Investigation of otulin and spexin immunereactivity in experimental ichemia-reperfusion damaged rat brain tissue

ABSTRACT INVESTIGATION OF OTULIN AND SPEXIN IMMUNEREACTIVITY IN EXPERIMENTAL ICHEMIA-REPERFUSION DAMAGED RAT BRAIN TISSUE Stroke is a syndrome characterized by focal cerebral function loss resulting from vascular causes. The clinical presentation can vary greatly, ranging from mild symptoms that can resolve within days to severe cases with significant disabilities or even fatal outcomes. Stroke is among the leading causes of death in the community and the primary cause of disability. The pathophysiology of stroke involves neuronal necrosis, apoptosis, and neuroinflammation. Although the mechanism of neuroinflammation is not fully understood, it is believed that OTULIN and spectrin molecules also play a role in neuroinflammation and their levels are thought to increase. Inhibition of excessive inflammatory response in the early stage of acute ischemic stroke may protect neuronal cells and improve neurological outcomes. This study aims to investigate the serum levels and immunoreactivity of OTULIN and spectrin in rat brain tissue with experimental ischemia-reperfusion injury. A total of 35 adult male Sprague-Dawley rats aged 8-12 weeks were randomly divided into five groups: control group, sham group, I/R 2-hour group, I/R 12-hour group, and I/R 24-hour group. The control group received no intervention throughout the experiment. In the sham group, after observing bilateral common carotid arteries through a midline cervical incision, they were closed and the rats were decapitated at the 4th hour. In the I/R 2-hour, I/R 12-hour, and I/R 24-hour groups, after observing bilateral common carotid arteries, a surgical thread was advanced into the internal carotid artery to occlude the middle cerebral artery for 120 minutes. Reperfusion was then achieved at 2 hours, 12 hours, and 24 hours, respectively. At the end of the experiment, all rats in each group were decapitated under anesthesia with ketamine and xylazine. The tissues were rapidly removed, fixed with appropriate fixatives, processed through histological follow-up series, and embedded in paraffin blocks. Immunohistochemical staining for OTULIN and spectrin and TUNEL staining for apoptosis were performed on tissue sections obtained from the paraffin blocks. In addition, blood samples were centrifuged to obtain serum, and the levels of OTULIN, spectrin, and total oxidative status (TOS) were measured using the ELISA method. In our study, when the serum levels of OTULIN and spectrin were biochemically examined in the control, sham, I/R 2-hour, I/R 12-hour, and I/R 24-hour groups, the control, sham, and I/R 2-hour groups were similar. However, significant increases were observed in the I/R 12-hour and I/R 24-hour groups compared to the control group, while there was no significant difference between the I/R 12-hour and I/R 24-hour groups. Brain tissue was immunohistochemically stained for OTULIN and spectrin to evaluate their immunoreactivity. Similar staining patterns were observed in the control, sham, and I/R 2-hour groups. In the I/R 12-hour and I/R 24-hour groups, immunoreactivity of OTULIN and spectrin was significantly increased compared to the control group. There was no statistically significant difference between the I/R 12-hour and I/R 24-hour groups. In conclusion, experimental ischemia-reperfusion injury increased the levels of OTULIN and spectrin in brain tissue, suggesting their potential role in the pathophysiology of ischemia-reperfusion-induced brain tissue damage. Further extensive studies on OTULIN and spectrin in ischemic stroke may contribute to understanding the pathophysiology of stroke and even the development of treatment. Key words: Brain, ischemia/reperfusion injury, OTULIN, speksin