Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Pharmacology and Physiology

Major Professor

Keith R. Pennypacker, Ph.D.

Keywords

Sigma receptors, Ischemia, Spleen, Inflammation, Microglia

Abstract

Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first attacks, and 185,000 are recurrent attacks (Carandang et al. 2006). Currently the only FDA approved treatment for ischemic stroke is recombinant tissue plasminogen activator (Alteplase) (Marler and Goldstein 2003). Unfortunately its use is restricted to a short, 4.5 hour, time window. Two promising therapies in the treatment of stroke at delayed timepoints are human umbilical cord blood cells (HUCBC) and the sigma receptor agonist DTG The first series of experiments were conducted to characterize the effects of sigma receptors on various aspects of microglial activation. Sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines. Stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with microglial activation.

Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. An ex vivo organotypic slice culture (OTC) model to was utilized to characterize the efficacy of sigma receptor activation and HUCBC therapy in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglia derived nitric oxide in slice cultures subjected to oxygen glucose deprivation (OGD) back to levels seen in the normoxia controls. The final experiments were performed to characterize the effects of the peripheral immune system on the brain over time and identify changes mediated by HUCBC and DTG. Labeled splenocytes were found in spleen, blood, and thymus, but not in the brain in appreciable numbers at any timepoint. IL10 and IFN?; levels were found to significantly increase by 96hours post MCAO.

This increase in IL10 and IFN? expression was blocked HUCBC or DTG. The experiments described here have shed light on the molecular mechanisms of stroke injury and the relative targets that DTG and HUCBC therapies exploit. These data suggest that the neuroprotection achieved by DTG or HUCBC is mediated by the ability of these treatments to modulate the peripheral immune systems response to injury.

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