Investigation of neuroprotective potential of visnagin at different doses in a cerebral ischemia reperfusion injury model

Introduction and Aim: Cerebral ischemia is the interruption of 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; It 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 MCA. Currently, two types of treatment approaches are prominent 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 use is limited. Therefore, its use is being studied on wider treatment protocols. A number of different mechanisms play a role in ischemic stroke. Neuroprotection is the effort to protect the brain against ischemia by preventing or slowing down these biochemical and molecular events. Many antioxidant, anti-inflammatory, antiapoptotic and anticytotoxic agents have been used for this purpose. Visnagin furanochromone, which is a compound derivative of cromon and furan, is found in high amounts in the hiltan plant, also known as toothpick grass among the people. The hiltan plant, whose Latin name is Ammi visnaga, is among the edible wild plants and is found in countries such as Turkey and Morocco and is used extensively in traditional medicine in the society. In the toxicological studies, it was reported that toothpick, which is the major source of visnagin, did not show a toxicological effect in experimental animals even at high doses. In recent studies with this herbal product, it has been reported that visnagin has anti-inflammatory potential and has a vasodilator effect. In our study, we investigated the protective effect of visnagin's neuroprotective effect after cerebral ischemia reperfusion in low, medium and high dose applications determined by considering the literature. Materials and Methods: In this study, randomly selected 35 female Sprague Dawley rats weighing 328.53 ± 47.20gr were used. After anesthesia with 10 mg/kg xylazine and 80 mg/kg ketamine, the suture directed towards the internal carotid artery by entering from the right common carotid artery was advanced from the internal carotid artery to oclude the MCA. After 90 minutes of ischemia, the suture was withdrawn and reperfusion was achieved. Rats were divided into 5 groups of 7. 1st Group: no treatment group, 2nd Group: ischemia-induced group, 3rd Group: 10mg/kg Visnagin group given as low dose after ischemia, 4th Group: 30mg/kg visnagin group which is considered medium dose after ischemia, and 5 Group: 60mg/kg Visnagin, which we accepted as high dose after ischemia, was determined as the group. After the first dose of reperfusion was achieved, other doses of visnagin were given 24 hours apart, in total, 3 doses. After all visnagin treatment doses were administered, the rats were re-anesthetized and their intracardiac blood was taken first by thoracotomy. Then the rats were sacrificed and the 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 Bax, TNF-α, CD86 and CD163 antibodies were dripped on them and the resulting signals were observed. Results: In our experiment, hemorrhagic areas in brain tissue, perineural edema, congestion in vascular beds, pycnosis in neuron and glial cell nuclei, Bax, TNF-α CD86 and CD163 expressions as immunohistochemistry were examined. Diffuse and focal and hemorrhagic areas, increase in the amount of pycnotic nuclei in neurons and glia, congestion in the vascular lumen, perineural edema, increase in Bax level, decrease in CD163 immunopositivity, increase in TNF-α and CD86 levels were found in the ischemia group. In the group given 10mg/kg visnagin after ischemia, the hemorrhage areas partially disappeared, the cells with pycnotic nuclei in neuron and glial cells were considerably reduced, the perineural edema was partially reduced, the Bax level, CD86, CD163 immunopositivity and TNF-α expression were similar to the ischemia group. In the group given 30mg/kg visnagin after ischemia, the areas of hemorrhage were significantly reduced, and although perineural edema, pycnosis in neurons and glial cells were observed, this rate was considerably reduced compared to the ischemia group. It was observed that Bax level decreased, CD86 immunopositivity decreased compared to the group given 10mg visnagin, CD163 immunopositivity increased partially, and TNF-α expression decreased. In the group given 60mg/kg visnagin after ischemia, it was observed that the subarachnoid hemorrhage traces were almost completely eliminated, the blood tissue cells 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. In the group given 60mg/kg visnagin treatment, Bax level was very close to the control group, CD163 immunopositivity was increased especially in edematous and hemorrhagic brain tissue areas, and it was similar to the control group, and there was a significant decrease in CD86 immunopositivity. It was observed that TNF-α level was expressed very low. Conclusion: It has been observed that visnagin treatment is neuroprotective, reduces post-reperfusion bleeding and prevents apoptosis in cerebral ischemic stroke reperfusion injury. Therefore, it was concluded that it can be used in ischemic stroke reperfusion injury treatment protocols. Key words: ischemia, reperfusion, apoptosis, visnagin, neuroprotective