Concussions are the injury and symptoms that can result from transmission of a biomechanical force to the brain. They represent a significant global health burden, and are the subject of a growing body of medical research. A concussion can only be definitively diagnosed by a medical professional based on symptoms, although advanced neuroimaging and biomarker-based approaches are promising future diagnostic tools. There is no treatment for concussion beyond following return-to-work or -play guidelines, which recommend avoiding strenuous physical and cognitive activities until they no longer exacerbate symptoms. Preclinical models of concussion have been used to examine pathophysiological processes underlying symptoms, which is an important step in developing tools for diagnosis and treatment. Historically the clinical translation of preclinical concussion research has been limited, and the use of anaesthesia, and preference for adult male rats may contribute to this. These means of reducing variability are justified, but preclinical research moving forward should address these limitations to translatability by including more clinically relevant subjects and avoiding anaesthesia. To this end, we developed a new preclinical model for pediatric concussion. Our awake closed head injury (ACHI) model is well-suited to this purpose because it produces a helmeted closed-head injury involving vertical and rotational displacement of the head, and does not require anaesthesia. Before the ACHI model can be used to investigate concussion mechanism, diagnosis, and treatment, it needs to be characterized to demonstrate that it produces clinically relevant neurobehavioral and pathological changes. We developed a modified neurologic assessment protocol to test neurologic function immediately after each injury. The Barnes maze, elevated plus maze, open field, and Rotarod were used to measure injury-related changes in cognition, anxiety, and motor function. The Barnes maze reversal task was used to detect more subtle cognitive impairments of executive function. Structural MRI was used to search for visible lesion, hemorrhage, or atrophy; and silver-stain histology was used to detect neurodegeneration. We determined repeated ACHI produced acute neurologic impairment with the NAP, and a mild spatial learning deficit potentially mediated impaired cognitive flexibility in the Barnes maze and reversal training. These were accompanied by neurodegeneration in the optic tract, hippocampus, and ipsilateral cortex during the first week of recovery. Thus, following the internationally recognised definition developed by the concussion in sport group, we demonstrated 1) an “impulsive” force transmitted to the head results in 2) the rapid onset of short-lived neurologic impairment that resolves spontaneously. This occurs 3) with normal structural neuroimaging, and 4) produces cognitive impairment, and LOC in a subset of cases. The ACHI model is the first in Canada to forego anaesthesia, and this is the first demonstration of neurocognitive impairment accompanied by diffuse neurodegeneration in the absence of structural MRI abnormalities after mild traumatic brain injury in juvenile male and female rats. / Graduate

Cyclooxygenase-2 (COX-2) is a prostaglandin-generating enzyme that exhibits low basal expression levels and is upregulated in the central nervous system (CNS) in response to inflammatory stimuli. COX-2 has been implicated in the microglial-mediated neuroinflammatory and neurodegenerative processes of multiple sclerosis (MS), a demyelinating autoimmune disease. To study COX-2 activity and the role it may play in demyelination, a novel PET radiotracer specific for COX-2, [11C]BRD1158, was developed and evaluated in the lysolecithin rodent model of focal demyelination with PET-MR imaging. Preliminary results of this pilot pre-clinical study confirmed our hypothesis that the properties of [11C]BRD1158 enable visualization and monitoring of COX-2 activity under pathological conditions induced by LPC. Radiotracer uptake correlated positively with disease progression at the site of LPC injection in male rats, peaking at day 7 and resolving by day 28. Treatment with an FDA-approved MS therapy, Siponimod, diminished the increase in COX-2 activity and tracer uptake at the lesion site and throughout the brain in both male and female rats. The results from the present study will inform future pre-clinical and translational work that validates the use of [11C]BRD1158 to image COX-2 activity as a marker of underlying inflammation in MS, leading to a better understanding of pathological and inflammatory processes in MS development and progression.

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences.

Through the Cenozoic, paleoclimate records show general trends of global cooling and increased aridity, and environments in North America shifted from predominantly forests to more open habitats. Paleobotanical records indicate grasses were present on the continent in the Eocene; however, paleosol and phytolith studies indicate that open habitats did not arise until the late Eocene or even later in the Oligocene. Studies of large mammalian herbivores have documented changes in ecomorphology and community structure through time, revealing that shifts in mammalian morphology occurred millions of years after the environmental changes thought to have triggered them. Smaller mammals, like rodents and lagomorphs, should more closely track climate and habitat changes due to their shorter generation times and smaller ranges, but these animals have received much less study. To examine changes in smaller mammals through time, we have assembled and analyzed an ecomorphological database of all North American rodent and lagomorph species. Analyses of these data found that rodent and lagomorph community structure changed dramatically through the Cenozoic, and shifts in diversity and ecology correspond closely with the timing of habitat changes. Cenozoic rodent and lagomorph species diversity is strongly biased by sampling of localities, but sampling-corrected diversity reveals diversity dynamics that, after an initial density-dependent diversification in the Eocene, track habitat changes and the appearance of new ecological adaptations. As habitats became more open and arid through time, rodent and lagomorph crown heights increased while burrowing, jumping, and cursorial adaptations became more prevalent. Through time, open-habitat specialists were added during periods of diversification, while closed-habitat taxa were disproportionately lost in subsequent diversity declines. While shifts among rodents and lagomorphs parallel changes in ungulate communities, they started millions of years earlier than in larger mammals. This is likely a consequence of the smaller mammal' greater sensitivity to environmental changes and more rapid evolution. These results highlight the importance of examining understudied members of vertebrate faunas for understanding the evolution of terrestrial communities through time.

Assessment of chemical mutagenicity is essential to protecting human health from genetic disease. Current assays are limited in their ability to provide mechanistic insight into the endogenous and exogenous processes involved in mutagenesis. Duplex Sequencing (DS), a novel error-corrected sequencing technology, overcomes many of the limitations faced by conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. Furthermore, customizable panels enable assessment of the endogenous genomic features that drive mutagenesis. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. The objectives of this study were to demonstrate the potential of DS as a robust in vivo mutagenicity test and to explore its rich data to gain a better understanding of spontaneous and chemically-induced mutagenicity in somatic and germ cells. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) and germ cells of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and tissues were sampled at least 28 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in MF and distinct spectra consistent with the known mutagenic mechanisms of PRC in both tissues. Mouse PRC doses at which significant effects were observed are in range with those used for chemotherapy, suggesting that similar effects may be observed in human patients. This supports the contribution of PRC towards secondary cancers following treatment. DS results were comparable to those obtained using the gold-standard lacZ TGR assay, with DS showing greater sensitivity to detect smaller changes in MF. Analysis of mutation spectra and the genomic features that drive the mutational response revealed intrinsic differences between BM and germ cells that may underlie differences in endogenous mutagenic mechanisms and/or DNA repair pathways. The results suggest that germ cells may have intrinsic mechanisms to reduce mutation burden relative to somatic cells. While historically analysis of germ cell mutagenicity has been neglected in favour of somatic cells, our work supports the independent assessment of germ cell mutagenicity during regulatory testing. Finally, we conducted power analyses to inform the optimal DS study designs for the two tissues. We found that low intra-group variability within BM samples allows a reduction in sample size to three animals per group whilst still maintaining 80% power to detect an effect. In contrast, the relatively high intra-group variability and low background MF in germ cells suggests a minimum of eight animals per group to detect an effect. Overall, our results support the use of DS as a mutagenicity test and highlight many of the advantages it holds over conventional assays. Moreover, our study reveals the potential for mutagenic effects in PRC-treated cancer patients. Further work to test DS with more chemicals and across a wider range of tissues is recommended for future implementation as a mutagenicity test.

Alcohol is the most abused drug in the United States and most frequently performed assay in forensic laboratories. Alcohol is routinely present in biological specimens from fatal residential fires and forensic toxicologists must interpret if these individuals are impaired by determination of their blood alcohol concentrations on post-incineration blood collected at autopsy. There is no known data available to confirm or refute blood alcohol concentrations and impairment in fire-related deaths. Ethyl glucuronide (EtG), a non-volatile minor ethanol metabolite, may provide a better biomarker for ethanol consumption prior to burn injury. The literature does not address the possibility that ethanol or EtG concentrations are altered in fire deaths. A Sprague Dawley rat model was employed to determine if ethanol and EtG concentrations in blood, liver, heart, and kidney were altered after burn injuries using two incineration models with varying durations and temperatures. Blood and tissues were analyzed for ethanol by gas chromatography and EtG by enzyme immunoassay. Other measurements including organ weights, lower hindquarter weights, and blood glucose concentrations were chosen for analysis to determine the mechanism by which the blood and organ ethanol and EtG concentrations are altered in burnt corpses. The rodent provided an excellent model for studying the biotransformation of ethanol to EtG and the effects of burn injury on ethanol and EtG concentrations. Our study revealed that blood ethanol concentrations were not significantly altered by burn injury but tissue ethanol concentrations were altered by burn injury. EtG concentrations were found to be altered in blood and tissue specimens in both incineration models. Our data suggest that the change in ethanol and EtG concentrations may be correlated to higher core body temperatures from burn injury and not changes in organ weight. Determining if blood ethanol concentrations are altered in burnt corpses is important for forensic toxicologists to conclude if victims were impaired at the time of death. The knowledge gained from these experiments will help forensic toxicologists by confirming the current interpretation that blood ethanol concentrations are not altered in fire deaths and provide a better understanding for the interpretation of impairment in burn deaths.

Introduction- Exercise-induced plasma volume (PV) expansion is typically associated with an increase in plasma albumin content. Increased hepatic albumin synthesis, a transcriptionally regulated process, is thought to contribute to the increase in albumin content. Objective- We tested the hypothesis that exercise training induces an increase in albumin gene expression in relationship to the increase in PV. Methods and Results- 40 adult male Sprague-Dawley rats weighing between 245-350 grams were randomly assigned to one of four groups: cage control (CC); sham exercise 10 min/day at 48% VO2max (NE); continuous exercise training, 60 min /day at 72% VO2max (LI); and high intensity, intermittent exercise training, 8 bouts of 4 min at 98% VO2max followed by 5 min at 48% VO2max (HI). The training period lasted for two weeks with 12 training sessions with equalized training volumes in the exercise groups. 24 hours after the last training session the rats were anesthetized and a jugular catheter was placed for collecting blood samples during PV determination by a dilution of a labeled-albumin molecule (Texas Red albumin). The liver and red quadriceps (RQ) muscle tissue was then removed, flash frozen, and stored for later analysis. The training protocol produced a significant increase in RQ citrate synthase activity (p < 0.05). PV increased in proportion to the exercise intensity (p < 0.05) averaging 23.6 ± 2.7 ml•kg-1 body weight in the CC group and 26.6 ± 1.3 ml•kg-1 body weight in the HI group. Albumin mRNA expression determined by real time polymerase chain reaction (PCR) increased 2.2 ± 0.1 and 2.9 ± 0.2 fold following LI and HI exercise training, respectively. Conclusion- These data support the hypothesis that, during exercise-induced PV expansion, albumin gene expression is increased and contributes to an increase in plasma albumin content and PV.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Trace elements are involved in many biological processes and serve important functions to maintain the normal development of the central nervous system (CNS). In the CNS, iron (Fe), copper (Cu), and zinc (Zn) are some of the most important elements that play critical roles as catalysts, cofactors, and structural components for many cellular enzymes and proteins. The deficiency or excess of these metals may lead to various neurological disorders. Demyelination is a condition of loss of myelin and leads to neurological diseases like Multiple Sclerosis. Myelin consists of transition metals and hence it would be interesting to study concentrations of these elements in normal and demyelinated models. X-Ray Fluorescence (XRF) is a popular non-destructive technique applied in trace element studies. The principle involves exciting a sample and detecting characteristic X-rays, which provide information on elemental concentrations in the sample. In the present studies the feasibility of XRF for trace element studies was explored. A total of 120 samples of brain and spinal cord tissues were collected from Long Evans (control) and Long Evans Shaker (dysmyelinated)–an incomplete formation of myelin sheaths–rats at ages of 3 weeks and 16 weeks. The samples were excited using x-rays from an Energy Dispersive X-Ray Diffraction (EDXRF) set-up. The spectral data was collected using an Silicon Drift Detector (SDD) and the resultant data were analysed to see if statistically significant changes in concentrations were present in the samples. The results were discussed and suggestions for future work were made. / Thesis / Master of Science (MSc)

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13578号 / 論医博第2300号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 中島 貴子, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM