Abstract
Background/Aim: Stroke is a cerebrovascular disease with high mortality and disability, causing motor dysfunction and cognitive impairments. Middle cerebral artery occlusion (MCAO) mouse models of ischemic stroke are used for identifying therapeutic targets. Ischemic insults to the brain alter brain-derived neurotrophic factor (BDNF) expression in cortical and hippocampal neurons. In the present study, we investigated BDNF expression in the cortex and hippocampus of mice following MCAO.
Materials and Methods: Monofilament sutures coated with silicone rubber were introduced into the common carotid artery and occluded middle cerebral artery. The filament was withdrawn for reperfusion after 0.5 h. BDNF protein expression was measured using western blot. Immunofluorescence was performed with anti-NeuN and anti-BDNF antibodies.
Results: BDNF expression in the cerebral cortex and hippocampus was decreased one and three days after MCAO, compared to the control group (unoperated mice). BDNF was expressed in NeuN-positive neurons in the dentate gyrus of the hippocampus and motor cortex of the MCAO and control groups.
Conclusion: MCAO in mice reduced the expression of BDNF in mature neurons of both the motor cortex and hippocampus at one and three days after surgery.
Introduction
Stroke is a major cerebrovascular disease characterized by high mortality, disability, motor dysfunction, and cognitive impairment (1, 2). Strokes can be classified into two types: ischemic and hemorrhagic. The pathology of cerebral ischemia is complex and involves inflammatory reactions, oxidative stress, and apoptosis (3-5). This phenomenon damages multiple brain cells, such as neurons, astrocytes, and oligodendrocytes (6, 7). Although stroke leads to various types of damage in the cerebral parenchyma, effective therapies for ischemic stroke include recombinant tissue-type plasminogen activators and surgical thrombectomy (8). Middle cerebral artery occlusion (MCAO) in mice is a useful animal model for identifying novel therapeutic targets (9).
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophic factor family, has been studied as a neuroprotective and neurogenic factor in the ischemic brain (10). BDNF is widely distributed throughout the brain, with high expression in the hippocampus, cortex, and striatum (11). Many studies have shown that BDNF inhibits inflammatory factors and neuronal apoptosis (12). BDNF has been reported to be involved in learning, memory impairment, and motor dysfunction in animal ischemic models (13, 14). Guang-Xiao Ni et al. showed that BDNF expression is decreased after MCAO surgery, and that neurobehavioral function in rats with MCAO-induced ischemia is improved by BDNF up-regulation (15).
Strokes are associated with motor dysfunction and cognitive impairment. Therefore, it is important to study the motor cortex and hippocampus, which is involved in cognitive functions, together. Ischemic insults to the brain alter BDNF expression in cortical and hippocampal neurons (16). In the present study, we investigated BDNF expression in the cortex and hippocampus following MCAO.
Materials and Methods
Experimental design. Seven- to eight-week-old male C57BL/6 mice were obtained from Damul Laboratory Animals (Daejeon, Republic of Korea). The mice were bred in a controlled environment at a regular temperature of 25°C and were fed a normal diet. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Chosun University. The surgery was performed on 30 mice (MCAO group) and 20 unoperated mice were assigned to the control group. Tissues for western blotting and immunofluorescence were obtained one and three days after MCAO.
MCAO surgery. Mice were anesthetized with 2.5% isoflurane and maintained with 2.0% isoflurane in a mixture of 30% O2 and 70% N2O. Body temperature was maintained at 37±0.5°C using a rectal probe and temperature controller (JD-OT-06DT, JEUNG-DO BIO, Seoul, Republic of Korea). A midline incision was made in the neck and the left common carotid artery (CCA) was separated from the sheath and vagus nerve. The CCA and external carotid arteries were ligated using 4-0 silk sutures. A 6-0 nylon monofilament suture coated with silicone rubber was introduced into the common carotid artery to occlude the middle cerebral artery. The filament was withdrawn for reperfusion after 0.5 h.
Western blot analysis. All mice were anesthetized using sevoflurane inhalation (1.0~2.0%, end-tidal concentration) and the left portion of the cortex and hippocampal tissue were isolated. Tissues were homogenized in RIPA buffer containing one tablet of protease inhibitor cocktail (Roche, Indianapolis, IN, USA) in 10 ml of lysis buffer. The supernatant was collected, and the total protein concentration was measured using the Bradford method. After denaturation with SDS, the proteins were separated using SDS-PAGE, transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA), and probed with an anti-BDNF antibody (1:1,000, ab108319, Abcam, Cambridge, UK). The ImageJ software was used to quantify the signals. Anti-actin (1:2,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) was used as an internal control, and data were normalized according to the beta-actin value.
Immunofluorescence (IF). To perform IF in formalin-fixed paraffin-embedded (FFPE) tissues, section slides were dewaxed as follows: three times in 100% xylene for 5 min, twice in 100% ethanol for 3 min, twice in 95% ethanol for 3 min, once in 90% ethanol for 3 min, once in 80% ethanol for 3 min, once in 70% ethanol for 3 min, and once in H2O. The slide rack was then transferred to a glass container filled with 10 mM citrate buffer (pH 6.0). Antigen retrieval was performed for 10 min in a microwave, and then kept at room temperature (RT) for 30 min. After several washes with TBS-0.1% Tween20 (TBST) buffer, slides were wiped around the section to create an ‘island’ with ImmEdge pen (hydrophobic barrier pen). Immunofluorescence labeling was conducted as follows: The sections were blocked with 10% FBS solution for 30 min at RT, primary antibodies were incubated with 5% FBS solution for 24 h at 4°C. After washing three times with TBST, the tissues were incubated with a secondary antibody conjugated to the appropriate fluorochrome for 1 h at RT. The following antibodies were used for IF labeling: anti-NeuN (1:50, MAB377, Millipore, Burlington, MA, USA) and anti-BDNF (1:50, ab108319, Abcam). Rhodamine and fluorescein isothiocyanate-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, Baltimore Pike, PA, USA) were used. Finally, tissue slides were mounted with fluoroshield mounting medium with DAPI and analyzed using the confocal microscopy LSM800 imaging system (Carl Zeiss, Oberkochen, Germany).
Results
One day after MCAO, the expression of mature BDNF protein (15 kDa) was examined in the parietal cortex and hippocampus of the mice. BDNF protein levels were significantly lower in the left cortex and hippocampus (damaged side) of the MCAO group compared to the control group (Figure 1). In the cortex, BDNF protein in both the control and MCAO group mice was expressed in NeuN-positive neurons in the external granular layer (Figure 2). In the hippocampus, BDNF was expressed in NeuN-positive neurons in the dentate gyrus of both groups (Figure 2).
BDNF expression in the parietal cortex and hippocampus one day after middle cerebral artery occlusion (MCAO). The results were quantified and presented normalized to actin-beta expression. *p<0.05. neg-ctrl: Negative control.
Immunofluorescent staining for BDNF in parietal cortex and dentate gyrus of hippocampus one day after middle cerebral artery occlusion (MCAO).
Three days after MCAO, BDNF protein expression in the left cortex and hippocampus of the MCAO group was lower than that in the control group, similar to the findings at one day after MCAO (Figure 3). In the external granular layer of the cortex and dentate gyrus of the hippocampus, BDNF was expressed in NeuN-positive neurons, similar to the pattern observed one day after MCAO (Figure 4).
BDNF expression in the parietal cortex and hippocampus three days after middle cerebral artery occlusion (MCAO). The results were quantified and presented normalized to actin-beta expression. *p<0.05. neg-ctrl: Negative control.
Immunofluorescent staining for BDNF in parietal cortex and dentate gyrus of hippocampus three days after middle cerebral artery occlusion (MCAO).
Discussion
We studied the expression of endogenous BDNF in the parietal cortex and hippocampus. The amount of exogenous BDNF that permeates the brain parenchyma is very small because it does not easily pass through the blood-brain barrier (BBB) (17). Endogenous BDNF expression plays a key role in the repair of damaged neurons (18). BDNF induces neuronal cell proliferation, maturation, and repair following ischemic insult (19, 20).
In the cortex, BDNF protein expression was lower in the MCAO group at one and three days after MCAO. One and three days after MCAO represent the acute phase of stroke (21). During this phase, neuroinflammation occurs due to BBB leakage (22). The NF-κB pathway regulates proinflammatory cytokine release through the BDNF/ERK/REB pathway (23). This study suggested that the BDNF reduction in the parietal cortex, similar to our results, is associated with inflammatory dysregulation. One study showed that BDNF expression levels and TrkB signaling in the cortex of rats after MCAO regulate apoptosis-related protein expression (24). Another study reported that BDNF activates PI3K/Akt signaling and antagonizes proapoptotic actions (25). Wu et al. reported that the anti-apoptotic effect of BDNF activation in an ischemic stroke model improves motor behavior (26). These studies have shown that BDNF in the cortex of the ischemic region is related to apoptotic reactions and affects neuronal cells in the motor cortex. We have previously reported that BDNF is expressed in mature neurons in the motor cortex. Zhang et al. showed that BDNF down-regulation affects the migration of neurons from the subventricular zone to the cortex via the AKT/mTOR signaling pathway (27). Another study showed that newborn cell (BrdU-positive) survival in the penumbra of the cortex after MCAO induction is affected by BDNF expression (28). Espinera et al. reported that BDNF activation enhances neurovascular regeneration and motor recovery after ischemic stroke in mice (29). We suggest that BDNF protein expression in the motor cortex regulates the survival of mature neurons and is associated with motor function.
In the hippocampus, the expression of BDNF protein was lower in the MCAO group at one and three days after MCAO, similar to that in the cortex. Interestingly, BDNF protein expression in the control group was very high in the hippocampus.
It has been noted that BDNF is highly expressed in the hippocampus region (30). Zhu et al. reported that the reduction in BDNF expression in rats subjected to MCAO involves neuroinflammation and oxidative stress (31). Yi et al. showed that BDNF levels decreased after MCAO, similar to our results, and up-regulation of the expression of BDNF and CREB proteins in the hippocampus improved cognitive dysfunction (32). Other studies have reported that MCAO induces neuronal apoptosis by altering the expression of BDNF and apoptotic genes in the hippo-campus (33).
We showed that BDNF is expressed in mature neurons in the dentate gyrus of the hippocampus. The dentate gyrus is a neurogenic region of the forebrain (34). Taei et al. reported that BDNF expression regulates neuronal survival in the dentate gyrus and neuronal cell migration (35). Another study showed that activation of the BDNF receptor was related to neurogenesis in the dentate gyrus and improved learning and memory dysfunction after MCAO (36). Our results suggested that MCAO reduces BDNF expression in the hippocampus and affects neuronal survival in the hippocampal dentate gyrus.
Although we examined the expression level of BDNF protein after MCAO, this study did not include motor or cognitive behavioral tests. Further studies are needed to examine behavioral changes after MCAO.
Conclusion
MCAO in mice reduced the expression of BDNF in mature neurons of the motor cortex and hippocampus one and three days after surgery.
Acknowledgements
This study was supported by research funds from the Chosun University (2023).
Footnotes
Authors’ Contributions
YHJ designed the study. DJK, HKS, and YYC performed the surgical procedures. HIH analyzed the data. HIH performed the western blot analyses. All the Authors approved the final manuscript.
Conflicts of Interest
The Authors reported that they have no competing interests in relation to this study.
- Received January 26, 2025.
- Revision received February 18, 2025.
- Accepted February 19, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
