reserve shrinkage, being replacedby endometriotic cyst, is a possible cause of infertility inwomen with this diagnosis (21), but there are a number offactors that could contribute to infertility in this disease (22).
CONCLUSIONSThe lack of consensus regarding the etiopathogenesis,
diagnosis and management of endometriosis, the impactthat this disease has on a significant number of patients, aswell as the stigmatization of menstrual cycle-related
discussions that have led to the normalization of symptoms
such as pelvic pain are the main reasons why I chose this
topic for my thesis. I believe that endometriosis is acomplex disease with diverse implications in women's lives
especially in terms of fertility, so the existence of predictive
factors in terms of correlation between the two pathologies
would be very beneficial in clinical practice.
We believe that many of the hypotheses studied wererelevant to clinical practice, especially as more informationis needed regarding predictive, diagnostic and prognostic
factors for this disease, and this work can be a starting
point for possible future more conclusive studies.
In terms of the questions we have tried to answer
since the beginning of the paper, our patient cohort showedthat there are clinical and paraclinical links between
endometriosis and infertility, but the exact pathophysiologyof this link remains unelucidated and a comprehensive
approach to the diagnosis and management of infertility inendometriosis is needed. So we can say that although the25https://emedicine.medscape.com/article/271899-overview#a5.
12. Parazzini F, Esposito G, Tozzi L, Noli S, Bianchi S.
Epidemiology of endometriosis and its comorbidities. Eur J
Obstet Gynecol Reprod Biol. 2017 Feb;209:3-7. doi:
10.1016/j.ejogrb.2016.04.021. Epub 2016 Apr 30. PMID:
27216973
13. Arumugam K, Lim JM. Menstrual characteristics associatedwith endometriosis. Br J Obstet Gynaecol. 1997
Aug;104(8):948-50.
doi:
10.1111/j.1471-0528.1997.tb14357.x. PMID: 9255089.
14. Burney RO, Giudice LC. Pathogenesis and pathophysiology
of endometriosis. Fertil Steril. 2012 Sep;98(3):511-9. doi:
10.1016/j.fertnstert.2012.06.029. Epub 2012 Jul 20. PMID:
22819144; PMCID: PMC3836682.
15. Treloar SA, Bell TA, Nagle CM, Purdie DM, Green AC. Early
menstrual characteristics associated with subsequentdiagnosis of endometriosis. Am J Obstet Gynecol. 2010
Jun;202(6):534.e1-6. doi: 10.1016/j.ajog.2009.10.857. Epub
2009 Dec 22. PMID: 20022587
16. Karimi-Zarchi M, Dehshiri-Zadeh N, Sekhavat L, Nosouhi F.
Correlation of CA-125 serum level and clinico-pathologicalcharacteristic of patients with endometriosis. Int J Reprod
Biomed. 2016 Nov;14(11):713-718. PMID: 28008424;
PMCID: PMC5153578.
17. Mu F, Harris HR, Rich-Edwards JW, Hankinson SE, Rimm
EB, Spiegelman D, Missmer SA. A Prospective Study of
Inflammatory Markers and Risk of Endometriosis. Am J
Epidemiol.
2018Mar
1;187(3):515-522.
doi:
10.1093/aje/kwx272.
PMID:
28992341;
PMCID:
PMC5859972.
18. Rokhgireh S, Mehdizadeh Kashi A, Chaichian S, Delbandi
AA, Allahqoli L, Ahmadi-Pishkuhi M, Khodaverdi S, Alkatout I.
The Diagnostic Accuracy of Combined Enolase/Cr, CA125,
and CA19-9 in the Detection of Endometriosis. Biomed Res
Int. 2020 Sep 2;2020:5208279. doi: 10.1155/2020/5208279.
PMID: 33062681; PMCID: PMC7545435.
19. Yang H, Lang JH, Zhu L, Wang S, Sha GH, Zhang Y.
Diagnostic value of the neutrophil-to-lymphocyte ratio and the
combination of serum CA-125 for stages III and IV
endometriosis. Chin Med J (Engl). 2013;126(11):2011-4.
PMID: 23769549.
20. Cho S, Cho H, Nam A, Kim HY, Choi YS, Park KH, Cho DJ,
Lee BS. Neutrophil-to-lymphocyte ratio as an adjunct toCA-125 for the diagnosis of endometriosis. Fertil Steril. 2008
Dec;90(6):2073-9. doi: 10.1016/j.fertnstert.2008.03.061. Epub
2008 Jun 13. PMID: 18555226.
21. Ramachandran A, Dhulkhed S, Bhakta R, Bhat RG, Rao AC,
Vasudeva A, Vishalakshi A, Kumar P. Influence ofendomeriotic cyst diameter and the severity of endometriosis
on the ovarian parenchyma excised during laparoscopic
cystectomy. J Clin Diagn Res. 2013 Oct;7(10):2241-3. doi:
10.7860/JCDR/2013/5730.3481. Epub 2013 Oct 5. PMID:
24298486; PMCID: PMC3843466.
22. Broi MGD, Ferriani RA, Navarro PA. Ethiopathogenic
mechanisms of endometriosis-related infertility. JBRA Assist
Reprod.
2019Aug22;23(3):273-280.
doi:
10.5935/1518-0557.20190029. PMID: 31091056; PMCID:
PMC6724396.

Fiziologia - Physiology • 2023.2(105)INFECȚIA ȘI COLONIZAREA BACTERIANĂ LA FEMEILE INSARCINATE ȘI LA NOU-NASCUȚIREZUMAT
Diferite tipuri de microorganisme, bacterii, paraziți, virusuri și fungi, pot coloniza și pot determina infecții la nivelul tractului
genito-urinar feminin. Mai mult decât atât, nu puține femei expuse la infecțiile genitale bacteriene rămân insărcinate, crescând astfel
semnificativ riscul de transmitere al infecțiilor la nou-nascuți. Infecțiile neonatale au o importanță deosebită datorită riscului crescut demorbiditate și mortalitate. Scopul studiului de față a fost identificarea tulpinilor bacteriene din culturi de probe cervicale provenite de lapaciente însărcinate pe perioada unui an de zile, determinarea prevalenței și a rezistenței microbiene pentru fiecare patogen și corelarea
rezultatelor cu etapele sarcinii, respectiv cu diferite patologii de sarcină. Datele studiului prezent au evidențiat următoarele: cel mai
frecvent patogen Gram-negativ detectat a fost E. coli, în timp ce streptococul de grup B a prezentat cea mai mare prevalență din grupul
bacteriilor Gram-positive. Important de menționat este faptul că rezultatele au arătat un număr substanțial de microorganisme depistate în
culturile pacientelor cu sarcină în evoluție/sarcină la termen. Cu toate acestea, nu puțini patogeni au fost identificați în culturile pacientelorcare au prezentat avort sau alte patologii de sarcină, unele tulpini având fenotipuri de rezistență care necesită o atenție sporită în ceea ceprivește îngrijirea medicală și tratamentul infecțiilor din timpul sarcinii sau din timpul travaliului la pacientele însărcinate. În mod expectativ,
achiziționarea și implementarea unei noi linii automate de prelucrare microbiologică a probelor ar putea ajuta la identificarea mult mai
rapidă a microorganismelor și ar putea crește vigilența în supravegherea infecțiilor la pacientele însărcinate și la nou-născuți.
Cuvinte cheie: infecții genitale, sarcină, patologii neonatale, fenotip de rezistență

Titlul proiectului: ROHU-339, „Romanian-Hungarian cross-border project with a wider focus on diagnostics related toinfertility, healthy pregnancy and newborn care”, acronim HEALTH-PREGN-RO-HU
Editorul materialului: Spitalul Clinic Județean de Urgență „Pius Brînzeu” Timișoara
Data publicării: decembrie 2023
Acest proiect este cofinanțat de Uniunea Europeană prin Fondul European de Dezvoltare Regională în cadrul Programului Interreg
V-A România-Ungaria.
Conţinutul acestui material nu reprezintă în mod necesar poziţia oficială a Uniunii Europene.

27

INTRACRANIAL ANEURYSM UNVEILING AS NEUROLOGICAL DEFICITS:
A LITERATURE REVIEW AND DIAGNOSTIC CHALLENGEEMILIA BURADA1#, MADALINA ALDEA 2#, DENISA PIRSCOVEANU3, BURDUSEL DAIANA2,4,
RALUCA ELENA SANDU 3,4,*, ROXANA SURUGIU 4*, CARMEN VALERIA ALBU3
1

2

3

4

*
#

Department of Phisiology, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;
Department of Psychiatry, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;
Department of Neurology, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;
Department of Biochemistry, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;
Correspondence: Raluca Elena Sandu, raluca.sandu@umfcv.ro; Roxana Surugiu, roxana.surugiu@umfcv.ro
These authors contributed equally

ABSTRACT:
Intracranial saccular or berry aneurysms are abnormal blood vessel formations within the brain, responsible for the
majority of nontraumatic subarachnoid hemorrhages (SAH). Advances in medical imaging have improved detection rates,
though the prevalence of these aneurysms increases with age, particularly in women. Moreover, studies suggest a higher
risk of de novo aneurysm formation in females and highlight the role of unstable blood pressure as a risk factor. Aneurysms
are also associated with variations in estrogen levels and cardiometabolic risk factors, including hypertension, body massindex, and diabetes. Smoking is identified as a significant risk factor, with smoking-related vascular endothelial dysfunction
contributing to aneurysm rupture. To illustrate the diagnostic and management challenges of intracranial aneurysms, a casereport is presented. The case involves a 29-year-old female patient who initially presented with symptoms mimicking
ischemic stroke. Subsequent evaluation revealed a right internal carotid artery aneurysm, cervical-lumbar discopathy, andthyroid pathology. The case highlights the importance of thorough assessment and consideration of atypical etiologies inpatients with stroke-like symptoms. The study supports the proactive management of symptomatic aneurysms, as well as
the role of embolization and direct arterial reconstruction in addressing aneurysms at risk of neurological complications andunderscores the need for a comprehensive approach to their diagnosis and management, particularly in cases with atypicalclinical presentations.
Keywords: intracranial aneurysm; risk factors; estrogen; cardioembolic; smoking; coffee; physical activity.
1.Epidemiology
Intracranial saccular or berry aneurysms areabnormal, bulging blood vessel formations in the brainthat are typically acquired and are responsible forapproximately 80% of nontraumatic subarachnoid
hemorrhages (SAH) [1]. The detection rate of theseaneurysms on arteriograms and magnetic resonanceimaging (MRI) studies ranges from 1 to 2% [2], whereas
autopsy studies indicate a slightly higher detection rateof 1 to 9% [3]. It's worth nothing that advances in medical
imaging techniques, such as cross-sectional computed
tomography (CT) and high-quality MRI, have improved
the chances of identifying these aneurysms in recent
years, contributing to the increase in their diagnosis [4,5].
Furthermore, the prevalence of unruptured
intracranial aneurysms seems to rise with age, which ispartly due to the aging of the population [6]. Surprisingly,

women may have a higher likelihood of developing
aneurysms compared to men, with a study on unruptured
aneurysms revealing a 3:1 ratio of women to men [7,8].
When it comes to the development of new aneurysms,
extensive long-term follow-up studies and meta-analyses
have revealed a significantly greater risk of de novo
aneurysm formation in females when compared to males[9,10]. Numerous studies have indicated that it's notnecessarily high blood pressure but rather fluctuations inblood pressure that pose a risk factor for aneurysm
rupture [11]. Irregular use of antihypertensive medication
has been associated with increased variability in bloodpressure [11].Additionally, research has shown that theincidence of aneurysm rupture is lower in white patientscompared to african -american patients [12].

Fiziologia - Physiology • 2023.2(105)2.Risk factors
2.1. Estrogen levels
The activation of different estrogen subtypes andreceptors has been demonstrated to have varying effects
on the formation and rupture of intracranial aneurysms in
mice. In female mice that underwent ovariectomy, theadministration of an estrogen receptor-β agonist notably
decreased the incidence of aneurysms. However, theadministration of an estrogen receptor-α agonist or
17β-estradiol did not lead to a reduction in theoccurrence of intracranial aneurysms [13].
In estrogen-deficient mice, there is an elevation in
the serum levels of IL-6, and this cytokine seems to be
involved in promoting the rupture of cerebral aneurysms
associated with estrogen deficiency in mice. This
promotion occurs through an increased infiltration ofmacrophages at the circle of Willis [14].
The connection between estrogen deficiency and theheightened risk of aneurysm development has also been
established in humans [15,16]. The difference in theincidence of unruptured intracranial aneurysms (UIA)
based on gender became increasingly noticeable inindividuals aged 50 and older, implying a potential
contribution of estrogen deficiency to this discrepancy[15]. Furthermore, a case-control study that investigated
the relationship between the age at menopause and thepresence of intracranial aneurysms found a trend
suggesting that an earlier age at menopause wasassociated with the presence of cerebral aneurysms [17].
2.2. Cardiometabolic risk factors
When it comes to cardiometabolic risk factors, priorresearch has consistently shown that hypertension islinked to an increased risk of aneurysmal subarachnoid
hemorrhage (aSAH) [18,19], supported by magnetic
resonance (MR) results for both systolic and diastolicblood pressure [20].
However, findings have been conflicting for factors
like body mass index and diabetes. For instance, theassociation between body mass index and aSAH varies
between genders [21]. On the contrary, a borderline
significant positive association between body mass index
and aSAH was noted in previous MR studies conductedin both women and men [20,22]. Notably, high bodymass index is a well-established risk factor for type 2
diabetes, which has shown an inverse association withaSAH in a meta-analysis of case-control studies and anon-significant inverse association in a pooled analysis
of cohort studies [21]. The relationship between GHB(glycated hemoglobin, a marker of long-term blood sugar
control) and the risk of intracranial aneurysm (IA) rupture
is not straightforward and exhibits a nonlinear pattern. In
cases where individuals with diabetes mellitus (DM) who
have a single IA achieve good glycemic control, there isa reduction in the risk of IA rupture. Conversely, if
glycemic control is poor, the risk of IA rupture increases[23].
Regarding lipid levels, the available observationaldata, primarily from case-control studies, suggest that
hypercholesterolemia and high levels of high-density
lipoprotein are associated with a reduced risk of aSAH
[24]. Genetically determined levels of HDL-C(high-density lipoprotein cholesterol) and LDL-C(low-density lipoprotein cholesterol) have been
associated with a reduced risk of intracranial aneurysms
and recurrent intracranial aneurysms (rIA) [25].
2.3. Modifiable risk factors
Findings of earlier research have consistently
highlighted smoking as a significant risk factor for aSAH
[21,22]. A multivariate analysis comparing individuals
who are current smokers to those who have neversmoked, demonstrates that smoking is significantly
correlated with multiple aneurysms, and an increased
prevalence of aneurysms located at the basilar apex [26].
Tobacco smoke contains nicotine as well as a multitudeof other compounds that have the potential to promote
dysfunction in the inner lining of blood vessels, known as
vascular endothelial dysfunction, and ultimately
contribute to the rupture of intracranial aneurysms [27].
Mechanistic studies further reveal that nicotine exposure
amplifies the risk of IA rupture by acting on vascular cell
nicotinic acetylcholine receptors that contain α7 subunits.
This action leads to elevated levels of vascularendothelial growth factor, platelet-derived growth factor-B,
and inflammatory cytokines, all of which are processes
that can compromise blood vessel integrity and increase
the likelihood of an IA rupturing [27,28].
Research on the relationship between coffee
consumption and the risk of aneurysmal subarachnoid
hemorrhage is limited, and the findings have been
inconclusive. In some studies, an inverse association
between coffee consumption and aSAH risk was
observed, such as in cohorts of Swedish women [29].
However, in other studies, a neutral association wasreported, as seen in Finnish male smokers [30]. The
latest research provides evidence suggesting that theconsumption of coffee may be linked to an increased riskof IA and the subsequent occurrence of hemorrhage [31].
Regular physical activity has been linked to alowered risk of aSAH in various population groups,
including men of Japanese ancestry [32], Finnish adults[33], and over 1 million UK women [34]. Physicalactivity's protective effect against aSAH may be
attributed to its ability to enhance endothelial function,
lower blood pressure, and reduce systemic inflammation
[20].
29

3.Case Presentation:
In the realm of neurology and vascular pathology,
patients often present with a diverse array of symptoms
that challenge the diagnostic acumen of healthcare
providers. While ischemic strokes are typically attributed
to a well-defined set of cardiovascular risk factors, suchas hypertension, diabetes, and atherosclerosis, there
exist uncommon scenarios that confound conventional
diagnostic expectations [35-38].
This article delves into a unique case that uncovers
the intricate relationship between intracranial aneurysms
and neurological symptoms. Unlike the moreconventional association of aneurysms with hemorrhagic
strokes, in this instance, the aneurysm itself is the root
cause of debilitating symptoms, as described in theliterature [39]. The patient's clinical presentation is notcharacterized by the usual manifestations of an ischemic
stroke but rather by neurological deficits directly resulting
from the presence of the aneurysm.
Patient Information:
A 29-year-old female with a known history of
endocrine pathology and a rural background presented
with sudden-onset right limb weakness.
Clinical Examination:
The patient was in good general condition, conscious,and cooperative. Neurological examination revealed:absence of neck stiffness, preserved ocular motility inboth axes, symmetric facial appearance with nonystagmus, right-sided hemiparesis, mild hypotonia,
bilaterally present osteotendinous reflexes, bilateralplantar flexion. Blood cell count data and biochemical
findings are stated in Table I and II.
Imaging:
Cranial CT scan revealed the absence of recent
vascular lesions. However, it did indicate the presence ofan aneurysmal dilation in the right infra-cavernousinternal carotid artery. Additionally, there was observed
thickening of the mucous membrane in the rightsphenoidal sinus, along with a pre-communicating arteryon the left side displaying a hypoplastic appearance.
Furthermore, the ventricular system was found to bealigned along the midline.
Cranial MRI demonstrated a lack of signal
abnormalities or diffusion in favor of hemorrhagic lesions
and showed no current demyelination. However, it did
reveal a saccular aneurysmal dilation in the internal
carotid artery, specifically in the C5-C6 clival segment,
measuring
8/9
mm.
Additionally,
theleft
pre-communicating artery displayed a hypoplastic
appearance, while the venous sinuses remained patent,
and no signs of pathological intraneural intra-axial
tumor-like contrast enhancements were identified (Fig.

1).
The MRI investigation reveals a lack of signal
abnormalities or diffusion in favor of hemorrhagic lesions
and showed no current demyelination, with a saccularaneurysmal dilatation in the internal carotid artery,
specifically in the C5-C6 clival segment, measuring 8/9
mm (Fig. 2).
Spine X-ray: The X-ray of the cervical spine revealeda slight anterior bending or compression in the C5-C6
intervertebral space in the lower half, along with astraightening of the cervical spine. The X-ray of thelumbar spine indicated an accentuated lumbar lordosis,
suggesting narrowing or compression of theintervertebral spaces in the L4-S1 region.
ANGIO MRI revealed the presence of a right ICA
aneurysm, encouraging the patient's transfer to the
Department of Vascular Neurosurgery, whereacerebral angiography of the 4 major vessels reveals
the presence of the aforementioned aneurysm (Fig. 3).
By combining the patient's medical history, generalclinical examination, neurological assessment, laboratory
results, and clinical explorations, we have directed ourdiagnosis towards: Right hemiparesis. Aneurysm in the
territory of the right internal carotid artery, supported by
the sudden symptomatology, manifested by a motor
deficit of 4 out of 5 in the right upper limb, 4 out of 5 in
the right lower limb, and mild hypotonia.
In the acute phase, the patient was prescribed
Aspenter 100 mg thrice daily, later reduced to aonce-daily regimen. To maintain stability, the patient
received 500 ml of NaCl twice daily, and proton pumpinhibitor therapy was initiated with Pantoprazole FI once
daily. In addition, Famotidine at a dose of 40 mg wasadministered once daily.
Subsequent interventions took place, with aninterventional procedure targeting the aneurysmal
formation in the ophthalmic segment of the right internal
carotid artery. This procedure involved a right femoral
approach, using a 6F sheath and a Chaperon 6F
catheter. The guiding catheter was placed within the rightinternal carotid artery, enabling access to the aneurysm
sac via a microcatheter PX Slim. While coil embolization
was attempted, it was temporarily postponed to facilitate
the placement of a flow diverter. A follow-up intervention
occurred to address the same aneurysmal formation in
the ophthalmic segment of the right internal carotid artery.
During the hospitalization period the patient received
Algocalmin 500mg/2 ml, NaCl 0.9% 500 ml,
Dexamethasone 8 mg/2ml, Esomeprazole 40 mg(powder for IV administration), Heparin 25,000 IU/5 ml,
Iodine 10% 1000 ml, Iomeron 350 mg/100 ml,
Omniopaque 350 mg/ml, Pantoprazole 40 mg (powder
for injection), Paracetamol solution 100 ml, Protecardin
Fiziologia - Physiology • 2023.2(105)75 mg tablets, and Ringer's solution 500 ml.
Upon discharge, the patient was advised to follow ahygienic and dietary regimen, including low sodium, lowlipid, and low carbohydrate intake. Additionally, thepatient was instructed to limit sun exposure, especiallyavoiding extreme temperatures, and engage in physicalactivities within their tolerance level. For chronic hometreatment, the patient was prescribed Aspenter 75 mg
once daily, Controloc 20 mg twice daily for 20 days permonth, and Brilique 90 mg once daily for six months, witha planned angiographic follow-up.
In conclusion, we presented the case of a
29-year-old female patient, without significant risk factors
for arterial hypertension, admitted for clinical andimaging evaluation to exclude endovascular treatment ofa saccular aneurysm located in the right internal carotid
artery (ICA) ophthalmic segment. The decision wasmade to perform embolization with stent-assisted coiling,
for which dual antiplatelet therapy with Aspirin andTicagrelor was initiated.

unruptured aneurysms [44]. In parallel, the managementof extracranial internal carotid artery aneurysms often
involves surgical treatment to mitigate the risk of cerebral
ischemia due to thrombus formation or rupture, as
discussed by Yamaguchi et al. (1997). This approach isconsistent with the interventional measures taken in ourpatient's case, where embolization was deemednecessary to address the symptomatic aneurysm [45].
Both sources advocate for a proactive approach toaneurysms that present with symptoms or carry asignificant risk of neurological complications.
Additionally, the treatment of high cervical carotid
aneurysms, with bypass and carotid ligation, provides
insight into the complex surgical strategies that may berequired when direct arterial reconstruction is not
feasible [46].
This case adds to the literature by highlighting theneed for a high index of suspicion for aneurysms inatypical presentations of neurological deficits andsupports a multidisciplinary approach to management, assuggested by the success seen in similar cases [47].
This case reinforces the importance of considering
intracranial aneurysms in the differential diagnosis ofacute neurological deficits and suggests that tailoredtherapeutic strategies are essential for optimal
outcomes.

4.Discussion
Intracranial aneurysms are typically asymptomatic
but can present with neurological deficits in rare cases,
as discussed by Wiebers et al. [40] note that small,
unruptured aneurysms rarely cause symptoms unlessthey press on adjacent neural structures or cause
microembolic events. The motor deficit observed in ourpatient aligns with findings from a study, which suggeststhat aneurysms in the carotid artery can present withfocal neurological symptoms due to compression or
microembolization [41]. For instance, bilateralextracranial aneurysms of the internal carotid artery,emphasize the rarity and potential severity of suchpresentations, underscoring the importance of surgicalintervention in preventing complications [42].
Furthermore, the association of carotid arteryaneurysms with systemic diseases such as Takayasu's
arteritis, as described by Funiu et al. (2004), highlights
the need for a comprehensive evaluation of thevasculature in patients presenting with atypicalsymptoms [43]. This is particularly relevant given the
rapid enlargement and high risk of rupture associatedwith such aneurysms.
The therapeutic approach in this case, involving
Aspenter and proton pump inhibitors followed byinterventional coiling, is supported by the ISUIA study(International Study of Unruptured Intracranial
Aneurysms), which found the intervention to bebeneficial in patients with symptomatic aneurysms,
particularly when the risk of rupture is considered high.
The ISUIA study highlights the importance of intervention
in preventing the potential complications associated with

References
1. Brown RD. Unruptured intracranial aneurysms.
Semin Neurol. 2010 Nov;30(5):537-44. doi:
10.1055/s-0030-1268858. Epub 2011 Jan 4. PMID:
21207346.
2. Vernooij MW, Ikram MA, Tanghe HL, et al. Incidental
findings on brain MRI in the general population. N
Engl J Med 2007;357(18):1821–1828
3. Jakubowski J, Kendall B. Coincidental aneurysms
with tumours of pituitary origin. J Neurol Neurosurg
Psychiatry 1978;41(11):972–979
4. Wardlaw JM, White PM. The detection andmanagement of unruptured intracranial aneurysms.
Brain. 2000 Feb;123 ( Pt 2):205-21. doi:
10.1093/brain/123.2.205. PMID: 10648430.
5. Menghini VV, Brown RD Jr, Sicks JD, O’Fallon WM,
Wiebers DO. Clinical manifestations and survival
rates among patients with saccular intracranial
aneurysms: population-based study in Olmsted
County, Minnesota, 1965 to 1995. Neurosurgery
2001;49(2):251–256, discussion 256–258
6. Jiang H, Weng YX, Zhu Y, Shen J, Pan JW, Zhan RY.
Patient and aneurysm characteristics associatedwith rupture risk of multiple intracranial aneurysms
in the anterior circulation system. Acta Neurochir(Wien).
2016
Jul;158(7):1367-75.
doi:

31

7.

8.

9.

10.

11.

12.

13.

14.

15.

10.1007/s00701-016-2826-0. Epub 2016 May 10.
PMID: 27165300.
Meissner I, Torner J, Huston J 3rd, Rajput ML,
Wiebers DO, Jones LK Jr, Brown RD Jr;
International Study of Unruptured Intracranial
Aneurysms Investigators. Mirror aneurysms: areflection on natural history. J Neurosurg. 2012
Jun;116(6):1238-41. doi: 10.3171/2012.1.JNS11779.
Epub 2012 Mar 9. PMID: 22404675; PMCID:
PMC3914146.
International Study of Unruptured Intracranial
Aneurysms Investigators. Unruptured intracranial
aneurysms—risk of rupture and risks of surgicalintervention. N Engl J Med 1998;339(24):1725–1733
Giordan E, Lanzino G, Rangel-Castilla L, Murad MH,
Brinjikji W. Risk of de novo aneurysm formation inpatients with a prior diagnosis of ruptured or
unruptured aneurysm: systematic review andmeta-analysis.
J
Neurosurg.
2018Jul
6;131(1):14-24. doi: 10.3171/2018.1.JNS172450.
PMID: 29979115.
Hu S, Yu N, Li Y, Hao Z, Liu Z, Li MH. A
Meta-Analysis of Risk Factors for the Formation of
de novo Intracranial Aneurysms. Neurosurgery.
2019
Oct
1;85(4):454-465.
doi:
10.1093/neuros/nyy332. PMID: 30085204.
Feigin VL, Rinkel GJ, Lawes CM, Algra A, Bennett
DA, van Gijn J, Anderson CS. Risk factors forsubarachnoid hemorrhage: an updated systematic
review of epidemiological studies. Stroke. 2005
Dec;36(12):2773-80.
doi:
10.1161/01.STR.0000190838.02954.e8. Epub 2005
Nov 10. PMID: 16282541.
Broderick JP, Brott T, Tomsick T, Huster G, Miller R.
The risk of subarachnoid and intracerebral
hemorrhages in blacks as compared with whites. N
Engl J Med. 1992 Mar 12;326(11):733-6. doi:
10.1056/NEJM199203123261103. PMID: 1738378.
Tada Y, Makino H, Furukawa H, Shimada K, Wada K,
Liang EI, Murakami S, Kudo M, Kung DK, Hasan
DM, Kitazato KT, Nagahiro S, Lawton MT,
Hashimoto T. Roles of estrogen in the formation of
intracranial aneurysms in ovariectomized female
mice. Neurosurgery. 2014 Dec;75(6):690-5;
discussion
695.
doi:
10.1227/NEU.0000000000000528. PMID: 25181430;
PMCID: PMC4399640.
Wajima D, Hourani S, Dodd W, Patel D, Jones C,
Motwani K, Fazal HZ, Hosaka K, Hoh BL.
Interleukin-6
Promotes
MurineEstrogen
Deficiency-Associated Cerebral Aneurysm Rupture.
Neurosurgery. 2020 Apr 1;86(4):583-592. doi:
10.1093/neuros/nyz220. PMID: 31264696; PMCID:
PMC7317988.
Turan N, Heider RA, Zaharieva D, Ahmad FU,
Barrow DL, Pradilla G. Sex Differences in theFormation of Intracranial Aneurysms and Incidence
and Outcome of Subarachnoid Hemorrhage: Review

16.

17.

18.

19.

20.

of Experimental and Human Studies. Transl Stroke
Res.
2016
Feb;7(1):12-9.
doi:
10.1007/s12975-015-0434-6. Epub 2015 Nov 16.
PMID: 26573918.
Tabuchi S. Relationship between Postmenopausal
Estrogen Deficiency and Aneurysmal Subarachnoid
Hemorrhage. Behav Neurol. 2015;2015:720141. doi:
10.1155/2015/720141. Epub 2015 Oct 11. PMID:
26538819; PMCID: PMC4619901.
Ding C, Toll V, Ouyang B, Chen M. Younger age ofmenopause in women with cerebral aneurysms. J
Neurointerv Surg. 2013 Jul;5(4):327-31. doi:
10.1136/neurintsurg-2012-010364. Epub 2012 Jun
13. PMID: 22700728.
Feigin VL, Rinkel GJ, Lawes CM, Algra A, Bennett
DA, van Gijn J, Anderson CS. Risk factors forsubarachnoid hemorrhage: an updated systematic
review of epidemiological studies. Stroke. 2005
Dec;36(12):2773-80.
doi:
10.1161/01.STR.0000190838.02954.e8. Epub 2005
Nov 10. PMID: 16282541.
Bakker MK, van der Spek RAA, van Rheenen W,
Morel S, Bourcier R, Hostettler IC, Alg VS, van Eijk
KR, Koido M, Akiyama M, Terao C, Matsuda K,
Walters RG, Lin K, Li L, Millwood IY, Chen Z,
Rouleau GA, Zhou S, Rannikmäe K, Sudlow CLM,
Houlden H, van den Berg LH, Dina C, Naggara O,
Gentric JC, Shotar E, Eugène F, Desal H, Winsvold
BS, Børte S, Johnsen MB, Brumpton BM, Sandvei
MS, Willer CJ, Hveem K, Zwart JA, Verschuren
WMM, Friedrich CM, Hirsch S, Schilling S, Dauvillier
J, Martin O; HUNT All-In Stroke; China Kadoorie
Biobank Collaborative Group; BioBank Japan
Project Consortium; ICAN Study Group; CADISPGroup; Genetics and Observational Subarachnoid
Haemorrhage (GOSH) Study investigators;
International Stroke Genetics Consortium (ISGC);
Jones GT, Bown MJ, Ko NU, Kim H, Coleman JRI,
Breen G, Zaroff JG, Klijn CJM, Malik R, Dichgans M,
Sargurupremraj M, Tatlisumak T, Amouyel P,
Debette S, Rinkel GJE, Worrall BB, Pera J, Slowik A,
Gaál-Paavola EI, Niemelä M, Jääskeläinen JE, vonUnd Zu Fraunberg M, Lindgren A, Broderick JP,
Werring DJ, Woo D, Redon R, Bijlenga P, Kamatani
Y, Veldink JH, Ruigrok YM. Genome-wide
association study of intracranial aneurysms
identifies 17 risk loci and genetic overlap withclinical risk factors. Nat Genet. 2020
Dec;52(12):1303-1313.
doi:
10.1038/s41588-020-00725-7. Epub 2020 Nov 16.
Erratum in: Nat Genet. 2020 Dec 22;: PMID:
33199917; PMCID: PMC7116530.
Karhunen V, Bakker MK, Ruigrok YM, Gill D,
Larsson SC. Modifiable Risk Factors for Intracranial
Aneurysm
andAneurysmal
Subarachnoid
Hemorrhage: A Mendelian Randomization Study. J
Am Heart Assoc. 2021 Nov 16;10(22):e022277. doi:

Fiziologia - Physiology • 2023.2(105)

21.

22.

23.

24.

25.

26.

27.

10.1161/JAHA.121.022277. Epub 2021 Nov 3.
PMID: 34729997; PMCID: PMC8751955.
Sundström J, Söderholm M, Söderberg S,
Alfredsson L, Andersson M, Bellocco R, Björck M,
Broberg P, Eriksson M, Eriksson M, Forsberg B,
Fransson EI, Giedraitis V, Theorell-Haglöw J,
Hallqvist J, Hansson PO, Heller S, Håkansson N,
Ingelsson M, Janson C, Järvholm B, Khalili P,
Knutsson A, Lager A, Lagerros YT, Larsson SC,
Leander K, Leppert J, Lind L, Lindberg E,
Magnusson C, Magnusson PKE, Malfert M,
Michaëlsson K, Nilsson P, Olsson H, Pedersen NL,
Pennlert J, Rosenblad A, Rosengren A, Torén K,
Wanhainen A, Wolk A, Engström G, Svennblad B,
Wiberg B. Risk factors for subarachnoid
haemorrhage: a nationwide cohort of 950 000 adults.
Int J Epidemiol. 2019 Dec 1;48(6):2018-2025. doi:
10.1093/ije/dyz163. PMID: 31363756.
Larsson SC, Bäck M, Rees JMB, Mason AM,
Burgess S. Body mass index and body composition
in relation to 14 cardiovascular conditions in UK
Biobank: a Mendelian randomization study. Eur
Heart J. 2020 Jan 7;41(2):221-226. doi:
10.1093/eurheartj/ehz388. PMID: 31195408; PMCID:
PMC6945523.
Su SX, Wang XT, Li XF, Duan CZ, Bi YM, Zhang X.
Nonlinear Association of Glycosylated Hemoglobin
With Single Intracranial Aneurysm Rupture in
Patients With Diabetes Mellitus: A Cross-Sectional
Study. Front Neurol. 2022 Mar 28;13:854008. doi:
10.3389/fneur.2022.854008. PMID: 35418940;
PMCID: PMC8995878.
Can A, Castro VM, Dligach D, Finan S, Yu S, Gainer
V, Shadick NA, Savova G, Murphy S, Cai T, Weiss
ST, Du R. Lipid-Lowering Agents and High HDL(High-Density Lipoprotein) Are Inversely AssociatedWith Intracranial Aneurysm Rupture. Stroke. 2018
May;49(5):1148-1154.
doi:
10.1161/STROKEAHA.117.019972. Epub 2018 Apr
5. PMID: 29622625; PMCID: PMC5915939.
Zhang B, Dong S, Miao Y, Song G, Yuan F, Liu L,
Xia S, Qin Y, Huo X, Wu Z, Miao Z, Mo D, Liu A;
International Stroke Genetics Consortium (ISGC)
Intracranial Aneurysm Working Group. Effects ofblood lipids and lipid-modifying drugs on intracranial
aneurysms.
Eur
J
Neurol.
2022
Oct;29(10):2967-2975. doi: 10.1111/ene.15471.
Epub 2022 Jul 5. PMID: 35726534.
Ho AL, Lin N, Frerichs KU, Du R. Smoking andIntracranial Aneurysm Morphology. Neurosurgery.
2015 Jul;77(1):59-66; discussion 66. doi:
10.1227/NEU.0000000000000735.
PMID:
25839377.
Kamio Y, Miyamoto T, Kimura T, Mitsui K, Furukawa
H, Zhang D, Yokosuka K, Korai M, Kudo D, Lukas
RJ, Lawton MT, Hashimoto T. Roles of Nicotine in
the Development of Intracranial Aneurysm Rupture.
Stroke.
2018
Oct;49(10):2445-2452.
doi:

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

33

10.1161/STROKEAHA.118.021706.
PMID:
30355112; PMCID: PMC6214667.
Yang C, Li Z, Yan S, He Y, Dai R, Leung GP, Pan S,
Yang J, Yan R, Du G. Role of the nicotinic
acetylcholine receptor α3 subtype in vascularinflammation.
Br
J
Pharmacol.
2016
Nov;173(22):3235-3247. doi: 10.1111/bph.13609.
Epub 2016 Sep 29. PMID: 27572927; PMCID:
PMC5071564.
Larsson SC, Virtamo J, Wolk A. Coffee consumption
and risk of stroke in women. Stroke. 2011
Apr;42(4):908-12.
doi:
10.1161/STROKEAHA.110.603787. Epub 2011 Mar
10. PMID: 21393590.
Larsson SC, Männistö S, Virtanen MJ, Kontto J,
Albanes D, Virtamo J. Coffee and tea consumption
and risk of stroke subtypes in male smokers. Stroke.
2008
Jun;39(6):1681-7.
doi:
10.1161/STROKEAHA.107.504183. Epub 2008 Mar
27. PMID: 18369170.
Zhang Z, Wang M, Yuan S, Larsson SC, Liu X.
Genetically predicted coffee and tea consumption
and risk of intracranial aneurysm. Eur J Clin Nutr.
2023
Aug;77(8):811-814.
doi:
10.1038/s41430-023-01295-7. Epub 2023 Jun 13.
PMID: 37311867.
Abbott RD, Rodriguez BL, Burchfiel CM, Curb JD.
Physical activity in older middle-aged men andreduced risk of stroke: the Honolulu Heart Program.
Am J Epidemiol. 1994 May 1;139(9):881-93. doi:
10.1093/oxfordjournals.aje.a117094.
PMID:
8166138.
Lindbohm JV, Rautalin I, Jousilahti P, Salomaa V,
Kaprio J, Korja M. Physical activity associates with
subarachnoid hemorrhage risk- a population-based
long-term cohort study. Sci Rep. 2019 Jun
25;9(1):9219. doi: 10.1038/s41598-019-45614-0.
PMID: 31239477; PMCID: PMC6592878.
Armstrong ME, Green J, Reeves GK, Beral V,
Cairns BJ; Million Women Study Collaborators.
Frequent physical activity may not reduce vasculardisease risk as much as moderate activity: large
prospective study of women in the United Kingdom.
Circulation. 2015 Feb 24;131(8):721-9. doi:
10.1161/CIRCULATIONAHA.114.010296.
Epub2015 Feb 16. PMID: 25688148.
Maaijwee NA, et al., Ischaemic stroke in young
adults: risk factors and long-term consequences.
Nat Rev Neurol, 2014. 10(6): p. 315–25.
Vijayan M and Reddy PH, Stroke, Vascular
Dementia, and Alzheimer’s Disease: Molecular
Links. J Alzheimers Dis, 2016. 54(2): p. 427–43.
Khoury JC, et al., Diabetes mellitus: a risk factor forischemic stroke in a large biracial population. Stroke,2013. 44(6): p. 1500–4.
O’Collins VE, et al., Hypertension and experimental
stroke therapies. J Cereb Blood Flow Metab,2013. 33(8): p. 1141–7.

39. Brown RD Jr, Broderick JP. Unruptured intracranial
aneurysms:
epidemiology,
naturalhistory,
management options, and familial screening. Lancet
Neurol.
2014
Apr;13(4):393-404.
doi:
10.1016/S1474-4422(14)70015-8. PMID: 24646873.
40. Wiebers DO, Whisnant JP, Huston J 3rd, Meissner I,
Brown RD Jr, Piepgras DG, Forbes GS, Thielen K,
Nichols D, O'Fallon WM, Peacock J, Jaeger L,
Kassell NF, Kongable-Beckman GL, Torner JC;
International Study of Unruptured Intracranial
Aneurysms Investigators. Unruptured intracranial
aneurysms: natural history, clinical outcome, andrisks of surgical and endovascular treatment. Lancet.
2003Jul
12;362(9378):103-10.
doi:
10.1016/s0140-6736(03)13860-3. PMID: 12867109.
41. Rinne, J., Hernesniemi, J., Niskanen, M., et al.
(1994). Management of incidental aneurysms: along-term follow-up study. Journal of Neurosurgery.
42. Rossi P, Mirallie E, Pittaluga P, Chaillou P, Patra P.
Bilateral extracranial aneurysms of the internal
carotid artery. A case report. J Cardiovasc Surg(Torino). 1997 Feb;38(1):27-31. PMID: 9128118.

Index
WBC (White Blood Cell Count)
BAS# (Basophil Count)
BAS% (Percentage of Basophil)
NEU# (Neutrophil Count)
NEU% (Percentage of Neutrophils)
EOS# (Eosinophil Count)
EOS% (Percentage of Eosinophils)
LYM% (Percentage of Lymphocytes)
MON# (Monocyte Count)
MON% (Percentage of Monocytes)
RBC (Red Blood Cell Count)
HGB (Hemoglobin)
MCV (Mean Corpuscular Volume)
MCH (Mean Corpuscular Hemoglobin)
MCHC (Mean Corpuscular Hemoglobin Concentration)
RDW-CV (Red Cell Distribution Width Coefficient of
Variation)
RDW-SD (Red Cell Distribution Width Standard Deviation)
HCT (Hematocrit)
PLT (Platelet Count)
MPV (Mean Platelet Volume)
PDW (Platelet Distribution Width)
PCT (Plateletcrit)
ESR (Erythrocyte Sedimentation Rate)

43. Funiu H, Kokubo Y, Kondo R, Saino M, Ohki M,
Kayama T, Orita H, Hirooka S. [A case of bilateralextracranial carotid artery aneurysms caused by
Takayasu's arteritis]. No To Shinkei. 2004
Nov;56(11):971-5. Japanese. PMID: 15678956.
44. The International Study of Unruptured Aneurysms.
Background.
2007.
May
9th
,
www.isuia.org/isuia/Background.htm
45. Yamaguchi S, Oki S, Ogasawara H, Hibino S, Sato
H, Ito Y, Okazaki H. [A case of a surgically treatedextracranial internal carotid artery saccularaneurysm]. No Shinkei Geka. 1997 Feb;25(2):181-5.
Japanese. PMID: 9027897.
46. Ausman, J., Pearce, J., Reyes, R. A., & Schanz, G.
(1983). Treatment of a high extracranial carotid
artery aneurysm with CCA-MCA bypass and carotid
ligation.
Case
report.
DOI:
10.3171/JNS.1983.58.3.0421
47. Qureshi AI, Tuhrim S, Broderick JP, Batjer HH,
Hondo H, Hanley DF. Spontaneous intracerebral
hemorrhage. N Engl J Med. 2001 May
10;344(19):1450-60.
doi:
10.1056/NEJM200105103441907. PMID: 11346811.

Value
7.96 x 10^9/L
0.02 x 10^9/L
0.2%
5.88 x 10^9/L
73.9%
0.06 x 10^9/L
0.7%
1.58%
0.42 x 10^9/L
5.3%
4.33 x 10^12/L
13.2 g/dl
93.3 fL
30.6 pg32.8 g/dl
12.4%

Reference range
4.0 - 11.0 x 10^9/Lless than 0.1 x 10^9/Lless than 1%
2.0 - 7.5 x 10^9/L
40 - 75%0.04 - 0.4 x 10^9/L
0.5 - 5%20 - 40%
0.2 - 0.8 x 10^9/L
2 - 8%
4.2 - 5.4 x 10^12/L
12.0 - 16.0 g/dl
80 - 100 fL
27 - 33 pg
32 - 36 g/dl
11.5 - 14.5%

45.5 fL
40.4%
269 x 10^9/L
10.5 fL
15.8%
0.282%
5 mm/30 min

39.0 - 46.0 fL
36 - 46%
150 - 450 x 10^9/L
7.4 - 11.0 fL
9.0 - 17.0 fL
0.15 - 0.35%
0-20 mm/30 min

Table I: Blood cell count
Index
GlucoseSerum Urea
Serum Creatinine

Value
86 mg/dl
18.7 mg/dl
0.83 mg/dl

Reference range
70 - 100 mg/dl
7 - 20 mg/dl
0.6 - 1.1 mg/dl

Fiziologia - Physiology • 2023.2(105)Total Serum Cholesterol
AST (Aspartate Aminotransferase)
ALT (Alanine Aminotransferase)
Cl (Chlorine)
K (Potassium)

139.2 mg/dl
11.7 U/L
19 U/L
102.7 mmol/L
3.75 mmol/L

Less than 200 mg/dl
10 - 40 U/L
7 - 56 U/L
96 - 106 mmol/L
3.5 - 5.0 mmol/L

Table II: Biochemical findings

Fig. 1: CT scan evaluation reveals an aneurysmal dilation in the right infra-cavernous internal carotid artery.

Fig. 2 MRI investigation

37

Fig. 3 Cerebral angiography of the 4 major vessels reveals

the presence of the aforementioned aneurysm.

ANEVRISM INTRACRANIAN DEZVALUIT PRIN DEFICIT NEUROLOGIC - O EXPLORARE A
LITERATURII ȘI O PROVOCARE DIAGNOSTICĂ
Rezumat:
Anevrismele intracraniene de tip sacular sau de tip boabă reprezintă formări anormale ale vaselor de sânge în interiorul creierului,
responsabile pentru majoritatea hemoragiilor subarahnoidiene nontraumatice (HSA). Progresele în domeniul imagisticii medicale auîmbunătățit ratele de detectare, cu toate că prevalența acestor anevrisme crește odată cu înaintarea în vârstă, în special la femei. Mai
mult, studiile sugerează un risc mai mare de formare a anevrismelor de novo la femei și evidențiază rolul tensiunii arteriale instabile ca
factor de risc. Anevrismele sunt, de asemenea, asociate cu variații ale nivelurilor de estrogen și factori de risc cardio-metabolici, inclusiv
hipertensiunea arterială, indicele de masă corporală și diabetul. Fumatul este identificat ca un factor de risc semnificativ, contribuind la
disfuncția vasculară endotelială asociată fumatului și la ruptura anevrismelor. Pentru a ilustra provocările diagnostice și de gestionare aleanevrismelor intracraniene, este prezentat un studiu de caz. Cazul implică o pacientă de 29 de ani care a prezentat inițial simptomeasemănătoare unui accident vascular cerebral ischemic. Evaluarea ulterioară a relevat un anevrism de arteră carotidă internă dreaptă, odiscopatie cervicală-lombară și o patologie tiroidiană. Cazul evidențiază importanța evaluării amănunțite și a luării în considerare a
etiologiilor atipice la pacienții cu simptome asemănătoare accidentului vascular cerebral. Studiul susține managementul proactiv alanevrismelor simptomatice, precum și rolul embolizării și reconstrucției arteriale directe în abordarea anevrismelor cu risc de complicații
neurologice și subliniază necesitatea unei abordări cuprinzătoare în diagnosticul și managementul acestora, în special în cazurile cu
prezentări clinice atipice.
Cuvinte cheie: anevrism intracranian; factori de risc; estrogen; cardioembolic; fumat; cafea; activitate fizică.

Fiziologia - Physiology • 2023.2(105)EXPLORING PREDICTORS AND OUTCOMES IN ISCHEMIC STROKEELENA ANCA PINOSANU1#, BURDUSEL DAIANA2#, ROXANA SURUGIU3*, MADALINA ALDEA2*,RALUCA ELENA SANDU1,3
1

Department of Neurology, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;2

Department of Psychiatry, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;3

Department of Biochemistry, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;*

Correspondence: Raluca Elena Sandu, raluca.sandu@umfcv.ro; Roxana Surugiu, roxana.surugiu@umfcv.ro#

These authors contributed equally

ABSTRACT
Stroke, a critical neurological event, often leaves an indelible mark on a patient's life, demanding a comprehensive understanding of
its multifaceted facets for effective management and prognosis. This study focuses on ischemic strokes, which account for a significant
proportion of stroke cases. With the prevalence of strokes escalating globally, identifying robust prognostic markers becomes imperative
for tailoring interventions and improving patient outcomes. Key risk factors, including advanced age, hypertension, and comorbid vascularconditions, play pivotal roles in shaping the trajectory of ischemic stroke outcomes. Recognizing the intricate interplay between thesefactors is vital for clinicians to devise targeted therapeutic strategies. Prognosis post-ischemic stroke remains a complex puzzle, withvariables such as the severity of the infarct, associated complications, and the efficacy of medical interventions influencing the clinicalcourse. This study conducted retrospectively on a cohort of 200 patients, delves into these nuances, shedding light on correlations andpatterns that can refine our understanding of ischemic stroke prognosis. In unravelling the intricacies of these factors, this research aims
to contribute valuable insights that can inform evidence-based clinical practices and enhance the overall management of ischemic stroke
patients.
Keywords: stroke, risk factors, prognosis;

1.

Introduction
Stroke is currently defined according to the World
Health Organization (WHO) definition, introduced in 1970and still in use, as: "the rapid development of clinicalsigns of focal (or global) disturbance of brain function,
lasting more than 24 hours or leading to death, with noapparent cause other than vascular origin" [1].
However, this broad definition encompasses strokes,
cerebral and subarachnoid hemorrhages, without
keeping pace with scientific progress that allows theirseparation based on imaging and histopathologicalcriteria. In 2013, the American Heart Association
proposed redefining these terms: stroke represents
secondary cell death due to focal ischemia in the
cerebral, spinal, or retinal tissue, based on pathological,
imaging, or other objective evidence of ischemic injury to
the cerebral, spinal, or retinal tissue in a defined vascularterritory or clinical evidence of focal cerebral, spinal, or
retinal ischemic injury, based on symptoms persisting for≥24 hours or leading to death, excluding other etiologies[2].
Ischemic stroke is defined as an episode ofneurological dysfunction caused by focal cerebral, spinal,
or retinal infarction. Limiting the definition of ischemic
stroke to a focal injury takes into account several
considerations.
There are significant differences between thepathological mechanisms of focal and global ischemia,with focal ischemia resulting from the loss of perfusion in
an arterial territory due to stenosis or obstruction, leading
to cell death in the tributary tissue. In focal ischemia, thelesions are limited, with an ischemic core and apenumbral zone where neuronal and supporting cells areaffected. Global ischemia results from a decrease in39

cerebral perfusion due to decreased blood pressure(cardiogenic shock, hypovolemia) or a sudden increase
in intracranial pressure (trauma), resulting in selective
neuronal loss in sensitive areas (hippocampus, basal
ganglia, thalamus, neocortex, cerebellum) [3].
Risk factors are any characteristics of an individualthat increase the risk of a stroke compared to individuals
who do not have that characteristic [4]. Some risk factors
are non-modifiable, such as family history of
cardiovascular diseases, age, male gender, and race,
while others can be modified, leading to a reduction in
the risk of the disease. These risk factors, which often
coexist, account for approximately 60-80% of the risk ofstroke in the general population [4]. Although complex
genomic studies attempt to identify genes susceptible tostroke, with the identification of certain loci, the clinicalrelevance is still modest [5].
Risk factors have a cumulative effect on the structureand function of blood vessels, both locally andsystemically. Many of these risk factors alter vascular
structure with a pro-atherosclerotic effect, leading toarterial stiffening, narrowing, and twisting of arterioles
and capillaries [6]. At the cerebral level, thesemorphological changes are often associated with
reduced cerebral blood flow due to alterations in
cerebrovascular regulation mechanisms.
Advanced age, hypertension, diabetes, andhypercholesterolemia disrupt vital adaptive mechanisms
responsible for adequate perfusion [7]. The endothelial
ability for microvascular regulation is compromised, while
the increase in blood flow secondary to neural activity issuppressed, resulting in a desynchronization between
energy demand and supply [8].
Hypertension and diabetes alter protective vascularmechanisms that maintain stable cerebrovascular flow
during periods of blood pressure decline, facilitating theoccurrence or increased intensity of ischemic events
during decreased perfusion [9]. These vascular changes
increase cerebral vulnerability to ischemia following
vascular occlusion by compromising the development ofcollaterals from adjacent unobstructed vessels, a crucial
factor in limiting the infarcted area [10].
In addition to vascular risk, advanced age anddiabetes increase the susceptibility of brain cells to injury,
amplifying the effects of ischemia, though the underlying
mechanisms for this are not yet fully understood [11].
The assessment of stroke severity is conducted
through neurological examination and is likely the most
crucial factor affecting short or long-term symptoms [12].
Increasingly, in both research studies and clinicalpractice, neurological deficit is measured using the
NIHSS (National Institutes of Health Stroke Scale), which
employs a 15-point score. Several conducted studies

have demonstrated that the NIHSS score effectively
assesses the prognosis of an ischemic stroke [13,14]. In
one report, over 1200 patients were analyzed within thefirst 24 hours of acute stroke symptoms onset, utilizing
the NIHSS score. Each added point reduced thelikelihood of a favorable outcome at 3 months by 17%[13]. An NIHSS score <6 signifies good recovery - theability to live independently, while a score >16 is
associated with an increased risk of death or severe
disability [14].
The relationship between the NIHSS score and theoutcome depends on the time elapsed since the onset ofthe first stroke symptom, partly because early deficits
tend to be unstable, and many patients experience
gradual recovery [14,15].
The Canadian Neurological Score (CNS) is also
useful in predicting the outcome after an acute ischemic
stroke. A CNS score <6.5 is associated with increased
mortality at 30 days and a poor outcome at 6 months.
However, compared to the CNS score, the NIHSS scoremore precisely relates to the prognosis at 3 months[16,17].
Following thrombolysis or thrombectomy according to
guideline recommendations, patients with ischemic
stroke have shown a dramatic reduction in neurologicaldeficits.
This study aims to correlate and analyze the clinical,
biological, and imaging parameters of a cohort of 200
patients who have experienced an ischemic stroke, toidentify prognostic factors that influence the favorable orunfavorable progression of ischemic stroke. The specificobjectives include highlighting prognostic factors
associated with ischemic stroke, analyzing common anddistinct aspects of the correlation between prognostic
factors and the evolution of ischemic stroke, examining
the influence of factors such as origin, gender, and age,
identifying vascular comorbidities, and conducting an
in-depth analysis and correlation of the results obtained
from the studied patient cohort.
2. Materials and Methods
2.1. Study design
This investigation adopts a retrospective approachby integrating observational records obtained from the
Neurology Clinic at the Clinical Hospital of
Neuropsychiatry in Craiova. The study focuses onpatients diagnosed primarily with Ischemic Stroke during
the timeframe spanning from January 1, 2018, to June30, 2018. To compile comprehensive insights, pertinent
diagnostic information was extracted from diverse
sources, encompassing patient histories, clinicalexaminations, neurological assessments, biological andimaging features, as well as details concerning
Fiziologia - Physiology • 2023.2(105)administered treatments and the subsequent progressionof patients. This method allows for a thoroughexamination of the multifaceted factors influencing thecourse of Ischemic Stroke within the specified period.

Regarding the analysis of the studied patient cohort
based on age, Table I illustrates that the highest
frequency of ischemic strokes occurs in the age groupbetween 70 and 95 years (119 patients, representing
59.5% of the total studied patients), followed by the agegroup of 60-69 years (59 patients, representing 29.5% of
the total 200 patients). In the age groups of 50-59 years,
there were 16 patients (8%), and in the 40-49 age group,
7 patients were identified, accounting for 3.5%.
Secondly, the distribution of patients by gender in thestudied group shows an approximately equal percentage
of males at 49.5% (99 patients) and females at 50.5%(101 patients), indicating a balanced prevalence ofischemic stroke between both genders. Examining theorigin environment, it highlights a higher incidence ofischemic stroke in rural areas (63%) compared to urban
areas, where the percentage is 37%. These percentages
suggest potential differences in medical accessibility andhealth education, possibly associated with an aging
population and lower living standards in rural areas.
The graphic representation of hospitalization days forthe 200 studied patients reveals that 61% of patientsrequired a hospitalization period of 1-9 days, 34%
needed a more extended period of 10-15 days, and only
5% required hospitalization for approximately 16-28 days.
This breakdown provides insights into the duration ofcare received by the patients.
Hypertension stands out as a major risk andprognostic factor for ischemic stroke. Out of these, 170
patients were diagnosed with hypertension before
admission, constituting 85%, while the remaining 15%
were normotensive. Within the hypertensive group, themajority had grade II hypertension (69.5%), followed bygrade I (11%) and grade III (4.5%).
Focusing on atrial fibrillation (AF), a potent risk andprognostic factor in ischemic stroke, 29.5% of the studied
patients (59 individuals) were identified with AF, while theremaining 70.5% (141 patients) did not exhibit atrial
fibrillation. The presence of AF underscores the need forcorrect treatment and frequent monitoring to prevent
complications or unfavorable outcomes associated withischemic stroke.
Among the total of 200 ischemic stroke patients, 62.5%(125 patients) had a history of chronic ischemic heartdisease, while the remaining 37.5% (75 patients) had no
known chronic ischemic heart disease. Additionally, 18%(35 patients) had a history of type II diabetes, and 34%(68 patients) had blood glucose levels exceeding 126
mg/dl. Patients with diabetes have a higher susceptibility
to atherosclerosis, leading to an increased risk ofischemic stroke. While 25.5% (51 patients) of the total
studied patients had dyslipidemia, the majority (74.5%)
had elevated cholesterol, LDL-cholesterol, or triglyceride

2.2 Methodology
The extracted data encompassed various
parameters across different assessment domains. Fromthe patient history (anamnesis), information included age,gender, origin, primary diagnosis, secondary diagnoses,
admission reasons, pathological personal history, andassociated risk factors. The clinical examination involved
evaluating language impairments such as dysarthria andaphasia, assessing expressive and spontaneous speech,
repetitive and automatic speech, color and object naming,
as well as expressive lexia through reading aloud,recognition of names, and explanation of read words.
Writing abilities were also evaluated by the patient's
ability to write their name or desired content. In theparaclinical domain, recorded parameters consisted ofserum creatinine, blood glucose, and cholesterol levels.
The CT examination included noting the presence orabsence of recent or residual vascular lesions,cranio-cerebral traumas, intracranial hemorrhages,
aneurysms, hematomas, intracranial tumors, affectedvascular territories, and sequelae. Neurologicalexamination parameters involved observing unique
attitudes, and assessing involuntary movements in termsof rhythm, recurrence, frequency, speed, amplitude,
coordination disorders, and balance examination results.
This comprehensive dataset provides a foundation forthe correlation and analysis of clinical, biological, andimaging parameters in understanding the prognosis ofischemic stroke.
3. Results
3.1. Cohort characteristics
In the initial phase of our study, our focus was on
examining the distribution of ischemic stroke patients
based on age, investigating the prevalence of ischemic
stroke within our patient cohort according to gender, andanalyzing the distribution of patients across rural andurban environments with a gender-based breakdown.
Additionally, we delved into the distribution of patients
based on the number of hospitalization days, aiming togain insights into the duration of care received.
Furthermore, we explored the distribution of patientsconcerning associated prognostic factors andpathological personal histories. These preliminary
analyses form the foundation for our comprehensive
examination of the clinical, demographic, and prognostic
landscape of ischemic stroke in our study population. We
present in Table I the cohort characteristics.
41

levels but did not meet the criteria for dyslipidemia.
The presence of chronic kidney disease (CKD)
among the studied patients is 9% (18 patients). CKD
serves as both a risk factor for ischemic stroke and anegative prognostic factor for the post-stroke evolution.
The impairment of renal function contributes tocomplications such as hypertension, anemia, sodium andwater retention, nitrogen retention, hyperlipoproteinemia,
and acidosis, negatively impacting the quality of life andsurvival of patients post-ischemic stroke.
3.2 Complications
3.2.1 Urinary tract infections
Among the myriad complications associated withstroke, we specifically directed our attention toward two
prevalent issues within the studied patient group: urinary
tract infections (UTIs) and pneumonia. These
complications are of significant concern due to theirfrequency and potential impact on the overall health andrecovery of stroke patients.
Urinary tract infections are a common complication
observed in stroke survivors. The compromised mobility
and altered neurological function often lead to challenges
in maintaining proper hygiene and bladder control,
contributing to an increased susceptibility to UTIs.
Furthermore, issues such as urinary retention and theuse of catheters can elevate the risk of bacterial
infections in the urinary tract. UTIs, if not promptly
addressed, can lead to systemic complications,exacerbating the recovery process for stroke patients.
Within the cohort of studied patients, 9.5%,
equivalent to 19 individuals, presented with urinary tract
infections (UTIs). Among these cases, 7.5% (15 patients)
exhibited UTIs caused by Escherichia coli (E. coli), 1.5%
(3 patients) were diagnosed with Klebsiella infection, and
0.5% (1 patient) had an infection attributed to Citrobacter.
The occurrence of urinary tract infections represents a
noteworthy prognostic factor that can unfavorably
influence the trajectory of ischemic stroke patients,
particularly among the elderly, those immobilized forextended periods, or those experiencing cognitive
impairments. The identification of specific pathogens
causing UTIs underscores the importance of tailoredinterventions and vigilant management to mitigate theimpact of these infections on the overall well-being andrecovery of stroke patients (Fig. 1).
3.2.2 Pneumonia
Pneumonia is another noteworthy complication
frequently encountered in the aftermath of a stroke.
Reduced mobility, impaired cough reflex, andcompromised swallowing function contribute to anincreased risk of aspirating oral or gastric contents into

the lungs, leading to pneumonia. Additionally, theimmobility associated with stroke may result in aweakened respiratory system, making stroke survivors
more vulnerable to respiratory infections. Pneumonia,
when coupled with the existing challenges posed bystroke, can significantly impede the rehabilitation andrecovery of affected individuals.
Within the studied patient cohort, 8.5% (17 patients)
manifested bacterial pneumonia. Bacterial pneumonia
stands out as a critical complication in individuals withstrokes, primarily attributed to aspiration. Aspiration ismost commonly observed in patients with swallowing
disorders and those experiencing altered consciousness.
Therefore, oral feeding should be postponed until thepatient can adequately ingest small quantities of water oreven cough effectively. This highlights the significance of
proactive measures in managing swallowing difficulties tomitigate the risk of bacterial pneumonia, a prominentconcern in the post-stroke scenario (Fig. 2).
3.2.3 Other associated pathology
Moreover, our investigation delved into additionalcomorbidities, as outlined in Table II.
3.3 Stroke distribution
The distribution of the patient cohort based on thesize of ischemic cerebral infarction visualized throughcomputer tomography is depicted.
Specifically, acute ischemia at the brainstem wasobserved in 2 patients (1%), while acute corticalischemia was present in 34 patients (17%). Additionally,
6 patients (3%) exhibited acute lacunar ischemia, 12
patients (6%) had acute subcortical ischemia, and 28
patients (14%) demonstrated acute cortico-subcorticalischemia. Acute cerebellar ischemia was identified in 3
patients, constituting 1.5%. Furthermore, 95 patients(47.5%) showed no recently constituted lesions. This
comprehensive analysis provides insights into the varied
manifestations of ischemic cerebral infarction in thestudied population.
The increased size and location of the infarct areassociated with an unfavorable prognosis in the evolutionof patients with ischemic stroke. Most patients
experienced cortical infarcts (17%), followed by 14% withcortico-subcortical infarcts and 6% with subcortical
infarcts. Large-sized infarcts were identified in a total of
74 patients, and the involvement of a large area of
cerebral tissue leads to various motor, sensory, mixed,
language, cognitive, behavioral, memory, andconsciousness disorders. In contrast, only 3% had
lacunar infarcts, 1% in the brainstem, and 1.5% in the
cerebellum—these being particularly severe infarcts witha severe evolution due to their impact on a multitude ofvital structures in the body. This detailed analysis
Fiziologia - Physiology • 2023.2(105)highlights the variety of manifestations andconsequences of infarcts among patients with ischemic
stroke (Fig. 3).
The distribution of patients based on the affectedcerebral territory is particularly crucial, as the evolution
and prognosis of ischemic stroke depend on the specificarea affected. Within the studied patient cohort, thedistribution is as follows: the majority of patients sufferedischemic strokes in the territory of the left carotid artery,accounting for 28% (56 patients); 21% experienced
ischemic strokes in the territory of the right carotid artery(42 patients); 14.5% in the vertebro-basilar territory (29patients); 17% in the territory of the right middle cerebral
artery (34 patients); 18.5% in the territory of the left
middle cerebral artery (37 patients); 2% in the territory of
the right posterior cerebral artery (4 patients); 1% in the
territory of the left posterior cerebral artery (2 patients); 1%
in the territory of the anterior cerebral artery (2 patients);
and 3% in the total carotid territory (6 patients). Thisdetailed breakdown underscores the diverse patterns of
cerebral involvement and their significance inunderstanding the impact of ischemic stroke on patients(Table III).

infarcts for 6%, cortico-subcortical infarcts for 14%,
lacunar infarcts for 3%, brainstem infarcts for 1%, andcerebellar infarcts for 1%.
Infarct location is another prognostic factor
determining the evolution of ischemic stroke [23,24].
Obstruction of the basilar, vertebral arteries, or any of the
major intracranial vessels is associated with a very highrisk of unfavorable outcomes. In this study, obstruction of
the right carotid artery occurred in 21%, obstruction of
the left carotid artery in 28%, and vertebro-basilar
territory involvement in 14.5%.
Additionally, the literature suggests that obstructionof the middle cerebral artery is associated with a very
high mortality risk due to vital structures at this level[25,26]. In the studied patient cohort, obstruction of theright middle cerebral artery occurred in 17%, while
obstruction of the left middle cerebral artery occurred in
18.5%.
Complications of ischemic stroke represent
prognostic factors for post-stroke outcomes [27-30]. In
the studied patient group, bacterial pneumonia andurinary tract infections were encountered at percentagesof 8.5% and 9.5%, respectively. These findings
contribute to a comprehensive understanding of themultifaceted factors influencing the prognosis of patientswith ischemic stroke.

4. Discussions
Prognostic factors for patients with ischemic stroke
include the patient's age, the severity of the infarct, themechanism of infarct production, associatedcomorbidities, and complications of ischemic stroke, allof which contribute to the current morbidity and mortality
rates for these patients. The clinical study aimed toanalyze a cohort of 200 patients diagnosed with ischemic
stroke at the Neurology Clinic II over 6 months.
Examining the patient group reveals that the majority ofischemic stroke patients were found in the 70-95 age
group, with a relatively equal distribution between males
and females, and a higher representation from rural
areas. These findings align with existing literature in thefield [18-20].
The study identified several associated comorbidities,out of which hypertension emerged as a major
aggravating factor, affecting 85% of the studied patients,
with a predominance of grade II hypertension. The next
aggravating factor was diabetes mellitus, present in 18%
of patients, followed by atrial fibrillation encountered in
29.5%, and chronic ischemic heart disease observed inover half of the patients.
Another important parameter aiding prognosis
evaluation is neuroimaging, which provides information
about infarct volume and location [21,22]. In the studied
patient group, the volume of the infarct correlated asfollows: cortical infarcts accounted for 17%, subcortical

References
1. K. Aho, P. Harmsen, S. Hatano, J. Marquardsen, V.
Smirnov, T. Strasser. Cerebrovascular disease in thecommunity: results of a WHO collaborative study.
Bull World Health Organ, vol. 58, p. 113–130, 1980.
2. R. Sacco, S. Kasner, J. Broderick, e. al. Council on
Nutrition. AnUpdated Definition of Stroke for the 21stCentury - A Statement for Healthcare Professionals
From the American Heart Association/American
Stroke Association. Stroke, vol. 44, pp. 2064 2089,
2013.
3. D. Ellison, S. Love, L. Chimelli, B. Harding, J. Lowe,
H. Vinters, S. Brandner, W. Yong. Neuropathology, 3ed., London: Mosby Ltd., 2013.
4. G. Hankey. Potential new risk factors for ischemic
stroke: what is their potential? Stroke, vol. 37, pp.
2181-2188, 2006.
5. R. Hegele, M. Dichgans. Advances in stroke 2009:
update on the genetics of stroke andcerebrovascular disease 2009. Stroke, vol. 41, pp.
63-66, 2010.
6. C. Allen, U. Bayraktutan. Risk factors for ischaemic
stroke. Int. J. Stroke, vol. 3, pp. 105-116, 2008.
7. C. Iadecola, L. Park, C. Capone. Threats to the mind:
aging, amyloid, and hypertension. Stroke, vol. 40, nr.
3, pp. 40-44, 2009.
8. D. Arrick, G. Sharpe, H. Sun, W. Mayhan.
nNOS-dependent reactivity of cerebral arterioles in
43

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

type 1 diabetes. Brain Res., vol. 1184, pp. 365-371,
2007.
Y. Kim, R. Immink, W. Stok, J. Karemaker, N. Secher,J. van Lieshout. Dynamic cerebral autoregulatory
capacity is affected early in Type 2 diabetes. Clin.
Sci. (Lond.), vol. 115, pp. 255-262, 2008.
G. Biessels, L. van der Heide, A. Kamal, R. Bleys, W.
Gispen. Ageing and diabetes: implications for brain
function. Eur. J. Pharmacol., vol. 441, pp. 1-14,
2002.
M. Moskowitz, E. Lo, C. Iadecola. The Science ofStroke: Mechanisms in 94 Search of Treatments.
Neuron, vol. 67, nr. 2, pp. 181-198, 2010.
Weimar C, König IR, Kraywinkel K, Ziegler A, Diener
HC; German Stroke Study Collaboration. Age andNational Institutes of Health Stroke Scale Score
within 6 hours after onset are accurate predictors ofoutcome after cerebral ischemia: development andexternal validation of prognostic models. Stroke.
2004
Jan;35(1):158-62.
doi:
10.1161/01.STR.0000106761.94985.8B. Epub 2003
Dec 18. PMID: 14684776.
Adams HP Jr, Davis PH, Leira EC, Chang KC,
Bendixen BH, Clarke WR, Woolson RF, Hansen MD.
Baseline NIH Stroke Scale score strongly predicts
outcome after stroke: A report of the Trial of Org
10172 in Acute Stroke Treatment (TOAST).
Neurology. 1999 Jul 13;53(1):126-31. doi:
10.1212/wnl.53.1.126. PMID: 10408548.
Frankel MR, Morgenstern LB, Kwiatkowski T, Lu M,
Tilley BC, Broderick JP, Libman R, Levine SR, Brott
T. Predicting prognosis after stroke: a placebo group
analysis from the National Institute of Neurological
Disorders and Stroke rt-PA Stroke Trial. Neurology.
2000 Oct 10;55(7):952-9. doi: 10.1212/wnl.55.7.952.
PMID: 11061250.
Saver JL, Altman H. Relationship between
neurologic deficit severity and final functional
outcome shifts and strengthens during first hours
after onset. Stroke. 2012 Jun;43(6):1537-41. doi:
10.1161/STROKEAHA.111.636928. Epub 2012 Apr
5. PMID: 22492517; PMCID: PMC3509751.
Censori B, Camerlingo M, Casto L, Ferraro B,
Gazzaniga GC, Cesana B, Mamoli A. Prognostic
factors in first-ever stroke in the carotid artery
territory seen within 6 hours after onset. Stroke.
1993 Apr;24(4):532-5. doi: 10.1161/01.str.24.4.532.
PMID: 8465357.
Sumer MM, Ozdemir I, Tascilar N. Predictors ofoutcome after acute ischemic stroke. Acta Neurol
Scand.
2003
Apr;107(4):276-80.
doi:
10.1034/j.1600-0404.2003.02008.x.
PMID:
12675701.
Potter TBH, Tannous J, Vahidy FS. A Contemporary
Review of Epidemiology, Risk Factors, Etiology, andOutcomes of Premature Stroke. Curr Atheroscler
Rep.
2022
Dec;24(12):939-948.
doi:

19.

20.

21.

22.

23.

24.

25.

26.

27.

10.1007/s11883-022-01067-x. Epub 2022 Nov 14.
PMID: 36374365; PMCID: PMC9660017.
Webb AJ, Lawson A, Mazzucco S, Li L, Rothwell PM.
Age and sex distribution of beat-to-beat bloodpressure variability after transient ischemic attack
and minor stroke: A population-based study. Int J
Stroke.
2021
Aug;16(6):683-691.
doi:
10.1177/1747493020971905. Epub 2020 Nov 9.
PMID: 33167788; PMCID: PMC8366176.
Azarpazhooh MR, Mandzia JL, Thrift AG, Sposato
LA, Morovatdar N, Amiri A, Kapral MK, Yassi N,
Bahit C, Kaul S, Alladi S, Nilanont Y, Coppola M,
Nucera A, Silver B, Werring D, Simister R, Swartz
RH, Owolabi MO, Ovbiagele B, Hachinski V;
ASSESS investigators. Age, sex, and setting in the
etiology of stroke study (ASSESS): Study design
and protocol. J Neurol Sci. 2019 Apr 15;399:209-213.
doi: 10.1016/j.jns.2019.02.024. Epub 2019 Feb 14.
PMID: 30851659.
Jadhav AP, Desai SM, Liebeskind DS, Wechsler LR.
Neuroimaging of Acute Stroke. Neurol Clin. 2020
Feb;38(1):185-199. doi: 10.1016/j.ncl.2019.09.004.
Epub 2019 Nov 7. PMID: 31761058.
Menon BK. Neuroimaging in Acute Stroke.
Continuum (Minneap Minn). 2020 Apr;26(2):287-309.
doi: 10.1212/CON.0000000000000839. PMID:
32224753.
Kazi SA, Siddiqui M, Majid S. Stroke Outcome
Prediction Using Admission Nihss In Anterior And
Posterior Circulation Stroke. J Ayub Med Coll
Abbottabad. 2021 Apr-Jun;33(2):274-278. PMID:
34137544.
Munsch F, Sagnier S, Asselineau J, Bigourdan A,
Guttmann CR, Debruxelles S, Poli M, Renou P,
Perez P, Dousset V, Sibon I, Tourdias T. Stroke
Location Is an Independent Predictor of Cognitive
Outcome. Stroke. 2016 Jan;47(1):66-73. doi:
10.1161/STROKEAHA.115.011242. Epub 2015 Nov
19. PMID: 26585396.
Abboud H, Berroir S, Labreuche J, Orjuela K,
Amarenco P; GENIC Investigators. Insular
involvement in brain infarction increases risk for
cardiac arrhythmia and death. Ann Neurol. 2006
Apr;59(4):691-9. doi: 10.1002/ana.20806. PMID:
16566012.
Colivicchi F, Bassi A, Santini M, Caltagirone C.
Prognostic implications of right-sided insulardamage, cardiac autonomic derangement, andarrhythmias after acute ischemic stroke. Stroke.
2005
Aug;36(8):1710-5.
doi:
10.1161/01.STR.0000173400.19346.bd. Epub 2005
Jul 14. PMID: 16020766.
Alloubani A, Nimer R, Samara R. Relationshipbetween Hyperlipidemia, Cardiovascular Disease
and Stroke: A Systematic Review. Curr Cardiol Rev.
2021;17(6):e051121189015.
doi:
10.2174/1573403X16999201210200342.
PMID:
33305711; PMCID: PMC8950504.

Fiziologia - Physiology • 2023.2(105)28. Banda KJ, Chu H, Kang XL, Liu D, Pien LC, Jen HJ,
Hsiao SS, Chou KR. Prevalence of dysphagia andrisk of pneumonia and mortality in acute stroke
patients: a meta-analysis. BMC Geriatr. 2022 May
13;22(1):420. doi: 10.1186/s12877-022-02960-5.
PMID: 35562660; PMCID: PMC9103417.
29. Navi BB, Kasner SE, Elkind MSV, Cushman M,
Bang OY, DeAngelis LM. Cancer and Embolic
Stroke of Undetermined Source. Stroke. 2021
Mar;52(3):1121-1130.
doi:
10.1161/STROKEAHA.120.032002. Epub 2021 Jan
28. PMID: 33504187; PMCID: PMC7902455.

30. Perera KS, de Sa Boasquevisque D, Rao-Melacini P,
Taylor A, Cheng A, Hankey GJ, Lee S, Fabregas JM,
Ameriso SF, Field TS, Arauz A, Coutts SB, Arnold M,
Mikulik R, Toni D, Mandzia J, Veltkamp RC,
Meseguer E, Haeusler KG, Hart RG; Young ESUS
Investigators. Evaluating Rates of Recurrent
Ischemic Stroke Among Young Adults With Embolic
Stroke of Undetermined Source: The Young ESUS
Longitudinal Cohort Study. JAMA Neurol. 2022 May
1;79(5):450-458.
doi:
10.1001/jamaneurol.2022.0048. PMID: 35285869;
PMCID: PMC8922202.

Table I – Cohort characteristics

Fig. 1 – Main pathologic agents involved in UITs

45

Fig. 2 – Pneumonia distribution among patients

Table II – Associated pathology

Fiziologia - Physiology • 2023.2(105)Fig. 3 - Representation of the CT examination results in the studied patient cohort.

Table III – Affected cerebral territory

EXPLORAREA FACTORILOR DE RISC ȘI PROGNOSTIC ÎN ACCIDENTUL
VASCULAR CEREBRAL ISCHEMIC
RezumatAccidentul vascular cerebral, un eveniment neurologic critic, lasă adesea o amprentă indelebilă asupra vieții unui pacient, solicitând oînțelegere cuprinzătoare a aspectelor sale multiple pentru managementul și prognosticul eficient. Acest studiu se concentrează asupra
accidentelor vasculare ischemice, care reprezintă o proporție semnificativă din cazurile de accident vascular cerebral. Cu prevalența
accidentelor vasculare în creștere la nivel global, identificarea unor markeri prognostici robusti devine imperativă pentru adaptareaintervențiilor și îmbunătățirea rezultatelor pacienților. Factorii de risc cheie, inclusiv vârsta înaintată, hipertensiunea și afecțiunile vasculare
comorbide, joacă roluri pivotale în modelarea traiectoriei prognosticului accidentului vascular cerebral ischemic. Recunoașterea
interacțiunii intricate dintre acești factori este vitală pentru ca medicii să elaboreze strategii terapeutice direcționate. Prognoza
post-accident vascular cerebral ischemic rămâne un puzzle complex, cu variabile precum severitatea infarctului, complicațiile asociate și
eficacitatea intervențiilor medicale influențând cursul clinic. Acest studiu, realizat retrospectiv pe o cohortă de 200 de pacienți, explorează
aceste nuanțe, aducând în prim-plan corelații și modele care pot rafina înțelegerea asupra prognosticului accidentului vascular cerebral
ischemic. Dezvăluind complexitățile acestor factori, acestă cercetare își propune să contribuie cu perspective valoroase care pot infuența
practicile clinice bazate pe dovezi și pot îmbunătăți în mod general managementul pacienților cu accident vascular cerebral ischemic.
Cuvinte cheie: accident vascular cerebral, factori de risc, prognostic.

47

EXTRACELLULAR VESICLES IN ISCHEMIC STROKE: POTENTIALBIOMARKERS AND THERAPEUTIC AVENUESROXANASURUGIU1*,BURDUSELDAIANA1,2#,
DUMITRESCU-GENUNCHE4*, RALUCA ELENA SANDU1,4
4

EMILIA

BURADA3*,

AMELIA

Department of Biochemistry, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;5

Department of Psychiatry, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;6

Department of Physiology, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;7

Department of Internal Medicine, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;8

Department of Neurology, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;*

Correspondence: Emilia Burada, emilia.burada@umfcv.ro; Amelia Dumitrescu-Genunche, amelia.genunche@umfcv.ro;#

These authors contributed equally

ABSTRACT
Ischemic stroke, a global health concern, demands comprehensive exploration due to its substantial impact. This review delves into
critical aspects, notably excitotoxicity and calcium dysregulation, which play pivotal roles in irreversible neuronal damage. Excitotoxicity,
initiated by glutamate pathways, underscores the urgency of understanding these intricate mechanisms. The focus shifts to calcium
dysregulation, a key contributor to ischemic cell death. Various ion channels, pumps, and receptors are intricately involved in this process,emphasizing the need for a nuanced understanding. Amidst these complexities, extracellular vesicles (EVs) emerge as crucial mediators
in intercellular communication. Once considered cellular debris, EVs now present promising potential in stroke therapy. However,challenges in EV isolation and analysis are acknowledged, emphasizing the importance of reproducible biomarkers. As EVs exhibit
multifaceted impacts on neuroinflammation, neurogenesis, and angiogenesis, their role in stroke pathophysiology becomes increasingly
complex. With ongoing studies that provide a comprehensive perspective, EVs are positioned as promising candidates for both
diagnostic biomarkers and therapeutic interventions in ischemic stroke.
Keywords: stroke, EVs, biomarkers.

1.

Introduction
Stroke stands as the primary contributor to long-term
disability and ranks as the fifth most prevalent cause ofdeath in the United States [1]. This debilitating condition,
characterized by the sudden disruption of blood flow tothe brain, not only poses a significant threat to life but
also has enduring consequences for those who survive[2]. The impact of stroke extends beyond its immediate
life-threatening nature, often resulting in long-term
impairments that affect individuals' quality of life andindependence [3]. The European Union is projected toexperience a 27% rise in the number of individuals
affected by stroke from 2017 to 2047. This increase is
primarily attributed to population aging and enhanced
survival rates [4].
The findings from the BASIC study (Brain Attack

Surveillance in Corpus Christi) spanning the years
2000-2010 revealed a noteworthy decline in theoccurrence of ischemic strokes among individuals aged
60 and above. In contrast, rates for those aged between
45 and 59 remained relatively stable. According to thestudy's statistics, the adjusted incidence of hemorrhagic
strokes, accounting for age and ethnicity during the same
period, decreased from 5.21 per 10,000 (95% CI,
4.36-6.24) to 4.30 per 10,000 (95% CI, 3.21-5.76) [5].
Every year, approximately 55,000 more women than men
experience a stroke (GCNKSS, NINDS - Greater
Cincinnati/Northern Kentucky Stroke Study, National
Institutes of Neurological Disorders and Stroke) [6].
Women face a higher risk of stroke compared to men. In
the FHS (Framingham Heart Study), the lifetime risk ofFiziologia - Physiology • 2023.2(105)stroke among individuals aged 55 to 75 was 1 in 5 forwomen (95% CI, 20% - 21%) and approximately 1 in 6
for men (95% CI, 14% - 17%) [7]. Age-correlated
incidence is significantly lower in women than in men inyounger and middle-aged groups. However, thesedifferences diminish, and in the most advanced age
group, incidence rates for women become approximately
equal to or even higher than those for men [8,9].
Upon the initiation of pathological pathways in theischemic cascade, irreversible neuronal damage occurs
in the ischemic core within minutes of onset [10]. The
severity of the injury depends on the degree and durationof ischemia and the brain's ability to recover and repair[11].
Due to high intrinsic metabolic activity and increased
concentrations of neuroexcitatory substances (such as
glutamate), brain tissue is vulnerable to ischemia [12].
This vulnerability may result from in-situ thrombosis or
occlusive embolic events, often stemming fromatheromatous plaques at the carotid or aortic arch levels.
Neurologic dysfunction can manifest within seconds tominutes, hours, or even days from the onset of theischemic event.

the ischemic context. The selective increase in synaptic
receptors promotes neuronal survival, while theactivation of extrasynaptic receptors promotes cell death,in part through the activation of protein kinase 1 [18].
The MAP kinase family plays a role throughphosphorylation-dependent regulatory mechanisms,
such as the NR2A and NR2B receptor subunits [19].
Under ischemic conditions, cell vulnerability is reducedby modifying postsynaptic proteins. Targeting glutamate
receptors early would lead to decreased channel
regulation by suppressing depolarization [20].
2.2. Calcium dynamics in ischemic conditions
The elevated calcium influx resulting from theoveractivation of glutamate receptors, along with calcium
ions released from mitochondria and other cellulardeposits, is nonetheless insufficient to offset the
intracellular calcium accumulation due to excitotoxic
stimulation. Other ion channels and pumps activatedduring ischemia are also involved in calcium
accumulation mechanisms, including the Na+/Ca2+
pump, hemichannels, volume-regulated anion channels,
and TRP channels [21-24]. ASIC1a, activated byischemia-induced acidosis, is implicated in calcium influx,
with its inhibition exhibiting a neuroprotective effect [25].
As these channels are activated at a specific pH
commonly encountered in ischemia, this could explain
the worsening prognosis associated with the onset of
acidosis. Disruption of the Na+/Ca2+ pump also leads tocalcium accumulation. The prostaglandin E2 EP1
receptor plays a significant role in altering Na+/Ca2+
exchanges during the ischemic period, and inhibiting
them has a neuroprotective effect [26,27].

2. Neuronal Vulnerability in Ischemic Cascade
2.1 Glutamate excitotoxicity
Excitotoxicity and calcium excess are major factors in
the early stages of ischemic cell death. The pathway is
glutamate-dependent,themostabundant
neurotransmitter that accumulates in the extracellularspace due to ion pump dysfunction and reuptake
mechanisms [13]. Excess glutamate leads to prolonged
stimulation of NMDA and AMPA ionotropic receptors,
increasing the influx of calcium, sodium, and water at theneuronal level. Massive calcium influx activates catabolic
processes mediated by proteases, lipases, andnucleases [14]. Additionally, the activation of
Ca2+-dependent enzymes leads to the production of
nitric oxide, arachidonic acid metabolites, acting as
triggers for cell death. Oxidative phosphorylation
becomes inefficient, followed by ATP depletion and thegeneration of reactive oxygen species (ROS), releasing
calcium from the mitochondria, accelerating the series of
events leading to cell death [15].
Despite laboratory studies demonstrating glutamate
toxicity and the positive impact of new therapies inneuroscience, clinical trials aiming to inhibit NMDA and
AMPA receptors have not demonstrated their benefits intreating stroke patients [16]. The NMDA-AMPA model
does not account for calcium influx independent ofglutamate secondary to oxygen and glucose deprivation[17]. Moreover, it cannot consider the death of other cell
types such as astrocytes, vascular cells, and microglia in

2.3. Reactive Oxygen Species {ROS}
Numerous factors associated with cardiovascular risk
contribute to an upsurge in the generation of reactive
oxygen species (ROS), fostering both a systemic andcerebral pro-inflammatory environment [28]. The
vascular origins of ROS encompass enzymes like
NADPH oxidase, xanthine oxidase, mitochondrial
enzymes, and nitric oxide synthase. The detrimental
repercussions of oxidative stress on blood vessels often
correlate with the biological inactivation of nitric oxide[29]. Consequently, the loss of nitric oxide'svasoregulatory effects on vascular endothelium leads tovasoconstriction, disrupting the regulation of
microvascular flow [29].
This loss further extends to the anti-aggregating,anti-proliferative, and cell adhesion maintenance effects,
resulting in platelet aggregation, leukocyte adherence toendothelial cells, and proliferation of smooth muscle
tissue – pivotal stages in the inflammatory response49within blood vessels [30]. ROS amplifies inflammation byelevating blood-brain barrier permeability throughheightened expression of vascular endothelial growth
factor, cytokines, matrix remodeling enzymes,
metalloproteinases, and proinflammatory genes [31].
Additionally, the alteration of vasomotor reactivity occurs
through the inactivation of crucial enzymes in endothelialand smooth muscle cells, coupled with the activation ofpoly-ADP-ribose polymerase, leading to ATP depletionand dysfunction of ion-dependent channels at thevascular level [32].
Simultaneously, vascular inflammation induces anaugmented production of ROS, establishing a cyclic
pattern that significantly intensifies vascular damage.
Plasminogen activation, triggered by oxidative stress anda pro-inflammatory state, facilitates matrix remodeling,smooth muscle cell migration, and intimal hyperplasia –factors that potentiate the development of
atherosclerosis [33-35]. Oxidative stress and vascularinflammation stand as predominant pathways throughwhich the detrimental impact on blood vessels is
manifested.
3.

The role of Extracellular Vesicles (EVs) in
ischemic stroke
3.1. Prognostic biomarkers
Extracellular vesicles (EVs) have become a focal
point of considerable interest in recent decades,
particularly within the realm of biomarker research.
Initially dismissed as cellular debris or casually labeledwith terms such as 'placental mist,' their significance has
evolved, and they are now recognized as crucial
mediators of intercellular communication. The spectrumof nomenclature continues to expand annually, propelledby the advent of novel isolation techniques andadvancements in methods for quantifying and visualizing
extracellular vesicles [36]. While numerous investigations
have delved into neuroprotective and recovery strategies
following ischemic stroke, the available data on
circulating biomarkers possessing prognostic anddiagnostic value remains somewhat limited. Blood-based
biomarkers have emerged as a focal point of intensive
research and are widely acknowledged as valuable tools
for predicting the prognoses and survival rates ofindividuals who have experienced a stroke [37].
Consequently, the utilization of readily accessible
biomarkers, detectable through quantitative polymerase
chain reaction (qPCR)-based techniques from biofluids,
holds the potential to significantly enhance our ability to
predict long-term sensorimotor recovery with a high levelof accuracy [38].
EVs exhibit a unique molecular profile contingent
upon their origin, reflecting the phenotypic composition of

the donor cell to some extent. In the context of stroke,
various cell types within the brain and circulatory systemhave been demonstrated to release EVs into thebloodstream. This phenomenon underscores thepotential of EVs to serve as informative biomarkers,
offering insights into the molecular characteristics of thecells involved in the pathophysiology of stroke [39].
In the context of acute ischemic stroke (AIS),
research has highlighted the correlation between theshedding of endothelial cell-derived extracellular vesicles(EVs) and the severity, lesion volume, and overalloutcomes of AIS [40]. Another study focused on AIS
revealed a general increase in EV release from various
blood and vascular compartment cells, including
endothelial cells, platelets, erythrocytes, leukocytes,
monocytes, lymphocytes, and neural precursor cells [41].
Additionally, platelet-derived EVs were found to beelevated in AIS subgroups with large arteryatherosclerosis (LAA) and small vessel disease (cSVD)[42]. Specifically, platelet EVs were significantly
increased in patients with AIS due to large vessel
occlusion (LVO) and cSVD [43].
Intracerebral hemorrhage (ICH) studies indicated a
distinct temporal profiling of EVs, with origins fromendothelial cells, erythrocytes, and neutrophils, revealing
dynamic changes over time [44]. Annexin V-positive EVs
were reported to be elevated in ICH patients compared
to controls, with variations depending on the specificorigin of EVs, including endothelial, leucocyte, anderythrocyte EVs [45-46]. Another study supported thefinding of increased annexin V-positive EVs in ICH
compared to controls at admission [47].
The technical hurdles linked to the consistentisolation of extracellular vesicles (EVs), coupled with the
intricate methodologies essential for their efficient
analysis, could hinder the utilization of 'pure' EV
populations as reliable biomarkers. While the discoveryof biomarkers is crucial for enhancing diagnosis,
monitoring,andpredicting
outcomesinneurodegenerative diseases, prioritizing reproducibility
and user-friendliness is paramount [48].
Although extracellular vesicles (EVs) demonstratepromising value in terms of prognosis, there remains a
critical need for more comprehensive studies tothoroughly assess the longitudinal and dynamic evolutionof the plasmatic response. It is essential to exercise
caution when interpreting the results, considering the
intricate nature of EV interactions within the circulatory
system. Further investigations with a focus on thetemporal aspects of EV release and their correlation withthe evolving clinical condition will contribute to a deeper
understanding of their diagnostic and prognostic potential
in the context of various medical conditions, including
Fiziologia - Physiology • 2023.2(105)stroke.

In a phase I/II clinical trial, Bang et al. administeredintravenous BM-MSC transplantation twice within 9
weeks after ischemic stroke to five patients. No deaths,
recurrences, or post-transplant abnormalities werereported, and significant improvement in Barthel’s Index
was observed during the first year compared to controls[53]. In a 5-year study, Lee et al. administered two
intravenous autologous BM-MSC doses to 85
post-ischemic stroke patients. The MSC group showed asubstantially higher survival rate (72%) than the control
group (34%), correlating with increased blood SDF-1
levels and reduced damage in the lateral ventricles [54].
4. Conclusion
In conclusion, the intricate pathophysiologicalprocesses underlying ischemic stroke involve a cascadeof events, from excitotoxicity and calcium influx tooxidative stress and inflammation. Despite significant
strides in understanding these mechanisms, translating
preclinical findings into effective clinical interventions
remains a formidable challenge. The multifaceted rolesof extracellular vesicles (EVs) emerge as a promisingavenue, with their potential to serve as both diagnostic
biomarkers and therapeutic agents. While studies haveelucidated the immunomodulatory and neuroprotective
properties of EVs in animal models, their application inhuman subjects presents a new frontier in stroke
research. The ongoing clinical trials and evolving
methodologies for EV isolation and analysis underscorethe dynamic nature of this field. As we navigate thisscientific landscape, the integration of EVs into stroke
management holds great promise, offering a glimpse into
the future of more effective diagnostic strategies andinnovative therapeutic interventions for ischemic stroke.

3.2. Treatment potential
Exosomes originating from multipotent mesenchymal
stem cells (MSCs) have proven effective in preserving
white matter integrity and fostering functional recovery ina murine model of subcortical hemorrhage, simulating
hemorrhagic stroke [49]. Additionally, these MSC
exosomes demonstrate the capability to alleviate brain
injury and curb neuroinflammation after middle cerebral
artery occlusion (MCAO) by inhibiting the differentiationof proinflammatory M1 microglia [50].
The infusion of extracellular vesicles (EVs) instroke-afflicted mice reveals an activation of the
TGF-β/Smad2/3 signaling pathway in the ischemic brain.
Additionally, EVs enriched with TGF-β1, secreted by
microglia preconditioned under oxygen-glucose
deprivation (OGD), induce M2 polarization in resident
microglia within the ischemic cerebral environment. This
process may play a role in regulating the early
inflammatory response in postischemic hemispheres[51].
Experimental investigations focusing on ischemic
stroke have revealed that extracellular vesicles (EVs)possess immunomodulatory and neuroprotective
properties. These findings suggest that EVs may play arole in stimulating neurogenesis and angiogenesis in thecontext of ischemic stroke [52].
In light of the promising outcomes observed in animalmodels, the exploration of extracellular vesicles (EVs)transitioned to human studies. This shift in focus reflects
the growing interest and optimism surrounding thepotential translational impact of EVs in the field ofischemic stroke.
References
1. Barthels D, Das H. Current advances in ischemic
stroke research and therapies. Biochim Biophys
Acta Mol Basis Dis. 2020 Apr 1;1866(4):165260. doi:
10.1016/j.bbadis.2018.09.012. Epub 2018 Sep 15.
PMID: 31699365; PMCID: PMC6981280.
2. Campbell BCV, De Silva DA, Macleod MR, Coutts
SB, Schwamm LH, Davis SM, Donnan GA.
Ischaemic stroke. Nat Rev Dis Primers. 2019 Oct
10;5(1):70. doi: 10.1038/s41572-019-0118-8. PMID:
31601801.
3. Katan M, Luft A. Global Burden of Stroke. Semin
Neurol.
2018
Apr;38(2):208-211.
doi:
10.1055/s-0038-1649503. Epub 2018 May 23. PMID:
29791947.
4. Wafa HA, Wolfe CDA, Emmett E, Roth GA, Johnson
CO, Wang Y. Burden of Stroke in Europe:
Thirty-Year Projections of Incidence, Prevalence,
Deaths, and Disability-Adjusted Life Years. Stroke.
2020
Aug;51(8):2418-2427.
doi:
10.1161/STROKEAHA.120.029606. Epub 2020 Jul
10. PMID: 32646325; PMCID: PMC7382540.

5.

6.

7.

1.
8.

51

Zahuranec D., Lisabeth L., Sánchez B., Smith M.,
Brown D., Garcia N., Skolarus L., Meurer W., Burke
J., Adelman E., and Morgenstern L. "Intracerebral
hemorrhage mortality is not changing despite
declining incidence." Neurology, vol. 82, pp.
2180-2186, 2014.
Kleindorfer D., Khoury J., Moomaw C., Alwell K.,
Woo D., Flaherty M., Khatri P., Adeoye O., Ferioli S.,
Broderick J., and Kissela B. "Stroke incidence isdecreasing in whites but not in blacks: apopulation-based estimate of temporal trends instrokeincidence
from
theGreaterCincinnati/Northern Kentucky Stroke Study." Stroke,
vol. 41, pp. 1326-1331, 2010.
Seshadri S., Beiser A., Kelly-Hayes M., Kase C., Au
R., Kannel W., and Wolf P. "The lifetime risk ofstroke: estimates from the Framingham Study."
Stroke, vol. 37, p. 345–350, 2006.
Reeves M., Bushnell C., Howard G. et al. "Sexdifferences in stroke: epidemiology, clinical

9.

10.
11.

12.
13.
14.

15.
16.
17.

18.

19.

20.

21.
22.

23.

24.

presentation, medical care, and outcomes." Lancet
Neurol, vol. 7, pp. 915-926, 2008.
Sealy-Jefferson S., Wing J., Sánchez B., Brown D.,
Meurer W., Smith M., Morgenstern L., and Lisabeth
L. "Age- and ethnic-specific sex differences in strokerisk." Gend Med, vol. 9, pp. 121-128, 2012.
Dirnagl U., Iadecola C., and Moskowitz M.
"Pathobiology of ischaemic stroke: an integrated
view." Trends Neurosci, vol. 22, pp. 391-397, 1999.
Lakhan S., Kirchgessner A., and Hofer M.
"Inflammatory mechanisms in ischemic stroke:
therapeutic approaches." Journal of Translational
Medicine, vol. 7, p. 97, 2009.
Choi D. "Excitotoxic cell death." J. Neurobiol., vol.
23, pp. 1261-1276, 1992.
Choi D. and Rothman S. "The role of glutamate
neurotoxicity in hypoxic-ischemic neuronal death."Annu. Rev. Neurosci., vol. 13, pp. 171-182, 1990.
Ankarcrona M., Bonfoco E., Zhivotovsky B.,
Orrenius S., Lipton S., and Nicotera P.
"Glutamate-induced neuronal death: a succession of
necrosis or apoptosis depending on mitochondrial
function." Neuron, vol. 15, pp. 961-973, 1995.
Lo E., Dalkara T., and Moskowitz M. "Mechanisms,
challenges and opportunities in stroke." Nat. Rev.
Neurosci., vol. 4, pp. 399-415, 2003.
Ginsberg M. "Current status of neuroprotection for
cerebral ischemia: synoptic overview." Stroke, vol.
40, pp. S111-S114, 2009.
Aarts M., Iihara K., Wei W., Xiong Z., Arundine M.,
Cerwinski W., MacDonald J., and Tymianski M. "A
key role for TRPM7 channels in anoxic neuronal
death." Cell, vol. 115, pp. 863-877, 2003.
Tu W., Xu X., Peng L., Zhong X., Zhang W.,
Soundarapandian M., Balel C., Wang M., Jia N.,
Zhang W., Lew F., Chan S., Chen Y., and Lu Y.
"DAPK1 interaction with NMDA receptor NR2B
subunits mediates brain damage in stroke." Cell, vol.
140, pp. 222-234, 2010.
Hardingham G., Fukunaga Y., and Bading H.
"Extrasynaptic NMDARs oppose synaptic NMDARs
by triggering CREB shut-off and cell deathpathways." Nat. Neurosci., vol. 5, pp. 405-414,
2002.
Cui H., Hayashi A., Sun H., Belmares M., Cobey C.,
Phan T., Schweizer J., Salter M., Wang Y., Tasker R.,
and others. "PDZ protein interactions underlying
NMDA receptor-mediated excitotoxicity andneuroprotection by PSD-95 inhibitors." J. Neurosci.,
vol. 27, pp. 9901-9915, 2007.
Dirnagl U., Iadecola C., and Moskowitz M.
"Pathobiology of ischaemic stroke: an integrated
view." Trends Neurosci, vol. 22, pp. 391-397, 1999.
Lakhan S., Kirchgessner A., and Hofer M.
"Inflammatory mechanisms in ischemic stroke:
therapeutic approaches." Journal of Translational
Medicine, vol. 7, p. 97, 2009.
Bano D., Munarriz E., Chen H., Ziviani E., Lippi G.,
Young K., Nicotera P. "The plasma membrane
Na+/Ca2+ exchanger is cleaved by distinct proteasefamilies in neuronal cell death." Ann. N Y Acad. Sci.,
vol. 1099, pp. 451-455, 2007.
Contreras J., Sánchez H., Véliz L., Bukauskas F.,
Bennett M., Sáez J. "Role of connexin-based gap
junction channels and hemichannels in
ischemia-induced cell death in nervous tissue."

25.

26.
27.
28.

29.

30.
31.

32.
33.
34.
35.
36.

37.

38.

39.

40.

Brain Res. Brain Res. Rev., vol. 47, pp. 290-303,
2004.
Simard J., Kent T., Chen M., Tarasov K., Gerzanich
V. "Brain edema in focal ischemia: molecularpathophysiology and theoretical implications."
Lancet Neurol., vol. 6, pp. 258-268, 2007.
Aarts M., Tymianski M. "TRPMs and neuronal cell
death." Pflugers Arch., vol. 451, pp. 243-249, 2005.
Simon R. "Acidotoxicity trumps excitotoxicity inischemic brain." Arch. Neurol., vol. 63, pp.
1368-1371, 2006.
Abe T., Kunz A., Shimamura M., Zhou P., Anrather J.,
Iadecola C. "The neuroprotective effect of
prostaglandin E2 EP1 receptor inhibition has a wide
therapeutic window, is sustained in time and is notsexually dimorphic." J. Cereb. Blood Flow Metab.,vol. 29, pp. 66-72, 2009.
Kawano T., Anrather J., Zhou P., Park L., Wang G.,
Frys K., Kunz A., Cho S., Orio M., Iadecola C.
"Prostaglandin E2 EP1 receptors: downstream
effectors of COX-2 neurotoxicity." Nat. Med., vol. 12,
pp. 225-229, 2006.
Faraci F. "Reactive oxygen species: influence on
cerebral vascular tone." J. Appl. Physiol., vol. 100,
pp. 739-743, 2006.
Schulz E., Jansen T., Wenzel P., Daiber A., Münzel T.
"Nitric oxide, tetrahydrobiopterin, oxidative stress,and endothelial dysfunction in hypertension."
Antioxid. Redox Signal., vol. 10, pp. 1115-1126,
2008.
Pepine C. "The impact of nitric oxide in
cardiovascular medicine: untapped potential utility."
Am. J. Med., vol. 122, no. 5, pp. S10-S15, 2009.
Marchesi C., Paradis P., Schiffrin E. "Role of therenin-angiotensin system in vascular inflammation."
Trends Pharmacol. Sci., vol. 29, pp. 367-374, 2008.
Pacher P., Beckman J., Liaudet L. "Nitric oxide andperoxynitrite in health and disease." Physiol. Rev.,
vol. 87, pp. 315-424, 2007.
Nicholl S., Roztocil E., Davies M. "Plasminogenactivator system and vascular disease." Curr. Vasc.
Pharmacol., vol. 4, pp. 101-116, 2006.
Witwer KW, Thery C. Extracellular vesicles or
exosomes? On primacy, precision, and popularity
influencing a choice of nomenclature. J Extracell
Vesicles.
2019;8(1):1648167.
doi:
10.1080/20013078.2019.1648167
Steliga A., Kowiański P., Czuba E., Waśkow M.,Moryś J., Lietzau G. Neurovascular Unit as a Sourceof Ischemic Stroke Biomarkers—Limitations ofExperimental Studies and Perspectives for ClinicalApplication. Transl. Stroke Res. 2020;11:553–579.
doi: 10.1007/s12975-019-00744-5.
Pfeiffer S., Sánchez-Lechuga B., Donovan P.,
Halang L., Prehn J.H.M., Campos-Caro A., Byrne
M.M., López-Tinoco C. Circulating MiR-330-3p in
Late Pregnancy Is Associated with Pregnancy
Outcomes Among Lean Women with GDM. Sci.
Rep. 2020;10:908.
doi: 10.1038/s41598-020-57838-6.
Stenz KT, Just J, Blauenfeldt RA, Drasbek KR.
Extracellular Vesicles in Acute Stroke Diagnostics.
Biomedicines. 2020 Jul 28;8(8):248. doi:
10.3390/biomedicines8080248. PMID: 32731351;
PMCID: PMC7459954.
Simak J., Gelderman M.P., Yu H., Wright V., Baird
A.E. Circulating endothelial microparticles in acute

Fiziologia - Physiology • 2023.2(105)

41.

42.

43.

44.

45.

46.

47.

ischemic stroke: A link to severity, lesion volume andoutcome. J. Thromb. Haemost. 2006;4:1296–1302.
doi: 10.1111/j.1538-7836.2006.01911.x.
Chiva-Blanch G., Suades R., Crespo J., Peña E.,
Padró T., Jiménez-Xarrié E., Martí-Fàbregas J.,
Badimon L. Microparticle shedding from neuralprogenitor cells and vascular compartment cells isincreasedinischemic
stroke. PLoS
ONE. 2016;11:e0148176.
doi: 10.1371/journal.pone.0148176.
Chen Y., Xiao Y., Lin Z., Xiao X., He C., Bihl J.C.,
Zhao B., Ma X., Chen Y. The Role of Circulating
Platelets Microparticles and Platelet Parameters in
Acute Ischemic Stroke Patients. J. Stroke
Cerebrovasc.
Dis. 2015;24:2313–2320.
doi: 10.1016/j.jstrokecerebrovasdis.2015.06.018.
Kuriyama N., Nagakane Y., Hosomi A., Ohara T.,
Kasai T., Harada S., Takeda K., Yamada K., Ozasa
K., Tokuda T., et al. Evaluation of factors associatedwithelevatedlevelsofplatelet-derived
microparticles in the acute phase of cerebral
infarction. Clin. Appl. Thromb. 2009;16:26–32.
doi: 10.1177/1076029609338047.
Sanborn M.R., Thom S.R., Bohman L.-E., Stein S.C.,
Levine J.M., Milovanova T., Maloney-Wilensky E.,
Frangos S., Kumar M.A. Temporal dynamics of
microparticle elevation following subarachnoid
hemorrhage. J.
Neurosurg. 2012;117:579–586.
doi: 10.3171/2012.6.JNS111163.
Huang M., Hu Y.-Y., Dong X.-Q. High concentrationsof procoagulant microparticles in the cerebrospinal
fluid and peripheral blood of patients with acutebasal ganglia hemorrhage are associated with pooroutcome. Surg.
Neurol. 2009;72:481–489.
doi: 10.1016/j.surneu.2008.12.016.
Lackner P., Dietmann A., Beer R., Fischer M.,
Broessner G., Helbok R., Marxgut J., Pfausler B.,
Schmutzhard E. Cellular microparticles as a marker
for cerebral vasospasm in spontaneous
subarachnoid hemorrhage. Stroke. 2010;41:2353–
2357. doi: 10.1161/STROKEAHA.110.584995.
Dong X.-Q., Huang M., Hu Y.-Y., Yu W.-H., Zhang
Z.-Y. Time course of plasma microparticle
concentrations after acute spontaneous basal
gangliahemorrhage. Acta
Neurol.

48.

49.

50.

51.

52.

53.

54.

53

Scand. 2011;123:280–288.
doi: 10.1111/j.1600-0404.2010.01399.x.
Couch Y. Challenges associated with using
extracellular
vesiclesasbiomarkers
inneurodegenerative disease. Expert Rev Mol Diagn.
2023
Nov
2:1-15.
doi:
10.1080/14737159.2023.2277373. Epub ahead ofprint. PMID: 37916853.
Otero-Ortega L., Gómez de Frutos M.C.,
Laso-García
F.,
Rodríguez-Frutos
B.,
Medina-Gutiérrez E., López J.A., Vázquez J.,
Díez-Tejedor E., Gutiérrez-Fernández M. Exosomes
promote restoration after an experimental animalmodel of intracerebral hemorrhage. J. Cereb. Blood
Flow Metab. 2018;38:767–779.
Zhao Y., Gan Y., Xu G., Yin G., Liu D. MSCs-derived
exosomes attenuate acute brain injury and inhibit
microglial
inflammationbyreversingCysLT2R-ERK1/2
mediated
microglia
M1
polarization. Neurochem. Res. 2020;45:1180–1190.
Zhang L, Wei W, Ai X, Kilic E, Hermann DM,
Venkataramani V, Bähr M, Doeppner TR.
Extracellular vesicles from hypoxia-preconditioned
microglia promote angiogenesis and repress
apoptosis in stroke mice via the TGF-β/Smad2/3
pathway. Cell Death Dis. 2021 Nov 9;12(11):1068.
doi: 10.1038/s41419-021-04363-7. PMID: 34753919;
PMCID: PMC8578653.
Dabrowska S, Andrzejewska A, Lukomska B,
Janowski M. Neuroinflammation as a target fortreatment of stroke using mesenchymal stem cells
and extracellular vesicles. J Neuroinflammation.
2019
Sep
12;16(1):178.
doi:
10.1186/s12974-019-1571-8. PMID: 31514749;
PMCID: PMC6743114.
Bang OY, Lee JS, Lee PH, Lee G. Autologous
mesenchymal stem cell transplantation in stroke
patients. Ann
Neurol. 2005;57(6):874–882.
doi: 10.1002/ana.20501.
Lee JS, Hong JM, Moon GJ, Lee PH, Ahn YH, Bang
OY, et al. A long-term follow-up study of intravenous
autologous mesenchymal stem cell transplantationin patients with ischemic stroke. Stem Cells Dayt
Ohio. 2010;28(6):1099–1106.
doi: 10.1002/stem.430.

VEZICULELE EXTRACELULARE ÎN ACCIDENTUL VASCULAR CEREBRAL ISCHEMIC: DE LA
BIOMARKERI LA PERSPECTIVE TERAPEUTICE
RezumatAccidentul vascular cerebral ischemic este o problemă globală de sănătate, ce necesită o explorare exhaustivă datorită impactului său
semnificativ. Această review investighează aspecte critice, în special excitotoxicitatea și alterarea homeostaziei calciului, care joacă roluri
cheie în deteriorarea ireversibilă a neuronilor. Excitotoxicitatea, inițiată de căile glutamatului, subliniază urgența înțelegerii acestor mecanisme
intricate. Se pune accentul pe alterarea calciului, un contribuitor cheie la moartea celulară ischemică. Diverse canale ionice, pompe și receptorisunt implicate în acest proces, subliniind necesitatea unei înțelegeri superioare. În acest context, vesiculele extracelulare (EV-uri) apar caelemente cruciale în comunicarea intercelulară. Odată considerate reziduuri celulare, EV-urile prezintă acum un potențial promițător în terapia
accidentului vascular cerebral. Cu toate acestea, se recunosc provocările legate de izolarea și analiza EV-urilor, subliniind importanța
markerilor biologici reproductibili. Rolul EV-urile este demonstrate in ceea ce priveste neuroinflamația, neurogeneza și angiogeneza, însăcomplexittea lor în fiziopatologia accidentului vascular cerebral rămâne încă incomplete elucidată. În ultimii ani, tot mai multe studii oferă operspectivă cuprinzătoare, poziționând EV-urile ca posibili candidați atât pentru markeri diagnostici, cât și pentru intervenții terapeutice înaccidentul vascular cerebral ischemic.
Cuvinte cheie: accident vascular cerebral, veziculele extracelulare, biomarkeri.

Fiziologia - Physiology • 2023.2(105)EXPLORING DIVERSITY: MURINE MODELS FOR INDUCING SEPSIS IN
PRECLINICAL RESEARCHALEXANDRA DANIELA ROTARU ZAVALEANU1#, MIHAI RUSCU1#, VENERA DINESCU2*, RAMONA VASILE1*, SORINDINESCU1
9Department of Epidemiology, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova, Romania;

2

Department of Preventive Medicine, University of Medicine and Pharmacy of Craiova, 200433, St. Petru Rares, no. 2-4, Craiova,
Romania;*

Correspondence: Venera Dinescu, venera.dinescu@umfcv.ro; Ramona Vasile, ramona.vasile@umfcv.ro#

These authors contributed equally

ABSTRACT
Murine sepsis models play a crucial role in preclinical research, providing an essential platform for mimicking and understanding
the complexity of sepsis pathogenesis. These models allow researchers to accurately reproduce critical aspects of the immune
response and microbial interactions in a controlled environment. The use of endotoxins or the cecal ligation and puncture (CLP)
method in murine models provides a systematic approach to studying the immune response, enabling the investigation of boththe early and late phases of sepsis. The advantages of these models include reproducibility, control over experimental variables,
and the ability to manipulate genetics to assess the specific role of genes. However, in selecting murine models for sepsis
studies, careful consideration of ethical aspects, minimizing animal suffering, and maximizing scientific validity is crucial. In thecontext of future research, emerging technologies such as advanced imaging and personalized models represent promising
directions to improve relevance and deepen our understanding of sepsis pathology at the molecular and functional levels.
Keywords: sepsis, CLP, LPS, murine models
Introduction

Importance of Murine Models in Sepsis Research.
Advantages of Murine Models in Preclinical Sepsis
Research
Murine models are indispensable tools in preclinicalresearch, offering several advantages that significantly
contribute to the understanding of sepsis pathogenesis.
Firstly, murine models offer precise control overexperimental conditions, facilitating the systematic
manipulation of variables to elucidate specific aspects ofsepsis progression (7). The genetic tractability of miceallows for the development of transgenic or knockout
models, providing researchers with the capability toinvestigate the role of specific genes in sepsis susceptibility
and outcomes (8). Moreover, the short reproductive cycleof mice allows generations of large cohorts, facilitating
robust statistical analyses and enhancing the
reproducibility of experimental findings (9). The anatomicaland physiological similarities between murine and humanimmune systems further enhance the translational
relevance of findings from murine models to clinical sepsis
scenarios (10).
Relevance of Murine Models in Studying Sepsis
Mechanisms
Murine models allow a controlled environment for the
meticulous dissection of immune responses, from the initialrecognition of pathogens to the downstream signalingcascades (4). For instance, the use of murine models has
been instrumental in delineating the roles of various

Sepsis investigation is critical when it comes topreclinical studies, considering its highly important andconsequential impact on global public health (1, 2). It is alife-threatening syndrome which arises from thedysregulated host response to infection, and it remains aleading cause of mortality and morbidity worldwide (3).
Sepsis elucidation pathogenesis requires a thoughtful
exploration of its intricate molecular and cellularmechanisms, a task that is optimally approached in thecontrolled environment afforded by preclinical research (4).
Murine models, owing to their biological similarities andgenetic tractability, play a pivotal role in this investigative
landscape (5). Murine models offer a controlled
experimental platform to simulate various aspects of sepsis,
while facilitating the systematic dissection of immune
responses, microbial interactions, and host tissue
dynamics (6). The purpose of this article is tocomprehensively examine the diversity inherent in murine
models utilized in preclinical sepsis research, thereby
enriching our understanding of sepsis pathogenesis.
Through a meticulous analysis of these models and theirapplications, this exploration aims to contribute to therefinement of experimental methodologies, ultimately
enhancing the translational relevance of preclinical findings
to the complex clinical landscape of human sepsis.
55

immune cells, such as macrophages and neutrophils, insepsis-induced tissue damage (11). Also, murine models
allow the study of host-microbe interactions, helping with
the identification of microbial factors that contribute tosepsis severity (12). The insights obtained from murine
studies enhance our understanding of sepsis at amolecular level, offering the information for thedevelopment of targeted therapeutic interventions (13).
Limitations and Challenges Associated with Murine
ModelsAlthough murine models offer invaluable insights, it isessential to acknowledge their limitations and challenges inaccurately recapitulating the complexity of human sepsis.
One notable limitation is the inherent variability in murine
immune responses, which may differ from those observedin humans (14). Additionally, murine models may
oversimplify the diverse etiologies and comorbidities
associated with human sepsis, potentially limiting thegeneralizability of findings (15). The artificial nature ofexperimental sepsis induction methods, such as cecal
ligation and puncture (CLP) or endotoxin administration,
may not fully replicate the heterogeneity of clinical sepsis
scenarios (16). Furthermore, the ethical considerations
surrounding the use of animal models in researchnecessitate a constant evaluation of the balance between
scientific advancements and animal welfare (17).
Addressing these challenges is crucial for the judicious
interpretation of murine model data and the meaningfultranslation of research findings to the complex clinicallandscape of human sepsis.
III. Diversity in Murine Sepsis Models
a. Endotoxin-Induced Models
Endotoxin-induced models represent a widely
employed approach in murine sepsis research, aiming tosimulate
bacterial
infectionbyadministering
lipopolysaccharide (LPS), which is an essential componentof Gram-negative bacterial cell walls (6). This model
leverages the ability of LPS to activate Toll-like receptors,
though triggering a robust inflammatory response
reminiscentofbacterial
infections(6).
The
endotoxin-induced model offers multiple advantages,
including reproducibility, controlled experimental conditions,but also the ability to investigate specific aspects of thehost response to bacterial components (18). Variations inthis model involve adjusting LPS dosages, administrationroutes, and timing, allowing researchers to modulate theintensity and duration of the inflammatory response (18).
These variations enable the exploration of diverse facets ofsepsis pathophysiology, from the early stages of immune
activation to the later phases characterized byimmunosuppression (19).
b. Cecal Ligation and Puncture (CLP)The Cecal Ligation and Puncture (CLP) method stands
as a gold standard in murine sepsis models, serving as arobust technique to induce polymicrobial sepsis by
mimicking the clinical scenario of intra-abdominal infection(13). The CLP model involves the ligation and puncture of
the cecum, leading to the release of fecal contents into theperitoneal cavity and subsequent systemic infection (13).
The relevance of CLP in inducing polymicrobial sepsis isparticularly significant, as it mirrors the complexity ofclinical sepsis scenarios where multiple pathogens may
contribute to the infection (13). Variations in CLP models
include adjustments in cecal ligation tightness, puncture
size, and the number of punctures, allowing for themodulation of sepsis severity and the investigation of
distinct clinical phenotypes (14). These variations enhance
the model's versatility and contribute to its widespread use
in understanding the dynamic aspects of sepsis
progression.
c. Other Models
In addition to endotoxin-induced and CLP models,alternative murine models offer unique insights into sepsis
pathophysiology. For instance, models involving theinfusion of live bacteria provide a more authentic
representation of bacterial infections, allowing the study of
microbial virulence factors and host responses in real-time(20). Alternatively, the peritoneal contamination andinfection (PCI) model involves the introduction of fecal
material into the peritoneal cavity, leading to apolymicrobial infection akin to clinical scenarios involving
abdominal sepsis (6). These models offer specificadvantages, such as mimicking the natural course of
infection, thereby providing a holistic understanding ofsepsis dynamics. Researchers can strategically choose
from this array of murine models based on the specificaspects of sepsis they aim to investigate, fostering acomprehensive exploration of sepsis pathogenesis.
IV. Considerations in Murine Model Selection:
a. Ethical Considerations
The ethical implications of utilizing murine models insepsis research are paramount, necessitating a carefulbalance between scientific advancement and animal
welfare. Ethical concerns primarily revolve around thepotential for pain, distress, and suffering experienced byanimals during experimentation. Efforts to address theseconcerns involve stringent ethical review processes,
ensuring that research protocols adhere to establishedguidelines and regulations (21). Moreover, refinements inexperimental design, including the use of analgesics andanesthesia, aim to minimize discomfort and distress inmurine subjects (21). Researchers are increasingly
adopting the principles of the 3Rs (Replacement,
Reduction, Refinement) to enhance the ethical dimensionsof murine studies, emphasizing the pursuit of alternatives,
reduction of animal numbers, and refinement ofexperimental procedures to maximize scientific validity
while minimizing the impact on animal welfare (22).
b. Relevance to Human Sepsis
Assessing the translational relevance of murine models
to human sepsis is crucial for the meaningful extrapolationof preclinical findings to clinical scenarios. While murine

Fiziologia - Physiology • 2023.2(105)models offer valuable insights, inherent differences
between murine and human immune systems posechallenges in directly translating experimental outcomes(23). A comprehensive understanding of the similarities anddisparities between murine and human sepsis is essential
for refining model selection. Despite limitations, murine
models remain indispensable for elucidating fundamental
aspects of sepsis pathophysiology (17). Advances ingenetic engineering and the development of humanizedmouse models, wherein murine immune components are
replaced with human counterparts, show promise inbridging the translational gap (12). However, the complexityof sepsis and the diverse patient populations affected pose
ongoing challenges for achieving a direct correlation
between murine models and the clinical manifestation ofhuman sepsis (24). Future improvements in model design,
incorporation of advanced technologies, and collaborativeinterdisciplinary approaches are imperative for enhancing
the translational relevance of murine models in sepsis
research.

VII. Conclusion
In conclusion, the exploration of murine sepsis models
has been marked by recent advances that leverage
cutting-edge technologies to enhance the precision anddepth of preclinical investigations. These innovations
contribute significantly to unraveling the complexities ofsepsis pathogenesis, offering unprecedented insights into
immune responses, microbial interactions, and therapeuticinterventions. The imperative for continued research inrefining and expanding murine models remains evident. By
embracing
emerging
technologies,
incorporating
personalized approaches, and adopting systems-level
analyses, the field is poised to overcome current limitations
and bridge the translational gap between murine models
and human sepsis. The ongoing commitment to rigorous,
ethically sound, and technologically sophisticated researchis paramount for advancing our understanding of sepsis
and translating preclinical insights into meaningful clinicalapplications.
References
1.
Angus DC. The lingering consequences of sepsis: a hidden
public health disaster? JAMA. 2010;304(16):1833-4.
2.
Rudd MD, Bryan CJ. The Brief Suicide Cognitions Scale:
Development and Clinical Application. Front Psychiatry.
2021;12:737393.
3.
Singer BH, Newstead MW, Zeng X, Cooke CL, Thompson
RC, Singer K, et al. Cecal Ligation and Puncture Results inLong-Term Central Nervous System Myeloid Inflammation. PLoS
One. 2016;11(2):e0149136.
4.
Hotchkiss RS, Moldawer LL, Opal SM, Reinhart K, Turnbull
IR, Vincent JL. Sepsis and septic shock. Nat Rev Dis Primers.
2016;2:16045.
5.
Calandra T, Cohen J, Conference ISFDoIitIC. Theinternational sepsis forum consensus conference on definitions ofinfection in the intensive care unit. Crit Care Med.
2005;33(7):1538-48.
6.
Zaghloul N, Addorisio ME, Silverman HA, Patel HL,
Valdés-Ferrer SI, Ayasolla KR, et al. Forebrain Cholinergic
Dysfunction and Systemic and Brain Inflammation in Murine
Sepsis Survivors. Front Immunol. 2017;8:1673.
7.
Seymour CW, Gesten F, Prescott HC, Friedrich ME,
Iwashyna TJ, Phillips GS, et al. Time to Treatment and Mortality
during Mandated Emergency Care for Sepsis. N Engl J Med.
2017;376(23):2235-44.
8.
Hughes CG, Patel MB, Pandharipande PP.
Pathophysiology of acute brain dysfunction: what's the cause of
all this confusion? Curr Opin Crit Care. 2012;18(5):518-26.
9.
Pandharipande PP, Girard TD, Jackson JC, Morandi A,
Thompson JL, Pun BT, et al. Long-term cognitive impairment after
critical illness. N Engl J Med. 2013;369(14):1306-16.
10. Rhee C, Jones TM, Hamad Y, Pande A, Varon J, O'Brien C,
et al. Prevalence, Underlying Causes, and Preventability of
Sepsis-Associated Mortality in US Acute Care Hospitals. JAMA
Netw Open. 2019;2(2):e187571.
11. Andonegui G, Zelinski EL, Schubert CL, Knight D, Craig
LA, Winston BW, et al. Targeting inflammatory monocytes insepsis-associated encephalopathy and long-term cognitive
impairment. JCI Insight. 2018;3(9).
12. Barichello T, Fortunato JJ, Vitali AM, Feier G, Reinke A,
Moreira JC, et al. Oxidative variables in the rat brain after sepsis

V. Recent Advances and Innovations
Recent years have witnessed significant strides inadvancing murine sepsis models, with novel techniquesand groundbreaking studies contributing to the refinementof preclinical research. One noteworthy development
involves the integration of advanced imaging technologies,
such as intravital microscopy and bioluminescence imaging,allowing real-time visualization of immune responses andmicrobial dynamics within living organisms (25). These
imaging modalities offer unprecedented insights into thespatial and temporal aspects of sepsis pathogenesis.
Additionally, the incorporation of genetically modified
murine strains with fluorescent markers enables the
tracking of specific immune cell populations, providing anuanced understanding of cellular dynamics during sepsis(26). Such technological innovations enhance thegranularity of data obtained from murine models, fosteringa more comprehensive comprehension of sepsis
progression.
VI. Future Directions
The evolving landscape of murine sepsis models opens
avenues for future research aimed at further refining andexpanding our understanding of sepsis pathophysiology.
One promising direction involves the development of
personalized murine models that consider individual
genetic variations and susceptibility factors (27). Thisapproach could enhance the model's relevance to theheterogeneity observed in human sepsis. Furthermore, theintegration of systems biology approaches, encompassing
omics technologies and computational modeling, holds
potential for unraveling the complex network interactions
underlying sepsis (26).

57

induced by cecal ligation and perforation. Crit Care Med.
2006;34(3):886-9.
13. Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA.
Immunodesign of experimental sepsis by cecal ligation andpuncture. Nat Protoc. 2009;4(1):31-6.
14. Flierl MA, Stahel PF, Rittirsch D, Huber-Lang M,
Niederbichler AD, Hoesel LM, et al. Inhibition of complement C5aprevents breakdown of the blood-brain barrier and pituitary
dysfunction in experimental sepsis. Crit Care. 2009;13(1):R12.
15. Stearns-Kurosawa DJ, Osuchowski MF, Valentine C,
Kurosawa S, Remick DG. The pathogenesis of sepsis. Annu Rev
Pathol. 2011;6:19-48.
16. Remick DG. Pathophysiology of sepsis. Am J Pathol.
2007;170(5):1435-44.
17. Barichello T, Martins MR, Reinke A, Constantino LS,
Machado RA, Valvassori SS, et al. Behavioral deficits insepsis-surviving rats induced by cecal ligation and perforation.
Braz J Med Biol Res. 2007;40(6):831-7.
18. Ow CPC, Trask-Marino A, Betrie AH, Evans RG, May CN,
Lankadeva YR. Targeting Oxidative Stress in Septic Acute Kidney
Injury: From Theory to Practice. J Clin Med. 2021;10(17).
19. Peng X, Luo Z, He S, Zhang L, Li Y. Blood-Brain Barrier
Disruption by Lipopolysaccharide and Sepsis-AssociatedEncephalopathy. Front Cell Infect Microbiol. 2021;11:768108.
20. Yang J, Lunde LK, Nuntagij P, Oguchi T, Camassa LM,
Nilsson LN, et al. Loss of astrocyte polarization in the tg-ArcSwe

mouse model of Alzheimer's disease. J Alzheimers Dis.
2011;27(4):711-22.
21. Basak JM, Ferreiro A, Cohen LS, Sheehan PW, Nadarajah
CJ, Kanan MF, et al. Bacterial sepsis increases hippocampal
fibrillar amyloid plaque load and neuroinflammation in a mousemodel of Alzheimer's disease. Neurobiol Dis. 2021;152:105292.
22. Moraes CA, Zaverucha-do-Valle C, Fleurance R, Sharshar
T, Bozza FA, d'Avila JC. Neuroinflammation in Sepsis: Molecular
Pathways of Microglia Activation. Pharmaceuticals (Basel).
2021;14(5).
23. Akrout N, Sharshar T, Annane D. Mechanisms of brain
signaling
duringsepsis.
Curr
Neuropharmacol.
2009;7(4):296-301.
24. Mai SHC, Sharma N, Kwong AC, Dwivedi DJ, Khan M, GrinPM, et al. Body temperature and mouse scoring systems as
surrogate markers of death in cecal ligation and puncture sepsis.
Intensive Care Med Exp. 2018;6(1):20.
25. Yu SC, Betthauser KD, Gupta A, Lyons PG, Lai AM, Kollef
MH, et al. Comparison of Sepsis Definitions as Automated
Criteria. Crit Care Med. 2021;49(4):e433-e43.
26. Annane D, Sharshar T. Cognitive decline after sepsis.
Lancet Respir Med. 2015;3(1):61-9.
27. Bolton CF, Young GB, Zochodne DW. The neurologicalcomplications of sepsis. Ann Neurol. 1993;33(1):94-100.

EXPLORAND DIVERSITATEA: MODELE MURINE PENTRU INDUCEREA SEPSULUI ÎN CERCETAREAPRECLINICĂ
RezumatModelele murine de sepsis reprezintă instrumente cruciale în cercetarea preclinică, oferind o platformă esențială pentru mimarea șiînțelegerea complexității patogenezei sepsisului. Aceste modele permit cercetătorilor să reproducă cu precizie aspecte critice ale reacției imune
și ale interacțiunilor microbienelor într-un mediu controlat. Utilizarea endotoxinelor sau a metodei de legare și perforare a cecumului (CLP) în
modelele murine furnizează o abordare sistematică în studiul răspunsului imun, permitând investigarea fazelor timpurii și târzii ale sepsisului.
Avantajele acestor modele includ reproducibilitatea, controlul asupra variabilelor experimentale și posibilitatea manipulării genetice pentru aevalua rolul specific al genelor. Cu toate acestea, în selectarea modelelor murine pentru studiul sepsisului, este crucială evaluarea atentă aaspectelor etice, minimizarea suferinței animalelor și maximizarea validității științifice. În contextul cercetării viitoare, tehnologiile emergente,
precum imagistica avansată și modelele personalizate, reprezintă direcții promițătoare pentru îmbunătățirea relevanței și aprofundareaînțelegerii patologiei sepsisului la nivel molecular și funcțional.
Cuvinte cheie: sepsis, CLP, LPS, murine

Fiziologia - Physiology • 2023.2(105)A PSYCHIATRY PERSPECTIVE OF COVID-19 DISEASE IN PATIENTHOSPITAL ADMISSION
MĂDĂLINA ALDEA1*, VICTOR GHEORMAN1*, ALEXANDRA DANIELA ROTARU ZĂVĂLEANU2,DAIANA BURDUȘEL3+, ROXANA SURUGIU3+, DANIELA-GABRIELA GLĂVAN1
1.
2.
3.

Psychiatry Department, University of Medicine and Pharmacy Craiova
Department of Epidemiology, University of Medicine and Pharmacy Craiova
Biochemistry Department, University of Medicine and Pharmacy Craiova*The authors have equal contribution+Corresponding authors: Roxana Surugiu – roxana.surugiu@gmail.com , Daiana Burdușel – daiana.burdusel@gmail.com
Abstract:
The Covid-19 pandemic has posed a significant challenge to society and the medical system, particularly impacting patients withpsychiatric conditions. Studies indicate a correlation between imposed restrictions and an increase in decompensations among these patients,
while access to specialized services has been limited. The pandemic's effects on the mental health of the general population are still underinvestigation, with signals of increased anxiety and depression. The SARS-CoV-2 virus has neurotropic potential, emphasizing the need foressential interdisciplinary collaboration for the proper treatment of patients. This research examines changes in the accessibility of patients inthe psychiatry ward during the pandemic, considering factors such as restrictions, types of pathologies, and socio-demographic characteristics
of patients. Comparative data reveal significant shifts in the management of psychotic, manic, and depressive disorders, suggesting thatpatients with psychiatric conditions have faced difficulties due to reduced access to psychiatry clinics, and ambulatory services have been
affected by the fear of potential Covid-19 infection. The elevated levels of anxiety and depression have been felt across the entire population.
Keywords: Covid-19, psychiatry admission, SARS-CoV-2

Introduction
In late December of 2019, an outbreak of mysterious
pneumonia emerged in the Huanan Seafood Wholesale
Market, located in Wuhan, Hubei, China. The symptoms
included fever, dry cough, fatigue, and occasional
gastrointestinal issues. This initial outbreak was first
noticed in December 2019 and affected approximately
two-thirds of the market's employees. The market was shut
down on January 1, 2020, following an epidemiologicalnotice issued by the local health authorities on December
31, 2019 [1,2].
However, during the subsequent month of January, thedisease continued to rapidly spread, impacting thousandsof individuals across various regions in China, including
provinces like Hubei, Zhejiang, Guangdong, Henan, Hunan,
as well as cities such as Beijing and Shanghai. The virus
also made its way to other countries, including Thailand,
Japan, the Republic of Korea, Vietnam, Germany, theUnited States of America, and Singapore. The first case in
our country was reported on January 21, 2019. Thisoutbreak was eventually identified as a new
beta-coronavirus, labeled as the 2019 novel coronavirus(2019-nCoV), drawing parallels with the severe acuterespiratory syndrome (SARS-2003) caused by anotherbeta-coronavirus that had emerged 17 years earlier [3,4].
In 2003, a novel coronavirus was identified insoutheastern China, specifically in Guangdong province,
and it was named the SARS coronavirus, confirming
Koch's principles of disease causation. This virus had amortality rate ranging from 10% to 15%. Despiteadvancements in medical facilities, there is still noeffective treatment or vaccine for SARS. Another outbreak,linked to a novel coronavirus, took place in the Middle East
in 2012, sharing many characteristics with the 2003
outbreak. Both of these outbreaks were caused bycoronaviruses, with MERS thought to have an intermediate
host in the dromedary camel, resulting in a fatality rate of
up to 37%.
Both SARS and MERS often present with non-specificinitial clinical symptoms, although most patients exhibit
fever and respiratory issues. Hospital staff who lack proper
protection and have contact with infected patients are at
risk of contracting the disease, leading to nosocomial
infections. Additionally, cases of SARS, MERS, and
COVID-19 related to travel have been documented.
COVID-19 presents a significant risk to Taiwan due toglobal travel and the prevalence of tourism [2-9].
Epidemiology
The first laboratory-confirmed case of 2019-nCoV
infection presented with symptoms on December 1, 2019,
in Wuhan, China (as shown in Table I).1 Initially, reports
emerged of an outbreak associated with a local market, theHuanan Seafood Market, involving at least 41
individuals.[10]On December 31, 2019, the local health authority
issued an "epidemiologic warning," and the market ceased
its operations on January 1, 2020. A total of 59 suspectedcases involving fever and a dry cough were referred to a
designated hospital, namely the Jin Yin-tan Hospital.
Among these cases, 41 patients had their suspected cases
confirmed through next-generation sequencing or real-timereverse transcription-polymerase chain reaction (RT-PCR).
It's worth noting that 27 out of these 41 patients (66 percent,
27/41) had a history of exposure to the Huanan Seafood59

Market.[10]However, there is an important caveat: the initial caseon December 1 did not have a history of exposure to the
Huanan Seafood Market, and additional cases began toemerge nine days later on December 10. In thesubsequent days, a wave of cases spread from Wuhan toother parts of Hubei province. This virus then affected
multiple cities and regions. One contributing factor may
have been the increased travel associated with the
Chinese Lunar New Year, which fell on January 25. On
January 13, 2020, the first documented case outside of
China was reported in Thailand. The illness, however,rapidly and widely disseminated. Not only were familial
clusters observed, but outbreaks occurred on cruise ships
as well. As of February 6, 2020, the World HealthOrganization (WHO) had reported a total of 28,276
confirmed cases and 565 fatalities worldwide, spanning atleast 25 countries.[40] On January 30, 2020, the WHO
declared a public health emergency of international
concern (PHEIC). To mitigate the spread, stringent
quarantine measures and fever surveillance systems were
implemented. Initially, the mortality rates for hospitalized
patients were estimated to be in the range of 11%–15%[10,11] but more recent data suggests a range of 2%–3%.
Person-to-person transmission is highly likely throughrespiratory droplets and physical contact. Nosocomial
infections have occurred in healthcare settings,
underscoring the critical importance of infection control
measures.
Clinical manifestations
Fever (83–98%), cough (76–82%), and shortness ofbreath (31–55%) are the most common clinicalmanifestations. Approximately 15% of patients may present
with all three of these symptoms.[1, 9] Notably, conjunctival
injection was not reported in the initial cases, and theoccurrence of the disease in individuals under 18 years ofage was rare. Following the onset of symptoms, thedisease typically progresses with moderate severity, andthe median time to the first hospitalization is around 7.0
days (ranging from 4.0 to 8.0 days). However, in roughly 39%
of patients, the condition may progress to shortness ofbreath (around 8 days), acute respiratory distresssyndrome (ARDS, approximately 9 days), and the need formechanical ventilation (about 10.5 days). Patients withsevere conditions may develop ARDS and experience
rapid deterioration, eventually succumbing to multiple
organ failures.[1,10] The initial death rate in the early seriesof hospitalized patients was estimated to be between 11%
and 15%, but subsequent data indicate a lower rate of 2%–
3%.[10,4-7]
SMELL AND TASTE DISORDERS
Anosmia and dysgeusia are commonly observed as
early symptoms in individuals with COVID-19, with morethan 80% of patients in one study reporting these issues. A
meta-analysis of 83 studies involving over 27,000
individuals found that 48% of patients experienced

olfactory dysfunction (with a 95 percent confidence intervalof 41.2-54.5). These symptoms can manifest as the initial
indicators of COVID-19, even in the absence of nasal
congestion or discharge. However, they are seldom thesole manifestations of COVID-19 [12].
Patients with COVID-19 have been reported to exhibit
MRI signal abnormalities in one or both olfactory bulbs,
which can be resolved with follow-up imaging. Pathologicalfindings in two autopsy cases revealed inflammatory
infiltration and axonal destruction in the olfactory tracts,
although it remains uncertain whether direct viral infectionis the root cause. Instead of direct damage to olfactory
neurons, transient anosmia might result from inflammatorychanges in the sustentacular cells of the nasal epithelium.
Limited evidence is available regarding the olfactory cleft of
the nasal canals, based on an MRI study of 20 anosmia
patients. Improvement in olfactory function was correlatedwith reduced blockage at the one-month follow-up [13].
Reliable evidence concerning the long-term prognosis
is lacking. In one study, the average duration of symptoms
was eight days among the 33% of affected patients who
regained their olfactory function. In an Italian study of
non-hospitalized patients with olfactory dysfunction, 83%
reported complete recovery 37 days after the onset ofsymptoms. Full recovery was observed in 84% and 96% of
51 individuals with anosmia who underwent objective
olfactory testing at four and eight months, respectively.
These symptoms can also be triggered by other viral
infections and various factors, with a separate explanation
provided for their evaluation [14].
ENCEPHALOPATHY
Encephalopathy is prevalent among critically ill
COVID-19 patients. Delirium was widespread in a cohort
study of 2088 COVID-19 patients admitted to an intensive
care unit, affecting 55% of them. In a study of 509
COVID-19 hospitalized patients, 31.8% experienced
encephalopathy, and those with encephalopathy tended tobe older (66 versus 55 years), had a shorter time fromsymptom onset to hospitalization (6 versus 7 days), weremore likely to be male, and had more risk factors thanthose without encephalopathy. These risk factors included
a history of any neurologic disorder, cancer,
cerebrovascular disease, chronic kidney disease, diabetes,
dyslipidemia, heart failure, hypertension, or smoking [15].
COVID-19 can manifest with encephalopathy as a
primary symptom. Encephalopathy was found in 28% of
817 older individuals (median age 78 years) evaluated in
the emergency department and diagnosed with COVID-19
infection. Thirty-seven percent of these patients did notexhibit classic COVID-19 symptoms such as fever ordyspnea. Risk factors for encephalopathy included older
age, visual impairment, a history of Parkinson's disease or
stroke, and prior use of psychoactive medications [16].
The 2019-nCoV virus can enter the host through therespiratory tract or mucosal surfaces, such as the
conjunctiva. There is no evidence to support oral-fecal

Fiziologia - Physiology • 2023.2(105)transmission. The virus exhibits a specific affinity for human
airway epithelial cells, and like SARS, it uses ACE2 as its
cellular receptor. Nevertheless, the clinical variations andpathophysiology of the disease in humans are not yet fully
understood. It remains unclear whether the virus canreplicate in other parts of the body.
Diagnosis
COVID-19 typically presents as an acute viral
respiratory tract infection, and it can be confused with othercommon viral pneumonias, including influenza,
parainfluenza, adenovirus infection, respiratory syncytial
virus infection, metapneumovirus infection, and atypical
pathogenslike
Mycoplasma
pneumoniae
andChlamydophila pneumoniae infections, among others.
Therefore, it is crucial to inquire about a suspectedpatient's travel history and exposure when they return froman epidemic area. Commercial respiratory syndromic
diagnostic kits, such as the Filmarray Respiratory Panel,
which can identify multiple causative agents, may aid inachieving a rapid differential diagnosis. [11,9]
Laboratory diagnosis of COVID-19 should be
performed in a well-equipped laboratory with biosafety level
3 containment. COVID-19 is classified as a fifth-category
notifiable communicable disease in Taiwan, and cases
must be reported to the Taiwan Centers for Disease Control(CDC) within 24 hours. COVID-19 is defined based on
specific criteria, as of February 7, 2020, including clinicalconditions, laboratory evidence, and epidemiologicalfactors.
Clinical conditions must include either a febrile illness(with a temperature of 38°C or higher) or an acuterespiratory infection with signs of pneumonia on clinical,
radiological, or pathological examinations.
Laboratory conditions may involve clinical specimensthat are isolated and identified as 2019-nCoV, or a clinicalspecimen that tests positive through RT-PCR.
Epidemiological factors should cover events occurring
within 14 days before symptom onset, including travel
history to epidemic areas, interaction with individuals
suffering from fever or respiratory symptoms in thefirst-class epidemic area (Hubei, including Wuhan andGuangdong), prior travel to or residence in other regions of
mainland China (including Hong Kong and Macau), orinteraction with suspected or confirmed COVID-19 cases.
If a patient meets any of the criteria mentioned above,
they should be reported to the CDC within 24 hours. Even
if the initial laboratory report is negative, if a patient's
symptoms persist without an identifiable cause, a second
sample should be analyzed 24 hours later to rule out afalse-negative result.
Laboratory diagnosis is typically carried out using
real-time reverse transcription polymerase chain reaction(RT-PCR) targeting specific regions of viral RNA, such as
the pan beta-CoV consensus E region, RdRp, or N region.
Imaging studies like chest x-rays and computed
tomography (CT) scans often reveal bilateral pneumonia(75–98%) with various patterns such as mottling andground-glass opacity. Routine laboratory findings in theearly stages of the COVID-19 epidemic are similar to thoseof common viral infections, including lymphopenia,
prolonged prothrombin time, elevated D-dimer, liver
enzymes (alanine aminotransferase), total bilirubin, andlactate dehydrogenase, with more severe abnormalities inintensive care unit cases. Leukocytosis may occur in casesof secondary bacterial infection. Physicians should
consider the necessity of regular blood sampling andaspiration while taking precautions to minimize the risk ofunintended exposure [11,12].
Treatment
As of now, there is no established therapy for
COVID-19. The primary approach involves providing
supportive and symptomatic treatment, which includes
monitoring vital signs, maintaining oxygen levels, andaddressing complications such as secondary infections ororgan failure.
Numerous investigational treatments are being
explored due to the potential severity of COVID-19:
Remdesivir: Gilead Sciences, Inc. is developing thisnew nucleotide analog prodrug, which is an experimental
antiviral medication initially studied for the treatment of
Ebola and SARS. A case report in the United States
showed a reduction in viral loads and clinical improvement
when remdesivir was administered on day 11 of the illness.
However, randomized controlled studies are necessary toestablish its safety and effectiveness in treating2019-nCoV-infected individuals.
Convalescent therapy (plasma from recovered
COVID-19 patients): This method involves passive
immunization, which has been explored in research related
to MERS. Potential treatment options include convalescentplasma, interferon-beta/ribavirin combination therapy, andlopinavir. Nevertheless, there is currently no experience
with COVID-19, and no randomized controlled clinicalstudies have evaluated these treatments.
Antiviral drugs: Lopinavir/ritonavir and ribavirin havebeen used to treat SARS with apparent clinical benefits. In
vitro studies have shown antiviral activity against the
SARS-associated coronavirus. Notably, recent researchfound similarities between the unique insertions in the
2019-nCoV spike protein and HIV-1 gp120 and Gag [17].
Prevention
Given the lack of effective therapies for COVID-19,
preventing infection and further transmission is crucial. The
general public is advised against traveling to COVID-19
epidemic areas, primarily in China, including Wuhan, Hong
Kong, and Macau, as well as contact with or consumptionof wild animals. For individuals returning from epidemicareas within the last 14 days, self-monitoring of bodytemperature and symptoms is recommended. If compatible
symptoms emerge, approved transportation should be
used to avoid unprotected exposure.
Healthcare professionals should wear personal61

protective equipment when caring for suspected orconfirmed patients and follow proper procedures for its
removal. Strict infection control measures should be
implemented for high-risk medical procedures. If healthcare
personnel are exposed to a patient's blood or bodily fluids
without protection, the exposed area should be thoroughly
cleansed. The patient's body temperature should bemonitored for 14 days following the exposure. Confirmed
cases should be isolated, preferably in a negative pressure
isolation room or, alternatively, in a single room with good
ventilation. Isolation may be discontinued if symptoms have
improved for 24 hours, and two consecutive negative tests
have been obtained. Deceased individuals should be either
cremated or buried deeply.
Effective disinfection methods include the use of active
ingredients like sodium hypochlorite (0.1–0.5%), 70% ethyl
alcohol, povidone-iodine (iodine%), chloroxylenol (0.24%),
50% isopropanol, 0.05% benzalkonium chloride, and 1%
cresol soap. For areas with blood or bodily fluid spills, a
1:10 dilution of 5.25% household bleach should be used forcleaning, following WHO recommendations for Ebola virus
disinfection.
In summary, 2019-nCoV is a zoonotic illness with arelatively low to moderate fatality rate. Currently, there is noestablished treatment for the condition, and only supportive
care is available. While various experimental studies areunderway, the best approach to prevent a widespreadoutbreak is the strict implementation of infection control
measures. Healthcare providers should consider the
possibility of 2019-nCoV infection in patients with relevant
travel or exposure history and presenting symptoms.
Frontline healthcare workers should be well-versed inappropriate infection prevention strategies for suspectedcases.
The period of the Covid-19 pandemic meant a real
challenge for society and the medical system considering
During the pandemic period, 01.03.2020 to 28.02.2021,
Clinic II Psychiatry, being a Covid support ward, limited its
admissions only to psychiatric emergencies andsuspected/confirmed patients with Covid-19 infection,
admitting 595 patients, and Clinic I Psychiatry 1133
patients. The percentages by pathology were as follows:
4.03% Dementia, 18.49% Organic personality disorder,
33.95% Disorders from the spectrum of schizophrenia andother psychotic disorders, 6.55% Bipolar affective disorders
and 29.92% Depressive disorders, and in Clinic I
Psychiatry – 1.85% Dementia, 8.91% Organic personality
disorder, 22.69% Schizophrenia spectrum disorders andother psychotic disorders, 8.12% Bipolar affective disorders
and 43.25% Depressive disorders.
In the period 01.03.2021- 28.02.2022, Clinic I
Psychiatry had a total of 1127 hospitalizations, thepercentages being as follows: 2% Dementia, 6% Organic
personality disorder, 25% Disorders from the spectrum of
schizophrenia and other psychotic disorders, 10%
Disorders bipolar affective and 57% Depressive disorders.

the multiple symptoms and forms of manifestation of theinfection with the SARS CoV-2 virus, each specialty being
involved in the diagnosis and treatment of this condition.
In multiple studies, the connection is made between the
period of national restrictions and the increase in thenumber of decompensations of patients with various
psychiatric conditions, but access to specialized services
was limited.
The effects of the pandemic on the population are still
under investigation, but several studies claim that among
them would be the remaining of anxiety, depression in thegeneral population, but also the decompensation of oldcases of psychiatric pathology.
Coronaviruses can cause multiple neuropsychiatriccomplications, being neurotropic, and interdisciplinary
collaboration is essential for the adequate treatment ofpatients.
We started from the hypothesis that the addressabilityof patients in the psychiatric ward changed during the
pandemic, several factors being involved.
We compared the hospitalizations of the Craiova
Psychiatry Clinics I and II from the period
01.03.2019-28.02.2022.
In the pre-pandemic year, 1845 patients were admitted
to the II Psychiatry Clinic. The percentages by pathology
were as follows: 4.44% Dementia, 18.75% Organic
personality disorder, 23.74% Disorders from the spectrumof schizophrenia and other psychotic disorders, 5.8%
Bipolar affective disorders and 46.07% Depressive
disorders, and in Clinic I Psychiatry, from a total of 1886
patients, the percentages were as follows: 1.80% Dementia,
9.54% Organic personality disorder, 16.91% Schizophrenia
spectrum disorders and other psychotic disorders, 5.51%
Bipolar affective disorders and 53.98% Depressive
disorders.
Clinic II Psychiatry had 793 admissions, and thepercentages by pathology were as follows: 5% Dementia,
18% Organic personality disorder, 32% Schizophrenia
spectrum disorders and other psychotic disorders, 9%Bipolar affective disorders and 36% Depressive disorders.
It is observed that psychotic disorders and manic
episodes in particular could not be managed in theoutpatient setting, but also the increase in the percentage
of depressed patients.
We conclude that psychiatric patients encountereddifficulties by reducing access to psychiatric clinics, theaddressability to outpatient services was also impacted by
the fear of a possible Covid-19 infection, in conditions
where the increased level of anxiety and depression was
felt by the whole population.

Fiziologia - Physiology • 2023.2(105)Bibliography
1. Pun BT, Badenes R, Heras La Calle G, et al.
Prevalence and risk factors for delirium in critically illpatients with COVID-19 (COVID-D): a multicentre
cohort study. Lancet Respir Med 2021; 9:239.
2. Kennedy M, Helfand BKI, Gou RY, et al. Delirium inOlder Patients With COVID-19 Presenting to theEmergency Department. JAMA Netw Open 2020;
3:e2029540.
3. World Health Organization. Novel Coronavirus(2019-nCoV).
Availableat
https://www.who.int/emergencies/diseases/novel-coronavirus-2019.
4.
World Health Organization. 2019-nCoV Situation
Report.
https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situationreports
5. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus
AD, FouchierRA. Isolation of a novel coronavirus froma man with pneumonia inSaudi Arabia. N Engl J Med
2012;367:1814–20.
6. Chan Jasper FW, Yuan S, Kok KH, To Kelvin KW, Chu
H, Yang J, et al. A familial cluster of pneumonia
associated with the 2019 novel coronavirus indicating
person-to-person transmission: a study of a family
cluster.
Lancet2020.
DOI:
https://doi.org/10.1016/S0140-6736(20)30154-9.
7.
Holshue ML, DeBolt C, Lindquist S, Lofy KH,
Wiesman J, Bruce H, et al.First case of 2019 novel
coronavirus in the United States. N Engl J Med 2020.
DOI: 10.1056/NEJMoa2001191.
8. Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl
G, Wallrauch C, etal. Transmission of 2019-nCoV
infection from an asymptomatic contact in Germany. N
Engl J Med 2020. DOI: 10.1056/NEJMc2001468.
9.
Pgab LT, Nguyen TV, Luong QC, Nguyen TV, Nguyen
HY, Le HQ, et al.Importation and human-to-humantransmission of a novel coronavirusin Vietnam. N Engl
J Med 2020. DOI: 10.1056/NEJMc2001272.

10. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu y, et al.
Clinical features ofpatients infected with 2019 novel
coronavirus in Wuhan, China. Lancet 2020. Doi:
10.1016/S0140-6736(20)30183-5.
11. Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al.
Epidemiological and clinical characteristics of 99 casesof 2019 novel coronavirus pneumonia in Wuhan, China:
a descriptive study. N Engl J Med 2020.
DOI:10.1016/S0140-6736(20)30211-7.
12. Lechien JR, Chiesa-Estomba CM, De Siati DR, et al.
Olfactory and gustatory dysfunctions as a clinicalpresentation of mild-to-moderate forms of thecoronavirus disease (COVID-19): a multicenter
European study. Eur Arch Otorhinolaryngol 2020;
277:2251.
13. Lin E, Lantos JE, Strauss SB, et al. Brain Imaging ofPatients with COVID-19: Findings at an Academic
Institution during the Height of the Outbreak in New
York City. AJNR Am J Neuroradiol 2020; 41:2001.
14. Paderno A, Mattavelli D, Rampinelli V, et al. Olfactoryand Gustatory Outcomes in COVID-19: A Prospective
Evaluation in Nonhospitalized Subjects. Otolaryngol
Head Neck Surg 2020; 163:1144.
15. Pun BT, Badenes R, Heras La Calle G, et al.
Prevalence and risk factors for delirium in critically illpatients with COVID-19 (COVID-D): a multicentre
cohort study. Lancet Respir Med 2021; 9:239.
16. Kennedy M, Helfand BKI, Gou RY, et al. Delirium inOlder Patients With COVID-19 Presenting to theEmergency Department. JAMA Netw Open 2020;
3:e2029540.
17. Mo Y, Fisher D. A review of treatment modalities for
middle east respiratorysyndrome. J Antimicrob
Chemother 2016;71:3340–50.

ADRESABILITATEA PACIENȚILOR CU AFECȚIUNI PSIHIATRICE ÎN CONTEXTUL PANDEMIEICOVID-19
Rezumat:
Perioada pandemiei Covid-19 a însemnat o adevărată provocare pentru societate și sistemul medical având în vedere multiplele simptome
și forme de manifestare ale infecției cu virusul SARS CoV-2, fiecare specialitate fiind implicată în diagnosticarea și tratamentul acestei afecțiuni.
În multiple studii se face legătura între perioada de restricții naționale și creșterea numărului de decompensari ale pacienților cu diverseafecțiuni psihiatrice, însă accesul la servicii de specialitate a fost limitat. Efectele pandemiei asupra populației sunt încă în investigare, dar mai
multe studii susțin că printre acestea s-ar număra reșterea anxietății, depresiei în populația generală, dar și decompensarea cazurilor vechi depatologie psihiatrică. Coronavirusurile pot da multiple complicații neuropsihice, fiind neurotrope, iar colaborarea interdisciplinară este esențială
pentru tratamentul adecvat al pacienților. Am pornit de la ipoteza că adresabilitatea pacienților în secția de psihiatrie s-a modificat în perioada
pandemiei, fiind implicați mai mulți factori. Am evaluat comparativ internările Clinicii II Psihiatrie Craiova din perioada 01.03.2019-28.02.2022,
anterioară pandemiei, dar și perioada ce a inclus starea de urgență și alertă națională după tipul de patologii și informațiile socio-demograficeale pacienților, cât și după numărul de zile de internare. În perioada martie 2020 – februarie 2021 clinica a fost unitate suport covid, fiind limitatnumărul de internări la urgențele psihiatrice și pacienții confirmați/suspecți Covid – 19 ce asociau patologie psihiatrică. Se observă că
tulburările psihotice și episoadele maniacale, în special nu au putut fi gestionate în ambulatoriu, dar și creșterea procentului de pacienți
depresivi. Concluzionăm că pacienții de psihiatrie au întâmpinat dificultăți prin reducerea accesului la clinicile de psihiatrie, adresabilitatea laserviciile de ambulatoriu a fost afectată și de teama unei posibile infecții cu Covid-19, în condițiile în care nivelul crescut de anxietate și
depresie a fost resimțit de întreaga populație.
Cuvinte cheie: Covid-19, spitalizare, pishiatrie, SARS-CoV-2.
632021-2022

2020-2021

2019-2020

0500
Psychiatry II

1000

1500

2000

Psychiatry I

FIG. 1 Total number of admitted patients

70

[%]

61.51

56.61
50.99

60
50
40
26.7425.29
19.27

30
20
10 2.052.192.13
0

10.8810.51
6.21
2019-2020

DEMENTIA

ORGANICPERS.
DISORDER

9.579.76
6.28
2020-2021
PSYCHOTICDISORDER

2021-2022
BIPOLARDISORDER

DEPRESSIVEDISORDER

FIG. 2 Percentage of admitted cases by diagnosis in Psychiatry I Clinic

Fiziologia - Physiology • 2023.2(105)46.63

50
[%]
36.53
31.90

40
30

19.89
18.98 18.16

20
10

36.32
32.19

24.03

7.05 8.70
5.87

4.34
4.50 4.92

0
DEMENTIA

ORGANIC PERS. DISORDERPSYCHOTIC DISORDER BIPOLAR DISORDER DEPRESSIVE DISORDER

2019-2020

2020-2021

2021-2022

FIG. 3 Percentage of admitted cases by diagnosis in Psychiatry II Clinic

67

ALCOHOL MISUSE – DEMOGRAPHY, PREVALENCE ANDCOMPLICATIONSMĂDĂLINA ALDEA1*, RALUCA-ELENA SANDU2*, DANIELA-GABRIELA GLĂVAN1#, ROXANA SURUGIU2#,TUDOR-ADRIAN BĂLȘEANU3
1- Psychiatry Department, University of Medicine and Pharmacy Craiova
2- Biochemistry Department, University of Medicine and Pharmacy Craiova
3- Physiology Department, University of Medicine and Pharmacy Craiova*- The authors have equal contribution#- Corresponding authors: Roxana
danaglavan@gmail.com

Surugiu–

Roxana.surugiu@gmail.com,

Daniela-GabrielaGlăvan–ABSTRACT
Alcohol abuse represents a pervasive global issue, with alcohol consumption being the most widespread among substancesused for addictive purposes. This makes alcohol intoxication the leading cause of acute intoxication. On a global scale, the risk ofdeveloping harmful alcohol consumption patterns is 15% for men throughout their lifetime, approximately double that for women.
The risk of developing alcohol dependence is lower, with a 10% risk for men and around 5% for women. The findings of thisstudy lead to the conclusion that "Harmful Alcohol Use" encompasses a range of conditions, including alcohol abuse, alcohol
dependence, alcohol use disorder, and alcoholism. It constitutes a significant contribution to the global burden of disease, beinga major preventable cause of death and a prevalent psychiatric disorder.
"Harmful Alcohol Use" is a medical condition characterized by the diminished capacity of an individual to control or cease
alcohol consumption, even in the face of adverse social, professional, or health consequences. Globally, alcohol consumption is
responsible for 3.3 million deaths each year, contributing to disabilities and the overall poor health of millions of people. Harmful
alcohol use accounts for 5.1% of the global disease burden.
In a study conducted on patients admitted to the Clinical Hospital of Neuropsychiatry in Craiova due to mental and behavioral
disorders resulting from alcohol, several key findings emerged: the majority of admitted patients were men, representing 88% ofcases. A significant portion of patients came from rural areas, constituting 78% of total admissions. The highest proportion ofpatients fell into the 45-54 years age group, representing 41% of cases. The most prevalent diagnosis among admitted patientswas "withdrawal state with and without delirium," followed by "harmful alcohol consumption" and "psychotic disorder."Keywords: alcohol consumption, psychiatric disorder, complications.
approximately double that for women. The risk ofdeveloping alcohol dependence is lower, with a 10% risk
Introduction
for men and around 5% for women.
Alcohol misuse is a widespread global issue. Among all
Numerous epidemiological studies conducted oversubstances used for addictive purposes, alcohol
periods ranging from 4 to 20 years have definitively
consumption is the most prevalent, making ethanol
established the variable nature of annual ethanol
intoxication the primary cause of acute intoxication.
consumption, making it impossible to predict the potential
The prevalence of alcohol-related disorders varies withtrajectory of alcohol consumption over time. In urban adults
age, with the highest rates occurring in individuals aged
aged 25-65, the complications resulting from excessive
18-29 and the lowest rates (only 1.5%) in those over thealcohol use rank alcoholism as the fourth leading cause ofage of 65 years. Approximately 3.6% of the populationmortality in the United States. Alcohol-induced
between the ages of 15 and 65 years experience ancomplications in the USA contribute to over 100,000 deaths
alcohol use disorder, with higher rates observed ineach year.
developed countries compared to developing ones.
In Romania, despite the absence of specificEastern European countries have the highest prevalence
epidemiological data, a concerning trend of continuous(10.9%), followed by America (5.2%) and Africa (1.1%). In
growth in daily alcohol consumption is evident. An increase
Europe, Eastern and Northernin unregulated alcohol production and the prevalence ofconsumption in increasingly larger quantities have also
been observed. Privately produced alcoholic beverages in
European countries exhibit the highest rates ofRomania frequently surpass the maximum allowablealcohol-related disorders, while Mediterranean countries
methanol limit (0.8%), with over 75% containing up to 5%
have the lowest rates.
methanol. These factors have led to the highest recorded
On a global scale, the lifetime risk of developing
mortality rate from ethanol-related liver cirrhosis in Europe.
harmful alcohol consumption patterns is 15% for men and

Fiziologia - Physiology • 2023.2(105)The rates of alcohol-induced deaths and disabilities were
estimated to be 3.8% and 4.6%, respectively.
Given this context, the implementation of proactive
measures to reduce alcohol consumption in Romania is notjust a recommendation but an imperative requirement.
Without such measures, the World Health Organization(WHO) predicts that in the coming years, Romania will
exceed an annual per capita consumption of 10 liters.
Onset, course, and prognosis of harmful alcohol
use disorders
Generally, the initiation of alcohol consumption takes
place during adolescence. It is widely acknowledged thatgenetic predisposition contributes to approximately 40-60%
of the development of alcohol consumption disorders, withthe remaining influence attributed to the interplay ofenvironmental factors affecting the individual [1].
Another factor that significantly impacts the progressionof the consumption pattern is the consumer's level of
impulsivity. Individuals characterized by high levels of
impulsivity tend to experience an earlier onset of
consumption-related disorders and often exhibit more
severe manifestations of these issues.
Typically, the peak level of alcohol consumption occurs
in the age range of 18-22. Abusive consumption often
begins or continues between the ages of 20 and 30,
leading to alcohol addiction emerging around the age of 40.
This extended journey, punctuated by multiple
unsuccessful attempts to halt or regulate consumption,
ultimately culminates in cumulative complications resulting
from chronic abuse, often leading to death around the ageof 60.
Maintaining sobriety for a duration exceeding one year
is likely the most significant indicator of a potentially
positive outcome. Although the long-term progression
following comprehensive rehabilitation therapy has notbeen extensively studied, it has been observed that themost effective therapeutic outcomes are achieved throughinitial hospitalization in a specialized center for a relatively
brief period (approximately 4-6 weeks), followed by the
implementation of individualized long-term therapeuticinterventions (lasting around 6-12 months) outside thehospital.
Chronic ethanol consumption leads to a multitude ofmorphological and physiological changes in the body,causing significant medical consequences. However, these
consequences are sometimes reversible once alcohol
consumption is ceased.
The harmful impact of ethanol on the liver can be
attributed to its preferential utilization as an energy sourceby hepatocytes at the expense of lipids. This leads to theaccumulation of lipids in the liver, subsequently released
into the bloodstream, resulting in hepatic steatosis andhyperlipidemia. Even minimal alcohol consumption disrupts
gluconeogenesis, affecting the conversion of carbohydrates
into fats. This disruption accounts for the initialdegenerative liver change, characterized by hepatic

steatosis, followed by the development of ethanolichepatitis, perivenular fibrosis, and ultimately, liver cirrhosis.
When ethanol consumption is associated with any
pre-existing liver condition, it invariably heightens liver
toxicity and reduces the effectiveness of therapeuticinterventions for the underlying pathology.
Alcohol consumption can facilitate the colonization of
the stomach by the Helicobacter pylori bacterium,suggesting that alcohol consumption is not the primaryfactor leading to ulcer disease. Furthermore, alcohol
consumption can exacerbate or prolong the healing
process of pre-existing ulcers, disrupt the normal patternsof gastrointestinal peristalsis, contribute to the developmentof gastroesophageal reflux, and lead to hemorrhagic
gastritis.
Additionally, ethanol is responsible for inducing
structural, metabolic, and circulatory changes in the small
intestine. It also contributes to the erosion of the intestinal
villi, which, combined with increased intestinal motility,
disrupts the absorption of nutrients. This can result in thedevelopment of hemorrhoids, often associated with portal
hypertension.
Recurrent episodes of pancreatitis, marked by
endocrine and exocrine pancreatic insufficiency, have thepotential to progress to malabsorption and diabetes
mellitus, or even death.
Cardiovascular complications in individuals whochronically consume ethanol stem from the direct toxic
impact of ethanol on striated muscle fibers. This damage
can result in myocardial inflammation, cardiomyopathy, left
ventricular impairment, and the onset of cardiac
arrhythmias. Typically, these cardiovascular complications
manifest in approximately 25% of chronic ethanol users [2].
It is crucial to emphasize the importance of diligent
cardiac monitoring during ethanol withdrawal, as
preexisting cardiac conditions can deteriorate, and they
may present with new signs and symptoms that differ fromtheir usual clinical manifestation [2].
Hematological complications arising from thedetrimental use of alcohol are quite diverse. To begin with,liver dysfunction results in sodium retention, which, in turn,increases plasma volume and leads to a reduction inhematocrit levels. Hemolytic anemia occurs due to thepresence of macrocytes, schizocytes, and red blood cells
displaying a "target" appearance in the bloodstream. Theseabnormalities in red blood cells are a consequence ofalterations in the lipid components of the erythrocyte
membrane stemming from liver dysfunction [3].
The occurrence of leukopenia, particularly lymphopenia,
is responsible for the heightened susceptibility to infections
observed in individuals who are chronic alcohol users [3].
At the pulmonary level, ethanol contributes to thedevelopment of obstructive sleep apnea by directly
inhibiting the relaxation of the respiratory tract. Additionally,
liver dysfunction can lead to the onset of respiratory
alkalosis.
69

Liver and pancreatic impairment result in disruptions incarbohydrate metabolism. Ethanol consumption reduces
tissue insulin response, while ethanol withdrawal reduces
tissue insulin sensitivity, leading to fluctuations in blood
glucose levels, which can be either too low or too high.
Depletion of essential ions such as K+, Na-, and Mg2+
disrupts the hydro-electrolytic balance and can trigger
ethanolic ketoacidosis and lactic acidosis. These conditions
often manifest clinically with symptoms like vomiting,nausea, abdominal pain, and abnormal blood glucose
levels.
Alcohol also impacts the circadian rhythm, as it acts asa stimulant, causing daytime fatigue and insomnia while
making it difficult to fall asleep and maintain sleep. Even
moderate ethanol consumption in individuals who are notchronic drinkers can reduce REM sleep [4].
Acute alcohol intoxication and alcohol withdrawal
The colloquial term "drunkenness" actually refers toacute intoxication, which presents clinically with verbal andbehavioral disinhibition, speech difficulties (dyslalia),
emotional instability, and a state of euphoria. Alcohol
overdose, on the other hand, happens when an individual
ingests (either voluntarily or involuntarily) a quantity ofalcohol exceeding what their body can handle. This
overdose is characterized by symptoms such as coma,
cyanosis (bluish skin discoloration), pulmonary edema,slowed breathing (respiratory depression), pinpoint pupils(miosis), lowered body temperature (hypothermia), low
blood pressure (hypotension), cardiac arrhythmias, andprofuse sweating.
Alcohol withdrawal typically occurs only in individuals
who have developed physical alcohol dependence. It
encompasses a combination of physical and psychologicaleffects that arise after abruptly discontinuing alcohol
consumption. The initial, uncomplicated withdrawal
symptoms generally emerge around 72 hours after the
cessation of drinking.
The first clinical signs, appearing in the early hours ofwithdrawal, include fever, high blood pressure (arterial
hypertension), rapid heart rate (tachycardia), and tremorsin the extremities. Within the following 24 hours, individuals
may experience illusions, visual hallucinations (lesscommonly auditory ones), psychomotor agitation, anxiety,mental confusion, and worsening of symptoms associatedwith sympathetic nervous system activation. This clinicalpresentation is especially prevalent in individuals withpreexisting comorbidities who have consumed significant
quantities of alcohol.
One of the most concerning complications during thewithdrawal process is known as delirium tremens. Despite
therapeutic advances, this condition still carries anextremely high mortality rate, particularly within the first 48
hours. Delirium tremens is a medical-psychiatricemergency that necessitates specialized intensive careand is characterized by severe disturbances in mental
function and overall bodily equilibrium [5].

Oneirism is linked to the occurrence of false
recognitions, along with visual, auditory, olfactory, andgustatory hallucinations. It also involves experiences ofallo-psychic phenomena, where individuals haveperceptions related to external stimuli and temporal-spatial
disorientation. In the context of delirium tremens, oneirism
is distinctive due to the zoopsic nature of the hallucinations.
This means that the hallucinations often involve animals,
and what sets it apart is the patient's direct involvement or
participation in the hallucinatory scenario, making theexperience particularly vivid and intense [5].
The information was extracted by processing statistical
data from the sanitary statistical forms, from the Clinical
Hospital of Neuropsychiatry Craiova, and represents all thedata and information related to the characteristics ofpatients admitted to the Psychiatry ward, who received
diagnoses following
the Manual of diagnosis andstatistical classification of mental disorders, revision 5(DSM-V) and Statistical Classification and International
Classification of Diseases and Related Health Problems,
Revision 10 (ICD-10). All information was taken in fullrespect of the patient's right to confidentiality. The
phenomenon was analyzed throughout five years(2017-2021), so the designed study is a retrospective one.
The total statistical collectivity analyzed is a dynamic typeand is made up of the 6392 patients admitted to thePsychiatry ward who associate different psychiatricdisorders, and the community partial statistics focusing on
the 501 patients with mental and behavioral disorders
induced by the harmful use of alcohol defines the main
panel.
Analyzing the gender distribution of psychiatricdisorders at the level of the general statistical community, ahigher frequency of them was found among female patients(56.1%) compared to male patients (43.8%). However,within the main panel, the cast by gender of mental andbehavioral disorders due to alcohol consumption isrecorded as an important predominance of disorders
among men. Percentage-wise wise it is a value of only
12% in the case of women and 78% in the case of men(Fig 1).
The female/male ratio of cases associated withmental and behavioral disorders due to alcohol
consumption is 1:7. The next qualitative type statisticalvariable that was analyzed is represented by the
distribution depending on the place of residence of thepatients from the general and partial statistical collective
respectively. Analyzing the distribution by the residential
environment of patients with different types of conditions
from the group of psychiatric disorders it was found that apercentage of 56.9% of cases come from the urban
environment, and the remaining 43.1% come from the rural
environment. In contrast to these data, in the case ofpatients with mental and behavioral disorders due toalcohol consumption (Figure 2) a percentage of 78.1% of
the total cases as patients from rural areas, and the

Fiziologia - Physiology • 2023.2(105)remaining 21.9% are represented by patients who live inthe urban environment. This phenomenon can be
explained by the growth of unregistered production of
alcohol in rural areas.
Analyzing the distribution of cases with different typesof conditions from the group of psychiatric disorders, it was
found that the age range between 55-64 years has thehighest frequency of cases, with a percentage ofapproximately 33.55% of the total. This interval is followedby the group aged between 45-54 years (30.22%), 35-44
years (17.19%), 25-34 years (8.12%), 65-74 years (5.76%),
15-24 years old (2.9%), 75-84 years old (1.71%) and by thegroup over 85 years old in a percentage of 0.49% of thetotal. Conversely, the lowest frequency of cases, of only
0.01%, is found in the group with age between 5-14 years.
We can state that the highest frequency of psychiatricconditions it is found in the group of patients aged between
55-64 years.
Following the analysis of the frequency by age group of
mental and behavioral disorders due to alcohol
consumption, it was found that most cases are registeredin the age range between 45-54 years, in a percentage ofapproximately 40.6% of the total. This interval is followedby those aged between 35-44 years in a proportion of
21.8%, then by those aged between 55-64 years in apercentage of 15.6%. The age ranges between 25-34
years and 65-74 years each associate a percentage ofabout 15.6% of the total, and the age category between
65-75 years has the lowest percentage, only 3,10%. Thus
we can affirm the fact that mental and behavioral disorders
due to alcohol consumption have the highest frequency inthe range of patients aged between 45-54 years.
Analyzing the distribution by year of the prevalence ofmental and behavioral disorders induced by harmful use ofalcohol, it was estimated that the share of cases in 2017 is
8.56%, in 2018 it is 7.13%, in 2019 it is 7.88%, in 2020 it is10.29% and in 2021 it is 5.29%. Table I reports the
dynamics of the absolute number of cases within the main
panel. Thus from the obtained data it appears that thedemonstrative statistical indicator for 2018 is 85.03%, for2019 it is 113.38%, for the year 2020 it is 63% and foryear 2021 it is 33.07%, all compared to the year 2017.
This was followed by the study of the frequency ofdifferent types of mental and behavioral disorders inducedby the use of harmful alcohol. It was found that the state ofwithdrawal amounts to a proportion of 47.3%, followed bywithdrawal complicated by delirium (26.1%), harmful use ofalcohol (10.9%), psychotic disorder (7.7%), addiction
syndrome (5.1%), non-specific mental disorders andbehavior (0.9%), acute intoxication (1.3%), nonspecificmental and behavioral disorders (0.9%), and lastly, theamnesic syndrome (0.1%) (Fig 3).

use is a global phenomenon. In 2016, 56% of males(equivalent to 1.46 billion individuals) and 32% of females(0.88 billion) aged 15 and older consumed alcoholworldwide. In that year, alcohol was responsible forapproximately 3 million deaths, accounting for 5% of all
global deaths. Specifically, it resulted in 3.2 million deaths
among men (8%) and 0.7 million deaths among women(3%). Gender disparities in alcohol use are present
universally, but they can also vary from one country toanother.
A study conducted at a neuropsychiatric clinic hospitalfrom 2017 to 2021 found that, except 2019, a majority ofadmitted patients were men. Moreover, the percentage ofmen admitted with mental and behavioral disorders related
to alcohol was significantly higher compared to women,with 88% of the admitted patients being men, while womenconstituted only 12% of the admissions.
The prevalence of Alcohol Use Disorders (AUDs) andMental Health Disorders (MHDs) has been reported inmany countries, and the WHO World Mental Health (WMH)
Survey Initiative has facilitated comparisons of AUD
prevalences across different nations to identify variations
and commonalities. Looking at data from France, Germany,
Italy, Romania, and Spain, the prevalence of past-year
alcohol use, alcohol abuse, and alcohol dependence
indicates variations in the number of individuals affected ineach country. For instance, in France, there were 2894
cases, in Germany: 3555, Italy: 4712, Romania: 2357, andSpain: 5473.
When examining past-year DSM-IV alcohol use
disorder in these countries among both men and women,
the prevalence rates differ. For example, in France, 2.4% of
men and 0.2% of women were affected, in Germany, therates were 1.9% for men and 0.4% for women, in Italy, it
was 0.3% for men and 0.1% for women, in Romania, 1.6%of men and 0.1% of women were affected, and in Spain,
1.4% of men and 0.1% of women were affected [6,7].
This study's findings lead to the conclusion that Alcohol
Use Disorder (AUD) encompasses a range of conditions,
including alcohol abuse, alcohol dependence, alcohol
addiction, and alcoholism. It is a significant contributor to
the global disease burden, a leading cause of preventable
deaths, and a prevalent psychiatric disorder.
AUD is a medical condition characterized by anindividual's impaired ability to control or cease alcohol
consumption, even in the face of adverse social,occupational, or health consequences.
Globally, alcohol consumption is responsible for 3.3
million deaths each year, contributing to disabilities andoverall poor health in millions of people. The harmful use ofalcohol accounts for 5.1% of the global burden of disease.
In the study conducted on patients admitted to thepsychiatric clinic hospital in Craiova due to mental andbehavioral disorders resulting from alcohol, several key
findings emerged:

Discussions. Conclusions
Alcohol consumption has historically been moreprevalent among men, and this gender difference in alcohol
71

●
●
●
●

Gender Distribution: The majority of admitted
patients were male, accounting for 88% of thecases.
Rural Residence: A significant portion of thepatients hailed from rural areas, constituting 78%of the total admissions.
Age Group: The largest proportion of patients fellwithin the age group of 45-54, representing 41%of the cases.
Most common diagnoses: The most prevalentdiagnosis among the admitted patients was"withdrawal state with and without delirium”,
followed by “harmful use of alcohol” and"psychotic disorder”.

Bibliography
1. Crews FT, Vetreno RP, Broadwater MA, Robinson DL.
Adolescent Alcohol Exposure Persistently Impacts
Adult Neurobiology and Behavior. Pharmacol Rev.
2016 Oct;68(4):1074-1109. doi:
10.1124/pr.115.012138.
2. Ihekire NL, Okobi OE, Adedoye EA, Akahara PF,
Onyekwere AO, Afrifa-Yamoah J, Akinyemi FB.
Heartache in a Bottle: Understanding Alcoholic
Cardiomyopathy. Cureus. 2023 Aug 3;15(8):e42886.
doi: 10.7759/cureus.42886.
3. Ballard HS. The hematological complications of
alcoholism. Alcohol Health Res World.
1997;21(1):42-52.
4. Arteel GE. Liver-lung axes in alcohol-related liverdisease. Clin Mol Hepatol. 2020 Oct;26(4):670-676.
doi: 10.3350/cmh.2020.0174. Epub 2020 Oct 1.
5. Becker HC, Mulholland PJ. Neurochemicalmechanisms of alcohol withdrawal. Handb Clin Neurol.
2014;125:133-56. doi:
10.1016/B978-0-444-62619-6.00009-4.
6. Nina B.L. Urban, Lawrence S. Kegeles, Mark Slifstein,
Xiaoyan Xu, Diana Martinez, Ehab Sakr, FelipeCastillo, Tiffany Moadel, Stephanie S. O’Malley, John
H. Krystal. Sex Differences in Striatal Dopamine
Release in Young Adults After Oral Alcohol Challenge:
A Positron Emission Tomography Imaging Study With
[11C]Raclopride. Biological Psychiatry, 2010; 68 (8):689
7. Donath, C., Gräßel, E., Baier, D. et al. Alcohol
consumption and binge drinking in adolescents:
comparison of different migration backgrounds andrural vs. urban residence - a representative
study. BMC Public Health 11, 84 (2011).
https://doi.org/10.1186/1471-2458-11-84

Fiziologia - Physiology • 2023.2(105)Fig 1. Percentage of patients admitted with alcohol disorders by sex

Fig 2. Distribution by environmental criteria

Year
2017
2018
2019
2020
2021

No. of cases
127
108
144
80
42
Table I - Dynamics of the absolute number of cases

73

Statistical index
100 - base
85.03
113.3862.99
33.07

Fig 3. Percentage representation of the prevalence of different types of mental disorders and behavior due to alcohol
consumption.

UZUL NOCIV DE ALCOOL, DATE DEMOGRAFICE, PREVALENȚĂ ȘI COMPLICAȚII
RezumatAbuzul de alcool este o problemă larg răspândită la nivel mondial. Dintre toate substanțele utilizate în scopuri de dependență,consumul de alcool este cel mai răspândit, făcând din intoxicația alcoolică cauza principală a intoxicației acute. La scară globală, riscul
de a dezvolta modele dăunătoare de consum de alcool este de 15% pentru bărbați, pe parcursul vieții și este aproximativ dublu fațăde femei. Riscul de a dezvolta dependență de alcool este mai mic, cu un risc de 10% pentru bărbați și în jur de 5% pentru femei.
Descoperirile acestui studiu conduc la concluzia că “Uzul nociv de alcool” cuprinde o serie de afecțiuni, inclusiv abuzul de alcool,dependența de alcool, dependența de alcool și alcoolismul. Este o contribuție semnificativă la povara globală a bolii, o cauză
principală a deceselor care pot fi prevenite și o tulburare psihiatrică prevalentă. “Uzul nociv de alcool” este o afecțiune medicală
caracterizată prin scăderea capacității unei persoane de a controla sau de a înceta consumul de alcool, chiar și în fața unorconsecințe sociale, profesionale sau de sănătate adverse. La nivel global, consumul de alcool este responsabil pentru 3,3 milioane dedecese în fiecare an, contribuind la dizabilități și la starea generală de sănătate precară a milioane de oameni. Consumul nociv dealcool reprezintă 5,1% din povara globală a bolii. În studiul efectuat pe pacienții internați în Spitalul Clinic de Neuropsihiatrie din
Craiova din cauza tulburărilor psihice și comportamentale rezultate din alcool, au reieșit câteva constatări cheie: majoritatea pacienților
internați au fost bărbați, reprezentând 88% din cazuri, o parte semnificativă a pacienţilor proveneau din mediul rural, constituind 78%
din totalul internărilor, cea mai mare proporție de pacienți se încadrează în grupa de vârstă 45-54 de ani, reprezentând 41% din cazuri,
diagnosticul cel mai răspândit în rândul pacienților internați a fost „starea de sevraj cu și fără delir”, urmată de „consumul nociv dealcool” și „tulburarea psihotică”.
Cuvinte cheie: consum de alcool, tulburare psihiatrica, complicatii.

Fiziologia - Physiology • 2023.2(105)EVALUATION OF POSSIBLE PSYCHOMETRIC CHANGES INDUCED BY
THE PRACTICE OF PRAYER IN THE ORTHODOX CHRISTIAN
TRADITIONADRIAN SORIN MIHALACHE1,2, 3, OANA-MARIA VICU, DELIA OANA POPA1, LEON ZĂGREAN1
1 Faculty of Medicine, ”Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
2 Faculty of Orthodox Theology, ”Alexandru Ioan Cuza” University of Iași, 700506 Iași, Romania
3 Centre for Research on Medicine and Christian Spirituality, Providenta Hospital, 707317 Iași, Romania

ABSTRACTThe present study investigates the possible psychological changes in the sphere of stress/anxiety induced by the practice of
prayer (in the Orthodox Christian Tradition) - POCT, after an individual practice of 30 minutes daily, for 8 weeks. Participants (n=34) weredivided into two groups (Study Group, n=16, average age 39.2 years and Control Group n=17, average age 38.6 years). The subjects of
the Study Group – faithful Orthodox Christians without the practice of daily prayer, were trained, in a 3-hour session, with a view to thepsycho-emotional, cognitive, spiritual and behavioural peculiarities of POCT. No intervention was performed on subjects in the Control
Group. Before and after the interval of 8 weeks of daily practice of OCT, 30 min, five psychometric instruments were applied. Afterwards,
the results obtained at the intra- and inter-group pre- and post-test evaluations were comparatively analyzed. The obtained results showedthat POCT practice, according to the experimental scheme, did not produce statistically significant differences between the global scoreson the Hamilton Anxiety Scale both recorded at SG in relation to CG, pre- and post-testing. For both groups of subjects there was adecrease in the Hamilton scale score, suggesting that, in the case of GS subjects, it cannot be said with certainty that the decrease in the
Hamilton score occurred as a result of POCT practice. On the contrary, the statistically significant differences in CG subjects, post-test,
regarding a9 item ("cardiovascular symptoms"), suggest that other factors may have contributed to improve the responses of CG subjects,
which necessitates the introduction of additional conditions for more careful administration of POCT practice among SG subjects during theexperimental interval. However, some significant results were obtained, regarding the SG subjects, at post-test, regarding item a3("fears/phobias"), of the Hamilton scale, suggesting that POCT practice according to the experimental plan could have reduced theintensity of fears/phobias among subjects in SG. Also, the change appearing in the post-testing SG regarding a14 item ("appreciation of
the anxious mood at the interview") is significant, suggesting a possible influence of the POCT practice. We also note, in the list of results,
the averages of some coping mechanisms, which were significantly higher in SG compared to CG. These are e act ("active coping") and eres ("refrain/control/unwillingness") - among SG subjects at pre-test, and e pos ("positive interpretation and growth") and e rel ("religious
approach/religious coping") at post-test compared to CG subjects, indicating a possible influence of POCT practice. The results needfurther studies that more closely correlate the changes in these items, but also in other psychometric ones, with those of a physiologicalnature.

Keywords: Orthodox Christian prayer, psychological changes in the sphere of stress/anxiety, coping strategies,
fears/phobias, religious coping.
Introduction
Many studies have shown that various spiritual practices can reduce anxiety, mindfulness [1], MBI(Mindfulness-based interventions, in the case of students [2]), MBSR (Mindfulness Based Stress Reduction - in the caseof students [3], or patients with depression [4]), cultivating self-compassion [5]. Also, practices such as
self-compassion-centered mindfulness [6, 7], compassion training [2, 8] or MBSR [9] reduce stress. Along with these,
other conscious techniques for reducing stress and anxiety, contemplative practices [10], ACT therapy - Acceptance andcommitment therapy, DBT - Dialectical behaviour therapy, MBCT - Mindfulness-based Cognitive Therapy [11], or thosethat use progressive muscle relaxation, deep breathing, or sensory-guided imagery techniques [12]. Older meta-analyses[13, 14], but also recent ones [15, 16], have examined studies of this kind, emphasizing the therapeutic impact ofpractices of this kind.
The present study is part of a research that elaborated three studies on the possible psycho-metric andelectro-physiological changes induced by the practice of Prayer in Orthodox Christian Tradition (POCT). The generalobjective of the present study is to highlight the psychological changes in the field of stress/anxiety induced by thepractice of POCT. For this, we designed an experimental plan that provides for 30 minutes of daily practice, for 8 weeks,
performed by the subjects of a study group (n=17), comparing the data provided by the psycho-metric evaluations, withthose obtained in the case of the subjects of a control group (n=17), following the psychological changes in the field of
manifestations related to stress/anxiety.
For this we have followed:
1. A statistical analysis of the psychological changes in the stress/anxiety sphere on the psychometricassessment tools used intra and inter-group (significant differences, correlations, etc.).
2. An interpretation of the significant results proposed "a priori" and highlighted in the basic analysis as well asother aspects observed during the analysis ("a posteriori").
75

For this study, we formulated the following working hypotheses:
Practicing POCT 30 minutes daily, for 8 weeks:- significantly reduces the global score on the Hamilton Anxiety Scale, applied by the evaluator (Hypothesis 1.1);- significantly reduces the GAD 7 score, obtained through self-assessment (Hypothesis 1.2);- significantly reduces subjectively perceived stress on the Visual Analog Scale of Perceived Stress, obtained
through self-assessment (Hypothesis 1.3)
7. - significantly reduces the Global Score on the perceived stress scale (PSS-10), applied by the evaluator(Hypothesis 1.4)
8. - strengthens coping mechanisms and increases the global COPE score, obtained through self-assessment(Hypothesis 1.5)
2.
3.
4.
5.
6.

1.

Participants and methods

The subjects of the present study were those who also participated in the study of physiological changes,
published in Physiology, 2023, 1(104). We mean the following composition of SG and CG.
PT
Code
1
2
3
4
5
6
7
8
9
11
23
25
24
10
14
26
31

STUDY GROUP
Gen
Age
der
F
F
F
F
F
F
F
F
F
F
M
M
M
F
F
M
M

42
21
41
57
33
37
39
51
35
40
37
41
32
48
41
26
47

o

Ag
e
45
20
38
56
30
38
39
49
34
42
38
42
32
46
42
27
47

CONTROL GROUP
Gen
PT
der
Code
F
F
F
F
F
F
F
F
F
F
M
M
M
F
F
M

12
13
18
15
16
17
22
19
20
30
27
28
32
21
33
29

Table I. Age and gender structure of CG and SG. The numbers in the table represent the subjects' work codes (PTcode) used in the psychometric tests.
In order to highlight the possible psychological changes induced by POCT, we used psychometric measurement tools.
The changes that occurred were highlighted by comparing the answers given by the GS subjects, pre- and post-test, but also
the answers given by the SG subjects compared to those of the CG subjects, pre- and post-test.
The psychometric evaluation tools used in Study 1 were:
1. Hamilton scale for anxiety;
2. The GAD 7 (General Anxiety Disorder) test measures the level of anxiety symptoms;
3. Visual analogue scale of perceived stress, or visual analogue scale (VAC);
4. Perceived stress scale (PSS-10);
5. The COPE questionnaire targeting forms of coping with stress.
The working protocol in the application of psychometric instruments was as follows:- Application of the Hamilton Anxiety Scale (HAM-A) by the psychiatrist (approx. 15 min.) – code A in the
Database);- Application of the GAD 7 Questionnaire – pencil and paper method, self-assessment (approx. 5 min.) - code
B in the Database;- Application of the Visual Analog Scale of Perceived Stress (approx. 2 min.) - code C in the Database;- Application of the perceived stress scale by the psychiatrist (approx. 5 min.) - code D in the Database;- Application of the COPE questionnaire - pencil and paper method, self-assessment (approx. 10 min.) - code

Fiziologia - Physiology • 2023.2(105)E in the Database.
(i) The Hamilton scale for anxiety is a tool for global assessment of anxiety or the severity of anxiety symptoms(such as mental tension, anxious mood) but also the somatic aspects that appear in anxiety (such as the
bio-physiological changes associated with it) [17]. The Hamilton scale (14 items, corresponding to some symptoms ofanxiety), is applied in the form of a semi-structured interview, the items being evaluated by qualified personnel (on ascale from 0 to 4, where 0 corresponds to the absence of the symptom, and 4 to a severe symptom and disabling) [17].
The Hamilton scale score ranges from 0 to 56, with panic disorders or generalized anxiety disorders corresponding toscores above 20, while scores below 20 indicate the absence of these disorders [17].
(ii) The GAD 7 test (General Anxiety Disorder – 7 – compiled by Robert L. Spitzer) contains 7 questions thatassess the level of anxiety symptoms experienced by a person in the last two weeks. To these questions, therespondents choose as answers numbers from 0 to 3, which frame the assessments regarding how often they felt
emotional problems (0 meaning "not at all", 1 meaning "a few days", 2 meaning "more than half the days" and 3 "almost
daily", during the 14 days.)(iii) The Visual Analogue Scale of Perceived Stress, or Visual Analogue Scale (VAS) is the visual representationof the level of stress a person thinks they are experiencing. The subjects represented, on an axis with values between 1
and 10, the answer to the question "How stressed do you feel?", represented in Fig. 1.

Fig. 1. Example of a visual analogue scale of perceived stress, where the subject, asked "How stressed do you
feel?", graded and graphed level 2 out of 10.
(iv) Perceived Stress Scale (PSS-10) constructed by Cohen & all (1983) [18], is a 10-item questionnaire thatassesses the degree to which a person perceives life as unpredictable, uncontrollable and difficult to manage during thepast month. The subject is asked how often he felt a certain state, placing it through a numerical value between 0 and 4,
0 meaning "never", 1 meaning "almost never", 2 meaning "sometimes", 3 meaning "often", and 4 "very often".
(v) The COPE questionnaire, developed by Carver, Scheier and Weintraub (1989) [19], evaluates 15 forms of
coping with stress, which are active or passive. The questionnaire includes 60 statements, each form of coping being
evaluated through 4 items, with answers on a scale from 1 to 4, where the value 1 corresponds to the statement "Iusually do not do this", 2 - for "I rarely do this" , 3 for "I sometimes do this", and 4 for "I often do this". For the evaluation,
the score from each of the 4 items corresponding to each of the 15 coping mechanisms, mentioned below, is added.
2.

Primary data

After the application of the questionnaires, which was done by a resident in psychiatry, the data were introducedin Excel format.

Fig. 2. Extracted from the primary data summary table. In the first column are the codes associated with each77

subject from GS and GC, from 1 to 33 (When applying the questionnaire, it was not known whether the subjects
belonged to GS or GC). Next are the columns corresponding to gender, age, and education level for each subject, andthe first five items (A1-A5) of the Hamilton scale, for which the subjects' responses are completed. The extracted
fragment contains all responses completed by the subjects (with values between 0 and 4), for all subjects, at the time ofpre-testing.
Next, after the columns intended for the Hamilton scale (A), columns B1-B7, C, D1-D10, E1-E60 and respectively
the corresponding codes for each of the 15 coping mechanisms assessed by the COPE questionnaire were introduced.
A = Hamilton Anxiety Scales
B = GAD 7 Questionnaire – paper-pencil method, self-assessment
C = Visual analogue scales of perceived stress
D = Scale of stress perceived by the psychiatrist
E = COPE QuestionnaireFig. 3. Extracts from the primary data summary table, with four and six columns associated with answers B7, c,
D1 and D2, respectively the last two answers from the COPE Test and the first three coping strategies e pos, e men, efoc, for subjects coded from 1 to 4. Their answers are between the values mentioned for each individual instrument,
corresponding to the pre-testing moment.
The 15 coping strategies/mechanisms assessed by the COPE questionnaire and their corresponding items are:
Nr.
crt.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
1
2.
3.

1
1

4.
5.

1

cod
e
e pos
e men
e foc
e use
e act
e den
e rel
e hum
e beh
e res
e emo
e
sub
e
acc
e
sup
e
pla

Significance
Positive reinterpretation and growth
Mental disengagement
Focus on and venting of emotions
Use of instrumental social support
Active coping
DenialReligious coping
HumourBehavioural disengagement
Restraint
Use of emotional social support
Substance use

Corresponding
items
1, 29, 38, 59
2, 16, 31, 43
3, 17, 28, 46
4, 14, 30, 45
5, 25, 47, 58
6, 27, 40, 57
7, 18, 48, 60
8, 20, 36, 50
9, 24, 37, 51
10, 22, 47, 58
11, 23, 34, 52
12, 26, 35, 53

Acceptance13, 21, 44, 54

Suppression of competing activities

15, 33, 42, 55

Planning

19, 32, 39, 56

3. Statistical analysis tools
The descriptive statistical elements initially processed, on the variables to be analysed, revealed distributions that
do not satisfy the condition of normality. This aspect, the small sample (N < 30, the Study Group having 17 subjects andthe Control Group 16 subjects) and the type of analysed variables led to the choice of non-parametric tests in thestatistical analysis [20].
The tools used for statistical analysis in the present study are non-parametric tests for the median (Wilcoxon
signed-rank test), t-tests for paired data and the Mann-Whitney test for independent samples.

Fiziologia - Physiology • 2023.2(105)(a) The median and the average are two statistical indicators of central tendency for a set of statistical data. If theobserved data have a small volume (usually below 30-40), tests for the average (called parametric tests) are not relevant,since the statistical assumptions under which they operate are not met. That is why, for the present situation, we usedalternative tests (called non-parametric), for the median, especially since the latter is a better indicator of the central
tendency of the data (compared to the mean), in case the data are asymmetric.
(b) The Wilcoxon signed-rank test is a nonparametric statistical test used to either test the median of a
population based on a sample of data or to compare the medians of two populations using two matched samples. The
test used here checks whether there are significant differences for the median value before and after the 8 weeks of
personal experience (POCT).
(c) The t-test for paired data is a statistical method used to test whether the average difference between pairs ofmeasurements (before and after the experiment) is zero or not. The test can be used when the data values are paired
measurements and the distribution of differences between paired measurements should follow the normal distribution. (Inthe case of the present research, paired data refers to intra-group pre- and post-test data.)(d) The Mann-Whitney test for independent samples is a tool applied to compare two independent samples when
few data are available and when they do not follow a normal distribution.
Test results (equal medians or equal averages, as appropriate) are described by P-values (Pv), which represent
the level of significance observed for each test. The Pv value is a probability calculated based on an appropriate statisticthat describes how likely/plausible it is to have found a given set of observations if the null hypothesis (equal medians orequal means, as appropriate) were true. The result of a test is done by comparing the values of Pv and alpha (thetheoretical significance level chosen by the user. By default/standard, alpha = 0.05, if Pv < alpha, then the hypothesis is
accepted (the tested averages or medians are not equal.)
4. Statistical analysis and interpretation of results. Hamilton Anxiety Scale
The intra-group analysis was initially done on the global scores obtained and then refined, starting from thesignificant differences observed within the two groups, SG and CG. Intra-group analysis was performed with the
Wilcoxon test, pre- and post-testing.

25
20
15
10
5
0
1

2

3

4

5

6

7

8

9

10

GC pre-testare

11

12

13

14

15

16

GC post-testareFig. 4. Hamilton global score for CG, pre and post-testing.
Considering the values, it can be said that there are significant differences within CG pre- and post-testing on
global Hamilton scale scores at P < 0.01 (p = 0.001, z = -3.423).

79

20
15
10
5
0
1

2

3

4

5

6

7

8

9

GS pre-testare

10

11

12

13

14

15

16

17

GS post-testareFig. 5. Hamilton global score for SG, pre and post-testing.
We mention that, in order to verify these results, we transformed the two variables sumA1 and sumA2 into theirsqrtsumA1 and sqrtsumA2 variants in order to obtain a distribution as normal as possible and to perform the t-test for
paired samples, which would confirm the significance of the observed differences - GC pre-test/post-test P < P = 0.01,
bilateral, df = 15, t = 6.689, GS pre-test/post-test P < P = 0.01, bilateral, df = 16, t = 7.177.
The inter-group analysis was performed with the Mann Whitney test for independent samples.

25
20
15
10
5
0
1

2

3

4

5

6

7

8

9

Grup de control - pre-testare

10

11

12

13

14

15

16

17

Grup de studiu - pre-testareFig. 6. Global Hamilton score for CG and SG pre-testing.

Fiziologia - Physiology • 2023.2(105)

20
15
10

5
0
1

2

3

4

5

6

7

8

9

10

GC post-testare

11

12

13

14

15

16

17

GS post-testareFig. 7. Global Hamilton score for CG and SG post-testing.
We can say that there are no significant differences between the two groups both at the pre-testing (p = 0.6 >
0.05, z = - 0.525) and at the post-testing (p = 0.663 > 0.05, z = - 0.436).
We use the variables described above, sqrtsumA1 and sqrtsumA2, to perform the independent samples t-test.
This confirms the non-significance of the observed differences in pre-test P = 0.335 > P = 0.05, two-sided, df = 31, t = 0.40 and post-test P = 0.587 > P = 0.05, two-sided, df = 31, t = -0.53.
We also present the variation in scores across the entire sample, pre- and post-testing.
25
20
15
10
5
0
1

3

5

7

9

11 13 15
17 19

scor anx pre-testare

21

23

25

27

29

31

33

scor anx post-testareFig. 8. Hamilton global scores on the entire sample pre- and post-testing
We notice that, although there are no significant differences between the global scores on the Hamilton scale
between the two groups, pre- and post-testing, there are still statistically significant differences between the global scores
recorded on both SG and CG pre- and post-testing. This similar evolution, of the subjects in SG and CG, by registering adecrease in the score in the Hamilton scale, does not highlight with certainty that the practice of POCT contributed to thedecrease in the level of anxiety in SG. (In the discussion section we will analyse this situation.)
We have further thoroughly examined the existence of changes on the scale items, in the two groups, pre- andpost-testing. The values of the 14 items, marked with "a", from a1 to a14, were analysed. Table II shows the significant
differences recorded. Significant differences are noticed in both groups on a1, a2, a5, a6, a8 and a10.
81

Anxious mood(a1)
Controlgroup

P = 0,005 < P =0,01, z = -2,804

Difficulty inrelaxing/muscle
tension (a2)
P = 0,013 < P =0,05, z = -2,496

Study
group

P = 0,005 < P =0,01, z = -2,81

P = 0,001 < P =0,01, z = -3,307

Prosexo-mnesic
difficulties (a5)Depression(a6)

P = 0,035 < P =0,05, z = -2,111
P = 0,034 < P =0,05, z = -2,121

P = 0,004 < P= 0,01, z =-2,889P = 0,007 < P= 0,01, z =-2,714

Changes ofanalysers(a8)
P = 0,02 < P =0,05, z =-2,333
P = 0,035 < P= 0,05, z =-2,111

Breathingdifficulties(a10)
p = 0,034 < P= 0,05, z =-2,121
P = 0,014 <
0,05, z = -2,46

Table II. Significant differences recorded between values of items a1, a2, a5, a6, a8, a10, among subjects in SG
and CG, pre- and post-testing.
From the above mentioned data as well as from the analysis of the items where insignificant differences wererecorded in both groups (a4, a7, a11, a12, a13), the relatively similar evolution of SG and CG can be noticed. For SG, thepost-testing changes that appeared regarding item a3 ("fears/phobias") and a14 ("evaluating the anxious mood at theinterview") are significant (Table III).
Fears/phobias (a3)
P = 0,102 > P = 0,05,
z = - 1,633
P = 0,046 < P = 0,05,
z = - 1,999

Control group
Study group

Evaluating the anxious mood at the interview (a14)
P = 0,414 > P = 0,05,
z = - 0,816
P = 0,002 < P = 0,01,z = - 3,162

Table III. Significant differences recorded between the values of the a3 item (fears/phobias) and a14 item
(appreciation of anxious mood), among the subjects of SG, compared, pre- and post-testing.
It is significant that in the case of SG subjects the values of a3 ("fears/phobias") decreased, which suggests a
possible effect of POCT. This aspect could constitute a study hypothesis for a future research. The changes in the a14
item ("evaluation of the anxious mood" at the interview, by the evaluator) are also important, but could have been biasedby the assessment conditions.

3
2.5
2
1.5
1
0.5
0
1

2

3

4

5

6

7

8

9

Grup de studiu pre-testare a3

10

11

12

13

14

15

16

17

Grup de studiu post-testare a3

Fig. 9. Values of item a3 ("fears/phobias") pre- and post-testing in SG.
At the same time, changes also appear in the CG, regarding the a9 item ("cardiovascular symptoms") – Table IV.
Control group (CG)

Cardiovascular symptoms (a9)P = 0,021 < P = 0,05, z = - 2,31
Fiziologia - Physiology • 2023.2(105)Study group (SG)P = 0,248 > P = 0,05, z = - 1,155

Table IV. Significant differences recorded between the values of the a9 item among CG subjects, comparatively,
pre- and post-testing.
The fact that these significant differences also appear in CG - without the subjects having participated in anexperimental intervention - suggests the possibility that other unknown factors, related to the plan of personal life, may
have influenced the evolution. Also, good Hamilton scale scores recorded by subjects in the post-test CG could also be
caused by the influence exerted by the subjects' experiences after participating in the stages of the study (preparatoryinteractions, general exposure to the project, EEG measurement sessions, pre and post-testing).
On the other hand, the fact that SG subjects do not register, post-testing, significantly greater changes in global
scores on the Hamilton scale compared to CG subjects, could also be caused by the fact that significant changes inanxiety require a longer period of psychotherapeutic or drug intervention [21] (suggesting that for further improved
research an experimental interval longer than 8 weeks could be introduced). Another possible reason for the significantly
close scores of the SG and CG subjects, post-test, could be that the SG subjects failed - during the 8 weeks - to comply
with the indications formulated for practicing POCT.
We can formulate the conclusion that the present research does not highlight changes in the experimental groupthat can be exclusively determined by the practice of POCT, changes being also reported in the case of CG subjects, sothat other causes, such as the placebo effect, cannot be excluded or responses biased by laboratory conditions, during
sessions dedicated to measurements.
5. Statistical analysis and interpretation of results. The GAD 7 test
The use of the GAD 7 Test did not reveal significant changes between the Control Group and the Study Group,
pre- and post-testing.
Intra-group, for both groups, analysis was performed with the Wilcoxon test for paired samples, without
identifying significant differences, pre- vs. post-testing, between the two groups at P = 0.05.

20
15
10
5

0
1

2

3

4

5

6

7

8

Grup control pretestare

9

10

11

12

13

14

15

16

Grup control post-testareFig. 10. Global GAD 7 score for CG pre- and post-testing.

10
8
6
4
2
0
1

2

3

4

5

6

7

8

Grup de studiu pretestare

9 8310

11

12

13

14

15

Grup de studiu post-testare

16

17

Fig. 11. Global GAD 7 score of SG, pre- and post-testing.
Inter-group, however, the result is surprising: significant differences between the two groups SG and CG in the
pre-test and the absence of these differences in the post-test were recorded.

20
15
10
5
0
1

2

3

4

5

6

7

8

9

Grup de control - pre-testare B1

10

11

12

13

14

15

16

17

Grupul de studiu - pre-testare B1

Fig. 12. GAD 7 Scores for the CG vs. SG in pre-testing.

12
10
8
6

4
2
0
1

2

3

4

5

6

7

8

9

CG - post-testing B2

10 11

12

13

14

15

16

17

SG - post-testing B2

Fig. 13. GAD 7 Scores for the CG vs. SG in post-testing.
The inter-group analysis, performed with the Mann-Whitney test for independent samples, revealed that thedifferences between CG and SG faded to non-significance in post-testing (at P = 0.05). The situation is novel from anexperimental point of view, with the initial differences between the groups fading during the experimental period.

B1/B2

Study group /Control grouppre-testingP = 0,012 < P = 0,05, z = -2,513

Study group /Control grouppost-testingP = 0,586 > P = 0,05, z = -0,545

Fiziologia - Physiology • 2023.2(105)

Table V. Significant differences in pre-testing between the Study Group and the Control Group - revealed byMann-Whitney test for independent samples - fade in post-testing (at P = 0.05).
The maximum score in the Control Group in pre-testing was 16, and in post-testing was 11, with group averageof 6.38 and 4.5, respectively. The maximum score in the Study Group in pre-testing was 6, and in post-testing was 9, withthe group average of 2.88 and 3.82, respectively. Therefore, it can be concluded that the practice of POCT, in
accordance with the chosen research plan, did not produce statistically significant changes in the global score of the
GAD 7 scale, and did not influence the intensity of the anxiety-type symptomatology explored with the help of this scale.
In pre-testing, in SG, the global values did not exceed the threshold of clinical intensity (>10), while in CG there
were 3 initial values that exceeded this critical threshold, making it possible that this fact should have contributed to thesignificant differentiation of the two groups in the pre-testing. On the other hand, the fading of the differences between
SG and CG in post-testing could be explained by the fact that CG subjects were influenced by the experiences ofparticipating in the study regarding spiritual life. These changes, which may have occurred individually for some subjects
in the CG, could have significantly influenced the data for the CG as a whole, as the number of subjects in the CG issmall.
The conclusion of this analysis would be that the experimental design provided for the study failed to revealchanges brought about by POCT practice at the level of anxious symptomatology explored with the help of the GAD
scale.
6. Statistical analysis and interpretation of results. Visual analogue scale
Analysis and interpretation of the data provided by the Visual Analogue Scale of Perceived Stress did not provide
conclusive results. Intra-group, for both CG and SG, was performed with the Wilcoxon paired-samples test, pre- andpost-testing, the differences being statistically insignificant.

10

5

0
1

2

3

4

5

6

7

8

9

10 11 12
13

CG - pre-testing

14

15

16

CG - post-testingFig. 14. Visual analogue scale for CG in pre/post-testing.

10
50
1 2 3 45 6 7
89 10 1112

SG - pre-testing

13 14 15
16 17

SG - post-testingFig. 15. Visual Analogue Scale for the SG, in pre/post-testing.
85

The Mann-Whitney analysis for independent samples, pre- and post-testing, was performed inter-group to assess
differences between SG and CG, with inconclusive results.

10
8
6
4

2
0
1

2

3

4

5

6

7

8

9

CG - pre-testing

10 11 1213 14 15

16

17

SG - pre-testingFig. 16. Visual analogue scale for CG and SG in pre-testing.

10
8
6
4
2
0
1

2

3

4

5

6

7

8

9

CG - post-testing

10 11 1213 14 15
16 17

SG - post-testingFig. 17. Visual analogue scale for CG and SG in post-testing.
The conclusion, resulting from the use of this tool, is that the chosen experimental plan did not reveal significant
changes produced by POCT regarding the level of subjectively felt (self-evaluated) stress.
7. Statistical analysis and interpretation of results. Perceived stress scale - evaluated by the psychiatrist
The data obtained regarding the Perceived Stress Scale - evaluated by the psychiatrist - did not provide
conclusive results.
The intra-group analysis, done with the Wilcoxon test for paired samples, revealed statistically insignificant
intra-group, pre- and post-testing differences for both SG and CG subjects.

Fiziologia - Physiology • 2023.2(105)

30
25
20
15

10
5
0
1

2

3

4

5

6

7

8

9

CG - pre-testing

10

11

12

13

14

15

16

CG - post-testingFig. 18. PSS-10 global score for GC subjects at pre- and post-testing.

35
30
25
20
15
10
5
0
1

2

3

4

5

6

7

8

9

10

SG - pre-testing

11

12

13

14

15

16

17

SG - post-testingFig. 19. PSS-10 global score for GS subjects at pre- and post-testing.
The Mann-Whitney analysis for independent samples, pre- and post-testing, intra-group, was performed toassess differences between SG and CG.

87

35
30
25
20
15
10
5
0
1

2

3

4

5

6

7

8

9

10

CG - pre-testing

11

12

13

14

15

16

17

16

17

SG - pre-testingFig. 20. PSS10 global score for CG subjects vs. SG in pre-testing.

30
25
20
15
10
5
0
1

2

3

4

5

6

7

8

9

CG - post-testing

10

11

12

13

14

15

SG - post-testingFig. 21. PSS10 global score for CG subjects vs. SG in post-testing.
The results obtained were inconclusive. The conclusion, resulting from the use of this tool, is that the chosen
experimental plan did not reveal significant changes produced by POCT regarding the level of subjectively felt stressassessed by the PPS-10.
At the moment, we can formulate the conclusion that no change induced by the practice POCT in relation to thestress level was highlighted.
8. Statistical analysis and interpretation of results. The COPE test

Fiziologia - Physiology • 2023.2(105)Results of interest were obtained from the analysis of data corresponding to the COPE Test. In the first instance,
there were no statistically significant differences in the intra-group analysis, which was performed using the Wilcoxon
paired-samples test.

200
150
100
50
0
1

200

2

3

4

5

6

7

8

150

9

10 11 12

13

14

15

16

17

15

16

100
50

CG - pre-testing (COPE)

SG - pre-testing (COPE)

0
1

2

3

4

5

6

7

8

9

CG - pre-testing (COPE)

10

11

12

13

14

17

CG - post-testing (COPE)

Fig. 22. The COPE global score for CG subjects at pre- and post-testing.

200
150
100
50
0
1

2

3

4

5

6

7

8

9

SG - pre-testing (COPE)

10 11

12

13

14

15

16

17

SG - post-testing (COPE)

Fig. 23. COPE global score for GS subjects in pre- and post-testing.
Also, the inter-group analysis, performed with the Mann-Whitney test for independent samples, revealed
statistically insignificant inter-group differences on global COPE scores.
89

200
150
100
50
0
1

2

3

4

5

6

7

8

9

10

CG - post-testing (COPE)

11

12

13

14

15

16

17

SG - post-testing (COPE)

Fig. 24. COPE global score for CG subjects vs. SG in post-testing.
However, some significant aspects were highlighted. The averages of several coping mechanisms are higher in
SG compared to CG:- e act (the active coping approach) in the pre-testing;- e res (control/restraint), in pre-testing;- e pos (positive interpretation and growth), post-testing;- e rel (religious approach/religious coping), post-testing.

Control group/Studygroup

e act pre-testingP = 0,025 < P = 0,05,
z = -2,256

e res pre-testingP = 0,041 < P = 0,05,
z = -2,067

e pos post-testingP = 0,023 < P = 0,05, z= -2,276

e rel post-testingP = 0,035 < P = 0,05, z =-2,112

Table VI. Coping items for which the averages are significantly higher in SG compared to CG.
In CG the average for e act ("active approach to coping") in pre-testing is 11.81, compared to SG, where theaverage is 14, which suggests that, before the testing period, the active approach as a coping mechanism is morepresent in SG than in CG. The situation can be explained by the fact that the subjects selected for SG had apreoccupation with Christian S/RE.
In CG the average for e res (“control/restraint/necessity”) in pre-testing is 9.38, while in SG it is 11.35. We cantherefore say that this coping mechanism is more present in subjects from SG compared to subjects from CG, in
pre-testing. However, these differences are no longer as large in post-testing, the difference between the average values
for e act resulting from the two groups diminishes, becoming statistically insignificant. The situation shows similarities
with the evolution of responses on the Hamilton scale for comparative anxiety, for CG and SG subjects, in post-testingand pre-testing.
Regarding the average for e pos ("positive interpretation and growth") in post-testing for CG, there was anaverage of 12.25, while for SG, the value was 13.94, values that suggest an increased presence of this coping
mechanism among subjects in SG compared to CG, a change that could come from the practice of POCT.
Similarly, the values for e rel ("religious approach/religious coping"), in post-testing, are significantly higher for
subjects in SG than for those in CG (13.82 vs. 11.75), which suggests an increased presence of religious approach as a
coping mechanism among SG subjects after the experimental interval, a situation to which POCT practice could havecontributed.

Fiziologia - Physiology • 2023.2(105)

20
15
10
5
0
1

2

3

4

5

6

7

8

9

10 11 12

Scor epos pentru GC - post-testare

13

14

15

16

17

Scor epos pentru GS - post-testareFig. 27. The score is poor for subjects from CG vs. SG in post-testing
In CG the average in e rel 2 is 11.75 and in the SG it is 13.82.

20

15
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0
1

2

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4

5

6

7

8

9

10 11 12

Scor erel pentru GC - post-testare

13

14

15

16

17

Scor erel pentru GS - post-testareFig. 28. The e rel score for subjects from CG vs. SG in post-testing
Although the study did not reveal a statistically significant influence of POCT practice regarding the global scoreon the COPE scale, however, in SG, in the post-test, changes in some values were observed regarding the two coping
strategies, namely the positive interpretation and the religious approach.

91

9. Remarks
The obtained data also allow some correlations and some important remarks. There is a positive, statisticallysignificant correlation between the Hamilton Anxiety Scale Global Score and the GAD 7 global score (B2), high in value(Spearman's rho = 0.546), corresponding to a close relationship between the two variables, p=0.024<p=0.05), a result
suggesting that the two vary in the same direction, confirming the decrease in anxiety.
Another statistically significant correlation (Spearman's rho=0.529) indicates a close connection between twoother variables (p=0.029<p=0.05), namely a3 pos (item 3 of the Hamilton scale, corresponding to "fears/phobias" ) and
the GAD 7 global score, a result that indicates that the two vary in the same direction, the decrease in the value on item
a3 correlating with the decrease in the intensity of the anxiety symptomatology evaluated by GAD 7, a coherent aspect inrelation to the other obtained results.
The positive, statistically significant correlation (Spearman's rho = 0.492 - which indicates an average connection
between two variables, p=0.029<p=0.05) is also noted, between the same a3 item (item 3 on the Hamilton scale for"fears/phobias ”) and the PSS-10 global score post-testing, which tells us that the two vary in the same direction, thedecrease in the intensity of fears/phobias correlates positively with the decrease in stress.
There is also a statistically significant negative correlation (Spearman's rho = - 0.547), indicating a close
relationship between two other variables (p=0.029<p=0.05) e rel (the coping mechanism corresponding to the religious
approach in the COPE test) post-testing and the PSS-10 global score (D2 in Appendix 10.4), which tells us that the twovary inversely proportionally, with perceived stress decreasing as the religious coping mechanism increases.
The results that we can consider the most important, (in relation to the significant statistical differences and thenumber of subjects in the experimental group, n = 17) - are those on a3 post-testing (the fear/phobias item from the
Hamilton scale), e2 post- testing (the positive interpretation type coping mechanism from COPE) and e rel 2 ("thereligious approach type coping mechanism from COPE"). We can support this interpretation with the assumption thateven in the case of a small sample volume, which weakens the test results, the result remains significant because itshows an important effect size [149].
However, the changes recorded in the Study Group, in post-testing, in the two coping strategies positive
interpretation (e pos) and religious approach (e rel) do not change the final conclusion, as no significant inter-groupdifferences are recorded in post-testing. They can only provide a picture of the dynamics within the experimental groupon the two coping mechanisms. Since neither did the overall scores significantly differ statistically speaking, nor did thetwo items vary significantly within the study group, the changes are not significant, but may represent a starting point for
future research. This could consider the assessment of the possibility that POCT strengthens the belief-based coping
mechanism and, likewise, the positive construal mechanism.
10. Discussions
Numerous studies have highlighted the beneficial impact of Christian prayers on anxiety and stress levels.
One study, which focused on subjects with a depressive episode (according to DSM-IV-TR criteria), is significant
from the point of view of the experimental design, for the present paper. The subjects (ages 18, 60 women and one man)participated in an intervention that included one hour of prayer each week for six weeks. The prayer was led by a cleric,with distance, avoidance of touching and personalized texts adapted to the specific needs of the subjects. Measurements
revealed, by applying the Hamilton Depression Scale and the Hamilton Anxiety Scale, a reduction in stress and anxiety
levels compared to the Control Group (p < 0.01 in all cases), with the status quo still valid even one month after theintervention [22].
Other studies highlight similar influences determined by the practice of Christian prayers, in coping strategies[23-26], but also on the general mood (in the case of doctors and patients, for example) [28].
A study, published in 2017, used the COPE test to assess changes following a spiritual intervention. Possible
links between stress coping strategies, spirituality and social support among patients with rheumatoid arthritis (RA) were
examined using the Mini-COPE Inventory (n = 250). The results showed that stress coping strategies (return to religion,
acceptance), spirituality and social support influenced the perception of pain among the participants [27], a fact also
highlighted in the present study.
Other studies highlight similar influences determined by the practice of Christian prayers, in coping strategies[23-26], but also on the general mood (in the case of doctors and patients, for example) [28].
In relation to these results, but also to the conclusions of the present study, it can be mentioned that the decreasein the level of anxiety and stress determined by the practice of POCT could also be related to the way the relationshipwith the Supreme Being is understood and felt, on a personal level , in the case of Christian subjects, prayer being a wayof dialogue with God, of entrusting the believer to God. This aspect is present in a study published in 2012, which took
into account the state of teaching the subject of God (often found in religious people) and its influences on the mood ofpractitioners, using the State Anxiety Inventory (STAI). Regression analysis of the data provided by the STAI, applied to asample of 460 people (including 306 women), showed that the posture of submission to God was inversely related to

Fiziologia - Physiology • 2023.2(105)stress, meaning that S/RE influences health, in the sense of lower stress levels [29].
11. Limits of the study
The present study has several significant limitations.
(a) Recruitment of subjects
Some of the limitations of the present research are related to the difficulties of recruiting the participating
subjects, this aspect being found in the inventory of limitations in all three studies carried out in the present research.
In general, the selection of participants for the scientific study of ES/R is difficult. The experimental interval, theindependent variable and the number of subjects), difficulties aimed at the composition of CG and SG, are also present
in this research:- The unusual topic in the field of empirical research in the field of medical sciences;- The recruitment of subjects for SG, in the present research, was made, due to necessity, from among thefaithful who are not practitioners, but willing to participate in the study;- CG subjects were chosen from among people who are interested in the spiritual dimension of life (to be willingto participate in the presentation meetings and all stages of the experimental plan) but who do not systematically practice
any kind of spiritual exercises (mindfulness, yoga, meditation, or other practices), in order not to introduce randomvariables into the experimental plan;- Identification of subjects to express their agreement to participate, for a research that provides for a longexperimental interval of 8 weeks, in which daily tasks are provided, for a duration of 30 minutes.
Constraints of this kind led to limitations in the configuration of SG and CG. The group of participants in thisresearch, 33 subjects, was made up of 24 women (73%) and 9 men (27%). For this reason, the ratio of women/men in
SG and CG does not reflect the known ratio at the population level, respectively 49%/51%. Applying the binomial test to
the entire sample shows us that men are significantly less, relative to their proportion in the population at
p=0.005<p=0.05. Applied, distinctly, to the two SG and CG, the binomial test shows that the proportion of men differs
significantly in CG relative to the existing proportion in the population (p=0.032<p=0.05), but does not differ significantly
in SG relative to the same proportion existing in the population (p=0.061<p=0.05), this is due to the numerical inequalityof the groups (CG = 16, CG = 17), the groups being a little numerous.
(b) Administration of the independent variable
Other limitations of the present research stem from the administration of the independent variable of task. On the
one hand, the experimental task provided for the SG subjects could have affected the quality of task performance(independent variable) contributing to inconclusive results:- The frequency provided for the fulfillment of the task (daily);- Duration of the task (30 min, without interruption);- The interval provided for the administration of the task-independent variable (8 weeks);- The indications for the performance of the task, which aimed to cover the spiritual, cognitive andpsycho-emotional criteria of POCT, which increase the degree of difficulty of the requirements for the execution of thetask by the SG subjects:- Choosing a quiet space;- Preliminary interventions, to prepare an appropriate state for POCT (calming, profile reading);- The best possible attention to the text of the prayers read, during the prayer.
On the other hand, the conditions of the experimental plan (which aimed to carry out the prayer in private, inprivate space, in privacy) did not allow the supervision of the subjects in the SG by the specialized staff, excluding theexchange of email messages, during the experimental interval.
(c) Administration of psychometric instruments
The used scales assess the level of anxiety, but the instruments used do not distinguish between state anxiety
and anxiety as a personality trait. Since SG and CG are not numerous, it is possible to use personality tests, which could
highlight whether the increased level of anxiety stems from the personality profile. As a possible way to improve futurestudies, in addition to the anxiety assessment scales, the STAI - State Trait Anxiety Inventory - a test to assess the levelof anxiety at a given moment (state anxiety), and the general tendency, could be used of being anxious (trait anxiety)[30], or using the Big Five Inventory, for the assessment of personality traits related to the tendency to be anxious [31].
Other limitations of the study may have arisen from the use of the English version of the Hamilton Anxiety Scale,even though it was administered by specialist staff. (Currently there is an authorized translation, which is used on the
population of Romania, as an integral part of the CES (clinical evaluation system), cited at the beginning of this chapter.)
We saw that the evolution of Hamilton scores between pre and post-testing in the two groups was in some93

situations similar and that in other respects the CG subjects had a favourable evolution without having an experimental
task, which suggests that other unknown factors could have influenced the evolution of conditions in subjects in SG and
CG, including placebo, especially since the experimental interval was considerable.
An important part of these limitations could be eliminated or significantly diminished if in further studies a numberof volunteers, clerics were introduced to participate in prayer with the subjects from SG, together (as in the mentioned
study) [22], for a more careful management of the conditions for the personal experience of POCT. On the other hand, an
experimental design that provides prayer together with a cleric, for each of the SG subjects is difficult to administer,requiring more volunteers but also more supervision, to control the variables (which could come from the interpersonal
relationships of the volunteers clerics and SG subjects).
Another intervention, for increased quality of task performance by SG subjects, could be provided by a morethorough instruction, during several meetings, further detailing the practical coordinates of POCT, as well as an interval
designed to accommodate participants with POCT (1-2 weeks), before the experimental interval.
Finally, since the visual stress scale (by self-assessment) did not provide useful data, in future research on
changes induced by POCT, this instrument could be abandoned.
Conclusions
In relation to the proposed working hypotheses, the results of this study are as follows:
Hypothesis 1: Practicing OCP daily, for 30 minutes, for 8 weeks, significantly reduces the global score on the
Hamilton Anxiety Scale, administered by the evaluator.
Result: The practice of OCP, according to the experimental scheme, did not produce statistically significant
differences between the global scores recorded both in SG in relation to CG, pre and post-testing. For both groups ofsubjects there was a decrease in the Hamilton scale score, suggesting that, in the case of SG subjects, it cannot be said
with certainty that the decrease in the Hamilton score occurred as a result of POCT practice.
The analysis of the values of the 14 items, a1 - a14, revealed significant differences in both groups, pre andpost-testing, regarding a9 ("anxious mood"), a2 ("difficulties relaxing/muscle tension"), a5 ("difficulties prosexo-mnesis"),a6 ("depression"), a8 ("changes on analyzers"), a10 ("breathing difficulties"). There were also statistically significant
differences in CG subjects only, who, in post-testing, showed changes in the a9 item (“cardiovascular symptoms”),
suggesting that other factors may have contributed to the improvement in CG subjects' responses.
The change appearing in the SG in post-testing regarding the a3 item ("fears/phobias") is significant, suggestingthat POCT practice after the experimental design could have reduced the intensity of fears/phobias among the subjects
in the SG. This result could be a starting point for a future study to evaluate the ways in which the practice of POCT
changes the intensity of fears/phobias. Thus, we could further explore whether the modification of this item in the SG
correlates with the decrease in the salivary cortisol level, an aspect that would strengthen the conclusion related to theeffect of prayer on the decrease in the intensity of fears/phobias.
Also, the change in SG in post-testing regarding the a14 item ("evaluation of anxious mood at the interview") isalso significant, suggesting a possible influence of the POCT practice, but a future study is needed to check whether thevariation of a14 could be biased by the evaluator [32].
Hypotheses 2, 3, and 4 - POCT practice every day, 30 minutes daily, for 8 weeks:- significantly reduces the GAD 7 score, obtained through self-assessment;- significantly reduces subjectively perceived stress on the Visual Analogue Scale of Perceived Stress, obtained
through self-assessment. The hypothesis was not confirmed;- significantly reduces the Global Score on the perceived stress scale (PSS-10), applied by the evaluator.
Result: working hypotheses 2, 3 and 4 were invalidated.
Hypothesis 5: Practicing POCT every day, 30 minutes daily, for 8 weeks strengthens coping mechanisms andincreases self-reported COPE global score.
Result: Intra-group analysis (Wilcoxon test) for CG and SG did not reveal statistically significant differences
post-testing, compared to pre-testing. Inter-group analysis (Mann-Whitney test for independent samples) revealed nostatistically significant differences in post-testing COPE global scores.
However, the means of some coping mechanisms were significantly higher in SG compared to CG. Increased
averages in e act (“active approach to coping”) and e res (“control/unwillingness”) – among SG subjects at pre-testingstem from subjects being faithful, preoccupied with the Christian S/R sphere. (It remains to investigate the fact, that at
the end of the intervention, the difference between the average values for e act corresponding to the two groups
decreases, a dynamic similar to the evolution of the subjects - in the pre and post-testing interval - discussed in relation
to the evolution of anxiety evaluated with Hamilton scale.)
The increased averages among SG subjects for e pos (“positive interpretation and growth”) and e rel (“religious
approach/religious coping”) in post-testing compared to CG subjects indicate a possible influence of POCT practice.
Having no significant inter-group differences, in post-testing, these increased averages do not significantly change thefinal conclusion but highlight the dynamics of SG in the experimental interval, on the two coping mechanisms. We cannot
consider this change significant because the global scores did not significantly differ statistically, nor did the two items
Fiziologia - Physiology • 2023.2(105)vary significantly within the SG, but this relevant aspect can represent a starting point for future research. POCT practiceis expected to strengthen the belief-based coping mechanism and, along with it, the positive interpretation mechanism.
The experimental plan in this study did not reveal a significant influence of the POCT practice on the global scoreon the COPE scale, but it was revealed that the SG subjects have, in post-testing, higher averages on two coping
strategies, the positive interpretation and the religious approach respectively.
The results that we may consider as the most important, (in relation to the significant statistical differences andthe number of subjects in the experimental group, n=17) refer to a3 ("fears/phobias") on the Hamilton scale, e pos(corresponding to the coping mechanism "positive interpretation" from COPE) and e rel (item for "religious approach"from COPE), for SG subjects, post-testing, even if the sample is small, as such a result could show the size of theimportant effect [149].
Further research could examine whether the practice of POCT strengthens coping strategies based on positive
interpretation and religious approach and causes a decrease in anxiety.
REFERENCES[2]. Andersson, C., Mellner, C., Lilliengren, P. et al: Cultivating Compassion and Reducing Stress and Mental
Ill-Health in Employees—A Randomized Controlled Study. Front. Psychol. 2022;12:748140. doi:
10.3389/fpsyg.2021.748140.
[3]. Bamber, M.D., Schneider, J.K.: Mindfulness-based meditation to decrease stress and anxiety in college
students: A narrative synthesis of the research. Educational Research Review. 2016;18:1-32.
[21]. Bandelow, B., Michaelis, S., Wedekind, D.: Treatment of anxiety disorders. Dialogues Clin Neurosci.
2017 Jun;19(2):93-107. doi: 10.31887/DCNS.2017.19.2/bbandelow. PMID: 28867934; PMCID:
PMC5573566.
[17]. Beck, T.A., David, D. (coord.): SEC (Sistemul de evaluare clinică). Cluj-Napoca, UniversitateaBabeș-Bolyai, Institutul de studii avansate de psihoterapie și sănătate mentală aplicată, 2007:3.
[30]. Bieling, P.J., Antony, M.M., Swinson, R.P.: The State-Trait Anxiety Inventory, Trait version: structure andcontent re-examined. Behav Res Ther. 1998 Jul-Aug;36(7-8):777-88. doi: 10.1016/s0005-7967(98)00023-0.
PMID: 9682533.
[8]. Bodini, L., Bonetto, C., Cheli, S. et al: Effectiveness of a Mindful Compassion Care Program in reducing
burnout and psychological distress amonGSt frontline hospital nurses during the COVID-19 pandemic: astudy
protocolforarandomized
controlled
trial. Trials 2022;23:734.
https://doi.org/10.1186/s13063-022-06666-2.
[22]. Boelens, P.A., Reeves, R,R., Replogle, W.H. et al: A randomized trial of the effect of prayer on
depression and anxiety. Int J Psychiatry Med. 2009;39(4):377-92. doi: 10.2190/PM.39.4.c. PMID: 20391859.
[10]. Bruce, M.A., Skrine Jeffers, K., King R.J. et al: Contemplative Practices: A Strategy to Improve Healthand Reduce Disparities. Int J Environ Res Public Health. 2018 Oct 15;15(10):2253. doi:
10.3390/ijerph15102253. PMID: 30326604; PMCID: PMC6210378.
[19]. Carver, C.S., Scheier, M.F., Weintraub, J.K.: Assessing Coping Strategies: A Theoretically BasedApproach.
Journalof
Personality
andSocial
Psychology
1989,
56,
267-283.
http://dx.doi.org/10.1037/0022-3514.56.2.267.
[29]. Clements, A.D., Ermakova, A.V.: Surrender to God and stress: A possible link between religiosity andhealth. Psychology
ofReligionandSpirituality.
2012;4(2):93–
107. https://doi.org/10.1037/a0025109.
[18]. Cohen, S., Kamarck, T., Mermelstein, R.: A Global Measure of Perceived Stress, Journal of Health andSocial Behavior 1983; Vol. 24, No. 4, 385-396.
[32]. Crasovan, D.: Bazele psihologiei experimentale. Timișoara, Eurostampa, 2010.
[26]. Elmholdt, E.M, Skewes, J., Dietz, M. et al: Reduced Pain Sensation and Reduced BOLD Signal in
Parietofrontal Networks during Religious Prayer. Front. Hum. Neurosci. 2017;11:337. doi:95

10.3389/fnhum.2017.00337.
[6]. Eriksson, T., Germundsjö, L., Åström, E. et al: Mindful Self-Compassion Training Reduces Stress andBurnout Symptoms Among Practicing Psychologists: A Randomized Controlled Trial of a Brief Web-Based
Intervention. Front Psychol. 2018 Nov 27;9:2340. doi: 10.3389/fpsyg.2018.02340. PMID: 30538656; PMCID:
PMC6277494.
[14]. Gu, J., Strauss, C., Bond, R. et al: How do mindfulness-based cognitive therapy and mindfulness-based
stress reduction improve mental health and wellbeing? A systematic review and meta-analysis of mediation
studies. Clinical Psychology Review. 2015;37:1-12. https://doi.org/10.1016/j.cpr.2015.01.006.
[25]. Harris, J.I., Erbes, C.R., Engdahl, B.E. et al: Coping functions of prayer and posttraumatic
growth. International Journal for the Psychology of Religion. 2010;20(1):26–
38. https://doi.org/10.1080/10508610903418103.
[15]. Hathaisaard, C., Wannarit, K., Pattanaseri, K: Mindfulness-based interventions reducing and preventing
stress and burnout in medical students: A systematic review and meta-analysis. Asian Journal of Psychiatry.
2022;69:102997. doi: 10.1016/j.ajp.2021.102997.
[1]. Hofmann, S.G., Sawyer, A.T., Witt, A.A. et al: The effect of mindfulness-based therapy on anxiety anddepression: A meta-analytic review. J Consult Clin Psychol. 2010 Apr;78(2):169-83. doi: 10.1037/a0018555.
PMID: 20350028; PMCID: PMC2848393.
[11]. Hofmann, S.G., Gómez, A.F: Mindfulness-Based Interventions for Anxiety and Depression. Psychiatr
Clin North Am. 2017 Dec;40(4):739-749. doi: 10.1016/j.psc.2017.08.008. Epub 2017 Sep 18. PMID:
29080597; PMCID: PMC5679245.
[4]. Hoge, E.A., Bui, E., Mete, M. et al: Mindfulness-Based Stress Reduction vs Escitalopram for theTreatment of Adults With Anxiety Disorders: A Randomized Clinical Trial.
JAMA
Psychiatry. 2023;80(1):13–21. doi:10.1001/jamapsychiatry.2022.3679.
[13]. Khoury, B., Lecomte, T., Fortin, G. et al: Mindfulness-based therapy: A comprehensive meta-analysis.
Clinical Psychology Review. 2013;33(6):763-771. https://doi.org/10.1016/j.cpr.2013.05.005.
[7]. Kotera, Y., Young, H., Maybury, S. et al: Mediation of Self-Compassion on Pathways from Stress to
Psychopathologies among Japanese Workers. International Journal of Environmental
Research and Public Health. 2022;19(19):12423. https://doi.org/10.3390/ijerph191912423.
[5]. O’Driscoll, D., McAleese, M.: The protective role of self-compassion on test anxiety among
adolescents. Pastoral Care in Education. 2022. doi: 10.1080/02643944.2022.2054021.
[23]. Pargament, K.I., Ano, G.G., Wachholtz, A.B. (2005). The religious dimension of coping: Advances intheory, research, and practice. In Paloutzian, R.F., Park, C. (Eds.), Handbook of the psychology of religion
and spirituality. New York, NY: Guilford Press; 2005, pp. 479–495.
[20]. Popa, M. Statistică psihologică și prelucrarea computerizată a datelor. Noţiuni statistice fundamentale.
Statistici descriptive. Universitatea din București, Credis, 2008.
[27]. Rzeszutek, M., Oniszczenko, W., Kwiatkowska, B.: Stress coping strategies, spirituality, social support
and posttraumatic growth in a Polish sample of rheumatoid arthritis patients. Psychol Health Med.
2017;22(9):1082-1088. doi: 10.1080/13548506.2017.1280174. Epub 2017 Jan 12. PMID: 28081614.
[24]. Schjødt, U.: Homeostasis and religious behavior, Journal of Cognition and Culture, 2007;3:313-340.
[28]. Schroder, D.M.: Presidential Address: Can prayer help surgery? Am J Surg. 2011 Mar;201(3):275-8. doi:
10.1016/j.amjsurg.2010.08.029. PMID: 21367363.
[9]. Sharma, M., Rush, S.E: Mindfulness-based stress reduction as a stress management intervention for
healthy individuals: a systematic review. J Evid Based Complementary Altern Med. 2014 Oct;19(4):271-86.
doi: 10.1177/2156587214543143. Epub 2014 Jul 22. PMID: 25053754.

Fiziologia - Physiology • 2023.2(105)[16]. Sosa-Cordobés, E., Ramos-Pichardo, J.D., Sánchez-Ramos, J.L. et al: How Effective Are
Mindfulness-Based Interventions for Reducing Stress and Weight? A Systematic Review andMeta-Analysis. International Journal of Environmental Research and Public
Health. 2023; 20(1):446. https://doi.org/10.3390/ijerph20010446.
[12]. Toussaint, L., Nguyen, Q.A., Roettger, C. et al: Effectiveness of Progressive Muscle Relaxation, Deep
Breathing, and Guided Imagery in Promoting Psychological and Physiological States of Relaxation. Evid
Based Complement Alternat Med. 2021 Jul 2;2021:5924040. doi: 10.1155/2021/5924040. PMID: 34306146;
PMCID: PMC8272667.
[31]. Widiger, T.A., Oltmanns, J.R.: Neuroticism is a fundamental domain of personality with enormous publichealth implications. World Psychiatry. 2017 Jun;16(2):144-145. doi: 10.1002/wps.20411. PMID: 28498583;
PMCID: PMC5428182.

EVALUAREA POSIBILELOR MODIFICĂRI PSIHOMETRICE
PRACTICAREA RUGĂCIUNII ÎN TRADIȚIA CREȘTINĂ ORTODOXĂ

INDUSEDE

REZUMAT
Studiul de față investighează posibilele modificări psihologice în sfera stresului/anxietate induse de practica rugăciunii (întradiția creștin ortodoxă) - POCT, după o practică individuală de 30 de minute zilnic, timp de 8 săptămâni. Participanții
(n=34) au fost împărțiți în două grupuri (Grupul de studiu, n=16, vârsta medie 39,2 ani și Grupul de control n=17, vârsta
medie 38,6 ani). Subiecții din Grupul de studiu - creștini ortodocși credincioși, fără practicarea rugăciunii zilnice, au fost
instruiți, într-o sesiune de 3 ore, în vederea particularităților psiho-emoționale, cognitive, spirituale și comportamentale ale
POCT. Nu s-a efectuat nicio intervenție asupra subiecților din Grupul de control. Înainte și după intervalul de 8 săptămâni
de practică zilnică a POCT, de 30 de minute, au fost aplicate cinci instrumente psihometrice. Ulterior, au fost analizate
comparativ rezultatele obținute la evaluările pre și post-test intra și inter-grupuri. Rezultatele obținute au arătat că practica
POCT, conform schemei experimentale, nu a produs diferențe semnificative statistic între scorurile globale la scala deanxietate Hamilton, ambele înregistrate la SG în raport cu CG, pre și post-test. Pentru ambele grupe de subiecți s-aînregistrat o scădere a scorului scalei Hamilton, ceea ce sugerează că, în cazul subiecților din SG, nu se poate afirma cu
certitudine că scăderea scorului Hamilton s-a produs ca urmare a practicii POCT. Dimpotrivă, diferențele semnificative dinpunct de vedere statistic la subiecții CG, post-test, în ceea ce privește un9 item ("simptome cardiovasculare"), sugerează
că alți factori ar fi putut contribui la îmbunătățirea răspunsurilor subiecților CG, ceea ce impune introducerea unor condiții
suplimentare pentru o administrare mai atentă a practicii POCT în rândul subiecților SG pe parcursul intervalului
experimental. Cu toate acestea, s-au obținut unele rezultate semnificative, în ceea ce privește subiecții SG, la post-test,
în ceea ce privește itemul a3 ("temeri/fobii"), din scala Hamilton, sugerând că practica POCT conform planului
experimental ar fi putut reduce intensitatea temerilor/fobiilor în rândul subiecților din SG. De asemenea, modificareaapărută în SG post-test, în ceea ce privește itemul a14 ("aprecierea stării de anxietate la interviu") este semnificativă,sugerând o posibilă influență a practicii POCT. Remarcăm, de asemenea, în lista de rezultate, mediile unor mecanismede coping, care au fost semnificativ mai mari în SG comparativ cu CG. Acestea sunt e act ("coping activ") și e res("abținere/control/nevoință") - în rândul subiecților SG la pre-test, și e pos ("interpretare pozitivă și creștere") și e rel("abordare religioasă/ coping religios") la post-test comparativ cu subiecții CG, indicând o posibilă influență a practicii
POCT. Rezultatele necesită studii suplimentare care să coreleze mai îndeaproape modificările acestor itemi, dar și ale
altora psihometrici, cu cele de natură fiziologică.
Cuvinte cheie: rugăciune creștin ortodoxă, schimbări psihologice în sfera stresului/anxietate, strategii de coping,frici/fobii, coping religios.

97