Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 11, pp. 1922-1929 © The Author(s) 2024. This article is an open access publication.

1922

Neuroimmune Characteristics of Animals

with Prenatal Alcohol Intoxication

Inna Yu. Shamakina

1,a

*, Petr K. Anokhin

1,2

, Ruslan A. Ageldinov

, Viktor S. Kokhan

Serbsky National Medical Research Center of Psychiatry and Narcology,

Ministry of Health of the Russian Federation, 119002 Moscow, Russia

Artificial Intelligence Research Institute, 121170 Moscow, Russia

Scientific Center for Biomedical Technologies of the Federal Medical and Biological Agency of Russia,

143442 Svetlye Gory, Russia

e-mail: shamakina.i@serbsky.ru

Received May 28, 2024

Revised July 22, 2024

Accepted July 29, 2024

Abstract— Neuroinflammation can be an important factor of many disorders in central nervous system (CNS)

including cognitive dysfunction, affective disorders, and addictive behavior associated with prenatal alcohol

exposure and presented in early adulthood. In this study we used an experimental rodent model of prenatal

alcohol (PA) exposure (consumption of a 10% ethanol solution by female Wistar rats throughout pregnancy),

multiplex immunofluorescence analysis of interleukins (IL-1α, IL-1β, IL-3, IL-6, IL-9, and IL-12), tumor necrosis

factor (TNF-α), and chemokine CCL5, as well as quantitative real-time PCR to assess the level of cytokine mRNAs

in the prefrontal cortex of the sexually mature (PND60) offspring– male and female rats with prenatal alcohol

intoxication and control animals. Significant decrease in the content of TNF-α and interleukins IL-1β, IL-3, IL-6,

IL-9 was detected in the prefrontal cortex of male, but not in the female PA offspring. Importantly, PA males

also showed decrease in the level of TNF-α mRNA in the prefrontal cortex by 45% compared to the control

males, which may underlie the detected decrease in its content. Taken together, our study demonstrates that

a number of neuroimmune factors are regulated in a sex-specific manner in the prefrontal cortex and are

differentially affected in males and females by the prenatal exposure to alcohol. Sex factor must be taken into

account when conducting further translational studies of the fetal alcohol spectrum disorders and developing

new methods for prevention and therapy.

DOI: 10.1134/S0006297924110063

Keywords: prenatal alcohol intoxication, neuroinflammation, prefrontal cortex, interleukins, tumor necrosis fac-

tor, mRNA expression

Abbreviations: CNS,central nervous system; IL,interleukin;

PA, prenatal alcohol; TNF-α, tumor necrosis factor α.

* To whom correspondence should be addressed.

INTRODUCTION

Consumption of alcohol by women during preg-

nancy leads to the development of a range of physio-

logical, mental, behavioral, and intellectual disorders

collectively termed “fetal alcohol spectrum disorders”

(FASD)  [1]. According to the averaged epidemiological

data from recent years, 10% of women consumed al-

cohol during pregnancy [2,  3], with the prevalence of

FASD in children varying from 3 to 31% across the

different world regions [4-6]. Severity of the disorders

resulting from prenatal alcohol exposure (PA) depends

on the dose, duration, and frequency of alcohol con-

sumption during pregnancy, as well as characteristics

of maternal metabolism  [7]. The most severe form –

fetal alcohol syndrome (FAS) – manifests in children

during the early postnatal period with the develop-

mental facial defects, growth retardation, and central

nervous system (CNS) dysfunctions [7]. However, most

common group of disorders is not associated with

the faciocranial dysmorphia or developmental delay,

but manifests as behavioral and cognitive disorders,

NEURIOIMMUNE CHARACTERISTICS OF ANIMALS 1923

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

aswell as high risk of substance abuse in adolescence

and adulthood [8]. Manifestation of these symptoms is

usually associated with the beginning of school educa-

tion, stress, and social interaction problems [9].

Numerous studies have shown that central mech-

anisms of dependence involve neuroadaptation in

the mesocorticolimbic dopamine (DA) system of the

brain, which originates in the ventral tegmental area

(VTA) and projects to the limbic structures, including

Nucleus accumbens (NAc), amygdala, hippocampus,

and prefrontal cortex [10]. In addition, dopamine,

beyond its neurotransmitter function, has the ability

to activate dopamine receptors located on astrocytes,

microglial cells in the CNS, and cells of the periph-

eral immune system [11, 12]. It has been shown that

dopamine modulates activity level of microglial cells

[13-15] and, conversely, cytokines play an important

role in regulation of the brain dopamine system [16].

Itcould be speculated that one of the mechanisms of

dopamine-dependent behavioral disorders in PA ani-

mals is associated with alterations in the functional

state of microglial cells and expression of neuroin-

flammatory factors. Aim of this study was to examine

expression of neuroinflammatory factors in the pre-

frontal cortex of PA and intact (control) animals. Pre-

frontal cortex is considered one of the most complex

functional structures of the mammalian brain, whose

primary role is to integrate inputs from cortical and

subcortical structures and generate goal-directed be-

havioral responses, including those oriented towards

obtaining rewards and suppressing risk behaviors

[17]. Considering that the balance between pro- and

anti-inflammatory cytokines is critically important for

neuronal functions [18], we hypothesized that chang-

es in this balance in PA animals might be one of the

factors causing development of behavioral disorders.

We conducted simultaneous measurements of six key

cytokine proteins from the interleukin family (IL-1α,

IL-1β, IL-3, IL-6, IL-9, and IL-2), tumor necrosis factor  α

(TNF-α), and chemokine CCL5 in the prefrontal cortex

of mature (PND60) male and female rats with PA in-

toxication and control animals. IL-1 belongs to pro-in-

flammatory cytokines with broad spectrum of effects

on normal CNS functions  [19]. Cytokines from the IL-1

family consist of two main related family members

(IL-1α, IL-1β), which demonstrate cell-specific patterns

of expression and release and are synthesized by both

glia and neurons. Traditionally considered pro-inflam-

matory, cytokines such as interleukin (IL)-1β, IL-6,

IL-2, and tumor necrosis factor-α (TNF-α), play a vi-

tal role in brain development [20]. It has been shown

that disruptions in the IL-6 functions in brain are

associated with anomalies in the shape, length, and

distribution of dendritic spines  [21]. TNF-α is a key

mediator affecting synaptic remodeling, processes of

long-term potentiation (LTP) and long-term depression

(LTD) in the brain [22, 23]. In addition to IL-2 being a

key cytokine in immune regulation, it may play a role

in the development and regulation of brain neurons

involved in spatial learning and memory. Studies have

shown that IL-2 knockout mice exhibit impaired spa-

tial learning, accompanied by reduction in the length

of hippocampal mossy fibers [24].

According to the literature data, IL-3, IL-9, and

CCL-5 could have neuroprotective effects  [25-27].

It has been shown that IL-3 is widely expressed in

the central nervous system and has a trophic effect on

cholinergic neurons of Septum pellucidum both in  vitro

and in  vivo  [25], although the mechanisms underlying

neurotrophic action of IL-3 are not fully understood.

IL-9 and its receptor are also actively expressed in

the neural cells and specifically control programmed

cell death of neocortical neurons in the newborn mice

[26]. It has been suggested that the IL-9/IL-9R signal-

ing pathway represents an endogenous anti-apoptotic

mechanism for cortical neurons [26]. The chemokine

CCL5 and its receptors perform many functions in the

central nervous system, including neuromodulation of

synaptic activity and protection against neurotoxins

[27]. Highest level of the CCL5 mRNA expression is

found in oligodendrocytes, astrocytes, and microglia

of the brain cortex, caudate nucleus/shell, hippocam-

pus, and thalamus  [27]. Interestingly, in the midbrain,

the CCL5 mRNA is detected in the tyrosine hydroxy-

lase (TH)-positive cells of the ventral tegmentum, in-

dicating that CCL5 is expressed by subpopulation of

dopaminergic neurons in the mesolimbic system  [27].

It has been hypothesized that this chemokine may par-

ticipate in ensuring interaction between neurons and

glial cells  [27].

MATERIALS AND METHODS

Animals. Experiments were conducted with out-

bred Wistar rats of both sexes (from the “Stolbovaya”

Laboratory Animal Breeding Facility of the Federal

State Budgetary Institution “Scientific Center of Bio-

medical Technologies of the Federal Medical Biological

Agency”). During acclimation, experimental rats were

kept under natural light conditions at temperature of

22  ±  2°C with free access to food and water.

Prenatal alcohol exposure. To obtain the F1

generation offspring, two sexually mature female rats

(PND60) were housed with a male for three days. There

were 10 males and 20 females in total. Pregnancy was

confirmed by detecting sperm in the vaginal smears of

the females. Females mated with a single male were

randomly divided into two groups: the experimental

group received a 10% ethanol solution as their sole

source of liquid throughout the pregnancy (from day

1 to day 21), while the control females were kept on

SHAMAKINA et al.1924

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

a pure water regime during pregnancy. Alcohol con-

sumption by the females was measured daily through-

out the pregnancy. In this series, average daily alcohol

consumption by the females was 13.6 ±  2.1 g/kg. Vol-

ume of the consumed fluid was 31.0  ±  4.2  ml in the

experimental group and 35.1  ±  2.7  ml in the control

group, with no significant differences in the daily

volumes of fluid consumed. Body mass of females in

both control and experimental groups did not dif-

fer significantly at the beginning (210.0  ±  23.8  g and

207.0  ±  15.7  g, respectively) and at the end of the

experiment (325.0  ±  28.4  g and 331.0  ±  32.1  g, respec-

tively). After giving birth, all females were switched

to a water regime during the nursing period. Thus,

the offspring were exposed to alcohol only during the

prenatal period, corresponding to the first and second

trimesters of human pregnancy  [28]. On day 30 of life,

the offspring were weaned, sex-separated, and sub-

sequently housed six per cage (type: T/4B) with free

access to food and water.

Among the obtained offsprings, 18 PA rats (9males

and 9 females) and 18 control (9 males and 9 females)

rats were selected randomly. At the age of 60 days

(PND60), the rats were decapitated, and prefrontal

cortex was dissected (4.2 mm to 2.8  mm relative to

Bregma) according to the Paxinos and Watson rat

brain atlas [29]. The dissected tissue samples were

frozen and stored at –70°C.

Protein extraction for immunofluorescent

analysis. Samples were homogenized using glass

beads in a MagNA Lyser 230B homogenizer (Roche,

Switzerland) in a buffer solution containing 20 mM

Tris-HCl (pH7.5), 150mM NaCl, 1mM PMSF, 0.05% v/v

Tween-20, and 1% v/v Protease Inhibitor Cocktail II

(ab201116, Abcam, USA). The homogenates were cen-

trifuged at 12,000g for 15min at 3°C, and supernatant

was collected for further analysis. Protein content was

determined using the Bradford method with a com-

mercially available Quick Start Bradford Protein Assay

kit (Bio-Rad, USA).

Multiplex immunofluorescent analysis was con-

ducted using commercially available kits for determi-

nation of cytokines in the rat brain tissue (Cloud-Clone

Corp., China) according to the manufacturer’s instruc-

tions. The analysis was performed using a Bio-Plex

MAGPIX Multiplex Reader equipped with a Bio-Plex

Pro Wash Station (Bio-Rad). Cytokine concentrations

in the samples were automatically determined us-

ing standard calibration dilutions with the Bio-Plex

Manager Software  v.6.1 and Bio-Plex Data Pro Soft-

ware  v.1.2 (Bio-Rad). Content of target proteins was

normalized to total protein content in the sample.

Total RNA extraction was conducted using stan-

dard guanidinium thiocyanate-phenol-chloroform ex-

traction method with a PureZOL RNA Isolation Re-

agent, Bio-Rad. Tissue samples were homogenized in

1  ml of lysis buffer, mixed with 200  µl of chloroform

(Fluka, USA), followed by centrifugation for 15  min

at 4°C and 12,000g (Eppendorf 5804R, Germany).

An equal volume of isopropanol was added to the

supernatant, incubated at -20°C for 2  h, then centri-

fuged at 6,000g for 5  min. The pellet was washed with

70% EtOH, dried, and dissolved in a RNase-free water.

RNA aliquots were frozen and stored at –70°C. Amount

of total RNA was determined spectrophotometrically

(Eppendorf BioPhotometer, Germany). The obtained

RNA samples were treated with DNase (Thermo Fish-

er Scientific, USA) according to the manufacturer’s

instructions. cDNA synthesis was performed using

a Mint revertase kit (Evrogen, Russia) and used as a

template for quantitative PCR.

Real-time PCR. Primer sequences were designed

using the Primer-BLAST online resource (https://www.

ncbi.nlm.nih.gov/tools/primer-blast). Oligonucleotide

primer sequences were the following: for TNF-α (for-

ward 5′-AAATGGGCTCCCTCTCATCAGGTTC-3′, reverse

5′-TCTGCTTGGTGGTTTGCTACGAC-3′); IL-1β (forward

5′-CACCTCTCAAGCAGAGCACAG-3′, reverse 5′-GGGTTC-

CATGGTGAAGTCAAC-3′); and β-actin (forward 5′-CACT-

GCCGCATCCTCTTCCT-3′, reverse 5′-AACCGCTCATTGC-

CGATAGTG-3′). Amplification was conducted in a 25 µl

mixture containing 25  ng of template (cDNA), primers

at a final concentration of 0.4 µM, and 5  µl of 5× re-

action mixture qPCRmix-HS SYBR with SYBR Green  I

intercalating dye (Evrogen) using a CFX96 Real-Time

System C1000 Thermal Cycler (Bio-Rad) with the fol-

lowing regime: initial denaturation of the template –

3  min at 95°C; denaturation– 95°C, 15 s; annealing of

primers – 60°C, 15  s; elongation – 72°C, 30 s. The re-

action was conducted for 40 cycles followed by analy-

sis of the melting curves of the PCR products. β-actin

was used as a reference gene for data normalization.

Quantitative assessment of relative mRNA expression

levels was conducted using the 2

–ΔΔCt

method [30].

Statistical analysis. Data were processed using

Statistica software v.12 (StatSoft Inc., USA). Normality

of data distribution in the sample was assessed using

the Shapiro–Wilk criterion. Considering that all the

obtained data followed Gaussian distribution, a para-

metric method of analysis – two-way ANOVA – was

used, with factors being sex  ×  PA. Data are presented

as a mean ± standard deviation (SD). Post-hoc pro-

cessing was performed where significant differences

between the groups were identified, p-value of less

than 0.05 was considered statistically significant.

RESULTS

Cytokine content in the prefrontal cortex. Data

processing of cytokine/chemokine levels, as summa-

rized in Table1, indicated significant effects of various

NEURIOIMMUNE CHARACTERISTICS OF ANIMALS 1925

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Table 1. Content of cytokines in the prefrontal cortex of prenatally alcohol-exposed and control animals

Group

Content of cytokines in the prefrontal cortex (pg/mg total protein) arithmetic mean ± SD

IL-1α IL-1β IL-2 IL-3 IL-6 IL-9 TNF-α CCL5

M_C

(n=9)

58.0 ± 21.2 9.1 ± 2.0 175.1 ± 27.5 3.4 ± 1.0 3.4 ± 1.0 3.3 ± 1.1 38.5 ± 19.9 58.6 ± 23.0

M_PA

(n=9)

36.5 ± 16.0

6.4 ± 2,7*

(p = 0,01)

135.0 ± 57.9

2.1 ± 0.8

(p = 0.009)

1.9 ± 1.09

(p = 0.002)

1.9 ± 0.9

(p = 0.005)

21.4 ± 8.7*

(p = 0.03)

36.0 ± 6.4

F_C

(n=9)

45.9 ± 27.2

5.9 ± 2.0

(p = 0,005)

138.6 ± 64.8

1.8 ± 0.4

(p = 0.003)

2.0 ± 0.7

(p = 0.003)

1.7 ± 0.4

(p = 0.002)

26.8 ± 16.7 45.8 ± 20.0

F_PA

(n=9)

57.9 ± 41.9 5.9 ± 2.0 116.4 ± 26.6 2.5 ± 1.3 1.4 ± 0.9 2.5 ± 1.2 21.7 ± 12.0 55.4 ± 33.4

Note. M_C,males, control group; M_PA,males, prenatal alcohol exposure; F_C,females, control group; F_PA,females, prenatal

alcohol exposure. *  p < 0.05 (M_PA relative to M_C);

  p  < 0.01 (F_C relative to M_C); Duncan’s post-hoc test.

considered factors. The prenatal alcohol (PA) factor

influenced TNF-α (F

1,32

  =  4.9; p  =  0.03), and the sex fac-

tor influenced IL-1β (F

1,32

  =  6.7; p  =  0.01). Interaction

between PA and sex factors was significant for IL-3

1,32

  =  10; p =  0.003). Both PA (F

1,32

  =  12.8; p =  0.001)

and sex (F

1,32

 = 9.9; p  =  0.004) factors had significant

impacts on IL-6. Additionally, notable interactions be-

tween PA and sex factors were found analyzing IL-9

1,32

  =  13; p =  0.001) and CCL5 (F

1,32

  =  4.5; p =  0.04).

The highest cytokine concentrations in the prefrontal

cortex of both sexes were observed for IL-2, IL-1α,

and CCL5. The results of multiplex analysis revealed

significant differences in the levels of several inter-

leukins in the prefrontal cortex between the males

and females of the control group. Specifically, females

in the control group (F_C) showed decreased level in

IL-1β (p  <  0.01), IL-3 (p  <  0.001), IL-6 (p  <  0.01), and

IL-9 (p  <  0.001) compared to the control males (M_C)

(Table 1). Notably, prenatal alcohol exposure did not

influence cytokine levels in the females. At the same

time in the PA-exposed males (M_PA) there was a

significant decrease in the content of IL-1β by 29%,

IL-3 by 38%, IL-6 by 45%, IL-9 by 42%, and TNF-α by

45%, compared to the control males. Despite signifi-

cant differences being noted in the variance analysis

for chemokine CCL5, the post-hoc results were incon-

clusive; 39% reduction was observed in the PA males

compared to the control males (p  =  0.06, Duncan’s test;

trend) (Table1). Importantly, in contrast to the control

groups, no significant sex differences were observed

in the PA animal groups.

To explore the reasons behind the decreased levels

of TNF-α and IL-1β in the prefrontal cortex of the males

exposed to alcohol prenatally, parallel investigations

ofTNF-α and IL-1β mRNA expression were conducted.

Expression of TNF-α and IL-1β mRNA in the

prefrontal cortex of prenatally alcohol-exposed

and control animals. The analysis revealed a sig-

nificant impact of prenatal alcohol exposure on the

TNF-α mRNA levels in the prefrontal cortex (F

1,32

  =  8.2,

p  =  0.007). Statistically significant reductions in the

TNF-α mRNA were observed in the prefrontal cortex

of the prenatally alcohol-exposed males compared

to the controls, with 45% decrease noted (p  =  0.03).

This reduction may account for the lower TNF-α lev-

els observed. However, no significant differences in

the IL-1β mRNA expression between the groups were

found (Fig. 1).

Fig. 1. Relative mRNA expression levels of IL-1β and TNF-α

in the prefrontal cortex of control and prenatally alcohol-

exposed female and male rats. M_C, males, control group;

M_PA,males, prenatal alcohol exposure; F_C,females, control

group; F_PA, females, prenatal alcohol exposure. *  p  <  0.05

(M_PA relative to M_C, Duncan’s post-hoc test).

SHAMAKINA et al.1926

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

DISCUSSION

It has been previously demonstrated that the pre-

natal alcohol exposure is a significant risk factor for

future addictive behaviors, leading to increased volun-

tary alcohol consumption and elevated anxiety levels

during alcohol withdrawal in the male F1 offspring,

unlike in the female offspring when compared to the

respective control groups [31]. In this study, we also

identified the sex-specific delayed effect of prenatal al-

cohol exposure on the cytokine levels in the prefrontal

cortex of adult animals. Notably, there was a decrease

in the TNFα content in the prefrontal cortex of adult

male rats prenatally exposed to alcohol. These findings

are in agreement with the views of some authors [32]

that alcohol could suppress rather than enhance TNFα

expression in the brain. TNFα, traditionally regarded

as a pro-inflammatory cytokine, has been implicated

as a pathogenic factor in various brain diseases. How-

ever emerging evidence suggests its neuroprotective

roles, which include reducing nitric oxide and free

radical production, modifying excitatory amino acid

neurotransmission, maintaining neuronal calcium ho-

meostasis, and promoting synthesis of neurotrophic

factors  [33]. Transmission of signals in the cells with

participation of TNFα is realized through binding to

two receptors, TNFR1 and TNFR2  [34] and activation

of transcription factors NF-κB and AP-1, which me-

diate cell survival and proliferation [34]. NF-κB is

a transcription factor that regulates expression of

multiple genes encoding pro-inflammatory cytokines,

chemokines, and their receptors. Unlike TNFR2, TNFR1

includes a “death domain” in its cytoplasmic part,

potentially leading to cell death upon binding with

TNFα [34]. TNFR1 is expressed in almost all cell types,

whereas TNFR2 is primarily found in neurons and oli-

godendrocytes [34]. Previously TNFR2 was considered

as less significant for homeostasis, however, it has

been shown recently that the regulatory effects on

TNFα are associated precisely with the signal trans-

duction through TNFR2 [35].

The delayed effect of prenatal alcohol exposure

may stem from the long-term epigenetic modifica-

tions, as indirectly suggested by the decreased level

of TNFα mRNA expression. The mouse models with

Tnf gene knockout (C57BL/6-TNF

–/–

and C57BL/6-TNF

+/–

)

offer promising avenues for investigating the role of

TNF in behavior and central nervous system function-

ing in both health and disease. Current research using

these mouse models points to the role of TNF role in

regulating anxious behaviors and functioning of the

brain dopamine system [36].

Furthermore, cytokine IL-1β, produced in the

brain by microglial cells and neurons, contributes to

maintenance of the integrity and function of blood-

brain barrier [37]. IL-1β has been shown to influence

GABAergic synapses in the prefrontal cortex via two

distinct pathways: an anti-inflammatory (“survival”

PI3K/Akt) or a pro-inflammatory (MyD88/p38 MAPK),

with shift towards the PI3K/Akt pathway in the ab-

sence of alcohol, promoting survival [38]. Addition-

ally, research involving rodents with the IL-1β gene

knockout has revealed various behavioral and affec-

tive disturbances in such animals [39, 40].

Reductions in the IL-3, IL-6, and IL-9 levels in

the prefrontal cortex of males with prenatal alcohol

exposure could be associated with the diminished

neuroprotective properties of microglia. Studies have

demonstrated pivotal role of IL-3 in proliferation and

maintenance of the progenitor neuronal cells and

neuron survival [41]. IL-3 protects against the Aβ-in-

duced cell death, mediated by activation of phospha-

tidylinositol 3-kinase (PI3K)/protein kinaseB (Akt) and

Janus kinase  2 (Jak2) [42]. IL-9 has been shown to ac-

tivate transcription factors STAT1, STAT3, and STAT5,

and downregulate activation markers in macrophages

such as CD45, CD14, CD68, and CD11b, induced by lipo-

polysaccharide and gamma-interferon, thus potentially

modulating other cytokine and chemokine expressions

and exhibiting anti-inflammatory and antiapoptotic

properties [43-45].

IL-6 plays a role in neurogenesis, affecting both

neurons and glial cells, and regulating activity of the

mature neurons and glial cells in health and disease

[46]. It functions similarly to neurotrophins, explain-

ing why this family of cytokines is referred to as

neuropoietins. Studies of the neuronal cultures have

demonstrated that IL-6 supports survival of various

neuronal types including cholinergic neurons of the

forebrain and septum, midbrain catecholaminergic

neurons, retinal ganglion cells, sympathetic neurons,

and dorsal root ganglion cells  [47,  48]. Examination of

the IL-6 KO mice has highlighted involvement of IL-6

in regulation of nociception, thermoregulation, emo-

tional reactivity, learning, and memory [49, 50].

Thus, our study indicates that both pro-inflamma-

tory and neuroprotective cytokine levels are reduced

in the prefrontal cortex of the prenatally alcohol-

exposed animals, with this effect being sex-specif-

ic and observed only in the sexually mature males.

Considering that neuroinflammation is a complex,

dynamic process involving changes in astrocyte and

microglial cell numbers, activation of cytokines, cel-

lular morphological alterations, migration, and gene

expression changes  [51], further research is needed to

understand dynamics of cytokine expression chang-

es at the mRNA and protein levels, their role in the

spectrum of disorders associated with prenatal alcohol

effects, and biological underpinnings of sex differenc-

es in these effects.

Study limitations. The “semi-forced” alcohol ex-

posure model for pregnant females presents challenges

NEURIOIMMUNE CHARACTERISTICS OF ANIMALS 1927

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

as a translational model in data interpretation. It is

crucial to accurately control the fluid intake in both

experimental and control animal groups when em-

ploying this model. Furthermore, this study did not

evaluate caloric intake of the diets provided to the

alcohol-consuming and control groups. Another lim-

itation concerns the age of offspring; the observed

biochemical alterations may be typical of late ado-

lescence and adulthood, but not of the prepubertal

period discussed by other researchers [52].

Contributions. I.Y.S. conceptualization, supervi-

sion, manuscript writing; V.S.K. experiment execution,

statistical analysis, research discussion, manuscript

editing; P.K.A. experiment execution, research discus-

sion, statistical analysis; R.A.A. experiment execution.

Funding. This research was financially support-

ed by the Russian Ministry of Health under the state

assignment titled “Study of pathogenetic mechanisms

of addiction to psychoactive substances using genet-

ic, biochemical, immunological, neurophysiological,

and neurocognitive approaches” (Reg. no. NIOKTR

AAAA-A18-118032390130-3).

Ethics declarations. This research adhered to

international guidelines for biomedical research in-

volving animals, the European Convention for the Pro-

tection of Animals used for Experimental and Other

Scientific Purposes (Strasbourg, 1986, with appendix

from 15.06.2006), the regulations of the Council of

the European Community (Directive 86/609/EEC from

14.11.2005 and Directive 2010/63/EU from 22.09.2010),

and the Principles of Good Laboratory Practice (Or-

der of the Ministry of Health of the Russian Federa-

tion no. 199n from 01.04.2016, GOST R 53434-2009).

All ethical guidelines were followed, including mini-

mizing the number of animals used to achieve reliable

scientific results. The experimental protocol was ethi-

cally approved and met the standards for conducting

biomedical research involving animals by the ethical

committees of the Federal Medical Research Centre for

Psychiatry and Neurology named after V. P. Serbsky,

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