Biomedical Sciences ETDs

Espen Walker

Publication Date

5-1-2009

Abstract

Transient global ischemia to the mouse results in hypoxic hypoperfusion to the brain and can lead to regional vulnerability, inflammation, and cell death. Characterization of this model with varying occlusion and reperfusion times allowed us to provide evidence for a consistent injury to the hippocampus and white matter regions of the brain. Following 30 minutes of bilateral carotid artery occlusion (BCAO) and 7 days of reperfusion, hippocampal injury to the CA2 region was consistently identified with neuronal loss and fluorojade positive cells. Three days following reperfusion, white matter injury was demonstrated by increased astrocyte reactivity and oligodendrocyte loss. With a consistent reduction in blood flow following BCAO and reliable cell loss, we confirmed this model for further investigation. In the hippocampus, we provide evidence for increased reactivity of astrocytes and microglia. With tissue inhibitor of metalloproteinase-3 (TIMP-3) expression in the astrocytes, we demonstrate a role for TIMP-3 in delayed neuronal death using a Timp-3 knockout (T3KO) mouse following 7 days of reperfusion. The increased activity of tumor necrosis factor (TNFα) activating enzyme (TACE) at 3 days correlated with reduced expression of the death receptor TNFR1 on the surface of the T3KO mouse neurons, leading to neuronal protection. Increased matrix metalloproteinase-3 (MMP-3) activity was observed and neuronal stereology in the CA2 region shows neuronal protection in the Mmp-3 KO mice. Increased reactive microglia and TNFα expression in the Mmp-3 WT was also observed. Delayed treatment with the broad-spectrum MMP inhibitor BB-94 led to neuronal protection in both the T3WT and T3KO mice, implicating TIMP-3 and MMP independent mechanisms of neuronal injury following global ischemia. The white matter has also shown sensitivity to global ischemia and reperfusion. To quantify oligodendrocyte (OLG) cell loss we have developed a method using a combination of morphology and immunohistochemistry to identify oligodendrocytes and apply it towards ischemic injury. We provide evidence for increased reactivity of astrocytes and microglia, with MMP-2 expression in the astrocytes. MMP-2 protein is increased at 1 day of reperfusion, with MMP-2 activity occurring at 3 days of reperfusion. This timing correlated with a breakdown in myelin basic protein (MBP). OLG loss also occurred at 3 days of reperfusion along with increased caspase-3 expression in the white matter. Treatment with BB-94 led to reduced astrocyte reactivity, less MMP-2 activity, and reduced MBP loss. Drug treatment did not result in changes in OLG loss or caspase-3 expression. These findings suggest a role for MMPs in the loss of myelin, with OLG loss occurring by an MMP-independent mechanism. We provide evidence of a role for TIMP-3 and MMP-3 in delayed neuronal loss as well as a potential role for MMP-2 in myelin degradation, but not OLG loss. We were able to inhibit the detrimental effect of these MMPs with BB-94 following BCAO induced global ischemia. With varying timelines of injury to the gray and white matter, it appears MMPs have cell specific expression that leads to regional sensitivity. Further investigation into the use of MMP inhibitors may allow for potential therapeutic uses as we investigate these delayed mechanisms of injury to the brain.

Keywords

matrix metalloproteinase, oligodendrocyte, myelin, hippocampus, BB-94

Sponsors

National Institutes of Health

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Cunningham, LeeAnna

Second Committee Member

Bizzozero, Oscar

Third Committee Member

Reichard, Ross

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