<p>In a mouse model of ischemic stroke, infarct volume is highly variable and strain dependent, but the natural genetic determinants responsible for this difference remain unknown. To identify genetic determinants regulating ischemic neuronal damage and to dissect apart the role of individual genes and physiological mechanisms in infarction in mice, we performed forward genetic mapping analyses of surgically induced cerebral infarct volume. We have identified multiple quantitative trait loci (QTL) that modulate infarct volume, with a major locus (<italic>Civq1 </italic>) on chromosome 7 accounting for over 50% of the variation, with a combined LOD score of 21.7. Measurement of infarct volume in chromosome substitution strains (CSS) and two additional intercrosses validate that <italic>Civq1</italic> on chromosome 7 is present in multiple inbred strains. Interval-specific ancestral SNP haplotype analysis for <italic>Civq1</italic> results in 5 candidate genes. A causative gene underlying <italic>Civq1</italic> may regulate collateral artery formation and genetic variations in the gene may result in the differential outcome of cerebral infarction. Additionally, we have identified a locus of large effect, <italic>Civq4</italic>, modulating infarct volume through a mechanism different from collateral circulation. In conclusion, the extent of ischemic tissue damage after distal middle cerebral artery occlusion (MCAO) in inbred strains of mice is regulated by genetic variation mapping to at least 4 different loci. A single locus on chromosome 7 determines the majority of the observed variation in the trait in multiple mouse strains. <italic>Civq1</italic> appears to be identical to <italic>Lsq1</italic>, a locus conferring limb salvage and reperfusion in hindlimb ischemia. The identification of the genes underlying these loci may uncover novel genetic and physiological pathways that modulate cerebral infarction and provide new targets for therapeutic intervention in ischemic stroke, and possibly other human vascular occlusive diseases.</p> / Dissertation

The growth plate is a cartilaginous tissue responsible for the longitudinal growth of long bones. It is a complex tissue composed of chondrocytes whose maturation and proliferation is tightly regulated by a biochemical feedback loop. Injury to this tissue can result in a limb length discrepancy or angular deformity that may lead to life long disability. Given the recent rise in the number of growth plate injuries and the variability in success of current therapies, there is a significant need for a greater understanding of growth plate injury pathology and the development of improved treatment strategies. Cartilage tissue engineering strategies offer an attractive alternative to regenerating growth plate tissue and restoring growth function. Bone marrow-derived stem cells (BMSCs) have been shown to be able to undergo chondrogenic differentiation and in vitro and in vivo and therefore offers an appealing and abundant cell resource for developing tissue engineering strategies for the treatment of growth plate defects. However, the dependence of chondrogenic differentiation and matrix accumulation on monolayer expansion protocols and three-dimensional (3D) culture environment has received little attention. Prior to developing treatment strategies for growth plate injury repair, it is essential to first understand the interconnection between alterations in growth plate morphology and subsequent limb deformities. To that end, we have established a surgical defect model of growth plate injury in Sprague Dawley rats and developed a novel technique to quantitatively monitor growth plate morphology in health and disease using microcomputed tomography (micro-CT) imaging. In an effort to develop a tissue engineering treatment strategy for growth plate injury, the role of monolayer expansion, 3D scaffold, and growth factor regimen in the chondrogenic differentiation of rat BMSCs was also examined. This research study has demonstrated the utility of micro-CT as a non-invasive imaging modality for assessing growth plate injury and repair. This work has also provided an improved understanding of the interrelationship of monolayer expansion, 3D culture environment, and growth factor regimen in BMSC chondrogenic differentiation. Finally, this work suggests that an injectable in situ gelling hydrogel is a feasible method for decreasing limb length discrepancies, however, neither implantation of agarose alone into the defect nor the inclusion of BMSCs fully corrects growth disruption.

In this thesis I used Caenorhabitis elegans as a model of Fetal Alcohol Spectrum Disorder (FASD) to study the physical and behavioral effects of ethanol exposure during embryonic development. Davis et al. (2008) found that ethanol exposure during larval development in C. elegans produced physical/developmental and behavioral effects; however, whether exposure during embryonic development might produce similar outcomes remained to be elucidated. Because the type and degree of effects caused by developmental ethanol exposure was dependent on the pattern of ethanol treatment, in the first part of the thesis I investigated the physical/developmental effects of embryonic exposure to various ethanol doses, exposure durations, onsets and frequencies. I found that exposure to >30% ethanol for an hour during embryonic development was necessary to lower hatch rate, delay reproductive onset, and reduce body size in C. elegans. Furthermore, exposure during early embryonic development caused a larger effect than exposure during later stages, and multiple exposures produced a worse outcome than a single exposure for a comparable duration. In the second part of the thesis, I investigated locomotory activities and habituation of adult C. elegans exposed to various patterns of embryonic ethanol treatment. I found that the rate of locomotion was altered differently by chronic and acute embryonic ethanol exposure, but I did not find any effect in short- or long-term habituation. In summary, I have characterized the pattern of embryonic ethanol exposure necessary to produce physical/developmental effects in C. elegans, and identified the types of exposure conditions that would cause worse outcomes than others; in addition, I have found that embryonic ethanol exposure affects the rate of locomotion in C. elegans. In this thesis, I have established a foundation for the future investigation into the physical and motor defects caused by embryonic ethanol exposure in C. elegans.

Porcine Periweaning Failure-to-Thrive syndrome (PFTS) is a clinical syndrome of newly weaned pigs with unknown etiology and characterized by anorexia, lethargy and progressive debilitation. The hypothesis of this thesis is that PFTS is an infectious disease. Investigation in an index farm affected by PFTS from Saskatchewan Canada ruled out most common swine pathogens as the etiology and identified several lesions that were consistent across many cases. A larger study including multiple farms in North America was then undertaken. A total of 8 farms were investigated, within which 5 met the clinical definition of PFTS. Gross and histological examinations were performed on 8 case and 4 control pigs on each farm. Detection of relevant porcine pathogens, complete blood count, serum chemistry, and serum cytokine analysis were performed on each pig. Thymic atrophy, superficial gastritis and small intestinal villous atrophy were significantly more prevalent in case pigs compared to control pigs. All case pigs had at least two of these three lesions. All case and control pigs were negative for porcine reproductive and respiratory syndrome virus, swine influenza virus and were free of porcine circovirus associated diseases. Although several pathogens, such as porcine cytomegalovirus, haemagglutinating encephalomyelitis virus, porcine enteric calicivirus, group A rotavirus, enteroviruses and Cystoisospora suis were detected in some of the case and control pigs, none were associated with clinical status. Clinical pathology findings of case pigs was consistent with anorexia and dehydration, such as increases in haematocrit, blood urea, serum bilirubin, albumin, beta-hydroxybutyrate and decreases in blood glucose, calcium and phosphorous. Case pigs had similar levels to IL1-β than control pigs, which suggested that PFTS was not a result of excessive cytokines. In subsequent experiments, a snatched-farrowed porcine-colostrum-deprived (SF-pCD) pig model was developed and tissue homogenates were used to inoculate SF-pCD pigs in an attempt to reproduce the clinical signs of PFTS. The SF-pCD pigs were immunologically characterized and shown to be suitable for inoculation studies. However, inoculation of tissue homogenate from PFTS pigs failed to reproduce the clinical signs of PFTS in SF-pCD pigs. All together, PFTS is a clinical syndrome with consistent pathological and serum analytical changes among affected pigs. Despite the efforts of this research to establish an infectious etiology, there is a lack of evidence that PFTS is an infectious disease.

PURPOSE: Psychotic (positive) symptoms are the most distinctive feature of schizophrenia, although negative symptoms such as emotional flattening, social withdrawal and cognitive disturbances are the most treatment resistant manifestation of the illness. Schizophrenia is a progressive degenerative illness that has been causally linked to environmental and neurodevelopmental factors, as well as dysfunctional redox balance. Validated animal models are useful in identifying and studying novel neurobiological targets for neuropsychiatric illnesses. Post weaning social isolation rearing (SIR) in rats has been proposed to model the neurodevelopmental aspects of schizophrenia. We validated the SIR model with respect to effects on sensorimotor gating and social interaction, deficits of which are core symptoms of schizophrenia. Following this, effects on the levels of oxidative stress were determined in the frontal cortex and striatum of rats exposed to SIR, two brain regions strongly implicated in the pathology of schizophrenia. Finally, in order to more closely relate these bio-behavioural changes to the human condition, we studied the overall effect of sub-chronic treatment with the atypical antipsychotic, clozapine, on the above described behavioural and neurochemical parameters. METHODS: Male Sprague-Dawley (SD) rats (10 rats/group) were used. In a non-treatment arm, four groups of rats were randomly separated at weaning and exposed to either 8 weeks SIR or 8 weeks social rearing. At the respective time point of 8 weeks two groups were subjected to behavioural testing of mean startle amplitude (at 120dB) and percentage prepulse inhibition (%PPI) of the acoustic startle (AS) reflex (at 72, 76, 80 and 86dB prepulse), and various social interactive and self-directed behaviours were accessed using the open field test (OFT). The remaining two groups were sacrificed at 8 weeks and brain tissue was harvested for analysis of superoxide dismutase activity, oxidized (GSSG) versus reduced (GSH) glutathione ratio, and levels of lipid peroxidation, in the frontal cortex and striatum. In the treatment arm, consisting out of eight groups of animals, four groups of SIR rats received either saline or clozapine (5mg/kg i.p.) for the last 11 days of SIR. The remaining four groups were socially reared and also received either saline or clozapine treatment as above. At 8 weeks, four groups were subjected to behavioural testing as described above and a parallel neurochemical study was performed using the same layout as above, except that after the 8 weeks, neurochemical redox analysis were done as described above. Mixed statistical modelling with repeated measures and appropriate post hoc tests were used to access the effects of SIR with and without treatment on PPI and mean startle. Social interaction in SIR and socially reared animals, with and without treatment, was analyzed using 1-way ANOVA with suitable post hoc testing. Mixed linear models with repeated measures and appropriate post hoc tests were used for analysis of the redox data in SIR and socially reared animals, with and without treatment. RESULTS: In the non-treatment arm, %PPI was significantly reduced in SIR versus socially reared rats. Deficits in various social interactive behaviours were observed in SIR versus group-housed rats, as well as increased locomotor activity and self-grooming. Superoxide dismutase activity and oxidized versus reduced glutathione ratio were significantly decreased, together with a significant increase in products of lipid peroxidation, in isolation reared versus socially reared rats. Following clozapine treatment, %PPI in isolates was significantly elevated by clozapine versus saline treatment (i.e. reversed the effect of SIR). %PPI was unaltered in socially reared animals receiving either treatment. As with the non-treatment group, social interactive behaviours were significantly impaired in isolates receiving saline, while locomotor activity and self-grooming were increased. SIR rats receiving only saline showed similar altered redox state as the non-treatment groups, while clozapine treatment effectively reversed deficits in %PPI, aberrant social behaviours and redox alterations in the SIR rats, with limited to no effects in the socially reared controls. CONCLUSION: SIR thus significantly disrupts sensorimotor gating and social behaviours in male Sprague-Dawley rats, while at the same time evokes a significant disruption of redox state in both the frontal cortex and striatum of these animals, with distinct evidence for increased oxidative stress in these brain regions. Importantly, both altered behaviour and redox state are reversed by sub-chronic clozapine treatment. SIR is therefore a useful, non-lesion and non-pharmacological neurodevelopmental animal model of schizophrenia that presents with robust face, predictive and possibly construct validity for schizophrenia. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2010.

PURPOSE: Psychotic (positive) symptoms are the most distinctive feature of schizophrenia, although negative symptoms such as emotional flattening, social withdrawal and cognitive disturbances are the most treatment resistant manifestation of the illness. Schizophrenia is a progressive degenerative illness that has been causally linked to environmental and neurodevelopmental factors, as well as dysfunctional redox balance. Validated animal models are useful in identifying and studying novel neurobiological targets for neuropsychiatric illnesses. Post weaning social isolation rearing (SIR) in rats has been proposed to model the neurodevelopmental aspects of schizophrenia. We validated the SIR model with respect to effects on sensorimotor gating and social interaction, deficits of which are core symptoms of schizophrenia. Following this, effects on the levels of oxidative stress were determined in the frontal cortex and striatum of rats exposed to SIR, two brain regions strongly implicated in the pathology of schizophrenia. Finally, in order to more closely relate these bio-behavioural changes to the human condition, we studied the overall effect of sub-chronic treatment with the atypical antipsychotic, clozapine, on the above described behavioural and neurochemical parameters. METHODS: Male Sprague-Dawley (SD) rats (10 rats/group) were used. In a non-treatment arm, four groups of rats were randomly separated at weaning and exposed to either 8 weeks SIR or 8 weeks social rearing. At the respective time point of 8 weeks two groups were subjected to behavioural testing of mean startle amplitude (at 120dB) and percentage prepulse inhibition (%PPI) of the acoustic startle (AS) reflex (at 72, 76, 80 and 86dB prepulse), and various social interactive and self-directed behaviours were accessed using the open field test (OFT). The remaining two groups were sacrificed at 8 weeks and brain tissue was harvested for analysis of superoxide dismutase activity, oxidized (GSSG) versus reduced (GSH) glutathione ratio, and levels of lipid peroxidation, in the frontal cortex and striatum. In the treatment arm, consisting out of eight groups of animals, four groups of SIR rats received either saline or clozapine (5mg/kg i.p.) for the last 11 days of SIR. The remaining four groups were socially reared and also received either saline or clozapine treatment as above. At 8 weeks, four groups were subjected to behavioural testing as described above and a parallel neurochemical study was performed using the same layout as above, except that after the 8 weeks, neurochemical redox analysis were done as described above. Mixed statistical modelling with repeated measures and appropriate post hoc tests were used to access the effects of SIR with and without treatment on PPI and mean startle. Social interaction in SIR and socially reared animals, with and without treatment, was analyzed using 1-way ANOVA with suitable post hoc testing. Mixed linear models with repeated measures and appropriate post hoc tests were used for analysis of the redox data in SIR and socially reared animals, with and without treatment. RESULTS: In the non-treatment arm, %PPI was significantly reduced in SIR versus socially reared rats. Deficits in various social interactive behaviours were observed in SIR versus group-housed rats, as well as increased locomotor activity and self-grooming. Superoxide dismutase activity and oxidized versus reduced glutathione ratio were significantly decreased, together with a significant increase in products of lipid peroxidation, in isolation reared versus socially reared rats. Following clozapine treatment, %PPI in isolates was significantly elevated by clozapine versus saline treatment (i.e. reversed the effect of SIR). %PPI was unaltered in socially reared animals receiving either treatment. As with the non-treatment group, social interactive behaviours were significantly impaired in isolates receiving saline, while locomotor activity and self-grooming were increased. SIR rats receiving only saline showed similar altered redox state as the non-treatment groups, while clozapine treatment effectively reversed deficits in %PPI, aberrant social behaviours and redox alterations in the SIR rats, with limited to no effects in the socially reared controls. CONCLUSION: SIR thus significantly disrupts sensorimotor gating and social behaviours in male Sprague-Dawley rats, while at the same time evokes a significant disruption of redox state in both the frontal cortex and striatum of these animals, with distinct evidence for increased oxidative stress in these brain regions. Importantly, both altered behaviour and redox state are reversed by sub-chronic clozapine treatment. SIR is therefore a useful, non-lesion and non-pharmacological neurodevelopmental animal model of schizophrenia that presents with robust face, predictive and possibly construct validity for schizophrenia. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2010.

Renal cell carcinoma (RCC) is the commonest urogenital tumor, characterized by increased expression of hypoxia inducible factors (HIFs). During normoxia, HIFα subunits are targeted for proteasomal degradation by the product of the von Hippel Lindau gene (pVHL). In RCC, mutations in the VHL gene allow the HIFα subunits to escape degradation and translocate to the nucleus where they activate transcription of their target genes. Although both HIF1α and HIF2α are upregulated in RCC, it has been suggested that HIF2α plays the dominant role. To further elucidate the function of HIF2α in RCC, we generated a transgenic mouse model that permits temporal stabilization of HIF2α in renal tubular cells. Induction of HIF2α results in the rapid development of renal cysts - a feature observed in RCC. Taken together, these results suggest that HIF2α is a key player in development of RCC and an excellent candidate target for therapy in this disorder.

While participation in youth sports bolster a myriad of health benefits, it can also pose a risk to the athlete's health from the increasing prevalence of repetitive mild traumatic brain injuries (TBI), often referred to as concussions. The adverse effects from repeated traumatic blows give a combination of acute symptoms, which may potentially develop into long-term complications. There is little known about the epidemiology of concussions, and thus the development of an animal model would help enhance our understanding of this potentially debilitating injury. An appropriate animal model should mimic the conditions of how concussions occur, in that there is not an invasive method to induce the injury and follows the same biomechanics. In our adolescent repetitive mild TBI model, we utilized a free-falling weight to deliver the traumatic blow to anesthetized mice that allowed free head rotation after impact. The injured group received one hit daily over the course of three days. The mice then underwent several behavioral tests to analyze the cognitive deficits, and the pathology of the tissue was analyzed via silver, Hematoxylin and Eosin (H&E), and Fluoro Jade-B staining. The injured mice developed both short- and long-term memory and spatial learning deficits, symptoms commonly found in concussed athletes, but failed to show deficits in anxiety and depression tests. The Fluoro Jade-B, silver and H&E staining resulted in negative signals for cell death. This study properly demonstrates repetitive mild TBIs in an adolescent mice model.

Cancer and thrombosis are common co-occurrences in healthcare today. Cancer is the second leading cause of death in the United States with thrombosis being the second leading killer of cancer patients behind tumor progression. Cancer patients as a whole are 4 to 7 times more likely to develop thrombosis and 20%-30% of all first-time thrombosis diagnoses are cancer-related. Risk assessment and treatment options have much room for improvement. The lack of success for conventional antithrombotic prophylaxis suggests that hyperthrombosis in cancer works through a discrete pathway. A discovery by our group in recent years correlated the increased activity of Aryl hydrocarbon receptor (AHR) in the body to increase thrombotic phenotypes in patients with chronic kidney disease. Another group had published a manuscript about Kynurenine (Kyn), an activating ligand of the receptor that was produced by tumor cells to promote tumor growth through an AHR pathway (Opitz 2011). The link between the findings of these two groups could show that Kyn—AHR pathway is causing the increase in thrombosis in cancer patients. We used an animal model of thrombosis in cancer and created a new variation of it to test the Kyn—AHR pathway. We hypothesized that cancerous animals would show an increase in thrombosis and increased levels of AHR and Kyn along with downstream elements such as Tissue Factor (TF). Cancer was induced on nude mice via xenograft injection of cancer cells and 4-5 weeks of incubation to allow the tumors to proliferate. After the incubation period, mice underwent inferior vena cava (IVC) ligation, and were then euthanized 48 hours later. Two types of cancer were tested: HT-29 colon adenocarcinoma and A549 non small cell lung adenocarcinoma. There were 4 animal groups: mice that were injected with cancer cells and operated on, mice that were injected with cancer cells but not operated on, mice that were not injected but were operated on, and mice that did not receive neither the injections or the operation. After euthanasia, blood, tumors, and major organs were harvested to assess markers and pathways of thrombosis associated with cancer. We were able to successfully grow xenograft tumors in the nude mice. The HT-29 tumors grew very aggressively while A549 tumors experienced a small latent period before starting to proliferate. Animals with HT-29 and A549 xenograft tumors displayed greater thrombosis, measured by the weight of the blood clot formed in the IVC due to ligation (p=0.04 and p=0.05, respectively). HT-29 also displayed significant increases in Kyn, AHR activity, indoxyl sulfate (IS), and showed increased staining of tissue factor with immunohistochemistry. A549 did not have significant p-values in these experiments, but did show upward trends in all categories besides IS sera levels. In summary, we developed a new animal model of thrombosis in colon adenocarcinoma and showed significant increases in thrombosis as well as multiple markers of thrombosis. This is an exciting and complex way to study thrombosis in cancer in an in vivo approach with opportunities for future therapeutic testing. / 2020-07-03T00:00:00Z

Traumatic Brain Injury (TBI) is a leading cause of death and disability in the United States for which there are no federally approved pharmacological treatments. Preclinical trials with nicotinamide (NAM) and progesterone (Prog) treatment demonstrate beneficial neuroprotection and recovery of function following TBI. The primary goal of this study was to assess both neuroprotection and recovery of function in an animal model of TBI after combination treatment of both NAM and Prog. Animals received a cortical contusion injury over the sensorimotor cortex and were treated with either nicotinamide (75 mg/kg, i.p. NAM loading dose, 12 mg/kg/hr NAM, s.c. over 72 hrs), Prog (10 mg/kg Prog, i.p. over 72 hrs), NAM and Prog(75 mg/kg, i.p. NAM loading dose, followed by continuous infusion of 12 mg/kg/hr NAM, s.c. over 72 hrs; 10 mg/kg Prog, i.p. over 72 hrs) or Vehicle (75 mg/kg, i.p. sterile saline loading dose, followed by continuous infusion 12 mg/kg/hr sterile saline, s.c. over 72 hrs; 10 mg/kg peanut oil, i.p. over 72 hrs), and compared to a craniotomy only (Sham) group. Following this regimen they were assessed in a battery of behavioral (fine and gross motor, sensory, and cognitive) tasks or a histological assessment at 24 hrs post-injury assessing lesion cavity size, degenerating neurons, and reactive astrocytes. Our results replicate the beneficial effects of treatment with either NAM or Prog demonstrating significant improvements in recovery of function, and a reduction in lesion cavitation, degenerating neurons and reactive astrocytes 24 hours post-injury. The combination treatment of NAM and Prog led to a significant improvement in both neuroprotection at 24 hrs post-injury and recovery of function in sensorimotor related tasks when compared to each individual treatment (NAM or Prog). It is suggested here that further preclinical trials using NAM and Prog as a combination treatment should be done to identify any drug interactions, pharmacokinetics, and a new window of opportunity and proper dosing of this combination treatment.