İnvastigation of the neuroprotective effects of combined treatment of riociguat and resveratrol after experimental cerebral ischemia i̇n rats

Introduction and Aim: Stroke, which is defined as functional dysfunction of the brain that occurs without any cause other than vascular pathologies, is among the most important causes of mortality and morbidity. It is divided into two groups as hemorrhagic and ischemic. The majority of ischemic strokes, which account for approximately 88% of strokes, occur in areas fed by MCA. Today, two treatment approaches come to the fore in the treatment of ischemic stroke. These are neuroprotection and recanalization. The only medical therapy approved in recanalization is rtPA. Since it can be used in the first 4.5 hours after ischemic stroke, its usage is quite limited. Therefore, studies are continuing for treatments that may have wider use. Many complex mechanisms play a role in ischemic stroke. Neuroprotection is the effort to protect the brain against ischemia by inhibiting or slowing down these molecular and biochemical events. For this purpose, anti-excitotoxicity, antioxidant, anti-inflammatory and anti-apoptotic agents were used in the treatment. Resveratrol, which is mainly found in grapes, red wine, and peanuts, is a phytoalexin with many effects such as neuroprotective, anti-inflammatory, anticarcinogenic, cardioprotective. Riociguat, a guanylatecyclase stimulator that causes relaxation of vascular smooth muscle, is a vasodilator that has not yet been studied in cerebral ischemia, which reduces the ischemic area in cardiac ischemia studies. We investigated the effects of these two drug combinations after cerebral ischemia. Materials and Methods: In our study, 35 Wistar albino female rats with randomly selected weights of 328.53 ± 47.20 were used. After anesthesia with 80 mg/kg ketamine and 10 mg/kg xylazine, MCA occlusion was created by advancing the suture directed to the internal carotid artery by entering through the right external carotid artery. After waiting for 2 hours and creating ischemia, the suture was withdrawn and reperfusion was provided. Rats were divided into 5 and each group involves 7 rats. Group 1 was the group had no procedure, Group 2 was the ischemia group, Group 3 was the group given riociguat after ischemia, Group 4 was the group given resveratrol after ischemia, and finally Group 5 was the group given resveratrol + riociguat after ischemia. Resveratrol (p.o.) was given at a dose of 30 mg/kg/day and riociguat (i.p.) was given at a dose of 10 mg/kg/day for 2 days. Intracardiac blood was taken by thoracotomy method before sacrification of the rats which were re-anesthetized after the treatments. Right hemispheres were fixed in 10% formaldehyde at room temperature. Sections taken were stained with hematoxylin & eosin. Pycnosis in the cell nuclei, perivascular and perineural edema, vascular dilatation, congestion, and bleeding foci after reperfusion were evaluated in the sections. In addition, the sections taken were embedded in paraffin blocks and bax and bcl-2 antibodies were dripped on them to observe signal formation. Results: In our study, hemorrhagic areas in brain tissue, pycnosis in neuron and glial cell nuclei, perineural and perivascular edema, congestion in vessel lumens, bax and bcl-2 expressions were examined. In the ischemia group, focal and diffuse hemorrhagic areas, increase in pycnotic nucleated cells in neuron and glial cells, perineural edema, congestion in the vessel lumen, over-expression of bax and increase in bcl-2 immunopositivity were observed. In the group given riociguat after ischemia, it was observed that hemorrhagic areas disappeared, pycnotic cells in neurons and glial cells were considerably reduced, vessels were dilated, congestion was not observed, perivascular edema was considerably reduced, bax immunopositivity was reduced and was similar to the control group, and bcl-2 immunopositivity increased especially in axons. In the group given resveratrol after ischemia, although hemorrhagic areas decreased significantly, congestion, perineural edema, and pycnosis in neurons and glial cells were observed, it was considerably reduced compared to the ischemia group. Bax immunopositivity was similar to the control group, while bcl-2 immunopositivity was localized similar to the control and ischemia groups, but positivity was found to be closer to the control group, especially in nerve fibers. In the group given riociguat and resveratrol after ischemia, it was observed that the vessels were dilated, the cells with pycnotic nuclei in neurons and glial cells decreased, and the perineural and perivascular areas disappeared. While bax immunopositivity was negligible in the combined treatment group, bcl-2 immunopositivity was found to be quite intense compared to the ischemia groups. Conclusion: It has been seen that riociguat and resveratrol combined treatment in ischemic stroke is neuroprotective, prevents apoptosis and reduces post-reperfusion bleeding, and it has been concluded that it can be used in ischemic stroke treatment protocols. Keywords: Ischemia, Riociguat, Resveratrol, Neuroprotective, Apoptosis