The effect of the sildenafil citrate on cerebral vasospasm and apoptosis following experimental subarachnoid hemorrhage in rats

After fulfilling many clinical and experimental studies about the mechanism of cerebral vasospasm following subarachnoidal hemorrhage, it is thought that this pathologic disease to become after many connecting reactions and it is multifactorial. It is widely accepted that oxyhemoglobine, appearing following hemolysis of erythrocytes in the subarachnoidal space as a result of bleeding after rupture of an aneurysm, is the most important factor in the pathogenesis of vasospasm because of acting in many reactions causing the pathological process against the vascular and neuronal tissues. However since the discovery that nitric oxide, an endothelial derived relaxing factor has 1000 times higher affinity for hemoglobin than oxygen, it is put forward that decreased NO availibility following SAH is one of the most important factor in vasospasm(1). Vasoconstriction, vasospasm of the vessels and ischemia in the neurons and vascular tissue cells is the cause of inflammation, free radical production, lipid peroxidation and apoptosis developing after oxyhemoglobin and other blood products. Because of the damage in the vascular endothelial cells and production of free radicals, either decreasing NO availibility or running out of the present NO, cause of conctriction of the vessels. As we take into consideration of decreased levels of NO and antiinflamatuar, vasodilatation, antioxidan and cerebral blood flow regulation effect of NO; we thought that increasing the amount of NO availibility would be a good option in the treatment of vasospasm. We carried out our experimental study aiming at searching out the effect of sildenafil citrate PDE V inhibitor on cerebral vasospasm, which are currently approved for the treatment of erectil dysfunction and pulmonary hypertension by increasing NO-cGMP pathway. In this study 7 male Sprague-Dawley rats weighting between 245-550 grams were grouped into 4 groups. In group 2 ,3 and 4 the cisterna magna was aseptically punctured with a needle and 0.5 ml/kg of autologous arterial blood obtained by directly puncturing the femoral artery was slowly injected intracisternally. Group 1 (Control) rats were sacrificed and submitted to biochemical study without surgical manipulation. Group 3 and group 4 after administering the blood intracisternally, received 5 mg/kg and 15 mg/kg sildenafil respectively for 2 days, respectively. Estrogen has a neuroprotective effect by decreasing excitotoxicity, glutamate receptor activity and immun inflamation and by viii increasing axonal myelinization, synaptogenesis and dendtritic branch so that we used male rats in the experiment. All rats were decapitated following sacrification using high dose anesthetic agent on the second day. The rats basillary artery were sectioned from three seperate zones and three sections were obtained from each rat. Basillary artery luminal sectional areas were measured using freeware computerized image-analyse systems (ImageJ, version 1.341; National Institutes of Health, Bethesda, MD). For lipid peroxidation study cerebellum section samples from the rats fed by basillary artery were taken. The concentration of lipid preoxides was measured by thiobarbituric acid test. Immunohistology using the ApopTag Peroxidase In Situ Apoptosis Cell Detection Kit, POD (Roche, Germany) was used to demonstrate apoptosis in a cross section of basillary artery. Apoptotic index was calculated as the number of the immunoreactive nuclei per total number of endothelial cells, expressed as a percentage. No positive staining of endothelial cells with the ApopTag have been expressed as Grade 0, 0-24% immunostaining of endothelial cells as Grade 1, 25-49% as Grade 2, 50-74% as Grade 3, 75-100% immunostaining of endothelial cells have been expressed by Grade 4. Considering possible false positive results in TUNEL reaction in necrotic cells, each section was immunostained using for p53 (Clone Pab 240, mouse monoclonal antibody, NeoMarkers), caspase 3 (CPP32, NeoMarkers), caspase 8 (FLICE, NeoMarkers) and cytochrome c (Clone 7H8.2C12, mouse monoclonal antibody, NeoMarkers). Under light microscope examination, the positivity for all of these antibodies was calculated as positive and negative in the endothelial cells of basillary artery. In our atudy we used cytochrome c, representing the apoptotic mithocondrial pathway; caspase 8 respresenting the caspase dependent pathway, caspase 3 representing the both caspase dependent and independent pathway and p53 playing the essential role in the whole apoptotic cascade. In the SAH group(Group 2) the basillary artery circumferences constriction rate was 61.7% compared with the control group (Group 1). In the SC (5mg/kg)-treated SAH group, the basillary artery circumference contriction rate was 16.8% and in the SC-treated 15mg/kg SAH group this rate was 7.1% compared with the control group (Group 1). For the comparison of the effect of SC treatment after SAH, a Post Hoc Tukey test was performed between the groups. P value was less than 0.05 was considered significant. The results of group 1 between group 2, group 1 between group 3 and group 1 between group 4 ix was regarded as statistically significant (p<0.0001). As we take a look at the lipid peroxide results , lipid peroxide values in SAH group (group 2) was significantly higher compared with gruop 1(p<0.0001). the lipid peroxide values in the treatment groups (group 3 and group 4) was significantly decreased compared with group 2 (p<0.0001). We observed that sildenafil has prevented lipid peroxidation. The apoptotic cells rate, the immunostaining with In Situ Cell Detection Kit (POD (Roche, Germany)in the groups were 0%, 88%, 83% and 81%, respectively. With the immunostaining using for p53, caspase 3, caspase 8 and cytochrome c, there was no actual apoptotic nuclei in group 1. In group 2, 3 and 4 there was no significant difference in the number of apoptotic nuclei among these groups. In conclusion no preventing effect of sildenafil has been detected against apoptosis. In conclusion, we believe sildenafil will take a part in the treatment of vasospasm in the future because of improving effect on the vessels and preventing effect of lipid peroxidation. We think that impeding apoptosis is not enough to cure vasospasm and not enough to keep the cells alive; the main aim of the treatment and the most important issue is to hinder the chemical reactions of the blood cells in the subarachnoid region and lipid peroxidation and to increase NO availibility. Key Words: Subarachnoidal hemorrhage, Vasospasm, apoptosis, lipid peroxidation, sildenafil.