Mechanism Of Matrix Metalloproteinase 9 Involved In Early Brain Injury After Experimental Subarachnoid Hemorrhage

ObjectiveTo investigate the mechanism of early brain injury after SAH in rats by observing the expression of MMP-9 and the apoptosis of neurons in hippocampus and endothelial cells in basilar artery in early phase after SAH and analyzing the association between the expression of MMP-9 and clinical assessment, permeability of BBB and BWC.Methods1. Induction of SAH model in rats: three methods of SAH model were induced, and the amount of blood in the subarachnoid space and the mortality and morbidity were compared in order to choose the suitable SAH model for our study.2. Expression of MMP-9 mRNA and active MMP-9 were examined by RT-PCR and gelatin-containing SDS-PAGE zymography at different time points in hippocampus and basilar artery. Meanwhile, total MMP-9 protein in hippocampus was also examined by western blot.3. Laminin expression was examined in hippocampus and basilar artery by immunohistochemistry, and TUNEL staining was used to observe the apoptosis.4. BBB permeability was determined at different time points by measuring brain EB contents, and the brain edema was also examined by measuring BWC.5. CVS in early phase after SAH was also observed by measuring the wall thickness and calibrated diameter of basilar artery, and the morphus of neurons were observed by HE staining in hippocampal CA1 region.6. The clinical assessment was carried out at 24 h after SAH.7. The specific MMP-9 inhibitor SB-3CT was used to inhibite the MMP-9 activity, and the relationship among MMP-9 expression, apoptosis, BBB and brain edema were also observed. Minocycline was also injected to inhibit MMP-9 activity, and the neuroprotection to EBI was observed.Results1. The subarachnoid blood volume was close to the injected amount after prechiasmatic SAH. The mortality and morbidity in the prechiasmatic model were close to the clinical incidence.2. The expressions of the total and active protein and mRNA of MMP-9 increased dramatically at 12 h and reached the peak at 24 h, then decreased gradually at 48 h and 72 h which were still higher than that of sham group in hippocampus and basilar artery (P<0.05 or P<0.01).3. The immunohistochemistry showed that the laminin expression began to decrease at 12 h, and reached to the lowest at 24 h, then began to increase at 48 h and 72 h in hippocampus and basilar artery which were still lower than that of sham group (P<0.01).The examination of TUNEL showed that the number of neuronal apoptosis of hippocampus and endothelial cells of basilar artery increased at 12 h after SAH, reached peak at 24 h and began to decrease at 48 h and 72 h (P<0.01).4. Compared to sham group, there is no significant change in brain EB content and BWC at 6 h after SAH, but the increases in brain EB contents and BWC were found from 12 h to 72 h after SAH with a peak at 24 h.5. The number of degenerative neurons increased in hippocampal CA1 region from 12 h to 72 h after SAH (P<0.05, P<0.01); The normal neuronal density decreased significantly compared to sham group from 12 h (P<0.05) to 72 h and reached to the lowest at 24 h (P<0.01). Meanwhile, the morphological change of basilar artery observed by light microscopy showed that the calibrated diameter began to decrease and the wall thickness increased from 12 h after SAH and the acute CVS occured most significantly at 24 h which began to relieve at 48 h. At 72 h, the calibrated diameter and the wall thickness were no significant difference compared to sham group.6. Only few rats had neurological deficits, and no significant difference was observed among the groups (P>0.05). The appetite and activity scores in SB-3CT group were better than those in SAH and vehicle groups at 24 h (P<0.05).7. Expression of active and total MMP-9 was activated by SAH and inhibited by SB-3CT and minocycline at 24 h after SAH (P<0.01). The number of TUNEL-positive neurons in hippocampus were also decreased compared to sham group (P<0.01). And the morphological changes of hippocampal neurons improved.Conclusions1. The prechiasmatic SAH model is suitable for study of the pathophysiological mechanisms of EBI after SAH due to stimulating the clinical SAH better and high reproducibility and stability.2. MMP-9 may be involved in the pathological process of EBI after SAH, through mediating extracellular apoptotic pathway that is to degradate laminin leading to anoikis in neurons of hippocampus and endothelial cells of basilar artery in rats.3. SB-3CT injected at 24 h after SAH can inhibit expression of active and total MMP-9 protein. It may be a potential direction for brain protection after SAH by reducing anoikis and BBB permeability and improving morphous of hippocampal neurons of CA1 region.4. Minocycline can relieve the EBI after SAH, and attenuate the neuronal apoptosis which may be related to the inhibition of MMP-9 expression and block the extracellular apoptotic pathway.