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<p><strong>Purpose:</strong> Microglia are the innate immune cells of the central nervous system. Activated microglia release nitric oxide, glutamate, and superoxide radicals, which are harmful to retinal ganglion cells (RGCs). They may also benefit surviving cells by removing toxic cellular debris or by secretion of neurotrophic factors. The paradoxical role of microglia remains controversial because the nature and time-course of the injury that determines whether microglia acquire a neuroprotective or pro-inflammatory phenotype is unknown. HAPI cells are an immortalized microglial cell line, whose phenotype can be manipulated <em>in vitro</em>. It is my HYPOTHESIS that pharmacological manipulation of microglia to acquire either a pro-inflammatory or pro-survival phenotype will exacerbate neuronal cell death or enhance neuronal survival after injury, respectively.</p> <p><strong>Method:</strong> Lipopolysaccharides (LPS) were used to hyper-stimulate the HAPI cells and minocycline to maintain the HAPI cells in a quiescent state. Prior to the experiments, the HAPI cells were labelled with Wheat Germ Agglutinin conjugated to Texas Red. The HAPI cells were cultured and exposed to minocycline (10 µg/mL for 1 hour) or LPS (1 µg/mL for 24 hours). Sprague-Dawley rats then recieved intraocular (30,000 cells) or tail vein (5 million cells) injections of either the minocycline treated HAPI cells or the LPS treated HAPI cells and an optic nerve crush. Retinas were examined at 4-14 days later and the number of surviving RGCs will be determined by Brn3a labelling of RGCs. BM88 antibody labelling was done to determine the severity of the injury and to determine molecular changes after neuroinflammation.</p> <p><strong>Results: </strong>Injection of untreated HAPI cells resulted in the greater loss of RGCs early after ONC when injected into the vitreous and later after ONC when injected into the tail vein. LPS activated HAPI cells injected into the vitreous resulted in greater RGC loss with and without injury. When injected into the tail vein with ONC there was no loss of RGCs 4 days after ONC but later there was greater loss of RGCs. Minocycline treated HAPI cells injected into the vitreous resulted in greater RGC survival than when untreated HAPI cells were injected. However, when injected into the tail vein with ONC there was greater loss of RGCs. There was also BM88 down regulation after injury and this was more pronounced after HAPI cell injection.</p> <p><strong>Conclusion:</strong> Neuroprotection or cytotoxicity of microglia depends on the type of activation, time course of the injury, and if the microglia act on the axon or cell body of the retinal ganglion cell.</p> / Doctor of Philosophy (PhD)

Traumatic brain injury (TBI) is a prominent healthcare concern in the U.S. as millions of TBI-related emergency department visits occur annually. Recent reports estimate more than 5 million Americans currently suffer from life-long disabilities and psychiatric complications associated with TBI. While the risk of TBI has conventionally been considered to be male dominated, analyses of sex-comparable sports indicate that rates of concussions are higher and recovery time is longer following brain injury in females. Following anxiety and depression, substance use disorder (SUD) is the third most common de-novo neuropsychiatric condition diagnosed in both male and female TBI patients. Importantly, during adolescence the primary neuronal networks that regulate reward behaviors and perception of drug-induced euphoria are not fully developed, corroborating epidemiological studies identifying TBI sustained during adolescence as a risk factor for problematic drug use. Yet, to date, little is known about how TBI-induced molecular changes affect brain structures essential for the perception of reward and processing drug-induced euphoria. The following experiments were designed to test the hypothesis that adolescent TBI-induced neuroinflammation in areas such as prefrontal cortex (PFC) and nucleus accumbens (NAc) results in remodeling of neuronal reward networks and affect how the rewarding effects of cocaine shift as a consequence of TBI. Notably, the extent of sex differences in SUD susceptibility in TBI has not be investigated. Therefore, we also investigated whether the immune response stimulated by early-life TBI alters maturation of reward neurocircuits, leading to increased SUD vulnerability in a sex-dependent manner. Following the induction of TBI using the controlled cortical impact (CCI) model of brain injury, we utilized a biased, three-phased cocaine conditioned place preference (CPP) assay to assess the behavioral response to the rewarding effects of cocaine following adolescent injury in male and female C57BL6 mice. Furthermore, we characterized the effect of CCI-TBI on the stimulation of neuroinflammation within the PFC and NAc, comprising the reward pathway. Specifically, our studies revealed a sex-specific increase in 1) sensitivity to the rewarding efficacy of a subthreshold doses of cocaine interpreted from significantly higher cocaine CPP shifts, 2) the activation and phagocytosis of microglia observed by the positive expression of neuronal synaptic proteins in microglia sorted using flow cytometry, 3) increase in permeability of the blood-brain barrier indicated by discontinuous and depleted expression of tight junction proteins that line microvasculature isolated from reward nuclei, 4) decreased neuronal complexity, arborization, and spine density quantified from Golgi-cox stained NAc neurons, 5) changes in expression of genes related to the dopamine system analyzed by qRT-PCR in only male mice injured during adolescence. Additionally, our results imply that high levels of female hormones can promote neuroprotection against increased sensitivity to the rewarding properties of cocaine following injury, associated with decreased neuroinflammatory profiles after TBI in adolescent females. The studies herein aimed to elucidate underlying neuropathological outcomes following TBI in the reward circuitry that could be contributing to increased risk of addiction-like behavior observed clinically. Our findings suggest that TBI during adolescence may enhance the abuse liability of cocaine in adulthood and vulnerability to the rewarding effects of cocaine could be higher as a result of brain injury. Key pathological findings in the NAc such as activated microglial phagocytosis, BBB changes, reduced neuronal complexity, and changes in dopamine gene expression in areas of the reward pathways support the notion that neuroinflammation may contribute to how the rewarding efficacy of cocaine are affected post-TBI during adolescence. The ultimate goal of this research is to 1) advance TBI and SUD literature with the potential to increase awareness and help health care providers inform TBI patients about the increased risk for SUDs, and 2) to translate identified correlated mechanisms into novel targeted therapies that would provide a launching point for the treatment of patients with TBI-related SUD. / Biomedical Sciences