Bacterial collagenase-induced intracerebral hemorrhage in mouse: The role of matrix metalloproteinases and cell death mechanisms following injury

Intracerebral hemorrhage (ICH) initiates an inflammatory response with secondary growth of the hemorrhage and cell death. Matrix metalloproteinases (MMPs) are increased in ICH, and synthetic inhibitors to MMPs reduce edema and hemorrhage size. Recently, we found that tissue inhibitor of metalloproteinase-3 (TIMP-3) is elevated following ischemia and co-localizes with terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-positive cells. Additionally, inhibition of MMPs by endogenous and synthetic MMP inhibitors enhances apoptosis by blocking the shedding of the TNF superfamily of death receptors/ligands by stromelysin-1 (MMP-3) in an in vitro model of neuronal apoptosis. The effect of TIMP-3 and synthetic MMP inhibitors on cell death in ICH is unclear. Therefore, we used the bacterial collagenase (BC)-induced intracerebral hemorrhage (CIH) model in C57Bl/6 mice and studied MMP expression by zymography and PCR, and measured hemorrhage volume and cell death with unbiased stereology. Real-time PCR showed similar expression of mRNA for Mmp-2 and -9 in BC- and saline-injected mice, but a significant increase in Mmp-3 mRNA. Protein levels of MMP-3 and gelatinolytic activity of MMP-2 and -9, as assessed by zymography, were elevated in BC-injected mice at 72 h. Caspase-3 levels were elevated at 2 and 72 h and active caspase-8 was detected by 2 and 24 h. We used the Timp-3 null mouse and its corresponding wild-type to determine the role of TIMP-3, and found no differences in hemorrhage size and TUNEL-positive cells. Conversely, the broad-spectrum MMP inhibitor, BB-94, increased both hemorrhage size and TUNEL-positive cells. Our results suggest that TIMP-3 is not induced in the CIH model and that in the absence of TIMP-3, a synthetic inhibitor of MMPs increases apoptosis in CIH, possibly by preventing the shedding of death receptors and/or their ligands by MMP-3.