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Marc J. Carmichael
B.S. Chemical Engineering 2001
Thesis Advisor: Richard Nowakowski, PhD
Graduate Program in Biomedical Engineering
RWJMS, Room V-10
Friday, June 11, 2010
An injury to the brain or spinal cord sets off a cascade of events that includes axonal degeneration, cell death, and inflammation. Associated with these events is the production of new cells, i.e., cell proliferation. Although there have been recent studies considering cell proliferation after SCI, much remains unknown regarding its diversity after injury and if it occurs in regions of the CNS distant from the lesion site. The initial aim of this research was to elucidate whether SCI elicits a proliferative response in the brainstem, with the hypothesis that there is such a response within regions of the CNS distant from the site of injury. The rationale for this hypothesis is twofold: 1) any damage to the CNS, including degenerative changes, should stimulate the proliferation of glial cells and/or microglia involved in damage control, cleanup of debris, and perhaps reorganization of motor and/or sensory pathways, and 2) the axons of many neurons damaged after SCI are projections from the brainstem. To characterize the changes in cell proliferation within the CNS of adult, male C57BL/6J mice 96 hrs after a thoracic hemisection of the spinal cord, a single injection of bromodeoxyuridine (BrdU) 30 minutes before sacrifice was employed to label cells within S-phase. Within the brainstem, there is nearly a 2-fold increase (173%) in the number of BrdU labeled cells in the caudal medulla; the region of the brainstem with the most labeled cells. The bulk of this increase is in the ventral region of the caudal medulla, which accounts for approximately 90% of the total number of BrdU labeled cells in the caudal medulla. Double-labeling of BrdU (S-phase marker) with markers for glial cell types, i.e., GFAP+ (astrocyte marker) and NG-2+ (glial progenitor/oligodendrocyte precursor marker), confirms that this response is glial, and predominantly comprised of NG-2 expressing cells. An analysis of the asymmetry of the spinal cord lesion and the proliferative response in the caudal medulla shows that the asymmetry of the total area of white matter spared at the lesion epicenter correlates positively with the asymmetry of the total proliferative response in the caudal medulla (R = 0.799, p < 0.02), and specifically in the spinocerebellar tract (SCT; R = 0.711, p < 0.05). In other words, these results show that the more asymmetric the spinal cord lesion, the more asymmetric the proliferative response is in the caudal medulla, and particularly in the SCT. In order to assess whether the increase in cell proliferation following SCI is specific to the caudal medulla, a second region of the CNS distant from the site of the spinal cord lesion was examined. In level 1 of the cervical spinal cord (C-1), there is a significant increase (162%) in the number of BrdU labeled cells following SCI as compared to sham-operated controls. To further assess the specificity of the increase in cell proliferation following SCI, a third region of the CNS was examined. The dentate gyrus (DG) was chosen because there are no direct connections with the spinal cord or any part of the somatosensory and motor systems. Moreover, the hilus of the DG has a small population of stem/progenitor cells that persists through the lifetime of mammals, including humans and the mice employed in this study. There is a significant decrease (~17%) in the number of BrdU labeled cells in the stem/progenitor cell population of the DG of mice with moderate or severe SCI as compared to sham-operated controls. Importantly, the decrease in the number of S-phase cells in the DG is relatively symmetric, even though the SCI (i.e., left hemisection) is asymmetric. This symmetry indicates that the decrease in the number of BrdU labeled stem/progenitor cells in the DG is likely due to a systemic signal elicited after SCI. In addition, the number of BrdU labeled cells in the DG of injured mice decreases in a severity-dependent manner. It is important to note that the direction of the change in cell proliferation in the DG is the opposite of what was found in the brainstem and cervical spinal cord of the same animals. In addition, there are neuronal, i.e., DCX+ (immature neuron marker), as well as GFAP+ and NG-2+ glial cells in S-phase within the DG following SCI. Since there are only glial cells in S-phase in the brainstem while the DG includes neuronal cells in S-phase after SCI, and the responses in these regions differ in direction and symmetry, this indicates that different mechanisms are involved in the regulation of cell proliferation in these distinct populations after SCI. In general, the results of this study indicate that within 96 hrs after SCI: 1) there are significant changes in the number of S-phase, i.e., BrdU labeled, cells in distinct regions of the CNS that are distant from the spinal cord lesion, 2) these changes differ quantitatively and qualitatively in the areas studied, and 3) different mechanisms are involved in the regulation of cell proliferation in these distinct regions. With respect to the significant decrease in cell proliferation within the DG of the hippocampus, this finding sheds light on an early biological event after SCI that is a potential precursor to emotional/mood disruption, particularly depression. Ultimately, the results of this study expand our knowledge of the underlying biological changes in regions of the CNS beyond the spinal cord that have the potential to lead to anatomical and functional changes following SCI. Therefore, it is important that they are considered in the development of treatments for patients with SCI.