Applying Pre-Clinical Methodologies to Better Understand the Genetic and Physiologic Mechanisms Underlying Brain Injury
Osier, Nicole D.
Dixon, C. Edward
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Session presented on Thursday, July 21, 2016 and Friday, July 22, 2016: Introduction: Traumatic brain injury (TBI) is a significant worldwide health problem associated with significant personal and financial cost. Despite the extensive research effort aimed at understanding the pathophysiology of TBI and targeting it therapeutically, none of the efforts to-date have resulted in an FDA-approved medication that improves outcomes at TBI. Thus, there remains an impetus to better understand TBI pathophysiology and apply this knowledge to test novel therapies. An important part of this effort involves using animal models, which afford a high-degree of control over potential confounding variables (e.g. age, sex, genotype, extent of injury); pre-clinical TBI research studies are necessary to establish safety and efficacy before translation to clinical trials and ultimately patient care. One promising potential therapeutic is melatonin, an endogenous substance produced in the brain; evidence suggests endogenous melatonin levels are deranged after injury. Before melatonin can be tested in clinical trials, additional evidence is needed regarding the mechanism of action. There are two melatonin-specific receptors (MT1 and MT2) found in mammalian brains but they have not been characterized after injury. It is known that human genetic variation in these receptors exists and may influence response to melatonin therapy, as has been demonstrated in pre-clinical models of other neurological conditions. This dissertation project addresses an important gap in the knowledge by characterizing MT1 and MT2 after preclinical TBI. Methods: During the course of the dissertation study two types of test animals were used: C57BL/6J mice and Sprague Dawley rats. The sample sizes for each of the assessments varied. In both arms of the studies the mice or rats were randomly assigned to one of two exposures: severe traumatic brain injury modeled using controlled cortical impact (CCI) or sham control. Briefly, animals were anesthetized and a craniectomy performed using a drill. A pneumatic CCI device (Pittsburgh Precision Instruments, Pittsburgh, PA, USA) was used to induce injury. After impact, the scalp was sutured closed, animals monitored post-operatively, and returned to their cages. Sham animals received identical treatment except for the impact itself. Cellular endpoints in this study were assessed using westeRNlot and normalized to actin to account for protein loading. The following proteins were probed using antibodies: MT1, MT2, and caspase 3. Results: Pilot work explored the effects of TBI on functional outcomes in the domains of learning, memory, and motor function and explored how these symptoms related to pathophysiological changes surrounding apoptotic cell death and brain receptor levels. Results from testing in mice found that, compared to sham animals, there was an increase in apoptosis (Figure 1) and a decrease in MT1 levels (Figure 2) in hippocampal tissue one day after TBI. Interestingly, these pathophysiologic changes were associated with only modest functional deficits (Figures 3-5) as assessed using reliable and valid measures (e.g. Morris water maze; beam balance task; novel object recognition); this suggests that even in the absence of overt symptoms, cellular processes are deranged. Results from testing in rats found a decrease in MT1 and MT2 levels at 6 hours post-injury in the hippocampus after TBI (Figures 6-7). Discussion & Conclusion: Although preliminary, this study suggests that changes occur to the endogenous melatonergic system after TBI. These changes correlate with cell death, though not necessarily functional outcomes. Additional efforts are needed to better understand the role of MT1 and MT2 after injury and explore how human genetic variation in these receptors correlates with clinical outcomes and response to therapy. Future directions: The ongoing portion of the applicant's dissertation study includes repeating these experiments using mice lacking the MT1 receptor due to having the gene encoding MT1 knocked out (KO) of the genome. It is hypothesized that MT1 KO mice will have poorer outcomes than their wildtype (WT) counterparts as presented in this poster. Beyond the scope of this dissertation, additional work needs to be performed to characterize the melatonergic system and explore the therapeutic role of supplemental MEL; part of this effort should include evaluating how genotype contributes to response to MEL therapy since genetic variation in MEL-specific receptors has been reported.