Date

May 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

The goal of my research has been to develop new treatments for ischemic stroke. In pursuit of this goal I have performed a preclinical investigation of osteopontin (OPN) and 3-iodothyronamine (T1AM), two novel drug candidates that could be developed into therapies for stroke patients. Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biological functions, including inflammation, cell migration, and anti-apoptotic processes. In this thesis I investigate the neuroprotective potential of OPN using both in vitro and in vivo models of ischemia. I show that primary cortical neuron cultures exposed to OPN are protected against cell death from oxygen and glucose deprivation (OGD) and that the effect of OPN depends on the Arg-Gly-Asp (RGD) motif, and an increase in Akt and p42/p44 MAPK phosphorylation. Intra-cerebral ventricular administration of OPN caused a marked reduction in infarct size following transient middle cerebral artery occlusion in a murine stroke model and together these data suggest that OPN is a potent neuroprotectant against ischemic injury. I sought to increase the neuroprotective potency of OPN and improve the method of delivery. Data presented here shows that thrombin cleavage of OPN improves its ability to ligate integrin receptors and improves its neuroprotective capacity in models of ischemia. I also tested whether OPN could be administered by intranasal administration and found that OPN is efficiently targeted to the brain via this delivery route and confers robust protection against ischemic brain injury. OPN mimetics based on the peptide sequences located N or C terminal to the thrombin cleavage site were generated and tested in models of ischemia. Treatment with successively shorter N terminal peptides and a phosphorylated C terminal peptide provided significant neuroprotection against ischemic injury. These findings show that OPN mimetics offer promise for development into powerful new drugs for the treatment of stroke. Mild hypothermia confers profound neuroprotection in ischemia. Two recently discovered natural derivatives of thyroxine, 3-iodothyronamine (T1AM) and thyronamine (T0AM) have been shown to lower body temperature in rodents for several hours without induction of a compensatory homeostatic response. Here I tested whether T1AM- and T0AM-induced hypothermia protects against brain injury from experimental stroke. I administered T1AM and T0AM 1 hour after stroke to test for an acute protective effect and 2 days prior to stroke to test whether they could be used as a preconditioning stimulus. T1AM and T0AM administration reduced body temperature from 37oC to 31oC. Mice given T1AM or T0AM after the ischemic period had significantly smaller infarcts compared to controls. Mice preconditioned with T1AM prior to ischemia displayed significantly smaller infarcts compared to controls. Pre- and post-ischemia treatments required a drop in body temperature to achieve subsequent neuroprotection. These findings show that T1AM and T0AM, are potent neuroprotectants in acute stroke and T1AM can be used as antecedent treatment to induce neuroprotection against subsequent ischemia. The failure of any single neuroprotectant to be advanced to clinical use has led to considerable interest in combinatorial therapy. This thesis concludes with a combinatorial experiment using both OPN and T1AM with T1AM applied as an antecedent treatment and OPN as an acute neuroprotectant.

Identifier

doi:10.6083/M4CF9N3B

School

School of Medicine

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