Dissertationen: „Medical Neurobiology“

1

Taliaferro, Linda Kay. „Psychiatric Disorders as Potential Predictors in Medical Disease Development“. ScholarWorks, 2011. https://scholarworks.waldenu.edu/dissertations/939.

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Millions of individuals suffer disability or death from immune-based inflammatory diseases. If psychiatric disorders could be empirically linked to the prediction of immune-based inflammatory diseases, there would be a basis for promoting disease prevention measures for individuals diagnosed with one of four psychiatric disorders. Psychoneuroimmunology provided the theoretical base for understanding emotionally induced medical disease development. In this quantitative study, a parallel archival research design was used to investigate the degree to which generalized anxiety disorder, posttraumatic stress disorder, major depression recurrent, and dysthymic disorder predicted the presence of atherosclerosis, cardiovascular heart disease, rheumatoid arthritis, cancer, and type II diabetes. There were 1,209 electronic medical records of adult patients obtained through purposive stratified sampling. A secondary data analysis was employed using descriptive cross tabulation, chi-square test of independence, and multinomial logistic regression. The findings revealed major depression recurrent was a statistically significant predictor for atherosclerosis, rheumatoid arthritis, type II diabetes and cancer. Generalized anxiety disorder was a statistically significant predictor for cancer. The results can promote positive social change by providing information that could be used to develop assessment plans that identity individuals who are at risk of developing the comorbid diseases. The prevention programs could effectively be used to minimize the subsequent development of inflammatory diseases, which in turn could decrease the onset of the medical diseases among individuals with psychiatric disorders.

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2

Salagic, Belma. „Regulation of COX-2 signaling in the blood brain barrier“. Thesis, Linköping University, Linköping University, Department of Physics, Chemistry and Biology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-18113.

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Upon an inflammation the immune system signals the brain by secreted cytokines to elicit central nervous responses such as fever, loss of appetite and secretion of stress hormones. Since the blood brain barrier, (BBB) protects the brain from unwanted material, molecules like cytokines are not allowed to cross the barrier and enter the brain. However, it is clear that they in some way can signal the brain upon an inflammation. Many suggestions concerning this signaling has been made, one being that cytokines bind to receptors on the endothelial cells of the blood vessels of the brain and trigger the production of prostaglandins that can cross the BBB. This conversion is catalyzed by the enzyme cyclooxygenase-2, (COX-2), which is induced by transcription factors like NF-κB in response to cytokines. One of the central nervous responses to inflammatory stimuli is activation of the HPA-axis whose main purpose is glucocorticoid production. Glucocorticoids inhibit the inflammatory response by suppressing gene transcription of pro-inflammatory genes including those producing prostaglandins through direct interference with transcription factors such as NF-κB or initiation of transcription of anti-inflammatory genes like IκB or IL-10. It has however not been clear if glucocorticoids can target the endothelial cells of the brain in order to provide negative feed-back on the immune-to-brain signaling, and in that way inhibit central nervous inflammatory symptoms. An anatomical prerequisite for such a mechanism would be that the induced prostaglandin production occurs in cells expressing GR. This has however never been demonstrated. Here I show that a majority of the brain endothelial cells expressing the prostaglandin synthesizing enzyme COX-2 in response to immune challenge also express the glucocorticoid receptor, (GR). This indicates that immune-to-brain signaling is a target for negative regulation of inflammatory signaling executed by glucocorticoids and identifies brain endothelial GR as a possible future drug target for treatment of central nervous responses to inflammation such as fever and pain.

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3

Whicker, Wyatt, W. Drew Gill und Russell W. Brown. „DISCOVERY OF A NOVEL ANTI-NEUROINFLAMMATORY TREATMENT FOR AUDITORY SENSORIMOTOR GATING IN TWO RODENT MODELS OF SCHIZOPHRENIA“. Digital Commons @ East Tennessee State University, 2018. https://dc.etsu.edu/asrf/2018/schedule/204.

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Schizophrenia is primarily treated with the use of antipsychotic medications. However, antipsychotics used have severe, dose-dependent side effects in schizophrenia patients. Therefore, there is a need for new adjunctive drugs that lower the effective dose of first line schizophrenia drugs and improve patient symptoms. Neuroinflammation is associated with microglial activation in schizophrenia, and increased tumor necrosis factor-alpha (TNF) has shown to be associated with Metabolic Syndrome in schizophrenia patients. A newly developed anti-neuroinflammatory, PD2024, reduces TNF-alpha action in vitro and in vivo, and has been shown to be well-tolerated in rat and dog studies with no adverse effects. The purpose of this research is to evaluate the effect of PD2024 in two well-defined schizophrenia models in rats. The neonatal quinpirole model has been established through administration of the dopamine D2-like agonist quinpirole (NQ) or saline control (NS) postnatally from days 1-21. NQ treatment results in increases of dopamine D2 receptor sensitivity throughout the animal’s lifetime without changing receptor number, mimicking a hallmark of schizophrenia. The polyinosinic:polycytidylic acid (Poly I:C) model is based on mimicking an increase immune response during early brain development, which has been shown to increase the prevalence of schizophrenia. Poly I:C (2 mg/kg) was administered during the neonatal period at postnatal days (P)5-7 to produce this effect. Both models were given PD2024 at 10mg/kg orally through the diet from P30-67. Prepulse inhibition (PPI) was used to test sensorimotor gating deficits in the rats. PPI has past research showing its use as a quantitative phenotype for evaluating schizophrenia-associated behavioral and neurobiological deficits. In our PPI test, rats are exposed to three different, randomly ordered noise trials. The trials included a pulse trial with a 120-decibel startle pulse, a prepulse trial with an auditory click at 73, 76, or 82-decibels, and a no stimulus trial without any additional noise. The rats were given 25 randomized trials, comprised of 5 pulse, 15 prepulse (5 each of 73, 76, and 82dB) and 5 no stimulus trials. Background noise was 70dB, and the rats were tested during adolescence (days 45-46) and adulthood (60-65). In NQ adolescent rats, PPI was significantly improved in the PD2024-treated compared to NQ controls. NQ-PD2024 and NS rats were statistically equivalent throughout the trials. These results were reflected in the NQ adult model as well. The Poly I:C adolescents treated with PD2024 also demonstrated improved PPI performance compared to Poly I:C controls. This improvement was also shown in the adult Poly I:C rats. Overall, the PPI deficits in both models improved between 15 to 30% in adolescence and adulthood. These results indicate that PD2024 is effective in treating schizophrenia-associated behaviors.

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Batten, Seth R. „GLUTAMATE DYSREGULATION AND HIPPOCAMPAL DYSFUNCTION IN EPILEPTOGENESIS“. UKnowledge, 2013. http://uknowledge.uky.edu/medsci_etds/1.

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Epileptogenesis is the complex process of the brain developing epileptic acitivity. Due to the role of glutamate and the hippocampus in synaptic plasticity a dysregulation in glutamate neurotransmission and hippocampal dysfunction are implicated in the process of epileptogenesis. However, the exact causal factors that promote epileptogenesis are unknown. We study presynaptic proteins that regulate glutamate neurotransmission and their role in epileptogenesis. The presynaptic protein, tomosyn, is believed to be a negative regulator of glutamate neurotransmission; however, no one has studied the effects of this protein on glutamate transmission in vivo. Furthermore, evidence suggests that mice lacking tomosyn have a kindling phenotype. Thus, in vivo glutamate recordings in mice lacking tomosyn have the potential to elucidate the exact role of tomosyn in glutamate neurotransmission and its potential relationship to epileptogenesis. Here we used biosensors to measure glutamate in the dentate gyrus (DG), CA3, and CA1 of the hippocampus in tomosyn wild-type (Tom+/+), heterozygous (Tom+/-), and knock out (Tom-/-) mice. We found that, in the DG, that glutamate release increases as tomosyn expression decreases across genotype. This suggests that tomosyn dysregulation in the DG leads to an increase in glutamate release, which may explain why these mice have an epileptogenic phenotype.

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Ighodaro, Eseosa T. „STUDYING VASCULAR MORPHOLOGIES IN THE AGED HUMAN BRAIN USING LARGE AUTOPSY DATASETS“. UKnowledge, 2018. https://uknowledge.uky.edu/neurobio_etds/19.

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Cerebrovascular disease is a major cause of dementia in elderly individuals, especially Black/African Americans. Within my dissertation, we focused on two vascular morphologies that affect small vessels: brain arteriolosclerosis (B-ASC) and multi-vascular profiles (MVPs). B-ASC is characterized by degenerative thickening of the wall of brain arterioles. The risk factors, cognitive sequelae, and co-pathologies of B-ASC are not fully understood. To address this, we used multimodal data from the National Alzheimer’s Coordinating Center, Alzheimer’s Disease Neuroimaging Initiative, and brain-banked tissue samples from the University of Kentucky Alzheimer’s Disease Center (UK-ADC) brain repository. We analyzed two age at death groups separately: < 80 years and ≥ 80 years. Hypertension was a risk factor in the < 80 years at death group. In addition, an ABCC9 gene variant (rs704180), previously associated with aging-related hippocampal sclerosis, was associated with B-ASC in the ≥ 80 years at death group. With respect to cognition as determined by test scores, severe B-ASC was associated with worse global cognition in both age groups. With brain-banked tissue samples, we described B-ASC’s relationship to hippocampal sclerosis of aging (HS-Aging), a pathology characterized by neuronal cell loss in the hippocampal region not due to Alzheimer’s disease. We also studied MVPs, which are characterized by multiple small vessel lumens within a single vascular (Virchow-Robin) space. Little information exists on the frequency, risk factors, and co-pathologies of MVPs. Therefore, we used samples and data from the UK-ADC, University of Kentucky pathology department, and University of Pittsburgh pathology department to address this information. We only found MVPs to be correlated with age. Lastly, given the high prevalence of cerebrovascular disease and dementia in Black/African Americans, we discussed the challenges and considerations for studying Blacks/African Americans in these contexts.

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Xing, Bin. „THE EFFECT OF PPARγ ACTIVATION BY PIOGLITAZONE ON THE LIPOPOLYSACCHARIDE-INDUCED PGE₂ AND NO PRODUCTION: POTENTIALUNDERLYING ALTERATION OF SIGNALING TRANSDUCTION“. UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/629.

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Microglia-mediated neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Uncontrolled microglia activation produces major proinflammatory factors including cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) that may cause dopaminergic neurodegeneration. Peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone has potent antiinflammatory property. We hypothesize pioglitazone protects dopaminergic neuron from lipopolysaccharide (LPS)-induced neurotoxicity by interacting with relevant signal pathways, inhibiting microglial activation and decreasing inflammatory mediators. First, the neuroprotection of pioglitazone was explored. Second, the signaling transductions such as jun N-terminal kinase (JNK) and the interference with these pathways by pioglitazone were investigated. Third, the effect of pioglitazone on these pathways-mediated PGE2 / nitric oxide (NO) generation was investigated. Finally, the effect of PPARγ antagonist on the inhibition of PGE2 / NO by pioglitazone was explored. The results show that LPS neurotoxicity is microglia-dependent, and pioglitazone protects neurons against LPS insult possibly by suppressing LPS-induced microglia activation and proliferation. Second, pioglitazone protects neurons from COX-2 / PGE2 mediated neuronal loss by interfering with the NF-κB and JNK, in PPARγ-independent mechanisms. Third, pioglitazone significantly inhibits LPS-induced iNOS / NO production, and inhibition of LPS-induced iNOS protects neuron. Fourth, inhibition p38 MAPK reduces LPS-induced NO generation but no effect is found upon JNK inhibition, and pioglitazone inhibits p38 MAPK phosphorylation induced by LPS. In addition, pioglitazone increases PPARγ phosphorylation, followed by the increased PI3K/Akt phosphorylation. Nevertheless, inhibition of PI3K increases LPS-induced p38 MAPK phosphorylation. Inhibition of PI3K eliminates the inhibitive effect of pioglitazone on the LPS-induced NO production, suggesting that the inhibitive effect of pioglitazone on the LPS-induced iNOS and NO might be PI3K-dependent.

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Fu, Weisi. „PROTEIN KINASE A AND EPAC MEDIATE CHRONIC PAIN AFTER INJURY: PROLONGED INHIBITION BY ENDOGENOUS Y1 RECEPTORS IN DORSAL HORN“. UKnowledge, 2016. https://uknowledge.uky.edu/physiology_etds/31.

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Inflammation or nerve injury sensitizes several populations of nociceptive neurons in the dorsal horn of the spinal cord, including those that express the neuropeptide Y (NPY) Y1 receptor (Y1R). Our overall hypothesis is that after tissue or nerve injury, these Y1R-expressing neurons enter a state of latent sensitization (LS) that contributes to vulnerability to the development of chronic pain; furthermore, LS is under the tonic inhibitory control of endogenous Y1R signaling. First, we evaluated the intracellular signaling pathways that become activated in Y1R-expressing neurons and participate in LS. To do this, we established behavioral models of inflammatory or neuropathic pain, allowed pain hypersensitivity to resolve, and then during this period of pain remission we administered the Y1R receptor antagonist, BIBO3304, by intrathecal injection. As observed previously with mu-opioid receptor antagonists/inverse agonists, we found that BIBO3304 reinstated pain hypersensitivity via an N-methyl-D-aspartate receptor (NMDAR)- and adenylyl cyclase type 1 (AC1)-dependent mechanism. Our subsequent behavioral pharmacological experiments then established two signaling pathways downstream of AC1 that maintain LS. The first pathway involves protein kinase A (PKA) and transient receptor potential cation channel A1 (TRPA1) and channel V1 (TRPV1). The second pathway involves exchange proteins activated by cAMP (Epac 1 and Epac 2). We next found that nerve injury decreases the co-expression of Y1R with markers of excitatory interneurons, suggesting that Y1R-expressing neurons acquire a pain-enhancing phenotype after peripheral nerve injury. In a separate set of experiments that utilized Y1R-receptor internalization as an index of NPY release, we found that nerve injury increased stimulus-evoked NPY release. We conclude that injury induces pain-facilitatory mechanisms of LS in the dorsal horn involving PKA→TRPA1 and PKA→TRPV1 at the central terminals of primary afferent neurons. Whether Epac mechanisms are located on these same presynaptic terminals and/or at Y1R-expressing excitatory interneurons remain to be determined. We also conclude that injury-induced LS is masked by a compensatory up-regulation of spinal NPY release that tonically inhibits pain. These results present a novel mechanism of injury-induced LS and endogenous control of the transition from acute to chronic pain by the NPY-Y1R system. Our work sheds light on novel targets for the treatment of chronic pain.

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Salmeron, Kathleen Elizabeth. „INVESTIGATIONS OF INTERLEUKIN-1 ALPHA AS A NOVEL STROKE THERAPY IN EXPERIMENTAL ISCHEMIC STROKE“. UKnowledge, 2018. https://uknowledge.uky.edu/neurobio_etds/20.

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Stroke is a leading cause of death and disability worldwide. Although rapid recognition and prompt treatment have dropped mortality rates, most stroke survivors are left with permanent disability. Approximately 87% of all strokes result from the thromboembolic occlusion of the cerebrovasculature (ischemic strokes). Potential stroke therapeutics have included anti-inflammatory drugs, as well as many other targets with the goal of mitigating the acute and chronic inflammatory responses typically seen in an ischemic stroke. While these approaches have had great success in preclinical studies, their clinical translation has been less successful. Master inflammatory cytokines, such as IL-1, are of particular interest. IL-1’s isoforms, IL-1α and IL-1β, were long thought to have similar function. While IL-1β has been extensively studied in stroke, the role of IL-1α during post stroke inflammation has been overlooked. Because IL-1 inhibitors have been unsuccessful in clinical application, we reasoned that IL-1α may provide previously unknown benefits to the brain after injury. We hypothesized that IL-1α could be protective or even accelerate reparative processes in the brain such as producing new blood vessels (angiogenesis) or neurons (neurogenesis). To test that IL-1α is protective after stroke, we tested IL-1α’s protective effects on primary cortical neurons in in vitro models of stroke. We showed that IL-1α was directly protective on primary cortical neurons in a dose-dependent fashion. We then performed mouse middle cerebral artery occlusion stroke studies to determine the safety of giving IL-1α in vivo. These studies showed that administering IL-1α acutely was neuroprotective. However, intravenous (IV) administration of IL-1α resulted in transient, hemodynamic changes following drug delivery. To minimize these systemic effects, we administered IL-1α intra-arterially (IA) directly into the stroke affected brain tissue, allowing us to significantly lower the concentration of administered IL-1α. In comparison to IV, IA IL-1α showed greater histological protection from ischemic injury as well as improved functional recovery following stroke, all without systemic side effects. To test that IL-1α could aid in neurorepair following stroke, we tested IL-1α’s ability to help damaged blood vessels repair in vitro. We found that IL-1α significantly increased brain endothelial cell activation, proliferation, migration, and capillary formation. We tested IL-1α’s proangiogenic properties in vivo by administering IL-1α three days following stroke. Delayed administration allowed us to separate IL-1α’s acute neuroprotective effects from potential subacute angiogenic effects. We found that mice receiving IL-1α performed significantly better on behavioral tests and also showed greater vascularization within the penumbra two weeks following stroke. We also found that IL-1α treated animals showed more endothelial activation than vehicle treated animals. Finally, our studies showed that IL-1α treated animals showed increased early-phase neurogenesis with evidence of increased proliferation at the subventricular zone suggesting that IL-1α’s beneficial effects are even more far-reaching than previously thought. In conclusion, our experiments suggest that the inflammatory cytokine IL-1α is neuroprotective and neuroreparative in experimental ischemic stroke and worthy of further study as a novel stroke therapy.

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Ivy, Devon. „DEFINING THE RADIORESPONSE OF MOSSY CELLS“. CSUSB ScholarWorks, 2018. https://scholarworks.lib.csusb.edu/etd/633.

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Clinical radiotherapy is used to treat a variety of brain tumors within the central nervous system. While effective, it can result in progressive and debilitating cognitive impairment that can diminish quality of life. These impairments have been linked to hippocampal dysfunction and corresponding deficits in spatial learning and memory. Mossy cells are a major population of excitatory neurons located within the dentate hilus and highly involved in hippocampal circuitry. They play critical roles in spatial navigation, neurogenesis, memory, and are particularly vulnerable to a variety of neurotoxic insults. However, their sensitivity to ionizing radiation has yet to be investigated in detail. I hypothesize that mossy cells are critical targets for ionizing radiation, whereby damage to these targets contributes to the mechanisms associated with radiation-induced hippocampal dysfunction. To test this idea, wild-type mice were exposed to clinically relevant doses of cranial x-ray irradiation and their hippocampi were examined 1 month and 3 months post treatment. A significant decline in both the number of mossy cells and their activity were observed. In addition, dentate granular cells demonstrated reduced levels of activity, as well as reduced proliferation within the subgranular zone. A second cohort of mice was introduced to a novel environment in order to induce the expression of immediate early genes. Analysis of c-Fos mRNA yielded a significant increase in control but not irradiated animals, suggesting that radiotherapy impaired immediate early gene expression and resultant functional behavioral outcomes. These findings support the proposition that radiation-induced damage to mossy cells contributes to hippocampal deficiencies which result in cognitive dysfunction.

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Bardgett, Megan Elyse. „NEURAL MECHANISMS OF SYMPATHETIC ACTIVATION DURING HYPERINSULINEMIA AND OBESITY-INDUCED HYPERTENSION“. UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/46.

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Obesity afflicts more than 30% of the U.S. population and is a major risk factor for the development of hypertension, type II diabetes, and cardiovascular disease. Studies in humans and animals indicate that obesity is associated with increased sympathetic outflow to the vasculature and kidneys. One mechanism postulated to underlie the increase in sympathetic nerve activity (SNA) in obesity is hyperinsulinemia. Little is known regarding the central circuitry underlying elevated SNA and arterial blood pressure (ABP) during hyperinsulinemia and obesity or if sympathoexcitatory circuits are still responsive to insulin in obesity. Hyperinsulinemic-euglycemic clamps elevate SNA to the hind limb vasculature in lean rodents but obesity is associated with resistance to the peripheral and anorexic effects of insulin. Therefore, the first aim was to determine whether diet-induced obesity causes development of insulin resistance in the central circuits mediating SNA. The sympathoexcitatory response to insulin was still intact in diet-induced obese rats indicating a role for insulin in the elevation in SNA and ABP in obesity. The second aim of this project was to identify the specific receptors in the rostral ventrolateral medulla (RVLM) that mediate the elevated SNA during hyperinsulinemia. The RVLM provides basal sympathetic tone and maintains baseline ABP. Glutamate is the major excitatory neurotransmitter and glutamate receptors of the RVLM are known to mediate multiple forms of hypertension. Blockade of RVLM NMDA-specific glutamatergic receptors reverses the increased lumbar SNA associated with hyperinsulinemia. In contrast, blockade of angiotensin II type 1 or melanocortin receptors in the RVLM had no effect on the sympathoexcitatory response to insulin. The goal of the third aim was to identify the cellular mechanisms within RVLM that mediate the elevated SNA and ABP in diet-induced obesity. Blockade of RVLM glutamate receptors reversed the elevated ABP and lumbar SNA associated with diet-induced obesity while it had no effect on rats on a low fat diet or those resistant to weight gain on the high fat diet. Similar to the findings during hyperinsulinemia, blockade of RVLM angiotensin II type 1 or melanocortin receptors had no effect on lumbar SNA or ABP during diet-induced obesity.

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Bowles, Olivia M. „Potential Treatments for Malformation Associated Epilepsy“. VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4412.

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Epilepsy has been previously attributed to either increased excitation or decreased inhibition. With this closed frame of mind, modern medicine has been unable to develop a permanent treatment against the mechanisms of epilepsy. In order to treat patients with intractable seizures, especially those caused by developmental malformations, it is essential to understand the entirety of mechanisms that could possibly play a role in the abnormal cortical function. One such developmental malformation is known as polymicrogyria. Epileptogenesis occurs in an area laterally adjacent to this malformation known as the paramicrogyral region (PMR). Past studies have narrowed down the potential cause of this increased network excitation to a certain type of inhibitory interneuron, the somatostatin (SS) interneuron. Additionally, previous studies have shown an increase in the mGlu5 receptor on this interneurons in the PMR region only and not in control tissue, meaning that targeting these receptors as treatment will not affect normal functioning tissue. These results lead to our hypothesis: blockade of the mGluRs will decrease the 2 activity of SS interneurons and thereby prevent the generation of epileptiform activity and increased SS output in malformed cortex. Utilizing the freeze-lesion model for microgyria in transgenic mice expressing Channelrhodopsin optogenetic channels in SS interneurons, we assessed the contribution of these SS interneurons in four different animal groups: control or PMR treated with either Gabapentin, a current AED (antiepileptic drug), or MTEP, an mGlu5 receptor antagonist. We tested the effects of these two drugs on SS interneuron output to determine whether they decrease the over activation in the PMR that has been previously studied. The following study revealed no correlation between Gabapentin-treated animals and a decrease in epileptiform activity. Additionally, no significant difference was seen between the MTEP-treated groups in the protocols that were measured.

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Gilmer, Lesley Knight. „AGE MAY BE HAZARDOUS TO OUTCOME FOLLOWING TRAUMATIC BRAIN INJURY: THE MITOCHONDRIAL CONNECTION“. Lexington, Ky. : [University of Kentucky Libraries], 2009. http://hdl.handle.net/10225/1135.

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Thesis (Ph. D.)--University of Kentucky, 2009.
Title from document title page (viewed on May 11, 2010). Document formatted into pages; contains: viii, 161 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 130-154).

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Davis, Laurie Michelle Helene. „THE UNDERLYING MECHANISM(S) OF FASTING INDUCED NEUROPROTECTION AFTER MODERATE TRAUMATIC BRAIN INJURY“. UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/673.

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Traumatic brain injury (TBI) is becoming a national epidemic, as it accounts for 1.5 million cases each year. This disorder affects primarily the young population and elderly. Currently, there is no treatment for TBI, which means that ~2% of the U.S. population is currently living with prolonged neurological damage and dysfunction. Recently, there have been many studies showing that TBI negatively impacts mitochondrial function. It has been proposed that in order to save the cell from destruction mitochondrial function must be preserved. The ketogenic diet, originally designed to mimic fasting physiology, is effective in treating epilepsy. Therefore, we have used fasting as a post injury treatment and attempted to elucidate its underlying mechanism. 24 hours of fasting after a moderate TBI increased tissue sparing, cognitive recovery, improved mitochondrial function, and decreased mitochondrial biomarkers of injury. Fasting results in hypoglycemia, the production of ketones, and the upregulation of free fatty acids (FFA). As such, we investigated the neuroprotective effect of hypoglycemia in the absence of fasting through insulin administration. Insulin administration was not neuroprotective and increased mortality in some treatment groups. However, ketone administration resulted in increased tissue sparing. Also, reduced reactive oxygen species (ROS) production, increased the efficiency of NADH utilization, and increased respiratory function. FFAs and uncoupling proteins (UCP) have been implicated in an endogenously regulated anti-ROS mechanism. FFAs of various chain lengths and saturation were screened for their ability to activate UCP mediated mitochondrial respiration and attenuate ROS production. We also measured FFA levels in serum, brain, and CSF after a 24 hour fast. We also used UCP2 transgenic overexpressing and knockout mice in our CCI injury model, which showed UCP2 overexpression increased tissue sparing, however UCP2 deficient mice did not show a decrease in tissue sparing, compared with their wild type littermates. Together our results indicate that post injury initiated fasting is neuroprotective and that this treatment is able to preserve mitochondrial function. Our work also indicates ketones and UCPs may be working together to preserve mitochondrial and cellular function in a concerted mechanism, and that this cooperative system is the underlying mechanism of fasting induced neuroprotection.

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Hinzman, Jason Michael. „DISRUPTIONS IN THE REGULATION OF EXTRACELLULAR GLUTAMATE IN THE RAT CENTRAL NERVOUS SYSTEM AFTER DIFFUSE BRAIN INJURY“. UKnowledge, 2012. http://uknowledge.uky.edu/neurobio_etds/2.

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Glutamate, the predominant excitatory neurotransmitter in the central nervous system, is involved in almost all aspects of neurological function including cognition, motor function, memory, learning, decision making, and neuronal plasticity. For normal neurological function, glutamate signaling must be properly regulated. Disrupted glutamate regulation plays a pivotal role in the acute pathophysiology of traumatic brain injury (TBI), disrupting neuronal signaling, initiating secondary injury cascades, and producing excitotoxicity. Increases in extracellular glutamate have been correlated with unfavorable outcomes in TBI survivors, emphasizing the importance of glutamate regulation. The aim of this thesis was to examine disruptions in the regulation of extracellular glutamate after experimental TBI. In these studies, we used glutamate-sensitive microelectrode arrays (MEAs) to examine the regulation of extracellular glutamate two days after diffuse brain injury. First, we examined which brain regions were vulnerable to post-traumatic increases in extracellular glutamate. We detected significant increases in extracellular glutamate in the dentate gyrus and striatum, which correlated to the severity of brain injury. Second, we examined the regulation of extracellular glutamate by neurons and glia to determine the mechanisms responsible for post-traumatic increases in extracellular glutamate. In the striatum of brain-injured rats, we detected significant disruptions in release of glutamate by neurons and significant decreases in the removal of glutamate from the extracellular space by glia. Third, we examined if a novel therapeutic strategy, a viral-vector mediated gene delivery approach, could improve the regulation of extracellular glutamate. Infusion of an adeno-associated virus expressing a glutamate transporter into the rat striatum produced significant improvements in glutamate clearance, identifying a novel strategy to reduce excitotoxicity. Lastly, we examined the translational potential of MEAs as novel neuromonitoring device for clinical TBI research. Overall, these studies have demonstrated the translational potential of MEAs to aid in the diagnosis and treatment of TBI survivors.

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Cebak, John. „MITOCHONDRIAL AND NEUROPROTECTIVE EFFECTS OF PHENELZINE RELATED TO SCAVENGING OF NEUROTOXIC LIPID PEROXIDATION PRODUCTS“. UKnowledge, 2015. http://uknowledge.uky.edu/neurobio_etds/12.

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Lipid peroxidation is a key contributor to the pathophysiology of traumatic brain injury (TBI). Traditional antioxidant therapies are intended to scavenge the free radicals responsible for either the initiation or propagation of lipid peroxidation (LP). However, targeting free radicals after TBI is difficult as they rapidly react with other cellular macromolecules, and thus has a limited post-injury time window in which they may be intercepted by a radical scavenging agent. In contrast, our laboratory has begun testing an antioxidant approach that scavenges the final stages of LP i.e. formation of carbonyl-containing breakdown products. By scavenging breakdown products such as the highly reactive and neurotoxic aldehydes (often referred to as “carbonyls”) 4-hydroxynonenal (4-HNE) and acrolein (ACR), we are able to prevent the covalent modification of cellular proteins that are largely responsible for posttraumatic neurodegeneration. Without intervention, carbonyl additions render cellular proteins non-functional which initiates the loss of ionic homeostasis, mitochondrial failure, and subsequent neuronal death. Phenelzine (PZ) is an FDA-approved monoamine oxidase (MAO) inhibitor traditionally used for the treatment of depression. Phenelzine also possesses a hydrazine functional group capable of covalently binding neurotoxic carbonyls. The hypothesis of this dissertation is that carbonyl scavenging with PZ will exert an antioxidant neuroprotective effect in the traumatically injured rat brain mechanistically related to PZ’s hydrazine moiety reacting with the lipid peroxidation (LP)-derived reactive aldehydes 4-hydroxynonenal (4-HNE) and acrolein (ACR). Data from our ex vivo experiments demonstrate that the exogenous application of 4-HNE or ACR significantly reduced respiratory function and increased markers of oxidative damage in isolated non-injured rat cortical mitochondria, whereas PZ pre-treatment significantly prevented mitochondrial dysfunction and oxidative modification of mitochondrial proteins in a concentration-related manner. Additionally, PZ’s neuroprotective scavenging mechanism was confirmed to require the presence of a hydrazine moiety based on experiments with a structurally similar MAO inhibitor, pargyline, which lacks the hydrazine group and did not protect the isolated mitochondria from 4-HNE and ACR. Our in vivo work demonstrates that subcutaneous injections of PZ following TBI in the rat are able to significantly protect brain mitochondrial respiratory function, decrease markers of oxidative damage, protect mitochondrial calcium buffering capacity, and increase cortical tissue sparing without decreasing neuronal cytoskeletal spectrin degradation. These results confirm that PZ is capable of protecting mitochondrial function and providing neuroprotection after experimental TBI related to scavenging of neurotoxic LP degradation products.

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Talauliker, Pooja Mahendra. „CHARACTERIZATION AND OPTIMIZATION OF MICROELECTRODE ARRAYS FOR GLUTAMATE MEASUREMENTS IN THE RAT HIPPOCAMPUS“. Lexington, Ky. : [University of Kentucky Libraries], 2010. http://hdl.handle.net/10225/1116.

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Thesis (Ph. D.)--University of Kentucky, 2010.
Title from document title page (viewed on May 17, 2010). Document formatted into pages; contains: xii, 180 p. : ill. (some col.). Includes abstract and vita. Includes bibliographical references (p. 152-173).

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Yonutas, Heather M. „NOVEL TARGETS FOR MITOCHONDRIAL DYSFUNCTION FOLLOWING TRAUMATIC BRAIN INJURY“. UKnowledge, 2016. https://uknowledge.uky.edu/neurobio_etds/15.

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Mitochondrial dysfunction is a phenomenon observed in models of Traumatic Brain Injury (TBI). Loss of mitochondrial bioenergetics can result in diminished cellular homeostasis leading to cellular dysfunction and possible cellular death. Consequently, the resultant tissue damage can manifest as functional deficits and/or disease states. Therapeutic strategies to target this mitochondrial dysfunction have been investigated for models TBI and have shown promising effects. For this project, we tested the hypothesis that mitoNEET, a novel mitochondrial membrane protein, is a target for pioglitazone mediated neuroprotection. To test this, we used a severe Controlled Cortical Impact (CCI) injury model in mitoNEET null and wild-type mice. We then dosed these animals with pioglitazone or NL-1, which is a compound that has a similar structure to pioglitazone allowing us to hone in one the importance of mitoNEET binding. Wild-type animals treated with the mitoNEET ligands, both pioglitazone and NL-1, had improved mitochondrial function, tissue sparing and functional recovery, compared to mitoNEET null animals. In addition to this specific hypothesis tested, our experiments provided insight casting doubt on the central dogma that mitochondrial dysfunction following TBI is the result of vast oxidative damage and consequential irreversible mitochondrial loss. The data from these studies show that when mitoNEET is targeted with pioglitazone at 12 hours’ post-injury, mitochondrial dysfunction can be reversed. Additionally, when bypassing proteins upstream of Complex I with an alternative biofuel, such as beta-hydroxybuterate (BHB), TBI related mitochondrial dysfunction is once again reversed. This leads to novel hypothesis for future work which posits mitoNEET as a redox sensitive switch; when mitoNEET senses changes in redox, as seen in TBI, it inhibits mitochondrial respiration. When targeted with an agonist/ligand or bypassed with a biofuel TBI mitochondrial dysfunction can be reversed. These studies support the role of mitoNEET in the neuropathological sequelae of brain injury, supporting mitoNEET as a crucial target for pioglitazone mediated neuroprotection following TBI. Lastly, these studies propose a mechanism of TBI related mitochondrial dysfunction which can reversed with pharmacological agents.

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Duggan, Alexandra. „Synaptic protein expression in human postmortem brain tissue of autism spectrum disorder“. Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/honors/549.

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It is estimated that one in 59 children in the US are affected by autism spectrum disorder (ASD). ASD is distinguished by social and communication deficits that can be displayed throughout a wide range of severity. This resulting spectrum of behaviors observed in ASD suggests that a complex etiology is involved. Previous studies have shown a genetic susceptibility to autism including paternal age, twin and sibling concordance. Genetic sequencing of those affected as well as first order relatives have identified alterations in genes associated with neuronal synaptic communication. However, very little information is available regarding the pathophysiology of synapses in ASD. Neuronal communication between anterior cingulate cortical neurons via synapses with other brain regions is vital in the execution of social behaviors in individuals. The aim of this study was to evaluate the protein expression of the synaptic marker spinophilin and post-synaptic density protein-95 (PSD-95) in postmortem ASD gray matter brain tissue from the anterior cingulate and frontal cortex to compare to typically developing (TD) control brain tissue. Postmortem brain tissue of ASD and TD subjects was acquired from nationally funded brain repositories previously matched by brain area, age and gender. Immunoblotting for spinophilin and PSD-95 was performed using anterior cingulate and frontal cortical gray matter brain tissue from matched ASD and TD brain tissue. Spinophilin and PSD-95 protein amounts for all donors were normalized using GAPDH. Frontal cortical tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=6). Anterior cingulate tissue demonstrated no significant differences in spinophilin protein expression between TD and ASD groups (N=5). PSD-95 protein expression levels did not result in any significant differences between ASD donors and their control pairs for either brain tissue region. Although no changes were detected in the frontal cortex or anterior cingulate cortex, more brain areas and subjects must be evaluated to determine if spinophilin or PSD-95 can be reliable markers for synaptic alterations in ASD. These data are critical in determining synaptic pathology in ASD which may lead to future treatments.

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Khaychuk, Vadim. „PRION CHARACTERIZATION USING CELL BASED APPROACHES“. UKnowledge, 2012. http://uknowledge.uky.edu/microbio_etds/2.

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Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources.

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Sonne, James H. „EFFECTS OF INTRANASALLY ADMINISTERED DNSP-11 ON THE CENTRAL DOPAMINE SYSTEM OF NORMAL AND PARKINSONIAN FISCHER 344 RATS“. UKnowledge, 2013. http://uknowledge.uky.edu/neurobio_etds/5.

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Due to the blood-brain barrier, delivery of many drugs to the brain has required intracranial surgery which is prone to complication. Here we show that Dopamine Neuron Stimulating Peptide 11 (DNSP-11), following non-invasive intranasal administration, protects dopaminergic neurons from a lesion model of Parkinson’s disease in the rat. A significant and dose-dependent increase in an index of dopamine turnover (the ratio of DOPAC to dopamine) was observed in the striatum of normal young adult Fischer 344 rats by whole-tissue neurochemistry compared to vehicle administered controls. Among animals challenged with a moderate, unilateral 6-hydroxy-dopamine (6-OHDA) lesion of the substantia nigra, those treated repeatedly with intranasally administered DNSP-11 exhibited greater numbers of tyrosine hydroxylase (TH) positive dopaminergic neuronal cell bodies in the substantia nigra and greater TH+ fiber density in the striatum when compared to animals treated intranasally with vehicle only or a scrambled version of the DNSP-11 sequence. Lesioned animals that received intranasal DNSP-11 treatment did not exhibit abnormal, apomorphine-induced rotation behavior, contrasted with animals that received only vehicle or scrambled peptide that did exhibit significantly greater rotation behavior. In addition, the endogenous expression of DNSP-11 from the pro-region of GDNF was investigated by immunohistochemistry with a custom, polyclonal antibody. Signal from the DNSP-11 antibody was found to be differentially localized from the mature GDNF protein both spatially and temporally. While DNSP-11-like immunoreactivity extensively colocalizes with GDNF immunoreactivity at post-natal day 10, the day of maximal GDNF expression, DNSP-11-like signal was found to be present in the 3 month old rat brain with signal in the substantia nigra, ventral thalamic nucleus, dentate gyrus of the hippocampus, with the strongest signal observed in the locus ceruleus where GDNF is not expressed. Results from immunoprecipitation of brain homogenate were not consistent with the synthetic, amidated 11 amino-acid rat DNSP-11 sequence. However, binding patterns in the literature of NPY, the only homologous sequence present in the CNS, do not recapitulate the immunoreactive patterns observed for the DNSP-11 signal. This study provides evidence for a potential easy-to-administer intranasal therapeutic using the DNSP-11 peptide for protection from a 6-OHDA lesion rat model of Parkinson’s disease.

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Hunt, Robert F. III. „LOCAL SYNAPTIC NETWORK INTERACTIONS IN THE DENTATE GYRUS OF A CORTICAL CONTUSION MODEL OF POSTTRAUMATIC EPILEPSY“. UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/94.

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Posttraumatic epilepsy is a common consequence of brain trauma. However, little is known about how long-term changes in local excitatory and inhibitory synaptic networks contribute to epilepsy after closed-head brain injury. This study adapted a widely used model of experimental brain injury as a mouse model of posttraumatic epilepsy. Behavioral seizure activity and alterations in synaptic circuitry in the dentate gyrus were examined in mice after experimental cortical contusion brain injury. Spontaneous behavioral seizures were observed in 20% of mice after moderate injury and 36-40% of mice weeks after severe injury. In the dentate gyrus, most mice displayed regionally localized mossy fiber reorganization ipsilateral to the injury that was absent in control mice or sections contralateral to the injury. Extracellular field and whole-cell patch clamp recordings were performed in acute brain slice preparations of the dentate gyrus. Dentate granule cells displayed spontaneous and evoked activity that was consistent with network synchronization and the formation of recurrent excitatory network only in slices that had posttraumatic mossy fiber sprouting. The excitability of surviving hilar GABAergic interneurons, which provide important feedback inhibition to granule cells, was examined at similar time points. Cell-attached and whole-cell voltage-clamp recordings revealed increased spontaneous and glutamate photostimulation-evoked excitatory input to hilar GABA neurons ipsilateral to the injury, versus control and contralateral slices. Despite increased excitatory synaptic input to interneurons, whole-cell voltage-clamp recordings revealed a reduction in inhibitory synaptic input to granule cells. These findings suggest that there are alterations in excitatory and inhibitory circuits in mice with posttraumatic mossy fiber sprouting and seizures after cortical contusion head injury.

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Sauerbeck, Andrew David. „TRICHLOROETHYLENE EXPOSURE AND TRAUMATIC BRAIN INJURY INTERACT AND PRODUCE DUAL INJURY BASED PATHOLOGY AND PIOGLITAZONE CAN ATTENUATE DEFICITS FOLLOWING TRAUMATIC BRAIN INJURY“. UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/133.

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The development of Parkinson's disease (PD) in humans has been linked to genetic and environmental factors for many years. However, finding common single insults which can produce pathology in humans has proved difficult. Exposure to trichloroethylene (TCE) or traumatic brain injury (TBI) has been shown to be linked to PD and it has also been proposed that multiple insults may be needed for disease development. The present studies show that exposure to TCE prior to a TBI can result in pathology similar to early PD and that the interaction of both insults is required for impairment in behavioral function, and cell loss. Following exposure to TCE for 2 weeks there is a 75% impairment in mitochondrial function but it has yet to be shown if the addition of a TBI can make this worse. If the exposure to TCE is reduced to 1 week and combined with TBI a 50% reduction in mitochondrial function is observed following the dual injury which requires both insults. These studies provide further support for the hypothesis that PD may result from a multifactorial mechanism. It had been established that regional differences exist in mitochondrial function across brain regions. The present studies indicate that previous findings are not likely to be the result of differences in individual mitochondria isolated from the cortex, striatum, and hippocampus. Further analysis of the effect of mitochondrial inhibitors on enzyme activity and oxygen consumption reveal that the different regions of the brain are similarly affected by the inhibitors. These results suggest that findings from previous studies indicating regionally specific deficits following systemic toxin exposure, such as with TCE, are not the result of regional differences in the individual mitochondria. Given that TBI results in significant dysfunction, finding effective therapeutics for TBI will provide substantial benefits to individuals suffering an insult. Treatment with Pioglitazone following TBI reduced mitochondrial dysfunction, cognitive impairment, cortical tissue loss, and inflammation. These findings provide initial evidence that treatment with Pioglitazone may be an effective intervention for TBI.

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Clark, Jonathan Darrell. „FUNCTIONAL CONNECTIVITY FOR CONFIGURAL AND FEATURAL FACE PROCESSING IN THE BROAD AUTISM PHENOTYPE“. UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/174.

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During normal development, face processing involves a gradual shift from a featurally oriented style to a mature configural style by adolescence. This shift may coincide with increased right hemispheric dominance for faces supporting configural processing. Previous studies suggest that individuals diagnosed with ASD continue to process faces using individual parts and features into adulthood. This continued bias may be due to deficits in configural processing abilities. The current study investigated measures of functional connectivity during featural and configural processing of faces in broad autism phenotype sibling (ASD-sibs) children compared to age, sex, and handedness matched normal developing (ND) controls and in children diagnosed with an Autism Spectrum Disorder compared to ASD-matched ND controls. Results indicate that children with ASD and ASD-sibs were capable of performing configural processing tasks at similar performance levels to those of ND children. Additionally, patterns of functional network connectivity for configural processing in ASD-sibs were similar to those observed in ND controls. Few network-wide hemispheric differences emerged between groups. While behavioral performance and overall network-wide patterns of connectivity suggest a face processing network that is capable of supporting configural processing in ASD and ASD-sibs, abnormalities were observed in specific regions. The amygdala and fusiform face area showed fewer interactions with the rest of the face processing network in ASD children compared to ND during configural, but not featural processing. Additionally, hemispheric comparisons show greater differences between ASD and ND controls in the right fusiform face area. The ability of these regions to communicate with other regions in the face network could be important for social motivation and attention during configural processing. Interestingly, network connectivity in ASD children during passive viewing of faces, objects, and textures without featural or configural manipulations showed a more functionally integrated, and less segregated network with a lower “wiring cost” during non-face conditions compared to ND children. ASD-sibs may demonstrate a similar milder pattern.

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Stephens, Michelle Lee. „GLUTAMATE REGULATION IN THE HIPPOCAMPAL TRISYNAPTIC PATHWAY IN AGING AND STATUS EPILEPTICUS“. UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/736.

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A positive correlation exists between increasing age and the incidence of hippocampal-associated dysfunction and disease. Normal L-glutamate neurotransmission is absolutely critical for hippocampal function, while abnormal glutamate neurotransmission has been implicated in many neurodegenerative diseases. Previous studies, overwhelmingly utilizing ex vivo methods, have filled the literature with contradicting reports about hippocampal glutamate regulation during aging. For our studies, enzyme-based ceramic microelectrode arrays (MEA) were used for rapid (2 Hz) measurements of extracellular glutamate in the hippocampal trisynaptic pathway of young (3-6 months), late-middle aged (18 mo.) and aged (24 mo.) urethane-anesthetized Fischer 344 rats. Compared to young animals, glutamate terminals in cornu ammonis 3 (CA3) showed diminished potassium-evoked glutamate release in aged rats. In late-middle aged animals, terminals in the dentate gyrus (DG) showed increased evoked release compared to young rats. The aged DG demonstrated an increased glutamate clearance capacity, indicating a possible age-related compensation to deal with the increased glutamate release that occurred in late-middle age. To investigate the impact of changes in glutamate regulation on the expression of a disease process, we modified the MEA technology to allow recordings in unanesthetized rats. These studies permitted us to measure glutamate regulation in the hippocampal formation without anesthetic effects, which showed a significant increase in basal glutamatergic tone during aging. Status epilepticus was induced by local application of 4-aminopyridine. Realtime glutamate measurements allowed us to capture never-before-seen spontaneous and highly rhythmic glutamate release events during status epilepticus. A significant correlation between pre-status tonic glutamate and the quantity of status epilepticus-associated convulsions and glutamate release events was determined. Taken together, this body of work identifies the DG and CA3 as the loci of age-associated glutamate dysregulation in the hippocampus, and establishes elevated levels of glutamate as a key factor controlling severity of status epilepticus in aged animals. Based upon the potential ability to discriminate brain areas experiencing seizure (i.e. synchronized spontaneous glutamate release) from areas not, we have initiated the development of a MEA for human use during temporal lobe resection surgery. The final studies presented here document the development and testing of a human microelectrode array prototype in non-human primates.

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Alterman, Julia F. „A CNS-Active siRNA Chemical Scaffold for the Treatment of Neurodegenerative Diseases“. eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1027.

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Small interfering RNAs (siRNAs) are a promising class of drugs for treating genetically-defined diseases. Therapeutic siRNAs enable specific modulation of gene expression, but require chemical architecture that facilitates efficient in vivodelivery. siRNAs are informational drugs, therefore specificity for a target gene is defined by nucleotide sequence. Thus, developing a chemical scaffold that efficiently delivers siRNA to a particular tissue provides an opportunity to target any disease-associated gene in that tissue. The goal of this project was to develop a chemical scaffold that supports efficient siRNA delivery to the brain for the treatment of neurodegenerative diseases, specifically Huntington’s disease (HD). HD is an autosomal dominant neurodegenerative disorder that affects 3 out of every 100,000 people worldwide. This disorder is caused by an expansion of CAG repeats in the huntingtin gene that results in significant atrophy in the striatum and cortex of the brain. Silencing of the huntingtin gene is considered a viable treatment option for HD. This project: 1) identified a hyper-functional sequence for siRNA targeting the huntingtin gene, 2) developed a fully chemically modified architecture for the siRNA sequence, and 3) identified a new structure for siRNA central nervous system (CNS) delivery—Divalent-siRNA (Di-siRNA). Di-siRNAs, which are composed of two fully chemically-stabilized, phosphorothioate-containing siRNAs connected by a linker, support potent and sustained gene modulation in the CNS of mice and non-human primates. In mice, Di-siRNAs induced potent silencing of huntingtin mRNA and protein throughout the brain one month after a single intracerebroventricular injection. Silencing persisted for at least six months, with the degree of gene silencing correlating to guide strand tissue accumulation levels. In Cynomolgus macaques, a bolus injection exhibited significant distribution and robust silencing throughout the brain and spinal cord without detectable toxicity. This new siRNA scaffold opens the CNS for RNAi-based gene modulation, creating a path towards developing treatments for genetically-defined neurological disorders.

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Griggs, Ryan B. „TARGETING METHYLGLYOXAL AND PPAR GAMMA TO ALLEVIATE NEUROPATHIC PAIN ASSOCIATED WITH TYPE 2 DIABETES“. UKnowledge, 2015. https://uknowledge.uky.edu/physiology_etds/24.

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Neuropathic pain affects up to 50% of the 29 million diabetic patients in the United States. Neuropathic pain in diabetes manifests as a disease of the peripheral and central nervous systems. The prevalence of type 2 diabetes is far greater than type 1 (90%), yet the overwhelming focus on type 1 models this has left the mechanisms of pain in type 2 diabetes largely unknown. Therefore I aimed to improve the current mechanistic understanding of pain associated with type 2 diabetes using two preclinical rodent models: Zucker Diabetic Fatty rats and db/db mice. In addition, I highlight the translational importance of simultaneous measurement of evoked/sensory and non-evoked/affective pain-related behaviors in preclinical models. This work is the first to show a measure of motivational-affective pain in a model of type 2 diabetes. I used methodological approaches including: (1) immunohistochemical and calcium imaging to assess stimulus-evoked sensitization; (2) measurement nociceptive behaviors and evoked sensory thresholds as well as pain affect using novel mechanical conflict avoidance and conditioned place preference/aversion assays; (3) pharmacological and genetic manipulation of methylglyoxal, TRPA1, AC1, and PPARγ. I hypothesized that the thiazolidinedione class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists would reduce neuropathic pain-like behavior and spinal neuron sensitization in traumatic nerve injury and type 2 diabetes. As PPARγ is a nuclear receptor, and already targeted clinically to promote cellular insulin sensitization to reduce hyperglycemia, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. In two separate research aims, I challenged this view and tested whether the PPARγ agonist pioglitazone would (1) rapidly alleviate neuropathic pain through a non-genomic mechanism and (2) reduce painful sensitization in nociceptive and neuropathic pain models independent from lowering blood glucose. I aimed to investigate the contribution of the glucose metabolite methylglyoxal to painful type 2 diabetes. I tested the hypothesis that methylglyoxal produces nociceptive, evoked, and affective pain that is dependent on activation of the sensory neuron cation channel TRPA1 and the secondary messenger enzyme AC1. I also tested whether pioglitazone or the novel methylglyoxal scavenging peptide GERP10 could alleviate painful type 2 diabetes.

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Simmons, Christopher Ryan. „GENOME-WIDE ASSOCIATION STUDIES AT THE INTERFACE OF ALZHEIMER’S DISEASE AND EPIDEMIOLOGICALLY RELATED DISORDERS“. UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/824.

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Genome-wide association studies (GWAS)s provide an unbiased means of exploring the landscape of complex genetic disease. As such, these studies have identified genetic variants that are robustly associated with a multitude of conditions. I hypothesize that these genetic variants serve as excellent tools for evaluation of the genetic interface between epidemiologically related conditions. Herein, I test the association between SNPs associated with either (i) plasma lipids, (ii) rheumatoid arthritis (RA) or (iii) diabetes mellitus (DM) and late-onset Alzheimer’s disease (AD) to identify shared genetic variants. Regarding the most significantly AD-associated variants, I have also attempted to elucidate their molecular function. Only cholesterol-associated SNPs, as a group, are significantly associated with AD. This association remains after excluding APOE SNPs and suggests that peripheral and or central cholesterol metabolism contribute to AD risk. The general lack of association between RA-associated SNPs and AD is also significant in that these data challenge the hypothesis that genetic variants that increase risk of RA confer protection against AD. Functional studies of variants exhibiting novel associations with AD reveal that the lipid-associated SNP rs3846662 modulates HMGCR exon 13 splicing differentially in different cell types. Although less clear, trends were also observed between the RA-associated rs2837960 and the expression of several BACE2 isoforms, and between the DM-associated rs7804356 and expression of a rare SKAP2 isoform, respectively. In conclusion, the overlap of lipid-, RA- or DM-associated SNPs with AD is modest but in several instances significant. Continued analysis of the interface between GWAS of separate conditions will likely facilitate novel associations missed by conventional GWAS. Furthermore, the identification of functional variants associated with multiple conditions should provide insight into novel mechanisms of disease and may lead to the identification of new therapeutic targets in an era of personalized genomic medicine.

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Gouda, Mazen M. „Axon Initial Segment Integrity in Aging and Traumatic Brain Injury“. VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5993.

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According to the Center for Disease Control’s (CDC) report to the Congress, there are 2.2 million emergency department visits; 80,000 hospitalizations; and 50,000 deaths each year due to traumatic brain injury. Adults 65 years and older account substantially for the majority of the hospitalization and deaths. Over 70% of the traumatic brain injuries of the older adults are classified as mild to moderate; however, even with these milder injuries, older adults present with a significantly higher morbidity and mortality compared to all other age groups (LeBlanc et al., 2006). With that in mind, it seems essential to develop a deeper understanding of the causes behind higher mortality and morbidity of traumatic brain injury in the elder population. It is well documented that increased age is accompanied by increased CNS inflammation. Recently, our laboratory showed that inflammation drives brain pathology. Specifically, we reported that the axon initial segment of cortical neurons was structurally and functionally compromised in an inflamed CNS environment. With this in mind, we proposed that age-related inflammation predisposes that brain to exacerbated pathologic consequence. To test this hypothesis, we administered a mild to moderate central fluid percussion brain injury in aged and young adult mice. Using immunocytochemical labeling against the axon initial segment protein ankyrinG combined with laser scanning confocal microscopy, we quantitatively compared axon initial segment number and length between age groups and within age groups with and without injury. Additionally, we also quantified global axonal pathology by immunolabeling for amyloid precursor protein (APP) positive swelling as an indicator of compromised axonal transport. We proposed that ankyrinG labeling will be both reduced in the aged injured mice compared against aged uninjured, young adult injured and young adult non-injured. We observed a significant increase in APP accumulations due to injury independent of aging, and due to aging independent of injury. No significant changes in the effect of injury between young and aged injured mice were observed. Although AIS length was not altered between age groups following injury, our results demonstrate that the elderly population presents with significantly shorter initial segments. The consequence of this shortening is not clear but may reflect compensatory changes in the brain to maintain homeostasis.

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Welleford, Andrew. „Autologous Peripheral Nerve Grafts to the Brain for the Treatment of Parkinson's Disease“. UKnowledge, 2019. https://uknowledge.uky.edu/neurobio_etds/23.

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Parkinson’s disease (PD) is a disorder of the nervous system that causes problems with movement (motor symptoms) as well as other problems such as mood disorders, cognitive changes, sleep disorders, constipation, pain, and other non-motor symptoms. The severity of PD symptoms worsens over time as the disease progresses, and while there are treatments for the motor and some non-motor symptoms there is no known cure for PD. Thus there is a high demand for therapies to slow the progressive neurodegeneration observed in PD. Two clinical trials at the University of Kentucky College of Medicine (NCT02369003, NCT01833364) are currently underway that aim to develop a disease-modifying therapy that slows the progression of PD. These clinical trials are evaluating the safety and feasibility of an autologous peripheral nerve graft to the substantia nigra in combination with Deep Brain Stimulation (DBS) for the treatment of PD. By grafting peripheral nerve tissue to the Substantia Nigra, the researchers aim to introduce peripheral nerve tissue, which is capable of functional regeneration after injury, to the degenerating Substantia Nigra of patients with PD. The central hypothesis of these clinical trials is that the grafted tissue will slow degeneration of the target brain region through neural repair actions of Schwann cells as well as other pro-regenerative features of the peripheral nerve tissue. This dissertation details analysis of the peripheral nerve tissue used in the above clinical trials with respect to tissue composition and gene expression, both of injury-naive human peripheral nerve as well as the post-conditioning injury nerve tissue used in the grafting procedure. RNA-seq analysis of sural nerve tissue pre and post-conditioning show significant changes in gene expression corresponding with transdifferentiation of Schwann cells from a myelinating to a repair phenotype, release of growth factors, activation of macrophages and other immune cells, and an increase in anti-apoptotic and neuroprotective gene transcripts. These results reveal in vivo gene expression changes involved in the human peripheral nerve injury repair process, which has relevance beyond this clinical trial to the fields of Schwann cell biology and peripheral nerve repair. To assess the neurobiology of the graft post-implantation we developed an animal model of the grafting procedure, termed Neuro-Avatars, which feature human graft tissue implanted into athymic nude rats. Survival and infiltration of human graft cells into the host brain were shown using immunohistochemistry of Human Nuclear Antigen. Surgical methods and outcomes from the ongoing development of this animal model are reported. To connect the results of these laboratory studies to the clinical trial we compared the severity of motor symptoms before surgery to one year post-surgery in patients who received the analyzed graft tissue. Motor symptom severity was assessed using the Unified Parkinson’s Disease Rating Scale Part III. Finally, the implications and future directions of this research is discussed. In summary, this dissertation advances the translational science cycle by using clinical trial findings and samples to answer basic science questions that will in turn guide future clinical trial design.

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Peretti, Madeline. „DNA methylation of the clusterin promoter: Associations with Alzheimer’s Disease risk and related phenotypes“. Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2019. https://ro.ecu.edu.au/theses/2172.

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Background In 2017 approximately 50 million people worldwide were living with dementia. With Alzheimer’s disease (AD), accounting for 50-70% of dementia cases making this debilitating disease, with no current effective prevention, treatment or cure, a critical healthcare concern. Genome wide association studies (GWAS) have identified a number of risk genes for late onset AD (LOAD); Apolipoprotein E (APOE), a gene involved in the cholesterol/lipid pathway is considered the gene with the greatest risk. The third most associated AD risk gene is Clusterin (CLU), is also involved in the cholesterol/lipid pathway. CLU has been implicated in both a protective and aggravating role in AD making it a compelling gene for further study. The identification of a fluid-based AD biomarker/s has become an increasingly important area of study to assist in the identification of individuals before substantial pathological or cognitive symptoms arise. Plasma clusterin is one such biomarker showing promise as levels differ between healthy individuals and those with mild cognitive impairment (MCI) and AD. Methylation of CLU, from brain samples, has been associated with Aβ pathology and lower risk of AD diagnosis. In addition, methylation of other AD associated risk genes has been shown to be altered in the periphery. These factors make CLU a prime candidate for peripheral methylation study. Aims The overarching aim of the study was to investigate methylation within the CLU promoter region in peripheral blood samples. Specifically, to determine whether there is a relationship between specific methylation sites and AD-related phenotypes. The first study aimed to crosssectionally assess CLU promoter region methylation and its association with clinical classification, genetic variation in CLU (rs9331888/rs11136000), and pathological biomarkers (Chapter 3.2). The second study aimed to analyse the influence of methylation on longitudinal cognitive performance (Chapter 3.3). Aims The overarching aim of the study was to investigate methylation within the CLU promoter region in peripheral blood samples. Specifically, to determine whether there is a relationship between specific methylation sites and AD-related phenotypes. The first study aimed to crosssectionally assess CLU promoter region methylation and its association with clinical classification, genetic variation in CLU (rs9331888/rs11136000), and pathological biomarkers (Chapter 3.2). The second study aimed to analyse the influence of methylation on longitudinal cognitive performance (Chapter 3.3). Methods Utilising DNA samples collected as part of the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL). Methylation analysis was conducted on an initial 768 samples comprised of healthy controls (HC), MCI and AD participants. Methylation data was collected using the Agena MassArray platform across the CLU promoter region. Percentage methylation was determined by the EpiTYPER software. ANCOVA’s were used to study association between methylation levels and clinical classifications as well as genetic variants, whilst Spearman’s Partial correlations were used to investigate the relationship between methylation and brain imaging and fluid biomarker data (Chapter 3.2). Linear mixed models (LMMs) were employed to assess associations between methylation and cognitive performance, both at baseline and over 7.5 years (Chapter 3.3). Analyses were first conducted across the whole sample, followed by HC only, then HC with high brain Aβ burden (Aβ+). All P-values were corrected for the false discovery rate (FDR). Results Analysis revealed an association between clinical classification and methylation. After FDR correction significant decreases in methylation from HC to MCI was seen at 3 loci, with CpG_14 and CpG_15 showing a significant decrease from HC to AD. CpG_25_26_27_28 also showed a decrease in methylation between HC and AD. When analysing methylation patterns with respect to rs11136000 a significant difference in methylation levels were observed between non-carriers and carriers of the protective T allele at CpG_14, CpG_16 and CpG_23_24. This was only observed when assessed across all participants, whilst no significant differences in methylation were observed with respect to rs9331888. An increased percentage methylation at CpG_16 was associated with elevated plasma clusterin (⍴=0.1858, p=0.0034, q=0.0157) within the HC group, which remained as a trend in the HC Aβ+ after FDR correction (β=0.2704, p=0.0043, q=0.0817). With respect to cerebrospinal fluid (CSF) biomarkers no sites survived FDR correction in the whole cohort. CpG_37 was inversely correlated with phosphorylated tau (P-tau; ⍴=-0.4585, p=0.001, q=0.0155) within the HC cohort. Whilst with the Aβ+ HC group higher percentage methylation at CpG_1 (⍴=-0.6584, p=0.0016, q=0.0028), CpG_8_9 (⍴=-0.4704, p=0.0363, q=0.0297) and CpG_10 (⍴=-0.4433, p=0.0503, q=0.0297) was associated with elevated A42 CSF levels. Increased methylation at CpG_1, CpG_2_3, CpG_8_9, CpG_14, CpG_16, CpG_19 and decreased methylation at CpG_20_21, CpG_23_24, CpG_25_26_27_28, CpG_34, CpG_36, CpG_38_39 and CpG_41 were associated with a higher brain Aβ burden across the whole cohort. Whilst methylation at CpG_15 positively correlated with cortical grey matter volume. Within HC’s, increased methylation at CpG_1, CpG_2_3, CpG_8_9, CpG_14, CpG_16, CpG_19, and decreased methylation at CpG_20_21, CpG_23_24, CpG_25_26_27_28, CpG_36, CpG_38_39 and CpG_41 were associated with a higher brain A burden. Whilst methylation at CpG_15, CpG_36 and CpG_41 positively correlated with cortical grey matter volume. Again, within the Aβ+ HC group, increased methylation at CpG_8_9, CpG_14, CpG_16, CpG_19, and decreased methylation at CpG_20_21, CpG_25_26_27_28, CpG_34, CpG_36, and CpG_41 were associated with a higher brain Aβ burden. Whilst a significant positive correlation between methylation at CpG_37 and cortical white matter volume was observed. Whilst several methylation sites within the CLU promoter reached a nominal level of significance with respect to both baseline cognitive performance and change in cognitive performance over 7.5 years no sites retained significance after FDR correction. This finding was consistent when investigated across the entire cohort, within the HC and within the Aβ+ HC group. Conclusions Differential methylation was seen across the CLU promoter region in relation to clinical classification, SNP’s and both fluid and brain imaging biomarkers. These observations seen in the periphery provide further evidence of methylation, and specifically of methylation in the CLU promoter region, having the potential to be a biomarker in early AD diagnosis. Of particular interest is the decrease in methylation seen between clinical classifications and the associated impact on gene expression. Another area of noted interest is the association seen between CLU promoter region methylation and Aβ burden in the brain. Further exploration is warranted to validate and further examine these results by generating longitudinal methylation data to assess whether changes in methylation track with changes in AD clinical and pathological characteristics.

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Deng, Ying. „ROLE OF THE REACTIVE OXYGEN SPECIES PEROXYNITRITE IN TRAUMATIC BRAIN INJURY“. UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/667.

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Reactive oxygen species (ROS) is cytotoxic to the cell and is known to contribute to secondary cell death following primary traumatic brain injury (TBI). We described in our study that PN is the main mediator for both lipid peroxidation and protein nitration, and occurred almost immediately after injury. As a downstream factor to oxidative damage, the peak of Ca2+-dependent, calpainmediated cytoskeletal proteolysis preceded that of neurodegeneration, suggesting that calpain-mediated proteolysis is the common pathway leading to neuronal cell death. The time course study clearly elucidated the interrelationship of these cellular changes following TBI, provided window of opportunity for pharmacological intervention. Furthermore, we conducted a pharmacological study to solidify our hypothesis. First of all, we tested the potency of a membrane permeable, catalytic scavenger of PN-derived free radicals, tempol for its ability to antagonize PN-induced oxidative damage. Tempol successfully inhibited PNinduced protein nitration at dosages of 30, 100 and 300mg/kg. Moreover, early single dose of 300mg/kg was administered and isolated mitochondria were examined for respiratory function and oxidative damage level. Our data showed that tempol reduced mitochondrial oxidative damage, and maintained mitochondrial function within normal limits, which suggested that tempol is efficiently permeable to mitochondrial membrane and mitochondrial oxidative damage is essential to mitochondrial dysfunction. Next, we found that calpainmediated proteolysis is reduced at early treatment with a single dose of tempol. However, the effect of tempol on calpain is short-lived possibly due to systematic elimination. In our multiple dose study, tempol showed a significant inhibitory effect on SBDPs. Consequently, we measured neuordegeneration with the de Olmos aminocupric silver staining method at 7 days post-injury and detected a significant decrease of neuronal cell death. Together, the time course study and pharmacological study strongly support the hypothesis that PN is the upstream mediator in secondary cell death in the CCI TBI mouse model. Moreover, inhibition of PN-mediated oxidative damage with the antioxidant, tempol, is able to attenuate multiple downstream injury mechanisms. However, targeting PN alone may be clinically impractical due to its limited therapeutic window. This limitation may be overcome in future studies by a combination of multiple therapeutic strategies.

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Xiong, Yiqin. „ROLE OF REACTIVE OXYGEN SPECIES PEROXYNITRITE IN TRAUMATIC SPINAL CORD INJURY“. UKnowledge, 2008. http://uknowledge.uky.edu/gradschool_diss/657.

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Peroxynitrite (PN, ONOO-), formed by nitric oxide radical (•NO) and superoxide radical (O2•-), plays an important role in post-traumatic oxidative damage. In the early work, we determined the temporal characteristics of PN-derived oxidative damage in a rat spinal cord injury (SCI) model. Our results showed 3-nitrotyrosine (3-NT), a specific marker for PN, rapidly accumulated at early time points (1 hr, 3 hrs), after when it plateaued and the high level was sustained to 1 week post injury. The co-localization of 3-NT and lipid peroxidation derived-4-HNE observed in immunohistochemistry indicates PN is involved in lipid peroxidative as well as protein nitrative damage. PN-oxidative damage exacerbates intracellular Ca2+ overload, which activates Ca2+ dependent calpain-mediated cytoskeletal protein (α-spectrin) degradation. The 145 kD fragments of α-spectrin (SBDP 145), which are specifically generated by calpain, increased dramatically as early as 1 hr after injury although the peak increase did not occur until 72 hrs post injury. The high level waned back toward sham level at one week post injury. We then carried out experiments to evaluate the beneficial effects of tempol, a scavenger of PN-derived radicals, following SCI. Three pathological events including PN-induced oxidative damage, mitochondrial dysfunction and cytoskeletal degradation were investigated. Immunoblotting and immunohistochemical studies indicated PN-mediated oxidative damage including protein nitration, protein oxidation and lipid peroxidation, were all reduced by a single dose of tempol (300mg/kg, i.p) after SCI. Spinal cord (SC) mitochondrial dysfunction in terms of the respiratory control ratio (RCR) significantly improved by both 150 mg/kg and 300 mg/kg tempol treatments. Moreover, calpain-mediated proteolysis was significantly decreased by tempol, with greater effects on calpain-specific SBDP 145 observed. Direct PN-scavenging effect of tempol was confirmed in vitro. Exposure of healthy SC mitochondria to SIN-1, a PN donor in vitro, impaired mitochondrial respiration in a dose-dependent manner. Tempol was able to protect mitochondria against SIN-1-induced damage by improving mitochondrial function and decreasing mitochondrial 3-NT formation. These findings strongly support the concept that PN is a crucial player in the secondary damage following SCI. And tempol, by scavenging PN-induced free radicals, provides a promising pharmocotherapeutic strategy for treating acute SCI.

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Russell, Nicholas H. „Heme Oxygenase 1 expression after traumatic brain injury and effect of pharmacological manipulation on functional recovery“. VCU Scholars Compass, 2017. https://scholarscompass.vcu.edu/etd/5525.

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Traumatic Brain Injury (TBI) is an increasingly diagnosed constellation of injuries derived from acute mechanical trauma to the brain. With the rise of advanced neuroimaging techniques recent focus has oriented primarily towards the mild-moderate range of TBI which previously was missed diagnostically. Characteristically, these advances have shown increasing areas of micro-hemorrhage in susceptible areas of the brain and to date there are no treatment modalities targeting micro-hemorrhages or their sequelae. This dissertation explores the effects of the resulting heme processing response in the days following injury with a particular focus on inducing early heme clearance from the parenchyma using a rat central fluid percussion injury model in the mild-moderate injury range. Since heme is released ~24-48 hours post-injury and is known to be cytotoxic we observed there may be a critical window for treatment to clear heme before it is spontaneously released and to increase the buffering capacity of the tissue. We targeted heme clearance by using drugs known to increased expression of Nrf2, an upstream transcriptional regulator of the canonical heme processing protein heme oxygenase 1 (HO-1), and tracking expression of HO-1, the iron sequestration/storage proteins Lipocalin 2 (LCN2) and Ferritin (FTL), as well as the activity of matrix metalloproteinases 2 and 9 (MMP2, MMP9). We examined both tissue known to be frankly hemorrhagic (the neocortex) as well as tissue lacking any identifiable bleed (the hippocampus). We demonstrated that using the HO-1 inducers Hemin and Sulforaphane in a single dose paradigm given 1 hour post-injury heme clearance was accelerated in the neocortex with the majority of heme pigment processed by 24 hours post-injury. Further there was significant attenuation of protein expression in HO-1 and ferritin as well as the enzyme activity of MMP2 and MMP9 in both the neocortex and the hippocampus. Behavioral attenuation was also seen in both rotarod and Morris water maze tests. While we intended to target hemorrhagic processing after injury, and indeed demonstrated improved clearance of heme from post-injury hemorrhagic regions of the brain, in both tissues studied we observed remarkably similar responses to the drugs utilized in protein expression, enzyme activity, and behavioral improvement which may suggest a globally improved pathologic state or that there are unidentified pathologic micro-hemorrhages or leaky vessels which extend further into the brain parenchyma than currently identified.

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Clark, Jordan Mills. „ROLE OF CYCLOPHILIN D IN SECONDARY SPINAL CORD AND BRAIN INJURY“. UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/730.

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In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.

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Joshi, Aashish. „SUBSTRATE AND REGULATION OF MITOCHONDRIAL μ-CALPAIN“. UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/80.

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μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to detect in rat liver mitochondria. In a mitochondrial fraction from SH-SY5Y cells, incubation with 5 mM Ca2+ resulted in the activation of mitochondrial μ-calpain but not in AIF truncation. Finally, in hippocampal neurons calpain activation did not induce AIF processing or nuclear translocation and AIF translocation to nucleus was calpain independent. The localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive. We observed that mitochondrial μ-calpain required high Ca2+ for activation. We examined the hypothesis that the endogenous calpain inhibitor, calpastatin, may be present in the neuronal mitochondria. Calpastatin was detected in the mitochondriaenriched fraction obtained from rat cerebral cortex and SH-SY5Y cells. The mitochondrial calpastatin was resistant to proteinase K digestion, indicating localization internal to the outer mitochondrial membrane. Submitochondrial fractionation revealed that the calpastatin was localized to the mitochondrial intermembrane space and mitoplasts (inner mitochondrial membrane and matrix) but not to the mitochondrial outer membrane fraction. Mitochondrial calpastatin was not detected when mitoplasts were incubated with proteinase K, suggesting that calpastatin is not present in the matrix. The N-terminus of XL domain of calpastatin, when fused to GFP and transfected to SH-SY5Y cells showed mitochondrial localization and thus confirmed the presence of a mitochondrial targeting sequence in calpastatin. Together, these results demonstrate the presence of calpastatin in the neuronal mitochondrial intermembrane space, the same submitochondrial compartment as mitochondrial μ-calpain. This finding explains the high Ca2+ requirements for mitochondrial μ-calpain activation.

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Maniskas, Michael E. „LOOKING TO THE FUTURE OF STROKE TREATMENT: COMBINING RECANALIZATION AND NEUROPROTECTION IN ACUTE ISCHEMIC STROKE“. UKnowledge, 2016. http://uknowledge.uky.edu/neurobio_etds/17.

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Stroke is the 5th leading cause of death in the U.S. with 130,000 deaths and around 800,000 affected annually. Currently, there is a significant disconnect between basic stroke research and clinical stroke therapeutic needs. Few animal models of stroke target the large vessels that produce cortical deficits seen in the clinical setting. Also, current routes of drug administration, intraperitoneal and intravenous, do not mimic the clinical route of intra-arterial drug administration. To bridge this divide, we have retro-engineered a mouse model of stroke from the current standard of care for emergent large vessel occlusion (ELVO) stroke, endovascular thrombectomy, to include selective intra-arterial pharmacotherapy administration. Using the tandem transient common carotid and middle cerebral artery occlusion (MCAo) model to induce stroke, we threaded micro-angio tubing into the external carotid artery (ECA) towards the bifurcation of the common carotid and internal carotid arteries (CCA/ICA) allowing for the delivery of agents to the site of acute ischemia. Our model was optimized through a flow rate and injection volume study using carbon black ink injected through the intra-arterial model at different flow rates and injection volumes. The purpose of this study was to demonstrate that our injections were arriving at the site of ischemia and to improve injection volumes for future dosing while mitigating systemic side effects by preventing or minimizing systemic distribution. We determined that a flow rate of 2.5 µl/minute and injection volume of 10 µl was optimal. Next, we tested potential neuroprotective compounds nitroglycerin, verapamil, and a combination of verapamil and lubeluzole. Compounds were chosen for drug synergy and to target specific pathways in either an acute or delayed manner. Acute treatments included nitroglycerin and/or verapamil while delayed treatment included lubeluzole. The known mechanism of action for FDA approved nitroglycerin is through vessel dilation that results in increased blood flow to the treated region. A secondary mechanism of nitroglycerin is the production of nitric oxide, which has demonstrated antioxidant and anti-apoptotic effects when processed and released from cells surrounding the blood vessels. Verapamil, a calcium channel blocker, also FDA-approved for cerebral artery vasospasm: is thought to act by blocking the L-type calcium channels on the cell membrane from opening following membrane depolarization after insult. Finally, lubeluzole, also FDA-approved, is proposed to work as an NMDA modulator inhibiting the release of glutamate and nitric oxide synthase and blocking sodium and calcium channels. Through our stroke model we were able to demonstrate that each drug(s) showed a significant decrease in infarct volume and improved functional recovery while simultaneously minimizing potential systemic side effects suggesting that our stroke model may improve the preclinical validation of potential stroke therapies and help bridge the bench to bedside divide in developing new stroke therapies.

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Littrell, Ofelia Meagan. „NIGROSTRIATAL DOPAMINE-NEURON FUNCTION FROM NEUROTROPHIC-LIKE PEPTIDE TREATMENT AND NEUROTROPHIC FACTOR DEPLETION“. UKnowledge, 2011. http://uknowledge.uky.edu/neurobio_etds/1.

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Trophic factors have shown great promise in their potential to treat neurological disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent neurotrophic factor for midbrain dopamine (DA) neurons in the substantia nigra (SN), which lose function in Parkinson’s disease (PD). GDNF progressed to phase II clinical trials, which did not meet proposed endpoints. The large size and binding characteristics of GDNF have been suspected to contribute to some of the shortcomings of GDNF related to delivery to target brain regions. Smaller peptides derived from GDNF (Dopamine-Neuron Stimulating Peptides – DNSPs) have been recently investigated and appear to demonstrate trophic-like effects comparable to GDNF. In the described studies, a time course study was conducted to determine in vivo DA-release characteristics 1-, 2- and 4- weeks after peptide treatment. These studies determined the effects on DA terminals within striatal sub-regions using microelectrodes. A heterogeneous effect on striatal sub-regions was apparent with the maximum effect in the dorsal striatum – corresponding to terminals originating from the SN. Dysregulation of GDNF or GDNF signaling is believed to contribute to motor dysfunction in aging and PD. Thus, it is hypothesized that GDNF is necessary for the maintenance and function of neurons. To extend this line of investigation, in vivo functional measures (DA-release and -uptake) and behavioral and cellular alterations were investigated in a transgenic mouse model (Gdnf+/-) with reduced GDNF protein levels. The described studies determined that both DA-uptake and -release properties were altered in middle-aged Gdnf+/- mice with only modest reductions in DA neurochemical levels. GDNF levels in Gdnf+/- mice were restored to levels comparable to wild-type (WT) counterparts by treatment with GDNF. GDNF protein supplementation led to enhanced motor behavior and increased markers for DA neurons in the SN of Gdnf+/- mice. Gdnf+/- mice appeared to show a heightened sensitivity to GDNF treatment compared to WT counterparts. Overall, this body of work examines novel synthetic peptides with potential to enhance DA-neuron function and expands upon the current understanding of GDNF’s role in the nigrostriatal pathway.

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Thummala, Suneel K. „Axon Initial Segment Stability in Multiple Sclerosis“. VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4038.

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Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by inflammation and demyelination. In addition to these hallmark features, MS also presents with axonal pathology, which is likely responsible for the signs and symptoms of the disease. Although prominent in MS, axonal pathology is frequently considered a consequence of demyelination and not a primary event. This conclusion is consistent with demyelination inducing the loss of specific axonal domains, known as the nodes of Ranvier that are responsible for the propagation of action potentials along the axon. In contrast, we propose that axonal pathology associated with MS is a primary pathological event, independent of demyelination, and not a product of it. In support of our hypothesis, we have analyzed a different axonal domain known as the axon initial segment. Whereas a single axon has numerous nodes of Ranvier uniformly distributed along the axon, each axon contains only a single axon initial segment that is positioned immediately distal to the neuronal cell body. The axon initial segment is responsible for action potential generation and modulation, and hence is essential for normal neuronal function. Background studies conducted by our lab, employing a murine model of demyelination/remyelination, revealed no correlation between axon initial segment stability and myelin integrity. Here we investigate the fate of the axon initial segment in human multiple sclerosis. While not statistically significant, we provide data demonstrating an apparent 40% reduction in AIS numbers in MS. We further provide qualitative evidence that AIS integrity in MS is not dependent on myelination suggestive that axonal pathology may be a primary event in MS, independent of demyelination. Our current findings are intriguing, but unfortunately this study is underpowered, and more samples will be required to determine whether this apparent reduction is statistically significant.

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Gonzalez, Eric James. „A Role For Transforming Growth Factor-Beta In Urinary Bladder Dysfunction With Cyclophosphamide-Induced Cystitis“. ScholarWorks @ UVM, 2016. http://scholarworks.uvm.edu/graddis/553.

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Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain disorder characterized by at least six weeks of lower urinary tract symptoms and unpleasant sensations (pain, pressure and discomfort) thought to be related to the urinary bladder and not meeting exclusion criteria. While the etiology is not known, BPS/IC may involve a "vicious circle" of uroepithelial dysfunction, inflammation and peripheral and central sensitization. We propose that the urinary bladder inflammatory insult partly mediates voiding dysfunction and visceral neurogenic pain characteristic of BPS/IC. Several studies from our laboratory have already demonstrated the role(s) of cytokines and their downstream targets in the functional alterations in micturition reflex pathways following chemically (cyclophosphamide, CYP)-induced cystitis. More recently, the pleiotropic protein, TGF-β, has been implicated in the pathogenesis of CYP-induced cystitis. TGF-β is activated locally at the initial site of injury by protease-dependent or protease-independent mechanisms to initiate a proinflammatory milieu. Depending on its contextual cues, TGF-β may then aid in resolving the primary immune response and support tissue repair. Though TGF-β is necessary to maintain normal immunological function, its aberrant expression and activation may have detrimental effects on responding tissues and cell types. A sustained increase in peripheral TGF-β reactivity, such as what may be observed in chronic inflammatory bladder conditions, may influence bladder afferent excitability to amplify nociceptive transmission and CNS input. The subsequent sensitization of peripheral afferent nociceptors at the level of the DRG or urothelium may promote spinal cord "wind-up" and cascade into visceral hyperalgesia and allodynia. In the first aim of this dissertation we investigated the functional profile of TGF-β isoforms and receptor (TβR) variants in the normal and inflamed (CYP-induced cystitis) urinary bladder with qRT-PCR, ELISA, IHC and in vivo cystometry. Our studies determined (i) the involvement of TGF-β in lower urinary tract neuroplasticity following urinary bladder inflammation, (ii) a functional role for TGF-β signaling in the afferent limb of the micturition reflex and (iii) urinary bladder TβR-1 as a viable target to reduce voiding frequency with cystitis. In the second aim of this dissertation we investigated the sensory components of the urinary bladder that may underlie the pathophysiology of aberrant TGF-β activation with bladder-pelvic nerve electrophysiology and luciferin-luciferase assays for ATP measurement. Our studies determined that TGF-β1 increased bladder afferent nerve excitability by stimulating ATP release from the urothelium via vesicular exocytosis mechanisms with minimal contribution from pannexin-1 channels. Furthermore, blocking aberrant TGF-β signaling in CYP-induced cystitis with TβR-1 inhibition decreased afferent nerve excitability with an equivalent decrease in ATP release. Taken together, these results establish a causal link between an inflammatory mediator, TGF-β, and intrinsic signaling mechanisms of the urothelium that may contribute to the altered sensory processing of bladder filling to facilitate increased voiding frequency. The distinct interactions of multiple mediators underscore the challenges for single target therapies and support the development of combinatory therapeutics for bladder dysfunction. Ultimately, these studies have increased our understanding of functional disorders and visceral pain and have the potential to improve the health of those suffering from inflammation-associated bladder syndromes.

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Martha, Sarah R. „NEUROCHEMICAL FACTORS ASSOCIATED WITH THE INITIAL PATHOPHYSIOLOGICAL REACTION TO LARGE VESSEL OCCLUSION STROKE“. UKnowledge, 2019. https://uknowledge.uky.edu/nursing_etds/43.

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Ischemic stroke is the leading cause of disability world-wide and affects over 800,000 people per year in the United States. The majority of these strokes are ischemic due to a blockage of blood flow to the brain. Damage to the brain occurs at the onset of stroke, neuronal cell death is irreversible and therefore, quick treatment to remove blockage is critical factor in the recovery from stroke. Mechanical thrombectomy as a treatment for ischemic stroke provides an ideal opportunity to collect blood distal and proximal to the cerebral thrombus to examine neurochemical changes occurring during stroke. The purpose of this dissertation was to explore the trajectory of neurochemical changes that occur in response to ischemic stroke during the first 72 hours and the physiological response from stroke patients to improve stroke outcomes. The specific aims were to: 1) to determine whether venous blood gases predict infarct volume and/or mortality in acute ischemic stroke in young male rats; 2) determine whether venous blood gases predict infarct and edema volume, and/or mortality in acute ischemic stroke in aged male and female rats; 3) compare the presence and relative concentrations of acid/base and electrolytes in static blood distal to thrombus and in peripheral blood drawn from adults who received thrombectomy for ischemic stroke and identify associations to postreperfusion functional outcomes. Specific Aim One was addressed by evaluation of young (three-month old) Sprague-Dawley rats that underwent permanent or transient middle cerebral artery occlusion (MCAO). Pre- and post-MCAO venous samples from permanent and transient models provided pH, carbon dioxide, oxygen, bicarbonate, glucose, hematocrit, hematocrit, and electrolyte values of ionized calcium, potassium and sodium. The analyses indicated that mean differences in the blood gas and electrolytes between pre- to post-MCAO and pH and iCa2+ were predictors of infarct volume in the permanent MCAO model. The second aim was addressed by evaluation of aged (18 month old) male and female rats pre-MCAO, post-MCAO, and at 72 hours of permanent MCAO venous blood gas samples (pH, carbon dioxide, oxygen, bicarbonate, glucose, hematocrit, hematocrit, and electrolyte concentrations of ionized calcium, potassium and sodium). Changes in pH (from pre-MCAO to post-MACO and post-MCAO to 72 hours) and changes in Na+ and iCa2+ (from post-MCAO to 72 hours) were predictors of infarct volume and edema volume, respectively in both sexes. Cox regression revealed there was a 3.25 times increased risk for mortality based on changes (cut-off range within -2.00 to - 7.00) in bicarbonate levels (pre- to post-MCAO). The third aim was addressed by evaluation of acid/base balance (pH, carbon dioxide, oxygen, bicarbonate, ionized calcium, potassium and sodium) of ischemic stroke patients who underwent mechanical thrombectomy. Our results suggests sex differences matter in ischemic stroke populations. Significant differences occur within proximal blood between the sexes. Additionally, females had approximately 2.5 hour increased time between stroke symptom onset to thrombectomy completion time (described as infarct time). Changes in bicarbonate and base deficit were predictors of infarct time, but only in our female population.

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Lee, Franklin A. „How the manipulation of the Ras homolog enriched in striatum alters the behavioral and molecular progression of Huntington’s disease“. ScholarWorks@UNO, 2015. http://scholarworks.uno.edu/td/2092.

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Huntington’s disease is an incurable, progressive neurological disorder characterized by loss of motor control, psychiatric dysfunction, and eventual dystonia leading to death. Despite the fact that this disorder is caused by a mutation in one single gene, there is no cure. The mutant Huntingtin (mHtt) protein is expressed ubiquitously throughout the brain but frank cell death is limited to the striatum. Recent work has suggested that Rhes, Ras homolog enriched in striatum, which is selectively expressed in the striatum, may play a role in Huntington’s disease neuropathology. In vitro studies have shown Rhes to be an E3 ligase for the post-translational modification protein SUMO. Rhes increases binding of SUMO to mHtt which competes for the same binding site as Ubiquitin. SUMOylation of mHtt leads to disaggregation and cellular death, whereas ubiquitination leads to aggregation and cellular protection. In a previous study we showed that deletion of Rhes caused a decrease in the Huntington’s disease phenotype in mice. We hypothesized that mice lacking Rhes would also show increased aggregation in the striatum and this increased aggregation would correlate in a rescue of behavioral symptoms. Despite the prior in vitro and in vivo evidence, deletion of Rhes in vivo did not alter the aggregation of mHtt in the striatum of mice however deletion of Rhes still showed a rescue from the diseased phenotype. This result would indicate that deletion of Rhes alters the neurobehavioral phenotype of Huntington’s disease through a different pathway than promoting aggregation in striatal cells.

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Suri, Nikita. „Superbursts: Investigation of Abnormal Paroxysmal Bursting Activity in Nerve Cell Networks In Vitro“. Thesis, University of North Texas, 2018. https://digital.library.unt.edu/ark:/67531/metadc1157655/.

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Superbursts (SBs) are large, seemingly spontaneous activity fluctuations often encountered in high density neural networks in vitro. Little effort has been put forth to define and analyze SBs which are paroxysmal bursting discharges. Through qualitative and quantitative means, I have described specific occurrences of superbursting activity. A complex of paroxysmal bursting has been termed a "superburst episode," and each individual SB is a "superburst event" which is comprises a fine burst structure. Quantitative calculations (employing overall spike summations and coefficient of variation (CV) calculations), reveal three distinct phases. Phase 1 is a "build up" phase of increasingly strong, coordinated bursting with an average of a 17.6% ± 13.7 increase in activity from reference. Phase 2, the "paroxysmal" phase, is comprised of massive coordinated bursting with high frequency spike content. Individual spike activity increases by 52.9% ± 14.6. Phase 3 is a "recovery phase" of lower coordination and an average of a 50.1% ± 35.6 decrease in spike production from reference. SBs can be induced and terminated by physical manipulation of the medium. Using a peristaltic pump with a flow rate of 0.4ml/min, superbursting activity ceases approximately 28.3 min after the introduction of flow. Alternatively, upon cessation of medium flow superbursting activity reemerges after approximately 8.5 min. Additionally, this study explored other methods capable of inducing superbursting activity using osmotic shocks. The induction and termination of SBs demonstrates that the cell culture environment plays a major role in generating this phenomenon. The observations that high density multi-layer neuronal networks in culture are more likely to enter paroxysmal bursting also supports the hypothesis that enrichment and depletion layers of metabolites and ionic species are involved in such unusual activity. The dynamic similarity of the SB phenomenon with epileptiform discharges make further quantification on the spike pattern level pertinent and important.

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Reneer, Dexter V. „BLAST-INDUCED BRAIN INJURY: INFLUENCE OF SHOCKWAVE COMPONENTS“. UKnowledge, 2012. http://uknowledge.uky.edu/neurobio_etds/3.

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Blast-induced traumatic brain injury (bTBI) has been described as the defining injury of Operations Enduring Freedom and Iraqi Freedom (OEF/OIF). Previously, most blast injury research has focused on the effects of blast on internal, gas filled organs due to their increased susceptibility. However, due to a change in enemy tactics combined with better armor and front-line medical care, bTBI has become one of the most common injuries due to blast. Though there has been a significant amount of research characterizing the brain injury produced by blast, a sound understanding of the contribution of each component of the shockwave to the injury is needed. Large animal models of bTBI utilize chemical explosives as their shockwave source while small animal models predominantly utilize compressed air-driven membrane rupture as their shockwave source. We designed and built a multi-mode shock tube capable of utilizing compressed gas (air or helium)-driven membrane rupture or chemical explosives (oxyhydrogen – a 2:1 mixture of hydrogen and oxygen gasses, or RDX – high order explosive) to produce a shockwave. Analysis of the shockwaves produced by each mode of the McMillan Blast Device (MBD) revealed that compressed air-driven shockwaves exhibited longer duration positive phases than compressed helium-, oxyhydrogen-, or RDX-driven shockwaves of similar peak overpressure. The longer duration of compressed air-driven shockwaves results in greater energy being imparted on a test subject than would be imparted by shockwaves of identical peak overpressures from the other sources. Animals exposed to compressed air-driven shockwaves exhibited more extensive brain surface hematoma, more blood-brain barrier compromise, more extensive reactive astrocytosis, and greater numbers of activated microglia in their brains than did animals exposed to oxyhydrogen-driven shockwaves of even greater peak overpressure. Taken together, these data suggest that compressed air-driven shockwaves contain more energy than their chemical explosive-derived counterparts of equal peak overpressure and can result in greater injury in an experimental animal model. Additionally, these data suggest that exposure to longer duration shockwaves, which is common in certain realworld scenarios, can result in more severe bTBI. The results of this study can be used to aid design of blast wave mitigation technology and future clinical intervention.

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Williamitis, Joseph M. „Using fMRI BOLD Imaging to Motion-Correct Associated, Simultaneously Imaged PET Data“. Wright State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=wright1620585748146734.

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Patel, Kaushal S. „Post-TBI Hippocampal Neurogenesis in Different TBI Models“. VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4134.

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Traumatic brain injury (TBI) leads to short-term and long-term consequences that can cause many different life-long disorders. Studies of TBI have generally focused on the acute stage; however, it is now becoming important to investigate chronic responses following TBI as clinical reports of dementia and cognitive impairments have been linked to a history of TBI. Recent data have established that cognitive function is associated with hippocampal neurogenesis. Chronic injury induced changes in the brain may affect this endogenous process. Chronic responses following TBI include cell death pathways and inflammatory responses that are persistent in the brain for months to years after injury. In this study we investigate the chronic consequences of TBI on adult neurogenesis and the possible involvement of chronic-inflammation in regulating adult neurogenesis. We used two popular TBI animal models, Control Cortical Impact (CCI) and Lateral Fluid Percussion Injury (LFPI) models, to examine focal and diffuse injury responses respectively. Adult rats received CCI, LFPI, or sham injury and were sacrificed at either 15 days or 3 months after injury to examine either subacute or chronic TBI-induced responses respectively. We found no change in levels of proliferation activity at both time points in both TBI models compared to sham animals. Using Doublecortin immunolabeling we found an enhanced generation of new neurons at 15 days after injury and by 3 months this activity was significantly reduced in both TBI models compared to sham animals. We also found persistent inflammation in the injured brains at both time points. Morphological assessment showed that LFPI model of TBI causes shrinkage of the ipsilateral hippocampus. Our results show that moderate TBI induced hippocampal neurogenesis in both models at the early time post-injury. However, at chronic stage, reduced hippocampal neurogenesis is observed in both models and this is accompanied by chronic inflammation. These results suggest that persistent inflammatory responses maybe detrimental to normal neurogenic activity, leading to cognitive impairment and neurodegeneration in long-term TBI survivors.

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Stephenson, Jeannie B. „Longitudinal Quantitative Analysis of Gait and Balance in Friedreich's Ataxia“. Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5623.

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Friedreich's Ataxia (FA) is an autosomal-recessive, neurodegenerative disease characterized by progressive lower extremity muscle weakness and sensory loss, balance deficits, limb and gait ataxia, and dysarthria. FA is considered a sensory ataxia because the dorsal root ganglia and spinal cord dorsal columns are involved early in the disease, whereas the cerebellum is affected later. Balance deficits and gait ataxia are often evaluated clinically and in research using clinical rating scales. Recently, quantitative tools such as the Biodex Balance System SD and the GAITRite Walkway System have become available to objectively assess balance and gait, respectively. However, there are limited studies using instrumented measures to quantitatively assess and characterize balance and gait disturbances in FA, and longitudinal, quantitative analyses of both balance and gait have not been investigated in this patient cohort. The purpose of the present study was to characterize gait patterns of adults with FA and to identify changes in gait and balance over time using clinical rating scales and quantitative measures. Additionally, this study investigated the relationship between disease duration, clinical rating scale scores and objective measures of gait and balance. This study used a longitudinal research design to investigate changes in balance and gait in 8 adults with genetically confirmed FA and 8 healthy controls matched for gender, age, height, and weight. Subjects with FA were evaluated using the Berg Balance Scale (BBS), the Friedreich's Ataxia Rating Scale (FARS) and instrumented gait and balance measures at baseline, 6 months, 12 months and 24 months. Controls underwent the same tests at baseline and 12 months. Gait parameters were measured utilizing the GAITRite Walkway system with a focus on gait velocity, cadence, step and stride lengths, step and stride length variability and percent of the gait cycle in swing, stance and double limb support. Balance was assessed using the BBS and the Biodex Balance System; the latter included tests of postural stability and limits of stability. At baseline, there were significant differences in gait and balance parameters, BBS scores and FARS total scores between FA subjects and controls as determined using paired t-tests (p This is the first longitudinal study to demonstrate changes over time in gait and balance of adults with FA using both quantitative measures and clinical rating scales. This study provided a detailed characterization of the gait pattern and balance of adults with FA. The GAITRite Walkway system proved to be a sensitive measure, and able to detect subtle changes in gait parameters over time in adults with FA. In addition, the BBS was an appropriate and sensitive assessment to detect changes in static and dynamic balance in this patient cohort. Finally, results revealed a strong and consistent relationship between clinical rating scale scores, postural stability indices, limits of stability scores, and step and stride length variability in individuals with FA.

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Buechel, Heather M. „CHANGES IN SLEEP ARCHITECTURE AND COGNITION WITH AGE AND PSYCHOSOCIAL STRESS: A STUDY IN FISCHER 344 RATS“. UKnowledge, 2013. http://uknowledge.uky.edu/pharmacol_etds/4.

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Changes in both sleep architecture and cognition are common with age. Typically these changes have a negative connotation: sleep fragmentation, insomnia, and deep sleep loss as well as forgetfulness, lack of focus, and even dementia and Alzheimer’s disease. Research has shown that psychosocial stressors, such as isolation from family and friends or loss of a loved one can also have significant negative effects on sleep architecture and cognitive capabilities. This leaves the elderly in a particularly vulnerable situation: suffering from cognitive decline and sleep dysregulation already, and more likely to respond negatively to psychosocial stressors. Taking all of these factors into account, it’s surprising that little research has been done to elucidate the mechanisms behind aged subjects’ enhanced vulnerability to new onset psychosocial stress. Our lab embarked on a series of studies to test the effects of age and psychosocial stress on sleep architecture and cognition. Our first study measured sleep stages in young adult and aged F344 rats during their resting and active periods. Animals were behaviorally characterized on the Morris water maze and gene expression profiles of their parietal cortices were taken. We confirmed previous studies that found impaired cognition and decreased resting deep sleep with age. However, it was increased active deep sleep that correlated best with poor cognitive performance. In the second study rats were subjected to immobilization (restraint stress) immediately preceding their final water maze task. Hippocampi were prepared for synaptic electrophysiology and trunk blood was taken for corticosterone measurement after post-stress sleep architecture data was collected. Young subjects responded to acute stress with decreased cognition, elevated CORT levels and altered sleep architecture. In contrast, stressed aged subjects were statistically indistinguishable from control aged subjects, suggesting that aged rats are less responsive to an acute psychosocial stress event. Together, these studies suggest that alleviating sleep dysregulation could therapeutically benefit cognition psychosocial stress resilience.

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Biayna, Rodríguez Josep. „Using Phosphorodiamidate Morpholino Oligomers (PMOs) to characterize the role of neurofibromin in cell physiology“. Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/378358.

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The role of neurofibromin, the product of the Neurofibromatosis Type 1 (NF1) gene, in cell physiology is still not well understood. Considering the different traits associated to NF1 it is clear that neurofibromin participates in processes of proliferation and differentiation of different cell types. The Ras-GAP activity of neurofibromin is its best characterized biochemical function, which is regulated by the alternative splicing of exon 23a (E23a). In this thesis we intended to better understand the role of alternative splicing of E23a during neuronal differentiation, with the aim that in the future it could provide information on the learning and cognitive issues related to this disease. Due to the large size of neurofibromin, the difficulty of manipulating it in vitro and the necessity to mimic, as much as possible, the physiological conditions of the cell, we used Phosphorodiamidate Morpholino Oligomers (PMOs) to modify the exonic composition of NF1 mRNA while preserving the endogenous neurofibromin expression levels. We developed a PMO-based system that successfully allowed to force the expression of type II (+E23a) or type I (-E23a) isoforms without altering the physiological expression levels of NF1 mRNA in PC12 cells, a neuronal differentiation model, in the presence or absence of Nerve Growth Factor (NGF). This PMO system could be used in other cellular models of NF1, and could be reproduced for studying the regulation of the alternative splicing in other genes. Furthermore, we set up a group of functional assays for assessing proliferation, differentiation, signaling and apoptosis in this PC12 neuronal model. Our results showed that forcing type I (-E23a) isoform was not sufficient for inducing PC12 neuronal differentiation in the absence of NGF. However, the results also demonstrate that any alteration of the NGF-induced ratio between type I/II isoforms, either in a quantitative or time-dependent manner, interfered with the correct neuronal differentiation process, in particular, altering the correct formation of neurites, as well as the proper regulation of the RAS/MAPK and cAMP/PKA signalling pathways. Moreover, the alteration of the natural E23a alternative splicing also impeded the proper neuronal differentiation process in other neuronal models, such as the H19-7 hippocampus cells. Our results also showed that the depletion of neurofibromin in PC12 induce a generalized process of apoptosis; suggest the existence of an early negative feed-back on the function of neurofibromin when type I isoform is abnormally expressed, and shows that the regulation of the cAMP/PKA pathway is also dependent on the GAP domain of neurofibromin. All together, the results of this thesis indicate that the regulation of the alternative splicing of exon 23a of the NF1 gene allows the fine-tuning of the RAS/MAPK and cAMP/PKA pathways through its GAP activity in a coordinate and opposite way along the time-dependent process of neuronal differentiation.
La neurofibromina és el producte del gen NF1, que mutat causa la Neurofibromatosis de tipus 1. Tot i que en l'actualitat, encara ens cal entendre millor el rol d'aquesta proteïna en la fisiologia cel•lular, l'activitat Ras-GAP és la funció bioquímica més ben caracteritzada de la neurofibromina. Aquesta funció està regulada per l'splicing alternatiu de l'exó 23a (E23a). En aquesta tesi ens vàrem proposar comprendre millor el paper d'aquest splicing alternatiu durant el procés de diferenciació neuronal, amb l'objectiu de proporcionar nova informació sobre els problemes cognitius i d'aprenentatge associats a aquesta malaltia. Degut a la gran grandària de la neurofibromina i a la dificultat de manipular-la in vitxo, es varen utilitzar Phosphorodiamidate Morpholino Oligomers (PMOs) per modificar la composició exònica del mARN (per tant l'estructura resultant de la neurofibromina) sense alterar les condicions fisiològiques d'expressió del gen NF 1 . Es va desenvolupar un sistema basat en PMOs per forçar l'expressió de l'isoforma tipus II (+E23a) o tipus I (-E23a) del gen NF1 en cèl•lules PC12, un model de diferenciació neuronal, en presència o absència de Nerve Growth Factor (NGF). A més, per entendre la importància de 1'E23a es va establir un grup de metodologies i assajos funcionals per poder determinar diferents respostes cel•lulars i valorar la funció d'aquest en el procés de diferenciació neuronal. Els nostres resultats van mostrar que forçar l'isoforma tipus I (-E23a) no era suficient per induir la diferenciació de les cèl•lules PC12 en absència de NGF. No obstant això, qualsevol alteració en la relació entre les isoformes tipus I/II en presència de NGF, ja sigui d'una manera quantitativa o dependent del temps, interferia en el correcte procés de diferenciació neuronal, en particular, alterant la correcta formació de neurites, així com l'adequada regulació de les vies de senyalització RAS/MAPK i cAMP/PKA. En conjunt, els resultats d'aquesta tesi indiquen que la regulació de l'splicing alternatiu de 1'E23a del gen NF1 permet un ajust fi de les vies RAS/MAPK i cAMP/PKA a través de la activitat GAP de la neurofibromina, d'una forma oposada i coordinada al llarg del temps durant el procés de diferenciació neuronal.

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Jayatunga, Dona Pamoda Wajirapanie. „Investigation of selected nutraceuticals to protect against mitochondrial dysfunction: Potential therapeutic role in Alzheimer’s disease“. Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2020. https://ro.ecu.edu.au/theses/2368.

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Alzheimer’s disease (AD) is a devastating neurodegenerative disorder that affects approximately 60-80% of all clinically diagnosed dementia cases worldwide. It is the second major cause of death in Australia. The progressive nature of the disease is characterized by cognitive deficits that worsen over time, usually leading to death within about a decade from their first manifestation. The AD brain is classically characterized by extracellular deposition of amyloid-β (Aβ) protein aggregates, as senile plaques, and intracellular neurofibrillary tangles (NFTs), composed of hyper-phosphorylated forms of the microtubule-associated protein Tau. There is no effective treatment for AD and the drugs currently being used to treat the disease only alleviate symptoms for a limited period in most cases. There has been limited success in clinical trials for some proposed therapies, so attention has been drawn towards using alternative approaches, including prevention strategies. As a result, nutraceuticals, the bioactive components of foods, have become attractive compounds for their potential neuroprotective capabilities. The overall objective of the present study was to derive a nutraceutical compound combination in vitro that has the potential to be used as a preventative therapy for AD. The first part of this project was aimed at screening eleven nutraceutical compounds derived from pomegranate, turmeric and fish oil, for their neuroprotective action using an in vitro cell model of Aβ cytotoxicity. Of these eleven compounds, punicalagin (PUN), docosahexaenoic acid (DHA), luteolin (LUT) and urolithin A (UA) were identified to effectively inhibit Aβ1-42-induced toxicity. The next aim was to determine whether Aβ1-42-induced toxicity could be blocked by the identified compounds in a synergistic manner. For this aim, the compounds PUN, DHA, LUT and UA were tested in two-compound combinations using the established cell model. In silico prediction (CompuSyn v.1.0 software) was used to predict suitable combinations based upon data from the initial screening phase. Experimental validation of these compound pairs against Aβ1-42-induced toxicity allowed the prediction of a three-compound combination. This three-compound combination made up of DHA, LUT and UA, (D5L5U5), was experimentally shown to be very effective at inhibiting Aβ1-42-induced toxicity. The third aim was to determine a pathway of action for the three-compound combination in inhibiting Aβ1-42-induced toxicity. It is well-established that Aβ1-42 causes mitochondrial dysfunction. Moreover, one of the compounds in this combination UA, is known to act as a mitophagy-inducing compound. Mitophagy is the selective degradation of dysfunctional mitochondria and is a key mitochondrial quality control mechanism in cells. Due to accumulating evidence of mitophagy deficits in AD, this represents a possible therapeutic target for AD. Therefore, part of this aim was to examine the effects of Aβ1-42 cytotoxicity in cells towards impairment in the mitochondrial quality control mechanisms. The results indicated that chronic Aβ1-42 exposure promotes mitochondrial dysfunction by increasing ROS levels and severely diminishing mitochondrial ATP levels, while affecting mitochondrial dynamics, mitophagy and mitobiogenesis processes. Additionally, it was revealed that these changes were time dependent due to the cell responses against the Aβ1-42 cytotoxicity. The final aim of this thesis was to compare the three-compound combination with the component compounds in their optimal concentrations against Aβ1-42-induced mitochondrial dysfunction. The findings indicated that the three-compound nutraceutical combination, D5L5U5 precedes its component compounds for its protective ability on all aspects tested, namely, by reducing ROS levels, increasing ATP levels, and inducing mitophagy and mitobiogenesis. The compounds, LUT, DHA and UA were also independently protective of mitochondria. Overall, it was determined that the three-compound nutraceutical combination, D5L5U5 has strong inhibitory effects against Aβ1-42-induced toxicity through its mitoprotective activities by minimizing oxidative stress and inducing mitophagy and mitobiogenesis. However, it warrants further investigations in other in vitro and in vivo AD models to confirm its potential to be used as a preventative therapy for AD.

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Masdeu, Camara Maria del Mar. „Papel de la tirosina quinasa Ack1 en la neuritogénesis y señalización regulada por neurotrofinas y moléculas de guía axonal“. Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/145719.

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Ack1 es una proteína tirosina quinasa citoplasmática que pertenece a la familia de proteínas Ack que está formada por 9 miembros. Ack1 está compuesta por varios dominios de interacción proteína-proteína y un dominio catalítico tirosina quinasa cuya autofosforilación regula parcialmente la función de la proteína (Lougheed et al., 2004). Ack1 se encuentra altamente expresada en cerebro (Urena et al., 2005; La Torre et al., 2006), principalmente en las zonas del hipocampo, corteza cerebral, bulbo olfativo y cerebelo (Urena et al., 2005). Además, se ha demostrado que Ack1 se localiza tanto en dendritas como en regiones presinápticas y su expresión se regula a la alza por un incremento de actividad neuronal (Urena et al., 2005). Las funciones fisiológicas de Ack1 son bastante desconocidas. En conjunto, los datos publicados hasta la actualidad destacan la capacidad de la proteína Ack1 para interaccionar con una gran variedad de proteínas, que indica que Ack1 puede participar en diferentes rutas de transducción de señales, y por tanto, participar en diferentes procesos fisiológicos de las células. Por ejemplo, se ha descrito que puede participar en la regulación del citoesqueleto de actina (Burbelo et al., 1995), en los procesos de endocitosis (Teo et al., 2001), en las cascadas de señalización que resultan de la adhesión celular (Bourdoulous et al., 1998), en la migración celular (Modzelewska et al., 2006), en la proliferación (Nur et al., 2005) y en la supervivencia (Mahajan et al., 2005). La mayoría de funciones de Ack1 conocidas se deducen de las interacciones moleculares de ésta y hasta la actualidad se dispone de muy pocos datos respecto a las competencias biológicas de esta proteína, especialmente a nivel de SNC. Por eso, en esta tesis hemos intentado determinar el papel de la tirosina quinasa Ack1 en las funciones del SNC mediante 5 capítulos de resultados. En el capítulo I explicamos la producción de un anticuerpo monoclonal contra Ack1 que produjimos con el objetivo de poder estudiar la proteína Ack1 a nivel funcional. Este anticuerpo lo produjimos contra la región rica en prolinas de Ack1 con el fin que nos permitiera aumentar la especificidad de nuestros experimentos. En el capítulo II caracterizamos la proteína Ack1 a nivel más funcional. Demostramos que Ack1 es un componente de la vía de señalización de las neurotrofinas, siendo fosforilada en respuesta a neurotrofinas e interaccionando con sus receptores Trk. Además, también describimos que cambios de expresión de Ack1 alteran la neuritogénesis en células PC12 y los patrones de ramificación axonal y dendrítico en neuronas de hipocampo y células granulares de cerebelo. En el capítulo III examinamos la interacción de Ack1 con las proteínas de la densidad postsináptica CaMKII y PSD-95, su fosforilación en respuesta a los estímulos excitadores NMDA y glutamato, y la afectación de los botones axonales de las ramas colaterales de Schaffer por una falta de expresión de Ack1. Los resultados obtenidos en este capítulo apuntan a una posible implicación de Ack1 a nivel de terminales sinápticos. En el capítulo IV nos centramos en estudiar la interacción de Ack1 con la proteína FAK y su participación en procesos de quimioatracción mediados por Netrina-1. Los datos obtenidos sugieren una interacción entre ambas proteínas, que Ack1 se fosforila en respuesta a Netrina-1 y una implicación de la proteína Ack1 en los procesos de quimioatracción mediados por Netrina-1 en células de hipocampo. Finalmente, siendo las proteínas Ack1 y FAK unas proteínas que dependen de su estado de fosforilación para regular su actividad, en el capítulo V de resultados de esta tesis estudiamos las dianas de fosforilación y posibles proteínas de interacción de ambas proteínas, mediante la técnica de espectrometría de masas en muestras de cerebro de ratón en desarrollo, de cerebro de ratón adulto y de cerebro de ratón adulto hiperestimulado. - Bibliografía Bourdoulous S, Orend G, MacKenna DA, Pasqualini R, Ruoslahti E (1998) Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression. The Journal of cell biology 143:267-276. Burbelo PD, Drechsel D, Hall A (1995) A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. The Journal of biological chemistry 270:29071-29074. La Torre A, del Rio JA, Soriano E, Urena JM (2006) Expression pattern of ACK1 tyrosine kinase during brain development in the mouse. Gene Expr Patterns 6:886-892. Lougheed JC, Chen RH, Mak P, Stout TJ (2004) Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1. The Journal of biological chemistry 279:44039-44045. Mahajan NP, Whang YE, Mohler JL, Earp HS (2005) Activated tyrosine kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquitination of tumor suppressor Wwox. Cancer research 65:10514-10523. Modzelewska K, Newman LP, Desai R, Keely PJ (2006) Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas. The Journal of biological chemistry 281:37527-37535. Nur EKA, Zhang A, Keenan SM, Wang XI, Seraj J, Satoh T, Meiners S, Welsh WJ (2005) Requirement of activated Cdc42-associated kinase for survival of v-Ras-transformed mammalian cells. Mol Cancer Res 3:297-305. Teo M, Tan L, Lim L, Manser E (2001) The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors. The Journal of biological chemistry 276:18392-18398. Urena JM, La Torre A, Martinez A, Lowenstein E, Franco N, Winsky-Sommerer R, Fontana X, Casaroli-Marano R, Ibanez-Sabio MA, Pascual M, Del Rio JA, de Lecea L, Soriano E (2005) Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain. The Journal of comparative neurology 490:119-132.
Ack1 is a cytoplasmic tyrosine kinase highly expressed in Central Nervous System (Urena et al., 2005; La Torre et al., 2006) that has several protein-protein interaction domains and a catalytic domain that is autophosphorylated, and this process regulates, at least in part, the action of this protein (Lougheed et al., 2004). Moreover, it has been demonstrated that Ack1 is localized in dendrites and presynaptic regions and that its expression is up-regulated by an increase of neuronal activity (Urena et al., 2005). Most of the known functions of Ack1 have been elucidated from interactions of Ack1 with several proteins. But, most of the physiologically functions of Ack1 remain to be described, especially in CNS. For this reason, the aim of this thesis has been to unravel some of these functions on CNS that are described through 5 chapters. In the 1st chapter we have explained the production of a monoclonal antibody against Ack1 that allowed us to improve the specificity of our experiments and to study Ack1 at a functional level. In the 2nd chapter we have demonstrated that Ack1 is a component of the neurotrophin pathway, because it is phosphorylated as a response to neurotrophins and interacts with Trk receptors. Moreover, we also have described how changes in Ack1 expression alter neuritogenesis and axonal and dendritic ramification. In the 3th chapter we have analyzed the interaction of Ack1 with the postsynaptical proteins CaMKII and PSD-95, its phosphorylation in response to excitatory stimulus such as NMDA or glutamate and we have described that a lack of Ack1 expression decrease the size of axonal buttons. These data suggest an implication of Ack1 at a synaptic level. In the 4th chapter we focused on the determination of the interaction of Ack1 and FAK, the Ack1 phosphorylation in response to Netrin-1 and the effect of Ack1 in chemoattraction regulated by Netrin-1. Finally, in the 5th chapter we studied the phosphorylation sites and the possible interacting proteins of FAK and Ack1 by mass spectrometry in samples of mice brain in development, of adult mice brain and of adult mice overstimulated brain. - Bibliography La Torre A, del Rio JA, Soriano E, Urena JM (2006) Expression pattern of ACK1 tyrosine kinase during brain development in the mouse. Gene Expr Patterns 6:886-892. Lougheed JC, Chen RH, Mak P, Stout TJ (2004) Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1. The Journal of biological chemistry 279:44039-44045. Urena JM, La Torre A, Martinez A, Lowenstein E, Franco N, Winsky-Sommerer R, Fontana X, Casaroli-Marano R, Ibanez-Sabio MA, Pascual M, Del Rio JA, de Lecea L, Soriano E (2005) Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain. The Journal of comparative neurology 490:119-132.

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Dissertationen zum Thema „Medical Neurobiology“

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