Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

Neurotrauma is a general term describing injury to the central nervous system (CNS); which comprises of the brain and spinal cord. The damage resulting from neurotrauma includes primary injury, which occurs from different sources such as compressed air hitting the brain (bTBI) or an object/bone contusing the spinal cord, resulting in a spinal cord injury (SCI). These various means of primary brain and spinal cord injury are further complicated by the many possible combinations of severity levels and frequencies. However, primary injuries are regarded in many cases as unavoidable with the immediate nerve damage being largely irreversible. Despite all this, primary injuries of the CNS are related by common biochemical pathways in secondary injury. Secondary injury is the cause of declining outcomes after neurotrauma and poor recovery. Secondary injury begins immediately after primary injury and can continue to trigger death of neurons for years later. Given this contribution to poor recovery and its slow progression, secondary injury provides an excellent window of opportunity for therapeutic intervention. A major factor and key link in secondary injury and its perpetuation is reactive aldehyde formation, such as acrolein, from lipid peroxidation. The common formation of acrolein in neurotrauma is attributed to the unique structure of the CNS: with neurons containing a high lipid content from myelin and heavy metabolic activity they are vulnerable to acrolein formation. Thus, acrolein in secondary injury is a point of pathogenic convergence between the various forms of neurotrauma, and may play a role as well in the development of neurotrauma linked disorders and related neurodegeneration. The overall goal of this thesis is to therefore develop better strategies for acrolein removal. We explore here endogenous clearance strategies and targeted drug delivery in SCI, investigate detailed cellular structure changes in bTBI, and acrolein formation and removal in Parkinson’s disease. These findings of pathology, and effectiveness of new or existing acrolein removal strategies, will allow us to better employ treatments in future studies.

Blast-induced neurotrauma (BINT) is a major societal concern due to the complex expression of neuropathological disorders after exposure to blast. Disruptions in neuronal function, proximal in time to the blast exposure, may eventually contribute to the late emergence of the clinical deficits. Besides complications with differential clinical diagnosis, the biomolecular mechanism underlying BINT that gives rise to cognitive deficits is poorly understood. Some pre-clinical studies have demonstrated cognitive deficits at an acute stage following blast overpressure (BOP) exposure. However, the behavioral deficit type (e.g., short term memory) and the mechanism underlying injury prognosis that onsets the cognitive deficits remains to be further investigated. An established rodent model of blast neurotrauma was used in order to study impaired behavioral and neuropathological outcomes following blast. Anesthetized rats were exposed to a calibrated BOP using a blast simulator while control animals were not exposed to BOP. Behavioral changes in short term memory and anxiety were assessed with standard behavioral techniques (novel objected recognition paradigm and light and dark box test) at acute and chronic stages (range: 3 hours -- 3 months). In addition, brains were assayed for neurochemical changes using proton magnetic resonance spectroscopy (MRS) and neuropathology with immunohistochemistry in cognitive regions of brain (hippocampus, amygdala, frontal cortex and nucleus accumbens) Early metabolic changes and oxidative stress were observed along with a compromise in energy metabolism associated with sub-acute (7 days following BOP exposure) active neurodegeneration and glial scarring. Data suggested GABA shunting pathway was activated and phospholipase A2 regulated arachadonic acid pathway may be involved in cellular death cascades. In addition, increased myo-inositol levels in medial pre-frontal cortex (PFC) further supported the glial scarring and were associated with impaired working memory at a sub-acute stage (7 days) following BOP exposure. Chronic working memory issues and anxiety associated behavior could be related to chronic activation of microglia in hippocampus and astrocytes in amygdala respectively. Furthermore, these results from MRS could be directly translated into clinical studies to provide a valuable insight into diagnosis of BINT, and it is speculated that gliosis associated markers (myo-inositol) may be a potential biomarker for blast-induced memory impairment. / Ph. D.

Traumatic brain injury (TBI) produces axonal damage and deafferentation, triggering injury-induced synaptogenesis, a process influenced by matrix metalloproteinases (MMP) and their substrates. Here we report results of studies examining the expression and potential role of two membrane-bound MMPs, membrane-type 5-MMP (MT5-MMP) and a disintegrin and metalloproteinase-10 (ADAM-10), along with their common synaptic substrate N-cadherin, during the period of reactive synaptogenesis. Protein and mRNA expression of MT5-MMP, ADAM-10 and N-cadherin were compared in two TBI models, one exhibiting adaptive plasticity (unilateral entorhinal cortex lesion; UEC) and the other maladaptive plasticity (fluid percussion injury + bilateral EC lesions; TBI+BEC), targeting 2, 7, and 15d postinjury intervals. In adaptive UEC plasticity, membrane-bound MMP expression was elevated during synaptic degeneration (2d) and regeneration (7d), and normalized at 15d. By contrast, N-cadherin expression was significantly decreased at 2 and 7d after UEC, but increased during 15d synaptic stabilization. In maladaptive plasticity, 2d membrane-bound MMP expression was dampened compared to UEC, with persistent ADAM-10 elevation and reduced N-cadherin protein level at 15d. These results were supported by 7d microarray and qRT-PCR analyses, which showed transcript shifts in both hippocampus and dentate molecular layer (ML) for each model. Parallel immunohistochemistry revealed significant MT5-MMP, ADAM-10 and N-cadherin localization within ML reactive astrocytes, suggesting a glial synthetic or phagocytotic role for their processing during recovery. We also investigated the effect of MMP inhibition on molecular, electrophysiological, behavioral and structural outcome at 15d following TBI+BEC. MMP inhibitor GM6001 was administered at 6 and 7d postinjury, during elevated MT5-MMP/ADAM-10 expression and synapse regeneration. MMP inhibition showed: 1) reduced ADAM-10 and elevated N-cadherin protein expression, generating profiles similar to 15d post-UEC, 2) attenuation of deficits in the initiation phase of long-term potentiation, and 3) improved hippocampal dendritic and synaptic ultrastructure. Collectively, our results provide evidence that membrane-bound MMPs and N-cadherin influence both adaptive and maladaptive plasticity in a time and injury-dependent manner. Inhibition of membrane-bound MMPs during maladaptive plasticity produces more adaptive conditions, improving synaptic efficacy and structure. Thus, targeting MMP function and expression have potential to translate maladaptive plasticity into an adaptive process, facilitating improved recovery.

Postmortem brains from young athletes with a history of repetitive concussive head injury and military service personnel with history of blast neurotrauma revealed evidence of parenchymal contusion, myelinated axonopathy, microvasculopathy, neuroinflammation, neurodegeneration, and phosphorylated tauopathy consistent with chronic traumatic encephalopathy (CTE) (L. E. Goldstein et al., 2012). The mechanisms by which head trauma induces acute concussion and chronic sequelae are unknown. To elucidate the mechanistic connection between traumatic brain injury (TBI), acute concussion and chronic sequelae, including CTE, require the use of animal models. This doctoral dissertation investigated the hypothesis that closed-head impact injury in mice triggers acute neurological signs associated with sport-related concussion as well as brain pathologies and functional sequelae associated with CTE. To test this hypothesis, we developed a mouse model of impact neurotrauma that utilizes a momentum transfer device to induce non-skull deforming head acceleration, triggering transient neurological signs consistent with acute concussion and traumatic brain injury (TBI) in unanesthetized C57BL/6 mice. The Boston University Concussion Scale (BUCS) was developed to assess neurological signs that are consistent with acute concussion in humans. Mice exhibited contralateral circling and limb weakness, locomotor abnormalities, and impaired gait and balance that recapitulate acute concussion in humans. Concussed mice recovered neurological function within three hours, but demonstrated persistent myelinated axonopathy, microvasculopathy, neuroinflammation, and phosphorylated tauopathy consistent with early CTE. Concussive impact injury also induced blood-brain barrier disruption, neuroinflammation (including infiltration peripheral monocytes and activation microglia), impaired hippocampal axonal conduction, and defective long-term potentiation (LTP) of synaptic transmission in medial prefrontal cortex. Kinematic analysis during impact injury revealed head acceleration of sufficient intensity to induce acute concussion, traumatic brain injury (TBI), early CTE-linked pathology, and related chronic sequelae. Surprisingly, the presence or degree of concussion measured by BUCS did not correlate with brain injury. Moreover, concussion was observed following impact injury but not blast exposure under conditions that induce comparable head kinematics. Empirical pressure measurements and dynamic modeling revealed greater pressure on the head and compression wave loading in the brain during impact compared to blast neurotrauma. These findings suggest acute concussion is triggered by focal loading of energy that transit the brain before onset of macroscopic head motion. By contrast, the forces associated with rapid head motion is sufficient to induce CTE-linked pathology. Our results indicate that while acute concussion and chronic sequelae may be triggered by the same insult, the pathophysiological responses underpinning these effects are engaged through distinct mechanisms and time domains. Our results indicate that concussion is neither necessary nor sufficient to induce acute brain injury or chronic sequelae, including CTE. / 2018-02-17T00:00:00Z

The development of traumatic brain injury (TBI) therapeutics and effective translation to clinic remains stubbornly elusive despite the high prevalence of TBI within the United States and across the globe. Interventions must be devised around testable targets, appropriately timed to intercede on secondary results. Here, we have utilized temporal neuroproteomics as an ideal approach to inform on the complex biochemical processing in order to address the well-recognized temporal evolution of TBI pathobiology and interrogate a novel therapeutic target in a mild-moderate rat Controlled Cortical Impact (CCI) within perilesioned somatosensory cortex. First, our findings revealed 2047 proteins significantly impacted within the first two weeks following TBI. Subsequent artificial neural network analysis revealed a delayed-onset cluster of proteins highly enriched in GABAergic neurotransmission and ion transport to reveal the prototypical target potassium/chloride transport 2 (KCC2 or SLC12A5) for further investigation with the KCC2-specific pharmacologic CLP290. Our tested therapeutic window guided by post-translational processing preceding one-day prior to protein loss revealed effective CLP290 restoration of KCC2 localization. We further demonstrated recovery in functional and behavioral assessments with one-day administration paradigm supporting the effectiveness of CLP290 treatment after brain injury. To better understand the underlying mechanism of CLP290, we utilized proteomic and bioinformatic approaches to tease out the biological response to treatment. Results demonstrate recovery of PKCδ-mediated phosphorylation of KCC2 and recovery of transporter activity. Additionally, findings reveal preservation of tyrosine kinase by reversing ubiquitin-mediated proteasomal degradation. Our functional assessment of secondary injury insults two-weeks following TBI revealed recovery in seizure threshold, reduction in lesion expansion and a decrease in cell loss suggesting maintained recovery of KCC2 and restored E/I balance. In conclusion, the presented studies in these two chapters propose a novel approach for development of therapeutics for TBI and test the selective manipulation via pharmacological intervention. These findings are promising for the development and treatment of other neurological disorders.

Indiana University-Purdue University Indianapolis (IUPUI) / Spinal cord injury (SCI) is devastating and debilitating, and currently no effective treatments exist. Approximately, 12,000 new cases of SCI occur annually in the United States alone. The central nervous system has very low repair capability after injury, due to the toxic environment in the injured tissue. After spinal cord trauma, ruptured blood vessels cause neighboring cells and tissues to be deprived of oxygen and nutrients, and result in the accumulation of carbon dioxide and waste. New blood vessels form spontaneously after SCI, but then retract as the injured tissue forms a cavity. Thus, the newly formed vasculature likely retracts because it lacks a structural support matrix to extend across the lesion. Currently, in the field of spinal cord injury, combinational treatment approaches appear to hold the greatest therapeutic potential. Therefore, the aim of these studies was to transplant a novel, non-immunogenic, bioengineered hydrogel, into the injured spinal cord to serve as both a structural scaffold (for blood vessels, axons, and astrocytic processes), as well as a functional matrix with a time-controlled release of growth factors (Vascular endothelial growth factor, VEGF; Glial cell line-derived neurotrophic factor, GDNF). The benefit of this hydrogel is that it remains liquid at cooler temperatures, gels to conform to the space surrounding it at body temperature, and was designed to have a similar tensile strength as spinal cord tissue. This is advantageous due to the non-uniformity of lesion cavities following contusive spinal cord injury. Hydrogel alone and combinational treatment groups significantly improved several measures of functional recovery and showed modest histological improvements, yet did not provoke any increased sensitivity to a thermal stimulus. Collectively, these findings suggest that with further investigation, hydrogel along with a combination of growth factors might be a useful therapeutic approach for repairing the injured spinal cord.

No description available.

Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts, which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments, which are driven by the underlying cellular and molecular sequelae of the injury. Development of effective treatment strategies is limited by the lack of understanding on the cellular and molecular level [2a]. The overall hypothesis of this work is that epigenetic regulatory mechanisms contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms, including DNA methylation and histone acetylation, are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown. To test this hypothesis, an established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury, which was accompanied by decreased expression of the enzyme responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to decline in memory. This change was observed in astrocytes and may provide a mechanistic understanding for a hallmark characteristic of the injury. Treatment with a specific enzyme inhibitor was able to mitigate some of the histone acetylation changes. This corresponded with reduced astrocyte activation and an altered behavioral phenotype, which was characterized by high response to novelty. The diagnostic efficacy of epigenetic changes following blast was elucidated by the accumulation of cell-free nucleic acids in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The clinical potential impact on diagnosis and therapeutic intervention has been demonstrated. / Ph. D. / Blast-induced neurotrauma (BINT) is a prevalent brain injury within both military and civilian populations due to current engagement in overseas conflict and ongoing terrorist events worldwide. In the early 2000s, 78% of injuries were attributable to an explosive mechanism during overseas conflicts which has led to increased incidences of BINT [1a]. Clinical manifestations of BINT include long-term psychological impairments which are driven by the underlying cellular and molecular sequelae of the injury. To date, the development of effective treatment strategies has been unsuccessful. The work described herein seeks to evaluate the specific cellular mechanisms that contribute to the progression of the BINT pathology and neurological impairments. Epigenetic mechanisms are regulatory mechanisms that coordinate DNA modifications and DNA storage to facilitate altered cellular phenotypes. DNA modifications often involves DNA methylation, which is the addition of methyl groups to the DNA backbone. DNA storage is regulated by specific modifications to histone proteins. Histone acetylation is a well-studied modification process that is capable inciting either chromatin relaxation or compaction. Both DNA methylation and histone acetylation are important processes by which cells coordinate neurological and cellular response to environmental stimuli. To date, the role of epigenetics in BINT remains largely unknown. An established rodent model of BINT was employed [3a]. Analysis of DNA methylation, which is involved in memory processes, showed decreased levels one week following injury which was accompanied by decreased expression of one of the enzymes responsible for facilitating the addition of methyl groups to DNA. The one week time point also showed dramatic decreases in histone acetylation which correlated to memory impairment. This change was observed in astrocytes which are support cells in the brain and are particularly vulnerable to blast-induced aberrations. Drug administration, targeting the histone acetylation equilibrium, successfully mitigated astrocyte activation and altered the behavioral phenotype. Diagnosis of BINT remains clinically challenging. An accumulation of cell-free nucleic acids was observed the in cerebrospinal fluid one month after injury. Concentrations of these molecules shows promise in discriminating between injured and non-injured individuals. These nucleic acids are susceptible to DNA methylation and may provide a platform for studying epigenetic biomarkers. To date, the diagnostic and therapeutic efforts of BINT have been limited by the lack of a mechanistic understanding of the injury. This work provides novel diagnostic and therapeutic targets. The potential clinical impact on diagnosis and therapeutic intervention has been demonstrated.