Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 7, pp. 1187-1199 © The Author(s), 2026. This article is an open access publication.

1187

The Levels of Neurotrophic Factors in the Rat

Hippocampus in the Acute Period after Middle

Cerebral Artery Occlusion Are Associated with the

Hypothalamic–Pituitary–Adrenal Axis Activation

Mikhail V. Onufriev

1,a

*, Yulia V. Moiseeva

, Margarita R. Novikova

Pavel A. Kostryukov

, and Natalia V. Gulyaeva

Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences,

117485 Moscow, Russia

e-mail: mikeonuf1@mail.ru

Received November 29, 2025

Revised June 2, 2026

Accepted June 3, 2026

Abstract— Ischemic stroke is one of the most prevalent neurological disorders, with primary injury typi-

cally localized to the neocortex. However, secondary damage often extends to anatomically distant regions,

particularly the hippocampus. One of the early neuroendocrine responses to cerebral ischemia is activation

of the hypothalamic–pituitary–adrenal (HPA) axis. Increased release of corticosterone (CORT) by the adrenal

glands into the bloodstream leads to its accumulation in the hippocampus. We hypothesize that this process

may contribute to the hippocampal damage and underlie delayed cognitive and affective impairments fol-

lowing stroke. To test this, we investigated the levels of neurotrophic factors reflecting the neuroprotective

potential of the hippocampus using two established middle cerebral artery occlusion (MCAO) models that

differ in their effects on CORT production: the Koizumi et al. (1986) model (MCAO1) and the Longa et al.

(1989) model (MCAO2). Although both models produced comparable neurological deficits, mortality rates,

and infarct volumes, they differed markedly in endocrine and hippocampal responses. Significant increases

in circulating CORT and its accumulation in both ipsilateral and contralateral hippocampus were observed

only in MCAO1. Moreover, correlations between the CORT levels (in the blood and hippocampus) and both

rat body weight and neurological deficit were stronger in MCAO1 than in MCAO2. In both acute models,

brain-derived neurotrophic factor (BDNF) levels were elevated in the ipsilateral hippocampus, whereas the

content of glial cell line-derived neurotrophic factor (GDNF) remained unchanged. In contrast, ciliary neu-

rotrophic factor (CNTF) increased in the ipsilateral hippocampus only in MCAO2. These findings suggest

that differential activation of the HPA axis and consequent variations in corticosteroid signaling in the

hippocampus selectively modulate the neurotrophic response during the acute phase following MCAO.

DOI: 10.1134/S0006297925604162

Keywords: ischemic stroke, middle cerebral artery occlusion, hypothalamic–pituitary–adrenal axis, corticoste-

rone, neurotrophic factors, BDNF, CNTF, GDNF

* To whom correspondence should be addressed.

INTRODUCTION

Stroke is a severe neurological disorder that

leads to persistent neurological deficits, as well

as delayed affective and cognitive impairments.

In humans, the majority of ischemic strokes occur

within the middle cerebral artery (MCA) territory/

basin and are commonly associated with its occlu-

sion (MCAO). The post-stroke damage is not confined

to the primary infarct area but extends to remote,

non-ischemic brain regions, resulting in secondary

neurodegeneration. For instance, focal injury to the

cerebral cortex and/or striatum has been shown

to induce secondary pathological changes in the

ONUFRIEV et al.1188

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026

thalamus, substantia nigra, hippocampus, and spinal

cord [1-3].

In rodent models, the primary infarct zone fol-

lowing moderate MCAO typically involves the neocor-

tex and striatum. However, accumulating evidence in-

dicates that delayed neuronal loss also occurs in the

ipsilateral hippocampus [4]. Moreover, MCAO in rats

has been shown to trigger progressive delayed mor-

phological alterations and pyramidal neuron death in

the ipsilateral hippocampus, accompanied by impair-

ments in the hippocampus-dependent spatial memo-

ry [5]. The hippocampus is a critical brain structure

involved in controlling cognitive functions and emo-

tional responses. Notably, it is highly susceptible to

stress factors, including ischemic injury, partly due to

the high density of corticosteroid receptors compared

with other brain regions. These receptors bind stress

hormones such as corticosterone (CORT) or cortisol,

thereby mediating stress-related signaling [6].

One of the earliest physiological responses to focal

ischemia is activation of the hypothalamic–pituitary–

adrenal (HPA) axis, which occurs within hours after

ischemia onset and results in a sustained elevation

of circulating glucocorticoids [7-9]. Both abnormally

high and abnormally low cortisol levels have been

associated with increased 28-day mortality following

stroke [10]. Dysregulation of the HPA axis has been

proposed to be caused, in part, by an imbalance in

central corticosteroid receptor signaling, which may

also contribute to predisposition to stress-induced

psychiatric disorders, including depression [11]. The

HPA axis is regulated, in particular, by the hippocam-

pus  [12], largely through corticosteroid receptors that

are highly expressed in the CA1, CA2, and CA3 sub-

fields and the dentate gyrus [13].

A growing body of evidence indicates that neu-

rotrophic factors are critical for the structural and

functional recovery of nervous tissue after ischemic

injury [14, 15]. It is well known that mature forms of

neurotrophins, including brain-derived neurotrophic

factor (BDNF) and nerve growth factor (NGF), ex-

ert their neuroprotective effects through binding to

high-affinity tropomyosin receptor kinase (Trk) recep-

tors (TrkB and TrkA, respectively) and promote neu-

ronal survival via regulation of cell differentiation

and synaptogenesis [16, 17]. In contrast, pro-neurotro-

phins, including proBDNF and proNGF, preferentially

bind to the low-affinity p75 neurotrophin receptor

(p75NTR) and are implicated in the regulation of neu-

rite outgrowth and apoptotic signaling during central

nervous system (CNS) development. Under pathologi-

cal conditions, they may contribute to nervous tissue

injury  [18]. Stress-induced activation of the HPA axis

and the associated increase in glucocorticoids can

reduce neurotrophin expression in the hippocampus

[19, 20], with effect depending on the intensity and

duration of stress exposure  [21]. However, a single

administration of the synthetic glucocorticoid dexa-

methasone failed to alter BDNF, NGF, or neurotroph-

in-3 (NT-3) levels in the cortex and hippocampus, or

in the corresponding cell cultures, while still activat-

ing Trk receptors via increased phosphorylation [22].

Compared with BDNF and NGF, other neurotrophic

factors such as ciliary neurotrophic factor (CNTF) and

glial cell line-derived neurotrophic factor (GDNF) re-

main less extensively studied in models of ischemic

stroke, particularly with respect to remote hippocam-

pal alterations and glucocorticoid-related effects.

Given these considerations, the aim of the pres-

ent study was to investigate a potential association

between the HPA axis activation, as reflected by

circulating CORT levels, and concentrations of the

neurotrophic factors BDNF, GDNF, and CNTF in the

hippocampus, as a brain region remote from the pri-

mary ischemic lesion during the acute phase follow-

ing MCAO in rats.

MATERIALS AND METHODS

Animals and surgeries. All procedures involv-

ing animals were conducted in accordance with the

ethical standards of the Institute of Higher Nervous

Activity and Neurophysiology of the Russian Academy

of Sciences and approved legal regulations of the Rus-

sian Federation and international organizations. The

experimental protocol was approved by the Ethical

Committee of the Institute of Higher Nervous Activity

and Neurophysiology (Protocol no.  10; December 10,

2012). Male Wistar rats aged 3 months were obtained

from the Stolbovaya Animal Facility of the Scientific

Center for Biomedical Technologies of the Federal

Medical and Biological Agency (Moscow Region, Rus-

sia) and housed in standard vivarium cages. Animals

were kept under a 12-hour light/dark cycle with

ad  libitum access to food and water.

Models of ischemic stroke. MCAO1. The surgery

was performed according to Koizumi et al. [23]. Wis-

tar rats weighing 200-300  g were anesthetized with

isoflurane. An incision was made in the neck area,

and the muscle tissue was retracted on the left side

to expose the common carotid artery. Ligatures were

placed around the common, external, and internal ca-

rotid arteries. A nylon filament (3-0) with a rounded

tip was inserted through the opening at the bifurca-

tion into the external and internal carotids arteries

and advanced through the internal carotid artery to

the origin of the middle cerebral artery. The ligature

on the internal carotid artery was then tightened to

secure the filament in place. Occlusion was main-

tained for 60 min with the common, external, and

internal carotid arteries ligated, while the animal’s

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026

Fig. 1. Experimental design.

body temperature was kept at 37  ±  0.5°C. After the

occlusion period, the filament was removed, and the

ligature on the internal carotid artery was tightened

(MCAO1 group). In the sham-operated group (Sham1),

all manipulations were performed identically except

for the filament insertion.

MCAO2. In the second model, ischemic stroke was

induced using the method proposed by Longa et al.

[24]. Wistar rats weighing 200-300  g were anesthe-

tized using with isoflurane. The common and external

carotid arteries were exposed through a neck incision

and ligated. After electrocoagulation and dissection

of a segment of the left external carotid artery near

the bifurcation, a filament was inserted through the

remaining part and advanced through the internal

carotid artery to the intersection with the middle ce-

rebral artery. The occlusion duration was 60  min; the

rat’s body temperature was maintained at 37  ±  0.5°C.

Following the ischemic period, the filament was re-

moved, and the blood flow was restored through the

ipsilateral common carotid artery after release of the

ligature (MCAO2 group). In the sham-operated group

(Sham2), all manipulations were performed identical-

ly except for the filament insertion.

Thus, in the MCAO1 model, left common, exter-

nal, and internal carotid arteries remained ligated af-

ter the procedure, and reperfusion occurred mainly

through the circle of Willis. In contrast, in the MCAO2

model, only the external carotid artery remained li-

gated, while the blood flow through the internal and

common carotid arteries was restored. The duration

of anesthesia was comparable between the two mod-

els, with animals maintained under isoflurane for ap-

proximately 15-20  min during the surgical procedure.

Experimental design and number of animals

in groups. To compare the two models of ischemic

stroke, two independent experiments were conduct-

ed. In the first experiment, group sizes were as fol-

lows: intact (n  =  8), Sham1 (n  =  10), MCAO1 (n = 9),

Sham2 (n  =  9), MCAO2 (n  =  11). In the second ex-

periment, group sizes were: intact (n  =  11), Sham1

(n  =  10), MCAO1 (n  =  12), Sham2 (n  =  10), MCAO2

(n  =  11). In both experiments, neurological deficit,

body weight, mortality, and CORT levels in the blood

and hippocampus were assessed within 3 days after

occlusion (Fig.  1). In the first experiment, BDNF pro-

tein expression was evaluated in the hippocampus,

while in the second experiment, expression of GDNF

and CNTF was assessed. Because no statistically sig-

nificant differences were observed between the cor-

responding groups across the two experiments, the

data for neurological deficit, mortality, body weight,

and CORT levels in the blood and hippocampus were

pooled for analysis.

Neurological deficit assessment. In rats sub-

jected to MCAO1 and MCAO2, neurological deficit

was assessed on days 0, 1, and 3 post-MCAO using

the 5-point scale and tongue protrusion tests. In the

5-point scale test, neurological functions were evalu-

ated using a 5-point behavioral scoring system  [25]

that assesses the functional status of the contralateral

forelimb, the presence of turning or circling behavior

toward the contralateral side, and the overall loco-

motor activity. The tongue protrusion test evaluated

the oromotor function based on the rat’s ability to

lick peanut butter from a glass cylinder. Performance

was quantified by recording the distance from the

cylinder’s opening to the remaining level of peanut

butter  [26].

Rats in the MCAO groups without signs of neu-

rological deficit on the day of surgery, with mini-

mal deficit, e.g., paresis of the contralateral forelimb

(score 1-2 on the 5-point scale), or with maximal

deficit (score 5, immobility) were excluded from the

study.

Infarct volume assessment. Some animals

were euthanized on day 3 after MCAO. Their brains

were extracted, and coronal sections (2.0  mm thick)

were prepared. The sections were incubated in 2%

2,3,5-triphenyltetrazolium chloride (TTC, Sigma, USA)

in 0.01  M phosphate-buffered saline for 20  min at

37°C followed by fixation in 4% phosphate-buffered

paraformaldehyde solution overnight. Both surfaces

of each section were photographed using a Camedia

C-5060 digital camera (Olympus, USA), and imag-

es were analyzed with the ImagePro-Plus software.

Infarct areas were quantified, and infarct volumes

was calculated as described by Lin et al. [27].

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Preparation of biological material. Blood was

collected from the tail vein before occlusion and on

days 1 and 3 after MCAO, after which the rats were

euthanized. After decapitation, additional blood was

collected and brain tissue was harvested from both

the ipsilateral and contralateral hippocampus rel-

ative to the occlusion site. The collected blood was

centrifuged at 1500g at 4°C for 15  min to obtain se-

rum, which was stored at −80°C until biochemical

analysis.

Isolated hippocampi were homogenized in a

Potter homogenizer in phosphate-buffered saline

(PBS) containing 0.1% Nonidet P-40 and a protease

inhibitor cocktail (Thermo Scientific, USA). The re-

sulting homogenates were centrifuged at 13,000g at

4°C for 30  min, and the supernatants (soluble protein

fractions) were aliquoted and stored at −80°C until

biochemical analysis.

Biochemical assays. CORT assay. CORT levels

in the blood serum and hippocampal supernatants

were determined by enzyme-linked immunosor-

bent assay (ELISA) using DRG Corticosterone ELISA

kits (DRG International, Inc., USA). These kits detect

both free and protein-bound CORT using competitive

ELISA.

Neurotrophic factor assays. The contents of BDNF,

GDNF, and CNTF in the hippocampal supernatants

were assessed using corresponding ELISA kits (Rat

BDNF DuoSet ELISA, Rat GDNF DuoSet ELISA, and Rat

CNTF DuoSet ELISA; R&D Systems, USA) according to

the manufacturer’s instructions. These assays detect

total levels of neurotrophic factors, including mature

proteins and their pro-forms.

Statistical analysis. Outliers in the studied

groups were identified using the Tukey’s method.

The resulting data were assessed for normality of

distribution using the Shapiro–Wilk W test, which in-

dicated no violation of the assumption of normality

across all analyzed parameters.

Statistical analyses were performed using analy-

sis of variance (ANOVA), including repeated-measures

ANOVA for parameters evaluated over time, factorial

ANOVA to examine the effects of the factors “time,”

“model,” and “surgery” and their interactions, and

one-way ANOVA to compare experimental groups

with the intact (control) group, followed by Tukey’s

HSD (honestly significant difference) post  hoc test for

multiple comparisons

Mortality in MCAO groups was analyzed using

the 2×2 contingency test. Associations between the

neurological deficit scores and biochemical parame-

ters were evaluated using the Spearman’s rank cor-

relation coefficient. Statistical significance was set at

p  <  0.05, while p  <  0.1 was considered indicative of

a trend toward significance. Data are presented as

mean ± standard error of mean (SEM).

RESULTS

Neurological deficit, infarct volume, and mor-

tality in two MCAO models. Assessment of neu-

rological deficit in rats using the 5-point scale was

performed on the day of surgery and on days 1 and

3 after MCAO (Fig.  2a). The neurological deficit in

rats was maximal on the day of surgery, reaching

3.52  ±  0.11 points for MCAO1 and 3.64  ±  0.10 points

for MCAO2. Subsequently, the deficit decreased,

and on day 3 after occlusion, it was 2.90  ±  0.14 and

2.77  ±  0.20 points for MCAO1 and MCAO2, respective-

ly. The “model” factor had no significant effect on

the neurological score (F

1.41

  =  0.85; p  =  0.36), and no

differences in this parameter were observed between

MCAO1 and MCAO2 at any time point. In contrast,

the “time” factor had a significant effect (F

2.82

  =  20.78;

p  <  0.001), whereas the interaction between “model”

and “time” was not significant (F

2.82

  =  1.36; p  =  0.26).

In the tongue protrusion test, the neurological

deficit did not differ significantly between the two

occlusion models (Fig.  2b). For MCAO1, tongue pro-

trusion decreased from baseline by 21.20  ±  5.90% and

23.27  ±  5.64% on days 1 and 3 after occlusion, respec-

tively, whereas in the MCAO2 group, the reduction

was 9.19  ±  3.05% and 15.50  ±  4.65%, respectively. The

“surgery” factor had a significant effect on this param-

eter (F

1.76

  =  404.08; p  <  0.001), with a trend toward a

significant interaction between the “model” and “sur-

gery” factors significance (F

1.76

  =  3.88; p  =  0.052).

Body weight on day 3 after MCAO was reduced

by 18.08% in the MCAO1 group and by 20.20% in

the MCAO2 group relative to the corresponding sh-

am-operated controls, but no differences were found

between the MCAO models themselves (Fig.  2c). The

“surgery” factor significantly affected body weight

1.71

  =  232.09; p  <  0.001), whereas neither the “mod-

el” factor (F

1.71

  =  0.10; p  =  0.75) nor the interaction

between the factors (F

1.71

  =  2.28; p  =  0.13) reached

statistical significance.

Based on the results of TTC staining, the lesions

were primarily localized to the neocortex and stria-

tum, while the hippocampus remained largely unaf-

fected. On day 3 after MCAO1, the infarct volume ac-

counted for 33.41  ±  3.09% (n  =  6) of the contralateral

hemisphere, while after MCAO2, it was 35.14  ±  2.09%

(n  =  5), with no statistically significant difference be-

tween the two models. For MCAO1, 19 rats were op-

erated on, of which 12 survived and 7 died (mortality

rate, 36.84%). In the MCAO2 group, 11 of 17 animals

survived and 6 died (mortality rate, 35.29%). A 2×2

contingency analysis revealed no significant differ-

ence in mortality between the groups (p  =  0.59).

Overall, the two ischemic stroke models did not

differ in the neurological deficit severity, infarct vol-

ume, or post-occlusion mortality.

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Fig. 2. Neurological deficit, body weight, and infarct volume (day 3) after MCAO1 and MCAO2: a)5-point scale test; b)tongue

protrusion test (% of baseline); c) animal body weight (% of baseline); d) infarct volume (% of contralateral hemisphere

and brain sections after TTC staining). ** p < 0.01, difference with the corresponding Sham group.

CORT levels in the blood and hippocampus

after MCAO1 and MCAO2. CORT level in the blood

from the rat tail vein increased only after MCAO1,

but not after MCAO2. On day 1 after MCAO1, CORT

showed a non-significant trend toward elevation com-

pared with baseline pre-surgical values (1.46-fold in-

crease, p  <  0.1), and by day 3, this increase became

statistically (1.64-fold; p  <  0.05) (Fig.  3a). No signifi-

cant changes were observed by day 3 in the Sham1,

Sham2, or MCAO2 groups. We also revealed a signif-

icant main effect of the “surgery” factor (F

1.30

  =  6.80;

p  <  0.05), as well as a significant interaction be-

tween the “surgery” and “model” factors (F

1.30

  =  4.85;

p  <  0.05) influenced this parameter.

In the post-decapitation blood, CORT levels in-

creased significantly only in the MCAO1 group,

reaching 1.42-fold higher values than in the Sham1

group (Fig.  3b). Of the factors analyzed, only “sur-

gery” exerted a significant effect on this parameter

1.74

  =  19.54; p  <  0.001), but not the interaction of the

“surgery” and “model” factors (F

1.74

  =  2.01; p  =  0.15).

Similarly, on day 3 after MCAO1, CORT levels sig-

nificantly increased in both ipsilateral hippocampus

(1.76-fold) and contralateral hippocampus (1.46-fold)

(Fig.  4). The “model” factor (F

1.70

  =  8.00; p  =  0.0062),

the “surgery” factor (F

1.70

  =  16.19; p  =  0.0001), and

their interaction (F

1.70

  =  5.06; p  =  0.027) had a statisti-

cally significant effect on the CORT levels in the ipsilat-

eral hippocampus. Their influence was weaker in the

contralateral hippocampus, where the “model” factor

reached marginal significance (F

1.72

  =  4.01; p  =  0.048),

while the “surgery” factor (F

1.72

  =  3.38; p  =  0.069) and

the interaction of these factors (F

1.72

  =  3.22; p  =  0.076)

did not reach statistical significance.

Correlation analysis in the combined Sham1 and

MCAO1 groups on day  3 after occlusion revealed a

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Fig. 3. Dynamics of changes in CORT levels in blood from the tail vein (a) and post-decapitation blood (b, day 3) after

occlusion. Open squares, intact animals; other group designations are the same as in Fig.1; -1, 1, 3 in panel a, days before

and after occlusion;

 p < 0.1, * p < 0.05, ** p < 0.01, differences with the corresponding control group.

Fig. 4. CORT levels in the ipsilateral (a) and contralateral (b) hippocampus on day 3 after occlusion. Group designations are

the same as in Fig. 2; * p < 0.05 and ** p < 0.01, differences from with the corresponding Sham groups.

significant positive correlation between the CORT lev-

els in the ipsilateral and contralateral hippocampus

(r  =  0.74, p  <  0.05). Interestingly, in the MCAO1 group,

CORT levels in the post-decapitation blood positive-

ly correlated with the hippocampal CORT levels in

both the ipsilateral and contralateral hemispheres

(r  =  0.48, p  <  0.05 and r  =  0.41, p  <  0.05, respectively).

In addition, several significant negative correlations

were observed: tongue protrusion inversely correlat-

ed with the CORT levels in the blood from the tail

vein (r  =  −0.34, p  <  0.05) and in the post-decapitation

blood (r  =  −0.52, p  <  0.05); CORT levels in the ipsi-

lateral hippocampus showed negative correlations

with tongue protrusion (r  =  −0.48, p  <  0.05) and body

weight (r  =  −0.39, p  <  0.05).

In the combined Sham2 and MCAO2 groups, the

associations between the ipsilateral hippocampal CORT

levels and tongue protrusion, as well as between the

circulating and hippocampal CORT levels, were not

present. Only a negative correlation between post-de-

capitation blood CORT levels and tongue protrusion

persisted, although this relationship showed only a

trend toward significance (r  =  −0.38, p  <  0.1).

Neurotrophic factors in the hippocampus af-

ter MCAO1 and MCAO2. On day  3 after MCAO,

BDNF levels increased significantly in the ipsilater-

al hippocampus in both MCAO1 and MCAO2 groups

compared to their respective Sham controls (Fig.  5a).

The magnitude of this increase was comparable be-

tween the models, reaching 1.43-fold in MCAO1 and

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026

Fig. 5. BDNF levels in the ipsilateral (a) and contralateral (b) hippocampus on day 3 after occlusion. Group designations

are the same as in Fig. 2; ** p < 0.01, difference with the corresponding Sham group.

Fig. 6. GDNF levels in the ipsilateral (a) and contralateral (b) hippocampus on day 3 after occlusion. Group designations

are the same as in Fig. 2.

1.50-fold inMCAO2. The “surgery” factor had a statis-

tically significant effect on BDNF levels (F

1.34

  =  27.08;

p  <  0.0001), but not the “model” factor (F

1.34

  =  2.64;

p  =  0.113).

MCAO1 and MCAO2 did not affect BDNF lev-

els in the contralateral hippocampus (Fig. 5b). Nei-

ther the “model” (F

1.35

  =  0.46; p  =  0.503) or “surgery”

1.32

  =  0.30; p  =  0.587) factors, nor their interaction

1.32

  =  0.13; p  =  0.714) showed a significant effect on

this parameter.

On day  3 after MCAO1 and MCAO2, no statistical-

ly significant changes in GDNF levels were detected

in the hippocampus, largely due to a high inter-in-

dividual variability in the studied groups (Fig.  6).

Although an apparent increase was observed in the

ipsilateral hippocampus after MCAO1, this difference

did not reach statistical significance. However, the

“model” factor showed a trend toward significance

1.35

  =  3.16; p  =  0.084), whereas the “surgery” fac-

tor (F

1.35

  =  1.91; p  =  0.175) or the interaction of these

factors (F

1.35

= 1.48; p  =  0.231) were not significant.

In the contralateral hippocampus, no statistically

significant changes in GDNF levels were observed,

and neither the “model” (F

1.37

  =  0.001; p  =  0.97) or

“surgery” (F

1.37

  =  0.057; p  =  0.81) factors, nor their

interaction (F

1.37

  =  2.148; p  =  0.12) had a significant

effect.

Changes in CNTF levels were observed only in

the ipsilateral hippocampus following both occlusion

models. A 1.52-fold increase in this parameter after

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Fig. 7. CNTF levels in the ipsilateral (a) and contralateral (b) hippocampus on day 3 after occlusion. Group designations

are the same as in Fig. 2; * p < 0.05, difference from the MCAO1 group; ** p < 0.01, difference from the corresponding

Sham group.

MCAO1 did not reach statistical significance, whereas

MCAO2 induced a significant 1.92-fold increase com-

pared with both the corresponding Sham group and

the MCAO1 group (Fig.  7). In contrast to the contra-

lateral hippocampus, in the ipsilateral hippocampus,

both the “surgery” (F

1.38

  =  18.50; p  <  0.001) and “mod-

el” (F

1.38

  =  5.58; p  <  0.05) factors had a statistically

significant effect on CNTF levels, but not the interac-

tion of these factors (F

1.38

  =  2.15; p  =  0.15).

Significant positive correlations were observed

between the CORT levels in the contralateral hip-

pocampus and BDNF levels in the ipsilateral hippo-

campus (r  =  0.52; p  <  0.05) in the combined Sham1  +

MCAO1 groups on day  3 after occlusion. In the same

group, CORT levels also showed a strong positive

association with CNTF levels in the contralateral

hippocampus (r  =  0.71; p  <  0.05). CNTF levels in the

ipsilateral hippocampus positively correlated with

GDNF levels in the contralateral hippocampus in

the Sham1  +  MCAO1 group (r  =  0.60; p  <  0.05). In the

Sham2  +  MCAO2 group, CORT levels in the post-decap-

itation blood positively correlated with CNTF levels

in the contralateral hippocampus (r  =  0.55; p  <  0.05).

Conversely, a significant negative correlation was ob-

served between the CORT levels in the contralateral

hippocampus and GDNF levels in the ipsilateral hip-

pocampus (r  =  −0.45; p  <  0.05).

DISCUSSION

Activation of the HPA axis is initiated within the

first hours after ischemic stroke and persists for a

long period of time, with elevated circulating corti-

sol/CORT levels remaining detectable for several days,

as reported in clinical and experimental studies [8,

28, 29]. Glucocorticoids are well known for their an-

ti-inflammatory and immunosuppressive properties

and are widely used therapeutically to suppress in-

flammatory responses. However, experimental evi-

dence is accumulating that under certain conditions,

glucocorticoids may exert paradoxical pro-inflamma-

tory effects in the CNS, particularly in the context of

focal brain injury [30-33].

In our study, circulating CORT levels increased

as early as day  1 and reached a peak by day  3 af-

ter MCAO1, whereas in the MCAO2 model, CORT lev-

els showed only a smaller, non-significant elevation.

More pronounced activation of the HPA axis in the

MCAO1 model was accompanied by CORT accumula-

tion in the ipsilateral and contralateral hippocampus,

an effect not observed in the MCAO2 model. These

findings are consistent with the results of our re-

cent studies demonstrating that, unlike MCAO2, the

MCAO1 model is characterized by a systemic rise

in blood CORT on day  3 post-occlusion, followed by

its accumulation in the ipsilateral and contralateral

brain regions (frontal cortex and hippocampus) both

during the acute phase and at later time points after

ischemic stroke, together with enhanced neuroinflam-

matory responses [34, 35].

Furthermore, we observed significant negative

correlations between the CORT levels in the blood and

hippocampus and the severity of neurological deficit,

with these relationships being more pronounced in

the MCAO1 model than in MCAO2. The contribution

of isoflurane anesthesia (which has neuroprotective

properties) to these differences is unlikely, as anes-

thesia duration was comparable across both experi-

mental models.

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026

The difference between the models in the degree

of post-MCAO HPA axis activation was not reflected

in the BDNF expression in the hippocampus, as the

elevated levels of this neurotrophin observed on

day  3 did not differ between the MCAO1 and MCAO2

groups. It has been demonstrated that the severity

of focal ischemia influences the expression of neuro-

trophin genes and their receptors. For instance, tran-

sient (2  h) MCAO induced early upregulation of BDNF,

NGF, TrkA, and TrkB mRNAs within hours in multi-

ple cortical regions and hippocampal fields; however,

by 24  h, expression levels typically returned to base-

line. In contrast, a milder ischemic insult (15-min

MCAO) produced a more limited response, with in-

creased BDNF mRNA levels observed at 2  h of reper-

fusion in the cingulate cortex and frontal cortex, but

not in the hippocampus [36].

Following focal ischemia induced by microsphere

injection, which results in hippocampal damage, ele-

vated BDNF levels in this region persisted for up to

one week  [37]. Similarly, after 4  h of MCAO, in  situ

hybridization showed increased expression of BDNF

and TrkB mRNAs in the dentate gyrus and CA1-CA4

hippocampal subfields bilaterally  [38].

In our study, upregulation of the total BDNF ex-

pression in the ipsilateral hippocampus on day  3 af-

ter MCAO appeared to be independent of local CORT

levels. Likewise, elevated CORT in the contralateral

hippocampus after MCAO1 did not translate into

changes in BDNF expression. This is consistent with

evidence that the effect of CORT on BDNF expression

is dose-dependent: a single high-dose CORT admin-

istration reduced BDNF mRNA levels in the dentate

gyrus and CA1 and CA3 subfields, whereas low-dose

CORT increased TrkB mRNA expression in CA3 and

the dentate gyrus without altering BDNF mRNA lev-

els [39].

Furthermore, impaired BDNF/TrkB signaling ob-

served in glucocorticoid receptor knockout mice sup-

ports a functional interaction between the corticoste-

roid and neurotrophin pathways [40]. Compared to

other neurotrophins, the levels of BDNF expression in

the CNS are quite high and maintained by multiple

cell types. Therefore, moderate alterations in CORT

levels and, consequently, in corticosteroid signaling

during the acute post-MCAO period are unlikely to

substantially modify the ischemia-induced neuropro-

tective response of BDNF in the hippocampus.

We found no changes in the total GDNF level

in the ipsilateral hippocampus on day  3 in both the

model with increased CORT production (MCAO1) and

model characterized by moderate CORT secretion. Itis

known that the post-ischemic GDNF expression follows

a biphasic pattern. Thus, after transient MCAO (2  h),

GDNF mRNA levels increased within 1-2  h of reper-

fusion in the cerebral cortex and striatum [41, 42].

However, the initial upregulation was followed by

a decline toward baseline, and later, by a second-

ary increase at approximately 72  h, which coincided

with the elevated GDNF expression in the peri-infarct

regions of the cortex and striatum in the ipsilateral

hemisphere  [41]. Early increase in GDNF was observed

in striatal and cortical neurons, while the secondary

increase colocalized with astrocyte and microglia/

macrophage markers in the same brain regions  [41].

These data suggest that the most pronounced chang-

es in GDNF expression occur at earlier times after

MCAO in directly injured or peri-infarct areas such

as the cortex and striatum, and not in more remote

structures, including the hippocampus. It is also pos-

sible that the absence of detectable changes in hip-

pocampal GDNF in our study may reflect the limita-

tion of a single selected time point, which may not

capture transient or delayed alterations. Studies using

the four-vessel transient occlusion model, in which

the hippocampus is a primary target of injury, have

also demonstrated a biphasic GDNF response, with

the early phase characterized by a rapid increase in

neuronal GDNF in both the cortex and hippocampus,

followed by a delayed (3-day) elevation predominant-

ly in reactive astrocytes [43].

It has been shown that glucocorticoids can

suppress GDNF expression and secretion [44,  45].

Dexamethasone, CORT, and aldosterone inhibit the

proliferation of cultured rat cortical astrocytes in

a dose-dependent manner. This inhibitory effect of

dexamethasone is abolished by RU-486, a glucocorti-

coid receptor antagonist. However, RU-486 also par-

tially attenuates the inhibitory influence of neural cell

adhesion molecule (NCAM) on astrocyte proliferation,

indicating the involvement of glucocorticoid signaling

in the regulation of astrocyte proliferation  [46].

Under physiological conditions, the level of CNTF

expression in the CNS is low, but it increases dra-

matically following focal injury. For example, after

ischemic stroke, CNTF expression is significantly up-

regulated, with cytokine levels in astroglial cells of

the striatum doubling within 1  hour and increasing

more than 30-fold within two weeks after MCAO

in mice  [47]. Moreover, elevated CNTF expression

in the subventricular zone has been implicated in

stroke-induced neurogenesis and attenuation of neu-

roinflammatory responses  [48]. In rats subjected to a

prolonged (2-h) MCAO, increased CNTF levels were

detected in both the neocortex and hippocampus as

early as two days post-occlusion [49].

In a modified MCAO paradigm, in which blood

flow was blocked at the level of the middle cere-

bral artery for 1  h and both common carotid ar-

teries were ligated, a gradual increase in the CNTF

mRNA expression was observed in the neocortex of

the ischemic hemisphere at 12, 24, 72, 168, and 336  h

ONUFRIEV et al.1196

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026

post- occlusion. In contrast, in the granular layer cells

of the dentate gyrus in the ipsilateral hippocampus, a

significant elevation in the CNTF receptorα (CNTFRα)

mRNA was detected as early as 4  h after occlusion,

despite the absence of overt cellular damage in these

neurons [50].

Whereas in focal ischemia the hippocampus is

typically distant from the primary lesion, transient

global ischemia induces selective damage to this

brain structure. In this context, increased CNTF im-

munoreactivity has been reported in reactive astro-

cytes in the CA1 subfield and dentate gyrus between

days  3 and 7 of the post-ischemic period. Consistent-

ly, in  situ hybridization studies have demonstrat-

ed elevated CNTFRα mRNA expression in the same

hippocampal regions, peaking around day  3 after

ischemia [51].

In our experiment, hippocampal CNTF levels

increased on day  3 following both MCAO variants.

However, the effect was more pronounced after

MCAO2, which is associated with a comparatively

lower degree of HPA axis activation than MCAO1.

These findings suggest that variations in corticoste-

roid signaling intensity may modulate both spatial

and temporal dynamics of astrocyte activation and

CNTF biosynthesis. Given the involvement of neuro-

trophic factors in neuroplastic processes, our results

are consistent with the concept of glucocorticoid-me-

diated regulation of hippocampal plasticity  [52]. The

observed molecular alterations in this region may

therefore contribute to distinct trajectories of subse-

quent cognitive and neurological outcomes (Fig.  8).

CONCLUSION

In the absence of differences in neurological

deficit and mortality between the two MCAO models

in the acute phase, significant correlations were ob-

served in the ischemic stroke model with hypercor-

ticosteronemia, where neurological deficit negatively

correlated with the CORT levels in both the blood

and the hippocampus. In contrast, in the model with

moderate CORT secretion into circulation and accu-

mulation in the hippocampus, these correlations were

absent. This may indicate a possible link between en-

hanced glucocorticoid signaling and the development

of functional impairments, suggesting that CORT may

serve more as a biomarker of injury severity rather

than a direct causal factor of neurological dysfunc-

tion.

Irrespective of hippocampal CORT levels, in-

creased expression of neurotrophic factors during

the acute post-MCAO period was observed both upon

pronounced CORT accumulation in the ipsilateral and

contralateral hippocampus (as seen for BDNF) and in

the absence of hippocampal CORT accumulation (as

seen for CNTF). The lack of changes in GDNF expres-

sion at this time point does not exclude GDNF involve-

ment in post-stroke processes in the hippocampus at

earlier or later stages. The bidirectional relationships

identified between the hippocampal CORT content

and neurotrophic factor expression suggest diverse

protective roles of neurotrophic signaling and indi-

cate that interactions with glucocorticoids differ de-

pending on the degree of HPA axis activation during

the early phase following ischemic stroke.

Abbreviations

BDNF brain-derived neurotrophic factor

CORT corticosterone

CNTF ciliary neurotrophic factor

GDNF glial cell line-derived neurotrophic

factor

HPA hypothalamic–pituitary–adrenal

MCAO middle cerebral artery occlusion

NGF nerve growth factor

TTC 2,3,5-triphenyltetrazolium chloride

Contributions

M.V.O. and N.V.G. developed the concept, supervised

the study, and discussed study results; M.V.O., Yu.V.M.,

M.R.N., and P.A.K. conducted experiments; M.V.O. wrote

the text of the article; N.V.G. edited the manuscript.

Funding

This study was supported by the Russian Science

Foundation (project no. 24-25-00458).

Ethics approval and consent to participate

All procedures involving animals complied with the

ethical standards of the Institute of Higher Nervous

Fig.  8. Summary of changes in the hippocampus during

acute period after MCAO ↑, upregulation; =, no changes.

NEUROTROPHIC FACTORS IN THE HIPPOCAMPUS AFTER ISCHEMIC STROKE 1197

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 7 2026

Activity and Neurophysiology and approved legal acts

of the Russian Federation and international organi-

zations.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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