2021.31.2(102)  Fiziologia - Physiology

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Fiziologia - Physiology  2021.31.2(102)CONTENTS
Fiziologia-Physiology 2021.31.2(102)
1. Chemical agents as antimicrobial factors
Carmen Tatu, Gabriela Tănasie, Cristina Gaspar, Marius Georgescu, Florina Bojin, Daniela Puscasiu, Carmen
Panaitescu
2. Evaluation of the ability of some multidrug-resistant bacterial strains to form biofilms
Cristina Mirabela Gaspar, Brigitta Kis, Carmen Tatu, Carmen Panaitescu, Csilla Zambori, Alina Georgiana Simina,
Camelia Tulcan, Ioan Hutu, Daliborca Cristina Vlad, Virgil Păunescu, Gabriela Tănasie
3. Spread and physiological ecology of nontuberculous mycobacteria
Biciusca Viorel, Sorina Ionelia Stan, Patricia Durand, Biciusca Teodora, Mara Balteanu, Beatrice Mahler
4. An unusual case of morbus ledderhose in a caucasian, young male patient. Case report and literature review
Diana Maria Chiorean, Beatrice Barac, Raluca Niculescu, Andreea Cătălina Tinca, Mihaela Cornelia Sincu, Iuliu Gabriel
Cocuz, Adrian Horatiu Sabău, Beatrice Carmen Patricia Friciu, Ovidiu Simion Cotoi
5. Post-stroke depression – insights on the pathophysiological mechanisms
Oliver Daniel Schreiner, Thomas Gabriel Schreiner
6. Solubility, stability and degradation kinetics of nicotinamide riboside borate, a vitamin b3 derivative, inphysiological solutions
Andreea Silvia Neamtu, Andrei Bită, Ion Romulus Scorei, George Dan Mogoșanu, Cătălina Gabriela Pisoschi
7. Current trends in the assessment of suicide attempts
Eduard Nicusor Bondoc, Marinescu Ileana, Ramona Mălin
8. Prognostic factors in ischemic stroke
Raluca Elena Sandu, Carmen Valeria Albu, Andreea Oana Florescu, Florentina Dumitrescu, Andreea Cristina Stoian,
Emilia Burada, Tudor Adrian Balseanu, Cristian Constantin, Florin Ananu Vreju, Andreea Lili Barbulescu
CUPRINS

1. Agenții chimici ca factori antimicrobieni

Carmen Tatu, Gabriela Tănasie, Cristina Gaspar, Marius Georgescu, Florina Bojin, Daniela Puscasiu, Carmen Panaitescu

2. Evaluarea capacității unor tulpini bacteriene multirezistente de a forma biofilme

Cristina Mirabela Gaspar, Brigitta Kis, Carmen Tatu, Carmen Panaitescu, Csilla Zambori, Alina Georgiana Simina,
Camelia Tulcan, Ioan Hutu, Daliborca Cristina Vlad, Virgil Păunescu, Gabriela Tănasie

3. Răspândirea și ecologia fiziologică a micobacteriilor nontuberculoase (ntm)

Biciusca Viorel, Sorina Ionelia Stan, Patricia Durand, Biciusca Teodora, Mara Balteanu, Beatrice Mahler

4. Un caz neobișnuit de morbus ledderhose la un tânăr pacient caucazian. Prezentare de caz și analiza literaturiide specialitate
Diana Maria Chiorean, Beatrice Barac, Raluca Niculescu, Andreea Cătălina Tinca, Mihaela Cornelia Sincu, Iuliu Gabriel
Cocuz, Adrian Horatiu Sabău, Beatrice Carmen Patricia Friciu, Ovidiu Simion Cotoi

5. Depresia dupa accidentul vascular cerebral – perspective asupra mecanismelor fiziopatologice
Oliver Daniel Schreiner, Thomas Gabriel Schreiner

2021.31.2(102)  Fiziologia - Physiology

36. Cinetica de solubilitate, stabilitate și degradare a boratului de nicotinamidă ribosidă, o derivată a vitamineib3, în soluții fiziologice
Andreea Silvia Neamtu, Andrei Bită, Ion Romulus Scorei, George Dan Mogosanu, Cătălina Gabriela Pisoschi

7. Tendinţe actuale în evaluarea tentativelor de suicide

Eduard Nicusor Bondoc, Marinescu Ileana, Ramona Mălin

8. Factorii de prognostic în accidentul vascular cerebral ischemic

Raluca Elena Sandu, Carmen Valeria Albu, Andreea Oana Florescu, Florentina Dumitrescu, Andreea Cristina Stoian,
Emilia Burada, Tudor Adrian Balseanu, Cristian Constantin, Florin Ananu Vreju, Andreea Lili Barbulescu

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Fiziologia - Physiology  2021.31.2(102)CHEMICAL AGENTS AS ANTIMICROBIAL FACTORSCARMEN TATU1,2, GABRIELA TĂNASIE1,2, CRISTINA GAȘPAR3, MARIUS GEORGESCU1, FLORINA BOJIN2,4, DANIELA PUȘCAȘIU5, CARMEN PANAITESCU1,2
Department of Functional Sciences, Physiology, Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of
Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara
2
OncoGen Research Center, Timisoara County Emergency Clinical Hospital, Liviu Rebreanu Boulevard, no. 156, 300723, Timișoara
3
Banat’s University of Agricultural Science and Veterinary Medicine “King Michael the Ist of Romania” from Timisoara, Aradului str no. 119, 300645,
Timișoara, Romania
4
Department of Functional Sciences, Immunology, Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of
Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara
5
Department of Microscopic Morphology, Cellular and Molecular Biology, “Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania,
Eftimie Murgu Sq. no. 2, 300041 Timișoara
1

ABSTRACT
A wide variety of chemicals can be used as antibacterial agents to prevent contamination. Antiseptics and disinfectants are widely usedin hospitals and are an essential element in combating and preventing nosocomial infections. A wide variety of active chemicals or biocides are present in these products. Disinfectants and antiseptics affect bacteria in many ways. The widespread use of antiseptics anddisinfectants has contributed to the development of microbial resistance, in particular cross-resistance to antibiotics. In medical practice,antimicrobial agents must be selected taking into account the specific microorganisms, pH, solubility, toxicity, organic material present
and last but not least the cost. No ideal antimicrobial chemical exists, however the correct choice and proper use of current substances
will result in an adequate level of disinfection. This paper aims to review the main antimicrobial chemical agents, the mechanisms bywhich they act and their importance in clinical practice.
Key words: antimicrobial chemicals, alcohols, halogens, aldehydes, quaternary ammonium compounds, silver.

INTRODUCTION

During life we ​​are constantly exposed to a wide variety ofgerms. Last year, the public health emergency surrounding
the COVID-19 pandemic highlighted the importance of goodbiosecurity measures and practices. To prevent the spread of
infection, disinfection and hygiene habits are crucial, especiallywhen the microorganism can persist and survive on surfaces.
Contaminated surfaces have been called fomites and sometimes
microorganisms can survive here even for months [1]. As aresult, fomites serve as an important reservoir of pathogens andfacilitate the transfer between hosts. To prevent contamination,a wide variety of chemicals can be used as antibacterial agents.
At present there are various classifications of them but the vast
majority take into account the mechanism of action by whichthese substances exert their bactericidal effect. Antiseptics anddisinfectants are widely used in hospitals and are an essential
element in combating and preventing nosocomial infections [2]. A
wide variety of active chemicals or biocides are present in theseproducts. Disinfectants and antiseptics affect bacteria in many
ways. Those that lead to bacterial death are called bactericidal
agents. Those that cause temporary growth inhibition arebacteriostatic agents. There is no single antimicrobial agent that ismost effective in all situations. That is why different situations may
require different agents. The increase in the potential for microbial
contamination and the risk of infection has led to an increase in

the use of antiseptics and disinfectants by the general public [3].
Despite this, knowledge about the mode of action of these agentsis still limited. It is, however, well known that their spectrum ofactivity is wider than that of antibiotics. While antibiotics tend
to have specific intracellular targets, antibacterial chemicals
may have multiple targets. A number of techniques such as:
lysis of intracellular constituents, examination by studying thedisruption of cellular homeostasis, study of enzyme inhibition,
electron transport and oxidative phosphorylation, interaction
with macromolecules, examination biocides are used to study
the mechanisms of action of antiseptics and disinfectants on
microorganisms[4, 5]. The widespread use of antiseptics anddisinfectants has contributed to the development of microbial
resistance, in particular cross-resistance to antibiotics [2]. A
number of factors affect the selection of the best agent for
any given situation. Antimicrobial agents should be selected
for specific micro-organisms and environmental conditions.
Additional variables to consider when selecting an antimicrobial
agent include: pH, solubility, toxicity, organic material present
and last but not least cost. The present paper aims to review
some of the antimicrobial chemical agents, the mechanisms bywhich they act and their importance in clinical practice.
1. Alcohols
The mechanism of action of alcohols is nonspecific, thebacteriostatic effect is explained by their ability to distort proteins.
They are also solvents for lipids and as a result, can damage lipid
complexes in the cell membrane. Cells will be lysed and cellular
metabolism will be disrupted [2, 6]. This is supported since the

Received 4th of July 2021. Accepted 11th of August 2021. Address for correspondence: Carmen Tatu, Department of Functional Sciences, Physiology, Centerof Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq.
no. 2, 300041 Timișoara e-mail: carmen.tatu@umft.ro2021.31.2(102)  Fiziologia - Physiology

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1950s by specific studies that have shown the denaturation of
the dehydrogenases of the bacterium Escherichia coli and thedelayed growth of Enterobacter aerogenes, actions explained byinhibiting rapid cell division [7]. In addition to these mechanisms,
alcohols are also known as dehydrating agents. Under certain
conditions, the elimination of water from the cells caused by theaction of alcohol has a bacteriostatic effect. This may explain
the relative inefficiency of absolute alcohol on “dry” cells. In
addition to these mechanisms, part of the effectiveness of
alcohol in disinfecting surfaces can be attributed to its cleaningor detergent action which results in the mechanical removalof microorganisms. It has been observed that the general
antimicrobial activity of alcohols increases with the length of the
carbon chain, the maximum being reached at six carbon atoms[8]. As a result, the most effective are: ethanol, isopropanoland n-propanol. Alcohols have broad-spectrum antimicrobialactivity against vegetative bacteria (including mycobacteria),
viruses and fungi, but have no sporicidal effect. However, it is
known to inhibit the sporulation and germination of spores, aneffect that is reversible [9]. Due to the lack of sporicidal activity,
alcohols are not recommended for sterilization, but are widelyused for disinfecting hard surfaces and skin antisepsis. Lower
concentrations can also be used to potentiate the activityof other biocides. Among the mentioned alcohols, isopropyl
alcohol is considered more effective against bacteria, while ethyl
alcohol is more powerful against viruses [2]. However, this isdependent on both the concentrations of the active agent andthe microorganism tested. Against S. aureus, E. faecium or P.
aeruginosa, the bactericidal activity of ethyl alcohol appears to
be 80% stronger than 95% [10].
Ethanol is also effective against various mycobacteria. Since
1947 it has been shown that ethanol at a concentration of
95% kills M. tuberculosis in sputum within 15 seconds, while aconcentration of 70% required a contact time of 30 seconds andat 50% 60 seconds [11]. The same very effective bactericidal
activity was observed with 70% ethanol against M. bovis [12].
In addition, ethanol has a wide bactericidal activity against mostfungi, including yeasts and dermatophytes [12, 13, 14]. As forthe activity of ethanol against viruses, it largely depends on theconcentration of ethanol. As expected, higher concentrations of
ethanol (95%) generally have better antiviral activity than low
concentrations (60 to 80%), especially against non-encapsulated
viruses [15]. Most non-encapsulated viruses such as poliovirus,
astroviruses, feline calicivirus, rotaviruses and ecoviruses arealso inactivated by ethanol [13, 16, 17, 18]. The hepatitis A virus
is perhaps the only virus that is not completely inactivated in thepresence of ethanol. Regarding encapsulated viruses, ethanol
is active against vaccinia virus, influenza A virus, togaviruses,
Newcastle disease virus, HIV, HBV and herpes simplex viruses[19, 20].
Isopropyl alcohol (isopropanol) has higher lipophilic
properties than ethyl alcohol and is less active against
hydrophilic viruses such as polio. The bactericidal activity of
isopropanol starts at a concentration of 30% and increases
with increasing concentration, but an interesting aspect is the
reduction of bactericidal activity to 90% [8]. The same is true
for the bactericidal activity of n-propanol. It appears that thebactericidal activity of isopropanol refers to 13 gram-positive
species, 18 gram-negative species. Tuberculocidal activity of
isopropanol was also tested and good results were observed at
6concentrations between 50 and 70% [8]. It has limited activity
against non-encapsulated viruses. However, if the exposure
time is prolonged, it has an effect against non-encapsulated
viruses such as ecovirus (90% isopropanol for 10 minutes), feline
calicivirus (50-70% isopropanol for 3 minutes) or adenovirus(50% isopropanol for 10 minutes). 10 minutes) [21]. Isopropanol
has no sporicidal activity against B. subtilis and Clostridium
novyi spores [8].
N-propanol has been discovered since 1904 and has been
described as an alcohol with a very strong bactericidal effectstarting from a concentration of 30% [22, 23]. The antimicrobialactivity of n-propanol is thought to be similar to that of isopropanol[24]. In general, the antimicrobial activity of alcohols issignificantly lower at concentrations below 50% and is optimalin the range of 60-90%. Ethanol has been the first recommended
for hand disinfection since 1888 and the antimicrobial activity of
isopropanol was first investigated in 1904 [23]. This was followedby many studies that supported the use of the two propanols forhand disinfection [8]. The following classification of bactericidal
activity was established: n-propanol is stronger than isopropanol
and isopropanol is stronger than ethanol. Also, the bactericidal
activity is higher at a temperature between 30 and 40 degrees
Celsius compared to the range of 20-30 [25].

2. Halogens
A halogen is any of the five elements (fluorine, chlorine, bromine,
iodine and astatin) in group VII A of the periodic table. They exist
as free diatomic molecules and form salt as compounds withsodium and most other metals. Chlorine or iodine are important
halogens used as potent antimicrobial agents either in their freeform or in the form of their compounds.
Chlorine and its compounds
Chlorine is most commonly used in gaseous form or as its
compounds. Chlorine in gaseous form is difficult to handle
unless special equipment is available for its distribution.
Therefore, chlorine gas is used in large-scale operations. Many
chlorine compounds are now available, which can be used
more conveniently than free chlorine and which, under properconditions of use, are as effective as free chlorine. Calcium andsodium chloride compounds in the form of calcium hypochlorite- Ca (OCl) 2 and sodium hypochlorite - NaOCl are used insmall-scale operations. In addition to hypochlorites, there areother chlorine compounds in the chloramine group. Chloramines
are more stable than hypochlorites in terms of prolonged
chlorine release and are often used as disinfectants, sanitizers
or antiseptics. Examples of chloramines are monochloramine(NH2Cl), chloramine-T and azochloramide. As a mechanism ofaction, it is known that chlorine acts on microbial cells throughhypochlorous acid formed as a result of the reaction between
free chlorine and water. However, hypochlorites and chloramines
undergo hydrolysis, resulting in the formation of hypochlorous
acid. The fate of the hypochlorous acid formed in each case is thesame, it undergoes decomposition resulting in HCl and oxygen.
Oxygen released during the decomposition of hypochlorous acidis a strong oxidizing agent. It acts on the cellular constituentsof microorganisms and leads to their death. The destruction of
microbial cells by chlorine and its compounds is achieved bydirect effects on cell membranes and enzymes.
Fiziologia - Physiology  2021.31.2(102)Iodine and its compounds
Iodine in its crystalline form is black-bluish with a metallic luster.
It is poorly soluble in water, but slightly soluble in alcohol andaqueous solutions of potassium or sodium iodide. Althoughless reactive than chlorine, iodine is bactericidal, fungicidal,
tuberculocidal, virucidal and sporicidal, being an extremely
effective bacterial agent, unique in that it is effective against
all types of bacteria [26]. Iodine solutions are already used 150
years ago as antiseptics, being considered one of the oldest andmost effective antimicrobial agents, traditionally used as “iodine
tincture”. Aqueous solutions are generally unstable, at least
seven species of iodine are present in the solution. Of these,molecular iodine is primarily responsible for antimicrobial efficacy[26]. More recently, iodine has been combined with an organic
compound to form iodophore. Iodophores are the so-called“iodine carriers” or “iodine-releasing agents”. The most used
are povidone-iodine and poloxamer-iodine both in antiseptics
and as disinfectants. They are considered less active against
certain fungi and spores [27]. The mode of action of iodine and its
compounds is not clearly understood. Iodine, being an oxidizing
agent, is considered to have the property of irreversibly oxidizing
and thus inactivating essential metabolic compounds, such asproteins with sulfhydryl groups.
3. Aldehydes
Most low molecular weight aldehydes are antimicrobial. Twoaldehydes, formaldehyde and glutaraldehyde, are the mosteffective and are most commonly used to kill spores, therefore
they are sporicidal. As a mechanism of action, both commonlyused aldehydes, formaldehyde and glutaraldehyde, are highly
reactive molecules that combine easily with organic nitrogencompounds, such as nucleic acids and proteins, and inactivate
them, probably by crosslinking and alkylating molecules.
Inactivation of nucleic acids and proteins disrupts the functionof cellular organs and, as a result, cells are killed.
Glutaraldehyde has a broad spectrum of activity
against bacteria, spores, fungi and viruses. Studies on its
mechanism of action have shown a strong adhesion of
glutaraldehyde to the outer layers of microorganisms such as
E. coli and Staphylococcus aureus, inhibition of the transportof gram-negative bacteria, inhibition of dehydrogenase andother enzymes, prevention of lysis induced by a number ofsubstances in S. aureus and in E. coli and inhibition of RNA,DNA and protein synthesis [2, 28, 29]. Glutaraldehyde is more
active at alkaline pH than acid. Because the external pH at thecell surface changes from acid to alkaline, a faster bactericidal
effect is reached. The antiviral capacity of glutaraldehyde has
also been demonstrated [30]. It reduces the activity of hepatitis
B surface antigen (HBsAg) and interacts with lysine residues on
the surface of hepatitis A virus (HAV) [2].
Formaldehyde
It is a monoaldehyde that exists as a free water-soluble gas.
It is generally used clinically as a disinfectant and sterilizer
with bactericidal, sporicidal and virucidal effect, but it works
slower than glutaraldehyde [28, 31]. As a mechanism of action
formaldehyde is an extremely reactive chemical that interacts
with proteins, DNA and RNA in vitro. It has the ability to penetrateinside bacteria, and to modify HBs Ag and HBcAg of HBV [2].

2021.31.2(102)  Fiziologia - Physiology

4. Aromatic alcohols
Aromatic alcohols have effective antimicrobial properties fordisinfection, even in the presence of biological fluids.
Phenol (C6H5OH) is an organic compound consisting of abenzene ring bearing a single hydroxy substituent. Phenol
exerts antimicrobial activity against Gram-positive and negative
bacteria, fungi and viruses, but is not as effective as a sporicide[1]. Although the specific mechanism of action of phenol
derivatives is not clearly known, there is consensus that thesecompounds cause physical damage to the plasma membraneof the microbial cell. As a result, the content of essential cell
metabolites drains causing cell damage with cell lysis, while
acting as a protoplasmic poison that causes the cytoplasm tocoagulate, with the death of microorganisms. The efficacy of
triclosan (a phenolic derivative) was investigated in vitro andwas found to have bacteriostatic effect at lower concentrations,and bactericidal activity at higher concentrations. The activity oftriclosan is higher against gram-positive microorganisms thanagainst gram-negative bacteria, especially P. aeruginosa, and thefungicidal activity is good, including yeasts and dermatophytes[8].
Hexachlorophenol is another bis-phenol whose mode ofaction has been investigated based on studies with Bacillus
megatherium. It has been found to be bactericidal at 0 ° C,
despite the fact that it causes minimal leakage through thebacterial membrane at this temperature. Despite the efficacy
and broad spectrum of hexachlorophen, concerns about its
toxicity, especially in newborns, lead to its limited use in antiseptic
products [2].
Chloroxyleneol is another phenol used in antiseptic or
disinfectant formulas. Chloroxylene is bactericidal against
certain bacteria, while others are very resistant, for example
P. aeruginosa. Surprisingly, its mechanism of action has been
little studied despite its widespread use over time. Due to its
phenolic nature, it would be expected to have an effect on
microbial membranes.
In conclusion, commonly used phenolic compounds haveantimicrobial efficacy against bacteria, fungi, viruses, includingHIV. However, literature also reports that some phenolicdisinfectants have a limited effect on Coxsackie B4, Enterovirus
11 and Polyovirus [32].
5. Quaternary ammonium compounds
Quaternary ammonium compounds are the most popular
cationic detergents. They are characterized by a positively
charged nitrogen and a long hydrophobic aliphatic chain.
Quaternary ammonium compounds have a structure similar toammonium chloride, but with some changes. The demand forthese disinfectants has increased over the decades, in addition,
their use is not limited only as a germicidal agent, but they havebeen widely used in a variety of industrial, agricultural, clinicaland consumer applications [33]. As mechanism of action, it has
been shown that microbicidal activity is due to their adhesion toacidic proteins or phospholipids in the membrane, which leads
to the formation of so-called hydrophilic voids. Denaturation of
essential cellular proteins will increase the permeability of themembrane and ultimately cause the destruction of the microbial
cell. In addition, it seems that they are also involved in inactivatingenergy production and can be linked to DNA [34]. As a result,they are used as solid bactericidal agents, especially against
7Gram-positive bacteria and against encapsulated viruses (egherpes simplex, adenovirus, vaccinia), not being sporicidal,
tuberculocidal or with an effect on hydrophilic viruses. Basically,
they are commonly used in sanitizing non-critical surfaces, suchas floors, furniture and walls. The scientific literature reports theeffectiveness of some quaternary ammonium compounds inremoving and / or inactivating S. aureus and P. aeruginosa fromthe computer keyboard. Moreover, a recent paper by Brown et al.
[35] demonstrated that the microbial reduction activity following
their application on glass, continues for a long time and in humid
conditions. A number of advantages of quaternary ammonium
compounds can be listed, such as: high stability, low color, noodor and relatively low toxicity, as opposed to phenols andchlorine-based bleaches.
6. Chlorhexidine
Chlorhexidine is a cationic biguanide and its quality as anantimicrobial agent was established in 1954 [8]. It exists as
acetate (diacetate), gluconate and hydrochloride salts and isa biocide commonly used in antiseptic products, especiallyfor hand washing and oral products, but also as a disinfectant
and preservative. It has a wide spectrum of action and is welltolerated by the skin, rarely causing irritation. A large numberof studies have been dedicated to elucidating the antimicrobial
mechanism of action of this important substance [2]. Thus, it
was noted that chlorhexidine gluconate is rapidly absorbed bybacteria such as E. coli and S. aureus and that this depended
on its concentration and pH. The outer layer of the microbial cell
deteriorates, though insufficiently to induce lysis or cell death,but then the agent crosses the cell wall - probably by passive
diffusion - and subsequently attacks the cytoplasmic bacterial
membrane, followed by leakage of intracellular constituents andmicrobial death. High concentrations of chlorhexidine cause
coagulation of intracellular constituents. The antimicrobial activityof chlorhexidine depends on its concentration. Thus, at lower
concentrations, chlorhexidine has a bacteriostatic effect against
most gram-positive bacteria, on many gram-negative bacteria
and bacterial spores. At very high concentrations, a bactericidal
effect can be expected, as well as activity against yeasts [8].
In liquid soap, chlorhexidine usually has a concentration of
4% and has a bactericidal activity against Gram-negative andGram-positive bacteria [36]. In a number of comparative studies,chlorhexidine suspension (4%) was less effective against MRSA
than against methicillin-resistant S. aureus [8]. Chlorhexidine
does not inactivate non-encapsulated viruses such as rotavirus,
hepatitis A virus or poliovirus, this is explained by its activity
limited to nucleic acid or the outer shell. It seems that the latter
is a more important target [2]. And compared to dermatophytes,
such as Trichophyton mentagrophytes, chlorhexidine (1.5%) has
no activity. Chlorhexidine testing in clinical practice demonstrated
in a study of 52 volunteers who washed their hands with a 4%
chlorhexidine liquid soap 24 times a day for 5 days, a significant
decrease in the number of resident bacteria of skin compared
to a batch that was washed with classic soap [8]. Another study
in which hands were artificially contaminated with MRSA liquid
chlorhexidine soap proved to be as effective as plain soap.
A similar result has been reported after contamination of thehands with S. aureus [37]. In conclusion, chlorhexidine (2-4%)
has a good activity against most vegetative bacteria, yeasts andencapsulated viruses but limited activity against mycobacteria,
8dermatophytes and non-encapsulated viruses. Washing yourhands with a chlorhexidine soap can reduce the number ofbacteria.
7. Heavy metals and their compounds
Most heavy metals and heavy metal compounds or metal salts
have some degree of toxicity to microorganisms. For many
years, heavy metal ions have been used as germicides, butmore recently, they have been replaced by other less toxic andmore effective heavy metals and their compounds. The most
toxic heavy metals are mercury, silver and copper, and theleast toxic are sodium and potassium. Mode of action: heavy
metals and metallic compounds combine with proteins, oftenwith their sulfhydryl (SH) groups and inactivate them. The highconcentration of metal salts, especially those of mercury, silver
and copper, coagulates cellular proteins that lead to damage ordeath of the microbial cell. Metal salts can also precipitate andin high concentrations can cause the death of a microbial cell.
Silver compounds have been used to prevent burn infections
and some eye infections. It appears that silver salts and otherheavy metals such as copper act by binding to functional groupsof fungal enzymes. The mechanism is similar in the case of
microbes, silver ions bind to enzymes important for microbialactivity, causing their inhibition [2]. In addition to its effects on
enzymes, silver ions produce other changes in microorganisms.
It has been demonstrated that silver nitrate is able to cause
a marked inhibition of Cryptococcus neoformans growth bydepositing in the vacuoles of this fungus and in its cell wall in
the form of granules [38]. Silver ions inhibit cell division anddamage the cell lining and contents of P. aeruginosa. Bacterial
cells increase in size and there are structural abnormalities of
all cellular components. In addition, the interaction of silver ionswith nucleic acids has been noted, preferably with DNA bases
rather than phosphate groups, although the significance of thisin terms of its lethal action is unclear [39]. Silver sulfadiazine is acombination of two antibacterial agents, silver and sulfadiazine.
The question was asked which of the two compounds is due tothe antibacterial effect or whether it occurs as a result of theinteraction of the two. Silver sulfadiazine has a broad spectrumof action and unlike silver nitrate, it causes damage to themembrane of sensitive bacteria, it binds to cellular components,
including DNA. Bacterial inhibition is likely to occur when silver
binds to sufficient base pairs in the DNA helix, thereby managing
to inhibit transcription. The complete mechanism of action ofsilver sulfadiazine has not yet been elucidated [40].
In conclusion, the perfect antimicrobial chemical probably does
not yet exist, but we have the opportunity to make the right choice
and proper use of current chemicals, so as to avoid both theincrease in antimicrobial resistance and environmental problems.
Thus, a deep knowledge of the antimicrobial agent together withthe type of surface on which it is to be applied, would result inan adequate level of disinfection.

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ijbiomac.2020.08.059

9AGENȚII CHIMICI CA FACTORI ANTIMICROBIENIREZUMATO mare varietate de substante chimice pot fi folosite ca agenti antibacterieni in vederea prevenii contaminarii. Antisepticele și dezinfectanții
sunt folosiți pe scară largă în spitale si reprezinta un element esențial de combatere si prevenire a infecțiilor nosocomiale. In aceste
produse sunt prezenti o mare varietate de agenți chimici activi sau biocide. Dezinfectanții și antisepticele afectează bacteriile în multe
feluri. Utilizarea pe scară largă a antisepticelor și a produselor dezinfectante a contribuit la dezvoltarea rezistenței microbiene, în speciala rezistenței încrucișate la antibiotice. In practica medicala, agenții antimicrobieni trebuie selectați având în vedere microorganismele
specifice, pH-ul, solubilitatea, toxicitatea, materialul organic prezent și nu in ultimul rand costul. Agentul chimic antimicrobian ideal probabil
ca nu exista, dar alegerea corectă și utilizarea adecvată a substanțelor actuale, va avea drept rezultat un nivel adecvat de dezinfecție.
Aceasta lucrare isi propune o trecere in revista a principalilor agentii chimici antimicrobieni, a mecanismelor prin care ei actioneaza si
a importantei lor in practica clinica.
Cuvinte cheie: agenții chimici antimicrobieni, alcooli, halogeni, aldehide, compusi cuaternari de amoniu, argint.

10

Fiziologia - Physiology  2021.31.2(102)EVALUATION OF THE ABILITY OF SOME MULTIDRUGRESISTANT BACTERIAL STRAINS TO FORM BIOFILMS
CRISTINA MIRABELA GAȘPAR 1, BRIGITTA KIS 2, CARMEN TATU 2,3, CARMENPANAITESCU 2,3, CSILLA ZAMBORI 2,4, ALINA GEORGIANA SIMINA 2, CAMELIA
TULCAN1, IOAN HUȚU1, DALIBORCA CRISTINA VLAD5, VIRGIL PĂUNESCU2,4,GABRIELA TĂNASIE2,3
Banat’s University of Agricultural Science and Veterinary Medicine “King Michael the Ist of Romania” from Timisoara, Aradului str no. 119, 300645,
Timișoara, Romania
2
OncoGen Research Center, Timisoara County Emergency Clinical Hospital, Liviu Rebreanu Boulevard, no. 156, 300723, Timișoara
3
Department of Functional Sciences, Physiology, Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of Medicine
and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara
4
Department of Functional Sciences, Immunology, Center of Immuno-Physiology and Biotechnologies (CIFBIOTEH), “Victor Babeș” University of
Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2, 300041 Timișoara
5
Department of Biochemistry and Pharmacology, “Victor Babeș” University of Medicine and Pharmacy Timișoara, Romania, Eftimie Murgu Sq. no. 2,
300041 Timișoara
1

ABSTRACTThe crystal violet (CV) based microtitre plate assay is one of the most widely used method in screening for biofilm forming bacterial strains
or in evaluating the efficacy of antibiofilm products. Despite the modifications made over the time in order to increase CV staining method’s
accuracy, this assay still shares the issue of ’’edge effect’’, phenomenon due mainly to evaporation of the liquid from the peripheral wells.
This allows the planktonic cells to stick to the walls, which in turn binds the CV dye and gives a false result as biofilm biomass, thusincreasing the rate of experimental errors. The present study aimed to improve the accuracy of the crystal violet staining assay, by reducing
the ’’edge effect’’ through certain modifications that involved either changing the incubation conditions, by using a climate chamber insteadof a classic incubator or the working technique, by filling the peripheral wells from lines A, B, G, H and columns 1, 2, 11 and 12 of a 96
well microtiter plate with sterile distiled water. Results showed that by filling the peripheral wells with water, the „edge effect” is reduced
and the OD values are relatively homogene with lower standard deviations, when compared with incubating in a classic incubator andin the climate chamber (p< .05). Therefore, this improvement to crystal violet based microtiter plate assay has shown to give minimum
variability in our results, mainly when the biofilm assay requires long incubation time involving more than 24 h.
Key words: biofilms, multidrug-resistant bacteria, bacterial strains, edge effect

INTRODUCTION

Biofilms are microbial communities attached to different
surfaces wherein bacterial cells are embedded in a self-produced
extracellular polymeric matrix [1]. Biofilms cause significant
problems in both medical (e.g. device-related infections) andnon-medical settings (e.g. food processing environments, drinking water distribution systems) [2, 3, 4].
Over the years, a broad range of in vitro models havebeen described for the study of biofilm formation and developement [5]. Two methods are currently used for the phenotypicidentification of biofilm-producing bacteria, namely the Congo
red agar test [6] and the microtiter plate assay [7]. While theCongo red agar test is mainly used for the identification of biofilmproducing staphylococci, the microtiter plate method is suitable
for any bacterial specie of interest. The first method consists inthe property of biofilm-producing staphylococci to bind the Congo
red dye and develop black colonies on the red agar, while thenon-biofilm-forming strains apear as red-coloured colonies [4].
Also, Congo red staining is largely used to identify the amyloid

fibres-producing bacteria. Amyloids are found in almost all natural
biofilms. These fibres contribute to bacterial aggregation and toprotective role of biofilms, serving also as virulence factors [8, 9].
One of the most intensively studied biofilm-associated amyloids
are curli fibres in E. coli and Salmonella enterica, which also occur in other enterics [10]. In E. coli, curli fibres are co-regulatedwith pEtN-cellulose [11] and in many strains are produced
below 30°C only, suggesting a major role in environmental
biofilms [12]. However, curli fibres and/or pEtN-cellulose canalso be produced at 37°C by certain commensal or pathogenic
E. coli strains [13]. In the human intestine, curli fibres promoteinflammation and act as a virulence factor and can even trigger
autoimmunity [9, 14, 15].
The microtiter plate assay was developed to replace
the test tube assay, the first method used for macroscopic assessment of bacterial biofilms on the surface of plastic tubes[16]. These technique produces quantitative results based on
the assessment of matrix and both living and dead cells (biofilm biomass tests), of viable cells (viability tests) and of matrix
components, based on specific staining (matrix quantification

Received 6th of July 2020. Accepted 15th of June 2021. Address for correspondence: Banat’s University of Agricultural Science and Veterinary Medicine“King Michael the Ist of Romania” from Timisoara, Aradului str no. 119, 300645, Timișoara, Romania, e-mail: gasparcristina99@yahoo.com

2021.31.2(102)  Fiziologia - Physiology

11tests) [17].
The crystal violet (CV) based microtiter plate assay
is an indirect method of biofilm biomass quantification in theentire well [7, 18]. CV is a basic dye, which binds to negatively
charged surface molecules and polysaccharides in the extracellular matrix, as well as to both living and dead cells [19, 20].
Despite the modifications made in order to increase CV staining method’s accuracy [18], this assay still shares the issue of’’edge effect’’. The ’’edge effect’’ is due mainly to two reasons;first, the peripheral wells are more ventilated thus can provide
more oxigen for bacterial growth. Secondly, when incubated innormal incubators for more than 24 h, water evaporates quickly
from peripheral wells. This phenomenon allows the planktonic
cells to stick to the walls, which in turn binds the CV dye andgives a false result as biofilm biomass [21].
The present study aimed to improve the accuracy ofthe crystal violet staining assay, by reducing the ’’edge effect’’through certain improvisations that involved either changing the
incubation conditions or the working technique.

MATERIALS AND METHODS

Multi-drug resistant Escherichia coli strains CO1165,
CP7921, DK0336, CU0181, DM3527, CO1453 and Staphylococcus aureus strains CP7438, CO0587, CP7170, CP8150, CL9002
were overnight cultured on blood agar. One or two colonies fromeach culture were suspended in 10 ml of Brain Heart Infusion(BHI) and adjusted to 0.5 Mc Farland. Different numbers of wellsfrom the 96 well microtiter plates were inoculated with 150 µL
of bacterial suspension, depending on the applied protocol.
Wells containing the same quantity of BHI served as blanks. For
biofilm formation, the microtiter plates were incubated for 72 h
at 37±0.5°C, in a classic incubator (protocol 1 – P1, 2 – P2 and
3 – P3) or in a climate chamber ensuring 95% relative humidity
and a temperature of 37±2°C (protocol 4 – P4). In P1, P2 andP4, columns 1 and 12 remained uninoculated, column 2 and line
H served for the blanks (17 wells) and the rest of the microtiterplate was inoculated with three stains (21 wells for each strain).
In P3, in order to reduce evaporation, the peripheral wells corresponding to lines A, B and H and to columns 1 and 12, werefilled with 200 µL physiological saline remaining 14 wells for theblanks and 12 wells for each of the three strains.
Following the incubation period, the supernatant wasremoved from each well and the wells were rinsed using 160
μL physiological saline and air-dried for several minutes. Subsequently, 160 μL of 1% CV solution was added to each well. After
10 minutes, the excess dye was removed and the plates wererinsed twice with 160 μL (P1 and P2), respectively thrice with
200 μL physiological saline (P3 and P4) and again air-dried forseveral minutes. The biofilm biomass bound CV was released byadding 160 μL of 96% ethanol. After 30 minutes the absorbancewas measured at 540 nm (Tekan).
In protocol 1 and 2 there have been used the E. coli
strains and in protocols 3 and 4, the S. aureus strains. The
difference between protocol 1 and 2 consisted in the type of
12

micropipette used; in protocol 1 (as well as in protocols 3 and 4)
has been used a single channel micropipette while in protocol 2,
a multichannel one, in order to be more time effective.
Each strain was incubated in duplicate because one of
the microtiter plates served for swabing the surface of the weels
after removing the supernatant and rinsing with physiologicalsaline, in order to assess the viable cells adherent to the biofilm.
The colony forming units per milliliter (CFU/ml) were determined
after incubation at 37°C overnight, by serial dilution techniqueand plating on nutrient agar. Only the plates displaying between
25 and 300 colonies were taken into account in the final formula:
CFU/ml= (no. of colnies x dilution factor) / no. of plates
x volume of the inoculum
Statistical analysis
The blank corrected absorbance values of E. coli and
S. aureus strains which fitted in the interval mean ± 1 standard
deviation were used for reporting biofilm production. Aftereliminating the outliers and the negative values obtained inseveral cases, the final means and standard deviations wererecalculated.
To determine whether the number of viable bacteria in thebiofilm (CFU/ml) had any influence on the total biofilm biomass(optical density OD), the Pearson correlation was applied.
In order to determine which of the four protocols provides
the most homogeneous results, respectively increases the accuracy of the CV based microtiter plate assay, there have beentaken into account the number (proportion) of the extreme values
needed to be eliminated and the mean value of the standard
deviations obtained in each protocol. To compare between the
proportion of outliers and between the mean values of standard
deviations in each protocol, there have been used the Chi-square
test with Yates correction, respectively the Mann-Whitney U
test. For each of the applied tests the level of significance wasseted at p< .05.

RESULTS AND DISCUSSIONSAs shown in Tables I-IV and in Fig. 1, there were somedifferences between the protocols in which the same strains
have been used (P1 vs. P2 and P3 vs. P4). Mack et al. (2000)
proposed a blank corrected mean absorbance value of 0.1 inorder to distinguish between weak and strong biofilm producing
bacterial strains [22].

Fiziologia - Physiology  2021.31.2(102)Table I. Mean, standard deviation, viable cell count andnumber of outliers, for the E. coli strains in protocol 1
Mean (OD)SD
CFU/mlNo. of outliers
Total wells /strainCO11650.115582
0.054273
56 x 105
9CP79210.880945
0.083929
104 x 105
9

DK0336
0.371791
0.137623
74 x 105
13

CU01810.132527
0.019853
71 x 105
11

DM35270.029232
0.015438
138 x 105
12

CO14530.219874
0.046503
81 x 105
8

TOTAL

21

21

21

21

21

21

126

CU0181

DM3527CO1453

Table II. Mean, standard deviation, viable cell count andnumber of outliers, for the E. coli strains in protocol 2
CO1165CP7921
DK0336
Mean (OD)SD
CFU/mlNo. of outliers
Total wells /strain0.363226
0.085919
77.5 x 104
90.671408
0.107018
36.5 x 104
8

1.264640.411332
40 x 104140.290696
0.045167
92 x 104100.054717
0.0461134 x 105
13

0.679466
0.282692
26 x 104
11

21

21

21

21

21

21

Table III. Mean, standard deviation, viable cell count andnumber of outliers, for the S. aureus strains in protocol 3
CP7438CP7170CP8150CL9002

CO0587

Mean (OD)SD
CFU/mlNo. of outliers
Total wells / strain

0.09568
0.039726 x 1044120.336069
0.028633
106 x 1047
12CL9002

CO05870.283805
0.023113
106,3 x 104
6120.040724
0.005345
3 x 1047120.408894
0.053726
62,5 x 103
512Table IV. Mean, standard deviation, viable cell count andnumber of outliers, for the S. aureus strains in protocol 4
CP7438CP7170CP8150Mean (OD)SD
CFU/mlNo. of outliers
Total wells /strain0.726884
0.039399
70,5 x 104
120.201412
0.069513
45,5 x 103
110.954736
0.114076
130,5 x 103
120.208999
0.069468
17,2 x 104
100.783432
0.055975
2,8 x 104
8

21

21

21

21

21

In the case of E. coli strains, in both protocols (P1 and P2),
four out of five strains were found to be strong biofilm producers and only one strain (DM3527) had an OD value lower than0.1. Excepting strain CP7921, all the rest exhibited higher OD
values in P2 comparative to P1 (p> .05), in the same incubation
conditions and working procedure. The average of the standard deviations in P2 was almost three times higher than in P1,
however the differences were statistically insignificant (p> .05)
(Fig. 4). The higher variability of the results obtained in protocol
2 might be due to the usage of multichannel micropipette. Ap2021.31.2(102)  Fiziologia - Physiology

62

TOTAL

65
126

TOTAL

29
60

TOTAL

53
105

plying a highly concentrated CV dye (1%) with a multichannel
micropipette requires a lot of attention and skill, so as
not to splash the walls of the wells. If this occurs, rinsing
the excess dye with the multichannel micropipette is not as effective as using the single channel one, because the first of themcannot be easily rotated so that the rinsing solution to reach on
the entire surface of the wells.
In the case of S. aureus strains, in protocol 3, two out of four
strains were found to be strong biofilm producers, while in13

protocol 4 all the four strains had OD values higher than 0.1. All
strains exhibited higher OD values in P4 comparative to P3 (p>.05). The average of the standard deviations in P4 was almost
two and a half times higher than in P3, with statistically significant
difference (p= .03) (Fig. 4).
Both P3 and P4 protocols were designed to reduce the „edge
effect” and thus to increase the homogenity of the results. It is
well known that the environmental conditions impacts biofilm
formation and developement [23, 24]. Protocol 4 was designed toprevent evaporation from the peripheral wells by using a climaticchamber that maintained 95% humidity during the 72 h period.
These equipments share the disadvantage of temperature oscillation within wider limits than classical incubators to maintain firstof all constant humidity. In this particular case the temperature
variations were of 2 degrees around 37°C. Many studies havereported the effect of factors, such as temperature and humidity changes, nutrient availability, oxygen level, pH and surface
type, on the biofilm formation of pathogenic bacteria such as S.
aureus [24]. However, the effect of temperature changes on thebiofilm formation of S. aureus remains unclear and there are
discrepancies between studies undertaken in this regard. Somehave found that the biomass of S. aureus biofilms grown at 37°C
was more important than those grown at 25°C on polystyrene[25, 26]. Other authors reported a higher cell count of the S.
aureus biofilm at 25°C in contrast to that obtained at 37°C on
stainless steel. Instead, Da Silva Meira et al. (2012) showed that
there is no clear effect of the incubation temperature (7 and 28°C)
on the biofilm formation of S. aureus [27]. However, in the present
study a temperature variation between 35 and 39°C for a periodof 72 h determined an increased biofilm biomass production andless homogen results comparative to incubating at 37±0.5°C,
when quantificating S. aureus biofilm in 96-well microtitre plates.
Assessment of the relationship between the viable bacteria countin the biofilm (CFU/ml) and the biofilm biomass (OD) showedvariable results between the protocols. Regarding the E. coli
strains subjected to P1 and P2, in the first protocol there wasno correlation between the two variables r(62)=0.1779, p> .05,
while in the second protocol it was found a moderate negative
correlation r(59) = -0.5955, p< .00001 between the viable bacteria
count and the biofilm biomass (Fig. 2). Regarding the S. aureus
strains subjected to P3 and P4, in protocol 3 it was found a weak
positive correlation between the two variables r(29)=0.3125, p>.05, but without statistical significance, while in protocol 4 there
was no correlation between the viable bacteria count and thebiofilm biomass r(50)=0.1722, p> .05 (Fig. 3).
Figure 4 shows that regardless of the applied protocol, approximately half of the OD values were outliers as a result of
not falling within a deviation from the normal distribution of themean, with variations from 48.33% in protocol 3 up to 51.59%, in
protocol 2. The differences between the proportion of the outliersin each protocol were not statistically significant Χ2 (3, N = 417)
= 0.2325, p = .9721.
As previously stated the primary cause for the „edge effect”
phenomenon is evaporation [21]. To reduce excessive water
loss and to maintain humidity during the incubation period,
14

physiological saline has been added in the peripheral wells, in
protocol 3. Results showed that by applying this protocol, the„edge effect” is reduced. As shown in Fig. 5, the OD values inprotocol 3 are relatively homogene with reduced standard deviations, mainly when comparing with protocols 2 and 4 (p< .05).
Moreover, protocol 3 is significantlly less time consuming than
the rest of the protocols.

CONCLUSIONSThis modification to crystal violet based microtiter plate assay
has shown to give minimum variability in our results and ismainly helpful when the biofilm assay requires long incubation
time involving more than 24 h.
Acknowledgement: This work was supported by the grant“Bioeconomic approach of the antimicrobial agents use andresistance BIO AMR”, PN III P1-1.2 PCCDI 2017 0361.

Fig. 1. Mean OD ± SD for all the bacterial strains subjected todifferent protocols

Fiziologia - Physiology  2021.31.2(102)Fig. 4. Percentage of outliers that needed to be excluded due
to not fitting in mean ± 1 SD

Fig. 2. Relationship between the number of viable bacteria inthe biofilm (CFU/ml) and the biofilm biomass (OD), in E. coli
strains: Protocol 1 – lack of correlation, Protocol 2 – moderate
negative correlation

Fig. 5. Average values of the standard deviations in each
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Fig. 3. Relationship between the number of viable bacteria inthe biofilm (CFU/ml) and the biofilm biomass (OD), in S. aureus
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17. Peeters E, Nelis HJ, Coenye T: Comparison of multiple methods for quantification of microbial biofilms grown in microtiterplates. J Microbiol Methods 2008; 72(2):157-65. doi: 10.1016/j.
mimet.2007.11.010
18. Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M: A
modified microtiter-plate test for quantification of staphylococcal
biofilm formation. J Microbiol Methods 2000; 40:175–179
19. Li X, Yan Z, Xu J: Quantitative variation of biofilms among strainsin natural populations of Candida albicans. Microbiology 2003;
149:353–362
20. Pitts B, Hamilton MA, Zelver N, Stewart PS: A microtiter-plate
screening method for biofilm disinfection and removal. J Microbiol
Methods 2003; 54:269–276
21. Shukla S, Toleti SR. An Improved Crystal Violet Assay for Biofilm
Quantification in 96-Well Micro-Titre Plate. 2017; 10.1101/100214
22. Mack D, Rohde H, Dobinsky S, Riedewald J, Nedelmann M,
Knoblock JKM, Elsner HA, Feucht HH: Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm
formation. Infect Immun 2000;68:3799-3807
23, Else TA, Pantle CR, Amy PS: Boundaries for biofilm formation: humidity and temperature. Appl Environ Microbiol 2003; 69(8):50065010. doi:10.1128/aem.69.8.5006-5010.2003
6.

24. Abdallah, M., Benoliel, C., Drider, D. et al: Biofilm formation andpersistence on abiotic surfaces in the context of food and medical environments. Arch Microbiol 2014; 196:453–472. https://doi.
org/10.1007/s00203-014-0983-1
25. Choi N-Y, Kim B-R, Bae Y-M, Lee S-Y: Biofilm formation, attachment, and cell hydrophobicity of foodborne pathogens under varied
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56:207–220. doi:10.1007/s13765-012-3253-4
26. Vazquez-Sanchez D, Habimana O, Holck A: Impact of food-related
environmental factors on the adherence and biofilm formation ofnatural Staphylococcus aureus isolates. Curr Microbiol 2013;
66:110–121. doi:10.1007/s00284-012-0247-8
27. da Silva Meira QG, de Medeiros Barbosa I, Alves Aguiar Athayde
AJ, de Siqueira-Júnior JP, de Souza EL: Influence of temperatureand surface kind on biofilm formation by Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers. FoodControl 2012; 25:469–475. doi:10.1016/j.foodcont.2011.11.030

16

Fiziologia - Physiology  2021.31.2(102)EVALUAREA CAPACITĂȚII UNOR TULPINI BACTERIENE
MULTIREZISTENTE DE A FORMA BIOFILME
Rezumat:Tehnica în plăci de microtitrare cu cristal violet este una dintre cele mai utilizate metode pentru evaluarea capacității tulpinilor bacterienede a forma biofilme, precum și pentru testarea preparatelor care împiedică formarea acestora. În ciuda încercărilor de îmbunătățire
întreprinse de-a lungul timpului, această metodă încă prezintă dezavantajul apariției „efectului de margine”, fenomen datorat evaporării
lichidului din godeurile marginale. Pierderea de lichid determină atașarea celulelor planctonice la pereții godeurilor, fixarea colorantului
și astfel, interpretarea eronată ca și biofilm. Scopul prezentului studiu a fost de a încerca reducerea evaporării din godeurile marginale,prin modificări care vizează fie condițiile de incubare (incubarea într-o cameră climatică), fie tehnica de lucru, prin umplerea godeurilorde pe liniile A, B, G, H și coloanele 1, 2, 11 și 12 ale plăcii de microtitrare, cu apă distilată sterilă. S-a evidențiat o reducere a „efectului demargine” atunci când godeurile periferice au fost umplute cu apă, cu obținerea unor densități optice mai omogene și a unor deviații standard
mai mici, comparativ cu rezultatele obținute în urma incubării în termostatul clasic și în camera climatică (p< .05). Astfel, îmbunătățirea
adusă tehnicii de evidențiere a biofilmelor bacteriene în plăci de microtitrare, cu cristal violet, scade variabilitatea rezultatelor, în special
atunci când este necesar un timp de incubare mai mare de 24 de ore.
Cuvinte cheie: biofilme, bacterii multirezistente, tulpini bacteriene, efect de margine

2021.31.2(102)  Fiziologia - Physiology

17

SPREAD AND PHYSIOLOGICAL ECOLOGY OF NONTUBERCULOUS MYCOBACTERIABICIUSCA VIOREL1*, SORINA IONELIA STAN2*, PATRICIA DURAND3*, BICIUSCA
TEODORA#4, MARA BALTEANU#5, BEATRICE MAHLER6
Department of Internal Medicine, Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Romania.
Department of Internal Medicine, Resident Ph, Emergency County Clinical Hospital Craiova, Romania.
3
Department of Internal Medicine, Resident Ph, ”Filantropia”Municipal Clinical Hospital Craiova, Romania.
4
Department of Radiology, ”Goethe” University, Faculty of Medicine, Frankfurt, Germany.
5
Department of Pneumology, Faculty of Medicine, ”Titu Maiorescu” University, ”Prof. Dr. Marius Nasta” Institute for Pneumology, Romania.
6
Department of Pneumology, Faculty of Medicine,”Carol Davila” University of Medicine and Pharmacy Bucharest, ”Prof dr Marius Nasta” Institute forPneumology, Romania.
*These authors have equal contribution
1
2

ABSTRACT
Nontuberculous mycobacteria (NTMs) are ubiquitous microorganism, which are not only found in the natural environment (water, soil anddust), but also in the human environment. Due to their morphological and functional properties, they can survive in all natural and artificial
environments. Water from distribution systems and sanitary installations is the ideal place for development and multiplication of NTMs, aswell as the main transmission route of NTM from nature in the artificial system. NTMs form biofilms on the surface of household objects andthe aerosolubilization and subsequent inhalation by humans is the main way of producing NTM lung disease. NTMs include saprophytic
and opportunistic microorganisms. Most of them have a low pathogenicity, which is why it requires an affected host to produce disease.
Key words: NTMs, opportunistic microorganisms, physiological ecology

INTRODUCTION

The incidence and prevalence of lung disease caused by NTM(LD-NTM) are increasing and their impact on human health is acurrent topic of great interest [1]. Generally, the term nontuberculous mycobacteria (NTM) refers to species of mycobacteria
othen than Mycobacterium tuberculosis and M. Leprae complex[2]. In the specialized literature, NTMs have been named forvarious names, including “environmental mycobacteria”, “atypicalmycobacteria” and “mycobacteria other than M. tuberculosis”. In
the past, they were considered minor pathogens, in relation to
the pathology caused by Mycobacterium tuberculosis.

Currently, due to the increase in the percentage of immunocompromised hosts, in particular due to the increasing incidenceof HIV/AIDS, NMTS have emerged as important pathogens
worldwide [3]. NTM infection mainly presents four distinct clinicaldiseases in humans: chronic pulmonary disease; disseminateddisease in severely immunocompromised patients; skin and soft
tissue for clinicians [4]. In addition, microbiological recurrence iscommon even after successful treatment infections; and superficial lymphadenitis, especially cervical lymphadenitis in children[5]. Pulmonary disease accounts for 80–90% of NTM-associateddiseases [6].The diagnosis and treatment of NTM lung diseaseremain challenging, with substantially high reinfection rate.

Received 25th of June 2021. Accepted 29th of July 2021. Address for correspondence: Biciusca Teodora, Goethe University, Theodor W Adorno Platz 1,
60323 Frankfurt am Main, Germany, phone:+4915202839885, email: teodorabiciusca96@gmail.com; Balteanu Mara, PhD, ”Titu Maiorescu” University,
Faculty of Medicine, Department of Pneumology, Gheorghe Petrascu Street no 67A, RO-031593 Bucharest, Romania, phone:+40213243013, email: mara.
balteanu@prof.utm.ro.

18

Fiziologia - Physiology  2021.31.2(102)Table I. Classification of mycobacteria. [5]Spread of NTMs

NTMs can be found anywhere in nature and at all altitudes. They
grow best in soil, but are also present in water [7]. NTMs are
represented by potentially pathogenic mycobacteria in humans
and animals, but also by normally saprophytic mycobacteria,
which are non-pathogenic or potentially pathogenic [8]. Potentially pathogenic mycobacteria are widespread throughout thewild and can become pathogenic under certain circumstances[9]. They are called opportunistic or occasionally pathogenic
mycobacteria in order to differentiate them from the strictly
pathogenic ones. Some of them are rarely (ex M malmoense)or never (M ulcerans) isolated in the environment, although
the epidemiological profiles of the diseases generated by them
indicate that they are present in nature [10]. Saprophytic or potentially pathogenic mycobacteria often refer to non-tuberculous
mycobacteria [11]. They form homogeneous groups that are easy
to identify in the laboratory. Although the clinical manifestations
produced by these mycobacteria are similar to those producedby the Koch bacillus, they are considered atypical, because the
etiological agent does not belong to the M tuberculosis complex[12]. People are constantly in contact with these mycobacteria,
which they can inhale or ingest. As a result, a temporary or
permanent colonization of the skin, respiratory and digestive
tracts is performed[13]. The natural environment is considered
to be the environment that was not created by humans or that is
not normally subject to human pollution (urban water systems,
urban rivers that flow into natural rivers or lakes) [14]. In theseenvironments the distribution of mycobacteria is different. If theurban environment seems to be conducive to the developmentof mycobacteria (ex M terrae), the environment located along
the water or savannah coasts seems to be conducive to theisolation of mycobacterial species of the MAC complex[15]. The
distribution of mycobacteria in the environment is influenced bya number of factors, such as: the chemical composition and2021.31.2(102)  Fiziologia - Physiology

pH of the soil, as well as the temperature of the environment.
Artificial environments (eg water sources, swimming pools) arecolonized by another spectrum of mycobacteria, some of whichappear to live only in man-made environments (eg M kansasii,
M xenopi) [7]. Some of the mycobacteria (M marinum, M chelonae, M avium) are rare in the wild, but are common in artificial
environments [16]. There is also a group of non-tuberculous
mycobacteria that have not been identified in the environment(M haemophilum, M genavense), but epidemiological data on the
diseases they cause suggest that they come from the environment. These mycobacteria require special growth media in vitro
or grow poorly in laboratory conditions, which may explain why
they have never been isolated in the environment [17].

Mycobacteria Ecology

The physiological ecology of a microorganism is defined by thosephysiological characteristics (features) of that microorganism,
which are determinants of their ecology and, therefore, of theepidemiology of the diseases determined by it [18].

Intraspecies genomic variation of NTMs is a property of

microorganisms that allows their phenotypic variability. Analysisof the mycobacterial genome revealed a number of genes
responsible for encoding virulence factors. The virulence genes
available in the chromosome provide flexibility for opportunistic
pathogens to infect a host when conditions become favorable.
The genetic elements responsible for these pathogenic traits canbe incorporated into the stable chromosome or are transferredthrough mobile plasmids. Plasmids contain resistance,
tolerance or persistence genes that have a beneficial potential
for maintaining and spreading mycobacteria. Mycobacteria
19incorporated plasmids independently and maintained theplasmids that helped each strain survive in its specific conditions.
It is necessary in the future to give a major emphasis to theinvestigation of the genome of various NTM species in order tobetter understand the genetic elements and genomic diversitythat may influence the ability of environmental mycobacteria to
become opportunistic pathogens. Specifically, the identification ofpotential virulence factors, drug resistance genes, and plasmids
using complete genome information sheds light on this area.
Also, the study and identification of genomes for each species arenecessary both for the investigation of the pathogenic potentialof NTMs and for use in diagnostic methods [19].
Slow growth and the underlying metabolism of NTM areimportant characteristics that allow them to adapt to stressfulenvironmental conditions [20] such as anaerobiosis [21],starvation [22], low pH [23], high temperature [24], osmotic
stress [25], but also less sensitive to antimicrobial agents anddisinfectants [26,27]. Thus, the adaptation of mycobacteria
to these conditions is achieved by reducing the metabolismthat protects the cell from harmful conditions together withthe mycobacterial cell wall [28]. The slow growth of NTM is
achieved by a small number of 16 S r RNA genes but also bylimiting resources for essential cellular processes, as well as
population growth that involves a lot of energy for the synthesisof long chain C60-C80 mycolic acids in the mycobacterial cellwall [29]. Protein synthesis, such as M spA in the case of M.
smegmatis, improves nutrient absorption and increases growthrate [30]; this property is also found in the case of M. fortuitum
but absent for M. tuberculosis [31].
The cell wall, made up of a glycoprotein layer, surrounded
by a lipid membrane is the fundamental structural feature that
ensures the resistance of mycobacteria to environmental factors.
Due to the lipid-forming outer cell membrane, mycobacteria arehydrophobic microorganisms and relatively impermeable tosubstances that cross the membrane of these cells. Although
the rates of transmembrane transport of nutrients are quite low
compared to other microorganisms, which limits mycobacterial
growth and replication, mycobacteria have been shown tobe particularly resistant to a number of antimicrobial agents,
especially the hydrophilic ones [ 32, 33, 34, 35]. The hydrophobic
character of the outer membrane favors the attachment of
mycobacteria on the surfaces, thus preventing the washing anddilution of these microorganisms inside the water systems (rivers
and drinking water distribution systems). Also, hydrophobicity
causes the concentration of environmental mycobacteria
at the air-water interfaces, where the high concentration oforganic compounds provides a good source of food for thesemicroorganisms [36]. For this reason, the best sampling sites forenvironmental mycobacteria are water surfaces and monsters.
The hydrophobic, waterproof outer lipid membrane must be
thought of as a 2-faceted structure: on the one hand it limits
mycobacterial growth, and on the other hand it offers a higher
environmental resistance, which has the effect of occupying
larger habitat areas [37]. In drinking water systems, disinfection(e.g. chlorination) reduces the number of most microorganisms,
20

leaving mycobacteria resistant to these disinfectants, a larger
habitat for nutrient consumption and attachment to surfaces in
the absence of competition [37]. The persistence and growthof mycobacteria in drinking water distribution systems andsanitary facilities are favored by their attachment to surfaces, aphenomenon favored by the hydrophobicity of outer membranes.
As a result, attached mycobacteria are less likely to be removed
by washing with high water flows, and mycobacterial growthand biofilm formation lead even to higher levels of microbial
resistance. On the other hand, the hydrophobicity of the cellsurface of mycobacteria will generate cell aggregation, a rather
annoying problem for researchers in laboratories, in the case ofculture growth of mycobacteria. The increased hydrophobicityof the external surface of opportunistic mycobacteria favors theformation of mycobacterial biofilms [37].
Biofilm refers to a macromolecular aggregate composed of
microorganisms, whose cells adhere to each other and alsoadhere to a surface [38,39]. Thus, the cells become adherent due
to an extracellular matrix composed of substances of polymericnature. Biofilm formation by Mycobacterium avium, which
requires divalent cations, is higher when cells are in nutrientrich and low-nutrient conditions and is inhibited by humic acid
[39,40]. Mycobacterium smegmatis [41] and glycolipoproteins
are required for biofilm formation in the case of M. avium on
polyvinyl chloride (PVC), but not on plastic or glass surfaces[42]. A surprising finding was that M. avium mutants, deficient inbiofilm formation, also showed deficiencies in the invasion of thecell epithelium [43]. In the future, it is particularly important thatthe interpretation of biofilm formation rates include a separate
analysis of the surface adhesion stage and the analysis of
the biofilm growth stage. Also, increased hydrophobicity of
mycobacteria is a determinant of aerosolization.
Aerosolization is the process by which mycobacteria aretransferred from water to air, thus favoring the inhalation of thesemicroorganisms in the human respiratory tree. Mycobacterial
cells, with hydrophobic outer membranes, attach to the walls of
air bubbles that rise from the water column and when the bubbles
reach the surface, they break and form a crater whose walls areenriched with mycobacteria. Following the disappearance ofthe crater, multiple very small drops of water appear, which arethrown at heights of 10-20 cm. These drops have a concentrationof mycobacteria over 10,000 times higher than in deep water[18]. In this regard, an enrichment factor with mycobacteria in
discarded drops has been described, which varies depending on
the species, strain and type of mycobacterial colony. Also, thisenrichment factor is closely correlated with the hydrophobicityof the mycobacterial cell surface [44]. Some of the droplets
formed are so small that they are able to penetrate the bronchi
and alveoli of the lungs [18]. In the future, it is necessary to findbetter strategies for collecting mycobacteria from water tanks. In
this regard, it is recommended that mycobacteria be harvested
either from the collection of aerosolized droplets [45], surface
microlayers [46] or fractional water samples [47].
Resistance to amoebae is an important feature of nontuberculous mycobacteria [48]. This parasitic relationship may
Fiziologia - Physiology  2021.31.2(102)explain the occurrence of human infections. Both microorganisms
have the same habitat, namely insufficiently treated waters. In thelast 2 decades, numerous community infections and outbreaks ofhealthcare associated (nosocomial) infections following surgeries
and endoscopic interventions caused by non-tuberculous
mycobacteria have been described. [49, 50, 51, 52, 53]. There
are studies that have shown the colonization of drinking water,even in hospitals, with vegetative forms of amoebae, but alsowith non-tuberculous mycobacteria [54]. Due to the particular
structure of the cell wall, environmental mycobacteria areresistant in the cytoplasm of living amoebae [55]. This feature
makes it possible for the mycobacterium phagocytosed by theamoeba to be found in the wall structure of the amoeba cyst,
realizing what are called pathogenic trophozoites [56]. These
practical and scientific observations have led to the developmentof a pathogenic ameobiantrophozoite model, which acts as a
“Trojan horse” and protects environmental non-tuberculous
mycobacteria against adverse environmental conditions[48]. This particular location allows both the protection of the
mycobacterium from adverse environmental conditions andtheir rapid release from the wall of the ameobian cyst, when theenvironmental conditions are favorable.
In conclusion, the morphological and functional peculiarities ofopportunistic mycobacteria are those that ensure their resistanceand spread in the environment. Therefore, understanding the
mycobacteria ecology is an essential condition in preventing
and treating these infections.

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[53]. Viana-Niero C, Lima KV, Lopes ML, Rabello MC, Marsola LR, et al.
Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures. J Clin
Microbiol2008;46:850–855.
[54]. Thomas V, Herrera-Rimann K, Blanc DS, Greub G. Biodiversityof amoebae and amoeba-resisting bacteria in a hospital water
network. Appl Environ Microbiol2006;72:2428–2438.
[55]. Greub G, Raoult D. Microorganisms resistant to free-living amoebae. ClinMicrobiol Rev 2004;17:413–433.
[56]. Ben Salah I, Drancourt M. Surviving within the amoebalexocyst:
the Mycobacterium avium complex paradigm. BMC Microbiol 2010;10:99.

Fiziologia - Physiology  2021.31.2(102)RĂSPÂNDIREA ȘI ECOLOGIA FIZIOLOGICĂ A MICOBACTERIILOR NONTUBERCULOASE (NTM)
REZUMAT
Micobacteriile nontuberculoase (MNT) sunt microorganisme omniprezente, care se gasesc nu numai în mediul natural, apa, sol și praf,dar și în mediul uman. Datorită proprietăților lor morfofuncționale, ele pot supraviețui în toate mediile naturale și artificiale. Apa din sistemele de distribuție și instalațiile sanitare reprezintă locul ideal de dezvoltare și multiplicare pentru MNT, precum și principala cale detransmisie a MNT din natură în sistemul artificial. MNT formează biofilme pe suprafața obiectelor din gospodării, iar prin aerosolubilizare și
inhalarea ulterioară de către oameni reprezintă principala cale de producere a bolii pulmonare MNT. MNT includ microorganisme saprofite
si oportuniste. Majoritatea dintre ele au o patogenitatea scăzută, motiv pentru care necesită o gazdă afectată pentru a produce boală.
Cuvinte cheie: MNT, microorganisme oportuniste, ecologie fiziologica

2021.31.2(102)  Fiziologia - Physiology

23

AN UNUSUAL CASE OF MORBUS LEDDERHOSE IN A CAUCASIAN,
YOUNG MALE PATIENT. CASE REPORT AND LITERATURE REVIEWDIANA MARIA CHIOREAN 1,3*, BEATRICE BARAC 5, RALUCA NICULESCU 1,2,3,
ANDREEA CĂTĂLINA TINCA1,2,3, MIHAELA CORNELIA ȘINCU1,3, IULIU GABRIELCOCUZ1,2,3, ADRIAN HORAȚIU SABĂU1,2,3, BEATRICE CARMEN PATRICIA FRICIU4,OVIDIU SIMION COTOI2,3
1
Doctoral School of Medicine and Pharmacy, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Târgu Mureș, 38
Gheorghe Marinescu Street, 540142, Târgu Mureș, România;2
Department of Pathophysiology, George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Târgu Mureș, 38 Gheorghe
Marinescu Street, 540142, Târgu Mureș, România;3
Department of Pathology, County Clinical Hospital of Târgu Mureș, 6 Bernády György Square, 540072, Târgu Mureș, România;4
Plastic Surgery Department, County Clinical Hospital of Târgu Mureș, 6 Bernády György Square, 540072, Târgu Mureș, România;5
Department of Pharmacology, Victor Babeș University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300041, Timișoara, România.

ABSTRACTIntroduction: Plantar fibromatosis also known as Morbus Ledderhose, first described in 1897 by Georg Ledderhose is characterized
by slow-growing benign extra-abdominal desmoid nodules on the plantar aponeurosis. The etiology of this disease is unknown but isoften associated with diabetes mellitus, liver disease, alcoholism, and repeated traumatic episodes of the soles. New possible causative
agents reported in the literature are: drugs from the class of beta-blockers, anticonvulsants, nutritional supplements high in glucosamineor chondroitin, and high doses of vitamin C. Case presentation: In this report, we present the case of a 33-year-old caucasian malewith a 5-year history of palpable nodule within the medial plantar region of the left sole, who denied any personal or family history that
could be related to this disease, medications or alcohol consumption. Instead, he did report daily intake of nutritional supplements highin vitamin C, which corresponds to the onset of disease. Conclusion: Because our patient did not have any other predisposing factorsthat could lead to this pathology, we intend to highlight the fact that prolonged consumption of nutritional supplements high in vitamin C
can influence the occurrence of Morbus Ledderhose. Further investigation between the long-term use of these nutritional supplements
and the development of this pathology is necessary.
Key-words: plantar fibromatosis, vitamin C, Morbus Ledderhose, nutritional supplements

INTRODUCTION

Plantar fibromatosis also known as Morbus Ledderhose,
was firstly described in 1897 by Georg Ledderhose andis characterized by slow-growing benign extra-abdominal
desmoid nodules on the plantar aponeurosis [1,2].This
condition is characterized by three developmental phases: thefirst phase with high proliferative cellular activity, a subsequentacquisition of myofibroblastic characteristics with tissue
contraction, and a final phase characterized by few mature
fibroblasts embedded in a dense matrix [3]. The etiology of thisdisease is unknown, but it is often associated with diabetes
mellitus, liver disease, alcoholism, and repeated traumatic
episodes of the soles. Recent studies report in the literature
possible new causative agents: beta-blockers, anticonvulsants,
and nutritional supplements high in glucosamine or chondroitin
and high doses of vitamin C, thus promoting the production ofexcess collagen [4]. The pathophysiological perspective statesthat growth factors play a role in increasing fibroblastic activity
causing the formation of the fibromas. The identified growth

factors are the platelet-derived growth factor, the transforming
growth factor-beta, free oxidized radicals, interleukin-1 alpha,and interleukin-1 beta[1,5,6].]. As a benign disease, treatment
has involved, in the last decades, symptomatic management,
as it follows: steroid injections, verapamil, radiation therapy,
extracorporeal shock wave therapy, and not least tamoxifen.
When conservative measures fail, surgical removal of the
fibromas and adjacent plantar fascia is often conducted,
although recurrence is common [7].
Case report
We present the case of a 33-year-old caucasian male witha 5-year history of palpable nodule within the medial plantar
region of the left sole, who presented to the plastic surgery
department of our institution. The patient reported increasing
size of the nodule and local tenderness over the past 10
months, particularly after standing for a long period. The
patient has denied any personal or family history that could be
related to this disease, medications, or alcohol consumption.
He did report daily intake of nutritional supplements high invitamin C, which corresponds to the onset of disease.

Received 12th of June 2021. Accepted 11th of August 2021. Address for correspondence: Chiorean Diana Maria, County Clinical Hospital of Târgu Mureș,6 Bernády György Square, 540072, Târgu Mureș, România. Telephone: +40760513978. E-mail: chioreandianamaria@yahoo.com

24

Fiziologia - Physiology  2021.31.2(102)Clinical examination revealed a 6 x 3 cm lesion of the plantar
surface, hyperalgesic and erythematous. At the time of thereferral, an MRI showed a large deep plantar mass locatedon the middle segment of the left sole (Figure 1A), imprecisely
contoured with contrast, highlighting the flexor muscles of
the toes. The subcutaneous adipose tissue has also been
infiltrated on the medial contour. Due to worsening pain
and limitations in daily living, the patient elected for surgicalintervention. Following the surgery, the tumor was sent to thepathology department of our institution for a definite diagnosis
to be established.
The examined fragments were represented by connectiveadipose tissue in which multinodular, fusiform fibroblastic
cells were observed (Figure 1B). No cytonuclear atypia wasidentified. For a more accurate diagnosis, we performed thepositive-expressed Vimentin immunohistochemical reaction,
which revealed the activity of fibroblasts (Figure 1C).
Regarding follow-up, the patient returned to our clinic 2 weeks
post-surgery, and the incision was noted to be healing well withno evidence of infection.

DISCUSSIONAs described in the previous lines, the diagnosis of Ledderhosedisease is usually clinical and does not require additional
testing. In our case, the clinical findings did not raise awarenessin the direction of this diagnosis. The tumor was present
unilaterally. This aspect led us to think of a malignant tumor, incontrast to data from the literature that supports the presenceof plantar fibromas on both soles, simultaneously. Therefore,
we included as differential diagnosis the following: plantar
fasciitis, leiomyoma, rhabdomyosarcoma, and liposarcoma,
as mentioned by other specialty studies [9-11]. Last but notleast, another aspect that has raised concern was the size ofour patient’s tumor. The National Institute for Health and Care
Excellence recommends the prompt referral to a sarcoma
treatment center for patients presenting with soft tissueswellings that are greater than 5 cm in diameter, painful, deep
to the fascia, increasing in size, or recurring after a previous
excision [12]. Histological features have highlighted the benign
nature of this disease.
Imaging Assessment
Magnetic Resonance Imaging proved to be the preferred option
for diagnosis and it has the added advantage of examining theanatomy of the structures involved, the adjacent soft tissue
planes, and identification of other pathologies. If imaging
characteristics are ambiguous or typical imaging features of2021.31.2(102)  Fiziologia - Physiology

a plantar fibromatosis are absent, tissue histology should be
performed to exclude other pathologies [10]. MRI is influential
in surgical planning and can help define the margins of thelesion allowing for better surgical resection [1].
The most important aspect, like in any disease, is to relieve
the pain, to get patients’ comfort, and to use therapeuticstrategies, which do not activate or aggravate underlying
chronic pathologies [13-15].Conservative therapies can beapplied in the early stages of the disease, without pain, walking
discomfort, or balance problems. Unfortunately, this Regarding
shock therapy produces direct trauma of the lesion unleashingan intensive healing response and lesion lysing, by increasing
its vascularity [16]. Furthermore, radiotherapy is effective in thefirst stages, targeting the disease progression and preservationof the feet function, but few studies support this hypothesis.
Ionizing radiation reduces the proliferative activity of the
fibroblasts and myofibroblasts by interacting with TGF-β
production [1]. This therapy presents acute side effects likelethargy, local edema, pain, or local skin reaction. Late side
effects are represented by fibrosis and local skin modifications.
It can produce a decrease in patients’ immunity status leading
to general complications. In the case of young patients, as inour case, we do not recommend the use of this therapy due
to the risk of promoting secondary malignancy like soft tissue
sarcoma or skin cancer.
Intralesional steroid injections, decrease the expression ofvascular cell adhesion molecule-1, thus dropping the productionof TGF-β, basic fibroblast growth factor, IL-1α, and IL-1β. As aresult, the proliferation of fibroblasts and collagen production isdecreased [16]. Another conservative therapy is represented
by collagenase Clostridium histolyticum injections, containing
two types of collagenase: AUX-1 and AUX-2. Both degradecollagen and appear to be a safe and effective alternative
treatment to surgery [18].
Surgical excision is usually used as a last resort, in caseswith inefficient conservative therapies, progressive lesions,advanced stages of the disease, unsupportable pain, walking
or balance problems, as our patient also reported. It can be
performed classically or endoscopic, with partial or complete
fascia resection. Some surgeons prefer the local excision,
removing only the nodules, but this method has a recurrence
rate from 57–100% [19]. The endoscopic plantar fasciectomy
has the benefit of reducing the wound size with better
cosmetics, decreasing the incidence of painful hypertrophic
scars, necrosis, or infection. In case of lesions invading themuscle, plantar skin, or neurovascular bundles, this technique
is contraindicated. Another impediment of this approach is,after all, technically demanding and should be performed only
by experienced arthroscopists [20]. A personalized therapeuticplan depending on the individual characteristics, presence,and intensity of the symptoms and the stage of pathologicalmodifications is, also, strongly recommended.

25

CONCLUSION

Because our patient did not have any other predisposing factorsthat could lead to this pathology, we intend to highlight the factthat prolonged consumption of nutritional supplements high invitamin C can influence the occurrence of Morbus Ledderhose.
However, a further investigation between the long-term useof these nutritional supplements and the development of thispathology is necessary.
Acknowledgments: Conflict of interest – none to declare. The
article is an original work and the final form was not published
before.

REFERENCES
1.

Carroll, P.; Henshaw, RM.; Garwood, C.; Raspovic K.; Kumar, D.
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2. Ledderhose G. Zur Pthologie der Aponeurose de Fusses und derHand. Arch Klin Chir 1897, 55:694-712.
3. Frizziero, A.; Barazzuol, M.; Vittadini, F.; Bellon, G.; Masiero, S.;
Meneghini, A. Plantar Fascial Fibromatosis: Two Cases TreatedWith Low-Energy Focused Shock Waves. J Clin Rheumatol
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4. Causes of Plantar Fibromatosis. Available online: https://www.
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5. Veith, N.T.; Tschernig, T.; Histing, T.; Madry, H. Plantar fibromatosis-topical review. Foot Ankle Int. 2013, 34(12):1742-6; doi:
10.1177/1071100713505535. PMID: 24043350.
6. Seegenschmiedt, M.; Keilholz, L.; Wielputz, M.; Schubert, C.;
Fehlauer, F. Long-term outcome of radiotherapy for early stage
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Diease and Related Hyperproliferative Disorders (Berlin) 2012,349-371.
7. Young, J.R.; Sternbach, S.; Willinger, M.; Hutchinson, ID.;
Rosenbaum, AJ. The etiology, evaluation, and managementof plantar fibromatosis. Orthop Res Rev 2018, 17;11:1-7; doi:
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8. Yun, D.H.; Bonaroti, A.R.; Lin, K. Ledderhose Disease With
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contracture of the foot: ledderhose disease. Surg J (NY) 2016,
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doi: 10.1016/j.foot.2020.101736. PMID: 33268229.
11. Chan, J; Chandrasekar, C; Yin, Q. A review of the pattern of referrals
for soft tissue sarcomas in the Mersey region. Orthop Proc. 94-B
12. Bone and soft tissue sarcoma – recognition and referral – NICE
CKS. 2020. Cks. Nice.Org.Uk.
13. Adib, O.; Noizet, E.; Croue, A.; Aubé, C. Ledderhose’s disease:radiologic/pathologic correlation of superficial plantar fibromatosis.
Diagn Interv Imaging 2014, 95(9):893–896.
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14. Neagu, T.P.; Cocolos, I.; Cobilinschi, C.; Tiglis, M.; Florescu, I.P.;
Badila, E.; Sinescu, R.D. The benefits of Botulinum neurotoxin
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(Bucharest) 2017, 68(12):2978–2983.
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Fiziologia - Physiology  2021.31.2(102)UN CAZ NEOBIȘNUIT DE MORBUS LEDDERHOSE LA UN
TÂNĂR PACIENT CAUCAZIAN. PREZENTARE DE CAZ ȘI
ANALIZA LITERATURII DE SPECIALITATEREZUMAT
Introducere: Fibromatoza plantară, cunoscută și sub numele de Morbus Ledderhose, descrisă pentru prima dată în 1897 de Georg
Ledderhose, se caracterizează prin apariția unor noduli desmoizi extra-abdominali cu caracter benign și creștere lentă, la nivelul
aponevrozei plantare. Etiologia bolii este necunoscută, însă este adesea asociată cu diabetul zaharat, bolile hepatice, consumul dealcool și episoadele traumatice repetate la nivelul tălpilor. În literatura de specialitate au fost raportați noi posibili agenți cauzali, precum:
medicamentele din clasa beta-blocantelor și anticonvulsivantelor, precum și suplimentele nutriționale bogate în glucozamină, condroitină
sau cu concentrații crescute de vitamina C. Prezentarea cazului: În acest raport dorim să prezentăm cazul unui bărbat caucazian în vârstă
de 33 de ani, cu un istoric de 5 ani a unui nodul palpabil în regiunea plantară mediană a tălpii stângi, și care a negat orice antecedente
personale sau familiale, consumul de medicamente sau alcool, care ar fi putut determina apariția bolii. În schimb, pacientul a raportat
consumul zilnic de suplimente nutritive bogate în vitamina C, fapt ce corespunde cu momentul declanșării bolii. Concluzie: Având în
vedere că pacientul nostru nu prezenta alți factori predispozanți, intenționăm să subliniem faptul că un consum prelungit de suplimente
nutritive bogate în vitamina C, poate influența apariția bolii Ledderhose. Pentru viitor, sugerăm efectuarea unor investigații suplimentareîn ceea ce privește legătura dintre utilizarea pe termen lung a acestor suplimente nutritive și dezvoltarea patologiei Ledderhose.
Cuvinte-cheie: fibromatoză plantară, vitamina C, Morbus Ledderhose, suplimente nutritive

2021.31.2(102)  Fiziologia - Physiology

27

P O S T- S T R O K E D E P R E S S I O N – I N S I G H T S O N T H E
PATHOPHYSIOLOGICAL MECHANISMS
OLIVER DANIEL SCHREINER1, THOMAS GABRIEL SCHREINER1,2,3,*
1 University of Medicine and Pharmacy „Gr. T. Popa”, Iași, Romania
2 Neurology Department, Clinical Rehabilitation Hospital, Iași, Romania
3 University of Medicine and Pharmacy „Carol Davila”, Bucharest, Romania
ABSTRACT
Post-stroke depression (PSD) is one of the most common disorders encountered in stroke patients, having an unfavorable impact on
the person’s recovery and, implicitly, on his/her quality of life. Numerous epidemiological studies have highlighted the significant impactof this phenomenon, both at the individual level (approximately 1 in 3 patients with stroke will suffer from depression) and at the socioeconomic level. Although intensively studied, the occurrence of depression in only a part of people who have had a history of stroke is
not fully explained, currently existing several incomplete and possibly interdependent theories. In this context, the aim of this paper is
to bring a new perspective on the phenomenon of depression associated with stroke, the authors presenting in the first part the latest
results from the literature regarding PSD’s pathophysiological mechanisms, while in the second part, current and future therapeutic
approaches are detailed.
Key words: stroke, depression, pathophysiology, neuroinflammation, oxidative stress

INTRODUCTION

Depression is common in neurology, with a significant percentageof neurological patients also suffering from major or minor
depression (1)(2). Stroke, the third cause of death and having
a great negative impact at individual and socio-economic level,is frequently associated with post-stroke depression (PSD),
this condition affecting approximately one third of survivors(3). Because of the important burden generated, PSD has
increasingly become a concern to researchers and clinicians,
with numerous studies and meta-analyzes having focused on
the epidemiology, risk factors and treatment possibilities of thispsychiatric pathology. Nowadays, it is known that the predisposing
factors for PSD can be divided into several categories, including
personal unchangeable (female gender, personal history ofpre-stroke depression), modifiable (cognitive and physicalimpairment, level of independence), and independent risk factors
such as lack of family and social support (4).
Regarding the pathophysiological mechanism of PSD, there
are still many unknowns, as the currently accepted theories
are not enough to completely explain the phenomenon. It isclear that depression diagnosed after stroke is a complex andmultifactorial process that has at least two generating elements:
ischemia-induced neurobiological dysfunctions induced by
the acute vascular event which also have chronic effects
and, additionally, the psychosocial distress. Recent research
considers that neurobiological changes are more important thanthe mental damage resulting from the sudden onset of disabilityor due to an unsatisfactory outcome following recovery therapy(5). In addition, other studies have shown a positive correlation
between certain genetic factors such as polymorphisms ofserotonin (5-hydroxytryptamine, 5-HT) transporter 5-HTTLPR,
5-HT2A receptor and of the Brain-Derived Neurotrophic Factor(BDNF) genotypes and the occurrence post stroke depression(6). In this context, the aim of this article is to offer a detailed
view of the most important pathophysiological theories that try
to explain the appearance of PSD syndrome, remembering also
possible therapies that are born from them, but also the missing
elements that require future research.
Most relevant pathophysiological mechanisms of poststroke depression
Nowadays, numerous pathophysiological mechanisms are being
discussed that partially explain the occurrence of PSD in stroke
patients, some of them common with the mechanisms of primary
depression. Among the theories regarding the neurotransmitter
imbalance as a cause of depression, we mention first of all the
monoaminergic hypothesis. Although one of the first hypotheses
raised by the medical world, it is no longer accepted as the
only pathophysiological explanation for the depressive mooddisorder. Considering this theory applicable in the case of PSD,
following the acute cerebral infarction, it is thought that there aredestructions of the aminergic neurons that connect the brainstem
and the upper levels of the cerebral nervous system (CNS),
especially the left cerebral cortex. Consequently, a low productionof serotonin and norepinephrine results in the temporal andfrontal lobes and in the basal ganglia. This serotonin depletion is

Received 12th of May 2021. Accepted 11th of June 2021. Address for correspondence: Thomas Gabriel Schreiner, University of Medicine and Pharmacy„Gr. T. Popa”, str. Universității, no. 16, 700115, Iași, Romania; Phone: +40744860474, schreiner.thomasgabriel@yahoo.com

28

Fiziologia - Physiology  2021.31.2(102)supported by studies that have demonstrated low concentrationsof 5-hydroxyindolacetic acid in CSF (7) and up-regulated PET
measured 5-HT2A receptors in patients with PSD compared with
non-depressive stroke patients (8).
Another neurotransmitter which CNS concentration is alteredin the case of PSD and could play an important role in thepathophysiology of this disorder is glutamate. The glutamateinduced toxicity hypothesis is based on the hyperactivity of theglutamatergic system manifested during the first moments tomonths after stroke. Paraclinical evidence from studies showsa correlation between the appearance of early-onset PSD and alow plasma glutamate level following acute ischemic stroke (9).
Along with the involvement of the neurotransmitters imbalanceat CNS level, there are also systemic theories that emphasize
a more complex imbalance, with possible implications forthe whole human body, thus considering PSD more as a“system disfunction”. Among these theories, we mention
neuroinflammation, a frequently encountered theory used to
explain many neurological pathologies. This hypothesis isbased on both cellular elements such as microglia activationand disturbance of the microenvironment of the neurovascularunit, and non-cellular elements, the heterogeneous group ofproinflammatory cytokines (IL-1β, IL-6, IL-18 and TNF-α) playing
an essential role in generating and sustaining inflamation. It is
known that upregulated activity of proinflammatory cytokines
is found both in response to ischemic stroke and in major
depression, thus becoming a common element that explains
the coexistence of the two conditions. Accordingly, IL-1β hasa clearly defined pathologic effect in stroke, different trials
assessing that this cytokine increases infarct size, while blocking
its activity through administration of recombinant human receptor
antagonist has protective effects (10).
Regarding IL-6, there are only a few reports on its modulating
functions in experimental stroke conducted mainly on rodent
models. IL-6 seems to have neuroprotective functions, byreducing infarct volume (11). A recent meta-analysis was
conducted in order to clarify the role of another important
cytokine, IL-18, after stroke, author’s findings revealing that
increased IL-18 level contributed to the development and severityof ischemic stroke, suggesting also the potential of IL-18 as an
early biomarker (12).
Regarding depression, a recent meta-analysis reveals the
positive association of this disorder with IL-1 and IL-6, both inclinical trials and in community samples. Moreover, time trackingof samples suggests a dose-response relationship between the
depressive phenomenon and inflammatory markers, body massindex being a mediating factor in this association (13).
TNF-α, one of the most extensively studied cytokines in humanand animal stroke models, seems to have both neurotoxic
and neuroprotective impact, demonstrating that the survival ofischemic neurons is modulated by the TNF (14). In support ofthis theory come studies that confirm the positive association
between elevated levels of TNF-α and IL-1 β and the early
appearance of PSD (at 2 weeks). Moreover, this association
also depends on the genetic polymorphism, Alleles associated2021.31.2(102)  Fiziologia - Physiology

with reduced anti-inflammatory cytokine function were associatedwith PSD (15).
In case of neuroinflammation, the release of cytokines
subsequently means the activation of the hypothalamic-pituitaryadrenal (HPA) connection, with the upregulation of corticosteroids
production. Elevated cortisol levels have been associated witha more unfavorable prognosis in terms of post-stroke recovery,
higher mortality, but also a higher risk of PSD. Glucocorticoids
found in high amounts for long periods of time have manyside effects on many organs and systems, including the CNS,
which cause decreased neurogenesis, neuronal survival anddysfunction of neural circuits, especially in the hippocampus (16).
These harmful effects have been highlighted in animal models
that simulate pre or post stroke (17). Older studies have alreadyshown an association between HPA axis deficiencies and theserotonergic system that has been shown to be affected in PSD(18). Specifically, it is a bidirectional relationship between thetwo seemingly unrelated systems, with serotonin binding andtransport in the CNS being influenced by HPA, while serotonintransporter polymorphism involves a chronic state of stress, i.e.
chronic hyperproduction of cortisol.
Another theory that is gaining more and more popularity is basedon brain damage associated with decreased performance ofcompensatory mechanisms, ultimately translating into decreased
neuroplasticity. Multiple evidence has shown a connection
between chronic stress in disorders such as PSD and localized
brain degeneration, especially in the hippocampus, where at theneuronal level is translated in axonal damage, downregulated
dendritogenesis and decreased synaptogenesis (6). At the coreof this theory lays the brain-derived neurotrophic factor (BDNF).
Also known as abrineurin, BDNF exerts its action to support theviability of existing neurons, and the growth and differentiationof new neurons and synapses in certain specific regions of thebrain, such as the hippocampus, the cortex or the basal ganglia.
Shortly after stroke, low levels of BDNF (detected in serum,
for example) were shown to correlate directly with early-onset
PSD, approximately 3 months’ post-cerebrovascular event,and indirectly with stroke severity (19). The explanations areincomplete. On the one hand, it is possible that in some patients,
the BDNF level before stroke is already low, thus creating a
predisposition for a depressive syndrome, and on the other
hand, the hypoxic neuronal microenvironment found in cerebral
ischemia could cause downregulation of BDNF expression.
Finally, two theories directly related to the ischemic episode andtilting the balance towards cerebrovascular pathology are worth
mentioning. We must not forget the vascular factors such as thelesions of the white matter which, by destroying some anatomical
tracts or structures with important neurological implications,
could support, at least from a structural point of view, the validityof the other hypotheses. Regarding other risk factors such as
hypertension, obesity or dyslipidemia, by promoting an initiallylocal chronic inflammatory environment and by partially known
and explained mechanisms, they may be able to represent anadditional condition for the occurrence of PSD. In the samecontext, researchers take into account the metabolic-energy
29theory, being already known that the CNS is a large energy
consumer, with an impressive number of mitochondria, and thesudden decrease in blood flow in ischemia means altered aerobic
metabolism and switch to anaerobic, with the detrimental impactof oxidative stress, which according to the latest research, could
support the occurrence of depression (20).
Current and future therapeutic approaches based on today’s
knowledge
Although incomplete, each of the aforementioned hypothesis
is a possible therapeutic direction which is worth to be followed
when thinking of novel treatments. Approaches have alreadybeen attempted in preclinical and clinical studies based on the
majority of the stated theories, and the use of drugs and othernon-pharmacological therapies with encouraging results in othercases of depression has provided at least a starting point for
future studies.
Thus, similar to primary depression and taking into accounthypotheses based on the lack of certain neurotransmitters,
a first natural step is to replace the missing substance fromexogenous sources. This is the explanation for the successful
use of selective serotonin reuptake inhibitors (SSRIs) which are
the first-line treatment in PSD, although there are no definite
data to show better efficacy compared to other drugs usedin depression (tricyclic antidepressants). In addition, SSRIs
were linked with a tendency of increased hemorrhagic risks,
including intracerebral hemorrhage (21). Of course, basedon the same clinical-pharmacological replacement principle,
other antidepressants have been tried in clinical practice,such as serotonin-norepinephrine reuptake inhibitors (SNRIs)
which, theoretically, by improving the level of two different
neurotransmitters (serotonin and norepinephrine) should be
more effective. However, meta-analyzes show similar results
between SNRIs and SSRIs, both in terms of beneficial effects
and in relation to their side effects, especially bleeding (22).
Another drug, mirtazapine, which modulates the noradrenergicand serotonergic systems, has been effective in preventing andtreating PSD after a cure of at least 8 weeks (23), but with theadverse effect of sedation by antagonizing histaminergic H1
receptors.
Among the hypotheses mentioned above and which so far havenot been exploited at all in clinical trials, we mention the theoryof neuroinflammation, including the cascade of pro-inflammatory
cytokines and oxidative stress. Even simple drugs such as
nonsteroidal anti-inflammatory drugs (NSAIDs), with clinically
proven effectiveness in case of various local and general
inflammations and depression, have not yet been exploredin PSD. From the heterogeneous group of NSAIDs, selective
COX-2 NSAIDs should be tried, given that the effects of Cox-2
at CNS level are known. Regarding cytokines, novel therapies
such as monoclonal antibodies and cytokine inhibitors could
focus selectively on one cytokine, or, with reduced selectivity,
inhibit more than one cytokine at a time. Anti-cytokine treatment,
especially anti-TNF-alpha drugs, when used as adjunctive
therapy combined with SSRIs, is also effective in cases oftreatment-resistant depression (24). However, to be effective,
30

anti-cytokine monoclonal antibodies must cross the blood-brain
barrier and properly modulate the entire cytokine pathway inorder to minimize possible adverse effects, thus, improved
pharmaco-engineering is required in order to obtain novel potent
drug particles.
When thinking of the HPA axis, although strong preclinical andclinical evidence already exists, no drug has been approved until
today that targets specifically this neuroanatomical structure.
Moreover, comparative to other hypotheses such as theneuroinflammatory one, where at least in clinical studies, cytokine
detection is more routinely used, with cytokines becoming
increasingly valuable biomarkers, regarding the HPA alteration,
no biological or imagistic test are yet available. One simple
explanation could be that not all depressed patients display
alterations of the HPA axis, as this fact raises another future
therapeutic concern, mainly personalized medicine.
In addition to drugs, non-pharmacological therapies could be
used additionally to treat PSD. Among these, the cognitive
behavioral therapy (CBT) has been tried in several clinical trials,
a recent meta-analysis of 23 studies with 1,972 participants
supporting the effectiveness of this procedure, with variations
depending on the mean age and gender of the patient, baseline
depression score, and elements related to duration, manner andnumber of CBT administration sessions (25). Alternative andcomplementary treatment methods should not be neglected,
for example acupuncture, which can be an additional way oftreatment, along with drug therapy, currently several trials areunderway whose results will be later systematized in order toconfirm or not the benefit of these therapeutic methods (26).
Finally, control of vascular factors remains important and,
although often neglected by both the patient and the physician,relatively simple non-pharmacological methods and drugs exist,
both having an acceptable safety and efficacy profile. Observing
the lack of international accepted consent on PSD therapies,
fundamental and clinical studies are urgently needed in order
to explain as completely as possible the other factors related to
PSD, such as the roles of mitochondria in the pathophysiologyof this disorder.

CONCLUSION

PSD, due to the increased prevalence and the significant impact
on the individual’s quality of life, remains a topic of great interestfor clinicians dealing with rehabilitation after stroke. It is clear that
we cannot focus only on motor or language parameters when
considering a good recovery of stroke patients, ensuring a life
without depression being a goal at least as important. Currently,
it is necessary to continue studying in this field, with an emphasison the most complete explanation of the pathophysiology of
PSD. Once the biological mechanisms are understood, it will
be possible to create highly effective treatments with much
lower side effects compared to those related to currently used
medication. Checking the effectiveness of alternative therapies
and other non-pharmacological means of treatment can only
bring an extra benefit to the patient, increasing the person’squality of life after stroke.
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31

DEPRESIA DUPA ACCIDENTUL VASCULAR CEREBRAL –PERSPECTIVE ASUPRA MECANISMELOR FIZIOPATOLOGICEREZUMAT
Depresia dupa accident vascular cerebral este una din cele mai frecvente tulburari aparute la pacientul cu accident vascular cerebral,avand un impact nefavorabil asupra recuperarii si, implicit, asupra calitatii vietii acestuia. Numeroase studii epidemiologice au evidentiat
impactul semnificativ al acestui fenomen atat la nivel de individ (aproximativ 1 din 3 pacienti cu accident vascular cerebral va suferi
de depresie), cat si la nivel socio-economic. Desi intens studiata, aparitia depresiei doar la o parte dintre persoanele care au avut unaccident vascular cerebral in antecedente nu este pe deplin explicata, existand la acest moment mai multe teorii incomplete si posibilinterdependente. In acest context, scopul acestei lucrari este de a aduce o noua perspectiva asupra fenomenului depresiei associate
accidentului vascular cerebral, autorii prezentand in prima parte cele mai recente rezultate din literature in ce priveste mecanisme fiziopatologice ale depresiei post accident vascular cerebral, iar in partea a doua, mijloacele terapeutice actuale si viitoare sunt detaliate.
Cuvinte cheie: accident vascular cerebral, depresie, fiziopatologie, neuroinflamatie, stres oxidativ

32

Fiziologia - Physiology  2021.31.2(102)SOLUBILITY, STABILITY AND DEGRADATION KINETICS OF NICOTINAMIDE RIBOSIDE BORATE, A VITAMIN B3 DERIVATIVE, IN
PHYSIOLOGICAL SOLUTIONSAndreea Silvia Neamţu1, Andrei Biţă2,3, Ion Romulus Scorei3, George DanMogoşanu2, Cătălina Gabriela Pisoschi4
Doctoral School, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Dolj County, Romania
Department of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349
Craiova, Dolj County, Romania
3
BioBoron Research Institute, S.C. Natural Research S.R.L., 31B Dunării Street, 207465 Podari Commune, Dolj County, Romania
4
Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349
Craiova, Dolj County, Romania
1
2

ABSTRACT
Nicotinamide riboside (NR), a natural nicotinamide adenine dinucleotide (NAD+) precursor, is a nutrient that has been shown to havesignificant therapeutic potential due to its activity on sirtuins’ signaling pathways. Moreover, esterification of borate with nicotinamide
nucleosides could be of physiological significance since the ribose-borate complex released after their hydrolysis could be an important
physiological boron reservoir. These are reasons to undergo investigations in order to evaluate the effect of nicotinamide riboside borate(NRB) supplementation in experimental models of various diseases and becomes critical to determine the solubility and stability of thecomplex NRB in different physiological pH conditions that are found in the human body. Solubility and degradation kinetics of nicotinamide
riboside borate (NRB) in the following physiological media, 0.1 N hydrochloric acid (pH 1.5), sodium acetate buffer (pH 5.0), water (pH7.0) and phosphate-buffered saline (pH 7.4), were investigated using high-performance thin-layer chromatography. Stability studies of NRB
were also performed to determine the effect of different pH media and temperature on the degradation kinetics. Solubility and stabilityof NRB varied significantly in tested biological solutions. Solubility of NRB was pH-dependent and augmented with the increase in pH.
Keywords: Nicotinamide riboside borate, solubility, stability, degradation kinetics.

INTRODUCTION
Vitamins, especially those included in B group,
have gained considerable attention due to several benefits over drugs, including a wide rangeof therapeutic activities, fewer side effects andstructural complexity [1]. More than a hundred
clinical trials are underway to evaluate the effectiveness of natural drugs. After oral administration, the level of vitamins in plasma depends on
the absorption through the gastrointestinal tract(GIT). The stomach and intestines are the mainsites for the absorption of a vitamin molecule.
Therefore, it becomes critical to determine thesolubility and stability of a drug at the different
physiological pH conditions that are found in
the GIT. Many researches have highlighted thetherapeutic effects of a nicotinamide nucleoside,
nicotinamide riboside chloride (NRCl), but onlya systematic and adequate study evaluates solu-

bility and stability under different pH conditions
in environments simulating the physiologicalconditions of the GIT and blood plasma [2].
Comparatively to this study, we investigate theeffect of pH and storage temperature on nicotinamide riboside borate (NRB) stability.
A simple and fast high-performance thin-layer
chromatography (HPTLC) method confirmsthat both NRB and nicotinamide riboside (NR)have an alkaline hydrolytic degradation to nicotinamide (NAM) and ribose–borate complex insimulated GIT solutions. Any formulation should
promote the stability of NRB and NR and protect
them from hostile environments to prevent theaccumulation of a potentially antagonistic degradation product. With the current study, we hopeto enrich the NR literature with the data obtained
useful for developing and processing a future
formulation of the new NR compound stabi-

Received 16th of June 2021. Accepted 11th of August 2021. Address for correspondence: Andrei Biţă,, Department of Pharmacognosy & Phytotherapy,
Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Dolj County, Romania, phone number:
0723133740, e-mail: andrei.bita@umfcv.ro2021.31.2(102)  Fiziologia - Physiology

33

lized with borate and not with hydrochloric acid
[3–6]. The aim of this study was to characterize
NR stabilized with borate versus NR regarding
the physicochemical properties, including pKa,
logP, solubility, degradation kinetics, its stability
and behavior under thermally and physiologically relevant conditions.
MATERIALS AND METHODS
Chemicals and reagents
NAM was purchased from Sigma-Aldrich(Taufkirchen, Germany), while NR stabilizedwith dextrin (21.23% content of 93.4% purity)
was obtained from BOC Sciences (Shirley, NY,
USA) and the boric acid was bought from Merck(Darmstadt, Germany). Hydrochloric acid, formic acid, acetic acid, phosphate-buffered saline(PBS), sodium acetate and ammonium acetate
were purchased from Merck. The solvents,
namely, ethanol, methanol and water were alsobought from Merck.
Analytical method
NR was analyzed in different solutions using
HPTLC (CAMAG, Muttenz, Switzerland) andultra-high performance liquid chromatography(UHPLC, ACQUITY Arc, Waters Corporation,
Milford, MA, USA) coupled with tandem quadrupole mass spectrometer (MS, ACQUITY TQD,
Waters) [7]. The CAMAG system consists of an
application device, Linomat V, capable of applying the samples in bands and a TLC Scanner 3
which was used to obtain the densitograms, bothdevices being controlled using the visionCATS
software package version 3.0.
The samples were applied with the CAMAG
Linomat V in 8 mm length bands on HPTLC
silica gel 60 F254 glass plates (Merck) at 8 mmfrom the bottom edge. The mobile phase used
was ethanol–1M ammonium acetate–formic
acid (7:1:0.1, v/v/v). The elution was conducted
in 20×10 cm glass twin-trough chamber on a 60
mm distance. After elution, the plate was dried
with the help of an air drier at room temperature
for about 5 minutes. After drying, the plates
were documented at 270 nm (the maximum absorbance peak of NR). The densitograms wereobtained with the CAMAG TLC Scanner 3 in34

absorbance mode optimized for resolution, witha scanning speed of 20 mm/s, a slit of 5×0.2 mm
and a data resolution of 100 μm/step. The lamps
used for detection were deuterium and tungsten.
The evaluation of the resulted densitograms were
processed using the visionCATS software package version 3.0.
The UHPLC method employed a CORTECS
C18 column (4.6×50 mm, 2.7 μm) and themobile phase consisted of an isocratic elution of methanol–10 mM ammonium acetate(40:60, v/v). The flow rate was kept at 1 mL/
min, at a column temperature of 60°C [8, 9].
The MS/MS parameters used for NR and NRB
were positive mode, collision energy 12 eV
and cone voltage 14 eV. The NR sample forthe stability testing was obtained by solubilizing in methanol an amount of the dextrin
stabilized product to obtain a concentration of
2 mg/mL NR. To compare the stability of NR
with the NRB, an additional sample was preparedby adding to the previous NR stock solution an
amount of boric acid to obtain a 1 mg/mL concentration. The concentration of the samples for
MS/MS analysis was 10 mM.
NRB determination
The coupling of NR with boric acid was investigated by the means of the UHPLC–MS/MS assay(see “Analytical method” subsection).
pH stability testingStability studies were performed in 0.1 N hydrochloric acid (pH 1.5), sodium acetate buffer(pH 5.0), water (pH 7.0) and PBS (pH 7.4) containing NR and NRB with the concentration as
mentioned in the “Analytical method” subsection(see above). All the solutions were incubatedat 37°C for 24 hours after which the HPTLC
method was employed to determine the stabilityof the compounds.
Temperature stability testing
The sample preparation used for the temperaturestability testing was described in the “Analytical method” subsection (see above). The temperatures at which the samples were kept for 15
minutes were 30°C, 40°C, 50°C and 60°C. The
reference temperature was 20°C. Aliquots were
Fiziologia - Physiology  2021.31.2(102)removed and prepared for the HPTLC analysis.
Statistical analysis
All experiments were performed in triplicate
and results are reported as a mean ± standard
deviation (SD). The p-values less than 0.05 wereconsidered as statistically significant.
RESULTSNRB synthesis
The synthesis of NRB was performed according
to the US Patent [6] and verified via the method
described in the “Materials and Methods” section. The obtained spectra for both NR and NRB
are shown in Figures 1 and 2.

Fig.1. NR mass spectrometry spectrum (NR – m/z 255; NAM – m/z 123). NAM: Nicotinamide; NR: Nicotinamide riboside

2021.31.2(102)  Fiziologia - Physiology

35

Fig. 2. NRB mass spectrometry spectrum (NRB: 11B – m/z 281; 10B – m/z 280). NRB: Nicotinamide riboside borate.
pH stability testing
The results regarding the pH stability testing are accurately presented in the graphs (Figures 3 and 4) and Table I from below. The RF
obtained for NAM and NR/NRB during the pH stability test were 0.62±0.01 and 0.255±0.01, respectively. The areas of each peak were
integrated using the visionCATS software package version 3.0 and shown in Table I. The graph was computed using the GraphPad
Prism 8 software and it shows that the NRB begins to degrade at pH 7.0 (Figure 4).

Fig. 3. Overview of pH stability test densitograms..

36

Fiziologia - Physiology  2021.31.2(102)Fig. 4. Graph showing pH stability for both NR and NRB.NR: Nicotinamide riboside; NRB: Nicotinamide riboside borate.

Table 1. Sample areas for the pH stability testing analysis
extracted from densitograms.

pH

NR

NRB

0.010640.01335

0.011130.01259

0.01169

0.005880.012900.00577

1.5
5.0
7.0
7.4

Temperature stability testing
The RF obtained for NAM and NR/NRB during the temperature
test were just slightly different (mainly due to the difference in thelaboratory environment temperature and humidity), 0.615±0.01
and 0.250±0.01, respectively (Figure 5).

NR: Nicotinamide riboside; NRB: Nicotinamide ribosideborate.

2021.31.2(102)  Fiziologia - Physiology

37

Fig. 5. Overview of temperature stability test densitograms.

Same as before, the areas of each peak were integrated using the visionCATS software package version 3.0 and shown in Table II.
Table 2. Sample areas for the temperature stability testing analysis extracted from densitograms.
Temperature [°C]
20
30
40
50
60

NR

NRB

0.009475

0.009410

0.009501

0.009695

0.009504

0.009482

0.009112

0.009208

0.007205

0.007322

NR: Nicotinamide riboside; NRB: Nicotinamide riboside borate.
The computed graph shows that both NR and NRB begin to degrade starting 50°C (Figure 6).

38

Fiziologia - Physiology  2021.31.2(102)Fig. 6. Graph showing the temperature stability of both NR and NRB. NR: Nicotinamide riboside; NRB:
Nicotinamide riboside borate.

Aqueous solubility of NRB
The solubility of NRB was determined by the shake-flask method
and Table III summarizes the solubility in different solvents, atroom temperature [10]. The solubility of NRB at pH 1.5, 5.0 and
7.4 was measured to be 1972.7±15.4 mg/mL, 1060.5±31.0
mg/mL and 926.0±34.4 mg/mL, respectively. NRB was highly
soluble in all three solutions with different pH, taking into accountthe recommendations of the United States Pharmacopeia [11].
Table 3. Nicotinamide riboside borate solubility in different
solutionspH

Solubility [mg/mL]

1.5

1972.7±15.4

5.01060.5±31.0

7.4

926.0±34.4

Gibbs free energy (ΔG) for comparative degradation wasdetermined to study the spontaneity of NR and NRB degradationin different pH solutions. ΔG was calculated using the following
equation:,where A and A0 are the absorbance values of the sample at 0 h
and 24 h, respectively.
Table IV and Figure 7 show the values for the pH-dependent NR
and NRB solutions under different storage conditions, to deduce
spontaneous NRB degradation. The ΔG values were -96.46 kcal/
mol and 2.43 kcal/mol for NR and NRB, respectively.
Table 4. ODs for storage temperatures
Temperature [°C]2021.31.2(102)  Fiziologia - Physiology

NRB

0.961

0.998

0.999

0.998

1

0.999

1

0.997

0.989

0.985

20
30
40

pKa determination and logP prediction
An isosbestic point is observed at the λmax 270 nm for all
tested pH solutions. A predicted logP of –6.25 was obtained by
MarvinSketch (ChemAxon Ltd., Hungary) inputting the chemical
structure of NR, value which suggest that NR is a very hydrophiliccompound. The pKa of NR was predicted to be 11.25, using thesame software package. The predicted logP for NRB was -4.17
and the pKa was 13.509.
Degradation kinetics of NRB

NR

50
60

NR: Nicotinamide riboside; NRB: Nicotinamide ribosideborate; ODs: Optical densities.
39

Fig. 7. Graph compiled from the ODs. NR: Nicotinamide riboside; NRB: Nicotinamide riboside borate; OD:
Optical density.

DISCUSSIONS

NR is a vitamin B3 derivative endowed with a significant
therapeutic potential and has undergone investigations toassess its benefits as a nutritional supplement. NR enhances
NAD+ biosynthesis and have beneficial effects in multiple
disorders [12–15]. Accumulating data has validated its efficiency
across numerous animal and human studies for the treatmentof cardiovascular, neurodegenerative, metabolic disorders,
and retina degeneration [16,17]. NR, like any other nutrient
administered to the body, can be exposed to a wide range of pH
conditions in various compartments. An intravenous injection of
NR would be exposed to pH 7.4 in the blood, not to mention the
more complex pH environment along the GIT [2]. When NRB istested as a nutritional supplement, the production of NAM (animportant vitamin involved in the same biochemical processes
as NR) and of ribose–borate complex (a sugar–borate that isa physiological boron reservoir) as degradation products it is asignificant advantage [6]. However, the release of NAM is notdesirable, since NAM may counteract some positive effects
attributed to NR and NRB supplementation. NAM and NR are
opposite in the regulatory mechanism of sirtuin action in vitro andin vivo [18]. It is known that NAM acts as a sirtuin inhibitor while
NR increases NAD+ supply for sirtuins actions [18-20]. That’s why
is of the outmost importance to understand the physicochemical
conditions under which NR and NRB are stable. It is known thatthe presence of the anionic borate can inhibit NR dissolution to
NAM and stimulates SIRT.
There are few scientific data that show a minor degradation of NR
in simulated gastric fluids but it seems that there is a significant
40

degradation in the intestinal environment due to its high pH [2].
Neutral conditions, such as pH 7.4 used in this study, have asignificant effect on NR degradation. In the Arrhenius model of
NRB degradation, the rate of NR degradation is higher at pH
7.4 compared to pH 1.5 and pH 5.0 and supports the hypothesisthat NRB degradation will be related to the proportion of hydroxylions in the solution. Given the fact that it undergoes hydrolysis,
care must be taken when one uses an aqueous solution of
NRB, especially at high pH values with excessive hydroxyl ionspromoting NRB degradation. Based on our results, we presume
that there is a slightly different mechanism of NR and NRB
degradation in acidic conditions compared to their degradationin neutral/alkaline conditions. So, this model can be used as a
general guide for understanding the stability of NRB in various
buffered solutions. Temperature also has an effect on NRBcontent in solution, a 10°C increase approximately doubling its
degradation in any pH condition.
logP estimation is important to predict NRB permeability. It
is known that the compounds with estimated logP values​​
less than 1.72 have a low permeability [21]. According to thebiopharmaceutical classification system (BCS), NRB should beclass III due to its high solubility but expected low permeability[22] related to a logP of -4.17. Physiologically, this could be
related to the proportion of degradation products released.
UHPLC spectra suggested that the degradation products of NRB
in aqueous solutions are NAM and ribose-borate complex. Whendegrades, each molecule of NRB forms a molecule of NAM andone of ribose-borate. For this reason, NAM concentration canbe used as a marker for NRB degradation as well as for NRCl
Fiziologia - Physiology  2021.31.2(102)[2], the commercial form existing on the market. HPTLC method
developed can separate and quantify NR and NAM peaks witha high separation resolution (R = 1.8) that far exceeds theacceptable resolution (R>1) recommended by the Food andDrug Administration [23]. This method provides rapid detectionof NR and its degradation product, NAM, as well as ribose-borate
complex.
Our research data provide, for the first time, an explicit upper
temperature limit for the processing of NRB and its products,
which may have an impact on the production of supplements inseveral stages. Optimizing the administration of NRB will havea significant impact on its effectiveness. Through this study, we
tried to reveal a mechanism by which NRB degradation forms aphysiological product when exposed to simulated physiologicalfluids in vitro. Development of the industrial pilot manufacturer
NRB will allow further research with a potential high impact on
its perspective therapeutic use. Therefore, under this condition,
NRB solutions and formulations could be very stable. The
results of this study will be helpful for pharmaceutical scientists
in handling NRB working solutions and designing appropriate
formulations for NRB delivery. Clear pH-dependent NRB
solubilization was observed. NRB was found to be extremely
unstable at pH greater than 8.0. However, it was metastable inan acidic and neutral environment.
CONCLUSIONSNRB could be better as a nutritional supplement when compared
to NRCl due to the blockade of the glycosidic bond between
the pyrimidine base and ribose by boric acid residue. The study
revealed the main physicochemical properties to be consideredwhen NRB is used for in vitro and in vivo experiments. The
HPTLC method described could be useful to discriminate
between NRB and its degradation products in media with pH
conditions similar to those from the GIT. The data obtained arealso important for processing, production, and storage of this
supplement.
Acknowledgments: This work was supported by a grant of
the Romanian Ministry of Education and Research, CNCS–
UEFISCDI, project number PN-III-P1-1.1-PD-2019-0214, within
PNCDI III.

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Neamţu AS, Biţă A, Scorei IR et al. Simultaneous quantitation of
nicotinamide riboside and nicotinamide in dietary supplements
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Trammell SA, Brenner C. Targeted LCMS-based metabolomics
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Carpi F, Cortese M, Orsomando G et al. Simultaneous quantification of nicotinamide mononucleotide and related pyridine
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2018;1(1):22-30.
10. Baka E, Comer JE, Takács-Novák K. Study of equilibrium
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11. Stegemann S, Leveiller F, Franchi D et al. When poor solubility
becomes an issue: from early stage to proof of concept. Eur J
Pharm Sci 2007;31(5):249-261.
12. Bieganowski P, Brenner C. Discoveries of nicotinamide riboside
as a nutrient and conserved NRK genes establish a Preiss–
Handler independent route to NAD+ in fungi and humans. Cell
2004;117(4):495-502.
13. Bogan KL, Brenner C. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitaminsin human nutrition. Annu Rev Nutr 2008;28:115-130.
14. Cantó C, Houtkooper RH, Pirinen E et al. The NAD+ precursor nicotinamide riboside enhances oxidative metabolism
and protects against high-fat diet-induced obesity. Cell Metab
2012;15(6):838-847.
15. Chi Y, Sauve AA. Nicotinamide riboside, a trace nutrient in foods,
is a vitamin B3 with effects on energy metabolism and neuroprotection. Curr Opin Clin Nutr Metab Care 2013;16(6):657-661.
16. Mehmel M, Jovanović N, Spitz U. Nicotinamide riboside – thecurrent state of research and therapeutic uses. Nutrients 2020;12(6):1616.
17. Pirvu AS, Andrei AM, Stănciulescu EC et al. NAD+ metabolism andretinal degeneration (Review). Exp Ther Med 2021;22(1): 670.
18. Avalos JL, Bever KM, Wolberg C. Mechanism of Sirtuin Inhibition
by Nicotinamide: Altering the NAD+ Cosubstrate Specificity of a
Sir2 Enzyme. Mol Cell 2005;17(6):855-868.
19. Brown KD, Maqsood S, Huang JY et al. Activation of SIRT3 by
the NAD+ precursor nicotinamide riboside protects from noiseinduced hearing loss. Cell Metab 2014;20(6):1059-1068.
20. Trammell SA, Schmidt MS, Weidemann BJ et al. Nicotinamide
riboside is uniquely and orally bioavailable in mice and humans.
Nature Commun 2016;7:12948.
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Dahan A, Miller JM, Amidon GL. Prediction of Solubility andPermeability Class Membership: Provisional BCS Classificationof the World’s Top Oral Drugs. AAPS J. 2009;11(4):740–746.
22. Amidon GL, Lennernäs H, Shah VP, Crison JR. A theoreticalbasis for a biopharmaceutic drug classification: the correlation ofin vitro drug product dissolution and in vivo bioavailability. Pharm
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23. Analytical Methods Technical Committee of the Chemistry Manufacturing Controls Coordinating Committee of the Center for Drug
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41

CINETICA DE SOLUBILITATE, STABILITATE ȘI DEGRADAREA BORATULUI DE NICOTINAMIDĂ RIBOSIDĂ, O DERIVATĂ
A VITAMINEI B3, ÎN SOLUȚII FIZIOLOGICEREZUMAT
Nicotinamid ribozida (NR), precursor natural al nicotinamid adenin dinucleotidei (NAD+), este un nutrient ce a dovedit un potențial
terapeutic important datorită acțiunii sale asupra căilor de semnalizare a sirtuinelor. În plus, esterificarea nicotinamid nucleozidelor cuacid boric poate avea semnificație fiziologică deoarece complexul riboză-borat eliberat după hidroliza acestora ar putea fi un important
rezervor fiziologic de bor. Acestea sunt câteva motive pentru studii pe modele experimentale ale unor afecțiuni variate pentru evaluarea
efectelor suplimentării cu nicotinamid ribozid borat fiind importantă cunoașterea solubilității și stabilității complexului NRB în condițiile
mediilor cu pH fiziologic variat din organismul uman. Au fost investigate solubilitatea și cinetica de degradare a NRB în medii fiziologicediferite, precum acid clorhidric 0.1 N (pH 1.2), tampon acetat (pH 5.0), apă (pH 7.0) și tampon fosfat salin (pH 7.4), prin HPTLC (highperformance thin-layer chromatography-cromatografie în strat subțire de înaltă performanță). Studiile de stabilitate au fost realizate pentru
a determina efectul mediilor cu pH diferit și al temperaturii asupra cineticii de degradare a NRB. Solubilitatea și stabilitatea NRB au variat
semnificativ în soluțiile biologice testate. Solubilitatea NRB a crescut odată cu creșterea valorilor pH-ului.
Cuvinte cheie: nicotinamid ribozid borat, solubilitate, stabilitate, cinetică de degradare.

42

Fiziologia - Physiology  2021.31.2(102)CURRENT TRENDS IN THE ASSESSMENT OF SUICIDE ATTEMPTS
EDUARD NICUȘOR BONDOC1, MARINESCU ILEANA2, RAMONA MĂLIN3
1Clinical psychologist , Phd Student, University Clinic of Neuropsychiatry Craiova
2University of Medicine and Pharmacy of Craiova, Romania
3University of Medicine and Pharmacy of Craiova, Romania
ABSTRACT
Scientific acquisitions in recent decades generate new perspectives in deciphering the suicidal phenomenon, defining it as a multi- andinterdisciplinary problem still under the sign of scientific interrogation, despite its efforts and its age.
This research, with a theoretical justification, starts from the hypothesis that suicidal behavior goes beyond pathological conditioning,extending into the individual system of moral values and the personal way of responding to psychostressing factors.
The risk for suicide in the context of somatic diseases is significantly correlated with the presence of a mental disorder, particularly
depression, but also involves the personality traits of the patient, his ability to adapt, availability for social support, the presence of
psychosocial stressors and history personal suicidal behavior.
From these results it can be concluded that the frequency of suicidal act could be influenced in the sense of reducing it, in the conditionsof primary, secondary and tertiary prophylaxis, emphasizing the therapeutic adequacy and socio-familial reintegration measures.
Keywords: suicidal phenomenon, pathological conditioning, moral values, psychostressing factors.

INTRODUCTION
Both the criteria for defining mental illness and the criteria
for considering a suicide or attempt differ and show instabilityof the approach. It is recognized that a fairly small numberof suicidal acts is related to clear, obvious mental illness.
Otherwise, it is up to the person carrying out the researchwhether to study a transient reaction, classified it as a diseaseor as a normal response to special situations. Secondly,
difficulties may also arise in the way in which the presence of amental illness is assessed at the time of committing the suicidal
act. On the other hand, data from subjects with suicide attempts
cannot be correlated with information obtained in the case ofcompleted suicides, as they represent two different groups.
This research, with a theoretical justification, starts fromthe assumption that suicidal behavior goes beyond pathologicalconditioning, extending into the individual system of moral
values and the personal way of responding to stressing
factors. At the same time, the approach of the ethiopathic
study of suicide in depression was motivated by the reality that
depressive disorder is the most common psychiatric syndrome
found in practice, with heterogeneous and multifactorial etiology
and a multitude of clinical types.
Scientific acquisitions in recent decades generate newperspectives in deciphering the suicidal phenomenon, defining
it as a multi- and interdisciplinary problem still under the sign ofscientific interrogation, despite its efforts and its age.

ETIOPATHOGENIC IPOTESIS IN SUICIDE
There are currently numerous arguments for involvement in
the ethiopathogeny of depression and schizophrenia, conditions
associated with increased suicide rates, neuroanatomical
circuits and neurotransmitter systems.[14]Neuroimaging studies CT and MRI have shownventriculomegaly associated with suicides with low L-tryptophan
levels and increased cortisolemy (presynaptic serotonin
depression). [25]PET studies in patients with recurrent suicide attempts
showed decreased cerebral blood flow and metabolism in theleft frontal cortex, in contrast to significant increase in righttemporal lobe activity. [29]
SPECT studies have shown in suicide patients asignificant increase in the activity of 5-HT2 receptors in the righthemisphere, frontal, parietal and temporal, with a decrease in
the number of GABA receptors. [16]Current trends in the assessment of suicide attempts are
based on different neurobiological models, with two variants:high level and low level of risk of death. These studies(Oquendo, 2003) are of particular clinical importance byidentifying differentiated neurobiological bases. [21]In subjects with high-risk suicide attempts, serotonin levels
and indirect biochemical indicators for serotonin function show
sharp decreases compared to depression levels and low-risk
lethal risk attempt.
The differences between the two groups relate to

Received 16th of July 2021. Accepted 28th of August 2021. Address for correspondence: Psych. Eduard Nicușor Bondoc, University Clinic of Neuropsychiatry
Craiova, Aleea Potelu no 24, Craiova, Dolj, Romania, tel 0723948913, email: edy.bondoc @yahoo.com

2021.31.2(102)  Fiziologia - Physiology

43the relative hypo functionality of the prefrontal cortex anddecreased activity in the anterior cingulate gyrus, significantly
correlated with frontal dysfunction, either in the bilateral frontal
median gyrus or in the right upper frontal gyrus.
These data allow the assessment of suicidal behavior andits lethal risk independently of impulsiveness. In this context
assessment of a suicide attempt will take into account a set ofcriteria:• Neuroimaging changes;• the clinical aspect of the depressive disorder;• the presence of impulsivity and the planning of thesuicidal act;
• hypercortisolemy;• cognitive deficit.
The association of low dopamine levels and markers in
CRL, as well as the history of extrapyramidal phenomena, areadditional risk factors for the severity of suicidal act.
From a psychopharmacological perspective theoccurrence of extrapyramidal symptomatology in a patient
treated with serotonin antidepressants (SSRIs) may be amarker for predicting suicidal risk. The same mechanism canbe considered in the case of patients with major psychotic
disorders, antipsychotic treatment and extrapyramidal side
effects.
Biological-biochemical vulnerability is the background on
which autolytic behavior can be carried out, the psychological,
cognitive and social components being favorable factors,
genuine triggers of suicidal drama.
In the ethiopaty of suicidal behavior we can speak of
a socio-genetic vulnerability in which neurobiochemicalvulnerability is distinguished. Rice has shown an increased risk
for monopolistic affective disease in subjects from families withdepression, alcoholism and suicidal or aggressive behavior
since 1987.
Extensive studies have evaluated the presynaptic releaseof monoamines, intrasynaptic or intracellular degradation,and the phenomenon of receptor "binding", the place androle of receptors in the mechanism of action of psychotropic
substances.
Serotonin deficiency
Post-mortem studies have objectified the deficiency of theserotonin system by:• decrease in 5-HT levels and 5-hydroxyindolacetic
acid (5-HIAA), predominately in the frontal cortex;• significant decrease in binding sites for tricyclic andparoxetine antidepressants, marked with radioactive isotopes(binding studies);• increase in the number of 5-HT2 receptors in thefrontal cortex, as an adaptive factor of the post synaptic floor
compared to the decrease in presynaptic 5-HT activity;• modification of pre and postsynaptic 5-HT receptors
in the hippocampus and basal ganglia.
These changes may or may not be associated withdiagnostic psychopathological disorders at the time ofsuicide. Postmortem studies take on a special dimension, as
44

argumentative elements, objectives that support or disprove
suicide, in unclear cases.
"In vivo" studies show a marked decrease in 5-HIAA
concentration in CRL, significantly associated with impulsiveviolent suicidal behavior and its recurrence. Dynamic trackingof 5-HIAA levels in the LCR reveals two types of activity:• Consistently low levels of 5-HIAA – translate a
primary presynaptic serotonin deficiency correlated with low
L-tryptophan levels and low platelet MAO activity (strong
genetic brand);• Variable levels of 5-HIAA whose decrease anticipatessuicidal behavior, being determined by the decrease in theeffectiveness of the system of second messengers in theserotonin neurotransmission chain, with inositol-phosphate at
the center (Shimon, 1998) [26]Altering the functioning of the system of second
messengers raises the problem of "collapse" of serotonintransmission at the postsynaptic level, by the inability of thereceptor system to decode the presynaptic signal.
The decrease in presynaptic serotonin transmission
appears to be significantly correlated with violent suicide andantisocial behavior, with a strong genetic determinism:• positive family history for antisocial behavior,correlated with low levels of 5-HIAA in CRL (Constantino, 1997)[7], faithful witness of aggression and hostility;• Cloninger noted in alcoholism type I, an impulsive
antisocial behavior and a tendency to violent suicide, that the
deficit of 5-HT is associated with the decrease in the level of
MAO platelet activity and 5-HIAA in the CRL.
• Serotonin central activity is also related to the weakhormonal response to the administration of a serotonin agent- d-phenfluramine (Coccaro,1996) [6]; prolactin secretion
does not increase significantly in people with violent self- or
heteroagressive violent behavior.
Recurrent aggressive and impulsive behavior is correlatedwith low values of 5-HIAA in CRL (Asberg, 1986; Virkkonen,1994) and low platelet levels of paroxetine binding (Coccaro,1996), and the seasonality of depression correlates withthe decrease in tryptophan (Neumeister A,, 1998) [20], with
presynaptic serotonin deficiency (Carroll BJ, 2007) [5]
Dopaminergic dysfunction
The objective of reversing the ratio between prolactinic
stimulation with d-phenfluramine and 5-HIAA in the LCR brings
into question the intrigue of 5-HT and DA activity in a genuine
functional balance of the two neurotransmission systems.
This view is supported by research, which has shown inpeople with behavior/ excitable (impulsively the existence of D4"variant" receptors D4.7 – compared to the normal D4 receptor
D4.4 (present in 66% of subjects).
The D4.7 receptor is much more common in people with
ADHD (Attention Deficit Hipereactivity Disorder) syndrome andexcitable – impulsive behavior.
The interference of serotonin and dopaminergic systems
in suicidal behavior seems all the more evident since, as early
as 1988, Lopez Ibor jr. correlated serotonin deficiency with
Fiziologia - Physiology  2021.31.2(102)attention deficit, hyperkinetic syndrome and aggressive and/or
suicidal behavior.
Consistent with those supported, depression by DA
deficiency (depressive dopaminedependence – Mouret, 1988)[19] is considered to have the most important suicide risk.
Noradrenergic dysfunction:
In severe forms of depression accompanied by suicidal
behavior/risk, there is an increased plasma level of NA
significantly influenced by electroconvulsive therapy.
It can be assumed that there is a NA/5HT junction, sincein major depression, the NA system behaves bimodally: NA
deficiency – inhibited depression, NA hyperactivity – depression
with aggression, hostility, anxiety and suicidal risk.
The involvement of the noradrenargic system by the NA/5HT junction in hyperkinetic syndrome and ADHD explains theobvious therapeutic results with amphetamine derivatives inthe specific treatment of these syndromes.
Tiihonen – 1997 [27], argues that the seasonality ofpathological crime is superposable over that of violent suicide,both manifestations being related to a primary "presynaptic"
defect; Bandecchi – 1994 [2], considers that the seasonal peakof pathological crime can also be influenced by the level of DA
transmission and testosterone secretion, which significantly
raises the rate of the aggressive behavior.

EPIDEMIOLOGICAL DATA
Unlike other medical clinical conditions, estimating thesuicide mortality rate is difficult to achieve due to epidemiologicalentropy factors, among which it is a masked way, suicide
is often recorded as an accident that remains etiologically
undetermined. In other cases, it cannot be determined whether
death is caused by suicide or murder (Cooper, J, , 2006) [10].
Among people whose deaths are recorded as accidental,
some had previously suffered from depression, drug or alcohol
dependence, thus presenting risk conditions for suicide (Haris,
Barraclough,1997) [13]. In England and Wales, official figuresdepend on verdicts in the courts, with a strict rule that suicidemust be proven by evidence, and if there is doubt about theverdict, investigations are resumed. In this situation, the sourceof error practically depends on the legal criteria. In othercountries, less stringent criteria are used.
Any national or international research on the suicide
rate raises the issue of the validity of data collected fromdescriptions, analyzes or even national mortality statistics.
The methods of accomplishment and the criteria for declaring
suicide vary greatly in different peoples of the globe, withdifferences in the calculation of mortality, cultural attitudes
towards suicide affecting official statistics to such an extent thatthey become inoperative (Cosman, 1999) [11].
Sex
A number of studies show that men commit suicide 3 times
more often than women, and the latter commit about 14 times
2021.31.2(102)  Fiziologia - Physiology

more suicide attempts. Regarding the comparative incidence
by sex of suicide attempts, most authors noted that this ratio is
not constant, but variable with age: thus under the age of 20,
the ratio is 10: 1, after which it shows a decrease continues, sothat in the 41-50s, this ratio reaches 3: 1 (Haw, 2005).
In countries with high suicide rates such as Lithuania,
Estonia, Russia, the male / female suicide rate is much higher
than average. An exception is China, for which studies havesuggested a suicide rate in women 25% higher than in men(O.M.S., 2006). The maximum value in men is after 45 years
and in women after 55 years.
In the United States, the suicide rate has risen, especiallyamong men aged 15-24, the third leading cause of death after
accidents and homicides, and suicide attempts in this age
group range from 1 to 2 million annually. Also, in the last 10
years, suicide has increased by 30% in men between 25 and34 years old (Sadock and Kaplan, 2007).
There is a common opinion that the increased suicide
rate in men is also due to the highly lethal methods used bythem: hanging, firearms, precipitation or defenestration fromhigh altitudes, as opposed to overdose intake of psychoactive
substances or poisons used by women. (Hawton, 2000). In
some European countries and in the USA, the most common
method of suicide remains hanging, a trend common toindustrialized countries.
In principle, suicide methods in the elderly are much more
violent in men than in women. The pattern of increasing suicide
rates with aging is similar in both sexes, although the rate ishigher in men probably in relation to the frequency of problems
related to alcohol or substance abuse, without depressive
symptoms.
One can discuss the existence of protective factors present
in women, such as pregnancy and the presence of children in
the family, but it must also be taken into account that womencommit more unsuccessful suicide attempts compared to men(Kutcher and Checil, 2009).
Age
There is unanimous recognition that the suicide rateincreases with age. In North America, Western Europe,
including the United Kingdom, and in most countries that reportvalidated data, the suicide rate increases with age, but there isa tendency for two peaks: adolescents and young adults.
Mortality declared by suicide before the age of 15 is little
recognized. Suicide is the second leading cause of deathamong young people between the ages of 15 and 19, and is oneof the most serious problems facing society today. A number of
specific risk factors with a favorable role were identified: family
factors (conflicting relationships with the family, family abuse,
divided families, parental divorce, death of a close person);school performance (children and adolescents feel pressured
by the family to get better results); social stressors (lack ofcommunication, emotional expression and relationship skills);
individual factors (emotional, cognitive, behavioral, health);family history of suicidal behavior, mental disorders or alcohol45dependence [1].
The risk of suicide, which accounts for 12% of adolescentmortality, is significantly associated after Sadock and Kaplan
with depressive disorders. However, the same authors statethat in the last 15 years, both the rate of attempted andcompleted suicide has decreased in adolescents, apparently
in relation to the introduction of antidepressant treatment in thegroup of selective serotonin reuptake inhibitors, prescribed toadolescents with mood and behavior disorders.
Regarding the differences in the suicide rate according toage, Sadock and Kaplan – 2002 [24], state that the highest
rate of 40 per 100,000 inhabitants is in men over 65 years.
Older people commit fewer suicide attempts, but have a higher
incidence of completed autolytic acts. Although in the USA it
represents only 10% of the population, the elderly commit 25%of the total suicides, the rate over 75 years being three times
higher than among young people.
The consideration that most suicides in the elderly are"rational, comprehensible" in response to irreversible life
situations is not supported by clinical trials, depression being
the most important predictor of suicide in old age.
Conwell – 1996 [8], studying the relationship between age
and Axis 1 on a sample of 141 complete suicides in people
aged 21-92 years, found affective disorders in 71% and 84% ofcases in the cohort 75-92 years and 55 respectively -74 years,
compared to 30% of the age group 21-34 years.
Major late-onset depression was diagnosed inapproximately 60% of suicides in old age, with non-affective
psychosis and addiction disorders being rare.
Barraclough – 1971 [3], outlined the symptomatic profile
of elderly people with suicidal depression, revealing: insomnia
in 90% of cases, weight loss 75%, guilt 50% and hypochondria
50% of cases. Isolated depressive symptoms are risk factors
for major depression and should not be considered as normal
in the aging process or as a natural consequence of the socialand economic difficulties faced by this category of patients.
Many countries have reported a decline in the suicide rate
among the elderly in recent years, with the exception of men
over the age of 85. The gender ratio in the elderly is 3: 1 formen.
Marital and professional status
The status of unmarried, divorced or widowed is a factor
that doubles the risk of suicide, compared to marriage withchildren.
Professional status is another factor that can influence
the risk of suicide, being recognized by many authors thefact that a high professional and social position increases
the risk of suicide but also its loss. Unemployed people andthe unemployed have a higher suicide rate. Work is generally
recognized as a protection factor against suicide. The suicide
rate increases during periods of economic recession anddecreases during times of war.
Depending on the occupational category, doctors are
traditionally considered to have a higher risk of suicide. The
46

conclusion of several studies is that in the US there is anincreased suicide rate in doctors of both sexes. Women doctors
have a higher risk of suicide than other women, 410/0000
compared to 120/0000 over 25 years.
Recent data from England and Scandinavia have shownthat the suicide rate in male physicians is 2 to 3 times higher
than in men of the same age in the general population.
Both women and men doctors commit suicide more
frequently by overdose and less by firearms than the general
population, this being determined by access to drugs andknowledge about their toxicity.
Among doctors, psychiatrists are considered to be at thehighest risk, followed by ophthalmologists and anesthetists, but
all specialties are vulnerable (Sadock, 2007) [18].
Somatic diseases
Somatic diseases may increase the risk of suicide
especially if they are associated with functional and / or
cognitive impairment, chronic pain, disfigurement, increased
dependence on others, or sensory impairment of sight or
hearing; Neurological diseases such as epilepsy, multiple
sclerosis, Huntington's chorea or brain or spinal injuries areassociated with an increased risk of suicide. Other somaticconditions recognized as being associated with an increased
risk of suicide include: AIDS / HIV, neoplasms, peptic ulcer,
disseminated lupus erythematous, heart disease, chronic
obstructive pulmonary disease (Kutcher and Chehil, 2009) [15].
The risk for suicide in the context of somatic diseases issignificantly correlated with the presence of a mental disorder,
particularly depression, but also involves the personality traitsof the patient, his ability to adapt, availability for social support,
the presence of psychosocial stressors and history personal
suicidal behavior. In patients with chronic somatic disorders,special importance should be given to depression especiallythat associated with suicidal ideation; there is a tendency tounderdiagnosed depression.
Ways to commit suicide
Hanging is one of the most common forms of suicide
worldwide with an increasing incidence over the past 30 years.
Studies conducted in England and the United States haveshown an increase in this suicidal modality in parallel with thedecrease in the number of suicides with firearms.
Only a small proportion, about 10% of hanging suicides
occur in controlled areas such as hospitals, prisons or police
custody, most of them in the community.
In England, hanging deaths account for about 70% of
attempts in this way, and most patients, 80-90%, who arrive at
the hospital alive, survive this trial.
Materials used, rope, cables, etc. they are easy to procure,for this reason prevention cannot be focused at this level asin the case of limiting access to firearms or the sale of toxic
substances, but on general measures of prophylaxis.
However, in the United States, gun suicide accounts
for more than half of all suicides. In 2005, an average of 46
Fiziologia - Physiology  2021.31.2(102)Americans committed suicide with firearms per day (Miller and
Hemenway, 2008) [18].

SUICIDAL BEHAVIOR IN PSYCHOLOGICALDISEASES
Suicide research has been conducted in a variety of ways,
the most important methods including studies on the generalor clinical population. An example would be the method of
psychological autopsy, in which survivors or other people cangive information about victims, and medical records are studied
in order to establish the mental state.
Of the general population studies conducted in the last 30
years, six provided similar information on Robins suicide - 1960,
Dorpat and Ripley - 1960, Rich - 1986, in the United States,
Baraclough - 1974 in the United Kingdom, Besskow - 1970 in
Sweden, Chynoweth - 1980 in Australia, despite differences inmethodology, diagnostic criteria, location of studies and year ofcompletion reached several common conclusions:• 90% of suicides suffer from a major mental disorderat the time of suicide, and about half had clinically manifest
depression and a third were chronic alcohol users.
• Mental disorders such as schizophrenia, organic
psychiatric disorders, anxiety disorders and those caused bysubstance abuse have been rare among suicides.
• Few suicides have been committed by people
considered without a mental disorder, suggesting that "rational"
suicide is rare.
• Men over 40, divorced make up 2/3 of the numberof suicides.
• Psychiatric diagnoses tend to differ depending on
age groups, so Dorpat and Ripley - 1960, found schizophrenia
as the most common diagnosis under 40 years, alcoholism
between 40-60 years and depression over 60 years; Rich - 1986
found substance abuse and antisocial personality disorders
predominant under the age of 30, and affective disorders over
the age of 30.
The six studies have shown the importance of major mental
disorders, but Murphy - 1983, noted that the intervention ofother factors is "necessary", the psychiatric diagnosis not being"sufficient" to determine suicide.
Studies on the clinical population performed on groups of
inpatients or outpatients have led to different conclusions:• Black - 1985, Kraft and Babigian - 1976, Martin- 1985, Pokorny - 1964, showed that most suicides among
patients with mental illness were committed by those withemotional disorders, schizophrenia or alcoholism.
• Schizophrenia tends to be responsible for mostsuicides in patient groups, unlike the general population.
• Suicide in the clinical population has a closer
distribution by sex, although men predominate.
• The risk of suicide tends to be higher in younger
patients compared to the general population.
• After 69 years there are only significant differences
2021.31.2(102)  Fiziologia - Physiology

in the suicide rate between the two populations studied (Black,1985).
In a study of 5,412 patients hospitalized Black - 1985,
found that 68 people committed suicide immediately after
discharge, 38% in the first 6 months, 75% in the first year and
79% within 2 years.
Almost 95% of all people who commit suicide or attempthave a diagnosis of mental disorder. Depressive disorders
account for 80% of these, schizophrenia 10%, dementia ordelirium 5%. Of all people with psychiatric disorders, 25% arealso addicted to alcohol and have 2 diagnoses. People withpsychotic depression have the highest risk of suicide. The
presence in the personal history of impulsive behavior or actsof violence increases the risk of suicide (Mousavi et al, 2004).
Also, previous hospitalizations in psychiatric wards for any
psychiatric disorder (Qin et al., 2003)[23].
The risk of suicide in psychiatric patients is estimatedby most authors to be 3-12 times higher than in the general
population.
The degree of risk is variable depending on age, sex,
diagnosis and whether or not they were admitted to a psychiatrichospital unit. The risk for those who were hospitalized,
regardless of sex, is estimated in the US as 5 to 10 times higher
than in the general population, and for outpatients who have
not had any hospitalization in the personal history is only 3- 4times higher. The highest risk for both sexes is mood disorders,
and within them major depression.
A study of 5,000 patients discharged from a psychiatrichospital in Iowa showed that in the first 3 months after
discharge, the suicide rate in women was 275 times higher
than in the female population in Iowa, and in men 70 times
bigger. Multiple studies have shown that one-third or evenmore of patients with depression commit suicide in the first 6
months after discharge, probably due to a relapse. The mostimportant risk groups for suicide are patients with depressive
disorders, schizophrenia and substance abuse. Patients withpanic disorder who call the emergency department are also at
increased risk (Sadock, 2007).
Suicide in depressive disorders
Depressive disorders are most commonly associated withsuicide, although advances in psychopharmacology shouldhave reduced the risk of suicide in depressives. Patients withdepression commit suicide more frequently during the onset ofthe disease, and the higher the risk, the greater the risk factors
for suicidal behavior. It is known that the risk of suicide does
not decrease with the relief of depression, but on the contrary
manifests itself more intensely after the patient has gone
through the depressive episode. The patient often requests
discharge and conceals his symptoms in order to implement
his suicidal plan. Patients with major depression may havedifferent types of self-aggressive behavior: refusal to eat, selfmutilation "symbolic suicide", elaborate suicide, suicidal frenzy(in depressions with marked anxiety), "altruistic" suicide (inwhich heteroagressive behavior has delusional motivation).
47

The data presented are arguments for the importance ofearly diagnosis of depressive disorders and the establishmentof antidepressant treatment according to the principle oftherapeutic adequacy. To this end, we consider it useful topresent information on the epidemiology of depressive disorder,
diagnostic criteria and clinical and evolutionary features ofclinical forms of depression.
The division of depression into the two categories seems to
be dependent on factors of genetic and biological vulnerability.
The figures of epidemiological studies have important variations,being also influenced by ethnic, cultural, social components.
Psychopharmacogenetic studies have confirmed the risk ofdispositional turn when administering psychotropic drugs. This
traditional nosographic dichotomy is all the more difficult as theinfluence of the biological brain factor is dependent on somecomorbidity that may be associated with the initial depressive
disorder: vascular, metabolic, traumatic and toxic. Depression
is estimated in current practice in primary care in 20% of all
patients with psychiatric disorders. The latest data suggest that
30% of depressed patients make a dispositional turn at somepoint in their evolution, becoming bipolar depression.
Ethnic variations can be significantly correlated withgenetic differences in different populations, explaining
different epidemiological values and indirectly confirming theneurobiological background. Thus, studies performed on the
Chinese population estimated prevalence values of 0.4% forbipolar disorder and 1.4% for major unipolar depression. For
incidence, rates of 2.3% per year were suggested for unipolar
depression, compared to annual incidence rates of 2.5% formanic episode and 10.3% for major depression [9].
Psychotic depression. It occupies an intermediate
position in clinical nosography between affective disorders
and schizophrenia, the presence and recognition of thisentity calling into question the unique psychosis hypothesis.
The prevalence of depression with psychotic symptoms is
12.5% - 16% in contrast to the prevalence of schizoaffective
disorder, estimated at 0.2% - 0.5%. Epidemiological data
show a significant difference between unipolar and bipolar
depressive disorder associated with psychotic symptoms andschizoaffective disorder, anticipating different neurobiologicalpatterns for psychotic depression in the spectrum of bipolaror unipolar disorder and depression in the spectrum of
schizophrenia.
Approximately 20% of patients with major depressive
disorder with psychotic symptoms, 2 years after the initiationof drug treatment with antidepressants show poor quality withpersistent depressive symptoms and worsening cognitive
dysfunction. After multiple drug interventions, 10% of thesepatients do not show significant improvements and an
unfavorable evolution, with a significant increase in suicide.
Incomplete remission of depressive disorders with psychotic
symptoms increases the risk of somatic manifestations,obscuring the prognosis (Marinescu D., 2009).
Suicide in bipolar disorder48

Bipolar affective disorder is a chronic condition
characterized by extreme and severe mood swings. Prospective
studies on the natural evolution of bipolar disorder have shown
that, most of the time, patients are in the depressive phase(30% - 50%), compared to the manic phase (1% - 10%) or themixed phase (2% - 6%). .
Patients with bipolar disorder have an increased risk ofsuicide. it is estimated that 20% - 25% had attempted suicide
during their lifetime. The risk for suicide is higher in bipolar
II than in bipolar I, in light of the predominance of recurrent
severe depression, although the rate of attempts is similar.
In a meta-analysis of English studies of mortality in mental
disorders, the average suicide rate in bipolar patients wasestimated at 0.40% per year, versus the average of 0.017%
in the international population. (Vieta, 2007). This leads to a
standardized mortality rate of 22, compared to about 20 inunipolar depression and 8.4 in schizophrenia.
Suicide attempts in patients with bipolar disorder havea high risk of lethality. One in five attempts are complete inbipolar versus one in 10-20 in the general population. Bipolar
patients may attempt earlier in a depressive episode than otherpsychic patients, and those who develop a rapid cycling patternalso have marked depressive symptoms and a very high riskof suicide attempts. Among the phases of bipolar disorder,
depressive episodes are burdened with the highest suicidal
risk, followed by mixed states and the presence of psychotic
states, with manic episodes having the lowest risk.
Suicide in schizophrenia
Bleuler - 1950 considers suicide to be "the worst symptomof schizophrenia"[22]. In contrast to therapeutic advances,suicide remains the leading cause of premature death in patientswith schizophrenia. The rate of suicidal behavior remains highwith an obvious upward trend. Suicide initially estimated at 7%(Johnston, 1986) then increases to 13% (Caldwell, 1990) and
18% (Baldwin; Sinclair, 2004), while (Meltzer, 1995) reports a
20% attempt rate - 40%.
Harris - 1998, estimates suicide as a mortality rate at
23%, and the main modalities are evaluated by Foster – 1999[17], differentiated by sex: hanging predominates in men, andintoxication in women.
Also Foster identifies a number of risk factors for patientswith schizophrenia: male gender, white race, lack of service,chronic relapses, pre-existing depression, a history of treatment
for depression, depression during the disease episode, andrecent discharge.
Most patients with suicidal schizophrenia are young.
In a many studies, there were no cases of suicide among
chronic patients over 40 years of age. The increased suicide
rate requires a reassessment of depressive symptoms inschizophrenia. Wiersma, 2000, considering that depressionin schizophrenia is of secondary type and describes 4
psychopathological sub models:a) depression as a reactive symptom to the ameliorationof positive psychotic symptoms (awareness of the disease andFiziologia - Physiology  2021.31.2(102)implicitly of its severity);b) depression secondary to loss of communication
skills directly proportional to autistic retraction and loss of selfindependence;c) depression as an active and independent psychotic
symptom, evolving even after the acute episode;d) prodromal symptoms such as atypical depression [28].
Elements of neurobiology may partially explain suicidal
behavior based on levels of primary vulnerability (biochemicaland genetic abnormalities), secondary (related to disease andtreatment), and cognitive vulnerability. At the first hospitalization,
Haw's meta-analysis - 2005, estimates the suicide risk at
5.6%; moreover, the completed suicide rate increases in direct
proportion to the evolution of the condition and the number ofreadmissions. Negative life events are associated with suicidal
behavior in schizophrenia in a proportion of 52% in outpatientsand 22% in hospitalized patients. The catamnestic study
performed by Siris - 1995, on 30 studies estimates the rate ofdepression as variable between 7% and 65%, with an averageof 25% [4].
The psychopathological inspiration model does notprovide sufficient explanations for suicidal behavior. Based on
the premise that in about 2/3 of patients with schizophrenia,
the prodromal period contains depression, the therapeutic
objectives of the acute episode of schizophrenia werereconsidered, improving cognition and preserving affectivity
occupying an important place, along with positive symptoms.
Suicide in substance use disorders
The relationship between alcoholism and suicidedepression has been revealed in the literature since 1896 by
many authors.
Chronic alcoholism is a predisposing risk factor for suicidal
behavior, but must be viewed in relation to a number of otherdemographic or psychopathological risk factors that modulate
its predictive value:• alteration of family and social relations;• diminishing work capacity;• job loss;• compromising psychophysical health;• progressive isolation.
According to Sadock and Kaplan, alcohol use and alcoholrelated disorders are associated in about 50% of homicide
cases and 25% of all suicides (Sadock B.J., Kaplan 2007) [24].
Alcohol-related disorders are found in people of all socioeconomic backgrounds. Frequently, alcoholism overlaps withsome pre-existing mental or organic disorders (personality
disorders, mood disorders, schizophrenia, organic brain
disorders).
Their association with somatic and psychopathologicaldisorders induced by alcohol abuse and the coexistence ofstressful socio-cultural and situational factors, determines acumul of risk factors for suicide. Suicidal behaviors are quite
common in subjects with excessive alcohol and / or druguse, regardless of gender. Often, however, suicidal acts are2021.31.2(102)  Fiziologia - Physiology

performed against the background of psychotic or depressive
decompensations (Grecu Gaboş M, 2000) [12].
Suicide in personality disorders
The suicide rate in Borderline Disorder has been comparedin multiple studies to that of bipolar disorder or schizophrenia,
despite the fact that most patients with borderline disorder live
longer than those with psychosis. Many patients after the age of
30 significantly improve their condition and no longer meet thecriteria for Borderline Disorder, so 8% or 10% of adolescentsor young adults with Borderline Disorder will develop various
forms of affective disorder (bipolar II or unipolar depression),
almost no one schizophrenia. Factors associated with a poor
prognosis, including suicide, include incest, parental cruelty,
comorbidity with antisocial disorder, and marked impulsivity(Grecu Gaboş M, 2000 [12]). Those who continue to abuse
alcohol have a suicide rate of 37%. The average suicide rate
was 10% and was usually accompanied by substance abuse.
Long-term studies have had encouraging results compared
to short-term studies suggesting that there are no majordifferences between patients with borderline disorder andschizophrenics.
We used the method of retrospective statistical study,
analyzing cases of suicide attempt with lethal end, admitted
to the Craiova Psychiatric Clinic between 2019-2020 and thenrecorded as completed suicide at IFM Craiova.
A lot B = 58 cases was selected from all admissions, theinclusion criteria being:• The presence of a suicide attempt as the main reason
for the admission recorded in the observation sheet;• Completeness of the data in the observation sheetson the items studied.
It should be noted that the suicide attempt is not codified
in the ICD-10 classification system used in psychiatry as adiagnosis in its own right, which has made our research morelaborious, since the attempt as such was mentioned only in thereferral diagnosis.

B Group Distribution of group B by sex
Table I. Distribution of group B by sex

Men

Women

Total

Abs.

%

Abs.

%

Abs.

%

20

34.5

38

65.5

58

(100)

49

Table III. Distribution of group B by sex, by profession
Relative values were calculated for the whole batch.
Sex
Profession

Men

Women

Total

Abs. %

Abs. %

Abs. %

Workers

2

3,4

11

19,0

13

22,4

Te c h n i c i a n s ,students, subengineers
4

6,9

7

12,1

11

19,0

Sex and age at the time of the suicideIntellectual

2

3,4

2

3,4

4

6,8

Table II. Distribution of group B by sex, by age at the time ofsuicide. Relative values were calculated for the whole batch

Without

8

13,9

15

25,8

23

39,7

Pensioners

4

6,9

3

5,2

7

12,1

Total

20

34,5

38

65,5

58

(100)

Fig. 1. Distribution of group B by sex. In accordance withthe data from the specialized literature, there is an increased
percentage (almost double) for females, which can becorrelated with the existential situation, as well as the statusof “inferiority” attributed to this sex. Also, the suicide rate in
women corresponds to the number of suicide attempts - muchhigher than the completed suicide (Table I, Fig. 1).

Sex
Age

Men
Abs.

Women

Total

%

Abs.

%

Abs.

%

Under 30 4

6,9

25

43,1

29

50,0

30-45

6

10,3

6

10,3

12

20,6

Over 45 10

17,3

7

12,1

17

29,4

Total

34,5

38

65,5

58

(100)

20

Fig. 2. Distribution of group B by sex, by age at the time ofsuicide The results obtained reveal - worryingly, the factthat half of the suicides belong to the age group under 30,
with a net predominance of females (Table II, Fig. 2). In
males, a progressive increase is observed with age, which
could be correlated both with the increase of family / social
responsibilities and with alcohol consumption.
Sex and profession
50

Fig. 3. Distribution of group B by sex, by profession The factthat suicide is much more common in the “disadvantaged”
categories (workers, farmers or unemployed) is consistent
with the socio-cultural level, but not with the moral-religious.
Intellectuals have a low rate of suicide in terms of depressive
disorder, the distribution by sex being equal, which cannot be
said in the other categories where females have “supremacy”(Table III, Fig. 3).

Sex and marital status

Table IV. Distribution of group B by sex, by marital status.
Relative values were calculated for the whole batch

Fiziologia - Physiology  2021.31.2(102)Sex
Marital status

Men

Women

Total

Abs.

%

Abs.

%

Abs.

%

Married

6

10,4

11

19,0

17

29,4

Unmarried

3

5,2

2

3,4

5

8,6

Divorced

9

15,5

20

34,5

29

50,0

Widower

2

3,4

5

8,6

7

12,0

Total

20

34,5

38

65,5

58

(100)

Fig. 5. Distribution of group B by sex, according toheredocolateral antecedents for mental illness or alcoholism.
Although the predominance of heredocolateral antecedents formental illness and alcoholism is obvious - 2/3 of the cases, thecorrelation of the data obtained by one’s own study may not
be relevant due to the difficulties in obtaining anamnestic data.
However, the results obtained are consistent with those in theliterature (Table V, Fig. 5).

Fig. 4. Distribution of group B by sex, by marital status. The
status of divorced obviously predominates, being explicable bysudden changes from a socio-economic point of view - which
justifies the much higher percentage of females. Interesting,and even contrary to the above, is the proportion of marriedpeople, marital incidents seem to play a decisive role. In thesetwo categories, the percentage distribution by sex is almost
unequal, which cannot be stated in the case of widowhood
status where the ratio in favor of the female sex is reversed(Table IV, Fig. 4).

Sex and hereditary history of mental illness or alcoholism
Table V. Distribution of group B by sex, according to heredocolateral antecedents for mental illness or alcoholism. Relativevalues were calculated for the whole batch
Sex
AHC

Men

Women

Abs.

%

Abs.

%

Abs.

%

Positive

14

24,1

24

41,4

38

65,5

Negative

6

10,4

14

24,1

20

34,5

Total

20

34,5

38

65,5

58

(100)

Sex and suicide
Table VI. Distribution of group B by sex, by suicide. Relativevalues were calculated separately by sex.
Sex
Method

Men

Women

Total

Abs.

%

Abs.

%

Abs.

%

Hanging

14

70,0

12

31,6

26

44,8

Defenestration

4

20,0

9

23,7

13

22,4

Drug ingestion

1

5,0

14

36,8

15

25,9

Other methods

1

5,0

3

7,9

4

6,9

Total

20

34,5

38

65,5

58

(100)

Total

2021.31.2(102)  Fiziologia - Physiology

51

Fig. 6. Distribution by sex, by suicide. There is an obvious
discrepancy between suicidal methods regarding the sex of
the suicide bomber. Thus, hanging is the method of choice formales, while in women drug ingestion predominates (comparedto males) - Table VI, Fig. 6. With the exception of drug ingestionand other methods - which may be the result of “unexpected”
accidents by the patient, it should be noted that unlike suicide
attempts, completed suicide occurs through brutal methods
that do not give a chance of survival. This reflects the intensityof the desire for death and, implicitly, of depression.

Fig. 7. Distribution by sex, after the onset-suicide interval. The
research shows the increased frequency of suicide between
4-10 years and over, as well as the impressive share of suicidein women after 10 years from the onset of the disease (Table
VII, Fig. 7). The fact that some men commit suicide less than
1 year after onset can be explained by alcohol consumption or
impulsive behavior. It should also be noted that between 4-10
years the distribution of suicide by sex is almost equal, which
may raise the issue of inadequacy or therapeutic compliance.

CONCLUSIONS

Sex and onset-suicide interval
Table VII. Distribution of group B by sex, after the onset-suicideinterval. Relative values were calculated for the whole batch
Sex
Suicide-Debut

Men

Women

Total

Abs.

%

Abs.

%

Abs.

%

Under 1 year

1

1,7

---

---

1

1,7

1-3 years

2

3,4

3

5,2

5

8,6

4-10 years

12

20,7

13

22,4

25

43,1

Over 10 years

5

8,7

22

37,9

27

36,6

Total

20

34,5

38

65,5

58

(100)

1. Following the study, the following risk factors (prediction) for
suicide in people with depressive syndromes were highlighted:• Female sex;
• Age under 45 years;• Acute onset of the disease;• Low level of training;• Divorced or married people with marital problems;• Positive hereditary collateral history of mental illness andalcoholism.
2. Regarding the suicidal modality, the share of suicide byhanging in men and drug ingestion in women was noted.
3. The interval between the onset of the mental illness and thecommission of the suicidal act clearly reveals the increase of
the suicide frequency after at least 4 years of evolution.
From these results it can be concluded that the frequency ofsuicidal act could be influenced in the sense of reducing it, inthe conditions of primary, secondary and tertiary prophylaxis,
emphasizing the therapeutic adequacy and socio-familial
reintegration measures.

52

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53

TENDINŢE ACTUALE ÎN EVALUAREA TENTATIVELOR DE
SUICIDREZUMAT
Achiziţiile ştiinţifice din ultimele decenii generează noi perspective în descifrarea fenomenului suicidar, definindu-l ca o problemă multi- şiinterdisciplinară rămasă încă sub semnul interogaţiei ştiinţifice, în ciuda eforturilor depuse şi a vechimii sale.
Cercetarea de faţă, cu o bază teoretică justificatoare, porneşte de la ipoteza că un comportament suicidar depăşeşte condiţionarea
patologică, extinzându-se în sistemul individual de valori morale şi la modalitatea personală de răspuns la factori psihostresanţi.
Vulnerabilitatea biologic-biochimică constituie fundalul pe care se poate derula comportamentul autolitic, componentele psihologică,cognitivă şi socială fiind factori favorizanţi, veritabili triggeri ai dramei suicidare.
Riscul pentru suicid în contextul bolilor somatice este corelat în mod semnificativ cu prezenţa unei tulburări psihice, în mod particulara depresiei, dar sunt implicate şi trăsăturile de personalitate ale bolnavului, capacitatea sa de adaptare, disponibilitatea pentru suportsocial, prezenţa stresorilor psihosociali ca şi antecedentele personale de comportament suicidar.
Din aceste rezultate se poate conchide că frecvenţa actului suicidar ar putea fi influenţată în sensul reducerii acestuia, în condiţiile unei
profilaxii primare, secundare şi terţiare, punând accentul pe adecvanţa terapeutică şi măsurile de reintegrare socio-familială..
Cuvinte cheie: fenomen suicidar, condiţionarea patologică, valori morale, factori psihostresanţi

54

Fiziologia - Physiology  2021.31.2(102)PROGNOSTIC FACTORS IN ISCHEMIC STROKERALUCA ELENA SANDU1, CARMEN VALERIA ALBU2, ANDREEA OANA FLORESCU3,
FLORENTINA DUMITRESCU4, ANDREEA CRISTINA STOIAN4, EMILIA BURADA5,TUDOR ADRIAN BALSEANU5, CRISTIAN CONSTANTIN6, FLORIN ANANU VREJU7,
ANDREEA LILI BARBULESCU8¹University of Medicine and Pharmacy of Craiova, Biochemistry Department, 2 Petru Rares Str., 200349, Craiova, Romania²University of Medicine and Pharmacy of Craiova, Neurology Department, 2 Petru Rares Str., 200349, Craiova, Romania³University of Medicine and Pharmacy of Craiova, PhD student, Radiology and Imaging Department, 2 Petru Rares Str., 200349, Craiova, Romania
4
University of Medicine and Pharmacy of Craiova, Infectious Diseases Department, 2 Petru Rares Str., 200349, Craiova, Romania
5
University of Medicine and Pharmacy of Craiova, Physiology Department, 2 Petru Rares Str., 200349, Craiova, Romania
6
University of Medicine and Pharmacy of Craiova, PhD student, Radiology and Imaging Department, 2 Petru Rares Str., 200349, Craiova, Romania
7
University of Medicine and Pharmacy of Craiova, Rheumatology Department, 2 Petru Rares Str., 200349, Craiova, Romania
8
University of Medicine and Pharmacy of Craiova, Pharmacology Department, 2 Petru Rares Str., 200349, Craiova, Romania
ABSTRACT
Stroke represents one of the most important public health problems nowadays as it is a major cause of both mortality and morbidity
worldwide, but especially in highly developed countries[1-2, 6-12]. The study is a retrospective one, carried out on a group of patientswith ischemic stroke admitted to the Neurology Clinic of the Clinical Neuropsychiatry Hospital in Craiova from January 1, 2020 to June30, 2020.Prognostic factors for patients with ischemic stroke are the age of the patient, the severity of the infarction, the mechanism of
infarction production, related comorbidities, complications of ischemic stroke, which currently contribute to the morbidity and mortalityof these patients.[1-4]
Keywords: ischemic stroke, prognostic factors, hypertension, atrial fibrillation, diabetes mellitus

Introduction

Stroke represents one of the most important public healthproblems nowadays as it is a major cause of both mortality andmorbidity worldwide, but especially in highly developed countries[1-4].All over the world it is expected that by 2030 the numberof patients with stroke will reach 23 million.[1-6] According
to both the European Commission and the Organization for
Economic Development and Cooperation (OCED), Romania ison the 10th place in the world in terms of stroke incidence andon the second position in what mortality caused by stroke isconcerned [1-6,37,48].Studies on ischemic stroke, performed onanimal models, highlight the importance of the proinflammatorycascade, the free radicals in generating ischemic stroke [16].Improving the management of risk factors is essential indecreasing mortality in patients presenting stroke [2]. The
presence of extracranial cerebral atherosclerosis, emphasizedon Doppler ultrasonography, affects the functional outcome andrepresents a risk factor for ischemic stroke [3]. The risk factors
for stroke also include higher blood glucose levels whichis linked
to a worse clinical outcome and early brain edema, however,pathophysiological pathways are not yet fully understood [4].
Moreover, the presence of cardiac comorbidities influences
the incidence and the prevalence of stroke. Compared withpatients that do not present cardiac comorbidities, patientswith hypertension, coronary heart disease and atrial fibrillation

associate a higher stroke risk (hypertension alone doubles therisk for stroke). Comorbidities such as coronary heart disease,cardiac failure, atrial fibrillation doubled the stroke risk in men
and trebled the risk in women. The elderly represent a vulnerable
category to stroke when atrial fibrillation is also present [5]. Stroke
is one of the major health problems our society is facing today.
1. Experimental part
The purpose of this work is to analyse the clinical, biologicaland imaging parameters of a group of 200 patients diagnosed
with ischemic stroke and furthermore to identify the prognostic
factors associated with this pathology.
Materials and methods
The study is a retrospective one, carried out on a group ofpatients with ischemic stroke admitted to the Neurology Clinicof the Clinical Neuropsychiatry Hospital in Craiova from January
1, 2020 to June 30, 2020.
Relevant data have been considered for the diagnosis resulting
from the anamnesis, clinical exam, neurological examination,
biological characteristics, but also imaging features. The
parameters resulting from the anamnesis refer to: age,gender, main diagnosis, secondary diagnoses, reasons for
hospitalization, personal pathological history, associated risk
factors.
During the clinical examination the following parameters

Received 25th of August 2021. Accepted 15th of September 2021. Address for correspondence: Carmen Valeria Albu, University of Medicine and Pharmacy
of Craiova, Neurology Department, 2 Petru Rares Str., 200349, Craiova, Romania, e-mail: carmenvaleriaalbu@yahoo.com

2021.31.2(102)  Fiziologia - Physiology

55

were noted: particular attitudes observed through inspection,
involuntary movements taken into consideration in terms ofrhythmicity, recurrence, frequency, speed and amplitude, thepresence or absence of coordination and balance disorders.
There has been an assessment of: the expressive, spontaneous,
repetitive, automatic speech, the name of the colours of theobjects, the shapes and the images, but also the expressive
speech was evaluated. Speech disorders have been observed,
such as dysarthria, aphasia.
From a paraclinic point of view, there has been monitoring of
the values of creatinine (mg/dl), blood glucose (mg/dl) andcholesterol (mg/dl). The computed tomography scan of thebrain highlighted: the presence or absence of tomodensitometricchanges in favour of certain recent or sequellar vascular lesions,craniocerebral traumas, intracranial haemorrhages, aneurysms,
hematomas, intracranial tumours, the affected vascular territory,
the presence of sequelae.
2. Results
In a first stage we followed the distribution of patients withischemic stroke according to age and we noted that the highest
frequency of ischemic stroke occurs in the age group between
70 and 95 years (119 patients representing 59.5% of the total
number of patients under study), followed by the age group of
60-69 years (59 patients namely 29.5% of the total number ofpatients).

Fig 1. The percentage and graphical distribution of patients according to age

With regard to the distribution of patients by gender, there has
been identified an approximately equal prevalence of ischemic
stroke for both genders, respectively 50%.

56

Fiziologia - Physiology  2021.31.2(102)Fig 2. Graphical and percentage distribution of patients according to gender
A.The distribution of the group of patients according to thereasons for hospitalization
A.I. The distribution of patients according to the presence ofhemiparesis / monoparesis
The highest incidence corresponds to left-sided hemiparesis,
respectively 24.5% (49 patients), followed by right-sided
hemiparesis with a percentage of 22.5% (45 patients). All theother types of hemiparesis had similar percentages: rightsidedpredominantly brachial hemiparesis 5.5% - 11 patients,right-sidedpredominantly crural hemiparesis 1.5% -3 patients,
left-sidedpredominantly brachial hemiparesis 3.5% -7 patients,
left-sided predominantly crural hemiparesis 1% - 2 patients,
left-sided predominantly brachial monoparesis 1.5% - 3 patients.
A.II. The distribution of patients according to the presence ofhemiplegia / monoplegia

Fig 3. The graphical and percentage representation of patients with hemiplegia / monoplegia

2021.31.2(102)  Fiziologia - Physiology

57

The incidence of right-sided / left-sided hemiplegia is quite highas shown in the figure 3. The right-sided hemiplegia was detected
in a number of 32 patients and the left-sided hemiplegia in 21
patients. The rest of plegia deficits were found in approximately
equal percentages.
B.The distribution of the group of patients considering thepresence of speech disorder
Of the 200 patients assessed, almost half of them, more precisely 42% of the patients presented dysarthria. A percentageof 31% (61 patients) presented mixed aphasia, 8% (15 patients)
presentedmixed predominantly motor aphasia and 2% mixed
predominantly sensitive aphasia.
C. The distribution of patients with ischemic stroke considering the results of the neurological examination.
The prevailing elements of the clinical picture in the studied groupwere the pyramidal syndrome with a percentage of 60% of entire
group and speech disorders with a percentage of 79.5%.

Fig 4.The graphical and percentage representation of the symptoms evaluated following the neurologicalexamination.

58

Fiziologia - Physiology  2021.31.2(102)

D. The distribution of patients with HBPconsideringthe
degree of hypertension
Of the total number of patients diagnosed with high blood pressure (170 patients), most of them presentedgrade IIhypertension,respectively 69.5%. A percentage of 11% presented grade I
hypertension and 4.5% grade III hypertension.
E. The distribution of patients with stroke depending on
the presence of atrial fibrillation in their history was also
discussed, atrial fibrillation being a high risk factor, but at thesame time a prognostic factor in ischemic stroke. Randomized
studies have shown that antiplatelet medication reduced theincidence of stroke in patients with nonvalvular AF. In the study
group, we encountered a significant percentage of 29.5% of
patients presenting atrial fibrillation, which certifies once more
the implications of this pathology and of cardiovascular pathology
in stroke ethiopathogeny.
F. The distribution of patients with stroke considering thepresence of diabetes mellitus in their history
Of the group of 200 patients with ischemic stroke, a percentage
of 18%, respectively 35 patients had type II diabetes mellitus in
their medical history and 34%, respectively, 68 patients had ablood glucose level> 126 mg/dl. Patients with diabetes mellitus
have a higher susceptibility to arteriosclerosis, prospective epidemiological studies confirming the increased risk of ischemic
stroke in patients with diabetes.
G.The distribution of patients with ischemic stroke considering the blood glucose levels
A significant percentage of 33.5% was represented by patientsregistering a blood glucose level above 126 mg/dl (67 patients).
Values of blood glucose ranging between 110.1-126 mg/dl were
found in 21.5% of patients, the remaining 42.5% being represented by patients with normal blood glucose levels: 70-110mg/
dl (85 patients).

We have also discussed the presence of chronic kidney diseasein the group of patients included in the study. Of the 200 patientsdiagnosed with ischemic stroke, 18 patients (9%) also sufferedfrom chronic kidney disease. Chronic kidney disease is a risk
factor for the onset of ischemic stroke, but also a negative
prognostic factor for the evolution of a patient who suffered anischemic stroke. By affecting the kidney function, the glomerular
filtration rate decreases, the decrease of excretion being accompanied by the accumulation in the body of toxic or metabolic
compounds and thus the appearance of complications: hypertension, anaemia, sodium and water retention, nitrogen retention,
hyperlipoproteinemia, acidosis.
K.The distribution of the group of patients consideringthe
presence of pneumonia as a prognostic factor
Of the group of patients included in the study, 8.5% (17 patients)
suffered from bacterial pneumonia. Bacterial pneumonia is oneof the most significant complications in patients undergoing astroke and it is mainly caused by aspiration. Aspiration occurs
most frequently in patients with swallowing disorders and thosewith impaired consciousness.
L. Other associated pathologies that represent a negative
prognostic factor in the patient with ischemic stroke: myocardial infarction in his/her medical history, vascular dementia,
COPD, asthma, acute chronic bronchitis, acute respiratory
failure, chronic renal failure, right latero-basal pachypleuritis,
congestive heart failure, decubitus ulcers, epileptic seizures,
gangrene in the lower limbs, chronic obliterative arteriopathy,
neoplasm of the colon operated on, prostate neoplasm, cervicalneoplasm.
M. The distribution of patients according to CT examination results

H. The distribution of patients with diabetes mellitus considering the average value of glycosylated haemoglobin
Of 35 patients with type II diabetes, we analysed the glycosylated
haemoglobin in 17 of them, resulting in an average value of
8.7%. This increased value is associated with an unfavourable
prognosis for the evolution of a patient with ischemic stroke.
I.The distribution of the group of patients considering thecholesterol values Given the fact that the values of serum
cholesterol ​​have a significant impact on the pathogenesis of
cerebrovascular diseases, we proceeded to measure it in thestudied group. Therefore, we identified119 patients (59.5%)
with cholesterol values ​​below 200mg/dl, 23.5% of patients withcholesterol values ​​ranging between 201-250 mg/dl and 15% of
patients with the value of cholesterol above 250 mg/dl.
J. The distribution of patients with chronic kidney disease2021.31.2(102)  Fiziologia - Physiology

59

Fig 5. Graphical and percentage representation of the CT examination results of the studied group ofpatients

Most of the patients in the group did not present anyrecently
established lesions, more precisely in a number of 95 patients
corresponding to a percentage of 47.5%.
Most patients suffered from ischemic cortical stroke, respectively
17%, followed by a percentage of 14% corresponding to patientswith cortical-subcortical infarctions and 6% with subcortical infarctions. Large infarctions were noted in a total of 74 patients.
By affecting a large area of ​​brain tissue, the patient suffers froma variety of disorders: motor, sensory, mixed, speech, cognitive,
behaviour, memory, awareness disorders etc.

60

The increased size and the location of the infarction are associated with an unfavourable prognosis on the evolution of thepatient suffering from ischemic stroke. It is also worth mentioning
the percentage of 1% infarctions in the brainstem and 1.5% in
the cerebellum, highly severe infarctions, with potentially severe
evolution given the fact that at these levels are found a lot ofstructures playing a vital role for the body.

Fiziologia - Physiology  2021.31.2(102)

N.The distribution of patients with ischemic
stroke depending on the affected areaFig 6. Graphic and percentage representation of patients depending on the area involved in ischemic
strokeThe distribution of patients depending on the affected cerebral
area is particularly important, because the affected area has an
influence on the evolution and the prognosis of ischemic stroke.
From this point of view most patients suffered an ischemic
stroke in the area of the left carotid artery, namely 28%. On the
2ndplace in terms of frequency was identified the area of the rightcarotid artery (18.5%), the following being left MCA (18.5%) andrespectively right MCA (17%).

3. DISCUSSIONS
Prognostic factors for patients with ischemic stroke are the ageof the patient, the severity of the infarction, the mechanism of
infarction production, related comorbidities, complications ofischemic stroke, which currently contribute to the morbidity andmortality of these patients[1-4,7-12,47,49]
The clinical study was carried out with a view to analyse anumber of 200 patients diagnosed with ischemic stroke in the
Neurology Clinic II over a 6-month period. By analysing the groupof patients, it can be noted that most patients with ischemic stroke
were found in the 70-95 age group, there is a relatively equal
distribution between men and women, and most patients were2021.31.2(102)  Fiziologia - Physiology

from the rural area. These data are in line with data found in thespecialty literature.[1-6,13-17,35,48]
The current study helped to identify a series of clinical signs or
symptoms suggesting ischemic stroke, of the type: right-sided
hemiparesis, left-sided hemiparesis, right-sided hemiplegia,
left-sided hemiplegia.
With regard to the presence of speech disorders, in this study weencountered: dysarthria, mixed aphasia, mixed motor aphasia,
mixed sensory aphasia.
Following the analysis of comorbidities associated with ischemic
stroke[2-4, 23-29,32,41-47], the main aggravating factor is highblood pressure. In the group of patients included in the study
85% were hypertensive, and of these, most presented grade II
hypertension. The next aggravating factor is diabetes found in apercentage of 18% of patients, then atrial fibrillation which was
found in a number of 29.5% of patients and chronic ischemic
cardiomyopathy encountered in more than half of the patients.
One of the parameters individualizing the group of patients in the
study is the severity of the ischemic stroke, which is assessed
after the neurological examination and represents one of the mostimportant prognostic factors affecting the symptoms in the shortor long term[2-4, 23-29]. Most patients presented a pyramidal
syndrome (120 patients), paresthetic syndrome (71 patients),
speech disorders (159 patients).
61

Another significant parameter that helps to evaluate theprognosis is brain imaging, which provides information on thevolume and location of the infarction[1-6,32-36]. In the group ofpatients included in the study the volume of the infarction wascorrelated as follows: cortical infarctions encountered in 17%,
subcortical infarctions 6%, cortical-subcortical infarctions 14%,
lacunar infarctions 3%, infarctions at the brainstem level 1%,
infarctions at the cerebellum level 1%.
The location of the infarction is another prognostic factor
with influence on the evolution of ischemic stroke[10,15,48].
Obstruction of the basilar, vertebral artery or any of the large
intracranial vessels is associated with a very high risk ofunfavourable outcomes[1,35,41-43]. In the study performed we
encountered the obstruction of the right carotid artery in 21%,the left carotid artery obstruction 28%, the vertebrobasilar area
14.5%.
The complications of ischemic stroke represent prognostic
factors for post-stroke evolution [8-10,43-49]. In the groupof patients included in the study we encountered bacterial
pneumonia in 8.5% of patients, urinary tract infection in 9.5%of patients.

4. CONCLUSIONS
Following the analysis of the results obtained by studying thegroup of patients diagnosed with ischemic strokes, we concluded:
1. Ischemic strokes were common in both genders, with similar
percentages: men 49.5% and women 50.5%.
2. The most affected age group was between 70-95 years,
followed by the age group of 60-69 years. The other age groups
represented a smaller percentage.
3. The main reasons for hospitalization were: right-sided
hemiparesis (45 patients), left-sided hemiparesis (49 patients),right-sided hemiplegia (32 patients) and left-sided hemiplegia(21 patients).
4. By analysing speech disorders we noted that most frequently
the patients suffered from dysarthria 42% and mixed aphasia
31%.
5. 85% of patients with ischemic stroke had high blood pressurein their personal pathological history, and most of them presented
stage II hypertension 69.5%.
6. 29.5% of the group patients presented atrial fibrillation.
7. Following the study carried out 18% of the patients had type
II diabetes and 25.5% had dyslipidaemia.
8. Following the analysis of the laboratory medical analyses,
it was found that most patients had normal value of creatinine
in 72% of patients and only 26% had increased values, thecholesterol value was normal in 59.5% of patients and increased
at a percentage of 38% patients. The blood glucose value was
between 70-110mg/dl at 42.5% and over 110 mg/dl at 55%.
9. The vascular areas were affected in various percentages: left
carotid artery (28%), right carotid artery (21%), vertebrobasilar
territory (14.5%), left middle cerebral artery (18.5%), right middle
cerebral artery ( 17%), ACA (1%), right PCA 2%, left PCA (1%)
and entire carotid area (3%).
62

10. Based on the CT exam, it was found that most patients
suffered from cortical infarctions (17%), followed by 14% of
patients whose ischemic strokes were located in the corticalsubcortical area, while only 6% had suffered from subcortical
infarctions, then 1.5% patients had cerebellar infarctions, 1%patients underwent infarctions at the brainstem level and 3% of
patients underwent lacunar infarctions.
11. As prognostic factors, along with age, the severity ofneurological impairment and the size of the infarction, shall be
added comorbidities that accentuate the negative prognosis
of ischemic stroke: urinary tract infection 9.5% of patients,
pneumonia 8.5%, chronic kidney disease 9%, myocardial
infarction 2.5%, dementia 3.5%, COPD 1.5%, tumours 2%
of patients, acute chronic bronchitis 2.5% of patients, acuterespiratory failure 1% of patients, chronic renal failure 1% of
patients, latero-basal pachypleuritis 3% patients, decubitus sores
2.5% of patients, epileptic seizures 1% of patients, congestive
heart failure 1% of patients.

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FACTORII DE PROGNOSTIC ÎN ACCIDENTUL VASCULARCEREBRAL ISCHEMICREZUMAT
Accidentul vascular cerebral reprezintă una dintre cele mai importante probleme de sănătate publică în zilele noastre, deoarece, lanivel mondial, dar mai ales în țările foarte dezvoltate este o cauză majoră de mortalitate cât și de morbiditate.[1-2, 6-12] Studiul este
unul retrospectiv, efectuat pe un lot de pacienti cu AVC ischemic internati in Clinica de Neurologie a Spitalului Clinic de Neuropsihiatrie
din Craiova in perioada 1 ianuarie 2020- 30 iunie 2020. Factorii de prognostic pentru pacienții cu accident vascular cerebral ischemic
sunt vârsta pacientului, severitatea infarctului, mecanismul de producere al infarctului,comorbiditățile asociate, complicațiile accidentului
cerebral ischemic, care contribuie în momentul actual la morbiditatea și mortalitatea acestor pacienți.[1-4]
Cuvinte cheie: accidentul vascular cerebral ischemic, factori de prognostic, hipertensiunea, fibrilatia atriala, diabetul zaharat

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Fiziologia - Physiology  2021.31.2(102)