FIZIOLOGIA
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FOUNDING EDITORCHIEF EDITOR
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 • 2024.1(106)CUPRINSROLURILE INSULINEI ÎN HOMEOSTAZIA GLUCOZEI LA NIVEL NEURONAL ȘI ASTROCITAR .................................... 4
Matei Daniela Mariana
ALGORITM SIMPLU ÎN MATLAB PENTRU INTERPRETAREA ANTIBIOGRAMELOR - REZULTATE PRELIMINARE......... 20
Gabriel Vinter, Denisa Alexandra Vereș, Florin Muselin, Beatrice Zembrod, Daniela Crînsic, Albert Ioiart, Alexandru Tîrziu,
Mihai Sărăndan, Damian Olei, Daniel Bonciog, David Comaniciu
POSIBILE MODIFICĂRI ELECTROFIZIOLOGICE INDUSE DE PRACTICAREA RUGĂCIUNII ÎN TRADIȚIA CREȘTINĂ
ORTODOXĂ ...........................................................................................................................................................27
Adrian Sorin Mihalache, Alexandru Paslaru, Marian Poboroniuc, Dănuț-Constantin Irimia, Iulian Stoleriu, Leon Zăgrean
MONOTERAPIA ANTIAGREGANTA VERSUS TERAPIA COMBINATA CU HEPARINA CU GREUTATE MOLECULARAMICA IN REDUCEREA RISCULUI DE APARITIE A PREECLAMPSIEI SI A RESTRICTIEI DE CRESTERE FETALAINTRAUTERINA ........................................................................................................................................ 52
Anghel Savciu Loredana, Patru Ciprian Laurentiu, Pana Razvan Cosmin, Balaceanu Dobromir Cristina,Cernea Nicolae
IMPLICAREA TROMBOFILIEI EREDITARE IN RESTRICTIA DE CRESTERE INTRAUTERINA DE ETIOLOGIENECUNOSCUTA ....................................................................................................................................... 57
Anghel Savciu Loredana, Patru Ciprian Laurentiu, Popa Adelina, Balaceanu Dobromir Cristina, Cernea NicolaeRĂSPUNSUL IMUN ÎN ELECTROCHIMIOTERAPIE. SCURTĂ SINTEZĂ........................................................................................61
Mircea Bogdan Matei, Christien Oktaviani Matei, Ali Sibel, Leon Zăgrean, Mihaela Georgeta Moisescu
REACTIVITATEA EEG LA STIMULI EXTERNI ÎN STĂRI DE CONȘTIENȚĂ ALTERATĂ…………………………………………69
Alexandru-Cătălin Pâslaru, Vlad-Petru Morozan, Andrei Bordeianu, Alexandra Mocanu, Mihai Stancu, Ana-Maria Zăgrean,
Mihai Moldovan, Leon Zăgrean

CONTENTS
THE ROLES OF INSULIN IN GLUCOSE HOMEOSTASIS AT THE NEURONAL AND ASTROCYTARY LEVEL .................... 4
Matei Daniela Mariana
SIMPLE ALGORITHM IN MATLAB FOR THE INTERPRETATION OF ANTIBIOGRAMS – PRELIMINARY RESULTS .......... 20
Gabriel Vinter, Denisa Alexandra Vereș, Florin Muselin, Beatrice Zembrod, Daniela Crînsic, Albert Ioiart, Alexandru Tîrziu,
Mihai Sărăndan, Damian Olei, Daniel Bonciog, David Comaniciu
POSSIBLE ELECTROPHYSIOLOGICAL CHANGES INDUCED BY THE PRACTICE OF PRAYER IN ORTHODOXCHRISTIAN TRADITION ............................................................................................................................... 27
Adrian Sorin Mihalache, Alexandru Paslaru, Marian Poboroniuc, Dănuț-Constantin Irimia, Iulian Stoleriu, Leon Zăgrean
ANTIPLATELET MONOTHERAPY VERSUS ANTIPLATELET AND ANTICOAGULANT THERAPY IN REDUCING THE RISKOF PREECLAMPSIA AND FETAL INTRAUTERINE GROWTH RESTRICTION........................................................... 52
Anghel Savciu Loredana, Patru Ciprian Laurentiu, Pana Razvan Cosmin, Balaceanu Dobromir Cristina,Cernea Nicolae
IMPLICATIONS OF HEREDITARY THROMBOPHILIA IN DEVELOPMENT OF FETAL INTRAUTERINE GROWTH
RESTRICTION OF UNKNOWN ORIGIN............................................................................................................ 57
Anghel Savciu Loredana, Patru Ciprian Laurentiu, Popa Adelina, Balaceanu Dobromir Cristina, Cernea Nicolae
A FOCUSED REVIEW OF THE IMMUNE RESPONSE IN ELECTROCHEMOTHERAPY..........................................................61
Mircea Bogdan Matei, Christien Oktaviani Matei, Ali Sibel, Leon Zăgrean, Mihaela Georgeta Moisescu
EEG REACTIVITY TO EXTERNAL STIMULI IN THE IMPAIRED CONSCIOUSNESS STATES………………………………69
Alexandru-Cătălin Pâslaru, Vlad-Petru Morozan, Andrei Bordeianu, Alexandra Mocanu, Mihai Stancu, Ana-Maria Zăgrean,
Mihai Moldovan, Leon Zăgrean
Fiziologia - Physiology • 2024.1(106)34Fiziologia - Physiology • 2024.1(106)THE ROLES OF INSULIN IN GLUCOSE HOMEOSTASIS AT THE NEURONAL AND ASTROCYTARYLEVELMATEI DANIELA MARIANA
University of Oradea, Faculty of Medicine and Pharmacy, Department of Preclinical Disciplines
ABSTRACTThe identification of insulin receptors on the plasma membrane of brain cells is unequivocal evidence that insulin acts in the brain,
performing functions related to brain energy metabolism, but not only. Insulin could facilitate the coupling of cerebral blood flow with the levelof neuronal activity, the expression of some neuronal and astrocytic glucose transporters, the uptake of glucose and its storage as glycogen,and the normal functioning of mitochondria in these cells. The aim of this work is to analyze aspects regarding the crossing of the blood brain
barrier by glucose and insulin, the energy substrate used by neurons and astrocytes in resting and activated conditions, the effect of blood
glucose concentration on glucose transporters, insulin, the insulin receptor and energy metabolism of the brain. From this perspective,
disturbances observed in diseases associated with brain insulin resistance, such as type 2 diabetes mellitus and Alzheimer`s disease, will
be highlighted.
Keywords: glucose transporters, insulin, astrocytes, energy metabolism, diabetes mellitus.

INTRODUCTION
Glial cells (GCs) represent a heterogeneous cell population in the nervous system, which, in addition to the structuralrole of supporting nerve cells and fibers, influence their metabolism and functioning, creating a microenvironment that allows
the normal development of neuronal activity. A type of GC is the astrocyte. It participates in the formation of the blood-brain
barrier (BBB). This barrier consists of tightly connected endothelial cells, the basement membrane of cerebral capillaries,
pericytes and the foot processes of astrocytes [1]. Being located between blood vessels and neurons, astrocytes couple thedelivery of blood-supplied nutrients with the neurons' required energy level [2-5]. One of the nutrients is glucose, which is theprincipal fuel needed by the brain`s energy metabolism [2, 4, 6-8]. Additionally, the brain can use lactate [4, 6, 9-11], mainly
when its blood level is elevated, as in strenuous exercise, ketone bodies [4, 6, 9-11] derived from fat catabolism, stimulated
during long-term starvation, and fatty acids [9,11].
Under normal living conditions, the concentration of glucose in the blood is tightly regulated to enable neurons to becontinuously supplied with an adequate amount of glucose per unit time. Too much glucose or depriving the nervous systemof glucose adversely affects neurons and GCs [12]. Hyperglycemia causes glucose neurotoxicity [13-15], either directly
damaging neurons or indirectly via GCs [13], whereas hypoglycemia ultimately leads to neuronal death.
ENDOTHELIAL, NEURONAL AND ASTROCYTIC GLUCOSE TRANSPORTERS
Meeting the energy demands of neurons requires the passage of glucose into the brain parenchyma and close
cooperation between neurons and astrocytes [4, 11, 16, 17]. Glucose is absorbed from the bloodstream by the endothelial cellsof the brain`s microvessels. Facilitated diffusion occurs via the glucose transporter (GLUT) 1 which has been identified inendothelial cells of the human BBB and other mammalian species [18-20]. GLUT1 has an essential role in providing glucose
to the brain, even though it is not the only type of GLUT expressed by the endothelium [20-23]. GLUT1 is located both in theluminal and abluminal membrane of vascular endothelial cells and in intracellular vesicles [20, 22, 24]. The transporter isasymmetrically distributed between these compartments in a proportion that varies with the species analyzed and the method
used for its identification [7, 20, 22]. GLUT1 from the intracellular pool could be recruited to the luminal surface of endothelial
cells to increase glucose uptake when neuronal energy needs are high [24].
Normally, the concentration of glucose in the blood perfusing the brain is higher than its concentration in the brain`sinterstitial fluid [22, 25]. This concentration gradient causes the movement of glucose to the brain cells [25]. It is assumed thatthe largest fraction of blood glucose is taken up by astrocytes and only a small amount reaches the interstitial space directly[22]. From the interstitial fluid of the brain, glucose is transported into neurons by GLUT3 [7, 21, 22, 24], the main GLUT
expressed by these cells [22, 26, 27]. GLUT3 has a low Michaelis-Menten constant that facilitates neuronal glucose uptake
even when the extracellular glucose concentration is reduced [7, 22]. Another isoform of GLUT, namely GLUT4, has been
identified in neuronal populations associated with high energy expenditure [26, 27] and cognitive behavior [28]. Its distribu tion
appears to be related to that of the insulin receptor (IR) [7, 22, 26, 29], suggesting that GLUT4 is an insulin-sensitive transporter[7, 28-30] that is regulated by insulin [7, 26, 28]. GLUT4 provides neurons with an extra amount of glucose during periods ofhigh energy demand, supplementing the glucose taken up by GLUT3 [22, 26, 28], whose expression is considered not to besignificantly influenced by neuronal activity [22, 31], although in a study [32] on cultured cortical and hippocampal neurons,
synaptic activation was proven to increase GLUT3 translocation to the neuronal plasma membrane and glucose transport into
neurons [32]. Increased synaptic transmission has also been shown to induce insertion of GLUT4 into the presynaptic plasma
membrane [29, 31, 33], and ablation of GLUT4 resulted in cessation of synaptic vesicle recycling during sustained neuronal
Fiziologia - Physiology • 2024.1(106)5firing [31]. GLUT4 has been identified in the rat hippocampus [26, 29, 31]. Impaired translocation of GLUT4 to the plasma
membrane of neurons might be a consequence of hippocampal insulin resistance and a cause of cognitive dysfunctions intype 2 diabetes mellitus (DM) and Alzheimer`s disease (AD) [26, 28, 34].
GLUT1 and GLUT3 have a heterogeneous distribution in the rat brain [7]. A direct correlation has been found between
resting blood flow in different brain areas, capillary density in those areas, and local density of GLUTs [7]. GLUT1 and GLUT3
density also positively correlates with local cerebral glucose utilization [7, 35] and conversely, glucose utilization in a particularpart of the brain influences the density of the two GLUTs in that brain structure [7]. Local glucose utilization was unchanged inacute hyperglycemia, but increased in chronic hyperglycemia (3 weeks) experimentally induced in rats by intraperitoneal
injection of streptozotocin compared with normoglycemic control animals [35].
In conditions of chronic hypoglycemia or hyperglycemia, GLUT density can change [7, 15]. Studies in rat brain
evidenced an increase in GLUT3 density in hypoglycemia [7, 36], but no change in GLUT1 density [7], and a moderate
downregulation of GLUT1 in hyperglycemia [7, 35]. Other studies observed an upregulation of GLUT1 and increased
expression of GLUT1 mRNA in chronic hypoglycemia in rats [19, 37], without changing the level of GLUT3 mRNA or GLUT3
protein [19]. The level of glycemia may be the main factor determining the expression of GLUT1 mRNA and GLUT3 mRNAand the level of GLUT1 and GLUT3 proteins in the brain [15]. It was unveiled that the expression of GLUT1 and GLUT3 wasmore reduced in diabetic rats compared to normal control rats and this was not due to lower cerebral microvessel density indiabetic rats [15]. Decreasing blood glucose by insulin administration may correct and reverse abnormal GLUTs expression in
the brain [15].
The downregulation of GLUT1 and GLUT3 expression in chronic hyperglycemia might be an adaptive and protective
reaction of the BBB endothelium and neurons to prevent their damage by intracellular glucose accumulation [15, 21], while thestimulation of the gene expression of the two GLUTs in chronic hypoglycemia [21] and upregulation of GLUT1 and GLUT3
have the role to enhance glucose uptake, providing the energy substrate necessary for cell metabolism [15]. Although it has
been stated in many studies that glucose transport via GLUT1 and GLUT3 is insulin-independent [13, 38-41] and the gene
expression of these two brain GLUTs is not regulated by insulin [15], others consider that GLUT3 is an insulin-sensitivetransporter whose activity is modulated by insulin [30, 42]. Clarification of this aspect through future research is crucial tounderstanding the role of insulin in glucose delivery to the brain cells.
Endothelial cells of the BBB directly transfer glucose to astrocytes. GLUTs expressed on the pericapillary processesof astrocytes carry glucose across the cell membrane. Morgello et al. [18] identified by immunoelectron microscopy thepresence of GLUT1 in the foot processes of astrocytes surrounding gray matter blood vessels and synapses in the humanbrain [18]. As a peculiarity, GLUT1 of perivascular astrocytic endfeet is not structurally identical to GLUT1 of BBB endothelial
cells. The latter is glycosylated and has a higher molecular weight (55 KDa versus 45 KDa) [7, 18-20]. GLUT1 is more
expressed in astrocytes than in BBB endothelium [19, 43]. In addition to the high expression of GLUT1, human astrocytes
significantly express GLUT3, very little GLUT2, and only traces of GLUT4 [22, 44]. GLUT1 and GLUT3 are insulin-independenttransporters [39, 40, 44], whereas GLUT4 is insulin-dependent [22, 44]. However, in rats, immunoflourescence analysis ofcortical astrocyte culture and astrocytes co-cultured with neurons obtained from the cerebellum and cerebral cortex did not
identify GLUT3, but only GLUT1 [45].
When GLUT mRNA content was compared between human astrocytes, myotubes and adipocytes, the determination
showed that the amount of GLUT1 mRNA and GLUT2 mRNA was higher in astrocytes than in myotubes or adipocytes, while
GLUT3 mRNA content was found at comparable values to those in myotubes and adipocytes [44]. Human astrocytic GLUT3
has a high affinity for glucose and was detected by Heni and co-workers even under basal conditions [44]. Therefore, they
considered that GLUT3 might have an important role in astrocytic glucose uptake [44].
While ablation of GLUT1 in BBB endothelial cells causes changes in brain vasculature and BBB integrity [46, 47],
ablation of astrocyte GLUT1 in mice was not followed by altered vascular morphology nor leakage of serum proteins(Immunoglobulin G and fibrin) from blood vessels, suggesting preservation of BBB integrity [46]. Astrocytes with GLUT1
ablation present structural changes characterized by increased branching of the processes, but without their elongation [46].
Hyperglycemia can promote structural alterations in astrocytes as well. Exposure to high glucose concentration (25 mM) of
primary cultured astrocytes was followed by irreversible inhibition of proliferation and modification of their morphology, with aflat appearance and fewer processes compared to astrocytes cultured in normal glucose medium [13]. Their viability was alsosignificantly reduced by hyperglycemic conditions compared to culture in a normoglycemic medium (5.5 mM) [13]. As aconsequence, in DM, inhibition of astrocyte proliferation is a disadvantage in the reconstruction process after brain damage[13]. Insulin was proved to stimulate the proliferation of cultured human astrocytes [44].
Research in mice overexpressing amyloid β-peptide (Aβ) precursor protein showed that GLUT1 deletion in theendothelium results in diminished BBB glucose transport, BBB disruption, capillary degeneration, reduced cerebral
microvasculature and perfusion, and Aβ accumulation that contributed to neuronal dysfunction and neuronal loss [47]. Reduced
expression of GLUT1 and GLUT3 in the cerebral cortex and other brain regions has been observed in AD patients, leading tolower glucose uptake and brain energy metabolism [22, 23, 30, 48] and, eventually, to neuronal degeneration [27, 30]. The
decrease in the concentration of GLUT3 in the cortex of these subjects was greater than that of GLUT1 and than the loss of

6

Fiziologia - Physiology • 2024.1(106)synapses [48].

ENDOTHELIAL, NEURONAL AND ASTROCYTIC INSULIN RECEPTOR
In addition to GLUTs, the cell membrane of astrocytes and neurons exhibits IR [39]. Expression of IR on mouse
astrocytes was demonstrated by Fernandez et al. [3], who, using IR immunogold labeling combined with electron microscopy,
located them in astrocytic endfeet ensheating the endothelial cells of the BBB [3]. The authors also uncovered that theinteraction of insulin with its receptor in astrocytes is responsible for coupling brain glucose uptake with blood flow to su stain
brain activity and that brain insulin resistance uncouples this process [3]. Immunoreactive IR deposits were also found in theendothelial cells of BBB capillaries [3]. Leclerc et al. [49] revealed that in humans and animals (murines), cerebral IRs,
especially the isoform B of IR, are more concentrated in microvessels than in the brain parenchyma [49].
Human astrocytes express IRs as well, quantitatively similar to myotubes [44]. Approximately two-thirds of humanastrocyte IR is in the isoform A and one-third in the isoform B [44]. According to other determinations, the predominant isoform
of human astrocytic IR is isoform B [39, 50, 51]. Studies in mice with astrocyte IR ablation (GFAP-IR KO mice) highlighted therole of astrocyte IR and insulin signaling for adequate glucose and insulin transport into the brain [43]. Neurons express only
the isoform A of IR [28, 39, 50-52], characterized by a high binding affinity for insulin and the ability to be stimulated by low
concentrations of insulin-like growth factor II (IGF-II) [28, 52]. Neurons have a higher IR density compared to astrocytes [52,53]. Immunochemical methods demonstrated that in the rat brain, neuronal IRs are concentrated at synapses [54], both inpresynaptic and postsynaptic plasma membranes [51], and IR is a component of postsynaptic density fractions [54]. IR is also
localized in nonsynaptic areas of dendrites [54].
Although there are still uncertainties regarding the pathway through which circulating insulin reaches the brain
interstitial fluid, probably either crossing the BBB or via the cerebrospinal fluid [38-40, 55], Gray and co-workers [56] showedthat the passage of insulin from the bloodstream to astrocytes occurs by crossing the endothelial cells of the BBB [56]. The
process of transcytosis has been evidenced in cultured human and rat brain endothelial cells, uncovering that at physiologicalconcentrations of insulin, its uptake is an IR-specific, vesicle-mediated transport [56]. Anyway, tight junctions between BBB
endothelial cells prohibit paracellular transport of insulin [38], except in non-BBB zones of the brain with fenestrated capillaries,
such as those in the circumventricular organs [39, 55]. Furthermore, the authors [56] observed that insulin transcytosis
increased when astrocytes were stimulated with L-glutamate and decreased by inhibiting nitric oxide synthase (NOS), fromwhich it follows that astrocytes could regulate the passage of insulin across the BBB [56]. In IR-deficient astrocyte mice,
injection of digoxigenin-labeled insulin into the carotid artery was not followed by its passage into the brain parenchyma, but
was retained in the BBB endothelium [3].
A previous study on microvessels obtained from autopsied brains showed that microvessels bind insulin with highaffinity, internalize it, and transport it into the medium [57]. However, it has been demonstrated in two models, one genetic(mice with endothelial-specific loss of the signaling-related IR) and one pharmacological (utilization of a selective antagonist of
IR – S961), that insulin can also be transported across the BBB in vivo, independent of endothelial cell signaling-related IRs,
albeit the amount of insulin bound to the endothelium was lower [58]. This research [58] indicates that there might be otherprotein or proteins involved in the transport of insulin across the BBB and suggests that signaling-related IR is not the only
mechanism for insulin to pass through the BBB into the brain interstitium [58]. Consequently, it is incompletely understoodwhether IR at the BBB endothelium only binds insulin and acts as a receptor, or whether it also mediates insulin transport
through the cytoplasm of endothelial cells [49, 58]. Nonetheless, an animal study provided evidence that IR at the BBB acts as
a receptor, not as a transporter, and circulating insulin interacts with IR at the luminal membrane of BBB vessels [49].
The source of insulin in the brain is most likely peripheral insulin secreted by the pancreas [39, 40, 56, 59]. Recently,
it has been indicated that insulin is also produced by the brain [22, 40, 60, 61]. Insulin influences the BBB transport of othermolecules, as well as its own transport rate [25, 59]. There are differences in insulin uptake between brain regions and between
the brain and spinal cord [25]. The rate of insulin transport across the BBB is influenced by many factors, including blood
glucose level [25].
The rate of insulin transport into the brain is altered in DM [59, 62] and AD [25, 49, 59]. Studies on the brain ofpatients who died of AD revealed that, compared to age-matched controls, IR and IGF-I receptor expression was decreased
in several brain structures [39, 63, 64]. Moreover, this abnormality has been associated with impaired insulin and IGF-I and
IGF-II signaling mechanism, decreased IR substrate mRNA level, and expression of genes encoding insulin, IGF-I and IGF-II[63]. Research in human and animal models of AD showed that there is an alteration in vascular IR-mediated response in thisdisease and that defects and low level of endothelial IR contribute to insulin resistance in AD [49].
GLYCATION OF INSULIN AND THE INSULIN RECEPTOR
Under hyperglycemic conditions, both insulin [65, 66] and IR can be glycated [65]. This interferes with the binding of
insulin to its receptor and may lead to insulin resistance [65-68]. IR shares identical amino acid sequences with insulin andbinds glucose like insulin [65]. Several glucose binding sites have been identified, located on both the A and B chains of insulin[65, 67]. The experiment conducted by Rhinesmith et al. [65], showed that insulin glycation is a two-step process, with an initial,
Fiziologia - Physiology • 2024.1(106)7very rapid glycation in the first few hours after exposure to high glucose levels (above 20 mM), followed by a slower glycation
over several days [65]. The same authors [65], working on IR-derived peptides, uncovered their rapid glycation [65].
Interestingly, the process was not observed for lower (5 mM) glucose concentrations, but only for hyperglycemic levels [65].
High glucose is associated with glycation of the insulin-binding insulin-like regions of the IR, and this, in addition toglycation of insulin itself, can cause in vivo a decrease in the binding affinity of insulin to its receptor and a significant reductionin IR activation [65], effects that have also been observed in other research [67, 68]. The molecular dynamics study of glyca ted
insulin showed that the glycation process of insulin results in conformational changes of the insulin B-chain, with the transition
from the open to the close conformation, the alteration in the secondary structure of insulin, the impairment of its binding to the
IR and signaling transduction [66]. IR glycation might have long-term consequences because the turnover of glycated IR isslower, of weeks or months [65]. Aside from insulin and IR, enzymes [68] and plasma proteins can be glycated [65, 67].
The presence of glycated insulin has been evidenced in human plasma from patients with type 2 DM and thatglycated insulin has a much lower ability to decrease glycemia in normal subjects compared to physiological concentrations of
native insulin [68]. The study carried out by Walke et al. [67], which aimed to evaluate the effect of insulin and glycated insulin
on both IR and receptor for advanced glycation end-products (RAGE) signaling in Chinese hamster ovary cells (CHO-IRGLUT4) showed that treatment of these cells with glycated insulin had several consequences, such as impaired insulin
signaling and translocation of GLUT4 to the cell membrane, reduced binding affinity of insulin to IR but increased binding
affinity to RAGE, activation of RAGE signaling, increased production of reactive oxygen species (ROS) and a higher level ofcell death compared to cells treated with nonglycated insulin [67]. These changes can ultimately determine increased insulin
resistance, decreased glucose uptake by cells, and cytotoxicity [67].
By stimulating ROS production and apoptosis, glycated insulin causes BBB damage, leading to a reduction in thenumber of astrocytes and alteration of tight junctions [67]. On the other hand, there is evidence that AGEs promote cerebral
small vessel dysfunction that affects cerebral perfusion and causes neuronal disturbances and cognitive alterations in type 2
DM patients [69, 70]. Hyperglycemia is associated with overproduction of AGEs which in turn stimulate ROS production byincreasing NADPH oxidase activity [71]. This enzyme is a considerable source of ROS (superoxide) in cerebral blood vessels,
neurons and GCs [72].
Brain insulin resistance has been shown in type 2 DM and AD [30, 39, 64, 73]. Type 2 DM is widely recognized as arisk factor for AD [14, 64, 73-75]. Peripheral insulin resistance in type 2 DM is also implicated in impaired BBB functionality [71].
Hyperinsulinemia causes downregulation of endothelial IR and decreased BBB permeability to insulin [71]. However, increased
BBB permeability was evidenced in a study of type 2 DM patients compared to age-matched healthy controls [74]. BBB
dysfunction was assessed by gadolinium magnetic resonance imaging [74].
Another element that is affected in type 2 DM and AD is the brain insulin-phosphoinositide 3-kinase (PI3K)-protein
kinase B (Akt) signaling pathway [63, 71, 75]. The level and activity of the components of this signaling pathway were
investigated in samples of frontal cortices obtained postmortem from patients with these diseases and controls [75]. They weresignificantly reduced in both type 2 DM and AD brains compared to the age-matched control brains [75]. In most cases, thedecrease was greater in brain samples from patients with type 2 DM than in those from patients with AD, and the greatest
reduction was noticed in the brains of patients who had both type 2 DM and AD [75]. Downregulation of components of theinsulin-PI3K-AKT signaling pathway in the brain of individuals with type 2 DM is a finding that supports the existence of brain
insulin resistance and that type 2 DM, through this alteration, may increase the risk of AD [64, 75].
NEUROVASCULAR COUPLING AND THE EFFECT OF INSULIN ON THIS PROCESS
Astrocytes have a very important role in coupling the level of neuronal activity with focal cerebral blood flow, the socalled neurovascular coupling or functional hyperemia [3-5, 12, 76, 77]. The aim of this process is to ensure that the brain
regions that are highly functionally active will receive a commensurate amount of oxygen and fuel when activated [77-79].
Neurovascular coupling requires precise coordination between neurons, astrocytes, and vascular wall cells [69, 79]. This
process is subject to change during development, correlated with the maturation of the anatomical elements that make up theneurovascular unit of the brain [76].
Several explanations have been proposed to decipher the underlying mechanism of neurovascular coupling [5, 76,80]. It was initially thought that the decrease in energy substrate and oxygen supply to the brain or the accumulation of was teproducts derived from neuronal metabolism causes vasodilation to improve blood flow and to compensate for the lack of ATP
and to remove byproducts and heat, but these mechanisms could not entirely explain the response of the vasculature to thechange in neuronal activity [5, 12, 76, 78]. Lin et al. [81], in research investigating activation-induced hyperemia by visual
stimulation in healthy human subjects, showed that the increase in cerebral blood flow triggered by neuronal activation waslarge and negatively correlated with the percentage change in cerebral metabolic rate of oxygen, instead, neuronal activation
caused a small increase in ATP production [81]. The authors found that there is a disproportion between the hyperemic
response and energy demand, the latter increasing much less than the hyperemic response, leading them to infer that theaugmentation in cerebral blood flow secondary to neuronal activation is not mediated by oxygen demand [81]. This observation
may also support the idea that neurovascular coupling could be regulated by a feedforward neurotransmitter-mediatedmechanism [12], not by a negative feedback mechanism driven by energy demand, because in the case of negative feedback
8

Fiziologia - Physiology • 2024.1(106)regulation, the increase in energy supply does not exceed the increase in energy consumption [12].
Information from studies investigating this issue has found that neurovascular coupling is very rapid, with the change
in blood flow occurring within less than a second [80, 82] or within a few seconds [77, 78, 83] of the onset of neuronal activation,
too early to allow the accumulation of byproducts, and encompassing areas from hundreds of microns to millimeters away fromthe center of the activated focus [80]. Since activation of both excitatory and inhibitory neurons in the brain causes increased
local blood flow [12, 69, 84, 85], it has been challenging to establish which type of neurons can trigger neurovascular coupling
and whether neurons signal directly to smooth muscle cells in the arteriolar wall to dilate or do so indirectly through other cell
types [12, 80]. One possibility is that neurons first signal to astrocytes and then these cells signal to smooth muscle cells torelax and alter blood flow [12, 78-80, 83, 86]. The other scenario is that neurons signal directly to microvessels [12, 70, 87].
There is evidence that, at least in the hippocampus, the connection between neurons and blood vessels is made directly byNO, released from active glutamatergic neurons, which diffuses into the blood vessels and causes them to dilate [70, 87].
The outer surface of capillaries and arterioles in the BBB is almost completely covered by perivascular astrocytic
endfeet, while the perisynaptic processes of astrocytes enwrap neuronal synapses. This architectural organization makes
astrocytes an ideal relay for transmitting signals related to changes in neuronal activity to blood vessels [5, 11, 76-78, 80].
In vivo and in vitro studies have shown that increase in astrocyte Ca2+ concentration as a result of neuronal activation
determines the release of a variety of astrocytic vasoactive agents that can trigger arteriolar relaxation and enhance cerebral
blood flow [4, 12, 78, 79]. In support of this observation is a study [86] performed on rat cortical slices showing that inhibitionof the astrocytic increase in Ca 2+ concentration mediated by glutamate release causes a decrease in activity-induced hyperemia[86].
Takano et al. [83] observed that cortical astrocytes in the somatosensory cortex of adult mice respond to photolysis
of caged Ca2+ in astrocytic processes covering the blood vessel wall by a marked increase in blood flow [83]. The latency ofthis response was only 1 - 2 seconds, and vasodilation was blocked by indomethacin and a cyclooxygenase-1 inhibitor SC560 [83]. Another in vivo study [82] turned out that in the primary somatosensory cortex of mice there is a subset of astrocytesthat present short-latency Ca2+ responses to sensory stimulation [82]. The peak of Ca2+ elevation was recorded within 0.5
seconds after stimulation [82]. This short latency is consistent with astrocyte modulation of the hyperemic response to neuronal
activation [82]. However, other experimental data collected from awake mice showed that functional hyperemia is a bimodal
phenomenon, with an early and a late phase [79]. The authors considered that astrocyte Ca2+ does not initiate or mediatefunctional hyperemia when neuronal activation is of short-lived (less than 5 seconds), but amplifies it when neuronal activation
is prolonged (30 seconds) [79]. Therefore, astrocytic Ca2+ might be involved in augmenting cerebral blood flow when neuronal
activation is sustained [79].
One hypothesis regarding local blood flow regulation by astrocytes posits that this occurs through activation of
metabotropic glutamate receptors on astrocyte foot processes ensheating synapses [4, 5, 69, 86]. These receptors bindglutamate released into the synaptic cleft during neuronal firing and cause an increase in intraastrocytic Ca 2+ concentration,
followed by stimulation of cytosolic phospholipase A2 and release of arachidonic acid [4, 5, 12, 86, 88]. Further, from thiscompound, vasodilator agents are generated, either via the cyclooxygenase pathway, such as prostaglandin E2 (PGE2), or via
the epoxygenase pathway, such as epoxyeicosatrienoic acids (EETs), which cause smooth muscle relaxation in the wall ofarterioles located near the active neurons [4, 5, 12, 76, 86, 88].
The level of neuronal activity and oxygen tension influence the tone of cerebral blood vessels [12, 88]. Studies in rat
brain slices have shown that neuronal activation causes relaxation of blood vessels when oxygen tension is low andvasoconstriction when the oxygen level is high [4, 88]. It is hypothesized that when oxygen supply is increased or when it is
not consumed quickly because neurons are not activated in certain regions of the brain, arachidonic acid from astrocytes
diffuses into arteriolar smooth muscle cells where it is converted to a vasoconstrictor metabolite, 20-hydroxyeicosatetraenoic
acid (20-HETE), reducing cerebral blood flow in less active foci [4, 5, 12, 76, 88]. Under low oxygen conditions, increased
astrocyte glycolysis results in increased production of lactate [88, 89, 90] and adenosine [88]. Lactate causes vasodilation by
inhibiting the prostaglandin transporter in astrocytes and thereby increases the local level of PGE 2 [4, 88], while adenosine
inhibits the vasoconstrictor effect of 20-HETE on smooth muscle in the arteriolar wall [4, 88].
Apart from metabotropic receptor activation, glutamate in the synaptic cleft is co-transported with Na+ into astrocyte
foot processes [89, 91] where it stimulates glucose uptake and utilization and production of lactate [5, 77, 88, 89, 91]. Glutamate
release from active synapses and its uptake by astrocytes may represent a signaling mechanism that couples neuronal activity
to the stimulation of astrocytic glycolysis, as demonstrated in primary culture of mouse cerebral cortical astrocytes [89].
Another vasoactive substance that participates in the regulation of neurovascular coupling is NO [12, 70, 87]. This
compound is synthesized and released by neurons in the hippocampus and acts on vascular smooth muscle, having animportant role in regulation of blood flow in this structure [70, 87]. Glutamate released by active neurons binds to thepostsynaptic N-methyl-D-aspartate (NMDA)-glutamate receptor, elevates intracellular Ca2+ concentration and activates
neuronal NOS (nNOS), which catalyzes the convertion of L-Arginine to NO [12, 87]. NO diffuses through the interstitial fluid tothe smooth muscle cells in the microvessel wall, where it stimulates guanylate cyclase to convert GTP to cGMP, thereby
increasing local blood flow and oxygen amount [12, 87]. A transient increase in NO concentration induced by glutamate release
Fiziologia - Physiology • 2024.1(106)9causes a synergistic transient increase in time, space, and amplitude with local cerebral blood flow in the rat hippocampus
seconds later [87]. Administration of the NOS inhibitor (NG -monomethyl-l-arginine) in humans substantially reduced theneurovascular coupling response, demonstrating that NO is a key messenger involved in this process [92].
Increased intracellular Ca2+ concentration in neurons can also activate phospholipase A2 and release arachidonic
acid, which is subsequently converted to a vasodilator prostaglandin (PG) by cyclooxygenase 2 [12]. Thus, through theconvergent action of NO and PG, blood vessel tone decreases [12]. It is possible that in vivo, both neurons and astrocytesintervene in the control of cerebral blood flow by releasing different vasoactive compounds through various pathways, between
which could be interactions, but the importance of these agents in mediating neurovascular coupling may be variable from a
region to another of the brain [12].
The role of insulin and IR in the regulation of neurovascular coupling [3, 8] and the fact that this process might bealtered in type 2 DM [69, 70, 93] is beginning to be increasingly investigated. Ablation of astrocytic IR in mice results inmitochondrial dysfunction and elevated ROS that trigger hypoxia-inducible factor-1α activation and angiogenesis [3]. These
disturbances were corrected by treatment with N-acetylcysteine, an antioxidant substance [3]. Astrocyte IR can regulate
neurovascular coupling by influencing glucose uptake and brain vascularity [3]. Disruption of neurovascular coupling in patientswith type 2 DM was demonstrated by functional magnetic resonance imaging [93]. Compared to healthy controls, type 2 DM
patients showed lower neurovascular coupling in several brain regions (hippocampus, putamen, amygdala), related to memory,emotions and movements [93].
Impairment of neurovascular coupling in type 2 DM is the result of reduced bioavailability of NO secondary tooxidative stress in the brain microvasculature [70]. Altered neurovascular coupling in the hippocampus, a potential causative
element of cognitive disturbance in type 2 diabetic rats was tested in a study using a rat model (Goto-Kakizaki rats) thatspontaneously develops DM [70]. Measurement of NO concentration and change in cerebral blood flow in the hippocampus in
response to glutamatergic stimulation of diabetic and control animals evidenced that in both groups stimulation caused atransient increase in NO concentration coupled in time and space to a transient increase in cerebral blood flow, but in diabetic
animals, the amount of NO and the hemodynamic response to the same stimulus were lower compared to controls [70].
Maladjustment of cerebral perfusion to brain activity, particularly in the hippocampus, contributes to neuronal
disturbances and cognitive alterations in type 2 DM [69, 70] and AD [5, 12, 94]. Increased levels of ROS and reactive nitrogenspecies (RNS) alter nNOS activity and function [95] and inhibit the production of vasodilator messengers [12, 85, 95]. This
leads to brain hypoperfusion in patients with type 2 DM [69] and AD [12, 85, 94]. The reduction of functional hyperemia isprobably the result of ROS production by the cerebral microvessels that occurs from the early stages of AD [94]. The correlation
was demonstrated in mice overexpressing amyloid precursor protein [85, 94]. These animals developed oxidative stress in
cerebral blood vessels long before the appearance of oxidative stress in neurons and GCs [94]. The effect of reducing cerebral
blood flow in response to activation of the somatosensory cortex by whisker stimulation was prevented by ROS scavengers,
indicating that oxidative stress contributes to the decrease in functional hyperemia [94]. AGEs impair neurovascular coupling
and promote cognitive alterations in type 2 DM [93]. Neurovascular uncoupling in type 2 DM may occur as a result of
microvessel dysfunction driven by hyperglycemia-induced oxidative stress [96].
THE INFLUENCE OF INSULIN AND GLUCOSE CONCENTRATION ON GLUCOSE UPTAKE BY ASTROCYTES AND
NEURONSThe role of insulin in glucose uptake by astrocytes and neurons is not fully established. The results, sometimes
divergent, of the studies that have focused on this issue could be explained by the fact that the studies were not carried out
under the same conditions, being either in vivo or in vitro studies on different animal species or cell cultures, with a different
density and distribution of GLUTs, using glucose or glucose analogs in different concentrations and under stimulation of neuralactivity or under resting conditions. Besides, there might be regional differences in insulin sensitivity on glucose uptake, andsome structures in the brain might be able to use different energy substrates, especially during starvation [55]. These facto rs
can influence the measured value of parameters related to brain energy metabolism.
The view that brain glucose uptake is insulin-independent stems from studies showing that hyperinsulinemia did notcause an increase in glucose transport across the BBB [55]. However, a study using a fluorescent glucose analog (18fluorodeoxyglucose) and positron emission tomography (PET) to evaluate the effect of basal insulin on global and regional
brain glucose uptake revealed that in healthy male subjects the global rate of brain glucose utilization was significantly higher
with insulin than without insulin, concluding that basal insulin influences glucose uptake in the human brain [55]. Regional
differences were demonstrated, with glucose uptake in the cerebral cortex being most influenced by insulin and significantly
less in the cerebellum [55]. It appeared that metabolic effects of insulin on the brain are greatest at basal (fasting) blood insulin
concentrations and brain glucose uptake could be partially insulin sensitive [55].
A research [44], which had as one of its objectives to evaluate the influence of insulin on glucose uptake by cultured
human astrocytes, did not prove an increase in glucose uptake after insulin stimulation, probably because the main GLUTs inhuman astrocytes, GLUT1 and GLUT3, are considered insulin-independent transporters [44]. However, another study on
cultured rat GCs showed that insulin, aside from being bound to GCs (astrocytes) by a specific IR, stimulated 2-deoxy-D-

10

Fiziologia - Physiology • 2024.1(106)glucose uptake [97].
The effect of insulin on glucose uptake was also assessed indirectly by using genetically modified astrocytes without
IR [8, 43]. Lack of astrocyte IR causes a decrease in GLUT1 expression, which is considered partially sensitive to insulin [8,55], and thereby a reduction in astrocyte glucose uptake, glycolytic rate and L-lactate efflux compared to control animals withIR [43], and an increase in astrocyte fatty acid metabolism to compensate for the reduction in glucose supply [8, 43]. Ablationof astrocyte IR leads to decreased glucose transport across the BBB [43].
Deletion of astrocyte IR results in insulin resistance [3, 43] and uncoupling of blood flow with glucose uptake [3],altered metabolism and energy production [8]. Consistent with the lack of astrocyte IR, deletion of GLUT1 has an analogous
effect. Glucose uptake was markedly decreased in primary cultured astrocytes obtained from mice in which deletion of GLUT1
was induced, compared to control astrocytes with GLUT1, which proves that GLUT1 is required for glucose uptake byastrocytes [46]. Additionally, these astrocytes showed increased IR expression compared to controls [46], and upregulation of
IGF-I receptor [8].
Regarding the effect of insulin on glucose uptake by neurons, an in vitro experiment on hippocampal neuronal culture
showed that insulin did not increase their glucose uptake [42]. In other studies on neuronal culture exposed to highconcentrations of insulin, increased glucose uptake by neurons could not be demonstrated either [51, 97]. Instead, insulin
stimulated the translocation of GLUT3 to the plasma membrane of neurons without causing the fusion of GLUT3 with theplasma membrane [42]. However, in neurons pre-exposed to insulin, depolarization with KCl determined increased fusion of
GLUT3 with the cell membrane and glucose uptake [42]. Contrary to the initial conception that neuronal glucose uptake is an
insulin-independent process, it appears that insulin influences neuronal glucose uptake, at least in vitro, in conditions ofneuronal stimulation, by inducing GLUT3 translocation [42]. Further research in this direction is needed to obtain a better
understanding of the influence of insulin on the process of glucose uptake by neurons.
The amount of glucose taken up by astrocytes is variable, depending on the concentration of glucose in theirenvironment [2]. When neonatal rat astrocytes are exposed to a medium containing a high concentration of fluorescent glucose
analog, they take up more glucose and store more glycogen compared to astrocytes exposed to a medium with a normal
concentration of glucose analog [2]. But, as a consequence of exposure to excess glucose, astrocytes showed an alteredmetabolism with decrease in maximal mitochondrial respiration and glycolytic capacity and broke down much more glycogenthan normal glucose astrocytes [2]. Another study on primary cultured astrocytes exposed to high glucose (25 mM) compared
to normal glucose (5 mM) reached the same conclusion regarding glycogen content and glucose uptake [13]. A similar effectcould not be evidenced in neurons [98]. If primary neurons in culture were incubated with increasing concentrations of glucose,
their glycogen content did not vary with glucose concentration, indicating that neurons compared to astrocytes may have somemechanisms that maintain intracellular glycogen at a low level [98]. Accumulation of glycogen in neurons above a certain
threshold is harmful and can cause their degeneration [99] and apoptosis [100, 101]. The mechanism by which intracellularglycogen is detrimental to neurons is not clearly understood [101].
The uptake of glucose and its analogs appears to be related to the state of activity and the part of the brain evaluated.
An in vivo experiment on male rats that assessed the uptake of a nonmetabolizable fluorescent glucose analog 6-deoxy-N-(7nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (6-NBDG) by astrocytes and neurons uncovered that during resting conditions,both astrocytes and neurons in the rat barrel cortex took up 6-NBDG at the same rate, but during intense sensory neuronal
activation, astrocytes increased their uptake more, while neuronal uptake remained almost unchanged [102]. It shows that
glucose is primarily taken up by astrocytes [102]. But when the uptake of glucose and 6-NBDG was assessed by cultured
neurons and astrocytes from mouse hippocampus and cerebellum, it was observed that cultured astrocytes took up glucose
faster than neurons, whereas uptake of the glucose analog was faster in neurons than in astrocytes [103]. The result could be
due to the different affinities and kinetics of GLUTs expressed by the two cell types in the evaluated structures [103].
In AD, the uptake of D-glucose [22, 41] and its analogs [30, 41] in specific parts of the brain (hippocampus and cortex)[41] is reduced, as a result of decreased cerebral blood flow and expression of GLUTs in BBB [22, 41]. This predisposes thebrain to insufficient glucose supply, functional impairment and deterioration of cognitive capacity [41]. Accumulation of amyloidβ may be responsible for the depletion of GLUT1 and GLUT3 in the hippocampus and cerebral cortex in AD subjects [41].
Altered glucose transport and metabolism precedes the onset of cognitive impairment in AD by several years [41].
GLYCOGEN STORAGE IN NEURONS AND ATROCYTESIn both astrocytes and neurons, glucose is phosphorylated and irreversibly converted to glucose-6-phosphate [2, 4].
Further, this compound can follow several metabolic pathways, being either stored in the form of glycogen or metabolized
through glycolysis [2, 4, 91, 104] and the pentose phosphate pathway [4, 91]. Glycogen is stored mainly in astrocytes [11, 16 ,98, 105] and in very small amounts in neurons [98, 105]. Stimulation of human astrocytes in culture with insulin was followed
by increased glucose incorporation into glycogen [44]. Glycogen is the only form in which energy is stored in the brain [6], andglucose can be rapidly released from this form without consuming adenosine triphosphate (ATP) [4]. The amount of glycogenin the brain is low compared to that deposited in the liver and muscle [44, 106]. For this reason, the brain depends on acontinuous supply of blood-born energy substrate to support its intense energy metabolism [4].

10

Fiziologia - Physiology • 2024.1(106)Based on the intravenous infusion of [1-13C] glucose combined with 13C nuclear magnetic resonance spectroscopy,
measurement of glycogen content and turnover in the human brain was carried out [106]. It was found that the amount of
metabolically active glycogen in the brain was quite low (3 - 4 µmol/g) compared to other organs (liver and muscle), but muchhigher compared to the concentration of free glucose in the brain under normoglycemic conditions [106]. Brain glycogenturnover was very low, complete turnover taking 3 - 5 days [106]. Prolonged visual stimulation did not cause a change in theglycogen content of the visual cortex in human subjects, nor a significant increase in its turnover [106]. On the contrary, in
studies on rat brains, the researchers indicated that the concentration of cerebral cortical glycogen in the unstimulated ani mals
was higher (11 - 12 µmol/g), but the measured value was variable with the method used for its extraction [107]. Glycogen level
decreased significantly after a brief sensory stimulation, and chronic inhibition of NOS substantially reduced resting glycogencontent of the rat brain [107].
It was previously thought that glycogen is not stored in neurons but only in astrocytes, and neurons either do notpossess glycogen phosphorylase to metabolize it or express this enzyme [98]. Nonetheless, Saez et al. [98], using a moresensitive method, demonstrated the presence of small but measurable amounts of glycogen in primary cultured neurons [98].
These cells express an isoform of glycogen synthase that is also expressed by muscle, called muscle glycogen synthase [98].
They also express the brain isoform of glycogen phosphorylase [98]. Neuronal glycogen protects cultured neurons fromhypoxia-induced death [98, 105]. On the other hand, a decrease in brain glycogen phosphorylase activity may result in an
abnormal accumulation of glycogen in neurons, followed by various brain diseases [99]. Primary mouse neurons in culture
have the ability to synthesize and accumulate glycogen under basal conditions and at the same time to degrade it [98]. It wasobserved that in normoxemia neuronal glycogen turnover is decreased, whereas in hypoxia it is increased, when both glycogensynthase and glycogen phosphorylase activity were increased [98]. Glycogen could protect neurons in vivo against hypoxic
stress [98].
Astrocytes express brain and muscle glycogen phosphorylase isoforms that degrade stored glycogen in response to
extracellular signals and their metabolic needs [98, 99]. The release of neurotransmitters during intense neuronal activity
induces the mobilization of glycogen stored in astrocytes [44]. Astrocytic glycogen protects neurons against lack of energy
substrate, as can occur in hypoglycemia [4, 44, 99, 108] and ischemia [99, 106]. It has been demonstrated experimentally that
if rats are subjected to moderate hypoglycemia by insulin administration, their brain glycogen content gradually decreases, andwhen plasma and brain glucose concentrations rise again, brain glycogen concentration begins to increase, exceeding several
times pre-hypoglycemic level [108]. Brain glycogen metabolism changes with variations in insulinemia [108]. Astrocyte
glycogen content is higher when cells are cultured under high glucose conditions compared to normal glucose levels [2, 13].
Because astrocytes lack glucose-6-phosphatase [105] to catalyze the release of glucose from glucose-6-phosphatefor export to neurons, astrocytic glycogen can be metabolized to lactate and supplied to neighboring neurons. This mechanism
probably operates when neurons have to deal with a glucose deficiency [2, 16, 105, 108]. At the same time, astrocytes
themselves could use their own glycogen for energy production to facilitate the utilization of interstitial fluid glucose by neurons
in situations of high energy requirements [17, 104, 105]. Other roles of astrocytic glycogen are related to providing energy tosustain glutamate synthesis, release and uptake [11, 108]. This prevents glutamate excitotoxicity [108]. By stimulating glycogenstorage in astrocytes [108], insulin might indirectly contribute to sustaining neuronal activity [44], providing neuroprotection[108].
ENERGY SOURCE IN NEURONS AND ASTROCYTES UNDER RESTING AND NEURONAL ACTIVATIONCONDITIONS
The release of neurotransmitters, the restoration of neuronal transmembrane potential differences, and the recyclingof synaptic vesicles consume a large portion of neuronal energy [4, 30, 91]. Thus, it is important to understand which pathwa yand substrate the brain uses to rapidly produce ATP when the firing rate of neurons suddenly increases [33, 91]. Questions
still persist regarding the importance of neuronal glycolysis in vivo in energy production and glucose metabolism through thispathway, as neurons are thought to derive most of their energy from oxidative phosphorylation [13, 91], and under physiologicalconditions rely less on glycolysis [11, 27, 30, 91], while astrocytes obtain energy mainly through glycolysis [4, 11, 30, 39, 91]
and acid β oxidation [39]. The preference for a certain metabolic pathway of these cells is related to the different expression ofgenes related to energy metabolism [4, 13]. When glycolysis is increased in neurons, less glucose will be metabolized throughthe pentose-phosphate pathway [30].
Li et al. [27], investigated in vitro and in vivo glucose uptake by neurons and their glycolytic capacity using cellularand mouse models, in which either the GLUT3 or pyruvate kinase were deleted [27]. They showed that human neurons cantake up glucose and metabolize it through glycolysis and use this pathway to produce most of the metabolites of the tricarboxylic
acid cycle [27]. In addition, glucose uptake is required to maintain ATP level at the synapses and for normal neuronal function
in vivo [27]. Glucose uptake was reduced in neurons lacking GLUT3, and cells were less responsive to variations in extracellularglucose [27]. Impaired glucose uptake was compensated by increased galactose metabolism [27].
On the other hand, contrary to the conclusion of the study by Diaz-Garcia et al. [109], Pellerin and Magistretti [89]considered that neuronal activation is associated with the stimulation of aerobic glycolysis in astrocytes, and the lactate derived
Fiziologia - Physiology • 2024.1(106)11from this process is transferred to neurons where is converted into pyruvate that enters the mitochondria to be further
metabolized by oxidative phosphorylation [89]. Inhibition of glycolysis caused a marked decrease in glutamate uptake byastrocytes, while hypoxia had less influence on this process [89, 110]. According to this model (ANLS – Astrocyte-Neuron
Lactate Shuttle), lactate provided by astrocytes is the energy substrate for ATP production in firing neurons, not glucose, an dthe metabolic pathway is oxidative phosphorylation and less glycolysis [33]. Consistent with this hypothesis is the study [90 ]performed on rat cortical neurons showing that lactate is preferred by active neurons to glucose for producing energy, and ATP
produced by cortical neurons derived mainly from oxidative phosphorylation and insignificantly from glycolysis [90]. Lactate
and pyruvate, but not glucose, increase the activity of cortical neurons [90].
Glutamate released in the synaptic cleft is taken by astrocytes via the Na+-glutamate transporter. Accumulation of
Na+ in astrocytes stimulates the Na+/K+-ATPase, increases ATP consumption [91], while decreasing the ATP/(ADP + AMP)
ratio [22] and increasing GLUT1 activity [22, 30]. This, in turn, activates glucose uptake and glycolysis in astrocytes, followed
by lactate production that is delivered to neurons [22, 30]. Upregulation of GLUT1 in astrocytes secondary to neuronal activation
occurs within seconds and is mediated by increasing in cytosolic Ca 2+concentration as a result of Na+-glutamate co-transport[22]. Experimental deletion of GLUT1 in mouse astrocytes results in significantly reduced glucose uptake and glycolytic rate,
lower extracellular lactate concentration, and reduced lactate release upon glucose stimulation after starvation compared with
control astrocytes [46].
In mixed cultures of hippocampal neurons and astrocytes, glutamate was observed to inhibit glucose transport into
neurons but stimulate its entry into astrocytes, causing within seconds a large increase in the ratio of astrocyte-to-neuron
glucose uptake rate [111]. This experiment showed that glutamate diverts glucose to astrocytes and indicates that activatedneurons reduce their glucose uptake in favor of astrocytes and cause them preferentially use lactate, provided by neighboring
astrocytes [111]. To determine the rate of glucose uptake by neurons and astrocytes, Jakoby et al. [103] assessed the transportand metabolism of two fluorescent glucose analogs in mouse hippocampal and cerebellar slices [103]. They found that, in both
structures, glucose uptake and metabolism were faster in the astrocyte-rich compartment than in the nearby neuronalcompartment [103]. This showed preferential glucose transport and metabolism by GCs than by neurons and may indirectly
support the ANLS hypothesis at rest [103].
There are biochemical arguments for the preference for glycolysis by astrocytes over neurons. Compared to neurons,
where some glycolytic enzymes are inhibited [30], astrocytes express glycolytic enzymes and an isoform of lactic
dehydrogenase (LDH), type 5, which catalyzes the conversion of pyruvate, the product of glycolysis, to lactate [30]. Moreover,the higher NADH to NAD+ ratio in astrocytes than in neurons stimulates the reduction of pyruvate to lactate [30], which can bedelivered to nearby neurons. Neurons have another isoform of LDH, LDH1, which catalyzes the conversion of lactate to
pyruvate [30] that will be further metabolized in mitochondrion. Neurons also express monocarboxylate transporter (MCT) type2, with high affinity for lactate whereas astrocytes express lower affinity MCTs, type 1 and 4 [30]. This favors the movement oflactate from producing cells (astrocytes) to consuming cells (neurons) in the direction of the lactate concentration gradient [30].
However, as mentioned, Diaz-Garcia et al. [109], in research on hippocampal slices and brains of awake mice,evidenced that increased neuronal energy demand is supported by the direct increase in glucose consumption by neurons andnot by the uptake of lactate released by astrocytes when stimulated by glutamate [109]. According to this view, neuronalglycolysis provides rapid energy supply when neurons are stimulated whereas astrocyte derived lactate can be used at rest,
not during neuronal activation [109]. During stimulation, neurons might export lactate rather than import it from astrocytes [109].
Although oxidative phosphorylation can produce more ATP than glycolysis, this pathway takes longer to release energy, making
it less efficient when there is an abrupt increase in energy requirement [109].
Glycolytic enzymes accumulate at the presynaptic ending of neurons in response to energy stress [112]. This location
has the role of metabolizing glucose to provide the energy needed for vesicle recycling [33]. Neuronal glycolysis has an
essential role in supplying neurons with ATP, as its blockade was followed by a slow reduction in ATP level when assessed in
resting neurons, but the reduction was accelerated when glycolysis was blocked during activity [113]. Decreased ATPavailability leads to impaired synaptic transmission [112, 113].
A study based on a theoretical model to assess changes in stored glycogen and the effect of glycogenolysis insustaining the energy demand associated with 360-seconds of brain stimulation showed that astrocytic glycogen has the roleof sparing extracellular glucose for neuronal use when stimulated, rather than as o source of lactate that is delivered fromastrocytes to active neurons [17]. Astrocytic glycogen is primarily broken down and used by these cells, decreasing their needs
for extracellular glucose [17]. In this way, more interstitial glucose can be taken by neurons. Therefore, there would be no needfor glucose to be released from astrocytic glycogen and subsequently converted to lactate, and this exported to neurons [17].
Activation of glycogen phosphorylase leads to rapid formation of glucose-6-phosphate which inhibits hexokinase during
stimulation, resulting in decreased glucose uptake and consumption by astrocytes during activation, but not during resting
conditions [17]. A small amount of astrocytic cytosolic glucose can pass into the interstitium and be taken up by neurons [17].
Another analysis based on the concentration and kinetic properties of GLUT1, GLUT3 and MCTs in BBB endothelial
cells, astrocytes and neurons allowed the development of a model for determining brain glucose and lactate levels during
neuronal stimulation [24]. Simulations using this model suggest that during neuronal activation, glucose is primarily taken up

12

Fiziologia - Physiology • 2024.1(106)and metabolized by neurons, which release lactate that may be taken up by astrocytes [24]. Calculations have demonstrated
that neurons are the primary site of glucose uptake and utilization, both in rest and activation state [24].
In conditions of hyperglycemia (25 mM), astrocytes have reduced maximal oxygen consumption rate and glycolytic
activity compared to normoglycemic conditioned astrocytes (5.5 mM), and due to this to be unable to deliver lactate to neurons
when energy requirements increase [2]. It was shown that the rate of oxygen consumption was variable depending on the time
the cultured astrocytes were exposed to high levels of glucose (25 mM) [13]. After 6 hours of exposure, no significant changein oxygen consumption rate or extracellular acidification rate was observed compared to cells expose to normal glucose level[13]. Instead, the rate of extracellular acidification increased significantly after 24-hour of high glucose exposure, that signifies
an activation of glycolysis and lactate production [13]. Hyperglycemic environment also resulted in increased expression of
MCT1 mRNA and hexokinase mRNA levels in cultured astrocytes compared to exposure to normal glucose level [13].
Experiments on rodent models of streptozotocin-induced DM showed that glycolysis and lactate concentration wereincreased in hippocampal extracts of diabetic rats compared to control rats [14]. The researchers hypothesized that in the earlystages of diabetes in rats, the elevated amount of lactate can be used by surrounding neurons, while in the later stages of thedisease the use of lactate by neurons is reduced due to mitochondrial dysfunction and thus lactate accumulates in theextracellular fluid of the brain, which over time induces impairment of neuronal function and cognitive decline in rats [14]. The
same authors working on primary cell culture exposed to high glucose concentrations found increased lactate levels in
astrocytes but not in neurons [14].
The normal functioning of the brain`s energy metabolism requires insulin. Both glucose uptake and normal
mitochondrial function have been shown to be dependent on astrocyte IR [3]. As mentioned, ablation of IR in hypothalamic
mice astrocytes resulted in decreased glucose uptake and glycolytic rate compared with controls, but higher basal
mitochondrial respiration and expression of the enzyme necessary for long-chain fatty acid transport in mitochondria (carnitine
palmitoyltransferase 1C) [43]. Since most of the energy required for neuronal function is produced in the mitochondria byoxidative phosphorylation [64, 91, 114], mitochondrial dysfunction will result in impaired oxidative phosphorylation, decreased
ATP synthesis, and abnormal neuronal metabolism, contributing to neurodegeneration [64]. Lack of astrocyte IR was also
associated with fewer and smaller mitochondria and increased autophagosomes in response to hyperglycemia compared tocontrols [43].
Increased energy demand due to neurotransmission enhances mitochondrial ROS production [91, 114]. At the sametime, it activates the transcription of antioxidant genes in astrocytes and the synthesis of glutathione, which confers
neuroprotection and facilitates neuron survival [91, 114]. Neurons have their intrinsic capacity to defend against oxidative stress,
but they are also assisted by astrocytes (astrocyte-neuronal glutathione shuttle) [91].
The binding of glutamate to its receptor in astrocytes activates a cascade of reactions that ultimately lead to thephosphorylarion of nuclear factor‐erythroid 2‐related factor‐2 (Nrf2) which translocates to the astrocytic nucleus whereinteracts with antioxidant responsive elements and causes the expression of genes related to antioxidant compounds, including
glutathione [91, 114]. In response to enhanced production of ROS, astrocytes synthesize and release reduced glutathione(GSH) into the extracellular fluid. GSH is degraded and the resulted compounds could be taken by nearby neurons toresynthesize GSH [114]. Astrocytes have a key role in supporting adjacent neurons to fight against oxidative stress, as theantioxidant capacity of neurons is less efficient [114].
In a study on cultured cortical neurons subjected to ascorbate/Fe2+ to induce oxidative stress, Duarte et al. [61],demonstrated that insulin inhibited lipid and protein oxidation, including GLUT3 oxidation and decrease in neuronal viability,
reduced cell death by necrosis and apoptosis, and exerted a neuroprotective action against oxidative insult [61]. Also, thehormone preserved the antioxidant defense capacity of neurons subjected to oxidative stress by stimulating glutathione
reductase and inhibiting glutathione peroxidase activities, thus maintaining the ratio between reduced (GSH) and oxidized(GSSG) forms of glutathione (GSH/GSSG) [61]. The decrease in intracellular uric acid level was also inhibited by insulin [61].
A lower level of cerebral GSH associated with increased GSSG was evidenced in conditions of brain insulin resistance and AD[71]. Insulin exerts a protective effect against oxidative injury of primary cortical neurons experimentally subjected to oxidative
stress by stimulating the neuronal uptake of glucose and its glycolysis to pyruvate, increasing the intracellular concentration ofmacroergic compounds, ATP and phosphocreatine, and the level of adenosine [60]. Through several steps, adenosine isconverted to uric acid [60].
Type 2 DM and AD are conditions with a complex pathogenetic mechanism. Altered insulin responsiveness [115],
impaired brain glucose metabolism, mitochondrial dysfunction and oxidative stress have been evidenced in both diseases [30,64, 73, 115, 116]. It has been hypothesized that neuronal oxidative stress may be causally related to peripheral or brain insulin
resistance [71]. Due to the fact neurons are cells with high energy consumption, any changes in the number, motility, structureand function of mitochondria can increase the production of ROS and cause oxidative stress and neurodegeneration, and thismay contribute to cognitive impairment in diabetic and AD patients [64].
CONCLUSIONS
Uncertainties persist regarding the metabolic peculiarities of neurons and astrocytes, the cooperation between
Fiziologia - Physiology • 2024.1(106)13these cells in normal and pathological circumstances, the effect of variation in blood glucose concentration on the function andstructure of brain cells, and the importance of insulin in modulating these aspects. Knowledge of brain energy metabolism andthe role of insulin in supporting this process are critical, as impaired glucose metabolism is associated with neurodegenerative
diseases. Insulin may fulfill several roles related to glucose homeostasis in the brain. Studies have shown that insulin coul dstimulate glucose uptake by neurons and astrocytes, especially under conditions of increased neuronal activity and theexpression of some GLUTs that might be sensitive to insulin. It could also enhance the storage of glucose in astrocytes in theform of glycogen and its use as an energy resource, mainly in case of cerebral ischemia and hypoglycemia, having aneuroprotective role. Astrocyte IR is required for coupling glucose uptake to cerebral blood flow. This ensures an adequate
supply of energy substrate in proportion to the metabolic demands of the brain. The normal activity and structure of brain cell
mitochondria could be influenced by insulin as well, including antioxidant protection against ROS.
A number of mechanisms might account for impaired activity of insulin in the brain: decreased in the number of IR inbrain cells, decreased in the rate of insulin transport in the brain parenchyma, glycation of insulin and IR in a high glucose
environment, alteration of components of the insulin signaling pathways, and oxidative stress. The effects of altered insulin
actions can lead to reduced brain glucose uptake, especially when energy requirements are high, decreased astrocytic
glycogen stores, uncoupling of neuronal activity with glucose and oxygen demand, and overproduction of ROS. These factors
may underlie brain cell degeneration in patients with type 2 DM and AD.
Future studies will shed light on the complexity of insulin`s actions in the brain, the link between brain insulin
resistance and cognitive impairment in DM and AD, uncovering its therapeutic benefits, particularly in AD.
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Fiziologia - Physiology • 2024.1(106)113. Rangaraju V, Calloway N, Ryan TA. Activity-driven local ATP synthesis is required for synaptic function. Cell. 2014 Feb
13;156(4):825-35. doi: 10.1016/j.cell.2013.12.042. PMID: 24529383; PMCID: PMC3955179.
114. Baxter PS, Hardingham GE. Adaptive regulation of the brain's antioxidant defences by neurons and astrocytes. Free Radic Biol
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115. De Felice FG, Ferreira ST. Inflammation, defective insulin signaling, and mitochondrial dysfunction as common moleculardenominators connecting type 2 diabetes to Alzheimer disease. Diabetes. 2014 Jul;63(7):2262-72. doi: 10.2337/db13-1954. Epub
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116. Belosludtsev KN, Belosludtseva NV, Dubinin MV. Diabetes Mellitus, Mitochondrial Dysfunction and Ca 2+-Dependent Permeability
Transition Pore. Int J Mol Sci. 2020 Sep 8;21(18):6559. doi: 10.3390/ijms21186559. PMID: 32911736; PMCID: PMC7555889.
ROLURILE INSULINEI ÎN HOMEOSTAZIA GLUCOZEI LA NIVEL NEURONAL ȘI ASTROCITARREZUMAT
Identificarea receptorilor pentru insulină pe membrana plasmatică a celulelor creierului este o dovadă fără echivoc că insulinaacționează în creier, îndeplinind funcții legate de metabolismul energetic al creierului, dar nu numai. Insulina ar putea facilita cuplarea fluxului
sanguin cerebral cu nivelul activității neuronale, expresia unor transportori neuronali și astrocitari de glucoză, captarea glucozei și stocarea
acesteia sub formă de glicogen și funcționarea normală a mitocondriilor din aceste celule. Scopul acestei lucrări este de a analiza aspecte
privind traversarea barierei hematoencefalice de către glucoză și insulină, substratul energetic utilizat de neuroni și astrocite în condiții derepaus și activare, efectul concentrației sanguine a glucozei asupra transportorilor de glucoză, insulinei, receptorului pentru insulină și
metabolismului energetic al creierului. Din această perspectivă, tulburările observate în bolile asociate cu rezistența la insulină a creierului,precum diabetul zaharat de tip 2 și boala Alzheimer, vor fi evidențiate.
Cuvinte cheie: transportori de glucoză, insulină, astrocite, metabolism energetic, diabet zaharat.

Fiziologia - Physiology • 2024.1(106)19

SIMPLE ALGORITHM IN MATLAB FOR THE INTERPRETATION OF ANTIBIOGRAMS –PRELIMINARY RESULTSGABRIEL VINTER1, DENISA ALEXANDRA VEREȘ2, FLORIN MUSELIN1*, BEATRICE ZEMBROD3, DANIELA
CRÎNSIC3, ALBERT IOIART3, ALEXANDRU TÎRZIU3, MIHAI SĂRĂNDAN4, DAMIAN OLEI2, DANIEL BONCIOG2 AND
DAVID COMANICIU3
1 University of Life Sciences „Regele Mihai I“ from Timișoara, Faculty of Veterinary Medicine, 119,Calea Aradului, 300645, Timișoara, Romania
2 Politehnica University of Timișoara, 2 Victoriei Square, RO-300006, Timișoara, România
3 University of Medicine and Pharmacy ”V. Babeș” Timișoara, 2 Eftimie Murgu Square, 300041,
Timișoara, România
4 Veterinary Sanitary and Food Safety Agency,
Arad, 98C, Liviu Rebreanu Street, 310414, România* Address for correspondence: Florin Muselin, PhD, University of Life Sciences „Regele Mihai I“ fromTimișoara, Faculty of Veterinary Medicine, 119, Calea Aradului, 300645, Timișoara, Romania; e-mail:florinmuselin@usvt.ro
ABSTRACTThe purpose of this work was to test a simple MATLAB algorithm for the automatic analysis of
Kirby-Bauer diffusion technique antibiograms. The experimental setup includes a digital camera
connected to a computer for acquisition of the Petri dish images and an algorithm developed in MATLAB
for the automatic interpretation of the image data. The workflow data required by this software includes
patient identification, sample identification and the list of the antibiotics tested. The growth inhibition area
corresponding to each of the antibiotics tested is identified, measured and interpreted automatically by
the software which also calculates the results and returns data on the strain’s sensibility to an antibiotic.
The results are also presented as a graphical chart ranking the antibiotics based on their efficiency, whichsimplifies the physician’s task of choosing the proper treatment for the patient. This algorithm represents
another fast and cost-effective way for the interpretation of the laboratory bacterial antibiotic resistancetest results.
Key words: Kirby-Bauer’s disk diffusion test, MATLAB, automated analysis of antibiogram, anti-microbial
resistance
INTRODUCTION
The extensive and often unjustified use of antibiotic treatments in both human and veterinary
medicine has led to the emergence of microbial strains resistant to common antibiotics. This evolution isworrisome in the long term because humanity could return to the pre-antibiotic era, when infectious
diseases of bacterial etiology generated a significant part of morbidity and mortality in the general
population [1]. In order to limit this phenomenon as much as possible, it is necessary to treat infectious
diseases caused by bacteria using the antibiotics to which the respective germ is sensitive and of course
taking into account the particularities of each patient. Also, the duration of the treatment must be adapted
to each individual case, the intention being the sterilization of the infectious lesion.
The antibiogram or the antibiotic susceptibility test is widely used in all microbiology laboratories
and completes the bacteriological diagnosis. The most frequently used procedure is the Kirby-Bauer
20

Fiziologia - Physiology • 2024.1(106)diffusion method. This method involves the cultivation of the microorganism isolated from the pathologicalproduct on the Mueller Hinton Agar medium, and the application of the antibiotics of interest on the Petri
dish in the form of commercially available disks impregnated with antibiotics in standardized
concentrations. The discs are positioned at equal distances, conveniently chosen so that the inhibitionareas are well defined, without overlapping. Antibiotics diffuse freely in the culture medium, theirconcentration decreases as we move away from the respective disc, and when the minimum inhibitory
concentration is reached, the microorganism develops on the rest of the culture medium. In this way, aseries of clear, well-defined circular areas are formed, with a diameter proportional to the effectiveness of
the respective antibiotic. The rest of the surface of the culture medium remains opaque due to thedevelopment of microorganisms.
The evaluation of sensitivity to antibiotics involves the measurement of these diameters and theircomparison with standardized values for each individual antibiotic, either manually or with the computer
through a data acquisition system. Currently, there are available software applications used for the
automatic interpretation of antibiograms. An example is AntibiogramJ, open-source that offers goodresults, comparable to those given by experienced microbiologists [2,3]. The software application
proposed by us has the advantage of being designed in a programming environment with great flexibility,
widely accepted by the academic community and which offers the possibility of further development using
the artificial intelligence and machine learning tools offered by MATLAB [4].
The potential of artificial intelligence in the field of microbiology is enormous, it will be possible touse genomic, proteomic and metabolomic data which, in association with the data provided by the usual
antibiograms, will be able to generate much more accurate predictions regarding antibiotic resistance andcertainly the development of effective health policies on long term regarding the management of bacterial
infectious diseases [5,6].
EXPERIMENTAL DATA
The experimental device (Fig. 1) consists of a web camera (Selecline model PPW-10) fixed on astand that allows photographing the Petri dish with the antibiogram on a black background and connected
to a computer. The positioning of the plate is done so that one of the antibiotic disks that will be considereddisk no. 1 to be positioned as precisely as possible at 9 o'clock on a fictitious clock face. This aspect isvery important in the process of identifying antibiotics in the list to be entered in the software application.

Fiziologia - Physiology • 2024.1(106)211Fig. 1. The experimental device for the automatic interpretation of the antibiogram using
MATLABThe raw image is acquired with the open-source YouCam 365 software and is saved in a directory
created especially for this purpose, preferably on the computer's D drive. The name under which thisimage is saved is identical to the one that will be entered in the Sample ID field in the MATLAB application.
The actual application is structured in the form of a main program to which is added the use ofsome functions that ensure the interface with the user and proper function of software. Figure 2 shows
the block diagram.

22

Fiziologia - Physiology • 2024.1(106)Fig. 2. Block diagram of the software application in MATLAB
In a first step (Fig.3), the identification data of the patient (surname and surname), the data related
to the sample number and the pathological germ previously identified through specific microbiologicaltests (isolation in pure culture on specific media) are entered. The following formally presented instructions
are used: userInput = inputdlg(prompt, dlgtitle, dims).

Fiziologia - Physiology • 2024.1(106)23

Fig.3. Entering input data into the software application
In order to be able to correctly identify each area of inhibition of the growth of microorganisms,
an intermediate stage of processing the original image acquired by the web camera is necessary. The
following formally presented instructions are used: imresize, rgb2gray, imadjust, im2bw, bwareaopen,
imcomplement. The identification of all the antibiotics (Fig. 4) used will be done starting with the first discpositioned according to the previously mentioned. The following predefined instructions in MATLAB are
used: imfindcircles (imag, [Rmin Rmax],'Object Polarity','bright', 'Sensitivity' value,'EdgeThreshold',value);reference Point; angles; [sortedAngles, sortedIndices]; sorted Centers; sortedRadi; viscircles (sorted
Centers, sorted Radis,'Edge Color','...'); viscircles (sorted Centers,sorted Radis,'Edge Color','...');
userInput.

Fig. 4. Identifying the position of the microtablets with the antibiotics to be tested
The last step in the data analysis consists in determining and sorting the radius of the zone of inhibitionof the growth zone of microorganisms. Thus, three categories of zones are obtained that correspond to
the sensitivity of the microorganism involved to the set of antibiotics considered: ineffective, effective andvery effective. The following predefined instructions in MATLAB are used: [Ctrs1,Radi1] =find_antibiotics(Rmin, Rmax, sortedRadi, sortedCenters); viscircles(Ctrs1,Radi1,'EdgeColor','b');
title(sprintf('Antibiotic sensitivity test = INEFFECTIVE ANTIBIOTIC',numel(Radi1))). The result ofapplying this algorithm is presented in the figure 5.

24

Fiziologia - Physiology • 2024.1(106)Fig. 5. The categories of microbial growth inhibition zones depending on their radius
RESULTS AND DISCUSSION
The result of the automatic interpretation of the antibiograms (Fig. 6.) with the proposed software
is displayed in a very friendly way for the doctor in the form of an image and saved as a pdf file. The majoradvantage of this approach is the significant reduction of the time needed to analyze the antibiograms
compared to the classic manual method of measuring the zones of inhibition of the growth of
microorganisms. The limitations of this software are related to the possible confluence of inhibition areas,
the existence of imprecisely delimited or inhomogeneous inhibition zones that raise the suspicion of theexistence of several types of microorganisms with different sensitivity to antibiotics. The wider use of deeplearning and machine learning methods will contribute decisively to the removal of these limitations.

Fig. 6. Presentation of the result of the automatic analysis of antibiograms using the proposed
MATLAB software
CONCLUSIONSManual analysis of antibiograms is generally a time-consuming process with a degree ofsubjectivity. The software proposed by us can bring improvements to this process, being useful both in
the daily activity of microbiology laboratories and for didactic purposes, being flexible and easy to use andimprove. The presentation of the results in a graphic way is more attractive and intuitive for the laboratory
doctor and the clinician.

Fiziologia - Physiology • 2024.1(106)25

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ALGORITM SIMPLU ÎN MATLAB PENTRU INTERPRETAREA ANTIBIOGRAMELOR - REZULTATE PRELIMINARE
RezumatScopul acestei lucrări a fost testarea unui algoritm simplu MATLAB pentru analiza automată a antibiogramelor realizate prin tehnica dedifuzie Kirby-Bauer. Configurația experimentală include o cameră digitală conectată la un computer pentru achiziția imaginilor plăcilor Petriși un algoritm dezvoltat în MATLAB pentru interpretarea automată a datelor. Softul propus include identificarea pacientului, identificarea
probei și lista antibioticelor testate. Zona de inhibare a creșterii microbiene corespunzătoare fiecărui antibiotic testat este identificată,măsurată și interpretată automat de software-ul care, de asemenea, calculează rezultatele și returnează date despre sensibilitatea tulpinii laun anumit antibiotic. Rezultatele sunt, de asemenea, prezentate sub forma unei diagrame grafice clasificând antibioticele în funcție deeficiența acestora, ceea ce simplifică sarcina medicului de a alege tratamentul adecvat pentru cazul respectiv. Acest algoritm reprezintă oaltă modalitate rapidă și rentabilă de interpretare a rezultatelor testului de rezistență bacteriană la antibiotice prin tehnica difuziei.
Cuvinte cheie: test de difuziune Kirby-Bauer, MATLAB, antibiograma automată, rezistență antimicrobiană

26

Fiziologia - Physiology • 2024.1(106)

POSSIBLE ELECTROPHYSIOLOGICAL CHANGESINDUCED BY THE PRACTICE OF PRAYER IN ORTHODOX CHRISTIAN TRADITIONADRIAN SORIN MIHALACHE1,2,5, ALEXANDRU PASLARU1, MARIAN POBORONIUC3, DĂNUȚ-CONSTANTIN IRIMIA3,
IULIAN STOLERIU4, LEON ZĂGREAN1
1 "Carol Davila" University of Medicine and Pharmacy, 050474 Bucharest, Romania
2 Faculty of Orthodox Theology, "Alexandru Ioan Cuza" University of Iași, Romania
3 Faculty of Electrical Engineering, “Gheorghe Asachi” Technical University of Iaşi, Romania
4 Faculty of Mathematics, "Alexandru Ioan Cuza" University of Iași, Romania
5 Medicine and Spirituality Research Center - Providența Hospital, IașiABSTRACTThe present study aims to highlight possible changes that the practice of prayer (in the Christian Orthodox Tradition)- PCOT - after a pattern of 30 minutes of daily practice, for 8 weeks - can induce in the electrical activity of the brain. The
participants (n=33) were divided into two groups: Study Group (SG), n=16, with an average age of 39.2 years and Control
Group (CG) n=17, with an average age of 38.6 years). The Study Group included Orthodox Christian faithful who do notpray daily, being trained in a 3-hour session on the psycho-emotional, behavioural and spiritual specifics of PCOT. No
intervention was performed on the subjects of the Control Group. The study included, before and after the period of 8
weeks of daily practice of PCOT, EEG measurements according to a measurement protocol. Applying the Wilcoxon signed
rank test revealed significant differences in median value in the case of SG subjects, pre/post-experience, in the alpha andgamma2 registers, but not in the beta and gamma1 registers. Further research and changes in the experimental design
are needed so as to allow for more accurate assessments. Results are discussed in the context of similar studies,
highlighting some specific limitations of studies that evaluate the physiological changes induced by spiritual/religious
experiences, especially of those that use EEG, while also formulating some recommendations for increasing themethodological quality of future studies.
Keywords: Orthodox Christian prayer, EEG changes, alpha, gamma, beta frequency spectrum
1.

GENERAL OBJECTIVE AND WORKING HYPOTHESESIn the present study we aimed to highlight possible changes in the electrical activity of the brain, in the case ofsome subjects who practice PCOT (prayer in the Christian Orthodox tradition). The research focused on threeinterconnected studies, which constitute the research section of the PhD paper The Neurosciences of Spiritual Life,
defended at UMPCD in July 2023.
The general objective of the research was to highlight possible changes induced by the practice of Christian prayer,in the Christian Orthodox Tradition, through three different measurements (psychometric tests, the levels of some salivary
markers and the inventory of changes in the electrical activity of the brain through EEG). In the first study, which inventoried
the physiological changes in the table of some salivary markers, respectively [1]: Cortisol, Salivary Alpha Amylase (sAA),
Immunoglobulin A (IgA), Oxytocin, Testosterone and cytokines interleukin 6 (IL-6) and interleukin 8 (IL-8), measurements
showed a statistically significant decrease in Salivary Cortisol and an increase in IL-6 in Study Group (SG) subjects at posttesting relative to levels measured at pre-testing, suggesting that PCOT may ameliorate the physiological response tostress and the inflammatory response. No statistically significant results were obtained in other salivary markers. The
second study, related to psychometric measurements [2], revealed significant results, regarding the subjects in SG at posttesting, regarding item a3 ("fears/phobias"), from the Hamilton scale and the change that appeared in SG at post-testing
regarding item a14 ("appraisal of anxious mood at interview"), suggesting a possible influence of PCOT practice.
The general objective of the third research study was to highlight the possible changes in the alpha, beta andgamma register that appeared as a result of the PCOT practice. The general working hypothesis is that PCOT practice for
8 weeks (30 min./day) will produce changes in the electrical activity in these three registers.
The electroencephalogram (EEG) is a non-invasive evaluation that detects, enhances and records the bioelectricalactivity of the brain. The study includes measurements of alpha, beta and gamma rhythms. The alpha (α) rhythm represents
the background electrical activity of the brain in a normal subject during a state of wakefulness and psychosensory rest.
The frequency of the alpha (α) rhythm ranges between 8 and 13 Hz. The beta (β) rhythm is the electrical expression of abrain in a state of activity. It has a fast frequency ranging between 14 and 30 Hz. Gamma (γ) waves are high-frequency
neural oscillations typically associated with cognitive functions such as memory, attention, and perception. In terms of27

Fiziologia - Physiology • 2024.1(106)electroencephalography (EEG), gamma waves generally have a frequency range of approximately 30 to 100 Hz. The
presence and modulation of gamma activity in the EEG can provide insights into various cognitive processes and are often
studied in the context of neuroscience and psychology. The Gamma1 (γ1) sub-band is usually considered to have afrequency range of approximately 30 to 60 Hz. The Gamma2 (γ2) sub-band typically covers frequencies above 60 Hz,
extending up to 100 Hz or more.
To test this hypothesis, we formulated four working hypotheses, which we detail below.
PCOT practiced 30 min./day, for 8 weeks triggers:- the increase in brain electrical activity in the alpha frequency spectrum in SG subjects, highlighted by the levelmeasured in post-testing compared to the level measured in pre-testing (Hypothesis 2.1). Subsequently, we also formulate
the hypothesis according to which the mean differences in the frequencies in the alpha register, between SG and Control
Group (CG) (calculated by subtracting from the mean value obtained after the experience, the mean value before theexperience, for each individual group), are statistically significant (Hypothesis 2.1. 1);- an increase in cerebral electrical activity in the beta frequency spectrum in SG subjects, highlighted by the levelmeasured post-testing compared to the level measured pre-testing (Hypothesis 2.2). Subsequently, we also formulate thehypothesis according to which the mean differences in the beta frequency spectrum, between SG and CG, are statisticallysignificant (Hypothesis 2.2.1);- increase in cerebral electrical activity in the gamma1 frequency spectrum in SG subjects, highlighted by the levelmeasured post-testing compared to the level measured pre-testing. (Hypothesis 2.3). Subsequently, we formulate thehypothesis according to which the mean differences in the gamma1 frequency spectrum, between SG and CG, arestatistically significant (Hypothesis 2.3.2);- the increase of cerebral electrical activity in the gamma2 frequency spectrum in SG subjects, highlighted by thelevel measured post-testing compared to the level measured pre-testing (Hypothesis 2.4). Subsequently, we formulate thehypothesis according to which the mean frequency differences in the gamma2 frequency spectrum, between SG and CG,
are statistically significant (Hypothesis 2.4.1).
Finally, the last two hypotheses concern possible changes induced by PCOT, 30 min. daily for 8 weeks in theelectrical activity in the prefrontal region (Ch1 – FP1, Ch2 - FPz, Ch3 – FP3, see Fig. 1), in all four registers (alpha, beta,
gamma1 and gamma2), among subjects from SG, compared to the evolution in the interval between the pre- and posttesting moments. For this, we follow a comparison between the median SG and that of CG, for the first three channels. For
this, we formulate the following hypothesis:- PCOT 30/8 triggers changes in electrical activity, in all registers, post-testing, compared to the time of pre-testing(Hypothesis 3.1);- at the time of post-testing, the median of SG subjects is different among SG subjects (Hypothesis 3.2).
Implicitly, we shall also investigate the possible changes that appear in the interval between pre- and post-testing,among the subjects in the CG, in all four registers, in the first three channels (Ch1 – PF1, Ch2 – PFz, Ch3 – PF2).
2.

PARTICIPANTS AND METHODSIn this study, subjects that had participated in the two previous studies were included [1,2].
EEG recordings were performed in two stages, pre- and post-testing, before and after the 8-week experimental
interval (individual PCOT) intended for the subjects in SG.
Electrical brain (EEG) and cardiac activity was recorded using the g.USBamp biosignal amplifier and braincomputer interface system (g.tec medical engineering GmbH, Graz, Austria) with 16 channels of signals being
simultaneously sampled at a maximum frequency of 37.4 kHz at 24-bit resolution.
12 EEG reference leads derivations were recorded with active electrodes in positions FP1, FP2, FPZ, FT7, FT8,
FC3, FC4, Cz, P3, P4, O1, O2 and the reference on the left mastoid process (Fig. 1), using electrodes with g.LadyBird gel(g.tec medical engineering GmbH) and a bipolar ECG lead (D1 lead) using special wet electrodes for ECG recordings.

28

Fiziologia - Physiology • 2024.1(106)Fig. 1 The layout of the electrodes on the scalp of the subjects was done according to the international 10-20
system.
Electrophysiological recordings were performed in a quiet room without background noise, having a constanttemperature of approx. 21℃. After attaching the electrodes, the subjects were placed in a comfortable chair with thebackrest in front of a white wall.
Electrophysiological recordings in Stages II and Stage V of the research followed a 10-interval (EEG Measurement
Protocol) sequencer, in the order presented in the EEG Measurement Protocol Intervals (Table 1).

29

Fiziologia - Physiology • 2024.1(106)Interval 1

Interval 2

Base line
Eyes Open(BLEO1)

Base line
Eyesclosed(BLEC1)

Interval 3Interval
4Interval 5Interval
6

Base line
Eyes Open(BLEO2)

Base line
Eyesclosed(BLEC2)

Image
Visualisation(VI
PHOTO)

Interval 7

Interval
9Interval 8
Cold Pressure/EGS

PERSONAL EXPERIENCE(PE)

Base line
Eyes Open(BLEO3)

Hand
IN

PAIN

Hand
OUT

Base line
Eyes Open(BLEO4)

Interval
10
Base
line
Eyes
closed(BLEC3)

2 min.

2 min.

12 min.

2 min.

2 min.

2 min.

2 min.

3 min.

2 min.

2 min.

At this stage, the subjects
were asked to proceed with thepersonal experience.
At this stage,
the subjects
were asked
to look at awhite walland relax,
without
thinking
about
anything inparticular.

At thisstage, thesubjects wereasked
toclose theireyes
andrelax, without
thinking
about
anything inparticular.

In this phase, CG subjects
were instructed to think of aperson they are fond of (either
from childhood or present),
while SG subjects practiced,
during the same interval, PCOT
in a way similar to that of theexperimental intervention
period.
Participants were instructedthat the laboratory staff, who
measured the EEG activity, did
not know which group the
subjects belonged to.

At thisstage, thesubjects
were asked
to look at awhite walland relax,
without
thinking
about
anything inparticular.

In thisstage thesubjects
were asked
to close theireyes andrelax, without
thinking
about
anything inparticular.

In thisstage thesubjects
were asked
to look at a
photograph.

In thisstage thesubjects wereasked to lookat a white walland relax,
without
thinking about
anything inparticular.

Table 1 EEG Measurement Protocol Intervals.

30

Fiziologia - Physiology • 2024.1(106)

At this stage they were asked toinsert a hand into a bowl of cold
water at -4o C, up to the wrist.
They were told to keep theirhand in the water as long as they
could and to signal (by verbally
announcing) when they felt pain.
They were told to remove their handfrom the water when the pain was
unbearable.

At thisstage, thesubjects
were asked
to look at awhite walland relax,
without
thinking
about
anything inparticular.

At thisstage, thesubjects
were asked
to closetheir eyes
and relax,
without
thinking
about
anything inparticular.

During the base line - eyes open intervals (1, 4, 7 and 9) the subjects were asked to remain relaxed, with their eyes
open, without thinking about anything specific. During the base line - eyes closed intervals (2, 5, and 10), subjects were at restwith their eyes closed, instructed to remain relaxed, without thinking about anything specific.
In interval 3 (personal experience) the laboratory team, not knowing the composition of CG and SG, made no reference
to PCOT. Laboratory personnel were instructed to guide subjects to settle into the personal experience without specifying its
nature. The volunteers from the two groups, CG and SG, were instructed to do the following when prompted that "personal
experience follows":- CG subjects thought of a loved one/pleasant childhood experience;- subjects from SG practiced, without written support, PCOT, without mentioning the specifics of their activity.
In interval 6 (photo), the subjects in the two groups were projected, on the white wall in front, a photograph (Photo 1),with the help of a video projector. The photograph was viewed by the subjects for 2 min. In post-testing, under similar conditions,the subjects looked at another photo for 2 minutes (Photo 2).

Photo 1 (Ahmet Sik via Getty Images)

Photo 2 (Pixabay, 2019)

In Stage I (pre-testing), participants from SG and
CG were shown Photo 1 on a frontal white wall for 2
minutes, during which EEG recordings were made.

In Stage V (post-testing), participants from SG and
CG were shown Photo 2 on a frontal white wall for 2
minutes, during which EEG recordings were made.

3.
SELECTION OF PHOTOS
The photos were chosen from a set of six initial photos following a selection based on the following criteria:- content without relevance to the personal life of the subjects;- emotional valences;- stimulation/request of empathy.
From a set of six photos with similar content, 36 people (who were not part of the CG or SG), aged between 18 and55, gave scores on a scale of 1 (a little impressive) to 5 (very impressive). Among the evaluated images, the photos with theclosest score were selected for the present research, respectively Photo 1, for the pre-test measurements and Photo 2 for the
post-test measurements.
For interval 8 of the EEG Measurement Protocol (cold pressor test), a 20 cm diameter cylindrical vessel was preparedwith a mixture of water and ice, being maintained at 4℃. The subjects had to put their right hand in, up to the wrist, and reportwhen pain started; then they were to remove their hand from the water when the pain became too much. These events weremarked by the technician as specific events on the electrophysiological recordings.
In all of these steps, from the EEG measurement Protocol, the subjects in the two groups were guided in the sameway, by the technician, who assisted all the electrophysiological recordings.
Two sets of electrophysiological recordings, which took place in two periods: April 2019, and subsequently May-June,
pre-test and, after the 8 weeks of practice of personal experience (PCOT), post-testing, were performed.
31

Fiziologia - Physiology • 2024.1(106)4.

EEG DATA COLLECTION
For the recording and processing of brain electrical activity, we used a high-performance and precision biosignalg.USBamp amplifier (g.tec medical engineering GmbH). The device allows the recording of physiological activity from the brain,eyes, heart, including respiration, galvanic skin response, temperature and other physiological and physical parameters. Dueto its technical specifications and extensive software environment, the device is widely used for neuropsychological andphysiological explorations and measurements.
The device used in the research is USB-compatible and supports 16 biosignal channels sampled simultaneously witha frequency of up to 37.4 kHz at 24-bit resolution. A total of 4 independent ground ports ensure that there is no interference
between recorded signals. Digital inputs and outputs allow the measurement of trigger channels along with biosignal channels
to easily transmit analysis results to external devices.
The g.USBamp system offers the possibility of detecting external events ('triggers') and correlating EEG data withinformation from sensors (in the case of our study, the one provided for the galvanic response of the skin). The data recorded
in real time were saved and later processed by means of a dedicated software, gBSanalyze.
5.

PROCESSING OF RECORDED DATA
The signals taken from the biosignal amplifier g.USBamp were dealt with in advance so as to eliminate measurement
noises (e.g. data filtering), but also to visually examine the recordings, to eliminate the recording fragments compromised due
to noises. A paradigm is used to correlate the characteristic results extracted from the EEG signal with external events. The
processing also ensured the extraction of the essential characteristics of the EEG signals in various frequency bands(exemplified, with a Simulink&Matlab program, in Fig. 2).

Fig. 2 Matlab Simulink program for extracting features in the field of alpha and beta frequencies.
The processing of the recorded signals was done with g.Bsanalyze (g.tec medical engineering GmbH), an interactive
software for multimodal processing and analysis of biosignals in the fields of clinical research, which includes a multitude offunctions, such as topographic diagrams or filters of different types (Fig. 3).

32

Fiziologia - Physiology • 2024.1(106)Fig. 3 The g.BSanalyze software main interface.
The files were saved in "*.hdf5" format, collecting a number of 2 files for each subject from CG and SG, with therecordings of the paradigm (EEG measurement protocol) in the Phase I of the research, pre-test, before the 8 weeks of PCOT
experience, and a second post-test file after the completion of the experimental period in Phase V of the research.
For each of the 10 intervals of the paradigm (the EEG measurement protocol), "triggers" were provided to mark the
beginning moment. The data were recorded working with a sampling frequency of 512 Hz. After the recordings, the acquired
data were visually examined, with noise-compromised sequences removed from the final determinations (Fig. 4).

Fig. 5 Biosignal fragment affected by noise, identified by visual examination, and removed from the final
33

Fiziologia - Physiology • 2024.1(106)determinations.
Each file, with data acquired through EEG measurements in the 10 intervals in the paradigm, contains recordings madeon the 14 channels (there is an example in Fig. 6).

Fig. 6 Display (time segment of 13 sec.) of the 14 channels, highlighting the moments of occurrence of the triggers(on the right of the figure) that mark the activities included in the 10 stages of the paradigm.
Primary data
The processing of the recorded signals provided numerical primary data, entered in Excel tables.

Fig. 7 The primary data, in the form of numerical values corresponding to the signals
in the Gamma1 and Gamma2 register. The extracted fragment represents the first two sets ofinterval 1 (eyes open – BLEO 1), for all channels (Ch1 – Ch12 – sensors) for one of the subjects,
at the time of pre-testing.

Fig. 8 For each of the 10 intervals, the processing of the recorded data provided a series of numerical values (oneto four). In the extracted table fragment, there are 4 numerical values corresponding to channels 11 and 12 (Ch 11 and Ch
34

Fiziologia - Physiology • 2024.1(106)12), during Intervals 1 (eyes open - BLEO1) and 2 (eyes closed - BLEC 1) of the Measurement Protocol, at one of thesubjects, at the time of pre-testing.

Fig. 9 Fragment of primary data table with 4 numerical values corresponding to the signals in the Gamma2 register,
captured by sensors 11, 12 and 13, during Interval 6 of the Measurement Protocol (PHOTO), in one of the subjects, at thetime of pre-testing.
Photo 7.5.4 For some of the intervals provided in
the Measurement Protocol, only a single numericalvalue was available, which will limit the statisticalanalysis of the data and the comparative evaluation
pre- and post-testing, intra-group and inter-group. In
the extracted fragment there are the numerical values
corresponding to the HAND IN, PAIN and HAND OUT
subintervals of Interval 8 - Cold Pressure/EGS, forsensors 11 and 12, in one of the subjects, during pretesting.

Fig. 10 In addition to the numerical values, the primary data contain graphs corresponding to these values, for eachindividual sensor. The extract shows a fragment of the graph corresponding to the electrical signals recorded in the alphaband, for intervals 4, 5, 6, 7, and 8 of the Measurement Protocol, for sensor 12, in one of the subjects, during pre-testing.
Such recordings were made for all 4 frequency bands (Alpha, Beta, Gamma1 and Gamma2), for all 12 channels (12sensors), for each subject in CG and SG, throughout the 10 Intervals of the Protocol EEG measurement, pre-testing and posttesting, respectively.
6.

STATISTICAL ANALYSIS TOOLSThe tools used for the statistical analysis of the primary data are non-parametric tests for the median (Wilcoxon signedrank test), t-tests for paired data, and bar charts with S.E.M. (Standard Error of the Mean). Non-parametric tests were used fordata that did not meet the normality requirement for using the corresponding parametric tests.
35

Fiziologia - Physiology • 2024.1(106)(a) The median and the mean are two statistical indicators of central tendency for a set of statistical data. If the observed
data have a small volume (usually below 30-40), tests for the mean (called parametric tests) are not relevant, since thestatistical assumptions under which they operate are not achieved. That is why, for the present situation, we used alternative
tests (called non-parametric) for the median. However, the latter is a better indicator of the central tendency of the data(compared to the mean) in case of asymmetric data.
(b) The Wilcoxon signed-rank test is a non-parametric test for statistical hypothesis testing used to compare thelocations of two populations using two matched samples.
(c) The t-test for paired data is a statistical technique that is used to compare two population means in the case of two
samples that come in pairs.
(d) Bar charts are bar representations of numerical values for the levels of a category characteristic. Levels are
represented on the horizontal axis of the chart and values are represented on the vertical axis. Each category value
corresponds to a bar, and the height of each bar corresponds (or is proportional to) to the magnitude of the value. To allow aneasy comparison of the values, the bars are represented in the same graph, of equal thicknesses (chosen by the user), without
being glued together.
(e) S.E.M. (Standard Error of the Mean) is the standard deviation (or root mean square error) of the sample mean. Thisis a measure of the precision (or variability) for an estimated mean of a statistical population characteristic. S.E.M. value
indicates how far a sample mean is likely to be from the population mean, using the original units of measurement. For a𝑠volume data set, S. E. M. = , where s is the standard deviation for a single data value.
√𝑛

7.

STATISTICAL ANALYSIS OF EEG DATA AND INTERPRETATION OF RESULTS
EEG measurements were valid for 30 volunteers grouped into two groups (GS - 16, and GC - 14). Acquired data: 12channels, 4 frequencies (α, β, γ1 and γ2), corresponding to a number of 12 moments (which include the mentioned 10 stages,
to which is added the moment of inserting the hand into the water and that of withdrawing the hand from the water, within therange of cold pressor tests).
The BLEO1, BLEC1, PE, BLEO2, BLEC2, VI FOTO, BLEO3, BLEO4 and BLEC3 features have 4 values each, andthe HAND IN, PAIN and HAND OUT features have a single value each (See Table 1 EEG Measurement Protocol Intervals).
For this data set, there are no missing values. In all these tests, E1 - E16 represent the experimental group (SG) volunteer
subjects, and N17 - N30 denote the CG subjects.
To test the working hypotheses of this study, we performed a first test, using the Wilcoxon signed-rank test, with which
we compared each of the four bands, taking into account the signals recorded by all channels at once, to highlight changes in
the CG and SG subjects medians, pre- and post-experience.
In this regard, we pursued an analysis of the data corresponding to each subject before the experience, in comparison
with the data obtained post-experience, after the 8 weeks of PCOT practice. For each volunteer (from both groups) and foreach of the α, β, γ1 and γ2 frequencies, the mean value of the 12 channels was calculated. Then, the Wilcoxon signed-rank
test was used to check whether there were statistically significant differences in the central values corresponding to eachfrequency (α, β, γ1 and γ2) before and after the experiment1.
The hypotheses of the Wilcoxon signed-rank test are:(H0): the median of the differences (for all subjects) between the two data sets is zero (or, equivalently, the two sets
have the same median value).
(H1): the median of the differences (for all subjects) between the two data sets is not zero. Table 2 shows the P-values
for all these tests. P-values written in color font correspond to statistically significant differences in the median value before
and after the experiment (the significance level is α = 0.05).

1

For this stage, the paired data t-test, which compares the mean values of the matched data (data collected in pairs, usually before
and after an experiment), was not chosen, as this t-test is based on the assumption that the data are normally distributed, condition which is
not met here. Instead, the Wilcoxon signed-rank, non-parametric test for matched or paired data was used. This test compares the medianvalues of two matched data sets, not their mean values. When the data are symmetrical, the median and mean values coincide.
36
Fiziologia - Physiology • 2024.1(106)Table 2

P-values for the Wilcoxon signed-rank test for the medians of each frequency. Volunteers E1-E16 are from
EG, N17 - N30 are from CG.

In order to test the working hypotheses 2.1, 2.2, 2.3 and 2.4 (PCOT practiced for 30 min./day, in 8 weeks causes anincrease in brain electrical activity in the alpha, beta, gamma1 and gamma2 register, in SG subjects), we used the Wilcoxon
signed-rank test, searching for significant differences in median value for SG subjects occurring between pre- and postexperience time points. For each given frequency (α, β, γ 1 or γ2), the mean value of the frequency within the SG, for all time
points, was calculated. Then, it was tested whether there were significant differences in the mean value before and after theexperiment. The corresponding P-values for the Wilcoxon signed-rank test are:Pα = 0.0001, Pβ = 0.9111,

Pγ1 = 0.1085, Pγ2 = 0.0015.

Significant differences (at significance level 0.05) in the medians (before and after the experiment, within the SG) are
noticed for the α and γ2 frequencies, confirming working hypotheses 2.1 and 2.4. Working hypotheses 2.2 and 2.3 are notconfirmed.
Figure 11 shows the mean frequency values within the SG (at all times). Figure 12 shows the total mean values ±S.E.M. of the four frequencies 2.

2 Here, and in the subsequent situations, total mean value is the mean of all means of the frequency group, considered in all

moments.
37

Fiziologia - Physiology • 2024.1(106)Fig. 11 Mean values for each frequency in SG before and after the experiment.

Fig. 12 Mean values ± S.E.M. of the four frequencies on all channels (SG).
Within the same hypothesis, we analysed the presence of statistically significant changes in CG as well. For this, weused the Wilcoxon signed-rank test to check for significant differences in median value in CG subjects, comparing pre/postexperience. For each given frequency (α, β, γ 1 or γ2) measured in subjects in the Control Group, we repeated the same
38

Fiziologia - Physiology • 2024.1(106)procedure. We calculated the mean value of the frequency within the group, for all time points, then tested whether there weresignificant differences in the mean value before and after the experiment. As a result, we obtained, for the four registers, theP-value corresponding to the Wilcoxon signed-rank test:
Pα = 0.0000,

Pβ = 0.0091,

Pγ1 = 0.5300,

Pγ2 = 0.2468.

The results show significant differences in the medians (within the CG), for the α and β frequencies, a result thatsuggests that the CG subjects also showed an improvement in their condition in the pre - post-testing interval, even if noexperimental intervention was performed on them. These results correlate with those of Study 1 (Chapter 6), which revealed
an improvement in anxiety levels (on the Hamilton scale) recorded during post-testing for CG subjects. Since the CG subjects
showed an increase in electrical activity in the alpha register, it cannot be said with certainty that the increase in brain activity
in the alpha register in the SG subjects was caused by PCOT. Figure 13 displays the mean frequency values within the CG (atall times).

39

Fiziologia - Physiology • 2024.1(106)Fig. 13 Mean values for each frequency within CG, pre- and post-testing.

Fig. 14 Mean values ± S.E.M. of the four frequencies on all channels (CG).
In order to test the subsequent hypotheses 2.1.1, 2.2.1, 2.3.1 and 2.4.1, which regard the mean differences of thefrequencies in the alpha, beta, gamma1 and gamma2 register between CG and SG, we applied the t-test - comparison,
between the SG median and CG median.
Specifically, for each given frequency (α, β, γ1 or γ2), we calculated the mean difference (subtracting from the meanvalue obtained after the experimental interval, the mean value obtained in the recordings before that interval) in the frequency
value, within each group, in all moments.
Whether these differences between SG and CG were statistically significant was then tested, with the P-values
corresponding to the two-sample t-test are:Pα = 0.3274, Pβ = 0.0359,
Pγ1 = 0.0403, Pγ2 = 0.1905.

Fig. 15 Mean difference (post-experiment – before the experiment) for each frequency and each group.
Significant differences for the β and γ1 frequencies are noticed, which confirms the subsequent hypotheses 2.21 and2.3.1 and refutes the hypotheses 2.1.2 and 2.4.1 regarding the α and γ2 frequencies.
For the last two hypotheses, we restrict the previous analyses to the data corresponding to the four registers, collectedfrom the front sensors 1, 2 and 3.
For each volunteer (from both groups) and for each of the α, β, γ1 and γ2 frequencies, we calculated the mean value
40

Fiziologia - Physiology • 2024.1(106)

of the first three channels. Then, we used a statistical test to check whether there were statistically significant differences in
the central values corresponding to each frequency (α, β, γ1 and γ2) pre- and post-testing.
The assumptions of the Wilcoxon signed-rank test are:(H0): the median of the differences (for the entire population) between the two data sets is zero (or, equivalently, thetwo sets have the same median value);(H1): the median of the differences (for the entire population) between the two data sets is not zero.
Table 3 below displays the P-values for all these tests. The red P-values correspond to statistically significant
differences (significance level 0.05) in the median, pre- and post-test.
βPvalue
s
E1

α
0.933
3

E2
E3
E4
E5
E6
E7
E8
E9
E1
0

E1
1

E1
2

E1
3

E1
4

E1
5

E1
6

0.737
7
0.878
0
0.295
3
0.675
5
0.000
1
0.091
3
0.046
0
0.001
0
0.028
5
0.008
0
0.022
1
0.494
1
0.142
8
0.000
6
0.000
0

γ1
0.128

0.078
7

2
0.000
2
0.000
1
0.000
3
0.003
7
0.008
0
0.000
1
0.665
3
0.050
7
0.006
0
0.595
9
0.364
4
0.121
4
0.000
5
0.047
5
0.000
1

β

Pvalues
γ2
0.088
7
0.000
0
0.013
0
0.000
1
0.015
2
0.158
7
0.000
0
0.451
1
0.000
6
0.005
0
0.295
3
0.003
1
0.004
8
0.000
1
0.000
0
0.071
8

---

α
0.944
4
0.013
5
0.000
0
0.557
8
0.023
8
0.000
2
0.209
1
0.000
0
0.576
7
0.485
3
0.002
0
0.000
2
0.024
7
0.308
3
---

---

---

N1
7

0.000
0
0.150
6
0.000
0
0.357
0
0.944
4
0.000
0
0.270
3
0.000
4
0.000
0
0.230
1
0.007
1
0.002
6
0.001
1
0.000
0
0.150
6

N1
8

N1
9

N2
0

N2
1

N2
2

N2
3

N2
4

N2
5

N2
6

N2
7

N2
8

N2
9

N3
0

0.000
4
0.022
9
0.000
2
0.214
2
0.001
5
0.000
0
0.007
1
0.000
0
0.001
6
0.282
6
0.167
1
0.000
1
0.357
0
0.018
4
---

γ1
0.006
0
0.006
5
0.019
8
0.769
5
0.000
9
0.000
5
0.011
5
0.000
0
0.000
0
0.041
6
0.059
6
0.000
0
0.977
7
0.003
9
---

γ2
0.029
5
0.019
8
0.219
4
0.955
5
0.023
8
0.000
7
0.041
6
0.000
0
0.000
1
0.000
2
0.094
0
0.000
0
0.856
0
0.001
0
---

---

---

---

Table 3 P-values for the Wilcoxon signed-rank test for the medians of each frequency. Volunteers E1 - E16 arefrom SG, and volunteers N17 - N30 are from CG.
41

Fiziologia - Physiology • 2024.1(106)To evaluate the last set of hypotheses, we made a comparative analysis of the data of the SG subjects with themselves,in the two moments, pre- and post-testing, corresponding to the three channels (Ch1 – PF1, Ch2 – PFz, Ch3 – PF2). We test
Hypothesis 3.1: PCOT 30/8 induces changes in electrical activity, in all registers, post-testing, compared to pre-testing, in SG
subjects, regarding the three frontal sensors.
Only GS is considered, for significant differences in the pre- and post-testing median value, (only regarding the first
three channels), using the Wilcoxon signed-rank test. For each given frequency (α, β, γ1 or γ2), we calculated the mean valueof the frequency within the group for the three frontal sensors at all times. Next, we tested whether there were significant
differences in the mean value before and after the experiment. The corresponding P-values for the Wilcoxon signed-rank testare:Pα = 0.0787, Pβ = 0.5392, Pγ1 = 0.0091, Pγ2 = 0.5672.
For SG, we notice significant differences in medians (pre- and post-testing) only for the γ1 frequency. Figure 16 shows themean frequency values within the SG (at all times). Figure 17 shows the total mean values ± S.E.M of the four frequencies, at
all moments. Here, by the total mean value we imply the mean of all the means of the frequency group, considered for all
moments.

Fig. 16 Mean values for each frequency in SG subjects, pre- and post-testing.

Fig. 17 Total mean values ± S.E.M. of the four frequencies, across all channels, for SG subjects.
An identical examination is carried out for the subjects of the Control Group, in order to identify significant differencesin the median pre- and post-testing value, only for the first three channels, corresponding to the frontal sensors, Ch1 – PF1,
Ch2 – PFz, Ch3 – PF2). We use the Wilcoxon signed-rank test to identify significant differences in pre- and post-testing median42

Fiziologia - Physiology • 2024.1(106)CG values for the first three frontal channels.
For the given frequencies (α, β, γ 1 or γ2), we calculated the mean value of the frequency within the Control Group, forthe three frontal sensors, at all times, testing whether there are significant differences in the pre- and post-testing mean value.
The corresponding P-values for the Wilcoxon signed-rank test, for CG subjects, arePα = 0.0021,

Pβ = 0.5030,

Pγ1 = 0.3570,

Pγ2 = 0.4103.

For CG, we notice significant differences in the medians (pre- and post-testing) only for the alpha frequency, asituation that correlates with the results obtained in Study 1, which identified statistically significant changes also in the caseof CG, in the absence of an experimental intervention.
Figure 18 shows the mean frequency values within the CG (at all times). Figure 19 shows the total average values± S.E.M of the four frequencies, for the first three channels. (By the total mean value we refer to the mean of all means of thefrequency group, considered at all times.)

Fig. 18 Mean values for each frequency in CG subjects, pre- and post-testing.
43

Fiziologia - Physiology • 2024.1(106)Fig. 19 Total mean values ± S.E.M. of the four frequencies, across all channels, for CG subjects.
Finally, for the last set of working hypotheses, we compare the median of the experimental group with the medianobtained for the control group (only for the first three channels) (Hypothesis 3.2). We use the two-sample t-test for this. For
each given frequency (α, β, γ 1 or γ2), we calculated the mean difference (subtracting the pre-testing mean from the post-testingmean) in the frequency value within each group at all time points. Next, we tested whether these differences weresignificant between SG and CG. The P-values corresponding to the t-test, for the two samples, are:Pα = 0.1304,

Pβ = 0.3816, Pγ1 = 0.3172,

Pγ2 = 0.5805.

One can notice no significant differences. Some observations regarding the limitations present in the current study will
be discussed in a later section within this chapter.

Fig. 20 To highlight the possible changes brought about by the PCOT experience, we render the mean difference(post and pre-testing), for each group and for each frequency.
8. DISCUSSIONS
Many studies highlight that S/RE trigger changes in brain electrical activity patterns [176], those focusing on Buddhist,

44

Fiziologia - Physiology • 2024.1(106)yoga or transcendental meditation practices being more numerous than those approaching Christian spiritual practices 3 [177].
In regard to the alpha frequency spectrum, we have seen, using the Wilcoxon signed-rank test comparitively, andevaluating the significant differences in the median value for the pre/post-experience SG subjects, statistically significant results.
Testing whether there are significant differences in the median value before and after the experiment, in SG, we have identified
statistically significant values of P, regarding the alpha log, respectively Pα= 0.0001, which suggests that the practice of PCOT
could cause an increase in the intensity of electrical activity of the brain in this register.
Increases in electrical activity in the alpha register (8–12 Hz), correspond to an idling state, with reduced processingof sensory information [178], a non-directive meditation [179], but also to various stages of rest, while this state of relaxation
does not mean an empty mind [179]. The intensification of the activity in the alpha band is present in some types of meditation[180] and in connection with mindfulness sessions [181] (M - technique also called open meditation OM, of non-directive
monitoring of thoughts).
Enhancement of the activity in the alpha register has also been reported in the case of transcendental meditation (TM)
[182, 183], in experienced practicing meditators [184, 185], but also during Zen meditation [186] and in the case of somemedical assistants, participants for 8 weeks, in an MBT (Mindfulness-based treatment) intervention [187].
An intensification of electrical brain activity in the alpha register has also been reported during the Muslim Dhuha prayer.
In one study, EEG recordings were made while subjects had their eyes open, under three different conditions (resting, fourcycles of prayer, reciting specific verses and supplications and, finally, performing gestures and body postures specific to prayer,
but without the associated recitations). The analysis of the alpha register variations showed a higher mean relative alpha po wer(RPa) during prostration positions (body posture in the Dhuha prayer) compared to the rest state [188]. Therefore, highlighting
the intensification of electrical activity in the alpha register in SG subjects, as a result of PCOT practice (understood asinteraction and dialogue with the Supreme Being) could correspond to the mentioned results.
The gamma frequency spectrum is related to the activity of the visual cortex [189], premotor, frontal and parietal regions[190]. At the same time, the electrical activity of the brain in the gamma band is also related to the unification of the perceptual
field [191], which enters the debate concerning the topic of the conscience [192].
Regarding S/RE, two studies report increases in the gamma register activity in subjects practicing Buddhist meditation[193] or Vipassana, Himalayan Yoga and Isha Shoonya (in this case it is the register between 60 and 110 Hz, corresponding
to the gamma2 band) [176].
Another study revealed differences in mean EEG power in the gamma register among practitioners of the Islamic ritualof Salat (a series of steps and bodily postures performed while reciting verses). The study compared subjects actually
performing the ritual with other subjects only miming the execution (performing gestures without reading the verses).
Recordings showed that the signal strength in the gamma register was higher (frontal and parietal regions) throughout theritual in subjects who actually performed it compared to the level recorded in subjects who only mimicked [194]. The result
suggests the contribution of cognitive and attentional processing in the S/R experience.
In the present study we have highlighted an increase in the activity in the gamma2 register, for subjects from SG,
comparing pre- and post-testing, respectively Pγ2 = 0.0015 . The fact could be significant, considering that Christian
prayer in general, and Christian-Orthodox prayer in particular, is not a relaxation technique, having an important cognitive andattentional component (the text and the task of attention), as well as specific bodily postures.
The beta register is present in EEG investigations of S/RE as well. First discovered by Hans Berger, beta waves havebeen associated in time with various functions, such as focused attention, inhibition of movement and motor planning,
maintaining the status quo, and signalling decision making [195]. It is now accepted that increased beta brain activity
corresponds to anxiety, inability to relax and stress, while suppression of beta signals corresponds to ADHD, daydreaming,
depression, and poor cognition [196].
Another EEG study comparing Buddhist loving kindness meditation (LKM) with Christian religious prayer is significant
for the discussion of the beta register results. The study revealed that the practice of LKM corresponds to increased activity inthe delta, alpha and beta register, while the practice of religious prayer is associated only with increased alpha and gamma
3 An explanation for this fact is proposed by David P. Barash, based on the distinct manner of Buddhism, on the one hand, and of
the three great Abrahamic religions (Judaism, Christianity and Islam) on the other, in relation to the results of experimental sciences (David
P. Barash, Is Buddhism the Most Science-Friendly Religion?, Scientific American, February 11, 2014) [ii]. Current Buddhism quickly
assimilates various results of science, without the intervention of processes of spiritual significance, while in the doctrines of Truth from thethree Abrahamic religions, hermeneutic processes intervene, for the formulation of spiritual interpretations that reconcile the scientific resultswith the doctrine of Truth, considered immutable.
45
Fiziologia - Physiology • 2024.1(106)waves [197].
The result is interesting, because in the present study as well, in the EEG evaluation of the impact trained by the PCOT
practice, at post-testing, the measurements performed on the SG subjects did not reveal intensifications of brain activity in thebeta register, a fact that could represent a starting point for further studies.
9.

LIMITS OF THE STUDY
The present study has several limitations. Some arise from the difficulties of recruiting subjects for the two groups,
aspects also mentioned in the previous chapter.
Other limitations stem from the absence of EEG recordings for theta and delta.
The inclusion of the theta register in the dependent variable package in the EEG exploration of PCOT may haveprovided new data. A study on mindfulness, for example, found that, unlike regular relaxation, non-directive meditation sessions
lead to increased electrical activity in both alpha band and theta register [179]. However, mind-wandering episodes, in addition
to the decrease in amplitude and increase in frequency of alpha oscillations (8-14 Hz), also seem to induce a decrease in thefrequency (increase in amplitude) of theta oscillations (4-8 Hz) [198]. An exploration of PCOT -induced changes in the theta
band compared to alpha could reveal new aspects, if we also take into account a recent study that, in regard to meditation,notes inverse associations between the dynamics in the alpha and theta registers, suggesting a possible functional dissociationof the two neural signatures (alpha and theta oscillations) [199].
Also, the inclusion of the delta register could have provided new information about possible EEG peculiarities of PCOT.
The comparative study mentioned above, which looked at loving kindness meditation - LKM and Christian prayer, reported thepresence of activity in the delta band in the case of LKM, but not in the practice of Christian prayer [197]. Future research,targeting PCOT, could include delta band in the set of dependent variables to verify this aspect.
Other limitations of the present study stem from the insufficiency of recorded data. For example, comparison ofindividual CG and SG data acquired pre/post-experience for three distinct stages (BLEO1, BLEC1 and PE) was not possible
due to insufficient data.

Table 4 P-values for differences between pre- and post-experiment median values (for SG subjects), for three time
moments, BLEO1, BLEC1 and PE.
Since the observed data have a relatively small volume (n = 16 +14), non-parametric tests (Wilcoxon tests, used totest the equality of medians) were used here to the detriment of t-tests for the equality of means. It is known that t-tests for themean are more refined tests (in the sense that the error of accepting a false hypothesis is smaller) compared to non-parametrictests, for the situations where they are applicable.
Something similar can be found in the numerical values corresponding to the intervals in the EEG Measurement
Protocol. The record provided 4 or fewer numeric values. For example:
Interval 3 – Personal Experience (PE) – 4 numerical values;
Interval 6 – Image view (VI) – 4 numerical values;
Interval 8 – Cold Pressure (PAIN/HIP) – 1 numerical value.
For the 4 numerical values of the PE, a standard deviation can be calculated, therefore for the S.E.M., as well, so as
to describe the variability/precision, whereas in the case of interval 8 (PAIN/HIP), for which we have only one numerical value,a standard deviation cannot be calculated, so no S.E.M. to describe the variability/precision.
46

Fiziologia - Physiology • 2024.1(106)For further refined studies, the number of participating subjects (SG and CG) should be higher. At the same time, foreach interval provided in the EEG Measurement Protocol (e.g., personal experience, cold pressor test, etc.) several values areuseful, so that a better assessment of the variability (standard deviation or S.E.M.) is possible. Thus, the number of intervals
in the EEG Measurement Protocol could be reduced (from 10, as provided in the current study, to 4 or 5), with the extensionof the duration of experience for each of the important intervals (PCOT, IV – image view).
Extending the range intended for PCOT (Personal Experience) for SG subjects could provide better conditions andmore data for further highlighting possible differences from CG subjects, and for a more detailed analysis, selectively including
channels corresponding to sensors located in certain regions of interest.
Finally, further research, which would also preserve the IV interval (Image View) could also look for possible correlations
between the practice of PCOT and empathy (by applying some questionnaires), since in many existing prayers in PCOT there
are parts that refer to helping other people. This change is also justified by the results of a 2015 study suggesting the existenceof a distinction regarding increased activity in the beta register in the experience of empathy, discriminating between thesituation in which subjects are asked to rate the emotional state of others and the situation in which they rate their own emotions
arising from reporting to the condition of others [iii].
10.

CONCLUSIONS
Compared to the proposed working hypotheses, the results of this study are as follows:
Hypothesis 2.1 – PCOT practised for 30 min./day, 8 weeks, causes an increase in brain electrical activity in the alpharegister in SG subjects during post-testing compared to pre-testing level.
Hypothesis 2.2 - PCOT practised for 30 min./day, 8 weeks, causes an increase in brain electrical activity in the beta
register in SG subjects during post-testing compared to pre-testing level.
Hypothesis 2.3 - PCOT practised for 30 min./day, for 8 weeks, causes an increase in brain electrical activity in the
gamma1 register in SG subjects during post-testing compared to pre-testing level.
Hypothesis 2.4 - PCOT practised for 30 min./day, for 8 weeks, causes an increase in cerebral electrical activity in the
gamma2 register in SG subjects.
RESULTS
Applying the Wilcoxon signed-rank test revealed significant differences in median value in the case of SG subjects,
pre/post-experience. The intended experimental design (PCOT, 30 min./day, for 8 weeks), revealed changes only in the alphaand gamma2 registers, but not in the beta and gamma1 registers.
For each given frequency (α, β, γ1 or γ2), the mean value of the frequency within the SG, for all time points, wascalculated. The existence of significant differences in the median values was then tested.
Corresponding to the significant differences in the median value before and after the experiment, the corresponding Pvalues for the Wilcoxon signed-rank test are:Pα = 0.0001, Pβ = 0.9111,

Pγ1 = 0.1085, Pγ2 = 0.0015.

The obtained values validate working hypotheses 2.1 and 2.4 (frequencies α and γ2) and invalidate working
hypotheses 2.2 and 2.3.
Working hypothesis 2.1.1 - The mean differences in alpha register frequencies between SG and CG are statisticallysignificant.
Working hypothesis 2.2.1 - The mean differences in beta register frequencies between SG and CG are statisticallysignificant.
Working hypothesis 2.3.1 - The mean differences in gamma1 register frequencies between SG and CG are statisticallysignificant.
Working hypothesis 2.4.1 - The mean differences in gamma2 register frequencies between SG and CG are statisticallysignificant.
Results
The mean difference in the frequency value (α, β, γ 1 or γ2) calculated for SG and CG and the application of the t-test
revealed statistically significant differences between SG and CG. The P-values corresponding to the t-test are:47

Fiziologia - Physiology • 2024.1(106)Pα = 0.3274, Pβ = 0.0359, Pγ1 = 0.0403,

Pγ2 = 0.1905.

The obtained values validate the working hypotheses 2.2.1 and 2.3.1 (frequencies 𝛽 and 𝛾1) and invalidate the working
hypotheses 2.1.1 and 2.4.1 regarding the alpha and gamma register2.
Finally, the last two hypotheses, which regard possible changes induced by PCOT practice, 30 min. daily for 8 weeks,in the electrical activity in the prefrontal region in all four registers (alpha, beta, gamma1 and gamma2) among subjects in SG
compared to CG evolution, the results are inconclusive. For this we compared the median SG with that of CG, for the first threechannels (Ch1 – FP1, Ch2 - FPz, Ch3 – FP3), hypotheses 3.1 and 3.2 not being supported. The research did not reveal thatthe practice of PCOT induces changes in the electrical activity in the prefrontal region, in all registers, comparing the posttesting moment and the pre-testing moment, in the SG subjects (Hypothesis 3.1). There were no differences between the
median SG and the median CG at the time of post-testing (Hypothesis 3.2), but we recorded some changes in the alpharegister, in subjects from CG, changes that appeared in the experimental interval, between the pre- and post-testing phases.
As we have mentioned, it would be important for further research to improve the experimental design in order to provide
sufficient primary data to allow more accurate assessments.
11. FINAL CONCLUSIONS
Together with two other previous studies [1,2], the present paper is part of a research project included in the doctoral
programme completed in July 2023 with the defence of the PhD thesis entitled Neuroscience of Spiritual Life at UMPCD
Bucharest. The other two previous studies aimed to highlight possible changes in some salivary markers [1], and some
psychometric indicators [2] in the same SG and CG subjects, within the same experiment that had as an experimental task thepractice of PCOT for 8 weeks.
The first study [1] revealed a decrease in the salivary Cortisol (SC) levels and changes in Interleukin 6 (IL-6 sal) levels
in SG subjects. However, regarding SC, the comparative analysis of SG dynamics in relation to CG, pre- and post-testing, did
not reveal significant differences. In addition, whether PCOT corresponds to an increase or decrease in IL-6 sal needs
clarification. The second study [2] revealed post-testing differences, in SG subjects, on the Hamilton scale, regarding the a3
item (“fears/phobias”), and the 14 item (“appreciation of anxious mood at the interview”), suggesting a possible influence of the
PCOT practice, in the sense of decreasing the intensity of fears/phobias and assessing the anxious mood. On the other hand,using the COPE test, higher means were revealed in SG subjects compared to CG subjects, post-testing, regarding e pos(“positive interpretation and growth”) and e rel (“religious approach/religious coping”).
Finally, in the present study, the third of the research project, we noticed that the application of the Wilcoxon signedrank test revealed significant differences in the median value in the case of SG subjects, pre/post-testing in the alpha andgamma2 register. These results suggest that it is reasonable to consider that PCOT practice induces electro-physiological andpsychometric changes, an issue that requires closer examination. Regarding the results obtained in these three studies, further
research is needed to more rigorously evaluate possible correlations between variations in salivary markers, psychometricmeasurements, and electrical activity in the alpha and gamma2 band. For this, improvements are necessary. The use ofsalivary markers requires protocols with multiple sampling times throughout the day to account for variations introduced by thecircadian rhythm. EEG measurements must allow more data to be collected for more rigorous statistical evaluations. On theother hand, the experimental design should provide for longer experimental intervals to allow subjects in SG to adapt to thecomplex experimental tasks. This is because S/RE in general, and PCOT practice in particular, present complex configurations,which aim at cognitive-emotional, behavioural and spiritual aspects.
All in all, transversal approaches and cross-evaluations are needed so as to allow the possibility of comparing
psychometric measurements with electro-physiological and imaging data. In more general terms, for future research,
methodological clarifications are needed, regarding how the demands of the scientific study and the specifics of S/RE can bereconciled. Rigour is essential in exploring S/RE, considering not only the many fundamental questions or epistemologicalchallenges surrounding the relationship between science and religion, but also the therapeutic potential of these practices.
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[ii] Barash, D.: Is Buddhism the Most Science-Friendly Religion?. Scientific American, February 11, 2014. Retrieved April
24, 2024 from https://bloGS.scientificamerican.com/guest-blog/is-buddhism-the-most-sciencefriendly-religion.
[197] Faber, E.S.L.: The neural correlates of two forms of spiritual love: an EEG study. BioRxiv. 2016:
https://doi.org/10.1101/045898.
[179] Lagopoulos, J., Xu, J., Rasmussen I. et al.: Increased theta and alpha EEG activity during nondirective meditation. J
Altern Complement Med. 2009 Nov;15(11):1187-92. doi: 10.1089/acm.2009.0113. PMID: 19922249.
[198] Rodriguez-Larios, J., Alaerts, K.: EEG alpha-theta dynamics during mind wandering in the context of breath focus
meditation: An experience sampling approach with novice meditation practitioners. Eur J Neurosci. 2021 Mar;53(6):1855-1868.
doi: 10.1111/ejn.15073. Epub 2020 Dec 18. PMID: 33289167.
[199] Katyal. S., Goldin. P.: Alpha and theta oscillations are inversely related to progressive levels of meditation depth.
Neurosci Conscious. 2021 Nov 29;2021(1):niab042. doi: 10.1093/nc/niab042. PMID: 34858638; PMCID: PMC8633885.
[197] Faber, E.S.L.: The neural correlates of two forms of spiritual love: an EEG study. BioRxiv. 2016:
https://doi.org/10.1101/045898.
[iii] Woodruff, C.C.: Neuroscience of Empathy and Compassion Lab. Northern Arizona University. Retrieved April 24 fromhttps://nau.edu/psychological-sciences/nec/[1] Mihalache, A.S., Zăgrean A-M, Bălan, D.G. et al.: Possible physiological changes induced by the practice of Prayer in
the Christian-Orthodox Tradition. Physiology. 2023 1(104), 28-46.
[2] Mihalache, A.S.; Vicu, O-M, Zăgrean, L: Evaluation of possible psychometric changes induced by the practice of Prayer
in the Orthodox Christian Tradition. Physiology. 2023 2(105), 75-46.
[1] Mihalache, A.S., Zăgrean A-M, Bălan, D.G. et al.: Possible physiological changes induced by the practice of Prayer in
the Christian-Orthodox Tradition. Physiology. 2023 1(104), 28-46.
[2] Mihalache, A.S.; Vicu, O-M, Zăgrean, L: Evaluation of possible psychometric changes induced by the practice of Prayer
in the Orthodox Christian Tradition. Physiology. 2023 2(105), 75-46.
[1] Mihalache, A.S., Zăgrean A-M, Bălan, D.G. et al.: Possible physiological changes induced by the practice of Prayer in
the Christian-Orthodox Tradition. Physiology. 2023 1(104), 28-46.
[2] Mihalache, A.S.; Vicu, O-M, Zăgrean, L: Evaluation of possible psychometric changes induced by the practice of Prayer
in the Orthodox Christian Tradition. Physiology. 2023 2(105), 75-46.
POSIBILE MODIFICĂRI ELECTROFIZIOLOGICE INDUSE DE PRACTICAREA RUGĂCIUNII ÎN TRADIȚIA CREȘTINĂ ORTODOXĂREZUMAT
Studiul de față își propune să evidențieze posibilele modificări pe care practica rugăciunii (în tradiția creștin-ortodoxă) - PCOT - după unmodel de 30 de minute de practică zilnică, timp de 8 săptămâni - le poate induce în activitatea electrică a creierului. Participanții (n=33) au
fost împărțiți în două grupuri: Grupul de studiu (SG), n=16, cu o vârstă medie de 39,2 ani și Grupul de control (CG) n=17, cu o vârstă mediede 38,6 ani). Grupul de studiu a inclus credincioși creștini ortodocși care nu se roagă zilnic, fiind instruiți în cadrul unei sesiuni de 3 ore cu
privire la specificul psiho-emoțional, comportamental și spiritual al PCOT. Nu s-a efectuat nicio intervenție asupra subiecților din Grupul decontrol. Studiul a inclus, înainte și după perioada de 8 săptămâni de practică zilnică a PCOT, măsurători EEG conform unui protocol demăsurare. Aplicarea testului Wilcoxon signed rank a relevat diferențe semnificative în ceea ce privește valoarea mediană în cazul subiecților
SG, înainte/post-experiență, în registrele alfa și gamma2, dar nu și în registrele beta și gamma1. Sunt necesare cercetări suplimentare și
modificări ale designului experimental, astfel încât să permită evaluări mai precise. Rezultatele sunt discutate în contextul unor studii similare,evidențiind unele limitări specifice ale studiilor care evaluează modificările fiziologice induse de experiențele spirituale/religioase, în special
ale celor care utilizează EEG, formulând în același timp și câteva recomandări pentru creșterea calității metodologice a studiilor viitoare.
Cuvinte cheie: "Experiențe spirituale": Creștinismul ortodox
50

Fiziologia - Physiology • 2024.1(106)ANTIPLATELET MONOTHERAPY VERSUS ANTIPLATELET AND ANTICOAGULANT THERAPYIN REDUCING THE RISK OF PREECLAMPSIA AND FETAL INTRAUTERINE GROWTH
RESTRICTIONANGHEL SAVCIU LOREDANA1, PATRU CIPRIAN LAURENTIU2, PANA RAZVAN COSMIN,2 BALACEANUDOBROMIR CRISTINA1,CERNEA NICOLAE2
Clinical Emergency County Hospital Craiova, Obstetric and Gynecology Departament , St Tabaci nr.1,Craiova,200642
Departament of Obstetrics and Gynecolog ,University of Medicine and Pharmacy,Craiova,St.Petru Rares, nr 2-4,200433
Correspondence PATRU CIPRIAN LAURENTIU , patru_ciprian@yahoo.com, 0761632298
ABSTRACTPreeclampsia is a major cause of increased maternal and perinatal morbidity and mortality with an incidence between 2% and 8% of all
pregnancies. The association of preeclampsia with fetal intrauterine growth restriction and prematurity often leads to long-term consequences.
Administration of low molecular weight heparin (LWMH) may improve placental development and inhibit the reactive pathways involved in
pre-eclampsia and intrauterine growth restriction (IUGR).
We conducted a retrospective observational study, over a 12-month period, in a third-degree maternity in the Clinical Emergency County
Hospital from Craiova. The study included a number of 1200 patients examined starting with the first trimester of pregnancy. A combined
screening algorithm was applied to these patients. This algorithm included: maternal history of preeclampsia, mean blood pressure values,
Doppler assessment of the uterine arteries and serum level of the placental growth factor (PIGF). During this screening we identified anumber of 82 patients at high risk of preeclampsia.
The objective of the study was to evaluate the impact of the antiplatelet therapy compared to combined antiplatelet and anticoagulant
therapy in the occurrence of preeclampsia and intrauterine growth restriction.
The results of the study showed an outcome improvement by reducing the risk of preeclampsia and IUGR in patients who were treatedwith combined therapy vs monotherapy.
Keywords: preeclampsia, IUGR, aspirin, LMWH

INTRODUCTION
Preeclampsia is defined as elevated blood pressure values above 140/90 mmHg with onset after 20 weeks of gestationaccompanied by proteinuria or other signs of target organ damage. It is also an important cause of increased maternal andperinatal morbidity and mortality. Although many studies aimed to identify preventive measures for preeclampsia, the incidenceof this condition has been relatively unchanged in the recent decades because the pathophysiology of this disease is not fully
understood. There is increasing evidence suggesting that suboptimal trophoblastic invasion causes an imbalance of angiogenic
and antiangiogenic proteins, ultimately leading to widespread inflammation and endothelial damage, increased platelet
aggregation and thrombotic events with placental infarctions. [1]The incidence of this disease ranges from 2% to 8% of all pregnancies and is a significant cause of maternal and perinatal
morbidity and mortality. [2],[3] The association of preeclampsia with IUGR and prematurity often leads to long-term
consequences, including increased risk of cerebral palsy and neurodevelopmental delay, respiratory disorders, hypertension,
renal dysfunction, insulin resistance, obesity, cardiovascular disease and reduced work capacity. [4],[5]Cases diagnosed with preeclampsia are also 2 to 5 times more likely to develop hypertension and long-term cardiovascularand cerebrovascular disease [6],[7]Aspirin belongs to the class of non-steroidal anti-inflammatory drugs and its analgesic, antipyretic and anti-inflammatory
effects are due to the inactivation of the cyclooxygenase enzymes COX-1 and COX-2 which leads to the suppression of
prostaglandin and thromboxane production. This reduction of thromboxane also leads to inhibition of platelet aggregation,
producing an antithrombotic effect [8],[9]Low molecular weight heparin (LMWH) has been suggested as an intervention to improve placental function by increasing
blood flow [10], differentiation and invasion of trophoblasts in vivo [11] preventing monocyte adhesion, inhibiting tumor necrosis
factor [12] and decreasing vascular resistance and endothelial function. [13] Therefore, it could improve placental development
and inhibit reactive pathways involved in preeclampsia and intrauterine growth restriction [14],[15],[16]. Although studies havefailed to demonstrate a benefit of low molecular weight heparin in patients at risk, its effect in preventing such conditions islikely to be greater than previously observed when high-risk groups are selected or if heparin treatment is combined with aspirin[17],[18].
METHOD
We conducted a retrospective observational study over a 12-month period between October 2022 and September 2023,
in a third-degree maternity in the Clinical Emergency County Hospital from Craiova. A group of 1200 pregnancies were
examined and subject to a combined screening algorithm that included: history of preeclampsia, blood pressure values,
Doppler velocities of the uterine arteries, serum level of placental growth factor (PIGF). From this group we identified 82
pregnancies at high risk of preeclampsia.
Our primary objective was to assess the impact of the antiplatelet therapy compared to combined antiplatelet and
51

Fiziologia - Physiology • 2024.1(106)anticoagulant therapy in the occurrence of preeclampsia in pregnancies at high risk of developing this condition.
The secondary objectives were to assess the impact of these two treatment options on preterm birth; to assess the impact
on the fetal growth and intrauterine growth restriction; to assess the number of days spent in the neonatal intensive care unit;
to assess the number of the intrauterine fetal deaths. Informed consent was obtained from all patients included in the study.
Inclusion criteria for the study group were: age over 18 years; pregnancy between 11-13 weeks; history of
preeclampsia/uterine artery Doppler changes (pulsatility index) and elevated serum PIGF values.
The exclusion criteria were: maternal age over 36 years, body mass index (BMI) >25 kg/m2; fetal chromosomal ormorphological abnormalities.
Pregnancies identified at high risk of developing preeclampsia were divided into two equal groups of 41 patients each. The
first group received 150 mg aspirin and the second group received a combined treatment with 75 mg aspirin and 1mg/kg body
low molecular weight heparin.
Patients were enrolled in the study in the first trimester at a gestational age between 11-13 weeks during the genetic andmorphological screening program. In the cases identified at risk of developing preeclampsia the antiplatelet and anticoagulan ttreatment was administered. Subsequently, the enrolled patients were monitored for the fetal growth curve, Doppler velocities
of both uterine arteries, umbilical artery velocities and fetal well-being criteria. Fetal assessments were performed at successiveintervals of approximately 4 weeks. Maternal biological parameters were also evaluated (blood pressure, proteinuria/24 hours,
hemoleukogram, uric acid, serum transaminases, lactate dehydrogenase). Depending on the pregnancy outcome, thematernal-fetal evaluation intervals were adapted to each specific pathology (presence of early/late IUGR, persistently elevated
BP values, altered platelet, liver and kidney function values).
RESULTS
According to our study results we detected in the study group 35 cases with preeclampsia. According to the pregnancy
onset of this disease these cases were distributed as follows: 27 cases (65.8%) between 34-37 weeks of gestation, 5 cases(12.19%) between 28-34 weeks of gestation and 3 cases (7.31%) between 24-28 weeks of gestation (Table 1).
In the second group, 29 cases of preeclampsia were diagnosed, also distributed according to gestational age: 24 cases(58.5%) between 34-37 weeks of gestation, 4 cases (9.75%) between 28-34 weeks of gestation and 1 case (2.43%) between
24-28 weeks of gestation (Table 1).
We identified a number of 23 cases (56%) that developed intrauterine growth restriction in the first group and 18 cases(43.9%) in the second group. Thus, in the first group, we had a number of 17 (41.4%) newborns with birth weight below the10th percentile, 5 (12.19%) newborns with birth weight below the 5th percentile and 2 (4.87%) newborns with birth weight
below the 3rd percentile (Table 1). In the second group we had 15 (36.5%) newborns with birth weight below the 10th percentile,
2 (4.87%) newborns with birth weight below the 5th percentile and 1 (2.43%) newborn with birth weight below the 3rd percentile.
Regarding the number of days spent in the neonatal intensive care unit, in the first group we identified 6 newborns that
required more than 14 days and 2 cases were identified in the second group.
We identified 3 intrauterine fetal deaths. Of these, 2 cases were identified in the first study group and 1 in the other group.
One of the fetal deaths identified in the first group was of a fetus diagnosed with early intrauterine growth restriction below the
3rd percentile; in this case, the diagnosis of preeclampsia was made at a gestational age of 26 weeks.
The second case identified with intrauterine fetal death was also from the first study group and occurred at a gestationalage of 29-weeks. This patient was admitted through the emergency department with a uteroplacental apoplexy diagnosis.
In the second group we identified one intrapartum fetal death in a patient with a history of hereditary thrombophilia who
developed severe HELLP syndrome at 32 weeks of gestation. Emergency Caesarean section was performed and still the fetus
died before extraction.
We also recorded a postnatal fetal death in a 30-week pregnancy diagnosed with severe preeclampsia that required
elective extraction. This case was identified in the first study group. Fetal death occurred because of intestinal necrosis due tovascular redistribution and intestinal ischemia. The diagnosis was immediately established and the newborn was transferredto the pediatric surgery clinic where emergency surgery was performed with an unfavorable outcome.
First group- Aspirin 150mg
EP between 34-37 weeks
EP between 28-34 weeks
EP between 24-28 weeks
IUGR <10 percentile
IUGR< 5th percentile
IUGR< 3rd percentile
Neonatal Intensive Care Unit >14
days
Fetal deaths
52

27
5
3
17
5
2
6
2

Second group -Aspirin 75mg
+LMWH 1mg/kgc
24
4
1
15
2
1
2
1
Fiziologia - Physiology • 2024.1(106)Table 1. Distribution of the preeclampsia and IUGR cases according to the specific treatment, gestational age, number ofdays spent in the intensive unit and perinatal deaths
Maternal biomarkers were no significant different in the two groups in terms of maximum blood pressure values. Still
increased values of proteinuria were identified in 18 cases (43.5%) the first group vs 15 (36.5) in the second group, increased
values of uric acid in 6 cases (14.3%) in the first group vs 4 cases (9.75%) in the second. Changes in liver function weredetected in only one case in the second group (case with HELLP syndrome) (Table 2).
First group - Aspirin 150 mg
BP values (systolic/diastolic)Proteinuria/24h >300mg
Uric acid > 6 mg/dl
Platelets no<150000/mm3
Liver enzyme >50 UI/L
LDH>225 U/L

170/100 mmhg
18
6
0
0
0
Table 2. Maternal biomarkers values in the two groups

Second group -Aspirin 75 mg+LMWH 1mg/kg.
165/95 mmhg
15
4
1
1
1

DISCUSSIONS
Due to pathophysiology not yet fully understood, the management of preeclampsia has remained significantly unchanged.
Correct management involves 3 directions - prevention, early detection and treatment. In order to identify and correct theserisk factors, susceptible patients should be referred for preconceptual counselling.
The most effective screening model for identifying women at increased risk of developing PE is to combine maternal factor
analysis, blood pressure measurement, Doppler velocities of the uterine arteries and PIGF levels at a gestational age between
11-13 weeks. [19]In our study, using this screening algorithm we identified 82 high-risk patients who developed preeclampsia before 37
weeks of gestation (62%). This result is similar to a study conducted by Nikos et al. who identified approximately 75% of casesof preterm preeclampsia (birth before 37 weeks gestational age). [1]In the ASPRE study, 1776 women at high risk of developing PE, identified on the basis of combined first trimester screening,were randomized to either aspirin (150 mg per day before bedtime) or placebo from 11-14 weeks to 36 weeks gestation.
According to this study results, aspirin reduced the risk of PE before 37 weeks by 62% (from 4.3% to 1.6%) [19]. In our study.
monotherapy with 150 mg Aspirin reduced the risk of preeclampsia before 37 weeks in 27 cases (65%).
The beneficial effect of aspirin appears to depend on the degree of compliance, with a major decrease of the observed riskin patients with ≥ 90% compliance. [19]Studies show that LMWH transcends anticoagulant properties by promoting differentiation and trophoblast invasion in vivo[11], preventing monocyte adhesion, inhibiting tumor necrosis factor [12] and decreasing vascular resistance and endothelial
function [13]. In addition to these effects, LMWH could have a favorable effect on endothelial function and angiogenesis [14].
Therefore, LMWH administration could improve placental development and inhibit reactive pathways involved in preeclampsia
and IUGR.
Also, 2 randomized trials in high-risk patients showed that adding enoxaparin to standard treatment did not reduce the riskof recurrence of preeclampsia and IUGR. [17],[18]However, the effect of heparin in preventing such conditions is likely to be greater than previously observed if specificgroups of high-risk women are selected or if heparin is combined with low-dose aspirin. [17],[18]In our study, combined treatment with aspirin 75 mg and LWMH 1 mg/kg decreased the risk of developing preeclampsia to
58.5% vs 65.8% (aspirin in monotherapy) between 34-37 weeks, 9.75% vs 12.19% between 28-34 weeks and 2.43% vs 7.31%
between 24-28 weeks of gestation.
According to some studies early IUGR has a prevalence of 1-2% and is associated with preeclampsia in more than 50%
of patients. [20] Doppler abnormalities of the uterine arteries, characteristic of cases associating IUGR and preeclampsia [2 1]
was addressed by Trudinger et al. in a randomized double-blind clinical trial involving aspirin at a dose of 150 mg daily. [21]The results demonstrated an increase in birth weight of 516 g as well as an increase in placental weight.
Leitich et al. conducted a meta-analysis based on 13 clinical trials to study the effects of prophylactic aspirin treatment. [22]He concluded that aspirin treatment at doses of 100-150 mg daily from at least 17 weeks of gestation decreased the occurrence
rate of intrauterine growth restriction by approximately 65%, while the perinatal mortality rate decreased by approximately 60%.
In our study we demonstrated the efficacy of combined treatment with LWMH vs aspirin as monotherapy in reducing theincidence of IUGR as follows: 43.9% vs 56% overall; 36.5% vs 41.4% newborns below the 10th percentile, 4.87% vs 12.19%
newborns below the 5th percentile and 2.43% vs 4.87% newborns below the 3rd percentile. [23],[24],[25],[26]53Fiziologia - Physiology • 2024.1(106)We also recorded a reduction in the number of days spent in the neonatal intensive care unit in patients receiving combined
therapy.
CONCLUSIONS
Although aspirin as monotherapy as well as combined therapy with aspirin and heparin are widely used in the preventionof preeclampsia and IUGR and their efficacy has been proven by meta-analyses, there is still no consensus on the onset of
this therapy or the doses needed or when to replace aspirin with LMWH in specific cases. Thus, further studies are needed to
discover new treatments and prophylaxis methods for preeclampsia and IUGR.
According to our data, we can conclude that combined therapy before 16 weeks of gestation to patients at high risk ofdeveloping preeclampsia decreases the recurrence of this disease, decreases the risk of developing intrauterine fetal growth
restriction and the risks for intrauterine fetal death compared to aspirin 150 mg alone.
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2. Duley L. The global impact of pre-eclampsia and eclampsia. Semin Perinatol 2009;33:130-7
3. Stevens W, Shih T, Incerti D, et al. Short-term costs of preeclampsia to the United States health care system. Am J Obstet
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8. Vane JR, Botting RM. The mechanism of action of aspirin. Thromb Res 2003;110:255-8.
9. Tóth L, Muszbek L, Komáromi I. Mechanism of the irreversible inhibition of human cyclooxygenase-1 by aspirin as
predicted by QM/MM calculations J Mol Graph Model 2013;40:99-109.
10. Kingdom JC, Drewlo S. Is heparin a placental anticoagulant in high-risk pregnancies? Blood 2011;118:4780-8.
11. Quenby S, Mountfield S, Cartwright JE, Whitley GS, Vince G. Effects of low-molecularweight and unfractionated heparinon trophoblast function. Obstet Gynecol 2004;104: 354-61.
12. Manduteanu I, Dragomir E, Voinea M, Capraru M, Simionescu M. Enoxaparin reduces H2O2-induced activation ofhuman endothelial cells by a mechanism involving cell adhesion molecules and nuclear transcription factors. Pharmacology
2007;79:154-62.
13. Torricelli M, Reis FM, Florio P, et al. Low-molecular-weight heparin improves the performance of uterine artery Doppler
velocimetry to predict preeclampsia and small-for-gestational age infant in women with gestational hypertension. Ultrasound
Med Biol 2006;32:1431-5.
14. Yinon Y, Ben Meir E, Margolis L, et al. Low molecular weight heparin therapy during pregnancy is associated withelevated circulatory levels of placental growth factor. Placenta 2015;36:121-4.
15. Grandone E, Brancaccio V, Colaizzo D, et al. Preventing adverse obstetric outcomes in women with genetic
thrombophilia. Fertil Steril 2002;78:371-5.
16. Rai R, Cohen H, Dave M, Regan L. Rando mised controlled trial of aspirin and aspirin plus heparin in pregnant womenwith recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies). BMJ 1997;314:253-7.
17. Llurba E, Bella M, Burgos J, et al. Early prophylactic enoxaparin for the prevention of preeclampsia and intrauterine
growth restriction: a randomized trial. Fetal Diagn Ther 2020;47: 1-10.
18. Roberge S, Demers S, Nicolaides KH, Bureau M, Côté S, Bujold E. Prevention of preeclampsia by low-molecular-weight
heparin in addition to aspirin: a meta-analysis. Ultrasound Obstet Gynecol 2016;47:548-53. 25. Cumpston M, Li T, Page MJ,
et al. Updat.
19. ISUOG Medical Practice Guidelines: The role of ultrasound in screening and monitoring pre-eclampsia
20. Crovetto F.et al.Performance of firt-trimester integrated screening for early and small for gestational age
newborns.Ultrasound Obstet Gynecol 2013
21. Trudinger BJ, Cook CM, Thompson RS, et al. Low-dose aspirin therapy improves fetal weight in umbilical placental
insufficiency. Am J Obstet Gynecol. Sep 1988;159(3):681-5. [Medline].
22. Leitich H, Egarter C, Husslein P, et al. A meta-analysis of low dose aspirin for the prevention of intrauterine growth
retardation. Br J Obstet Gynaecol. Apr 1997;104(4):450-9. [Medline].
23. Sinkey, R.G.; Battarbee, A.N.; Bello, N.A.; Ives, C.W.; Oparil, S .; Tita, A.T.N. Prevention, diagnosis and managementof hypertensive disorders of pregnancy: a comparison of international guidelines. Curr. Hypertens. Rep. 2020, 22, 66.
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24. Hamulyák, E.N.; Scheres, L.J.; Marijnen, M.C.; Goddijn, M.; Middeldorp, S. Aspirin or heparin or both for improving
pregnancy outcomes in women with persistent antiphospholipid antibodies and recurrent pregnancy loss. Cochrane Database
Syst. Rev. 2020, 5, CD012852. [CrossRef]
25. Cruz-Lemini, M.; Vázquez, J.C.; Ullmo, J.; Llurba, E. Low-molecular-weight heparin for prevention of preeclampsia andother placenta-mediated complications: A systematic review and meta-analysis. Am. J. Obstet. Gynecol. 2022, 226, S1126S1144.e17. [CrossRef]
26. Wu, C.; Li, L.; Zhang, J.; Song, Y. Efficacy and safety of low-dose aspirin combined with low-molecular-weight heparinin treatment of preeclampsia: A meta-analysis and systematic review. Arch. Med. Sci. 2021, 18, 1525-1534. [CrossRef][PubMed]MONOTERAPIA ANTIAGREGANTA VERSUS TERAPIA COMBINATA CU HEPARINA CU GREUTATE MOLECULARA MICA IN
REDUCEREA RISCULUI DE APARITIE A PREECLAMPSIEI SI A RESTRICTIEI DE CRESTERE FETALA INTRAUTERINAREZUMAT
Preeclampsia reprezinta o cauză importanta de crestere a morbidității și mortalității materne și perinatale, cu o incidenta cuprinsa intre
2% până la 8% din toate sarcinile. Asocierea preeclampsiei cu restricția de creștere fetală intrauterina și prematuritatea duce adesea laconsecințe pe termen lung. Administrarea de heparina cu greutate moleculara mica (LWMH) ar putea îmbunătăți dezvoltarea placentei șiinhiba căile reactive implicate în preeclampsie și restrictia de crestere intrauterina (RCIU).
Am realizat un studiu retrospectiv observational, pe o perioada de 12 luni , intr-o maternitate de grad 3 din cadrul spitalului Cllinic De
Urgenta Craiova ,ce a inclus un numar de 1200 paciente examinate incepand cu primul trimestru de sarcina. La aceste paciente, s-a aplicat
un algoritm de screening combinat ce a inclus : istoric de preeclampsie, valori ale tensiunii arteriale medii , Doppler la nivelul arterelor uterine,
nivelul seric al factorului de crestere placentara (PIGF). Dintre pacientele screenate am identificat un numar de 82 cu risc crescut depreeclampsie.
Obiectivul studiului a fost evaluarea impactului tratamentului antiagregant plachetar comparativ cu tratamentul combinat antiagregant si
anticoagulant asupra aparitiei preclampsiei, si a restrictiei de crestere intrauterine(RCIU).
Rezultatele studiului arata o imbunatatire a outcome-ului prin reducerea riscului de aparitie a preeclampsiei si a RCIU la pacientele lacare s-a administrat tratament combinat vs aspirina ca monoterapie.
Cuvinte cheie: preeclampsie, RCIU, aspirina, LMWH

55

Fiziologia - Physiology • 2024.1(106)IMPLICATIONS OF HEREDITARY THROMBOPHILIA IN DEVELOPMENT OF FETAL INTRAUTERINEGROWTH RESTRICTION OF UNKNOWN ORIGINANGHEL SAVCIU LOREDANA1, PATRU CIPRIAN LAURENTIU2, POPA ADELINA1, BALACEANU DOBROMIR CRISTINA1,
CERNEA NICOLAE2
1 Clinical Emergency County Hospital Craiova, Obstetrics and Gynecology Department, St Tabaci nr.1, Craiova,200642
2 Department of Obstetrics and Gynecology, University of Medicine and Pharmacy, Craiova, St. Petru Rares, nr 2-4,200433

Correspondence PATRU CIPRIAN LAURENTIU, patru_ciprian@yahoo.com, 0761632298
ABSTRACT
Intrauterine growth restriction (IUGR) is defined as a fetal failure to reach the genetic potential for intrauterine growth. IUGR
complicates up to 10% of pregnancies, representing a primary cause of morbidity and mortality. Hereditary thrombophilia has
been detected in about 65% of pregnancies and because of the association with IUGR, preeclampsia, uteroplacental apoplexy,recurrent miscarriage and fetal intrauterine death, prenatal testing is recommended.
Our study included a number of 122 patients without pre-existing pregnancy pathology (study group) who were comparedwith a control group that included 122 patients with normal birth weight fetuses. Patients were evaluated at about 6-7 weeks
postnatally when we tried to correlate the intrauterine growth restriction with the profile of hereditary thrombophilia(determination of factor II gene mutations, factor V Leiden, MTHFR gene, PAI gene, factor XIII mutation, antithrombin III, protein
C, protein S).
The results of the study showed the presence of hereditary thrombophilia in 82 cases (67.2%) in the study group and in 21
cases (17.21%) in the control group. Among the genetic analyses performed we found a predominance of PAI-1 gene as well
as a positive association with intrauterine growth restriction in case of concomitant presence of factor V Leiden and PAI-1 geneor MTHFR gene.
Key words: hereditary thrombophilia, IUGR, MTHFR, PAI-1
INTRODUCTION
Intrauterine growth restriction (IUGR) is defined as a fetal failure to reach the genetic potential for intrauterine growth. Thismay be due to fetal, maternal or placental factors. [1]Chromosomal abnormalities (most commonly trisomy 13, 18, 21), structural abnormalities, intrauterine infections(cytomegalovirus, rubella virus, varicella-zoster virus infection, toxoplasma infection) can be implicated as fetal causes. [1]Of the maternal factors, the most frequently implicated may be: hypertension, insulin-dependent diabetes mellitus, kidneydisease, autoimmune diseases (systemic lupus erythematosus, collagenases), severe anemia, inherited or acquired
thrombophilia, chronic malnutrition, drug use (tobacco, heroin, cocaine, alcohol), use of teratogenic drugs, depression and highstress levels. [1],[2],[3]Placental factors include placental insufficiency, uteroplacental infarction or apoplexy, placental hemangioma or
chorioangioma, umbilical cord abnormalities (marginal or velamentous umbilical cord insertion, single umbilical artery).
[4],[5],[6],[7]IUGR complicates up to 10% of pregnancies and is a primary cause of morbidity and mortality. [8] These cases may facepostnatal increased risk of hypoglycemia, hypothermia, hyperbilirubinemia, necrotizing enterocolitis, intraventricularhemorrhage, seizures, respiratory distress syndrome, sepsis, and neonatal death. [9]In the long term, cognitive or neurological deficits may be associated, as well as cardiovascular (stroke, coronary heartdisease) and endocrinological disorders (dyslipidemia, diabetes mellitus). [4],[10]Thrombophilia may be associated with IUGR, PE, uteroplacental apoplexy, recurrent miscarriage, MIUF, due to impaired
maternal-fetal circulation including damage to the decidua and spiral arteries also causing a decrease in placental weight;
hypercoagulable state and secondary thrombosis may cause placental infarction through an increased number of syncytial
nodes and "accelerated villous maturation". [11],[12],[13],[14],[15],[16]Because of identification in 65% of pregnancies [4], testing for hereditary thrombophilia associated with IUGR is
recommended by the American College of Obstetrics and Gynecology (ACOG) [17]METHOD
We conducted a retrospective observational study over a 1-year period in the Obstetrics and Gynecology Clinic of theEmergency Clinical County Hospital from Craiova between February 2022 and January 2023.
In our study, we initially extracted from the total number of births completed in the Clinic (3820 births), the cases resulted
with IUGR (400 cases). From these cases we selected a group of 122 patients without pre-existing pregnancy pathologies andcompared with the control group which included 122 normal birth weight fetuses. Patients from the selected cases were
evaluated at 6 weeks postnatally when we tried to correlate the intrauterine growth restriction with the profile of hereditary
thrombophilia. We used a protocol that included determination of mutations of factor II (prothrombin), factor V Leiden, MTHFR
56

Fiziologia - Physiology • 2024.1(106)gene, PAI gene, factor XIII mutation, antithrombin III, protein C, protein S.
The patients selected in the study group presented the following inclusion criteria: age between 18 and 35 years,
primiparous patients, non-smokers and who presented a first trimester ultrasound pregnancy dating scan, patients without apreexisting pathology (diabetes mellitus, hypertension, preeclampsia, systemic lupus erythematosus, antiphospholipid
syndrome).
During the study, fetal monitoring was performed according to the study protocol approved by the Romanian Society of
Obstetrics and Gynecology. This included a detailed morphological scan at the end of the first trimester that assessed the fetal
skull (choroid plexuses, thalamus, median septum), thorax and fetal heart (lung areas, diaphragm, 4-chamber view, cardiac
situs and cardiac area, ejection tracts of the great vessels, confluence of the arches to the left of the spine), abdomen (presenceof stomach, bowel, integrity of the anterior abdominal wall, presence of the bladder and confirmation of the paravesical vessels)
and assessment of the upper and lower limbs (evidence of trisegmented limbs, assessment of the fingers and toes);
assessment of resistivity index of both uterine arteries. Ater the morphological scan, patients were genetically screened bymaternal serum determination of PAPA and HCG. From the patients screened by this first trimester protocol, we selected those
who met the study inclusion criteria mentioned above.
Subsequently, these patients were evaluated at a 4-week interval. Depending on the time of onset of complications (onset
of IUGR related to the gestational age), this study interval was modified and applied to each patient. Pregnancy follow-up
referred to a morphological reassessment scan in the second trimester (20-22 weeks of gestation), fetal growth curvemonitoring, assessment of the fetal intrauterine wellbeing criteria (Manning score) and resistivity index in both uterine arteries
and umbilical artery.
RESULTS
400 births were complicated by intrauterine fetal growth restriction. Of these, 122 cases fulfilled our study inclusion
criteria.

3820
births400 IUGR

3420 NON
IUGR

inclusion criteria

inclusion criteria

122
tested for thrombophilia

82278
untested

122

3298

tested for thrombophilia

untested

21

hereditary thrombophilia
hereditary thrombophilia

Figure 1 Patients sampling.
According to the gestational age we identified a distribution of IUGR as follows: between 30-34 weeks of gestation we
identified a number of 31 cases, between 34-36 weeks of gestation we identified a number of 73 cases and over 36 weeks ofgestation we identified a number of 18 cases (Figure 2).

57

Fiziologia - Physiology • 2024.1(106)Figure 2 Distribution of IUGR cases according to gestational age
Genetic and biological analysis for hereditary thrombophilia was performed at 6 weeks postpartum. Following these results,
we identified that hereditary thrombophilia was present in 82 cases (67.2%) in the group that developed intrauterine growth
restriction and in 21 cases (17.21%) in the group with normal weight newborns.
Among the genetic analyses we found the predominance of the PAI-1 gene (polymorphic variant 4G/5/G) which was foundin 58 cases in the group with restriction and in 24 cases in the group with normal-weight newborns. We also found a significant
association of the MTHFR gene in 55 cases in the first group and in 15 cases in the second group. Homozygous factor V
Leiden was present in 36 cases in the first group and in 4 cases in the second group, while the heterozygous variant waspresent in 32 cases in the first group and in 30 cases in the second group.
As for multiple thrombophilia (presence of more than one thrombophilic factor) a positive association with intrauterine
growth restriction was detected in the case of concomitance between factor V Leiden and the PAI-1 gene or the MTHFR gene.
(Table I)

PAI-1 GENEMTHFR GENE
FACTOR V LEIDEN HOMOZYGOUS
TYPE
FACTOR V LEYDEN HETEROSYGOT
TYPE
FACTOR V LEYDEN + PAI -1 GENE
FACTOR V LEIDEN + MTHFR GENE
PROTEIN S
PROTEIN C

Group I(IUGR)
58 (48%)55 (45%)36 (30%)Group II (normal weight)
24 (20%)15 (12%)4 (5%)

32 (26%)30(24,5%)

85 (69%)80 (65%)2
1

12 (9,8)
10 (8,1%)00Table I Distribution of thrombophilic mutation
DISCUSSION
Thrombophilia is a group of conditions that increase the risk of thromboembolic disease in the general population.[18] The
risk increases in pregnant women due to physiological hypercoagulability caused by certain changes in clotting factors. [19]This state of hypercoagulability and secondary thrombosis can lead to changes in maternal-fetal circulation resulting in adverse
pregnancy outcomes.
IUGR is a frequent cause of increased intrauterine fetal deaths, perinatal morbidity and mortality and long-term
consequences, but in most cases its etiology remains unknown. [20]In the current literature there is no consensus on the strongest association between the type of thrombophilia and IUGR.
Differences could result from the ethnic heterogeneity of the groups included in the study (for example, factor V Leiden is themost common thrombophilia mutation in Europe; in the United States the prevalence is between 3% and 8%, while in Asia it
is a rare mutation [21], [ 22] and also from the types of thrombophilia tested in each study.
Identification of the type of thrombophilia is opportune in order to diagnose a pregnancy at risk of IUGR, as it could minimize
or avoid maternal-fetal complications including decreasing the risk of antepartum fetal death.
In the study group, we identified 82 (67.2%) patients with IUGR fetuses without pre-existing maternal-fetal pathologies,
58

Fiziologia - Physiology • 2024.1(106)while in the control group we identified 21 (17.21%) patients with normal weight fetuses who had hereditary thrombophilia. This
result is similar to a study conducted by Dugalic et al [23] who also found that 69.68% cases with IUGR of unknown origin and18.88% of cases with normal fetal growth had associated hereditary thrombophilia. Also, the results of our study are inagreement with Jamal et al and Kupforminc et al who demonstrated a significant association between IUGR and thro mbophilia.
[24],[25]Depending on the subtype of the tested thrombophilia, we found a predominance of PAI-1(4G/5/G phenotype) and MTHFR(C677T, A1298C variants) gene mutations. Thus, in the IUGR group we had 58 cases (48%) with PAI-1 gene mutation andin the control group we identified 24 cases (20%). In the study group, we identified 55 patients (45%) with MTHFR genemutations and 15 patients (12%) in the control group.
The results of a study by Mirzaei et al [26] BUC show a prevalence of thrombophilia of 32% in the control group and 68%
in the IUGR group. A statistically significant association between types of hereditary thrombophilia and IUGR was present incases of MTHFR mutations, (48% in the restriction group versus 20% in the control group) protein S deficiency (32% versus
8%) and multiple thrombophilia (24% versus no cases in the control group).
Dugalic S et al found these mutations present in 36% for the PAI-1 gene and 27% for the MTHFR gene in cases of IUGRof unknown origin and in 7% of cases in the control group. [23]Mutlu et al [27] examined 204 cases with poor previous obstetric findings for thrombophilia mutations and discovered only
one association with MTHFR mutation (90.9%) related to IUGR and hereditary thrombophilia.
On the other hand, there are studies that do not confirm the association of IUGR and MTHFR mutation. In a study publishedin 2018 that included a total of 303 participants with MTHFR and IUGR mutations, Del Gobbo et al [28], did not find an
association between MTHFR 1298CC genotype and IUGR, but only found a trend of increasing MTHFR 677TT in IUGR.
In our study, the presence of factor V Leiden mutations was for the homozygous variant in 30% of the cases in the study
group and 5% of the cases in the control group. The presence of factor V Leiden mutations was for the heterozygous variant
was detected in 26% of the cases in the study group and in 24.5% of the cases in the control group.
We also identified various thrombophilic combinations. The most relevant was the association between factor V and the
PAI gene which was identified in 69% of cases in the study group versus 9.8% in the control group. We also identified an
association with the MTHFR gene present which was present in 65% of cases in the study group versus 8.1% in the control
group.
A retrospective study published by Vicoveanu et al [29] in 2021, which included 179 pregnancies with inherited
thrombophilia, analyzed neonatal outcomes and concluded that IUGR was statistically significantly associated withhomozygous factor V Leiden mutation (33.3%) and combined MTHFR and factor V Leiden mutations (83.4%).
CONCLUSIONS
According to our study results, we can conclude that there may be a causal relationship between hereditary thrombophilia
and IUGR, especially in relation to PAI, MTHFR, factor V Leiden gene mutations.
There is no consensus on the different types of thrombophilia; their classification should be made both in terms ofthrombogenic risk and in terms of pregnancy risks. The most important thrombophilic risk is the development of IUGR.
The relationship between the different types of hereditary thrombophilia and IUGR is needed to be further studied in order
to identify new ways of intervening to prevent recurrence.
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Y. Leitner et al., “Neurodevelopmental outcome of children with intrauterine growth retardation: Alongitudinal, 10-Year prospective study,” J Child Neurol, vol. 22, no. 5, pp. 580–587, May 2007, doi:
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I. D. Walker, “Thrombophilia in pregnancy,” 2000. [Online]. Available: www.jclinpath.com
H. Pinar, C. J. Sung, C. E. Oyer, and D. 6 Singer, “REFERENCE VALUES FOR SINGLETON AND TWIN
PLACENTAL WEIGHTS.”
B. B. Rogers et al., “Avascular villi, increased syncytial knots, and hypervascular villi are associated withpregnancies complicated by factor V leiden mutation,” Pediatric and Developmental Pathology, vol. 13, no. 5, pp.
341–347, Sep. 2010, doi: 10.2350/09-05-0657-OA.1.
R. W. Redline, “Thrombophilia and Placental Pathology.”“Prevalence of Common Inherited Thrombophilias Factor V Leiden.”
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02, 2019. doi: 10.23750/abm.v90i10-S.8758.
M. J. Paidas, D. H. W. Ku, and Y. S. Arkel, “Screening and management of inherited thrombophilias in the settingof adverse pregnancy outcome,” Clinics in Perinatology, vol. 31, no. 4. pp. 783–805, Dec. 2004. doi:
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1–16, 2011. doi: 10.1097/GIM.0b013e3181faa0f2.
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G. F. Del Gobbo, E. M. Price, C. W. Hanna, and W. P. Robinson, “No evidence for association of MTHFR 677C>T
and 1298A>C variants with placental DNA methylation,” Clin Epigenetics, vol. 10, no. 1, Mar. 2018, doi:
10.1186/s13148-018-0468-1.
P. Vicoveanu, “The association between factor V Leiden, MTHFR C667t/A1298c polymorphisms and pregnancy
outcomes,”
Revista Medico-Chirurgicala, vol. 125, no. 4, pp. 563–569, Dec. 2021, doi: 10.22551/MSJ.2021.04.11.
IMPLICAREA TROMBOFILIEI EREDITARE IN RESTRICTIA DE CRESTERE INTRAUTERINA DE ETIOLOGIENECUNOSCUTA REZUMAT
Restricția de creștere intrauterina(RCIU) este definita ca o incapacitate fetala de atingere a potentialului genetic decrestere intrauterina. RCIU complică până la 10% din sarcini, reprezentând o cauză primară de morbiditate și mortalitate
Datorita identificarii intr-un procent de 65% dintre sarcini si a asocierii cu RCIU, preclampsie, apoplexie utero-placentara,
avorturi recurente si moarte fetala in utero, se recomanda testarea trombofiliilor ereditare associate RCIU.
Studiul nostru a inclus un numar de 122 de paciente fara patologii asociate preexistente sarcinii (lotul de studiu) care au
fost comparate cu un lot martor ca a inclus 122 paciente cu feti cu greutate normala la nastere. Pacientele au fostevaluate la controlul de 6 saptamani postpartum pentru a corobora restrictia de crestere intrauterina cu profilul de trombofilie

60

Fiziologia - Physiology • 2024.1(106)ereditara (determinarea mutatiilor genelor factorului II, a factorului V Leiden, a genei MTHFR , a genei PAI, mutatia
factorului XIII , antitrombina III, proteina C, proteina S.
Rezultatele studiului au aratat prezenta trombofiliei ereditare intr-un numar de 82 de cazuri (67,2%) in lotul se studiu si la
21 de cazuri (17,21%) in lotul martor. Dintre analizele genetice efectuate am constatat predominanta genei PAI-1 cat si oasociere pozitiva cu restrictia de crestere intrauterina in cazul concomitentei prezentei factorul V Leiden si a genei PAI-1
sau gena MTHFR.
Cuvinte cheie: trombofilie ereditara, RCIU, MTHFR, PAI-1

A

FOCUSEDREVIEW

OF

THE

IMMUNERESPONSE

IN

ELECTROCHEMOTHERAPY
1,2*

2,3

2

1

Mircea Bogdan Matei , Christien Oktaviani Matei , Ali Sibel , Leon Zăgrean , Mihaela Georgeta
2,3
Moisescu
1
Physiology and Neuroscience Division., University of Medicine and Pharmacy Carol Davila, Bucharest,
Romania2
Biophysics and Cellular Biotechnology Division., University of Medicine and Pharmacy Carol Davila,
Bucharest, Romania
3
Excellence Center for Research in Biophysics and Cellular Biotechnology, University of Medicine andPharmacy Carol Davila, Bucharest, Romania* Corresponding author
Mircea Bogdan Matei, Physiology and Neuroscience Division and Biophysics and Cellular Biotechnology
Division,
University of Medicine and Pharmacy Carol Davila
Bucharest, Romania
e-mail: bogdan.matei@umfcd.roAbstract
Electrochemotherapy has been established since 2018 as an mainstream major cancer treatment.
Electrochemotherapy is based on reversible electroporation and concomitant low-dose chemotherapy
administration to target both cutaneous and subcutaneous tumor types . Standardized protocols,
including the revised ESOPE (2018)[1] and NICE (2014)[2] guidelines, support electrochemotherapy(ECT), which is practiced in over 150 medical centers across Europe. The Insp-ECT (International
Network for Sharing Practices on Electrochemotherapy) study[3] reported an average 80% objective
response rate for multiple cutaneous tumor types following ECT treatment. The emergence of checkpoint
inhibitors in immunotherapy, such as PD-L1 molecules, has highlighted the immune system's role incombating malignancy[4]. Consequently, the immune system's contribution to ECT's effectiveness,
including impacts on untreated distant tumors, has garnered interest. Some studies attribute the lack of
local recurrence after ECT to immunological mechanisms involving DAMPs molecules like calreticulin and
HMGB1, while others argue that this response is insufficient for a systemic abscopal effect[5]. Ongoing
research explores combining ECT with immuno-stimulants (including gene electro-transfer of immune
molecules) to enhance therapeutic outcomes[6]. This review summarizes reports on the immune
response's role in ECT's clinical efficacy and recent electro/immune therapy combinations.
Keywords: electrochemotherapy, abscopal effect, gene electrotransfer, electroporation
Overview
Electrochemotherapy (ECT) is a local procedure that combines reversible electroporation (EP) of cellular
membrane with low-dose chemotherapy. The intrinsic cytotoxicity of a non-permeant or poorly permeantchemotherapeutic drug delivered intravenously or injected intratumorally, is highly increased by the
exposure of cells to controlled electric pulses which permeabilize the cell membrane and allow anincreased diffusion of the drug inside the cell[7]. To be treated efficiently, the whole tumor must be
exposed to the drug molecules and simultaneously to electric field pulses having an intensity above a
specific threshold value which reversibly permeabilize the cytoplasmic membrane. The steps of an ECT

61

Fiziologia - Physiology • 2024.1(106)procedure are presented in Fig 1.

Fig 1 Schematic representation of ECT procedure: the drug is injected in bolus, after a waiting period of
20 minutes electroporation is performed on one or multiple nodules, after the cell membrane reseals, thedrug exerts the cytotoxic effect ending with the apoptotic/mitotic cell death. (Adapted after Miklavcic, D.,2012) [8]ECT is an effective treatment approach for a wide range of tumor histologies as malignant melanoma,
non-melanoma skin cancers, breast cancer, cervical leiomyosarcoma, basal cell and Merkel cell
carcinoma, Kaposi sarcoma and skin metastases of any histology which are symptomatic due to bleedingor pain. One unique advantage of ECT over conventional surgery or other ablation therapies is thepossibility to treat inoperable primary cancers or metastases like those found near major neuro-vascularpathways [9]. As a therapeutic method for cutaneous tumors, ECT is accepted as a standardized
procedure by ESOPE (European Standard Operating Procedures on Electrochemotherapy) and included
in National Institute for Health and Care Excellence (NICE) guidelines. This standardized treatment
requires the use of medical approved devices (like CliniporatorTM, IGEA [10]) which ensure a minimal
invasiveness, a fast application of electric field, and a simple and clear usability. The method became
more widely used after the recent reports from Insp-ECT (International Network for Sharing Practices on
Electrochemotherapy) about the remarkable efficiency of ECT in large cohorts of patients: 80% objective
response rate in the case of all oncological histologies, and 60–70% complete remission rate following
only one treatment[11].
Some of the major benefits of ECT include: the scarce chance of drug resistance occurrence in case of
repeated treatments, a reduction of chemotherapy dosage and the subsequently increased quality of life
index.
During EP pulses application the cells within the targeted tumor area are subjected to transient structuralchanges which can be modulated by using electric pulses with precisely controlled parameters (number,shape, duration, and repetition frequency, direction of the electric field), electrode geometry, andelectrode position with respect to the tumor. The pulsing procedure given in ESOPE consists in 8 squarewave pulses of 100 µs, resulting in an electric field of 1000V/cm between electrodes at adequate
distances, with variations in protocol depending on tumor size, drug administration pathway, electrodetype and the CliniporatorTM machine used[11]. When choosing the geometry and positioning ofelectrodes one must consider that the whole tumor should be exposed to an electric field above thepermeabilizing threshold and as homogenously as possible[8]. The normal tissue around the tumor
exposed to ECT is less affected since the division rate of healthy cells is slower compared to cancer cells
and the membrane repair capacity is better preserved[12].
During ECT, therapeutic drug concentrations are obtained in tumor cells, resulting in a several foldincreased cytotoxicity[13].This allows the use of lower dosages of bleomycin and cisplatin, compared tostandard chemotherapy, enabling to repeat the procedure several times and avoiding the risk ofoccurrence of major side effects of the drugs (for bleomycin: nausea and vomiting, pneumonia,
pulmonary fibrosis, hematological toxicity, skin reactions; for cisplatin: nausea and vomiting, hair loss,

62

Fiziologia - Physiology • 2024.1(106)impeded wound healing, temporary vision loss, infections, hematological toxicity).
The implications of immune activity during ECT administration have been investigated through various in
vitro, in vivo, and clinical studies, which indicate that the response to ECT differs significantly between
immunodeficient and immunocompetent cases. Additionally, the role of the immune system in the
progression of various solid tumors following combined ECT-immunotherapy treatment is being
increasingly studied, with positive outcomes such as the absence of local recurrence being observed.[14].
ECT and immune response
A series of studies have demonstrated that electrochemotherapy induces immunogenic cell deathmarkers, such as adenosine triphosphate, calreticulin, heat-shock proteins, and high-mobility group box1, which are crucial for generating tumor-specific cytotoxic T cells[15, 16]. The CD8+ T cells involved are
specifically generated and can potentially target cancer cells that are resistant to other treatments.
Studies on ECT in immunodeficient mice also support the immune response theory: the oedema andtumor regression after ECT delivery was significantly reduced in T lymphocyte-deficient mice compared toimmunocompetent ones. ECT-induced local oedema encourages the infiltration of dendritic cells andlymphocytes into the affected area [17], which are found in all tumors treated with electrochemotherapy
post-therapy. Furthermore, electrochemotherapy aids in the maturation of existing Langerhans cells
within the tumor, prompting their movement to the tumor-draining lymph nodes and triggering a robust
peripheral release of anti-tumor monocytes. It was previously thought that electrochemotherapy only
provoked a localized anti-tumor immune response and had no effect on distant untreated nodules,
indicating that the immunogenic cell death induced by electrochemotherapy was not strong enough toeradicate distant tumors. However, recent findings indicate that combining ECT with immunostimulants
like IL-2-based immunotherapy can potentially treat both ECT-targeted and distant nodules18.
Inflammatory responses
Reversible electroporation (EP) alone, without the addition of chemotherapy, does not result in significant
cell death or non-thermal tumor ablation[14]. However, it does induce immunological effects. The
secretion of pro-inflammatory mediators, such as TNF-α, IL-1β, and other cytokines from theelectropermeabilized cells, drives the recruitment of immune cells to the electroporated regions. This
results in an inflammatory microenvironment in the treated area [17], characterized by a highinflammatory cellular infiltration (such as macrophages, dendritic cells, and polymorphonuclear
leukocytes). Additionally, the presence of antigen-presenting cells in the electroporated area leads to theupregulation of APC maturation markers such as the F4/80 antigen or molecules of the type II major
histocompatibility complex class. Interestingly, studies have shown that APC and PMN infiltration also
occurs when electroporation is combined with DNA injection[18], enhancing immunization in DNA
vaccination contexts. Additionally, the release of ATP following EP supports the hypothesis that EP
promotes the chemoattraction of DCs and their precursors, facilitating their differentiation and maturation
into antigen-presenting DCs[17]. Another pro-immunological effect of EP is observed at the vascularlevel, where acute vasoconstriction of blood vessels[14], particularly prolonged in tumor capillaries,
facilitates the extravasation of immune cells and erythrocytes.
Induced immunogenic cell death
The pathway of cell death triggered by a treatment is crucial for its therapeutic success, as it influences
the effectiveness of the systemic anti-tumor immune response. The way in which tumoral cells die due toelectrochemotherapy depends on the specific mechanism of action of the chemotherapy. In this course ofactions the host's immune response is vital in overcoming tumor resistance [14]. Four main types of cell
death have been classified based on the tumor type and chemotherapeutic drug used: (i) mitotic cell
death (with bleomycin), (ii) apoptotic cell death (with bleomycin), (iii) necroptosis (with bleomycin,
cisplatin, and oxaliplatin), and (iv) pyroptosis-like immunogenic cell death (with bleomycin) [19].
One apoptosis type generated by various oncological treatments (such as chemotherapeutic drugs,
oncolytic viruses, physicochemical therapies (cryotherapy), photodynamic therapy or radiotherapy) is theimmunogenic cell death (ICD) [20, 21]. The ICD main mechanism of action is based on the release ofdamage-associated molecular patterns (DAMPs) from affected tumor cells. Critically during immunogenic
cell death, the increase of reactive oxygen species and the subsequent stress in the endoplasmic
reticulum (ER) leads to the increased level of (DAMPs). These DAMPs include the cell surface display ofcalreticulin (CRT) and heat-shock proteins (HSP70 and HSP90), as well as the extracellular release ofmolecules such as adenosine triphosphate (ATP), high-mobility group box-1 (HMGB1), type I interferons(IFNs), C-X-C motif chemokine ligand 10 (CXCL10), and members of the interleukin-1 (IL-1) cytokine
family.)[22]. These molecules act as activators and thus are able to boost tumor-specific immune

63

Fiziologia - Physiology • 2024.1(106)responses. ECT-released DAMPs bound on a series of risk-detecting receptors (or pattern recognition
receptors (PRRs) such as toll-like receptors (TLRs) or NOD-like receptors (NLRs)[23]. These receptors
are constitutive found on innate immune cells (such as dendritic cells (DCs), monocytes, andmacrophages), and their activation leads to the activation of T lymphocytes that are thus able to eliminate
tumor cells via phagocytosis. These processes may be supported and further enhanced by additional
molecular events, such as the exposure of other ER chaperones on the cancer cell surface (including
HSP90), and the secretion of various immunostimulatory cytokines like IL-1β and IL-17 by different
immune cell types, thus facilitating DC recruitment into the tumor bed (also stimulated by ATP), tumor
antigen engulfment by DCs (also stimulated by calreticuline), and optimal antigen presentation to T cells(also stimulated by HMGB1) [15]. ECT-treated melanoma patients have shown dendritic cell infiltration as
a local response to the oncological treatment [24].
This ICD process discussed above can be complementary described as a balance between calreticulin(CRT) and CD47 upregulation on the tumor cell membrane. The CD47 is a widely expressed cell surface
protein that regulates phagocytosis by innate immune cells, acts as a non- phagocytosis main signal
addressed to macrophages and opposes the CRT which acts here as one of the phagocytosis main
signal, as seen in Fig 2. [25].
To conclude, electrochemotherapy (ECT) induces mitotic death in the regions with both electric field anddrug concentration coverage, with the apoptosis cascade triggered by tumor-associated antigens releasefrom electroporated cells. The balance between calreticulin and CD47, as a crucial role/ and/or key
mechanism of action played in the immune response should be a focus of future studies on this ECT
driven immunogenic cell death. It is worth noted that/Notably, cells that receive suboptimal ECT, such as
surviving cancer stem cells or chemo-resistant cells, are later identified and eliminated by the cellularimmune system, specifically by primed effector T cells p(via their earlier exposure to TAAs)[6]. Thesefindings highlight the activation of the immune system following ECT, which may explain the absence of
local recurrence and support the rationale for combining ECT with immunotherapy.
Emerging co-therapies applied with ECT
Although electrochemotherapy has an efficient direct effect for solid tumors, the anti-tumor immune
response it elicits is insufficient to eradicate distant tumors. Emerging combination treatments, including
checkpoint inhibitors and immunomodulators, are now being employed to enhance the immunologicalresponse induced by ECT, thereby promoting a systemic anti-tumor effect.
ECT addresses immune evasion mechanisms when combined with immunotherapy
While immunogenic cell death (ICD) induced by electrochemotherapy (ECT) promotes a beneficial
immune response, tumors also have developed immune evasion mechanisms that are crucial for cancer
progression.
Studies have highlighted the role of tumor neoantigens in the immune recognition of cancer cells. Tumor
cells that present specific mutant antigens or fail to express antigens can evade immune detection.
Immune editing shape tumor cell antigenicity during tumor attacks and reductions creating surviving
resistant cells that contribute to the development of new cancer cell sub-populations.
Among several strategies employed by the tumoral cells to silence the immune response : (i) reducing theexpression of MHC-1 to prevent recognition by immune cells, (ii) diminishing NK cell attacks by lowering
the expression of NK cell receptors and activators, (iii) immune suppressor release that create an
immunosuppressive microenvironment, iv) immunoediting ability of immunocompetent T cells creating
cancerous resistant cell sub-populations[18, 27]. Examples of mechanisms by which tumor cells escapeimmune detection are illustrated in Fig. 3.

64

Fiziologia - Physiology • 2024.1(106)Fig. 2 Schematic representation of ECT as an ICD inducer in the micro-tumoral environment and thecomplex process of generation of tumor-specific cytotoxic T cells : 1) ECT application; 2) alterations in thetumor cell membrane inducing a specific immune reaction followed by the subsequent release of soluble
mediators into the tumor microenvironment which in turn are bounding on dedicated receptors expressedby APC such as dendritic cells, and 3) presentation of tumor-associated antigens, leading to activationand proliferation of type T cytotoxic lymphocytes (CD8+)Note: the number of various immune cells types
in place at the time of electric pulses application influences the impact of ECT in efficient removal oftumor cells. (Adapted after Terenzi et al., Journal of Inorg. Biochem. 2016[26])The immunosuppressive microenvironment is realized mainly by altering the induction of immune
checkpoint receptors (example given CTLA-4, PD-L1) and secretion of anti-inflammatory cytokines bytumoral cells (such as interleukins (IL-10) or metabolic enzymes like indoleamine 2,3-dioxygenase (IDO)[28]). A weak antitumor T cell activity is thus created which promotes the expansion of regulatory T cells(Treg) and myeloid-derived suppressor cells (MDSCs). Furthermore, macrophages are polarized towards
the tumor-promoting M2 phenotype, also known as tumor-associated macrophages (TAMs). ).[28].
Therefore, treatments aimed at enhancing the immune response have become a promising approach in
the oncology field.
Recent advances in immunotherapy have shown promising results in cancer treatment. By combining
immunotherapy with electrochemotherapy (ECT) new possibilities have been offered for long-term cancer
eradication by enhancing and sustaining anti-tumor immunity.[29]. Immunotherapy has recently become
one of the primary treatment options for melanoma. Most of the data on the efficacy of combining
immunotherapy with other treatments have been gathered from patients with this low-survival-rate
disease[30].
In a case report, combining BLM-ECT with ipilimumab, a monoclonal antibody targeting cytotoxic T
lymphocyte-associated protein 4 led to a complete clinical response in a patient with multiple cutaneous
melanoma metastases. Additionally, the appearance of vitiligo-like lesions specifically around the ECT
treatment sites indicated that ipilimumab enhanced the immune activation initially triggered by ECT [11].
In another case report, the combination of BLM-ECT with nivolumab, an anti-programmed cell death
protein 1 (PD-1) inhibitor, used as a fifth-line treatment, showed no evidence of cutaneous or visceraldisease at a 4-year follow-up. This resulted in an extended remission period for a patient with advanced
metastatic melanoma[31].
In a retrospective analysis patients treated with both ipilimumab and BLM-ECT, an objective response
was observed in 67% of them (n=15), with 27% achieving a complete response and 40% a partial
response. However, this study's limitation was the lack of a control group receiving only ipilimumab[32].

65

Fiziologia - Physiology • 2024.1(106)Another retrospective trial compared the effectiveness of BLM-ECT combined with ipilimumab to BLMECT combined with PD-1 inhibitors (pembrolizumab or nivolumab) in treating unresectable or metastaticmelanoma. The ipilimumab group showed a systemic overall response rate (ORR) of 19.2%, while theanti-PD-1 group exhibited a systemic ORR of 40%. The study concluded that ECT combined with PD-1
inhibitors was more effective than ECT with ipilimumab in terms of overall response[33]. These findings
are promising for the treatment of malignant melanoma, highlighting the need for more prospective trials
to fully assess this combination's potential. Currently, a phase II interventional trial of ECT combined withpembrolizumab is underway[34]Fig. 3 Schematic representation of the ways in which tumor cells escape the immune system detection.
(Abbreviations: NK, natural killer T lymphocyte; MHC-1, major histocompatibility complex molecules)
ECT and electrogene therapy
DNA vaccination and cytokine-based anti-cancer therapies represent the most advanced strategies ofgene electrotransfer (GET), also known as electrogenetherapy (EGT), which aims to stimulate anti-tumor
immunity. GET involves delivering plasmid DNA (pDNA) or small interfering RNA (siRNA) molecules tovarious tissues, including tumors, through electroporation. This process allows for the transfer oftherapeutic genetic material, enabling gene therapy to restore specific cellular functions and instruct cells
to produce therapeutic or immunogenic proteins endogenously[35].
GET, which can be administered either intratumorally or peritumorally into the skin, involves in vivo geneelectrotransfer of plasmid DNAs (pDNAs) encoding immunomodulatory molecules like cytokines,
chemokines, and adjuvant sequences. Additionally, DNA vaccines carrying tumor-specific or tumorassociated antigens (TAAs) can be used alone or combined with chemotherapeutics for tumor treatment.
The choice of approach depends on the immunogenicity of the tumors and the immune status of theorganism[36, 37].
Plasmids up to 15 kilobases, edited with a Kozak sequence, are used for electrotransfer-based genetreatments in eukaryotic cells. Gene expression is regulated by environmental and developmental factors,
with regulatory elements located at various distances from the gene. In gene therapy, coding sequencesmust be minimized. Tissue-specific promoters, which limit expression to specific tissues and reduce offtarget effects, are commonly used in clinical trials, though their expression levels are typically lower thanthose of viral promoters. Cell-specific targeting is also achievable through nuclear import sequences[38].
Regulable promoters, which can be controlled by an external factor, have significant potential but should
be designed for inducible rather than repressible expression to minimize patient exposure to the inducing
agent.
Electrotransfer of mRNA is commonly used to engineer antigen-presenting cells in vitro, inducing
antitumor activity when injected into a patient. RNA interference can suppress gene expression after
direct electroporation-mediated delivery of sequence-targeted small RNAs. Double-stranded smallinterfering RNA and stem-loop containing microRNA, derived from endogenous RNAs, are 20–30 basepair oligonucleotides that are processed in the cytoplasm to cleave and degrade specific target mRNAs.
Since ubiquitous and resilient ribonucleases degrade cytoplasmic RNA, this must be considered when

66

Fiziologia - Physiology • 2024.1(106)using electroporation as a delivery method. Pattern recognition receptors identify specific pathogen
components, such as bacterial endotoxin and nucleic acids, triggering an inflammatory response and/or
cell death. Gene therapies, including those involving viruses, plasmids, and oligonucleotides, can bedetected by these receptors and may be mistaken for pathogen invasion, leading to unregulated
inflammatory responses.[36].
Immune stimulation through EGT has shown significant promise for cancer treatment due to its safe
administration [39], enhanced DNA uptake, and adjuvant effects. The stress condition induced by EGT
stimulates local production of inflammatory cytokines, activating the innate immune response andrecruiting M1 macrophages with anti-tumor activity. It also enhances adaptive immunity by attracting
lymphocytes and inflammatory cells to the electroporated areas [27].
One of the most studied applications of EGT is the transfer of an immune gene coding for IL-12, which
promotes the cytotoxicity of immune cells in both the innate and adaptive immune systems, fostering anantitumor type 1 cytokine environment. IL-12 promotes the secretion of IFN-γ from NK cells and T
lymphocytes, enhancing the function of antigen-presenting cells by increasing class I and II MHC
molecule expression. IL-12 also has anti-angiogenic properties[27]. Although recombinant IL-12 has
shown systemic toxicity during clinical testing, the first clinical trial of GET with a plasmid encoding IL-12
in 2008 proved this method is safe and controlled. Studies using IL-2 or IL-12 have demonstrated
antitumor effectiveness on distant untreated tumors and long-term anti-tumor memory [40].
Combining ECT (electrochemotherapy) with immunostimulatory EGT offers a promising strategy toenhance local antitumor response and induce an abscopal effect. Recent studies [36] have focused on
combining BLM-ECT with peritumorally administered IL-12 GET. A study on three murine tumor models
showed that IL-12 GET enhances the antitumor effect of ECT in poorly immunogenic B16F10 melanoma,
while ECT alone is more effective in achieving an abscopal effect and long-term immunity in moreimmunogenic 4 T1 and CT26 tumors[41].
Investigating the immune response associated with ECT and immunomodulatory GET should include
examining regulatory T cells (Tregs). In the tumor environment, Treg-induced immune suppression
significantly hinders anticancer responses targeted by immunotherapeutic strategies[39] . Recent findings
suggest that combining Treg depletion with immunotherapy based GET in the B16F10 melanoma model
could reduce systemic metastasis and improve survival rates[41].
Short interfering RNA (siRNA) is also being explored as a viable oncological treatment. For example,
siRNA targeting VEGF, an angiogenic factor, has been used to suppress tumor growth[42]. Similarly,
siRNA targeting cyclin B1 has shown promising effects in inhibiting tumor cell proliferation and could beclinically validated in combination with other strategies, such as T cell targeting and downregulation of
PD-1 or CTLA-4 [43].
ECT and calcium electroporation
Calcium electroporation (Ca EP) is a safe, cost-effective cancer treatment that introduces high calcium
concentrations into cells via electroporation, causing ATP depletion and cancer cell death. A randomized
double-blind phase II study showed its safety and efficacy in treating cutaneous metastases and recurrent
head and neck cancer[44]. The efficacy is comparable to BLM-ECT for small tumor metastases, with 84%
objective response for BLM-ECT and 72% for Ca EP, with no statistical difference at a one-year followup. Further clinical trials are needed to validate these findings[7].
Although primarily a local therapy, calcium electroporation (Ca-EP) has been shown to trigger a systemic
immune response. In vivo studies revealed that Ca-EP treatment of colon cancer tumors inimmunocompetent mice led to a complete response and long-lasting anti-tumor effects, while
immunocompromised mice did not exhibit the same response. Additionally, in vivo Ca-EP increased thesystemic release of pro-inflammatory cytokines, and in vitro Ca-EP elevated the release of High MobilityGroup Box 1 protein (HMGB1), an important DAMP associated with immunogenic cell death (ICD)[45].
These findings indicate that Ca-EP can activate immune stimulators and induce a systemic immune
response. Another in vivo study showed that combining calcium with irreversible electroporation (whichpermanently permeabilizes cell membranes with high-intensity electric fields) resulted in more T-cells andfewer suppressor cells[46]. Furthermore, a case report documented a systemic response following
treatment with BLM-ECT and Ca-EP in a patient with disseminated malignant melanoma[47].
These studies suggest that Ca-EP can induce a systemic immune response, either alone or in

67

Fiziologia - Physiology • 2024.1(106)combination with ECT. However, further research is necessary to understand the mechanisms of immune
activation and to explore the potential of Ca-EP and immunotherapy combinations.
Conclusions
The immune system plays a crucial role in cancer progression and treatment response, making therapies
that activate the immune response a promising approach in oncology. EP-based therapies have gained
attention in recent years due to their high efficacy rates and lack of severe side effects, showing local
immune activation and markers of systemic response. However, there is no consensus on the abscopal
effect of immune activation from ECT.
Various immunotherapies have been recognized as effective treatments for many solid tumors, including
melanoma. Both immunotherapy and ECT have achieved efficacy levels that warrant their inclusion in thestandardized anti-tumor treatment panel. Consequently, combining immunotherapy with ECT requires
more attention to further improve patient clinical response, quality of life, long-term local and systemic
remission, and life expectancy.
The combination of ECT with gene electrotransfer (GET) of immunomodulatory molecules is currently
under study and showing encouraging results. Promising in vitro, in vivo, and clinical case reports indicate
the potential of calcium electroporation (Ca-EP) to stimulate the release of specific molecules that trigger
immunogenic cell death (ICD).
Despite these advances, the complex mechanisms behind the systemic immune response implications in
ECT and Ca-EP are not yet fully understood, necessitating further research to determine their long-term
impact on patient clinical outcomes.
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RĂSPUNSUL IMUN ÎN ELECTROCHIMIOTERAPIE. SCURTĂ SINTEZĂREZUMATElectrochimioterapia (ECT) a fost stabilită din 2018 ca unul din tratamentele principale împotriva cancerului. ECT sebazează pe electroporarea reversibilă a membranelor celulare și administrarea concomitentă de chimioterapie îndoze mici pentru a viza atât tipurile de tumori cutanate, cât și cele subcutanate. Protocoalele standardizate, inclusiv
ghidurile revizuite ESOPE (2018) și NICE (2014), susțin electrochimioterapia (ECT), care este astfel practicată în
peste 150 de centre medicale din întreaga Europă. Studiul Insp-ECT (International Network for Sharing Practices on
Electrochemotherapy) a raportat o rată medie de răspuns obiectiv de 80% pentru mai multe tipuri de tumori cutanatedupă tratamentul ECT. Apariția inhibitorilor de puncte de control în imunoterapie, cum ar fi moleculele PD-L1, aevidențiat rolul sistemului imunitar în combaterea malignității. S-a dezvoltat aşadar interesul acordat contribuției
sistemului imunitar la eficacitatea ECT, inclusiv impactul acesteia asupra tumorilor îndepărtate netratate. Unele studii
atribuie lipsa recurenței locale după ECT mecanismelor imunologice care implică participarea moleculelor DAMPs
precum calreticulina și HMGB1, în timp ce altele susțin că acest răspuns este insuficient pentru un efect abscopal
sistemic. Cercetările în curs explorează combinarea ECT cu imuno-stimulante (inclusiv electro-transferul genelor al
moleculelor imune) pentru a îmbunătăţi rezultatele terapeutice. Această sinteză rezumă rapoartele privind rolulrăspunsului imun în eficacitatea clinică a ECT și combinațiile recente între electrochimioterapie / imunoterapie.
Cuvinte cheie: electrochimioterapie, efect abscopal, transfer electro-genic, electroporare

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Fiziologia - Physiology • 2024.1(106)EEG REACTIVITY TO EXTERNAL STIMULI IN THE IMPAIRED CONSCIOUSNESSSTATES11

1

1

Alexandru-Cătălin Pâslaru , Vlad-Petru Morozan , Andrei Bordeianu , Alexandra Mocanu , Mihai
1,2
1
1,3,4,5
1
Stancu , Ana-Maria Zăgrean , Mihai Moldovan, Leon Zăgrean
1

Carol Davila University of Medicine and Pharmacy, Division of Physiology - Neuroscience, Department of
Functional Sciences, Bucharest, Romania
2
Ludwig-Maximilian University, Neurobiology, Munich, Germany
3
University of Copenhagen, Neuroscience, Copenhagen, Denmark
4
Rigshospitalet, Department of Neurology, Copenhagen, Denmark
5
Rigshospitalet, Department of Clinical Neurophysiology, Copenhagen, Denmark
ABSTRACTElectroencephalography (EEG) is an important functional assessment tool for patients with an impaired
state of consciousness. Besides brain imaging, EEG brings useful information and is easier to be appliedat the bedside. Sensorial stimulation of patients determines EEG changes referred to as reactivity. Visual,auditory, or somatosensory stimuli, can be used in medical practice or research to measure EEG
reactivity. Reactivity might be impaired in comatose patients, and quantifying the EEG reactivity can
improve the prediction of comatose patients' outcomes. However, no consensus or guidelines presently
exist regarding the methodology of EEG reactivity assessment.
Key words: EEG, reactivity, coma
The electroencephalography (EEG) has long been a cornerstone in the study of brain activity,offering key insights into the neural substrates underpinning various processes and brain oscillatory
rhythms [1,2]. EEG is the simple and non-invasive process of recording the brain's electrical activity andplotting it in a bidimensional time-voltage space.
Over the past four decades, significant advances in EEG technology and analytical methods have
expanded our understanding of how the brain analyses and responds to external stimuli [3]. Due to novel
EEG processing and analysis methods, the technique has gained more and more popularity and utility
both in the clinical setting and research. In the clinical setting, EEG analysis has become an important
tool used to diagnose and monitor patients suffering from sleep disorders, epilepsy, brain injuries, andcoma [4].
Coma, or an impaired state of consciousness, is clinically defined as the absence of clinically
relevant responses to external stimulation. A patient’s level of consciousness is usually assessed using
clinical scales, such as the Glasgow Coma scale (GCS), FOUR score, and Coma Recovery ScaleRevised (CRS-R), together with brain imaging techniques such as fMRI (Functional magnetic resonanceimaging), CT (Computed tomography) and EEG [5–7].
However, imaging techniques may prove impractical at the bedside and there is no consensus on
standardised assessment [7]. An appealing emerging field is tied to the use of EEG reactivity as aprognostic tool for patients who suffer brain lesions that alter the state of consciousness, providing vital
information that can guide clinical decision-making and patient care [8].
EEG reactivity is defined as the change in the background electrical activity of the brain in
response to external stimuli such as auditive, visual, nociceptive or mixed [9,10]. These changes inbaseline activity may be useful in assessing the state of consciousness and the prognosis in clinicalsettings [7]. Moreover, the baseline activity in itself can offer clues regarding the severity of the disorder ofconsciousness. Some patterns are associated with worse outcomes such as spike-and-wave complexes,
burst-suppression patterns, or EEG flatline [11]. Physicians might use it together with other assessment
tools in order to better understand the clinical status of the patient, for example, to differentiate between
the outcome of two comatose patients [6].
There are, however, problems with using EEG reactivity as a measure, or predictor, of thecomatose state in the clinical setting. Despite the versatility of EEG reactivity as a measure or predictor ofcomatose states, the method also poses some difficulties. First and foremost, raw visual EEG
interpretation in the clinic remains subjective [12]. Second, even though quantitative methods formeasuring EEG reactivity in comatose patients have been described [11,13,14], there is a lack of astandardised protocol for assessing EEG reactivity: multiple non-standardised stimulus modalities exist

71

Fiziologia - Physiology • 2024.1(106)and there is still no consensus on what a “sufficient” reactive EEG looks like. With the advance ofmachine learning and computing power, there are a plethora of methods to compute the EEG reactivity
measure, although few have been validated or reached clinical practice. Guidelines for standardised
recording and analysis of EEG are available and in continuous update by the European Academy of
Neurology, American Clinical Neurophysiology Society, and International Federation of Clinical
Neurophysiology [10,15–18].
Considering the fact that patients may have impaired consciousness due to the different types ofbrain lesions, it is intuitive why multiple sensory modalities must exist for assessing the EEG reactivity. It
is therefore essential that the stimulus reaches the brain in order for the EEG to change in response tothat stimulus. One of the first aspects that must be checked when assessing the EEG reactivity is theprimary response to stimuli. This can be done by triggering evoked potentials, which informs on thecapacity of the receptor to react to the stimulus, the integrity of the sensory pathways and the integrity ofthe primary sensory cortex [19,20].
We reviewed established stimulation protocols used in assessing the EEG reactivity, and theirclinical usability from the perspective of our own work. Furthermore, we reviewed novel research trends
using EEG reactivity to gain insights into the brain’s default mode network.
TYPES OF EXTERNAL STIMULI
Visual stimuli
Visual stimulation used in assessing the EEG reactivity in comatose patients involves passive eye
opening, or intermittent photic stimulation (e.g. simple light flashing in the eye). Event-related potentials(ERPs) such as the P300 and N170 components can be used to check if the stimulus reached the cortex.
Normal evoked potentials ensure the integrity of the retina, optic pathway and the primary visual cortex.
These can further trigger the changes in EEG that can be seen as reactivity. Reactivity can be falsely
lower if stimuli fail to reach the cortex [21,22].
Although visual pathways are more difficult to assess in comatose patients, as compared with theauditory or somaesthetic pathways, visual stimulation can be a relevant modality for measuring EEG
reactivity in comatose patients [8]. For example in [13], the authors managed to successfully useintermittent photic stimulation to assess coma severity in a small group of 5 comatose children withacquired brain injury, while in [23], the authors used IPS (intermittent photic stimulation) to assess EEG
reactivity in a study with 32 patients.
Auditory stimuli
Auditory stimuli usually involve actions such as clapping or calling the patient’s own name, and it
is considered more effective than visual stimulation [24]. One advantage of auditory stimulation overvisual stimulation can be that auditory stimuli are delivered correspondingly even if the patient has its
eyes closed or is facing away from the sound source [25]. Paradoxically, whereas visual stimulation has
been cited to be used on its own as a predictor for EEG reactivity, auditory stimulation seems to bemostly used in stimulation protocols that also include other types of sensory modalities such as
nociceptive or tactile stimuli [8,26].
Multiple studies which used stimulation setups that included both auditory stimuli and other typesof stimuli have shown a correlation between a nonreactive EEG measure with a poor outcome in patientswith impaired consciousness [24,27–29], showing that auditory stimulation should be taken into accountas a significant modality when considering a stimulation protocol.
A source of difficulty and lack of reproducibility can be due to the absence of a clinical standard,
as shown by the fact that auditory stimulation can involve different, slightly complex mechanisms such as
clapping or calling the patient’s own name [26], which can involve different hearing ranges and auditory
patterns. As such, to assess the EEG reactivity in a more stable and reproducible way, the authors from[30] used a text-to-speech synthesiser to obtain an audio recording of the subject's own name and thenreversed the same recording to obtain the audio wave of the name spelt in reverse. They used bothrecordings as an auditory-only stimulation protocol to obtain statistically relevant results of their EEG
reactivity measure when compared to controls.
In order for the stimuli to reach the cortex, all the structures involved in stimulus transduction,
conduction and processing should be within physiological working parameters. These structures include
the inner ear, the auditory nerve and the auditory pathway including the bulbopontine junction, lateral

72

Fiziologia - Physiology • 2024.1(106)lemniscus, inferior colliculus, thalamus and up to the primary auditory cortex [31]. Moreover, one canassess the auditory cortical evoked potentials to check for stimulus processes at the primary auditory
cortex: the P1 with a latency of 50ms, N1 latency of 100 ms and P2 with a latency of 180 ms[32]. Alongwith the cortical evoked potentials, there are also brainstem auditory evoked potentials [8].
Difficulties can still arise due to lesions in the auditory pathways or cortex, and/or inner ear
problems either due to age or trauma. Another source of confusion, if the reactivity measurement is donein the ICU can be the noisy baseline environment [8]. In the ICU there are machines that track the heart
rate and produce rhythmic beeps as well as ventilators, aspiration devices that can produce elevatedbackground noise levels, thus disturbing the recording setup. As mentioned previously, no consensus isin place regarding what a reactive EEG actually looks like, the main findings in a reactive EEG being
amplitude and frequency changes. Unfortunately, there are still a significant number of studies thatprovide no definition of what a reactive EEG means, as summarised by M.M. Admiraal et al [26].
Somatosensory stimuli, nociceptive and chemical stimuli
EEG reactivity was shown to respond to somatosensory stimulation in humans both during deep
anaesthesia [33], as well as during a comatose state, as the EEG reactivity to somatosensory stimulation
was shown to predict the outcome of patients who are in a coma after recovering from cardiac arrest or
stroke [34].
The integrity of neural pathways is a sine qua non condition to test the reactivity to these stimuli.
In the case of somatosensory stimulation, the integrity of both the somatosensory pathways and thesomatosensory cortex is of utmost importance.
In the clinical setting, median nerve stimulation is used together with assessment of thesomatosensory evoked potentials: N9, N13, P14, N20, and P25 [8]. No standard electrical stimulation
protocol exists regarding amplitude, waveform and frequency.
The nociceptive modality was proved to be the most effective method to assess the EEG
reactivity [24]. Nociceptive stimuli vary greatly from nipple pinching, periosteal rub, or compression of thefingernail bed [8].
Chemical stimuli could also be used to check EEG reactivity, as for example, olfactory stimuli
produce changes in the EEG [35]. The setup might prove technically more complex, although theolfactory pathway has the advantage of having the fewest synapses until it reaches the cortex.
BURST-SUPPRESSION EEG REACTIVITY
Burst-suppression (BS) is an EEG pattern that is characterised by alternating periods of electricalactivity with high amplitude (burst) and periods of low voltage electrical activity (suppression) [36]. This
type of activity can occur physiologically during foetal life, during coma of different aetiologies or deep
anaesthesia [37,38]. This pattern of activity can be seen as an on-off electrical activity so that an index
can be derived, the burst-suppression ratio. BS ratio is defined as the time spent in suppression from thetotal interest trial time. BS ratio varies from 0 (continuous EEG) to 1 (isoelectric line). The BS patternpresent on an EEG of a patient with acquired brain injury (irrespective of the aetiology), is usuallyconsidered a negative prognostic marker, which is obviously in the absence of any sedation drugs
administered beforehand [39].
Physiological burst-suppression patterns can occur during progressive deepening of anaesthesia,
where the EEG evolves from continuous to BS, with suppression periods getting longer and longer until
an isoelectric line is seen. The isoelectric line does not necessarily involve no brain activity [40]. Thisphenomenon can be completely reversible [40].
There are studies that show that BS patterns change in response to external stimuli, and thischange can be used to assess EEG reactivity. After stimulations, the number of bursts usually increases,
and the time when the EEG is in suppression decreases. An index can be derived using the suppression
ratio during the stimulus-free period and stimulus period, and changes of this index can be used toassess EEG reactivity [33,41]. While BSR depends on the recording setup, more precisely on the
distance between electrodes [42], it can be used to predict the outcome of a patient in a comatose state[13].
CONTINUOUS EEG REACTIVITY IN COMATOSE PATIENTSEEG reactivity was proposed as a marker for patient outcome also in continuous EEG recordings,
improving patient prediction outcome after cardiac arrest [43,44]. An altered reactivity can be seen after a73

Fiziologia - Physiology • 2024.1(106)cardiac arrest, while the reactivity can recover slower or faster depending on the use of hypothermia as
neuroprotection [44,45] as the recovery was observed to be faster in hypothermic patients [44]. As
described previously, there is still no consensus on what a reactive EEG actually looks like. Visuallyobservable changes in amplitude or frequency, or even waveforms, can be used to classify an EEG asreactive, while some studies use standard terminology for EEG assessment [15].
It is considered that an increase in activity following stimulation is a predictor of a good outcome
in regards to a patient’s state, while a decrease in EEG activity is a predictor of a poor outcome [24],while the alpha and theta bands of the EEG signal have a more discriminative power.
DEFAULT MODE NETWORK AND EEG REACTIVITY
The human brain has an extensive network composed of different areas that were found to havea reduction in its activity while performing different tasks [46]. Functional magnetic resonance imaging
and positron emission tomography mapped these areas and named the resting state of this network “A
Default Mode of Brain Function” [47] or default mode network (DMN). Structurally the default mode
network, as known at this time, is composed of three major structures, the dorsal medial prefrontal cortex,
the ventral medial prefrontal cortex and the posterior cingulate cortex with the adjacent precuneus andthe lateral parietal cortex, the area 39 (Brodmann area) [48]. The DMN is active during introspection, selforiented thinking, perceptual decoupling, task-irrelevant thoughts, unconscious thought, or stimulusindependent thought [49], and it is deactivated during task-oriented activity [47,50]. Studies show
deactivation of DMN, a phenomenon which can be called DMN reactivity, to both visual and auditory
stimulation [51].
Functional MRI studies indicate that there is a positive link between cognitive performance andthe functional interconnectivity of the DMN [52–55]. Alterations of DMN functionality are seen in neuropsychiatric diseases like Alzheimer’s disease, but to this time no biomarker that strongly correlates with
Alzheimer’s was observed [56]. A negative correlation can be seen in the functionality of DMN inschizophrenia [57]. Disruptions in DMN connectivity as seen for dementia can be also seen in autism
spectrum disorder patients [58]. EEG assessment of DMN functionality shows a correlation between DMN
connectivity and social anxiety disorder [59,60]. One study also revealed, using both fMRI and EEG
analysis, that out of the major structural components of the DMN, only some correlate with alpha power the medial prefrontal cortex and the posterior cingulate cortex[61].
A method of measuring the most reactive EEG macrostate to photic stimulation (the default EEG
macrostate - DEM), considered the EEG activity of DEM, demonstrated the impairment of DMN in
comatose patients after stroke [23].
CONCLUSIONS AND FURTHER DIRECTIONSThe EEG is a standard assessment method used in comatose patients [62,63]. EEG reactivity ismore and more used either as a standalone method or more frequently in association with other clinicaltools, as stated above, to evaluate the status and assess the outcome of these patients [34,64–66]. EEG
reactivity study and assessment gains more and more popularity as can be seen in Figure 1.
Somatosensory reactivity seems to be a good choice over the auditory reactivity assessment especially inthose with low or impaired auditory reactivity [67]. Pain stimuli are frequently used in the clinicalassessment of coma patients and this can also be easily integrated into EEG reactivity [68]. Althoughvisual reactivity is not the first to be chosen in the clinical setting, different methods of assessing the EEG
reactivity to visual stimuli emerged in both children and adults with an impaired state of consciousness[13,23,69]. Somatosensory evoked potentials and EEG reactivity are good predictors of patient outcome,and combining them with other modalities such as motor response, GCS, or neuroimaging techniques,
can increase the prediction accuracy [70–72].

74

Fiziologia - Physiology • 2024.1(106)Fig 1. A total number of 2,604 articles were found in PubMed, from 1952 to July 2024 that contain “EEG
reactivity”
There is high variety in how EEG reactivity data is acquired, tested, analysed, and interpreted, as
we have shown that multiple simulation protocols exist, and multiple EEG reactivity measures have beendeveloped [10]. At this time, to the best of our knowledge, a consensus for standardised practice for EEG
reactivity in clinical practice has not been reached [67]. Although multiple studies indicate theeffectiveness of EEG reactivity assessment in patient prognostic and the use of EEG reactivity as aprognostic marker, more profound and unified research is needed to understand the basis of reactivity
and to define better protocols that can be used in clinical practice for the prognostication of patients[10,39,73].

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Fiziologia - Physiology • 2024.1(106)Reactivitatea EEG la stimuli externi în stări de conștiență alteratăREZUMATElectroencefalograma (EEG) este o metodă funcțională important pentru monitorizarea pacienților a căror stare deconștiență este alterată. Alături de imagistica cerebrală, EEG adduce informații importante, fiind o metodă mai ușorde utilizat la patul pacientului. Stimularea senzorială a pacienților determină schimbări ale EEG cunoscute careactivitate. Stimulii vizuali, auditivi sau somatosenzoriali pot fi folosiți în clinică sau în cercetare pentru a măsura
reactivitatea EEG. Reactivitatea EEG poate fi modificată la pacienții comatoși, iar cuantificarea acesteia poate oferi
un prognostic mai bun la acești pacienți. La momentul actual, nu există un consens sau ghiduri în ceea ce privește
metodele de evaluare a reactivității EEG.
Cuvinte cheie: EEG, reactivitate, comă79

Fiziologia - Physiology • 2024.1(106)