Abstract
Background/Aim: Neurogenesis is an important process in the recovery from neurological damage caused by ischemic lesions. Endogenous neurogenesis is insufficient to restore neuronal damage following cerebral ischemia. Dexmedetomidine (DEX) exerts neuroprotective effects against cerebral ischemia and ischemia/reperfusion injury. DEX promotes neurogenesis, including neuronal proliferation and maturation in the hippocampus. In a previous study, we showed that early neurogenesis increased 3 days after bilateral common carotid artery occlusion (BCCAO). In this study, we investigated the effect of DEX on neurogenesis 3 days after BCCAO. Materials and Methods: Male Sprague–Dawley (SD) rats (7-8 weeks old) were used as a BCCAO model. Right and left common carotid arteries of the rats were occluded using 4-0 silk sutures. Two hours after surgery, an intracranial DEX injection was administered to rats that underwent surgery using a stereotaxic injector. Brains were obtained from control and BCCAO rats 3 days after surgery. Immunohistochemistry was performed on the cortex and dentate gyrus of the hippocampus using a NeuN antibody. Western blot was performed with HIF1α and brain-derived neurotrophic factor (BDNF) antibodies. Results: The number of mature neurons decreased 3 days after BCCAO, but DEX treatment alleviated neural loss in the parietal cortex and hippocampus. Up-regulation of BDNF was also observed after dexmedetomidine treatment. Conclusion: Stereotaxic injection of dexmedetomidine alleviates neural loss following BCCAO by up-regulating BDNF expression.
The bilateral common carotid artery occlusion (BCCAO) model has been used to study chronic cerebral hypoperfusion (1, 2). Cerebral hypoperfusion causes neuronal loss as well as learning and memory impairment (3, 4). Neurogenesis is an important process for recovery from neurological damage caused by ischemic lesions (5, 6). Neurogenesis is a complex process involving the proliferation, differentiation, and maturation of neural progenitor cells (7). Endogenous neurogenesis occurs in the hippocampus and cortex during cerebral hypoperfusion (8). However, neurogenesis is insufficient to restore neuronal damage after cerebral ischemia (9).
Dexmedetomidine (DEX) is an α2-adrenoceptor agonist used in general anesthesia, sedative, and intensive care (10, 11). DEX exerts neuroprotective effects against cerebral ischemia or ischemia/reperfusion injury (12). DEX-induced neurogenesis protects against cerebral hypoperfusion (13). DEX induces hippocampal neurogenesis and improves spatial learning and memory in neonatal 7-day rats (14). In another study, DEX promoted hippocampal neurogenesis, including neuronal proliferation and maturation (15). Lu et al. showed that DEX inhibited apoptosis and improved cell proliferation in ketamine-induced injury (16).
In a previous study, we showed that early neurogenesis increased 3 days after BCCAO (17). In this study, we investigated the effect of DEX on neurogenesis 3 days after BCCAO.
Materials and Methods
Animal surgery. Male Sprague–Dawley (SD) rats (7-8 weeks old) were supplied by a certified breeder (Damul Laboratory Animals, Daejeon, Republic of Korea). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Chosun University. BCCAO was performed as previously described (18). Briefly, sevoflurane was used for inhalation anesthesia (1.0%-2.0%, end-tidal concentration), the neck midline was dissected, and the carotid artery position was confirmed. The right and left common carotid arteries were occluded using 4-0 silk sutures. Two hours after surgery, an intracranial DEX injection was administered to rats that underwent surgery using a stereotaxic injector. The concentration of DEX was 0.1 μg/μl and the total volume was 5 μl. The location of the injection was 1.0 mm lateral and 1.0 mm posterior to the bregma and at a 3.0 mm depth from the skull surface. The injection duration was 5 min, as in the previous study (19). The rats that did not undergo surgery were included in the control group (n=22). The rats which underwent surgery comprised the BCCAO group (n=22). Rats treated with DEX were designated as the DEX-treated group (n=18).
Immunohistochemistry. Brains were obtained from control and BCCAO rats 3 days after surgery (control group n=17, BCCAO group n=15, DEX-treated group=12). Brains were fixed with a 4% paraformaldehyde solution for 3 days at 4°C. Paraffin sections were prepared using fixed brains as described previously (20). The cerebrums were separated from the brain stem and were cut at sagittal plane intervals of 7-8 μm. The sections were deparaffinized with xylene and washed with 0.1 M phosphate-buffered saline (pH 7.4). Antigen retrieval was performed with 0.01 M sodium citrate buffer (pH 6.0) in a microwave oven for 1 h. After cooling, the sections were placed in a jar filled with 0.3% hydrogen peroxide to block the endogenous peroxidase activity. The brains were incubated with mouse anti-neuronal nuclear protein (NeuN, 1:100; Millipore, Burlington, MA, USA) overnight at 4°C. On the second day, an avidin-biotin-peroxidase (ABC) detection system (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA, USA) was used to detect immuno-reactivity. Slides were counterstained with thionine and mounted (Polysciences, Warrington, PA, USA). The NeuN-immunoreactive cells (IR) were viewed under a light microscope (BX41; Olympus). The number of NeuN-IR cells in the cortex and hippocampus were counted and quantified as previously described (21). Two investigators manually calculated the number of NeuN-IR cells.
Western blot analysis. The cerebral cortex and hippocampus of Sprague-Dawley rats in the control and BCCAO groups were isolated under anesthesia 3 days after surgery (control group n=5, BCCAO group n=7, DEX-treated group=6). The isolated tissues were homogenized within RIPA buffer. The total proteins were separated on SDS-PAGE, transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA), and then probed with the primary antibodies, mouse anti-β-actin (1:1,000; Santa Cruz, CA, USA), and rabbit anti-BDNF (1:1,000; Abcam, Cambridge, UK) rabbit anti-HIF1α (1:1,000; Abnova, Taipei, Taiwan, ROC), followed by HRP-conjugated secondary antibodies.
Cytotoxicity analysis. Neuroblastoma (SH-SY5Y) cells were treated with 200 μM of Cobalt (II) chloride hexahydrate (CoCl2•6H2O) and DEX (SML0956, sigma, St. Louis, MO, USA) by concentration and incubated at 37°C, 5% CO2 for 24 h to test cytotoxicity. The viability of SH-SY5Y cells was determined by using the EZ-Cytox Assay Kit (EZ-500, DOGENBIO, Seoul, Republic of Korea), according to the manufacturer’s instructions. The absorbance was measured at 450 nm by microplate spectrophotometer.
Statistical analysis. All data were analyzed using the Statistical Package for Social Sciences (Information Analysis Systems, SPSS). All data were expressed as the mean±standard error of the mean. Statistical significance was set at 0.05. We performed the Mann-Whitney U-test with Bonferroni correction to compare the mean values between the control, BCCAO, and DEX-treated groups.
Results
The viability of SH-SY5Y cells was decreased after treatment with CoCl2•6H2O. However, after DEX treatment, the viability of SH-SY5Y cells increased (Figure 1).
Effects of dexmedetomidine on the viability of SH-SY5Y cells. The viability of SH-SY5Y cells is increased with respect to cells treated with CoCl2•6H2O. The results are shown as mean±SEM.
Cortex findings. Expression of HIF1α, a hypoxic status marker was increased in BCCAO and DEX-treated group 3 days after surgery (Figure 2). Similarly, the proportion of cells (NeuN-IR cells/total cells) in the parietal cortex was increased compared to that in the BCCAO group (Figure 3). BDNF expression in the western blot was induced in the DEX-treated group 3 days after surgery (Figure 2).
HIF1A and BDNF expression measured by western blot in the cortex. NC: Negative control. BCCAO: Bilateral common carotid artery occlusion. DEX: Dexmedetomidine.
Representative photograph and proportion of NeuN-positive cells on immunohistochemistry in the cerebral parietal cortex (A). Data are expressed as mean and standard error values (B). Significantly different at *p<0.05 compared to control and BCCAO. BCCAO: Bilateral common carotid artery occlusion. DEX: Dexmedetomidine.
Hippocampus findings. Similar to the cortex, the expression of HIF1α in the western blot was increased in BCCAO and DEX-treated groups (Figure 4). In the dentate gyrus, the proportion of cells (NeuN-IR cells/total cells) in the DEX-treated group was higher than that in the BCCAO group (Figure 5). BDNF expression was higher in the DEX-treated group than that in the BCCAO group (Figure 4).
HIF1A and BDNF expression measured by western blot in the hippocampus. HIF1A and BDNF expression in the western blot are induced in the DEX-treated group 3 days after surgery. DEX: Dexmedetomidine.
Representative photograph and proportion of NeuN-positive cells on immunohistochemistry in the dentate gyrus of the hippocampus (A). Data are expressed as mean and standard error values (B). Significantly different at p<0.05 compared to the *control and BCCAO. BCCAO: Bilateral common carotid artery occlusion. DEX: Dexmedetomidine.
Discussion
The expression of HIF1α in the cortex and hippocampus was increased in the BCCAO and DEX-treated groups. HIF1α increased response to chronic cerebral hypoperfusion (22, 23). Adult neurogenesis including neuron maturation occurred in the dentate gyrus of the hippocampus and cortex after chronic cerebral hypoperfusion (24, 25).
In the DEX-treated group, the proportion of NeuN-IR cells was higher than that in the BCCAO group. NeuN has been used as a marker to confirm mature neurons (26) and study neurogenesis in adults (27). As mentioned above, DEX exerts neuroprotective effects against cerebral hypoperfusion injury. Chen et al. showed that DEX treatment increases the number of newly formed neurons in the subventricular zone of hypoxic-ischemic neonatal rats (28). Another study reported that the systemic administration of DEX increases the number of NeuN-positive cells in the dentate gyrus (29). The increase in neurons induced by DEX treatment in neurogenic zones, such as the cortex and hippocampus, is related to the improvement in neurogenesis after DEX administration. Sha et al. reported that pretreatment with a high dose (10 μg/kg) of DEX induced the proliferation and differentiation of neuronal stem cells after repeated ketamine exposure (30). Taha et al. reported that DEX alleviated memory deficits by improving hippocampal neurogenesis via the ROCK-1/ERK1/2/CREB/BDNF pathway (31).
Studies have shown that dexmedetomidine reduces neuronal injury by up-regulating BDNF expression (32, 33). This reduction was related to improving adult neurogenesis and cognitive impairment (34, 35). In our study, BDNF expression was higher in the DEX-treated group than in the BCCAO group. In a permanent BCCAO model, cerebral blood flow decreased within 2-3 days after surgery (36). Li et al. showed that treatment with DEX increased BDNF expression after 3 days of ischemia/reperfusion injury but not after 6 h or 1 day (37). In another transient cerebral ischemia/reperfusion model, DEX induced neuronal survival in the hippocampus and cortex by up-regulating the PI3K/AKT and ERK pathways (38). In our previous studies, both AKT and ERK expression decreased with BDNF down-regulation under prenatal hypoxic conditions (20, 39). We propose that DEX treatment induces high BDNF expression in BCCAO.
Conclusion
The number of mature neurons decreased 3 days after BCCAO, but DEX treatment alleviated neural loss in the parietal cortex and hippocampus. Up-regulation of BDNF was also observed after DEX treatment. Regarding the effect of BDNF on neuronal survival, stereotaxic injection of DEX alleviated neural loss following BCCAO by up-regulating BDNF expression.
Footnotes
Authors’ Contributions
HSK, YHJ and YYC designed the study. DJK, YJS, and SYC performed the surgical procedures. HIH and HKS analyzed the data. HIH performed the western blot analyses. All the Authors have approved the final manuscript.
Conflicts of Interest
The Authors declare no competing interests regarding this study.
- Received September 25, 2023.
- Revision received October 26, 2023.
- Accepted October 27, 2023.
- Copyright © 2024 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).
