Anti-apoptotic Effect Of Recombinant Osteopontin On Cerebral Vasospasm Following Subarachnoid Hemorrhage In Rats

Objective:To investigate the anti-apoptotic effect of recombinant OPN (r-OPN) agrainst cerebral vasospasm (CVS) following SAH in rats.Methods:Eighty male Sprague Dawley rats were randomly assigned to four groups (n=20): (1) sham+vehicle (n=20), (2) SAH+vehicle (n=20), (3) SAH+OPN0.03 (0.03μg) (n=20), (4) SAH+OPN0.1 (0.1μg) (n=20). The double injection method of cisterna magna was performed to establish the animal model of CVS after SAH. r-OPN was administered intraventricularly nearly 30 min after the first SAH. r-OPN (0.03 μg in 1μl phosphate buffer saline (PBS)) and r-OPN (0.1μg in 1 μ PBS) were injected respectively in the SAH+ OPN0.03 group and the SAH+OPN0.1 group, 1μl PBS was injected respectively in the sham +vehicle group and the SAH+vehicle group. At pre-SAH,24h,48h and 72h after the first SAH, neurological score assessment was performed respectively. At 72h after the first hemorrhage induction, rats were perfused through hearts before the brainstems were removed and postfixed. The cross-sectional area and thickness of basilar arteries (BA) were measured under Hematoxylin-eosin (H&E) staining to assess CVS. Apoptosis of endothelial cells and smooth muscle cells of basilar arteries were identified by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. Immunohistochemistry were used to assess the expression of p-Akt and cleaved caspase-3 in BA. Western blot analysis was applied to evaluate the expression of p-Akt, cleaved caspase-3, Bcl-2-associated X protein (Bax) and Bcl-2 in BA.Results:Compared with the sham+vehicle group, neurological scores in the SAH+vehicle group was significantly poorer at 24h,48h and 72h (P<0.01). High dose r-OPN significantly improved scores compared with the SAH+vehicle group at 48h and 72h (P<0.01). The cross-sectional area of BA significantly decreased in the SAH+vehicle group compared with the sham+vehicle group (P<0.01), while it was significantly increased by administration of 0.1μg r-OPN (P<0.01) and 0.03μg r-OPN (P<0.05) respectively compared with the SAH+ vehicle group. The thickness of the vessel walls significantly increased in the SAH+vehicle group compared with that in the sham+vehicle group (P<0.01). Both high dose and low dose r-OPN significantly decreased the thickness respectively compared with the SAH+vehicle group (P<0.01). In the SAH+vehicle group, the apoptotic index in BA endothelial cells was significantly elevated as compared to that in the sham+vehicle group (P<0.01), and was significantly declined in both SAH+OPN0.03 group and SAH+OPN0.1 group compared with that in the SAH+vehicle group(P0.01). Compared with the sham+vehicle group, the expression of cleaved caspase-3 and Bax were significantly increased and the expression of Bcl-2 was significantly decreased in the SAH+vehicle group (P<0.01), and the expression of p-Akt was significantly increased in the SAH+OPN0.1 group (P<0.01). Compared with the SAH+vehicle group, high dose r-OPN significantly reduced the expression of cleaved caspase-3 (P<0.01) and Bax (P<0.01) in BA following SAH, and increased the level of p-Akt (P<0.01) and Bcl-2 (P<0.01), low dose r-OPN also significantly decreased the expression of cleaved caspase-3 (P<0.01), and increased the expression of p-Akt (P<0.05).Conclusion:These results demonstrate that early administration of r-OPN can attenuate CVS after SAH. The underlying mechanism may involve the anti-apoptotic effect of r-OPN in endothelial cells and smooth muscle cells via the activation of Akt signaling pathway, the subsequent decreased expression of cleaved caspased-3 and Bax, the increased expression of Bcl-2.