Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 6, pp. 956-966 © Pleiades Publishing, Ltd., 2026.

956

Association of Neutrophil Elastase Activity,

Oxidative Stress, Energy and Glutamate

Metabolism Parameters with Behavior and Learning

in Wistar Rats in a Postnatal Valproate Model

of Autism Spectrum Disorder

Anna A. Stakhanova

1,a

*, Olga G. Voskresenskaya

, Olga K. Savushkina

Tatyana A. Prokhorova

, Elena B. Tereshkina

, Ekaterina V. Semina

Svetlana A. Zozulya

, Natalia V. Kost

, and Yulia A. Chaika

Federal State Budgetary Scientific Institution, Russian Mental Health Research Center,

115522 Moscow, Russia

Lomonosov Moscow State University, 119234 Moscow, Russia

e-mail: anna.stahanova@inbox.ru

Received January 26, 2026

Revised June 17, 2026

Accepted June 19, 2026

Abstract— To study biochemical mechanisms underlying the development of autism spectrum disorders

(ASD), an experimental model based on early postnatal administration of valproic acid (VPA) to Wistar rats

was used. Social behavior impairments characteristic of ASD was accompanied by cognitive impairments

and were associated with the increased neutrophil elastase (NE) activity in the serum and cerebellum of

the rats treated with VPA. Increased NE activity in the cerebellum may indicate development of neuroin-

flammation. Impaired antioxidant defense under the influence of VPA was manifested by the increased

glutathione S-transferase (GST) activity in the serum and cerebellum of rats. Correlation analysis revealed

a link between the NE activity and formation of social behavior in animals. Enzymes of glutamate metab-

olism (glutamate dehydrogenase and glutamine synthetase) and antioxidant defense enzymes that regulate

oxidative stress (OS) levels correlated with formation and maintenance of the acquired skills in both control

and experimental animals.

DOI: 10.1134/S0006297926600158

Keywords: postnatal valproate model of autism spectrum disorders, rats, social behavior, memory, neuroinflam-

mation, neutrophil elastase, oxidative stress, energy metabolism, glutamate metabolism, glutathione S-trans-

ferase, creatine phosphokinase, cerebellum

* To whom correspondence should be addressed.

INTRODUCTION

Autism spectrum disorders (ASD) are character-

ized by complex impairments in social interaction,

stereotyped behavior, as well as changes in the reg-

ulation of cognitive functions and emotions. In the

individuals with ASD, pronounced memory impair-

ments have been described: episodic memory and

facial recognition are affected, while simple verbal

and mechanical memory may remain relatively pre-

served. Studies link these deficits to dysfunction of

the default mode network and atypical connectivity

between the hippocampus and posterior cingulate

cortex  [1].

In the studies on animals and humans, it has

been shown that the main symptoms of ASD, associ-

ated with executive function impairments, difficulties

in emotion processing, social interaction, and learn-

ing, could be linked to the changes in the structure

and function of the cerebellum, as well as disruption

BIOCHEMICAL MARKERS AND BEHAVIOR OF RATS IN THE ASD MODEL 957

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

of connections between the cerebellum and brain re-

gions involved in the pathophysiology of autism, in-

cluding prefrontal cortex, amygdala, and hippocam-

pus  [1]. The development of cerebellum begins in

the embryonic stage, continues throughout prenatal

development, and is not completed after birth, mak-

ing it vulnerable to the development of disorders  [2].

During the prenatal period, expression of the genes

associated with autism in the cerebellum increases,

and its damage during this period significantly in-

creases the risk of developing ASD  [3]. Inthe patients

with autism, numerous changes in the cerebellum are

observed, including loss of Purkinje cells, increased

oxidative stress (OS)  [4], and dysfunction of GABAergic

and glutamatergic systems  [5]. These changes in the

cerebellum play a key role in the pathophysiolo-

gy of the main symptoms of autism, such as mo-

tor and sensory impairments, memory and speech

disorders.

To study biological mechanisms of these disor-

ders and search for their pathogenetically significant

biochemical markers, experimental studies on ani-

mals are necessary. One of the experimental models

of ASD uses valproic acid (VPA). VPA is used in clin-

ical practice as an antiepileptic and mood-stabilizing

drug, but at high concentrations, it disrupts gene ex-

pression by blocking histone deacetylase. This leads

to epigenetic modifications that affect neurodevelop-

ment processes, synaptic plasticity, and behavioral

responses. Possible neurotoxic mechanisms of VPA

action in animal models of ASD include mitochondri-

al dysfunction, oxidative stress, glutamate metabolism

disorders, modulation of GABAergic and glutamater-

gic neurotransmission [6, 7].

Experimental exposure to VPA in animals can be

prenatal or early postnatal. Both options cause the

development of fetal valproate syndrome, which is

considered a valid model of ASD  [8]. Postnatal ad-

ministration of VPA affects the developing nervous

system during the critical period of cerebellum for-

mation  [9], which is of particular interest given the

role of this structure in the pathophysiology of ASD.

Studying behavior and cognitive abilities of animals

in this model would not only help to understand

better neurobiological foundations of autism but also

to develop potential therapy strategies. In particular,

the reward-based learning is of special interest, as it

allows assessing the ability to adaptive behavior, for-

mation of associative connections, and preservation

of learned skills in the absence of significant stress

load  [10].

In recent years, increasing data indicate the

role of neuroinflammation in the ASD pathogenesis.

In particular, in the cerebellum of the patients with

ASD, increased activation of microglia and astrocytes,

as well as pronounced accumulation of macrophages

and monocytes, have been detected  [1]. One of the

significant markers of inflammatory processes, in-

cluding in the nervous system, is neutrophil elastase

(NE) – a proteolytic enzyme involved in regulation

of the immune response. It has been shown that

high NE activity in ASD could lead to disruption of

the blood-brain barrier permeability, activation of

microglia, and dysfunction of synaptic connections,

which, in turn, affect cognitive functions and various

behavioral manifestations  [11]. Development of the

nervous system in ASD depends on the complex in-

terplay between the activity of the glutamate system,

intensity of OS, and mitochondrial energy processes,

mediated by signaling cascades associated with more

than 20 functional pathways and 22 genes associ-

ated with ASD  [12]. One of the central biochemical

pathways of convergence in various forms of ASD is

considered to be the mTOR pathway  [13]. Close rela-

tionships between inflammation and oxidative stress

processes are well known  [11].

Objective of this study is to analyze the effect

of postnatal administration of VPA on social behav-

ior and reward-based learning, as well as biochemi-

cal markers of inflammation, oxidative stress, energy,

and glutamate metabolism in the serum and brain

of rats.

MATERIALS AND METHODS

The study was conducted on 38 white Wistar

rat pups (3  litters). Rat offsprings were obtained by

mating males and females obtained from the Stolbo-

vaya nursery. Rats were kept in standard vivarium

conditions with free access to water and food. Using

artificial lighting, a 12-h light period (9:00-21:00) was

maintained in the room. Experiments on rats were

conducted from 10:00 to 18:00. Each litter was divid-

ed into 2 groups: control animals (n  =  18, including

10 females and 8 males) received intraperitoneal in-

jections of water, and experimental animals (n = 20,

including 10 females and 10 males) received VPA

(sodium valproate, Sigma) at a dose of 150  mg/kg

daily from the 6th to the 12th postnatal days (PND).

Dose and administration regimen of VPA were cho-

sen based on the literature data  [9]. Although the ex-

periment used animals of both sexes, sex difference

analysis was not performed due to the small sample

size.

The study was conducted in compliance with the

bioethical norms for the handling of experimental an-

imals in accordance with the “Rules of Good Labora-

tory Practice” (Order of the Ministry of Health of the

Russian Federation no.  107-d) and the requirements

of Directive 2010/63/EU of the European Parliament

dated September 22, 2010.

STAKHANOVA et al.958

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

Fig. 1. Experimental design.

Conditions for keeping animals and experimen-

tal procedures used in the study were approved by

the Bioethics Commission of Moscow State University

(Reg. no.  12.5-sod dated October 24, 2024).

Standard physiological and behavioral tests were

used in the work. The study design is presented in

Fig.  1.

Pain sensitivity testing. Pain sensitivity in rats

was determined using the “Hot Plate” test on PND  25.

Animals were placed on a surface heated to 55°C, and

time until licking of the hind paw was measured.

Testing was stopped if this time exceeded 30  s  [14].

Complex food maze test. To assess cognitive

abilities, the “Complex Food Maze” test was used,

which allows accounting for the speed of learning

and preservation of the acquired skill. Testing was

conducted from PND 40 to 45. Before the start of test-

ing, rats were fasted for 24  h. On the next day, the

animals were adapted to the experimental conditions

for 45  min (small pieces of bread were evenly scat-

tered throughout the maze). Then, for 4 days, the rats

were trained: each day, the rats had 5 attempts of

3  min each to perform the reaction. A rat was placed

in the section of the maze closest to the research-

er (point  A, Fig.  1), and the reaction was considered

completed if the rat took a piece of bread located at

the end of the maze (point  B, Fig.  1). During the ex-

periment, the rats were fed once a day after the ex-

periment. The retention test was conducted one week

after the last training session  [15].

The following parameters were recorded in the

experiment:

• Latency period (time to exit the starting corri-

dor), sec;

• Reaction time (RT), sec;

• Number of completed reactions (NCR);

• Number of errors – number of deviations from

the optimal trajectory, which includes the short-

est path from the starting section (point  A) to the

target section with food (point  B);

• Rearing;

• Grooming (number of times the animal touched

its muzzle with its front paws).

Social behavior test in the “Sibs/Non-Sibs”

modification (PND  55). Social behavior of rats was

studied on PND  55 in a T-shaped maze  [16], consist-

ing of squares. Each square measured 14×14  cm,

with walls 30  cm high. Six squares were located in

the main part, and two were in the starting section.

Before the start of testing, sibs and non-sibs were

placed in the side sections behind a grid as objects

of social interaction. The study was conducted un-

der red light. A test rat was placed in the starting

section of the maze and separated by a partition for

1  min – adaptation period. Within 5  min after re-

moving the partition, the following parameters were

recorded:

• Latency period of exiting the starting section;

• Locomotion – total number of squares the rat

passed through in 5  min;

• Rearing – vertical motor activity;

• Grooming – touching the muzzle with the front

paws;

• Latency period of approaching the side sections;

• Number of approaches, contacts (sniffing acts),

climbs onto the grid, as well as the time  (T) spent

by the test animal in the square near the objects

of social interaction, from which the novelty co-

efficient was calculated using the formula  (1):

Novelty coefficient =

TNon-Sibs

TSibs + TNon-Sibs

. (1)

Biological material collection. On PND  57, the

animals were decapitated, blood was collected in

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

tubes with a separating gel for serum (BD Vacutainer®

SST™II Advance), centrifuged at 2000g, and the serum

was frozen and stored at −78°C. Brain was extracted

immediately after decapitation and dissected on ice.

Cerebellum was weighed, frozen in liquid nitrogen,

and stored at −78°C.

To obtain cytosolic fraction of brain tissue (cer-

ebellum), the tissue was thawed and homogenized

(1  :  5) in a chilled 50  mM Tris-HCl buffer, pH  7.2,

with 0.32  M sucrose, and centrifuged first at 1000g

for 15  min at 4°C, next the supernatant was centri-

fuged at 60,000g for 60  min at 4°C using an Optima

L-90 ultracentrifuge (Beckman Coulter, USA). The re-

sulting supernatant was used for biochemical param-

eter analysis.

Protein concentration in cerebellum samples and

blood serum was determined using the Lowry meth-

od (Bio-Rad DC Protein Assay, USA).

Determination of enzyme activity. Neutrophil

elastase (NE) activity was determined based on the

rate of hydrolysis of a substrate (N-tert-butoxycarbon-

yl-alanine-β-nitrophenyl ester (N-t-BOC-Ala-p-NP, BOC))

(Sigma, USA)  [17]. The substrate was first dissolved

in acetonitrile and added to the incubation medium

at concentration of 0.2  mM. Incubation was carried

out at room temperature in 50  mM  PBS, pH  6.5. Con-

centration of the formed product (nitrophenol) was

measured spectrophotometrically at 347  nm. Reaction

rate was determined from the initial section of the

kinetic curve and expressed in conventional units

(U,  μM/min).

Activity of glutamate dehydrogenase (GDH), glu-

tamine synthetase (GS), creatine phosphokinase (CPK),

glutathione reductase (GR), and glutathione S-trans-

ferase (GST) was determined using spectrophotomet-

ric kinetic methods with a FlexA-200HT plate spectro-

photometer (Allsheng, China).

GDH activity was determined by measuring the

rate of NADH absorption decrease (PanReac Appli-

Chem, USA) at 340  nm using the Fisher’s method with

modifications, as described previously [18].

Enzymatic activity of GS was determined based

on the rate of formation of γ-glutamyl hydroxamate

using hydroxylamine as a substrate according to the

Iqbal method with minor modifications  [19].

CPK activity was determined using a “Creatine

Kinase-Novo” kit (Vector-Best, Russia).

Activity of glutathione metabolism enzymes was

determined based on the methodological recom-

mendations of A.  I.  Karpishchenko  (2013)  [20]. GST

activity was determined based on the rate of for-

mation of chromogenic glutathione conjugates with

1-chloro-2,4-dinitrobenzene (increase in absorption

recorded at 340  nm). Glutathione peroxidase (GPx) ac-

tivity was measured by a colorimetric method based

on the decrease in reduced glutathione concentration

with Ellman’s reagent monitored at 412  nm. GR ac-

tivity was determined based on the rate of NADPH

oxidation (decrease in absorption of the reaction me-

dium recorded at 340  nm) in the reaction of reducing

oxidized glutathione. After determining protein con-

centration in the samples, specific activity was calcu-

lated for all enzymes.

Concentration of superoxide dismutase-1 (SOD-1)

was determined using a “sandwich” version of the sol-

id-phase enzyme-linked immunosorbent assay (ELISA).

An “ELISA-SOD” kit (Cytokine, Russia) was used.

Statistical analysis. Statistical processing of the

test results was carried out using standard analysis

methods with the “Statistica  10.0” and “GraphPad

Prism  8.0” software packages. For behavioral test-

ing and biochemical studies, an individual animal

was used as the unit of analysis [n  (control)  =  18;

n  (VPA)  =  20]. At the initial stage of analysis, the data

were tested for normality of distribution using the

Shapiro–Wilk test. In the case of normal distribution,

data were compared using the parametric criterion

(Student’s t-test), and in the case of a distribution

different from normal, comparative analysis was

performed using the non-parametric Mann–Whitney

criterion. To detect relationships between biochemi-

cal parameters and behavioral indicators, correlation

analysis was performed using the Spearman rank

correlation coefficient (RS). On the graphs, the results

are presented as a median, confidence interval, min-

imum, and maximum. The significance level was set

at p  <  0.05.

RESULTS AND DISCUSSION

Effect of postnatal administration of valproic

acid (VPA) on physiological development of ani-

mals. The study of physiological development of the

animals was conducted to confirm effectiveness of the

ASD modeling using VPA in this study. According to

the literature, slow weight gain in the early postna-

tal period in this model is considered a physiological

disorder corresponding to those in ASD  [21]. In our

study, monitoring the weight of rat pups from PND  6

to  21 showed that on PND  12 (median 17.4 (15.2-20.4);

median 16.1 (13.6-18.3) for the control and experi-

mental groups, respectively), PND  14 (18.6 (16.2-22.8);

16.7 (14.5-19.2)), PND  15 (20.4 (18.5-25.4); 18.1 (16.0-

21.4)), and PND  16 (22.2 (20.0-28.0); 20.0 (18.0-23.8)),

the weight of animals receiving VPA was lower than

in the control (p  <  0.05). As a result, in the experi-

mental group, there was a reduction in weight gain

both from PND  6-12 (p  =  0.0042) and from PND  14-21

(p  =  0.0335) (Fig.  2a).

According to the literature, children with ASD

may exhibit a tendency toward excessive body

STAKHANOVA et al.960

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

Fig.  2. Effect of early postnatal administration of VPA on physiological development and behavior of rats. a)  Weight gain;

b)  latency period of licking the hind paw in the “Hot Plate” test; c)  social novelty coefficient in the “Social Behavior” test

in the sibs/non-sibs modification. *  Significant differences from control, p  <  0.05; **  Significant differences from control,

p  <  0.01; n  (control)  =  18; n  (VPA)  =  20 (Mann–Whitney U  test).

weight (33.9%) and obesity (18.2%), which is associ-

ated with selective appetite and low energy expendi-

ture. However, in some patients (5-9%), weight loss

has been also detected, associated with reduction in

the diversity of the gut microbiome and metabolic

disorders.

Reduced pain sensitivity is also characteristic of

ASD  [22]. In this study, it was shown that on PND  25,

in the rats that received VPA, the latency period of

paw licking in the “Hot Plate” test was increased

compared to the control (p  =  0.038) (Fig.  2b). The “Hot

Plate” test assesses changes in nociception at the cen-

tral level, so reduction in pain sensitivity under the

influence of VPA may be a result of its effect on the

central nervous system (CNS)  [14].

Thus, physiological disorders caused by the post-

natal administration of VPA confirm validity of the

experimental model used.

Effect of postnatal administration of VPA on

social behavior of animals. One of the main signs of

ASD is impairment of social interaction. In the “Social

Behavior” test, the animals that received VPA, had a

significantly increased latency period of exiting the

starting section (p  =  0.0016), as well as increased time

of approaching both sibs (p  =  0.029) and non-sibs

(p  =  0.00006) compared to the control individuals.

Disruption of social behavior in the animals that

received VPAwas also evidenced by the relative de-

crease in the time spent with non-sibs, as a result of

which the social novelty coefficient in this group was

lower than in the control (p  =  0.03) (Fig.  2c).

Thus, based on the analysis of the results of so-

cial behavior testing, it was shown that early postna-

tal administration of VPA causes behavioral disorders

characteristic of ASD.

Effect of postnatal administration of VPA on

learning in animals. In children with ASD, reduced

spatial working memory and complex visual mem-

ory are detected, which could cause difficulties in

processing socially significant information and orien-

tation in the stimulus-rich situations. It is assumed

that absence of the automatic “cross-talk” between

reasoning and memory systems makes it difficult to

identify significant elements of experience and their

thematic organization, which makes formation of

the integrated episodic representations less effective.

Therefore, there is a reason to assume close relation-

ship between the social dysfunction and cognitive im-

pairments in ASD. In particular, in the experimental

models of ASD in rats, specific impairments in epi-

sodic and social memory associated with changes in

hippocampal function are described [1].

Learning of rats in the food maze is a re-

ward-based learning, based on natural food search

strategies and high spatial memory capacity. Positive

reinforcement allows minimizing stress compared to

the tasks with negative reinforcement (e.g., Morris

water maze) [23,  24], which reduces contribution of

anxiety and makes the measurement of spatial learn-

ing and motivation to explore more “pure”. Positive,

predictable reinforcement also shifts the balance of

behavior toward approach and exploration, while un-

predictable or aversive events enhance defensive re-

sponses and avoidance of new environments. Hence,

rewarded navigation in the complex maze functions

as a repeated experience of controlled success, form-

ing stable expectations of achieving reward and re-

ducing uncertainty in the animal.

In our “Complex Food Maze” test, the animals

that received VPA spent more time completing the

task than the control individuals, both on the day  3 of

training (median 9.0 (7.4-12.4); median 14.4 (10.2-32.4)

for the control and experimental groups, respectively,

p  =  0.02) and during the retention test (median 9.9

(6.4-13.48); median 15.6 (10.9-42.5), p  =  0.011). Differ-

ences between the experimental and control animals

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

Fig. 3. Changes in biochemical parameters in the postnatal valproate model of autism spectrum disorders. a) Neutrophil

elastase activity in blood serum; b) glutathione S-transferase activity in blood serum; c) neutrophil elastase activity in the

cerebellum; d) glutathione S-transferase activity in the cerebellum. * Significant differences from control, p < 0.05; ** Sig-

nificant differences from control, p < 0.01; n (control) = 18; n (VPA) = 20 (Mann–Whitney U test).

in the number of completed reactions were observed

on the day 3 of training (median 7.0 (3.0-10.0); medi-

an 9.5 (7.0-18.0), p  =  0.046) and during the retention

test (median 4.0 (1.0-11.5); median 10.6 (10.0-17.0),

p  =  0.038). In addition, on the day  1 of training, the

experimental animals made fewer rears than the con-

trol animals (p  =  0.008).

Thus, physiological and behavioral deviations

characteristic of ASD, caused by early postnatal ad-

ministration of VPA, are accompanied by cognitive

impairments.

Effect of postnatal administration of VPA on

activity of inflammation factors, antioxidant de-

fense, energy, and glutamate metabolism in the

serum and cerebellum of rats. Biochemical param-

eter analysis showed that, compared to the control

group, in the serum and cerebellum of experimental

animals, there was a significant increase in the en-

zymatic activity of NE and GST (Fig.  3). The values of

other studied parameters (activity of GDH, CPK, GR)

in the experimental group did not differ from the

values in the control group.

Neutrophil activation is characteristic of inflam-

matory processes, during inflammation, elastase from

the azurophilic granules of neutrophils is secreted

into the extracellular space. NE activity is considered

one of the markers of inflammatory process in the

body. In this study, NE activity in the blood serum of

animals receiving VPA was higher than in the control

individuals (Fig.  3a).

The obtained results indicate development of in-

flammation in response to postnatal administration of

VPA to the rats and correspond to the data of clinical

studies, where it was shown that NE activity in the

plasma and serum of the patients closely correlates

with the severity and type of ASD  [25]. A similar cor-

relation could indicate a link between the NE activity

in the blood and severity of the pathological process

in the brain. Indirect confirmation of this hypoth-

esis may be the fact that in the cerebellum of ani-

mals receiving VPA, NE activity was higher than in

the control (Fig.  3c). It is assumed that physiological

and behavioral disorders induced by VPA adminis-

tration in the early postnatal period of development,

characteristic of ASD, could be associated with ac-

tivation of the inflammatory reactions both in the

brain and blood of the experimental rats. Increase

in NE activity could serve as an indicator of these

processes. Apparently, VPA administration leads to

the damage in the immature brain, where noticeable

vulnerability is observed, as well as to infiltration

of neutrophils into the brain and their activation,

STAKHANOVA et al.962

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

leading to the development of neuroinflammation

and behavioral disorders, as shown in the experimen-

tal brain ischemia  [11].

At the same time, no direct correlation was found

between the NE activity in the cerebellum and in the

blood of rats in any of the studied groups. Absence of

such relationships in general could be explained by

the relative independence of the development of in-

flammatory processes in the brain and in the periph-

ery  [26]. In addition, it is important to consider that

in this work, we determined activity of NE present

in the serum as a result of neutrophil degranulation,

while activity of the free and intracellular NE in the

cerebellum was determined. Long-term consequence

of the effect of VPA on antioxidant defense in the

early postnatal period could be the increased activ-

ity of GST in the serum and cerebellum of the rats

(Fig.  3, b  and  d), which may be associated with the

changes at the level of expression and is consistent

with the results reported by some researchers  [27].

For each studied parameter, absence of the sig-

nificant correlational relationships between the level

of its activity/content in the cerebellum and serum

was established.

Interrelationship of biochemical and behavior-

al parameters in the experimental model of ASD.

When assessing inflammation by the activity of se-

rum markers, it was found that the more active is the

inflammatory process, the lower is the pain sensitiv-

ity. In contrast, the latency period of paw licking on

the “Hot Plate” correlates negatively with NE activity

in the cerebellum (RS  =  −0.8, p  =  0.00006). After VPA

administration, this correlation changes to the oppo-

site (RS  =  +0.6, p  =  0.006), which once again indicates

disruption of the central mechanism of pain sensi-

tivity regulation in ASD. The observed reduction in

the pain sensitivity in the “Hot Plate” test could be

a consequence of activation of the opioid system due

to inflammation. It has been shown that the periph-

eral inflammation and neuroinflammation are accom-

panied by the increased levels of IL-1β, TNF-α, and

IL-6, which activate the HPA axis and enhance β-en-

dorphin secretion by the pituitary gland, causing a

systemic effect, including reduction in pain sensitivity

[28]. Opioid system also regulates peripheral nocicep-

tion, which leads to the reduction in response to the

painful stimuli  [29].

Different directions are observed in the correla-

tions between the latency period of paw licking in

the “Hot Plate” test and CPK activity in the serum and

cerebellum of the intact animals (RS  =  −0.7, p  =  0.0007

and RS  =  +0.6, p  =  0.016, respectively). As a result of

VPA administration, the correlation with serum CPK

activity changes to the opposite (RS  =  +0.5, p  =  0.018),

i.e., the higher is the CPK activity, the lower is the

pain sensitivity.

Connection of the latency period of paw licking

in the “Hot Plate” test with glutamate metabolism is

observed only in the control group. Correlations were

found with the GDH activity in the serum (RS  =  −0.6,

p  =  0.030) and with the GS activity in the cerebellum

(RS  =  +0.6, p  =  0.017).

As for the antioxidant defense parameters, pos-

itive correlation was shown between the latency pe-

riod of paw licking in the “Hot Plate” test and GR

activity in the cerebellum (RS  =  +0.5, p  =  0.017) in the

control group, and with the SOD content in the serum

(RS  =  +0.4, p  =  0.049) in the experimental animals.

Involvement of inflammation in the regulation of

social behavior in animals is evidenced by multiple

correlational relationships (Table  1). In particular, in

normal conditions (control group), most of the pa-

rameters of the “Social Behavior” test positively cor-

relate with NE activity in the blood serum of rats.

Negative role of neutrophils penetrating the brain in

the formation of behavioral reactions may be indi-

rectly indicated by the negative correlation between

the social activity parameters of the control rats and

NE activity in the cerebellum (Table  1). The VPA ex-

posure disrupts this relationship.

Positive correlation between the social behav-

ior parameters and CPK activity in the cerebellum

demonstrates that the higher is the enzymatic activity

of CPK (i.e., the more effective the energy metabo-

lism), the greater is social activity the animal exhib-

its. In the ASD model group, these relationships are

absent.

Positive correlation between the GS activity in

the cerebellum and the number of contacts and ap-

proaches to sibs (RS  =  +0.5, p  =  0.04) and grooming

near non-sibs (RS  =  +0.5, p  =  0.04) is observed in both

the control group and the ASD model.

To study biological mechanism of the develop-

ment of cognitive impairments caused by the early

postnatal administration of VPA, a correlation analy-

sis was performed (Table  2). The pathogenetic role

of neuroinflammation, energy, and glutamate metab-

olism in this experimental model of ASD has been

confirmed once again. The higher is the NE activity

in the cerebellum of rats that received VPA, the slow-

er is their learning and the weaker is preservation

of the acquired skill. Similar correlations were not

found in the control group. A similar pattern is ob-

served for the CPK activity (Table  2).

Enzymes involved in glutamate (GDH and GS)

and glutathione (GPx) metabolism are inversely re-

lated to the learning parameters, mainly in the exper-

imental animals. Positive correlational relationships

between the activity of antioxidant defense enzymes

(GR) and the parameter reflecting preservation of the

acquired skill are observed only in the control group

of animals.

BIOCHEMICAL MARKERS AND BEHAVIOR OF RATS IN THE ASD MODEL 963

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

Table 1. Relationship of social behavior parameters with the activity of biochemical markers in the serum

and cerebellum of control rats

Parameter Serum Cerebellum

NE CPK GDH NE CPK GS

Locomotion +0.6, 0.02 −0.56, 0.03 −0.6, 0.017 −0.7, 0.003

Climbing onto the grid

near non-sib

+0.55, 0.02 −0.5, 0.04

Grooming near non-sib +0.75, 0.002 +0.67, 0.007 +0.5, 0.04

Time in the center −0.5, 0.04

Time near non-sib +0.5, 0.04

Time near sib +0.5, 0.04

Contacts with sib +0.7, 0.002 −0.6, 0.02

Contacts with non-sib +0.6, 0.01 −0.5, 0.04 −0.55, 0.03

Total number of contacts +0.7, 0.003 −0.6, 0.02

Contacts + approaches

to non-sib

+0.65, 0.011 −0.55, 0.04 −0.57, 0.04

Contacts + approaches to sib +0.7, 0.002 −0.6, 0.02 +0.5, 0.04 +0.5, 0.04

Contacts + approaches +

climbing to non-sib

+0.7, 0.005 −0.6, 0.03

Contacts + approaches +

climbing to sib

+0.7, 0.002 −0.6, 0.02

Total number of climbs

onto the grid

+0.6, 0.02 −0.65, 0.006

Note. Data are presented as Spearman correlation coefficient (RS) and significance level (p).

CONCLUSION

Early postnatal administration of VPA to Wistar

rats causes physiological (slow weight gain and re-

duced pain sensitivity) and behavioral (social behav-

ior disorders) changes characteristic of ASD, and is

accompanied by the cognitive impairments. For the

first time, it was shown that these changes are as-

sociated with the increase in activity of the inflam-

mation markers (NE) in the serum and cerebellum

of the rats exposed to VPA, which presumably could

indicate infiltration of the brain with neutrophils,

i.e., development of neuroinflammation. The ob-

tained data are consistent with the results of clinical

and biological studies  [25] and confirm the hypoth-

esis about the involvement of central inflammatory

mechanisms in the development of autistic disor-

ders and negative impact of neuroinflammation on

learning and preservation of acquired skills, which

could be manifested by the increase in NE activity

in the blood. For the first time, significant changes

in the activity of the antioxidant defense and xeno-

biotic metabolism enzyme GST were detected in the

serum and cerebellum of the rats. Absence of the

changes in the activity/content of other biochemical

parameters may be associated with activation of the

compensatory mechanisms aimed at restoring dam-

age  [10] caused by the postnatal administration of

VPA. The identified correlations indirectly confirm

the link between inflammation (NE) and social be-

havior disorders in animals. The enzymes of gluta-

mate metabolism (GDH and GS) and antioxidant en-

zymes that regulate intensity of oxidative stress play

an important role in the formation and preservation

of acquired skills in both control and experimental

animals.

STAKHANOVA et al.964

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

Table  2. Correlational relationships of rat learning parameters in the “Complex Food Maze” test with activity of biochemical markers in the serum

and cerebellum of rats

Parameter NE

(Cerebellum)

CPK (Serum) CPK

(Cerebellum)

GDH

(Serum)

GDH

(Cerebellum)

SOD

(Cerebellum)

GPx

(Cerebellum)

(Control)

+0.8, 0.0005

(VPA)

+0.6, 0.03

(Control)

+0.6, 0.02 −0.6, 0.03

(VPA)

−0.6, 0.02

(VPA)

−0.7, 0.0016 −0.6, 0.006

T11

(Control)

−0.6, 0.009 −0.6, 0.011

T11

(VPA)

+0.5, 0.04 −0.5, 0.03

NCR 1

(Control)

−0.5, 0.04

NCR 1

(VPA)

+0.7, 0.002

NCR 2

(Control)

+0.5, 0.04

NCR 3

(Control)

+0.5, 0.04

NCR 11

(Control)

+0.5, 0.03

NCR 11

(VPA)

−0.6, 0.03 −0.6, 0.03

Note. (Control) – control group; (VPA) – experimental group receiving VPA; (Cerebellum) – cerebellum; (Serum) – serum; NCR – number of completed reactions from

days 1 to 4 of training and on day 11 (retention test); T – time to complete the reaction from days 1 to 4 of training and on day 11 (retention test).

BIOCHEMICAL MARKERS AND BEHAVIOR OF RATS IN THE ASD MODEL 965

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 6 2026

Abbreviations

ASD autism spectrum disorder

CPK creatine phosphokinase

GDH glutamate dehydrogenase

GPx glutathione peroxidase

GR glutathione reductase

GS glutamine synthetase

GST glutathione s-transferase

NE neutrophil elastase

OS oxidative stress

PND postnatal day

SOD superoxide dismutase

VPA valproic acid

Contributions

A.  A.  Stakhanova: Data acquisition, data processing,

discussion of the research results; O.  G.  Voskresenska-

ya: Conducting experiments, literature analysis;

N.  V.  Kost: Formulation of the idea, concept and super-

vision of the work, writing the manuscript; O.  K.  Sa-

vushkina: Data acquisition and statistical processing,

manuscript editing; T.  A.  Prokhorova: Conducting

experiments, literature analysis, writing the text;

E.  B.  Tereshkina: Conducting experiments, literature

analysis, manuscript editing; S.  A.  Zozulya: Study de-

sign development, conducting the experiment, manu-

script editing; E.  V.  Semina: General scientific supervi-

sion, discussion of the research results; Yu.  A.  Chaika:

General supervision, ensuring financial support.

Funding

The work was financially supported by the state as-

signment of the Federal State Budgetary Scientific

Institution, Mental Health Research Center (theme

FURU-2024-0016 (no.  124020700032-9) “Biological

markers of mental and neurodegenerative diseases:

fundamental and applied aspects”) and the state as-

signment of the Department of Human and Animal

Physiology, Faculty of Biology, Lomonosov Moscow

State University: 28-3-21 (CITIS number: 121032300071-8

“Mechanisms of physiological adaptations”).

Ethics approval and consent to participate

The study was conducted in compliance with the bio-

ethical norms for handling of experimental animals

in accordance with the “Rules of Good Laboratory

Practice” (Order of the Ministry of Health of the Rus-

sian Federation no.  107-d) and the requirements of Di-

rective 2010/63/EU of the European Parliament dated

September  22, 2010.

The conditions for keeping animals and the experi-

mental procedures used were approved by the Bio-

ethics Commission of Moscow State University (reg.

no.  12.5-sod dated October 24, 2024).

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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