Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 5, pp. 816-835 © Pleiades Publishing, Ltd., 2026.

816

Dependence of Antioxidant and Transcriptional

Responses to Periodic Hypoxia on Sex and Age in Rats

Sofia D. Kabiolskaya

1,a

*, Olga I. Patiavina

, Ilia A. Kabiolskiy

Olga K. Savushkina

, Elena B. Tereshkina

, Tatiana A. Prokhorova

Elena A. Sebentsova

1,3

, and Natalia G. Levitskaya

1,3

Faculty of Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

Mental Health Research Centre, 115522 Moscow, Russia

National Research Centre “Kurchatov Institute”, 123182 Moscow, Russia

e-mail: sofi.simonenko@mail.ru

Received January 26, 2026

Revised May 4, 2026

Accepted May 6, 2026

Abstract— Periodic hypoxia is a condition characterized by alternating episodes of oxygen deprivation

(hypoxia) and periods of normal or elevated oxygen levels (reoxygenation). Depending on severity and du-

ration of exposure, periodic hypoxia can activate both protective and pathological mechanisms. The aim of

this study was to evaluate dependence of the effects of acute and periodic hypoxia on sex and age of rats.

Male and female Wistar rats aged 2 and 4 months were used. The animals were exposed to normobaric

hypoxia (8% O

, 2 h) either once or daily for 5 consecutive days. Subsequently, changes in body weight,

activity and content of antioxidant system enzymes in blood plasma, as well as expression levels of the

HIF-1α, GPx4, BDNF, and caspase-3 genes in the frontal cortex, hippocampus, and striatum of the brain

were assessed. It was shown that daily hypoxia exposure leads to the decrease in body weight in both male

and female rats of both ages. In the males, age-dependent changes in activity of antioxidant enzymes and

increased expression of hypoxia marker genes in the brain were observed after a single hypoxia exposure.

After multiple exposures, the recorded parameters did not differ from the control values. In the females,

exposure to hypoxia did not affect activity of antioxidant enzymes, and increase in the expression of the

studied genes was observed only after five daily exposures to hypoxia. The experiments revealed significant

differences in the response to acute and periodic hypoxia exposure between male and female rats. These

data should be considered when developing experimental models of periodic hypoxia and studying the

mechanisms of adaptive and pathological reactions of the body to repeated hypoxia/reoxygenation episodes.

DOI: 10.1134/S0006297926600249

Keywords: periodic hypoxia, age, sex, body weight, antioxidant enzymes, glutathione system, HIF-1α, BDNF,

caspase-3

* To whom correspondence should be addressed.

INTRODUCTION

Hypoxia is a pathological condition characterized

by reduced oxygen saturation in organs and tissues.

It triggers numerous reactions that alter cellular me-

tabolism and is a risk factor for the development

of various disorders associated with dysfunctions of

different body systems, including metabolic disorders,

chronic heart and kidney diseases, and reproductive

disorders  [1,  2]. Brain is particularly sensitive to ox-

ygen deficiency due to its high metabolic activity

and active oxygen consumption  [3]. Hypoxia exerts

a complex influence on metabolic pathways and

signaling cascades in the brain. Metabolic changes

in the brain lead to energy metabolism disorders,

oxidative stress, dysregulation of neurotransmitter

systems, and other structural and functional im-

pairments. Hypoxia activates adaptive or pathologi-

cal signaling cascades, including activation of tran-

scriptional factors and modulation of ion channels.

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

A significant role in the formation of cellular re-

sponse is played by the signaling pathways regulated

by the hypoxia-inducible factors (HIFs)  [4]. Activa-

tion of the HIF-dependent transcription leads to re-

arrangement of metabolic processes, enhancement of

antioxidant defense, and modulation of cell survival

and apoptosis programs. These processes involve in-

teraction of the metabolic pathways associated with

mitochondrial function and formation of reactive ox-

ygen species (ROS) with the signaling cascades that

regulate expression of neurotrophic factors and an-

tioxidant system enzymes. Consequences of hypoxia

depend on its intensity and duration, as well as on

the age and sex of the organism  [5]. Hypoxia caus-

es dysfunction and death of nerve cells, which could

lead to the development of neurodegenerative dis-

eases  [6,  7].

Insufficient oxygen supply to the cells causes

dysfunction of the mitochondrial electron transport

chain and disruption of oxidative phosphorylation

processes, leading to the decrease in ATP synthesis,

depletion of cellular energy reserves, and increase in

formation of ROS, along with the decrease in activ-

ity of the cellular antioxidant systems  [8,  9]. There-

fore, oxidative stress develops in the cells, which is

a process leading to cell damage and death  [10,  11].

Oxidative stress is one of the main damaging factors

for the central nervous system (CNS)  [12,  13]. Hypox-

ia also triggers cytotoxic processes such as glutamate

excitotoxicity, which could lead to necrosis and apop-

tosis. Active glutamate uptake into the cell leads to

excessive accumulation of Ca

ions, which serves

as a signal for initiation of apoptosis and necrosis.

Additionally, formation of ROS and suppression of

antioxidant defense lead to the increased expres-

sion of pro-apoptotic proteins and activation of the

cell death processes  [9,  14]. Hypoxia also induces in-

flammation  [12]. ROS formed during hypoxia serve

as inducers of pathways that increase production of

pro-inflammatory factors, which, in turn, leads to the

increased ROS production and activation of apoptosis

pathways  [15]. Thus, oxygen deficiency exerts a com-

plex damaging effect on cells, activating pathophysi-

ological processes such as oxidative stress, excitotox-

icity, inflammation, necrosis, and apoptosis  [16,  17].

These processes could lead to irreversible changes

in the body tissues, especially in the CNS  [16]. Subse-

quent reoxygenation could exacerbate metabolic dis-

orders in the brain, and during this period, structural

and functional cell damage is often more pronounced

than during the hypoxia period  [8].

One of the key events leading to the cell damage

during hypoxia is excessive formation of ROS  [11].

Under physiological conditions, there is a balance

between the ROS formation and their elimination

by antioxidant defense systems. The main antioxi-

dant enzymes include superoxide dismutase, cata-

lase, and enzymes of glutathione and thioredoxin

systems  [18-20]. Glutathione system includes gluta-

thione peroxidases, glutathione reductase, and gluta-

thione-S-transferase, as well as glutathione peptide;

thioredoxin system includes thioredoxin reductase

and thioredoxin protein. Glutathione and thioredox-

in can be oxidized and subsequently reduced by the

action of corresponding enzymes, thereby neutraliz-

ing a large number of ROS, thus maintaining redox

balance in the cell  [21,  22]. Various studies have reg-

istered both increases and decreases in the activity of

glutathione system under hypoxia  [8, 23,  24]. Changes

in the activity of antioxidant enzymes could serve as

a marker of oxidative stress, not only in the dam-

aged tissue but also at the systemic level. It has been

shown that the changes recorded in blood reflect the

overall antioxidant status of the body  [25]. Dysfunc-

tion of antioxidant enzyme systems, particularly the

glutathione system, could lead to neurodegenerative

diseases (Alzheimer’s disease, Parkinson’s disease,

etc.) and mental disorders (schizophrenia, bipolar

disorder, autism spectrum disorders, etc.)  [26-30].

In response to hypoxic exposure, adaptive mech-

anisms that help maintain cell viability under oxy-

gen deficiency are activated in the body  [31]. These

mechanisms are based on activation of the hypox-

ia-inducible factors (HIFs)  [4]. HIFs are transcription-

al factors that trigger expression of many cytoprotec-

tive proteins. Among the HIF targets, brain-derived

neurotrophic factor (BDNF) occupies an important

place  [32]. BDNF regulates development and survival

of nervous system cells, plays a critical role in neu-

roplasticity processes, which determines its neuropro-

tective action and affect cognitive functions  [33,  34].

This neurotrophin exerts a protective effect on the

brain cells under hypoxia  [35]. HIF can activate anti-

oxidant processes, in particular, expression of gluta-

thione peroxidase  [36], as well as cascades associated

with the cell survival and mechanisms of apoptosis

modulation  [4,  37]. However, recent reports have

emerged reporting negative effects of HIF-1 activa-

tion, the mechanism of which is associated with the

increased oxidative stress  [38,  39].

Depending on duration and periodicity of expo-

sure, hypoxia is divided into continuous and periodic.

Periodic hypoxia is characterized by alternating peri-

ods of hypoxia/reoxygenation of varying duration and

frequency  [40,  41]. Periodic hypoxia exposure is ob-

served in sleep apnea, chronic lung diseases, non-in-

vasive mechanical ventilation, and altitude sickness

[6, 42, 43]. In healthy individuals, this form of hypoxia

could be associated with professional activities (min-

ers, firefighters, doctors, people working in “clean”

zones in production), as well as with the long-term

use of medical masks  [44,  45].

KABIOLSKAYA et al.818

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Direction of the effects of periodic hypoxia de-

pends on duration, intensity, and periodicity of the

exposure, parameters that determine the “dose” of

hypoxia [31, 46-48]. The “dose” of hypoxia, as well as

characteristics of an individual (sex, age), determine

acute and long-term reactions of the body, which

could be either protective or maladaptive/pathological

[17,  31,  40]. For example, moderate hypoxia (short-

term repeated sessions with frequency of 3-15 cycles

per day and oxygen content in the mixture of 10-16%)

is usually used as a protective exposure, while longer

or more frequent exposure (48-2400 cycles per day)

with oxygen concentration of less than 10% is con-

sidered as a severe hypoxia, capable of leading to

persistent negative consequences for the body [5, 40,

49-51]. In medicine, dosed periodic hypoxia is used

to improve health and increase performance and is

a non-drug treatment for a number of diseases [31,

40]. Duration of hypoxia exposure sessions and each

hypoxia/reoxygenation cycle, as well as number and

frequency of such cycles, vary significantly. Each ses-

sion could include several hypoxia/reoxygenation cy-

cles or a single exposure [31, 48, 50].

Animal experiments using various hypoxia proto-

cols revealed both pathological effects and beneficial

impacts  [51-53]. It has been shown that the short-

term, repeated exposures to mild or moderate hypoxia

trigger cellular and physiological adaptation  [31,  54].

Negative effects of periodic hypoxia have been stud-

ied mainly in sleep apnea syndrome models, in which

short hypoxia/reoxygenation cycles are repeated over

a long period. However, systemic hypoxia occurs in

various pathological conditions, including infections,

lung diseases, ischemic heart disease, and can also be

associated with professional activities  [44,  51]. Unlike

intermittent hypoxia associated with sleep apnea, in

these cases, people are exposed to hypoxia for several

hours a day over several days or weeks  [55]. To study

the effects of such hypoxic exposure, we used a mod-

el of moderate periodic continuous hypoxia in rats,

in which animals were exposed to daily normobar-

ic hypoxia (8%  O

, 2  h) for 5  days. Using this model,

increase in anxiety, decrease in learning ability, and

activation of the antioxidant system in blood plasma

were recorded in the male rats  [56]. Similar exposure

in females led to improved cognitive functions with-

out affecting anxiety levels  [57]. Thus, we revealed

a bidirectional effect of hypoxia on the behavior of

male and female rats, which confirms important role

of the individual differences in establishing a balance

between the adaptive and non-adaptive responses to

hypoxia  [54,  58].

Despite the large amount of accumulated data on

the effects of periodic hypoxia on the body, especially

on the CNS, the mechanisms determining pathological

and protective effects of multiple hypoxic exposures

are insufficiently studied. Additionally, influence of

individual characteristics such as sex and age on re-

sponse of the body to periodic hypoxia has not been

studied  [43,  59]. The aim of our work was to evaluate

changes in the expression levels of a number of pro-

teins – markers of hypoxic exposure in the brain –

as well as activity of antioxidant system enzymes in

the blood of male and female rats of different ages

that were exposed to acute and periodic hypoxia.

MATERIALS AND METHODS

The study was conducted with male and female

white Wistar rats obtained from the Stolbovaya nurs-

ery (Moscow Region, Russia). Animals aged 2  months

(“young”) and 4  months (“adult”) were used. The rats

were kept under standard vivarium conditions with

free access to water and standard laboratory feed,

maintaining a 12-h light/dark cycle. Total number of

animals was 96. Before the experiment, all rats un-

derwent a 7-day adaptation period – daily handling

for 1-2  min. Before the experiment, rats of each sex

and age were randomly divided into 3  groups: “con-

trol,” “single exposure to hypoxia,” and “five daily

exposures to hypoxia”. Body weight of the rats in

the “control” and “five daily exposures to hypoxia”

groups was recorded on the 1st, 2nd, and 5th days of

the experiment before the hypoxia exposure. Estrous

cycle stage in females was not controlled.

Modeling hypoxic exposure. To model normo-

baric hypoxia, a sealed chamber was used, into which

a gas mixture containing 8%  O

and 92%  N

was sup-

plied. Rats were exposed to hypoxia during the light

phase of the day, from 10:00 to 17:00. The animals

were placed in the chamber in individual cells with-

out access to water or food. Duration of the hypox-

ia session was 2  h. To study the effects of multiple

hypoxic exposures, the animals were placed in the

chamber daily for 5 consecutive days; for single expo-

sure, only on the last day of the experiment. Control

animals during the hypoxia session were kept un-

der similar conditions at normoxia (21%  O

). All rats

survived the hypoxia exposure used in the experi-

ments.

Immediately after the hypoxia session (in the

case of multiple hypoxia exposures, after the last

session), the rats were euthanized, blood samples

were collected, and brain structures (frontal cortex,

striatum, hippocampus) were isolated. Correspond-

ing samples were simultaneously obtained from

the control animals. Brain structure samples were

placed in a fixative solution (“IntactRNA”, Evrogen,

Russia) after isolation. Blood was collected in vacu-

um tubes containing 3.2% sodium citrate, then cen-

trifuged (15  min, 16,000g), and plasma was obtained.

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

The samples were next frozen in liquid nitrogen. All

samples were stored at –80°C.

In the brain structures, expression levels of the

genes for hypoxia-inducible factor (HIF-1α), glutathi-

one peroxidase  4 (GPx4), brain-derived neurotrophic

factor (BDNF), and caspase-3 (CAS-3) were evaluat-

ed. In blood plasma, activity of antioxidant enzymes

(glutathione-S-transferase (GST), glutathione reductase

(GR), glutathione peroxidase (GPx), and content of su-

peroxide dismutase (SOD)) were measured.

Assessment of antioxidant enzyme activity and

content. Activity of glutathione system enzymes was

determined based on methodological recommenda-

tions  [60]. GPx activity was measured using a colori-

metric method with Ellman’s reagent (Sigma-Aldrich,

USA). Reduced glutathione (Sigma-Aldrich) and 0.14%

tert-butyl peroxide (Sigma-Aldrich) were added to the

sample as a substrate, and peroxidase reaction was

carried out for 1  min. The reaction was next stopped

by  20% TCA. Reduced glutathione was determined

in the supernatant using Ellman’s reagent based on

absorption at 412  nm measured with an Allsheng

FlexA-200HT spectrophotometer (China).

GR activity was determined spectrophotomet-

rically from the rate of NADPH oxidation (PanReac

AppliChem, USA) (assessed by the decrease in absor-

bance recorded at 340  nm) in the reaction of oxidized

glutathione reduction (NeoFroxx, Germany) with a

Beckman DU 800 spectrophotometer (USA).

GST activity was determined spectrophotometri-

cally from the rate of formation of chromogenic glu-

tathione conjugates with 1-chloro-2,4-dinitrobenzene

(Acros, India) (assessed by the increase in absorbance

recorded at 340  nm with a Beckman DU 800 spectro-

photometer  [61].

SOD-1 content was determined using a “sand-

wich” version of solid-phase enzyme-linked immuno-

sorbent assay. An “IFA-SOD” kit (Tsitokin, Russia) was

used, containing rabbit polyclonal affinity-purified

antibodies against SOD-1 immobilized on the surface

of the wells of the plate and peroxidase-conjugated

antibodies against SOD-1. Optical density was deter-

mined at 450  nm with a Allsheng FlexA-200HT spec-

trophotometer (China).

Assessment of gene expression in brain struc-

tures. Relative expression levels of HIF-1α, GPx4,

BDNF, and CAS-3 genes were evaluated using real-time

reverse transcription polymerase chain reaction

(RT-PCR). Total RNA was isolated using an “ExtractRNA

Reagent” (Evrogen), followed by RNA purification

from genomic DNA using a “DNase  I, RNase-free” re-

agent (Thermo Fisher Scientific, USA). An RNAse in-

hibitor (Sintol, Russia) was also used for higher qual-

ity isolation. Reverse transcription reaction to obtain

cDNA samples was performed using a “MMLV  RT

kit” (Evrogen). Real-time PCR was performed using a

“5X  qPCRmix-HS SYBR” reagent (Evrogen) with a Bio-

Rad amplifier. Final volume of the reaction mixture

was 20  μL. Data were processed using the 2

−ΔΔCt

meth-

od, normalizing to the level of expression of β-actin

gene as a housekeeping gene. Nucleotide sequences of

the primers used are presented in Table  1.

Statistical analysis. Statistical analysis of the

data was performed using the software packages

“Statistica 10” and “GraphPad Prism 8.0.2.” For each

sample, normality of distribution and homogeneity

were assessed using the Shapiro–Wilk and Brown–For-

sythe tests, respectively. The results were evaluated

using factorial analysis of variance (ANOVA). Chang-

es in body weight were analyzed using the 4-factor

ANOVA with repeated measures (intergroup factors:

SEX, AGE, and HYPOXIA; intragroup factor: DAY OF

EXPERIMENT). For the analysis of other indicators,

a 3-factor ANOVA was used (intergroup factors: SEX,

AGE, and HYPOXIA). In the case of a significant in-

fluence of the selected factors or their interactions,

a post  hoc analysis was performed using the Tukey’s

test. The data in the figures are presented as individ-

ual mean values and standard deviation. Differences

between the groups were considered statistically sig-

nificant at p  <  0.05.

RESULTS

Effect of periodic hypoxia on body weight

changes in rats. Comparison of the initial body

weight of rats revealed a statistically significant

Table  1. Nucleotide sequences of primers used in

real-time RT-PCR

Gene Forward and reverse

primer sequences

Product

size (bp)

β-Actin 5′-CGGTCAGGTCATCACTATCG-3′ 165

5′-AGCACTGTGTTGGCATAGAG-3′

HIF-1α 5′-CGTGCCCCTACTATGTCGCTTTCT-3′ 205

5′-GGTTTCTGCTGCCTTGTATG-3′

BDNF 5′-GCCCAACGAAGAAAACCA-3′ 98

5′-CCAGCAGAAAGAGCAGAGGA-3′

GPx4 5′-CAGCAAGATCTGTGTAAATGGGG-3′ 112

5′-CTTGGTGAAGTTCCATTTGATGG-3′

CAS-3 5′-GCGAAGAAAAGTGACCATG-3′ 205

5′-GCGAAGAAAAGTGACCATG-3′

KABIOLSKAYA et al.820

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  1. Effect of periodic hypoxia on body weight changes in the male and female rats of different ages. Number of rats in

groups: n = 8. Statistically significant differences from the corresponding control are marked with *  (p  <  0.05, Tukey’s test).

influence of the factors SEX and AGE (F

1,56

  >  210;

p  <  0.0001). No significant influence of the HYPOXIA

factor or interactions between the factors was ob-

served (F

1,56

  <  3.0; p  >  0.30). No differences in the ini-

tial body weight were recorded between the groups of

rats of the same sex and age (p  >  0.25). Further analy-

sis compared changes in the body weight relative to

the baseline values (before the first hypoxia session).

A 4-factor repeated measures ANOVA revealed a sta-

tistically significant influence of the factors HYPOXIA,

AGE, and DAY OF EXPERIMENT (F

1,56

  >  45; p  <  0.0001),

as well as significant interaction of the factors SEX  ×

HYPOXIA, DAY OF EXPERIMENT  ×  HYPOXIA, DAY OF

EXPERIMENT  ×  AGE, DAY OF EXPERIMENT  ×  SEX, and

DAY OF EXPERIMENT  ×  HYPOXIA  ×  SEX.

Further analysis showed that in the young con-

trol rats of both sexes, body weight increased signifi-

cantly over 4 days (p  <  0.0001), with a greater weight

gain in the males than in the females (p  <  0.0001).

Body weight of the adult control males also increased

(p  <  0.0001), although the gain was less than in the

young animals (p  <  0.0001); in the adult control fe-

males, body weight did not change relative to the

baseline values (p  >  0.10).

In the young males and females subjected to

either one or four hypoxia sessions, no significant

changes in the body weight relative to the baseline

values were observed (p  >  0.13), but the weight gain

was significantly lower than in the corresponding

control groups (p  <  0.01). In the adult males, both

single and multiple hypoxia exposures led to the sta-

tistically significant decrease in the body weight rela-

tive to the baseline values (p  <  0.0003) and compared

to the adult control males (p  <  0.0002). In the adult

females, body weight after one hypoxia session did

not differ from the baseline values or corresponding

control values (p  >  0.35), but after four sessions, it

tended to be lower than the baseline and the control

values (p  <  0.08). In the adult rats, weight loss was

more pronounced in the males than in the females

(p  <  0.01) (Fig.  1).

Thus, the used regime of exposure to hypoxia

leads to slowdown in the weight gain in the young

males and females and decrease in the body weight

in the adult animals.

Effect of periodic hypoxia on antioxidant sta-

tus of the body. To assess the effect of hypoxia on

antioxidant status of the body, we measured activity

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  2. Effect of single and periodic hypoxia exposures on antioxidant enzyme activity in the blood plasma of rats of different

ages. Number of rats in groups: n  =  7-8. Statistically significant differences between the groups are marked with* (p  <  0.05,

Tukey’s test). Statistically significant influence of the AGE factor is marked with #  (p  <  0.05, 2-way ANOVA); SEX factor  –

with &  (p  <  0.05).

or content of antioxidant enzymes (GR, GPx, GST,

SOD) in the blood plasma of the male and female

rats of different ages under normal conditions, af-

ter a single and five daily exposures to hypoxia. The

use of 3-factor ANOVA revealed significant sex-as-

sociated differences in the enzyme activity. GR and

GST activity in the females was significantly higher

than in the males (F

1,84

  >  10.0; p  <  0.001), while the

GPx activity was higher in the males than in the fe-

males (F

1,84

  =  1.8; p  <  0.001). No influence of the SEX

factor on the SOD content in plasma was observed

1,84

  <  0.1; p  =  0.970).

We also recorded significant influence of the

AGE factor (F

1,84

  >  5.4; p  <  0.02) and interaction of the

KABIOLSKAYA et al.822

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

AGE  ×  SEX factors (F

1,84

  >  11.0; p  <  0.001) for the GST,

GR, and GPx activity indicators. In the young males,

GR activity was higher (p  <  0.001), and GPx activity

was lower (p  <  0.03) than in the adult rats. In the

young females, GR activity exceeded that in the adult

animals (p  <  0.005).

Assessment of the effect of hypoxia on antioxi-

dant enzyme activity in the males showed that single

exposure leads to the increase in GST and GPx activ-

ity in the young rats (p  <  0.04); in the adult males,

increase in GR activity was recorded after one hy-

poxia session (p  <  0.05). No significant influence of

the HYPOXIA factor or factor interactions on the

SOD content in the rat blood plasma was observed

2,84

  <  0.9; p  >  0.45) (data not shown). After five hy-

poxia sessions, the indicators in both young and adult

males did not differ from the control values. In the

females, no significant changes in indicators were de-

tected after the hypoxia exposure (p  >  0.15) (Fig.  2).

Thus, in response to single hypoxia exposure,

both young and adult male rats showed activation of

the antioxidant system. After multiple hypoxic expo-

sures, no significant changes in the antioxidant activ-

ity indicators relative to the control values were ob-

served. The used protocols of hypoxia exposure (both

acute and periodic) did not lead to the changes in the

antioxidant system activity of the female rats aged 2

and 4 months.

Effect of periodic hypoxia exposures on ex-

pression levels of hypoxia marker proteins in the

brain structures. Using real-time PCR, relative ex-

pression levels of the BDNF, HIF-1α, GPx4, and CAS3

genes were measured in the frontal cortex, hippo-

campus, and striatum of the male and female rats

of different ages under normal conditions, after one

and five hypoxia sessions. The use of 3-factor ANOVA

showed significant influence of the SEX factor on the

expression levels of the studied genes. In the frontal

cortex of the males, mRNA levels of the HIF-1α and

GPx4 genes exceeded the corresponding values in the

female subgroup (F  >  3.9, p  <  0.04). In the hippocam-

pus and striatum of the males, expression level of

the GPx4 gene was lower, and of the CAS-3 gene was

higher compared to the females (F  >  3.0, p  <  0.05).

Additionally, for a number of indicators (BDNF in

the frontal cortex and striatum, CAS-3 in the stria-

tum and hippocampus, HIF-1α and GPx4 in the fron-

tal cortex), a significant interaction of the SEX and

HYPOXIA factors was recorded (F > 3.6, p < 0.03),

indicating that the effects of hypoxia depend on the

sex of the animals. A significant influence of the AGE

factor on the expression levels of a number of genes

was also revealed. In particular, in the young rats, ex-

pression level of the GPx4 gene in the hippocampus

was higher, and expression levels of the HIF-1α and

GPx4 genes in the frontal cortex, BDNF, and CAS-3

in the striatum were lower than the corresponding

indicators in the adult rats. No significant interaction

of the AGE and HYPOXIA factors was recorded.

Next, we performed a 2-factor ANOVA for the

HYPOXIA and AGE factors separately in the subgroups

of males and females. In the male rats, a significant

influence of the AGE factor (F

1,40

  =  19.7; p  <  0.0001)

and a trend-level influence of the HYPOXIA factor

2,40

  =  3.1; p  =  0.056) on the HIF-1α gene expression

in the frontal cortex were recorded. Further analy-

sis showed a significant increase in the HIF-1α gene

mRNA levels in the group of adult males after one

hypoxia session relative to the males that underwent

five daily exposures to hypoxia (p  =  0.03). In other

studied brain structures of the male rats, no signif-

icant influence of the HYPOXIA and AGE factors or

their interaction was recorded (F  <  1.5; p  >  0.25) for

the HIF-1α gene mRNA levels (Fig.  3).

In the females, a significant influence of the

HYPOXIA factor on the HIF-1α gene expression in

the frontal cortex (F

2,37

  =  5.8; p  =  0.006) was not-

ed, as well as interaction of the HYPOXIA and AGE

factors for this indicator in the striatum (F

2,37

  =  6.5;

p  =  0.004). Intergroup comparison showed significant

increase in the HIF-1α mRNA levels in the frontal

cortex of the adult females that underwent multiple

hypoxia exposures compared to the other groups of

female rats (p  <  0.01), as well as increase in this in-

dicator in the striatum of the young females after

five daily exposures to hypoxia relative to the young

females that underwent a single exposure (Fig.  3).

In the hippocampus of the young and adult female

rats, no significant changes in the HIF-1α gene expres-

sion were observed after hypoxia (F  <  1.4; p  >  0.25).

In the subgroup of male rats, a significant influ-

ence of the AGE factor on the GPx4 gene expression

levels in all studied structures was noted (F  >  4.5;

p  <  0.05). Further analysis showed that expression of

this gene is higher in the frontal cortex and stria-

tum but lower in the hippocampus of the adult rats

compared to the young ones (Fig.  4). No influence

of the HYPOXIA factor or factor interactions on the

GPx4 gene expression levels in the brain structures of

the male rats was recorded (F  <  1.2; p  >  0.30). In the

subgroup of female rats, significant influence of the

HYPOXIA factor on the GPx4 gene expression in the

frontal cortex (F

2,36

  =  4.8; p  =  0.014) was noted, as

well as a trend-level influence of this factor on the

expression of this gene in the striatum (F

2,38

  =  2.9;

p  =  0.065). Additionally, in the striatum, a signifi-

cant interaction of the HYPOXIA and AGE factors

for this indicator was recorded (F

2,38

 = 3.8; p = 0.03).

Subsequent analysis showed significant increase in

the GPx4 gene mRNA levels in the frontal cortex of

the adult females and the striatum of the young fe-

male rats that underwent multiple hypoxia exposures

DEPENDENCE OF RESPONSE TO HYPOXIA ON GENDER AND AGE 823

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  3. Effect of single and periodic hypoxia exposure on the HIF-1α gene expression in the frontal cortex, hippocampus,

and striatum of the rats of different sex and age. Number of rats in groups: n = 6-8. Statistically significant differences

between the groups are marked with * (p  <  0.05, Tukey’s test). Statistically significant influence of the AGE factor is marked

with #  (p  <  0.05, 2-way ANOVA); SEX factor – with &  (p  <  0.05).

relative to the corresponding controls and rats after

one hypoxia session (p  <  0.02) (Fig.  4).

In the male rats, a significant influence of the

HYPOXIA factor on the BDNF gene mRNA levels in

the frontal cortex, hippocampus, and striatum was re-

corded (F  >  3.9; p  <  0.026). Additionally, a significant

influence of the AGE factor (F

1,40

  =  9.9; p  =  0.003) on

the expression of this neurotrophin in the striatum

was noted, as well as significant interaction of the

HYPOXIA and AGE factors for the BDNF gene expres-

sion in the frontal cortex and hippocampus (F  >  3.2;

p  <  0.05). Subsequent analysis showed that single

hypoxia exposure causes significant increase in the

BDNF gene mRNA levels in the frontal cortex and

KABIOLSKAYA et al.824

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  4. Effect of single and periodic hypoxia exposures on the GPx4 gene expression in the frontal cortex, hippocampus,

and striatum of the rats of different sex and age. Number of rats in groups: n  =  6-8. Statistically significant differences

between the groups are marked with* (p  <  0.05, Tukey’s test). Statistically significant influence of the AGE factor is marked

with #  (p  <  0.05, 2-way ANOVA); SEX factor – with &  (p  <  0.05).

striatum of the adult males, as well as in the hip-

pocampus of the young males compared to the cor-

responding indicators in the control group and the

group of male rats that were subjected to multiple

exposures (p  <  0.03). In the female rats, interaction

of the HYPOXIA and AGE factors at the trend level

was recorded in the striatum (F

2,34

  =  3.2; p  =  0.054);

further analysis revealed significant decrease in

the expression of this gene after five daily hypoxia

exposures compared to the acute single exposure

(p  =  0.03). No differences were found in the frontal

cortex and hippocampus of the female rats (Fig.  5).

DEPENDENCE OF RESPONSE TO HYPOXIA ON GENDER AND AGE 825

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  5. Effect of single and periodic hypoxia exposures on the BDNF gene expression in the frontal cortex, hippocampus,

and striatum of the rats of different sex and age. Number of rats in groups: n  =  6-8. Statistically significant differences

between the groups are marked with* (p  <  0.05, Tukey’s test). Statistically significant influence of the AGE factor is marked

with #  (p  <  0.05, 2-way ANOVA).

In the subgroup of male rats, assessment of the

CAS-3 gene expression levels showed significant influ-

ence of the HYPOXIA and AGE factors in the frontal

cortex (F  >  3.6; p  <  0.04) and of the AGE factor in the

striatum (F

2,39

  =  8.4; p  =  0.006). In the hippocampus,

significant influence of the HYPOXIA factor (F

2,39

  =  6.6;

p  =  0.008) and interaction of the HYPOXIA and AGE

factors (F

2,39

  =  4.9; p  =  0.013) was noted. When con-

solidating the sample by the AGE factor, a significant

increase in the CAS-3 gene expression in the frontal

cortex of the male rats that underwent single expo-

sure was revealed compared to the control animals

and animals that underwent five daily hypoxia expo-

sures (p  <  0.05). In the hippocampus of young males,

KABIOLSKAYA et al.826

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Fig.  6. Effect of single and periodic hypoxia exposures on the CAS-3 gene expression in the frontal cortex, hippocampus,

and striatum of the rats of different sex and age. Number of rats in groups: n  =  6-8. Statistically significant differences

between the groups are marked with* (p  <  0.05, Tukey’s test). Statistically significant influence of the AGE factor is marked

with #  (p  <  0.05, 2-way ANOVA); SEX factor – with &  (p  <  0.05).

similar changes in the expression of this gene were

recorded (p  <  0.002) (Fig.  6). No significant differenc-

es between the groups of males were noted in the

striatum. In the female rats, significant influence of

the HYPOXIA factor on the CAS-3 gene expression in

the frontal cortex was recorded (F

2,39

  =  3.6; p  =  0.038).

Nosignificant influence of the HYPOXIA factor on ex-

pression of this gene in other brain structures, as well

as the influence of the AGE factor and interaction of

these factors on the CAS-3 gene expression levels in

the brain structures of female rats, was observed.

Further analysis showed that multiple hypoxia ex-

posures lead to the significant increase in the CAS-3

mRNA levels in the frontal cortex of the adult females

compared to the control group and the group sub-

jected to a single hypoxia exposure (p  <  0.04) (Fig.  6).

DEPENDENCE OF RESPONSE TO HYPOXIA ON GENDER AND AGE 827

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Thus, in response to both acute and multiple hy-

poxia exposures, expression levels of the genes in-

volved in implementation of the body’s response to

oxygen deficiency change in the rat brain. The ob-

served changes depend on the sex and age of the

rats, as well as on the brain structure. The most pro-

nounced changes were recorded in the frontal cor-

tex – a region that plays an important role in reg-

ulation of cognitive functions and emotional status.

We noted significant influence of the animals’ age

on expression levels of the genes of interest. In par-

ticular, content of the BDNF mRNA in the striatum

and HIF-1α mRNA in the frontal cortex was higher

in the adult rats than in the young ones, while the

level of the GPx4 mRNA decreased with age. Howev-

er, we did not record significant interaction between

the HYPOXIA and AGE factors for any of the record-

ed indicators. The responses to hypoxia exposure dif-

fered significantly between the male and female rats.

In the males, changes in the gene expression levels

were mainly observed after single, rather than multi-

ple, exposures (HIF-1α, BDNF, and CAS-3 in the fron-

tal cortex, BDNF in the striatum and hippocampus,

CAS-3 in the hippocampus). In the female subgroup,

changes in the gene expression levels were recorded

only after multiple hypoxia exposures (HIF-1α, CAS-3,

and GPx4 in the frontal cortex, GPx4 and HIF-1α in

the striatum), and no significant changes in the con-

tent of mRNA of the genes of interest were observed

after one hypoxia session.

DISCUSSION

Effect of periodic hypoxia on body weight

changes in rats. The task of this study was to eval-

uate dependence of the effects of acute and periodic

hypoxia on the sex and age of rats. During the ex-

periment, we monitored body weight of the animals

and recorded changes in this parameter in response

to hypoxia. In the young animals subjected to hypox-

ia exposure, body weight increased more slowly than

in the control group, and in the adults, it even de-

creased, with values after 4 hypoxia sessions being

lower than after one session. Such changes were ob-

served in both males and females, and, therefore, did

not depend on the sex of the animals. Decrease in the

body weight after hypoxia exposure has been record-

ed in many studies. In the animal studies, decrease

in the body weight was recorded in the models of

intermittent and chronic hypoxia; authors associate

these changes with the reduction in the volume of

adipose tissue  [62,  63]. Moreover, slowdown in the

weight gain in the 2-month-old male rats after peri-

odic hypoxia exposure was shown  [64], which is fully

consistent with our results. Similar results were ob-

tained when studying the effect of hypoxia on human

metabolism: decrease in the weight gain and loss of

appetite were observed both during interval hypoxic

training  [65] and during chronic oxygen deficiency in

the individuals living at high altitudes  [66].

Effect of periodic hypoxia on antioxidant sta-

tus of the body. It is known that one of the main

damaging factors in hypoxia is oxidative stress. Inthis

regard, increase in the ROS content and change in the

antioxidant status of the body in response to hypoxia

exposure are expected, which was confirmed in the

studies with periodic hypoxia models  [64,  67]. In our

study, we measured activity of the glutathione sys-

tem enzymes (GPx, GR, GST) and SOD content in the

rat blood plasma. In response to hypoxia exposure,

no changes in any of the studied parameters were

observed in the female rats. In the males, increase

in the activity of glutathione system enzymes was re-

corded in response to single hypoxia exposure, and

the enzyme activity did not differ from the control

values after five daily hypoxia exposures.

Changes in the antioxidant status of the body

were noted in various models of hypoxia exposure.

Moreover, direction of these changes could vary. In

particular, in the model of acute severe hypoxia,

increase in the GPx activity in the brain was re-

corded, which persisted for one day after hypoxia

exposure  [38]. At the same time, other studies have

shown decrease in the GPx, GR, and SOD activities

in a similar model  [67]. In the sleep apnea syndrome

models, decrease in the GPx and SOD activities in the

brain  [68] and decrease in the SOD content in blood

serum  [69] were noted. When studying consequences

of chronic hypoxia, it was shown that, on the con-

trary, the activity of GPx and SOD in the rat brain in-

creased after hypoxic exposure  [70]. Presumably, such

differences in these indicators are due to the differ-

ent modifications of the hypoxia exposure protocols

used – primarily, differences in the ratio of hypoxia

and reoxygenation periods.

As for age differences, it is known that activity

of the glutathione system enzymes changes with age

in animals  [71]. It has been shown that the activity

of GPx increases with age, and GR activity decreas-

es with age in the animals, which is consistent with

our results  [72]. It can be assumed that in the adult

(4-month-old) rats, GPx activity is increased; and un-

der normoxia, the enzyme successfully neutralizes

hydrogen peroxide molecules, and in the case of hy-

poxia exposure, there is a need to quickly replenish

the pool of reduced glutathione, which is why GR ac-

tivity increases. In the young animals, on the other

hand, GR activity is higher than in the adults, so even

under conditions of oxygen deficiency, restoration of

the glutathione pool in the 2-month-old rats occurs

quite effectively.

KABIOLSKAYA et al.828

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

Considering sex differences in antioxidant activ-

ity in the rat blood plasma, it could be noted that

in the females, this parameter does not change in

response to hypoxia. We suggest that this is due to

the intrinsic antioxidant activity of estrogens, which

effectively neutralize ROS during oxidative stress  [73].

It has previously been shown that in the ovariecto-

mized females in the model of intermittent hypoxia,

there was a decrease in the activity of antioxidant

enzymes, while administration of estradiol returned

the values of these parameters to control levels  [68].

Effect of periodic hypoxia on expression lev-

els of hypoxia marker proteins in brain structures.

Inour study, we evaluated changes in the expression

of HIF-1α, GPx4, BDNF, and CAS-3 genes. These pro-

teins were selected as main markers of hypoxia expo-

sure. According to our data, the HIF-1α gene expres-

sion increased in response to hypoxia exposure in the

frontal cortex of 4-month-old male and female rats.

In the males, the value of this parameter increased

in response to the single hypoxia exposure, and it

returned to control levels after five daily hypoxia

exposures, while in the females, changes occurred

only after the 5th hypoxia session. Moreover, in the

females, increase in the expression of this indicator

in the striatum was observed after 5 daily hypoxia

exposures, and these differences were recorded in the

group of young females. Effect of age was also noted:

in the 4-month-old males, the level of the HIF-1α gene

expression was higher than in the 2-month-old males.

No changes in the HIF-1α mRNA expression were de-

tected in the hippocampus. It is known that hypoxia

inhibits the action of prolyl hydroxylases, which nor-

mally leads to degradation of HIF-α, and under con-

ditions of oxygen deficiency, the content of HIF-α in

the cells increases  [4]. This was confirmed in a num-

ber of studies on various hypoxia models  [8,  39,  67].

As for the expression of mRNA of this protein in the

brain, studies with rats also note an increase in this

parameter in response to hypoxia  [74,  75]; moreover,

even after a period of reoxygenation, the level of the

HIF-1α mRNA remains elevated for some time  [76].

As mentioned earlier, the glutathione system

enzymes are key factors in antioxidant cell defense

during hypoxia, especially in the brain. There is evi-

dence of a decrease in the content of the protein and

expression of the GPx4 gene in response to hypoxia

exposure in the brain cell culture  [77]. However, in

our study, the level of GPx4 gene expression did not

change in response to hypoxia exposure in the male

rats in any of the studied structures. At the same

time, in the females, increase in expression of this

gene was observed after 5 daily hypoxia exposures

in the frontal cortex and striatum, with changes, in

the first case, being recorded in the adults and, in the

second case, in the young animals. In the males, only

influence of age on this indicator was noted: in the

frontal cortex and striatum, the GPx4 gene expres-

sion was higher in the adult males, and in the hip-

pocampus, it was higher in the young males. It has

been shown that the level of GPx4 mRNA expression

significantly increases in the brain of older rats  [78],

which partially agrees with our results.

Brain-derived neurotrophic factor (BDNF) is one

of the most important regulators of cell metabolism

and could have a protective effect under oxygen

deficiency  [32]. In our study, it was shown that the

expression level of the BDNF gene increased after

the single hypoxia exposure and decreased again af-

ter the five daily hypoxia exposures in the frontal

cortex and striatum of the adult males and in the

hippocampus of the young males. As for females,

they showed decrease in the BDNF gene expression

in response to the five daily hypoxia exposures in

the striatum. Many studies have shown that expres-

sion levels of the BDNF gene in the brain increase in

hypoxia and play an important role in adaptation to

this exposure  [79,  80]. There is also evidence of the

differences in the BDNF expression during hypoxia

depending on sex and age. In particular, it is known

that the level of expression of the BDNF gene mRNA

in the brain decreases in the old rats compared to

the younger ones  [81]. There is also clinical evidence

that the BDNF levels in the blood of the patients

with sleep apnea syndrome decrease with age  [32].

In other studies, however, no effect of age on the

BDNF gene expression after hypoxia exposure was

found, but significant effect of sex was noted: in the

female rats, expression was higher than in the males

[82]. Our data on the influence of age and sex on

the BDNF gene expression do not agree with the lit-

erature. Probably, the reason is that in our study, we

compare the rats aged 2 and 4 months, while older

animals were used in other works  – 12 months and

older  [81].

The cascade of cell damage caused by hypoxia

could lead to apoptosis. One of the key apoptotic en-

zymes is CAS-3; it is this enzyme, when activated by

CAS-9, that mediates DNA cleavage in the cell through

the caspase-activated DNase  [83]. Therefore, in our

work, CAS-3 was chosen as a marker of cell death

caused by hypoxia in the brain tissue. We showed

that in the males of both ages, the expression level of

CAS-3 gene increased in response to the single hypox-

ia exposure and decreased again after 5 daily hypox-

ia exposures in the frontal cortex and hippocampus.

In the females, expression of this gene increased only

in the frontal cortex and in response to multiple hy-

poxia exposures. In addition, in the males, the influ-

ence of age on this indicator was noted: in the fron-

tal cortex, it was higher in the young animals, and

in the striatum, it was higher in the adult animals.

DEPENDENCE OF RESPONSE TO HYPOXIA ON GENDER AND AGE 829

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

It is known that the CAS-3 content in the rat hippo-

campus increased after chronic hypoxia exposure  [84].

Similar data exist on the increase in mRNA expres-

sion of this protein  [75]. In other studies, however,

the CAS-3 content in the rat brain tissue increased

after acute hypoxia exposure but did not change after

periodic exposure  [67]. The CAS-3 levels in the brain

depend on age: content of the pro-apoptotic proteins,

including CAS-3, increases in the hippocampus of rats

with age  [85]. In our work, no unambiguous depen-

dence of the changes in the CAS-3 gene expression on

the age of animals was revealed.

Analyzing the obtained results, we identified

a number of patterns. First, the most significant

changes in the expression of the target genes were

recorded in the frontal cortex of animals. It is be-

lieved that the most vulnerable structure to hypoxia

is hippocampus  [86], which contradicts our results.

Presumably, such discrepancy may be due to the use

of different hypoxia exposure protocols. In addition,

the effects of periodic hypoxia could vary depending

on the brain structure  [53]. Second, it is impossible

not to note the significant differences in the respons-

es to single and periodic hypoxia exposures in the

male and female rats. In the females in our study,

antioxidant status of the body did not change, and

there were fewer changes in the gene expression lev-

els than in the males. This is consistent with the liter-

ature data that females are more resistant to oxygen

deficiency due to the antioxidant and neuroprotective

effects of estrogens  [68,  87]. In the hippocampal neu-

ron culture, it was shown that the male cells survived

hypoxia to a lower degree than the female cells, and

when the culture was treated with estradiol, the cell

death during hypoxia was not observed  [87]. In the

conducted study, males showed response to the sin-

gle hypoxia exposure, and after multiple exposures,

the values returned to the control levels; and this

pattern was observed for all studied parameters. In

the females, changes were recorded only after 5 daily

hypoxia exposures. According to our previous studies,

the used protocol of periodic hypoxia exposure had

negative effect on the behavior of the male rats: it

increased anxiety levels and reduced learning abili-

ty [56]. In the females, such exposure did not cause

behavioral disorders; moreover, it had a positive ef-

fect on cognitive functions  [57].

It is known that, depending on the “dose,” pe-

riodic hypoxia could cause either physiological ad-

aptation or pathological damage. With decrease in

oxygen supply to the cells, adaptive mechanisms are

activated that facilitate survival of the cells under hy-

poxia. However, when intensity of the hypoxia expo-

sure exceeds certain threshold, a cascade of reactions

is triggered that lead to the changes in cell function

or their death  [54]. Our data on gene expression and

antioxidant enzyme activity do not allow us to char-

acterize the response to hypoxia as unambiguously

adaptive or maladaptive, but they indicate differenc-

es in the temporal organization of the response to

hypoxia exposure in the male and female rats. Inthe

males, changes in the expression of the HIF-1α, BDNF,

CAS-3, and GPx4 genes, as well as increase in the ac-

tivity of antioxidant system enzymes, were recorded

mainly after a single hypoxia exposure, while after

the five daily hypoxia exposures, the corresponding

indicators, as a rule, did not differ from the control

values. In the females, on the contrary, changes in

the gene expression levels were detected after multi-

ple hypoxia exposures in the absence of pronounced

shifts in antioxidant activity. Considering functional

role of the studied genes, this pattern may reflect

differences in the timing of the involvement of tran-

scriptional and post-transcriptional mechanisms as-

sociated with the hypoxia response, neuroplasticity,

regulation of cell death, and antioxidant defense.

Under hypoxia, ROS production increases, which,

when excessively produced, could lead to the devel-

opment of oxidative stress. Oxidative stress deter-

mines metabolic disorders such as energy deficiency,

lactate accumulation, acidosis, and others. Inflamma-

tory reactions activated by the increased oxidative

stress enhance metabolic, cognitive, and other disor-

ders  [88]. At the same time, activation of the HIF-de-

pendent signaling cascades leads to the induction of

the antioxidant defense genes and neurotrophic fac-

tors, as well as modulation of apoptosis processes.

Thus, the level of oxidative stress is one of the main

factors determining direction of the hypoxia effects,

including improvement or impairment of cognitive

functions  [51]. It has been shown that reduction of

oxidative stress reduces neuropathological disorders

caused by periodic hypoxia  [52]. In our experiments,

hypoxia exposure caused increase in the activity

of antioxidant enzymes in the male rats, while no

such changes were observed in the female group.

Increase in antioxidant activity in the male rats is

likely a compensatory response to the increased ox-

idative load. Many researchers consider increase in

the antioxidant enzyme activity (including SOD and

glutathione-dependent enzymes) under hypoxia as a

component of the response to oxidative stress  [89,

90]. Estrogens have their own antioxidant and neuro-

protective properties and could affect activity of the

signaling cascades associated with HIF-1α and regu-

lation of cell metabolism  [68]. Probably, these are the

mechanisms that may underlie the differences in the

responses of male and female rats to hypoxia expo-

sure. However, to clarify the mechanisms of protec-

tive and pathological effects of periodic hypoxia and

influence of sex on its consequences, further research

is needed.

KABIOLSKAYA et al.830

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026

CONCLUSION

This study aimed at investigating the effects of

non-lethal normobaric hypoxia in male and female

rats of different ages. An assessment was made of the

influence of acute and periodic hypoxia exposure on

the animals’ body weight, antioxidant enzyme activi-

ty in blood plasma, and expression levels of a num-

ber of proteins – markers of hypoxic exposure – in

the brain structures. The experiments showed that

the used protocols of exposure to hypoxia lead to a

slowdown in the weight gain of rats, and direction

of this effect does not depend on the sex and age of

the animals. We did not record a significant influence

of the age of rats on neurochemical changes caused

by hypoxia exposure, which may be due to the use

of animals close in age. At the same time, our stud-

ies revealed dependence of the effects of both acute

and periodic hypoxia on the sex of the animals. In

the males, changes in antioxidant enzyme activity

and increased expression of the genes of the mark-

ers of hypoxia exposure in the brain were observed

after a single hypoxia exposure. After multiple expo-

sures, the recorded indicators did not differ from the

control values. In the females, the used protocols of

exposure to hypoxia did not affect activity of anti-

oxidant enzymes, and increase in the expression of

the studied genes was observed only after multiple

hypoxia exposure. The obtained results, as well as the

data on bidirectional effect of the used protocols of

hypoxia exposure on the cognitive functions of male

and female rats obtained earlier, indicate greater vul-

nerability of the males to hypoxia compared to the

females. These data should be taken into account

when developing experimental models of periodic hy-

poxia and studying the mechanisms of adaptive and

pathological reactions of the body to multiple hypox-

ia/reoxygenation sessions.

Abbreviations

BDNF brain-derived neurotrophic factor

CAS-3 caspase-3

CNS central nervous system

GPx glutathione peroxidase

GPx4 glutathione peroxidase  4

GR glutathione reductase

GST glutathione-S-transferase

HIF-1α hypoxia-inducible factor 1-alpha

ROS reactive oxygen species

RT-PCR reverse transcription polymerase

chain reaction

SOD superoxide dismutase

Contributions

N.  G.  Levitskaya and E.  A.  Sebentsova – conception

and supervision of the work; S.  D.  Kabiolskaya,

O.  I.  Patiavina, I.  A.  Kabiolskiy, O.  K.  Savushkina,

E.  B.  Tereshkina, and T.  A.  Prokhorova – conducting

experiments; S.  D.  Kabiolskaya and N.  G.  Levitskaya–

discussion of the research results; S.  D.  Kabiolskaya,

I.  A.  Kabiolskiy, E.  A.  Sebentsova, and N.  G.  Levit-

skaya – writing and editing the text.

Funding

The work was financially supported by the State Bud-

get assignment of the Department of Human and Ani-

mal Physiology, Faculty of Biology, Lomonosov Moscow

State University: 28-3-21 (no.  121032300071-8) “Mecha-

nisms of Physiological Adaptations”; and by the State

Budget assignment of the Mental Health Research

Centre: FURU-2024-0016 (no.124020700032-9) “Biolog-

ical Markers of Mental and Neurodegenerative Diseas-

es: Fundamental and Applied Aspects.”

Ethics approval and consent to participate

The study was conducted in compliance with bioethi-

cal norms for the handling of experimental animals in

accordance with the “Good Laboratory Practice Rules”

(Order of the Ministry of Health of the Russian Feder-

ation No.  199 dated 01.04.2016) and the requirements

of Directive 2010/63/EU of the European Parliament

dated 22.09.2010. The conditions for keeping animals

and experimental procedures were approved by the

Bioethics Commission of Lomonosov Moscow State

University (applications no.  157-zh-2 dated 22.06.2023

and no.  12.5-sod dated 24.10.2024).

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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