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. 2026 Oct 29;40(6):3457–3466. doi: 10.21873/invivo.14489

Increased GluA1 Expression, c-Fos Activation, and MAP2 Loss During Early Cerebral Ischemia-reperfusion

HYOIN HWANG 1,2, JUNGHEE PARK 1,3, HYEKYOUNG SHIN 2, YOONYOUNG CHUNG 2, DONGJOON KIM 4, YONGHYUN JUN 2,5
PMCID: PMC13723964  PMID: 42957249

Abstract

Background/Aim

The disruption of excitatory and inhibitory neurotransmission is a key pathological event in ischemia/reperfusion injury. However, the temporal relationship among glutamatergic activation, inhibitory responses, and neuronal activity in the early phase of reperfusion remains unclear. The present study investigated changes in GluA1 signaling, GABAA receptor α1 expression, neuronal activation, and neuronal integrity following transient cerebral ischemia.

Materials and Methods

Transient focal cerebral ischemia was induced in male C57BL/6 mice by middle cerebral artery occlusion (MCAO). Mice were sacrificed immediately following ischemia (0 h) or 3 h after reperfusion. The expression levels of phosphorylated GluA1 (pGluA1), total GluA1, and GABAA receptor α1 in the ipsilateral cortex were analyzed by western blotting. Neuronal activation and integrity were evaluated using immunofluorescence staining for c-Fos and MAP2, respectively.

Results

The expression levels of pGluA1 and total GluA1 increased following MCAO and remained elevated after 3 h of reperfusion, whereas the pGluA1/total GluA1 ratio did not differ significantly among the experimental groups. GABAA receptor α1 expression did not differ significantly among the groups, although it tended to be higher after reperfusion. Immunofluorescence revealed a focal reduction in MAP2 immunoreactivity in the reperfusion group, while no obvious changes were observed immediately after ischemia. In addition, c-Fos immunoreactivity increased in both the ischemic and reperfusion groups compared with the sham group.

Conclusion

Early cerebral ischemia-reperfusion is characterized by progressive activation of GluA1 signaling, increased neuronal activity, and the onset of neuronal injury. GABAA receptor α1 expression showed a non-significant tendency to be higher after reperfusion, and the functional significance of this trend remains uncertain. These findings indicate that early cerebral ischemia-reperfusion is associated with altered GluA1 expression, increased neuronal activity, and early neuronal injury.

Keywords: Ischemia-reperfusion injury, GluA1, GABAA receptor α1, c-Fos, middle cerebral artery occlusion

Introduction

Ischemic stroke is the leading cause of mortality and long-term disability worldwide (1). Although timely reperfusion is essential for salvaging ischemic brain tissue, it can initiate a cascade of secondary injuries that contribute to neuronal dysfunction and cell death (2). Among the various pathological mechanisms involved in ischemia-reperfusion injury, disruption of excitatory and inhibitory neurotransmission is a critical determinant of neuronal fate during the acute phase of stroke (3, 4).

Glutamate-mediated excitotoxicity is a recognized hallmark of cerebral ischemia (5). The excessive release of glutamate during ischemia results in the overactivation of ionotropic glutamate receptors, leading to intracellular calcium overload and neuronal injury (6). In particular, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 plays a central role in regulating synaptic strength and neuronal excitability (7). Prior studies have demonstrated that phosphorylation of GluA1 enhances receptor trafficking and synaptic incorporation, thereby facilitating excitatory synaptic transmission (8, 9). Although such mechanisms contribute to physiological synaptic plasticity under normal conditions, excessive activation of GluA1 signaling can promote pathological hyperexcitability following cerebral ischemia.

In contrast to enhanced excitatory signaling, impaired inhibitory neurotransmission may further exacerbate neuronal dysfunction after stroke (10). γ-Aminobutyric acid type A (GABAA) receptors are the principal mediators of rapid synaptic inhibition in the adult brain (11). Among the various subunits, the α1 subunit is highly expressed in cortical neurons and is essential for maintaining inhibitory tone and network stability (12). Alterations in GABAA receptor expression or function following ischemia can contribute to an imbalance between excitation and inhibition, thus amplifying neuronal vulnerability during the reperfusion period.

The consequences of an excitatory-inhibitory imbalance are reflected at the network level. Immediate early genes, such as c-Fos, are widely assessed as markers of neuronal activation and provide insights into activity-dependent responses following brain injury (13). Increased c-Fos expression is associated with enhanced neuronal firing and network hyperactivity in various neurological disorders, including ischemic stroke (14). However, the temporal relationship among excitatory receptor activation, inhibitory receptor alterations, and network activity during the early phase of ischemia-reperfusion injury currently remains unclear. Recent studies using middle cerebral artery occlusion (MCAO) models have demonstrated that early molecular responses following ischemia-reperfusion are closely associated with neuroprotective signaling pathways, including BDNF-mediated mechanisms (15, 16).

Therefore, the present study investigated changes in phosphorylated GluA1, total GluA1, and GABAA receptor α1 expression during the early phase of cerebral ischemia-reperfusion. In addition, neuronal activation and neuronal integrity were evaluated using c-Fos and MAP2 immunofluorescence, respectively. We hypothesized that early ischemia-reperfusion injury would be associated with alterations in excitatory and inhibitory signaling, accompanied by increased neuronal activity and the onset of neuronal injury.

Materials and Methods

Animal surgery.All experimental procedures were approved by the Institutional Animal Care and Use Committee of Chosun University (Approval No. CIACUC2024-A0007) and conducted in accordance with institutional guidelines for animal research. Male C57BL/6 mice (7-8 weeks old) were obtained from Orient Bio (Seongnam, Republic of Korea). A total of 21 male mice were included in the final analyses. The animals were assigned to three groups: Sham, Post-MCAO 0 h, and MCAO/R 3 h. Western blot analysis was performed using three mice per group, whereas immunofluorescence analysis was performed using four mice per group, corresponding to nine mice for western blotting and twelve mice for immunofluorescence. Mice were anesthetized with 2.0% sevoflurane, and body temperature was maintained at 37 ± 0.5°C. Cerebral blood flow (CBF) was continuously monitored using a laser Doppler flowmeter (Periflux 6000, Perimed, Stockholm, Sweden). A silicone-coated 6-0 nylon monofilament (ETHICON, Raritan, NJ, USA) was prepared in-house and inserted through the external carotid artery into the internal carotid artery to occlude the origin of the middle cerebral artery while carefully avoiding the pterygopalatine artery. Successful MCAO was defined as a reduction in CBF of at least 80% of baseline. Animals showing less than an 80% reduction in CBF were excluded from the study. After 45 min of occlusion, mice were sacrificed immediately (0 h group) or underwent filament withdrawal to induce reperfusion. Reperfusion was confirmed by recovery of CBF immediately after filament withdrawal, although no predefined reperfusion threshold was applied.

Western blot. All mice were anesthetized using sevoflurane inhalation (1.0-2.0 %, end-tidal concentration), and the left cortical tissue was isolated. The tissue was homogenized in five volumes of RIPA buffer [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS] containing a protease inhibitor cocktail (Roche, Basel, Switzerland; Cat. No. 11836153001) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland; Cat. No. 04906837001). The supernatant was collected, and the total protein concentration was measured using the Bradford method. After boiling in sodium dodecyl sulfate (SDS), the protein extract was separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Cat. No. 1214498). Rabbit polyclonal anti-phospho-GluR1 (Ser831) antibody (Merck Millipore, Burlington, MA, USA; Cat. No. AB5847), rabbit polyclonal Glutamate Receptor 1 antibody (Cat. No. ab183797), rabbit polyclonal GABAA receptor α1 antibody (Abcam, Cambridge, UK; Cat No. ab33299), and mouse monoclonal beta-actin antibody (Santa Cruz Biotechnology, Dallas, TX, USA; Cat. No. sc-47778) were used at a dilution of 1:1,000. Image analysis was conducted using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Protein expression levels were normalized to beta-actin, which served as internal control.

Immunofluorescence. Mice were deeply anesthetized with sevoflurane and transcardially perfused with phosphate-buffered saline (PBS), followed by perfusion with 4% paraformaldehyde (PFA) in PBS. Brains were harvested, post-fixed in 4% PFA at 4°C overnight and cryoprotected in 30% sucrose solution until they sank. The tissues were embedded in optimal cutting temperature (OCT) compound and sectioned coronally at a thickness of 10 μm using a cryostat (Leica Microsystems, Wetzlar, Germany). Cryosections were then air-dried in a chemical fume hood for 30 min at room temperature and washed three times for 5 min each with PBS containing 0.1% Tween-20 (PBST). The sections were subsequently fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature, followed by three additional washes with PBST for 5 min each. A hydrophobic barrier was drawn around the tissue sections using a hydrophobic barrier pen (Abcam, Cambridge, UK; Cat No. ab2901) to create a confined, stained area.

The sections were subsequently blocked with 5% normal horse serum in PBST for 30 min at room temperature. After blocking, sections were incubated overnight at 4°C with chicken polyclonal anti-MAP2 antibody 1:2,000 (Thermo Fisher Scientific, Waltham MA, USA; Cat. No. PA1-10005) and mouse monoclonal anti-c-FOS antibody 1:1,000 (Hello Bio, Bristol, UK; Cat. No. HB8006) diluted in the blocking solution. Following primary antibody incubation, sections were washed three times for 5 min each with PBS containing 0.1% Tween-20 (PBST). The sections were then incubated with Alexa Fluor 488-conjugated anti-chicken IgY secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; Cat. No. 303-545-003) and Rhodamine (TRITC)-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch; Cat. No. 115-025-003) for 30 min at room temperature in the dark. Following incubation, the sections were washed three times for 5 min each with PBST. After the final washes, the sections were mounted with Fluoroshield™ mounting medium containing DAPI (Abcam; Cat. No. ab104139) and covered with coverslips. Fluorescence images were acquired using a confocal microscope (Leica STELLARIS 5; Leica Microsystems, Wetzlar, Germany). Quantitative analysis of immunofluorescence images was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For each animal, three regions of interest (ROIs) of identical size were randomly selected within the ipsilateral parietal cortex. The mean fluorescence intensity (arbitrary units, A.U.) from the three ROIs was averaged and used for statistical analysis. Four mice were analyzed in each experimental group.

Statistical analysis. Statistical analyses were performed using SPSS software (version 18.0; SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean (SEM). Comparisons among the experimental groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. A p-value of <0.05 was considered statistically significant.

Results

To investigate changes in excitatory signaling during the early phase of cerebral ischemia, the expression levels of phosphorylated (pGluA1) and total GluA1 were examined in the ipsilateral cortex. Western blot analysis revealed that pGluA1 expression increased in both the 45 min MCAO group and the 3 h reperfusion group compared with the sham group. Furthermore, pGluA1 expression was higher in the 3 h reperfusion group than in the ischemia-only group. Similar changes were observed in total GluA1 expression, indicating progressive activation of glutamatergic signaling during the early reperfusion period (Figure 1). To further determine whether the increase in pGluA1 reflected enhanced phosphorylation independent of total GluA1 expression, the pGluA1/total GluA1 ratio was analyzed. However, the pGluA1/total GluA1 ratio did not differ significantly among the experimental groups. (Figure 1).

Figure 1.

Figure 1

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Increased pGluA1 and total GluA1 expression following transient cerebral ischemia. Mice were subjected to middle cerebral artery occlusion (MCAO) for 45 min and sacrificed immediately after ischemia (Post-MCAO 0 h) or following 3 h of reperfusion (Post-MCAO/R 3h). Protein expression levels of phosphorylated GluA1 (pGluA1, Ser831), total GluA1, and the pGluA1/total GluA1 ratio in the ipsilateral cortex were analyzed by western blotting. Representative immunoblots and densitometric analyses normalized to β-actin are shown. Both pGluA1 and total GluA1 expression were increased in the MCAO groups compared with sham controls, with greater expression observed after 3 h of reperfusion. However, no significant differences were observed in the pGluA1/total GluA1 ratio among the experimental groups. Data are presented as mean ± SEM (n=3 mice per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01.

To determine whether inhibitory signaling was altered following ischemia-reperfusion injury, expression of GABAA receptor α1 was analyzed by Western blotting. No obvious difference in α1 expression was observed in the 45 min MCAO group compared with sham controls; However, GABAA receptor α1 expression tended to be higher in the 3 h reperfusion group than in the sham group, although the difference did not reach statistical significance (Figure 2). Neuronal integrity was evaluated by MAP2 immunofluorescence staining. Quantitative analysis of MAP2 fluorescence intensity in the ipsilateral parietal cortex showed no significant difference between the sham and ischemia-only groups. In contrast, MAP2 fluorescence intensity was significantly reduced after 3 h of reperfusion. Representative immunofluorescence images were consistent with the quantitative analysis and demonstrated reduced MAP2 immunoreactivity in the reperfusion group (Figure 3).

Figure 2.

Figure 2

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GABAA receptor α1 expression during early reperfusion following transient cerebral ischemia. Mice were subjected to middle cerebral artery occlusion (MCAO) for 45 min and sacrificed immediately after ischemia (Post-MCAO 0 h) or following 3 h of reperfusion (Post-MCAO/R 3 h). Protein expression levels of GABAA receptor α1 in the ipsilateral cortex were analyzed by western blotting. Representative immunoblots and densitometric analyses normalized to β-actin are shown. No significant differences in GABAA receptor α1 expression were observed among the experimental groups, although expression tended to be higher in the 3 h reperfusion group than in the sham group. Data are presented as mean ± SEM (n=3 mice per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 3.

Figure 3

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Reduced MAP2 immunoreactivity following early reperfusion. Brain sections obtained from sham-operated mice, mice subjected to 45 min middle cerebral artery occlusion (MCAO) (Post-MCAO 0 h), and mice subjected to 45 min MCAO followed by 3 h of reperfusion (Post-MCAO/R 3h) were immunostained for microtubule-associated protein 2 (MAP2). Representative immunofluorescence images and quantitative analysis of MAP2 fluorescence intensity in the ipsilateral parietal cortex are shown. Fluorescence intensity was quantified using ImageJ from three regions of interest of identical size per animal. MAP2 fluorescence intensity was significantly reduced after 3 h of reperfusion, whereas no significant difference was observed immediately after ischemia. Data are presented as mean ± SEM (n=4 mice per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. ****p<0.0001. Whole-image scale bar=1000 μm; ROI scale bar=200 μm.

To evaluate neuronal activation, c-Fos immunofluorescence staining was performed. Quantitative analysis of c-Fos fluorescence intensity in the ipsilateral parietal cortex revealed significantly increased c-Fos immunoreactivity in both the ischemia-only and reperfusion groups compared with sham controls (Figure 4).

Figure 4.

Figure 4

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Increased c-Fos immunoreactivity following cerebral ischemia-reperfusion. Brain sections obtained from sham-operated mice, mice subjected to 45 min middle cerebral artery occlusion (MCAO) (Post-MCAO 0 h), and mice subjected to 45 min MCAO followed by 3 h of reperfusion (Post-MCAO/R 3h) were immunostained for c-Fos (red). Representative immunofluorescence images and quantitative analysis of c-Fos fluorescence intensity in the ipsilateral parietal cortex are shown. Fluorescence intensity was quantified using ImageJ from three regions of interest (ROIs; 1300 × 1300 μm) of identical size per animal. c-Fos fluorescence intensity was significantly increased in both the ischemia-only and reperfusion groups compared with sham controls. Data are presented as mean ± SEM (n=4 mice per group). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. ***p<0.001. Whole-image scale bar=1000 μm; ROI scale bar=200 μm.

Discussion

The present study investigated the early alterations in excitatory and inhibitory signaling following transient cerebral ischemia. We demonstrated that both phosphorylated and total GluA1 expression were increased after MCAO and were further elevated during the early reperfusion period. Although both pGluA1 and total GluA1 expression increased following ischemia and reperfusion, the pGluA1/total GluA1 ratio did not change significantly. Therefore, the present findings are more consistent with increased GluA1 abundance accompanied by an increased absolute pGluA1 signal than with enhanced phosphorylation per receptor. GABAA receptor α1 expression showed a non-significant tendency to be higher 3 h after reperfusion. These molecular changes were accompanied by increased c-Fos expression and reduced MAP2 immunoreactivity, indicating enhanced neuronal activity and the onset of neuronal injury. Collectively, these findings suggest that early reperfusion is associated with altered excitatory signaling and increased neuronal activity, whereas the functional significance of the non-significant trend in GABAA receptor α1 expression remains uncertain.

Glutamate-mediated excitotoxicity is considered the primary mechanism underlying ischemic brain injury (17). Excessive glutamate release during ischemia activates ionotropic glutamate receptors and promotes intracellular calcium accumulation (18). Among the various AMPA receptor subunits, GluA1 plays an important role in regulating synaptic strength through activity-dependent trafficking and incorporation (19, 20). In the present study, both phosphorylated and total GluA1 expression increased following MCAO and further increased after reperfusion. These findings indicate that excitatory signaling remains active during the early reperfusion period and may contribute to the progressive activation of cortical neurons.

In contrast to our initial expectation, GABAA receptor α1 expression showed a tendency to be higher during the early reperfusion period, although this difference did not reach statistical significance. Because this observation was based on total-protein western blot analysis and was not statistically significant, it cannot be interpreted as evidence of enhanced inhibitory function. Further studies with larger sample sizes and functional analyses are required to determine the biological significance of this trend.

Despite an increase in inhibitory receptor expression, c-Fos immunofluorescence intensity was significantly increased in both the ischemia-only and reperfusion groups compared with sham controls. As c-Fos is widely recognized as a marker of neuronal activation (21, 22), these findings indicate that neuronal activity remains elevated throughout the early reperfusion phase. The coexistence of increased GluA1, increased GABAA receptor α1, and elevated c-Fos expression suggests that enhanced GABAA receptor α1 expression alone was not accompanied by normalization of neuronal activity. Whether this increase reflects a functional adaptive inhibitory response remains to be determined.

MAP2 immunofluorescence analysis revealed focal reductions in MAP2 staining 3 h after reperfusion, while no obvious alterations were observed immediately after ischemia. MAP2 is a dendritic cytoskeletal protein highly sensitive to ischemic injury and thus commonly used as an indicator of neuronal structural integrity (23, 24). This reduction in MAP2 immunoreactivity indicates that neuronal damage begins to develop during the early reperfusion period. Together with the increased c-Fos expression, these findings indicate that persistent neuronal activation may be associated with the initiation of structural neuronal injury following reperfusion.

Study limitations. Firstly, it focused exclusively on an early reperfusion time point and, therefore, did not provide any information regarding the long-term changes in excitatory and inhibitory signaling. Secondly, functional electrophysiological analyses were not conducted. Thus, alterations in receptor expression could not be directly correlated with synaptic activity. Thirdly, the mechanisms underlying the increase in GABAA receptor α1 expression remain to be determined. Future studies examining receptor localization, synaptic function, and longer reperfusion periods are required to clarify the role of compensatory inhibitory signaling during ischemia-reperfusion injury (25). Recent experimental studies have demonstrated that pharmacological interventions targeting early ischemia-reperfusion injury can improve neurological outcomes, supporting the importance of identifying early molecular alterations (26). Another limitation of this study is that only male mice were used. Although male animals were selected to reduce biological variability, sex differences in ischemia-reperfusion responses and AMPA receptor biology have been reported (27). Therefore, the present findings may not be directly generalizable to female animals, and future studies including both sexes are warranted.

Conclusion

In conclusion, the present study demonstrated that transient cerebral ischemia is associated with increased total GluA1 abundance accompanied by an increased absolute pGluA1 signal, elevated neuronal activity, and early neuronal injury. Although GABAA receptor α1 expression tended to be higher after reperfusion, no statistically significant differences were observed among the experimental groups, and the biological significance of this trend remains uncertain. Collectively, these findings contribute to a better understanding of the early molecular events underlying cerebral ischemia-reperfusion injury.

Conflicts of Interest

The Authors declare no competing interests.

Authors’ Contributions

YHJ and HIH designed the study. HKS and DJK performed surgical procedures. YYC and JHP analyzed the data. HIH and HKS performed western blot analyses. All authors approved the final manuscript.

Acknowledgements

This research was supported by a grant from the National Research Foundation of Korea to the Global Learning and Academic Research Institution for Master’s and PhD students and the Postdoctoral Fellows (LAMP) Program funded by the Ministry of Education (Grant Number: RS-2023-00285353).

Artificial Intelligence (AI) Disclosure

No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

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