Inhibition of gelatinase activity reduces neural injury in an ex vivo model of hypoxia-ischemia.

Perinatal hypoxia-ischemia (H-I) often manifests as cognitive and/or motor disturbances that appear early in development. Growing evidence indicates that neuroinflammation may exacerbate H-I injury. Resident microglia release proinflammatory cytokines and proteases in response to ischemia. Matrix metalloproteinases (MMPs), in particular, activate cytokines and degrade basement membrane proteins. These actions ultimately permit entry of peripheral leukocytes into the CNS neuropil, enhancing neuroinflammation and cell death. Currently, the relative contributions of resident and peripheral immune cells to ischemic brain injury are unclear. The present study employed an ex vivo model of H-I through oxygen glucose deprivation (OGD) to identify the cellular localization of MMP-9 in organotypic hippocampal slices from rat, and to determine whether inhibiting gelatin-degrading MMPs affords neuroprotection in the absence of peripheral immune cells. Immunohistochemistry revealed ubiquitous neuronal MMP-9 expression in both normoxic and hypoxic slices. Increased MMP-9 expression was detected in CD11b-positive microglia after 48 h exposure to OGD relative to normoxic controls. Consistent with these data, in situ zymography showed increased gelatinolytic activity after OGD. Gelatin-cleaved fluorescence localized to astrocytic processes and somata of various cellular morphologies. Treatment with either the MMP inhibitor AG3340 (prinomastat) or minocycline dampened OGD-induced gelatinolytic activity and neural injury, as measured by Fluoro-Jade staining, relative to vehicle controls. These results show that resident microglia, in the absence of peripheral immune cells, were sufficient to enhance neural injury after OGD in the organotypic hippocampal slice. Additionally, these effects were associated with upregulation or secretion of MMP-9, and were blocked after treatment with either the gelatinase-selective compound AG3340 or the anti-inflammatory compound minocycline. These data, coupled with the effectiveness of these compounds previously shown in vivo, support the selective targeting of gelatin-degrading MMPs and activated microglia as potential therapeutic approaches to combat neonatal H-I injury.