Investigation of neuroprotective effects of urapidil and resveratrol combined treatment after experimental cerebral ischemia in rats

Introduction and Aim: Cerebral ischemia is the inability to maintain the oxygenation required for neural tissues secondary to decreased blood flow to the brain. Ischemic stroke is among the most important causes of morbidity and mortality. Stroke is divided into hemorrhagic stroke and ischemic stroke. Ischemic stroke accounts for approximately 88% of strokes. The majority of ischemic strokes occur in areas fed by MCA. Currently, two treatment approaches stand out in ischemic stroke. These are recanalization and neuroprotection. The only accepted medical treatment option in recanalization is rtPA. Since rtPA can be used in the first 4.5 hours in ischemic stroke, its usage is limited. Therefore, treatments that may be used more broadly are being studied. A number of complex mechanisms play a role in ischemic stroke. Neuroprotection is the effort to protect the brain against ischemia by inhibiting or slowing down these biochemical and molecular events. Many antioxidant, antiapoptotic, antiexcitotoxic and anti-inflammatory agents have been used for this purpose. Resveratrol, which is mainly found in grapes and peanuts; is a phytoalexin with many effects such as anticarcinogenic, anti-inflammatory, neuroprotective and cardioprotective. Urapidil, a sympatholytic antihypertensive drug, is a vasodilator, although there are findings in the studies that it can be used as a protective agent when applied before reperfusion in ischemic cases with its vasodilator effect, and there are also findings that urapidil treatment in ischemia is protective by showing antioxidant, anti-inflammatory and antiapoptotic activities at the cellular level has not yet been studied in cerebral ischemia,. We also investigated the effects of the combination of these two drugs after cerebral ischemia. Materials and Methods: In our study, 35 randomly selected female Sprague Dawley rats with a weight of 328.53 ± 47.20gr were used. After anesthetization with 10 mg/kg xylazine and 80 mg/kg ketamine, the suture which was directed towards the internal carotid artery by entering from the right external carotid artery was advanced through the internal carotid artery and MCA occlusion was achieved. After waiting for 2 hours, ischemia was created, the suture was withdrawn and reperfusion was provided. The rats were divided into 5 groups of 7. The first group was the group that did not undergo any procedure, the second group was the ischemia-induced group, the third group was given urapidil after ischemia, the fourth group given resveratrol after ischemia, and the fifth group was given urapidil + resveratrol combined after ischemia. Resveratrol (i.p.) was given at a dose of 30 mg/kg/day and urapidil (i.p.) was given at a dose of 5 mg/kg/day for 3 days. After the treatment, the rats were re-anesthetized and their intracardiac blood was taken by thoracotomy. The rats were sacrificed and their brain tissues were removed under sterile conditions. The excised tissues were fixed in 10% formaldehyde at room temperature. The obtained sections were stained with hematoxylin & eosin. Congestion, perineural and perivascular edema, pycnosis in the cell nuclei, and bleeding foci after reperfusion were evaluated in the sections. The sections taken were also embedded in paraffin blocks, and the signals formed by dropping Bcl-2, TNF-α and caspase-3 antibodies on them were observed. Results: In our experiment, hemorrhagic areas in brain tissue, perineural edema, congestion in vascular beds, pycnosis in neuron and glial cell nuclei, caspase-3, Bcl-2 and TNF-α expressions were examined. In the ischemia group, diffuse and focal and hemorrhagic areas, an increase in the amount of pycnotic nuclei in neurons and glia, congestion in the vascular lumen, perineural edema, an increase in caspase-3 levels, a decrease in Bcl-2 immunopositivity and an increase in TNF-α levels were detected. In the group given urapidil after ischemia, it was observed that hemorrhage areas disappeared, pycnotic nucleated cells in neuron and glial cells were considerably reduced, rerineural edema was considerably reduced, caspase-3 and Bcl-2 immunopositivity decreased, and TNF-α expression increased. After ischemia, it was observed that the hemorrhage areas were significantly reduced in the group given resveratrol, and although perineural edema and pycnosis were observed in the neurons and glial cells, this rate was considerably lower compared to the ischemia group. Caspase-3 immunopositivity was similar to the control group, while Bcl-2 and TNF-α expression were decreased. In the group given urapidil and resveratrol combined treatment after ischemia, it was observed that the traces of subarachnoid hemorrhage were almost completely eliminated, the blood tissue cells that transferred to the brain tissue were largely eliminated, the perineural edema disappeared, and the pycnotic nucleated cells in the neurons and glial cells were decreased. It was observed that the caspase-3 level was very close to the control group in the combined treatment group, Bcl-2 expression was partially increased, especially in the edematous and hemorrhagic brain tissue areas; also the TNF-α level was detected as very low. Keywords: Ischemia, Urapidil, Resveratrol, Neuroprotective, Apoptosis