Publication Date

2009-12-18

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2009-08-26

First Committee Member

Edward Green - Committee Chair

Second Committee Member

Kenneth Muller - Committee Member

Third Committee Member

James Guest - Committee Member

Fourth Committee Member

Jacqueline Sagen - Mentor

Abstract

Persons with spinal cord injury (SCI) suffer life-long consequences including paralysis, loss of involuntary bodily functions, and chronic pain. A subset of SCI patients develop neuropathic pain (NP), a chronic condition resulting from damage to the spinal cord. Hyperexcitability of spinal cord sensory neurons near damaged tissue is believed to underlie SCI-related NP. Although many therapies have been employed clinically to combat SCI-NP, few give satisfactory long-term relief. Transplantation of cells that release GABA, a molecule that inhibits neuronal activity, is being explored as an alternative to current SCI-NP therapies. My experiments made progress toward preclinical modeling of GABA cell therapy for SCI-NP. First, I sought to determine whether quisqualic acid (QUIS)-induced SCI altered responses to tonic pain stimuli or altered GABAergic neural circuitry in rats. Second, I sought to determine whether a combination of genetic and trophic manipulations could promote a GABAergic phenotype in rat embryonic neural precursor cells (NPCs) in an in vitro culture system. The results revealed that QUIS-SCI rats exhibit unusually prolonged nocifensive responses to hind paw formalin injections. There was no significant difference between QUIS-SCI and sham surgery rats in c-Fos immunolabeling of spinal cord sensory neurons after formalin-induced neuronal activity. However, immunohistochemistry revealed substantial decreases in staining for markers of GABA presynaptic vesicles in injured spinal cord tissue. NPCs were enriched for a neuronal phenotype by combining withdrawal of the growth factor FGF-2 from culture media and overexpression of the transcription factor MASH1 in transfected cells. Although glial marker expression was suppressed in NPCs by these manipulations, expression of neuronal markers none the less declined through time. MASH1-overexpressing NPCs exhibited greater clonal expansion and decreased stress-induced PDI expression after FGF-2 withdrawal as compared to naïve. In light of existing data, these results suggest that the QUIS-SCI model may be useful for testing the efficacy of GABAergic NPC transplantation to reduce neuropathic pain. MASH1 overexpression and FGF-2 withdrawal could serve as a first step toward enriching GABA in NPCs for transplantation. Although the mechanism for MASH1 cytoprotection remains unclear, MASH1 may enhance survival of NPCs grafted into the spinal cord. These experiments contributed to the preclinical basis for application of therapeutic GABAergic stem cell transplantation for NP in human SCI patients.

Keywords

Pain; Spinal Cord; Injury; Stem Cell; Transplant; GABA; Transcription Factor

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