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theses

The mammalian brain retains the ability to produce new neurons and glia throughout life. The retention of stem cells in the adult brain is phylogenetically conserved from rodents to humans and probably exists in all mammals. Their functional role and the factors that control their proliferation, migration, and differentiation is currently an intense area of investigation and debate. Although there are broad species-specific differences in brain anatomical and neurochemical structure, two primary neurogenic regions in the adult appear to be consistent: the subventricular zone (SVZ) lining the lateral ventricles which gives rise to new olfactory interneurons, and the subgranular zone (SGZ) of the hippocampus dentate gyrus which gives rise to new granule cell neurons. There is accumulating evidence that SVZ-derived stem cells are capable of responding to environmental cues within the brain, including injury, to influence both their migratory behavior and their lineage fate. Response to injury has been explored in various rodent models, some of which have shown that SVZ-derived cells can proliferate and migrate to the injury site and differentiate into neurons or glia, though they may lack in number and appropriate phenotypic characteristics to qualify as a fully functional repair mechanism. SGZ stem cells are responsive to environmental stimuli involving learning behavior and stress, as well as upregulate proliferation in response to injury. The lineage potential of SGZ neural stem cells is generally limited to producing new granule cell neurons that integrate into the granule cell layer. Deviation from this pattern can occur under pathological condition such as when granule cell precursors ectopically migrate into the hippocampal molecular layer following chronic epileptic seizures. It is not clearly defined whether SVZ-derived cells in the adult can migrate to the injured hippocampus or contribute to hippocampal repair. We hypothesized that trimethyltin-induced hippocampal injury in mice would induce the response of endogenous subgranular zone stem cells and subventricular zone-derived cells as part of a repair mechanism. Trimethyltin is a limbic system neurotoxicant that preferentially damages granule cells in the mouse hippocampus dentate gyrus. Stem cell response to TMT injury was examined in vivo in C3H mice. Mice were injected intraperitoneally with TMT to determine both dose-response and time course of TMT -induced granule cell injury. Brains were harvested at appropriate timepoints for cryosection and immunostaining. Mice were also injected with bromodeoxyuridine to quantify proliferating cells and follow differentiation of newly born cells. To trace migrating SVZ-derived cells, mice were injected with the fluorescent dye spDiI directly into the lateral ventricle. A steep dose-dependent induction of cell death in the granule cell layer was evident within 48 hours post-TMT injection based on pyknotic nuclei and immunostaining for activated caspase-3, as well as fluorojade C labeling of dying granule cells. The peak period of cell death was at three days post-TMT, with a gradual decline in number of dead or dying cells to near control levels at 8 days post-TMT. No pyknotic or caspase-3+ cells were detected at 28 days post-TMT. Glial activation with was prominent and limited to the hippocampus, and coincident with the induction of granule cell death. Immunostaining using markers for microglia (cd11b) and astrocytes (GFAP) indicated microglial activation persisted out to 8 days post-TMT injection before returning to control level, while elevated GFAP expression and astrocyte branching persisted out to 28 days post-TMT. Co-localization of nestin expression was present in some activated astrocytes in TMT-exposed brain, possibly indicative of conversion to a more primitive phenotypic state with reparative functional roles. TMT injury induced increased Ki-67+ staining of cycling cells in the SGZ in a dose-dependent manner, with a decline above 2.8 mg/kg suggesting possible toxicity to either stem cells or dividing progeny. Quantitative analysis showed that induction of proliferation peaked at 3 to 5 days post-TMT injection and persisted out to eight days post-TMT. Expression of TBR2, a specific marker for neuronal precursors, was significantly increased from 2 to 5 days after TMT injection, indicating induction of a neurogenic response as opposed to gliogenesis. Proliferation of cells in response to TMT injury appeared to be limited to the hippocampus since there was no upregulation in the number of Ki-67+ cells in the subventricular zone of the lateral ventricles. Because neural stem cells occupy a neurovascular niche that may regulate stem cell activity, blood vessel parameters were quantified to determine whether TMT injury induces any overt morphological alterations. In TMT-injured brain there was a prolonged and progressive reduction in blood vessel thickness in the hippocampus, suggesting dysregulation of vessel constriction and dilation that could potentially influence stem cell activity. Icv injection of spDiI exclusively labeled the ependymal/subependymal cells lining the lateral ventricles. 28 days following TMT-induced injury of the hippocampus, spDiI+ cells were detected in the hippocampus dentate gyrus and molecular layer. Very few spDiI+ cells expressed markers of neuronal (NeuN) or glial cells (GFAP, cd11b) and may represent an undifferentiated population of cells. spDiI+ cells were absent in intact uninjured brain, and at the early time point (7d) after TMT injection. To determine whether new cells were critical for recovery from TMT injury, neurogenesis was blocked by dosing mice with 10 Gray gamma radiation prior to TMT exposure. Mice were euthanized 7 days and 1 year after TMT treatment and analyzed for stem cell activity. Time of onset of TMT-induced tremoring activity was unchanged by radiation treatment, but tremors were more severe and prolonged in mice that were irradiated. Hippocampal neurogenesis was reduced by 75% in irradiated mice. Irradiated mice retained the capacity to upregulate hippocampal neurogenesis following TMT-induced injury but it was significantly reduced compared to unirradiated mice. Only 3% of the newly-born BrdU+ cells in hippocampus generated after TMT injury survived at 1 year post-treatment and co-localized primarily with the neuronal marker NeuN. The number of Ki-67+ cells in the 1yr hippocampus was only approximately 2% of that measured in 7day post-treatment groups. Overall, the data suggest that stem cells in both subgranular and subventricular zones respond to TMT-induced hippocampal injury and may contribute to behavioral and cellular recovery. The vast majority of newly-born cells generated at the time of injury are not present one year after treatment, though the dentate gyrus in TMT-treated mice are morphologically indistinguishable from that of untreated mice at this time point.

ETD_7625

Ph.D.

Includes bibliographical references

by Blair Christopher Weig

doi:10.7282/T3T155ZD

ETD doctoral

The author owns the copyright to this work.