Background: Child maltreatment is a serious health concern in the United States (U.S.) affecting as many as one in four children throughout their lifetime (Finkelhor, Turner, Ormond, & Hamby, 2013). In 2013, a reported 678, 932 victims of child maltreatment were reported to Child Protective Services (CPS), and of those cases 1,520 were fatal (CDC, 2015a). Out of all the various types of child maltreatment, Shaken Baby Syndrome (SBS) is the leading cause of child abuse deaths in the U.S. (CDC, n.d.). While current research has focused on validating the effectiveness of educational interventions, very few studies have analyzed the efficacy of individualized, patient-centered action plans. Such data would be beneficial to assess the usefulness of action plans in preparing caregivers for coping with an inconsolable infant at home. Purpose: To enhance caregiver knowledge about SBS and to provide parents with the skills and resources necessary to cope effectively and efficiently at home when unable to console their infant. Methods: This study utilized a quasi-experimental pre-test/post-test design. Participants were recruited from the Franciscan Women’s Health Associates located at St. Joseph Medical Center in Tacoma, Washington and were members of the Centering prenatal groups. The entirety of the study was completed during these groups including the pre-test, intervention, action plan, and post-test. Data was analyzed through the utilization of descriptive statistics as well as a paired t test. Results: Overall, results revealed that participant (n=26) knowledge significantly improved after the educational intervention (p=0.000) with a mean score of 87.56% on the pre-test and a mean score of 95.38% on the post-test. In addition, a majority of participants (57.5%) found both the action plan and the education to be extremely useful. Discussion: The results of this study were consistent with current evidence indicating that education on SBS, the dangers of shaking, and healthy coping mechanisms significantly impacts caregiver knowledge. In addition, a majority of participants viewed the action plans favorably identifying that they would be beneficial if they felt frustrated. Future research is warranted to gather more information on the long-term outcomes of educational interventions as well as individualized action plans.

Blast wave induced traumatic brain injury (bTBI) is a modality of injury that has come into prominence at the current time due to the large number of military and civilian personnel who have experienced the localized shock wave produced by explosive devices. The shock wave will travel concentrically outward from the explosive center, being absorbed and transmitted thru soft objects, such as tissue, and reflecting off stationary obstructions. Transmission and absorption in tissues can result in a number of physiological measureable injuries, the most common of which being what is frequently called “blast lung”. Blast lung involves the spalling effect at air-tissue interfaces. Another documented effect involves the asynchronous motion of tissue, particularly in the cranium, as the shock wave passes by. This predominately manifests itself in what is believed to be diffuse axonal injury and initiation of secondary injury mechanism. This study is designed to explore the relationship between shock waves and bTBI. A blast device was constructed for generating a free field shock wave through the high pressure rupture of a polycarbonate membrane. Air pressure in a small chamber is increased to a value several orders of magnitude greater than ambient air pressure and is held in place with the polycarbonate member. At the rupture of this membrane a shock wave is created. Measurements of this blast event, carried out with a piezoelectric pressure transducer, have shown that this shock wave is reproducible for the different membrane materials tested and is symmetrical with respect to the central axis of the high pressure chamber and exit nozzle. Having characterized the shock wave properties in the blast field, a location was chosen at which maximum shock wave pressure could be applied to the cranium for inducing bTBI. Experiments involving blast wave exposure were performed on two separate groups of animals in an attempt at establishing injury. One group was placed at a fixed distance directly below the blast nozzle, thereby experiencing both the shock wave and the associated air blast from the residual air in the chamber, and one placed at a defined distance off-axis to avoid the air blast, yet receiving two sequential blast exposures. All animal studies were approved by the VCU Institutional Animal Care and Use Committee. The degree of injury was then assessed with the use of magnetic resonance imaging (MRI) and spectroscopy (MRS). Image Data was acquired on a 2.4 Tesla magnet for assessing changes in either the total percent water concentration or the apparent diffusion coefficients (ADC) of selected regions of interest in the brain of rats. Localized proton spectroscopic data was acquired from a voxel placed centrally in the brain. The baseline values of these parameters were established before the induction of bTBI. After the blast exposure, the animals were followed up with MRI and MRS at defined intervals over a period of one week. The first group of animals received blast exposure directly underneath the blast device nozzle and the MR data does suggest changes in some of the measureable parameters from baseline following blast exposure. This blast wave data though is confounded with additional and undesirable characteristics of the blast wave. The second group of animals that received a pure shock wave blast exposure revealed no remarkable changes in the MR data pre- to post- blast exposure. The percent water concentration, ADC and spectroscopic parameters were for statistical purposes identical before and after the blast. The resolution of this negative result will require reconsideration of the free field blast exposure concept.

Traumatic brain injury (TBI) affects 3 out of every 1000 Americans each year, and is the leading cause of morbidity and mortality after trauma, accounting for as many as 56,000 deaths per year (Dutton and McCunn, 2003). The Centers for Disease and Control and Prevention found that TBI most commonly occurs in adolescents and young adults aged from 15 to 24 years and in the elderly (75 years and older). Following injury, the secondary injury begins almost immediately after the primary injury and is the result of a number of cascades where once activated, exacerbate the already altered homeostasis of the injured brain. Brain trauma leads to complex secondary injury responses that trigger many cellular and molecular pathways, especially inflammation. The cerebral inflammation that occurs after TBI has been described through the processes of glial activation followed by leukocyte recruitment, and upreglation and secretion of cytokines and chemokines. With aging there is a decrease in the production of anti-inflammatory cytokines along with increasing amounts of pro-inflammatory cytokines by peripheral blood monoculear cells, microglia, and astrocytes. Studies have shown that inflammation has a strong negative effect on neurogenesis in the adult brain due to the impact of the pro-inflammatory cytokines that are released following the acute injury or disease. In this study, we first examined the differences in glial cells responses in young adult brain and aged brain following a moderate lateral fluid percussion injury and the correlation of glial cell activation with hippocampal neurogenesis. We then examined the effect of anti-inflammation treatment on glial cell response in the young and aged brain. The levels of astrocytic and microglial responses in the DG of the hippocampus following injury at 3, 7 or 28 days post-injury were measured using densitometry image analysis on GFAP or Iba1 immunofluorescent labeled brain tissue sections. We found that injury increased both astrocyte and microglial activation and proliferation in both young and aged brain. The young injured animals exhibited greater levels of GFAP while the aged injured animals exhibited greater levels of Iba1 expression at all three time points. We also found that short time anti-inflammatory treatment with minocycline decreased levels of Iba1 expression while increased levels of GFAP expression in both young and aged brain following injury. Our data suggests that there are differences in glial response in the injured young and aged brain that may contribute to the differences in the regenerative and recovery potential in the two age groups following injury.

Traumatic brain injury (TBI) is a worldwide epidemic as the number of victims living with the resulting cognitive and physical impairment continues to rise, principally due to limited treatment options which fail to address its multifaceted sequelae. By approaching TBI therapy from a molecular standpoint, we have the opportunity to develop a better understanding of the mechanisms which prevent effective recovery. With this information, we can move toward the identification of novel therapeutic treatments which target specific molecules to improve patient outcome following TBI. Here, we have focused on the therapeutic potential of osteopontin (OPN), an extracellular matrix (ECM) protein which is a substrate of several matrix metalloproteinases (MMPs), and capable of acting as both a cytokine and modulator of axonal outgrowth during synaptic recovery. The ECM and its components are of particular interest with respect to selecting novel TBI therapeutics since this network has been implicated in neuronal plasticity during both development and following central nervous system (CNS) insult. In this dissertation study, the temporal and spatial profile of OPN expression, its protein and transcript localization within reactive glia (IBA1 positive microglia or GFAP positive astroglia), and its interaction with the cytoarchitectural protein (microtubule associated protein 1B, MAP1B) after injury were each compared under conditions of deafferentation induced synaptogenesis. Two TBI models were employed: one exhibiting adaptive synaptic plasticity (unilateral entorhinal cortex lesion, UEC), and the other generating maladaptive synaptic plasticity (central fluid percussion injury followed by bilateral entorhinal cortex lesions, TBI+BEC), in each case targeting 1, 2, and 7d postinjury intervals. In addition, we examined the potential for converting the adaptive response to one of maladaptive plasticity by attenuating immune reactivity through acute administration of the tricyclic antibiotic minocycline, utilizing a dosing paradigm previously demonstrated to reduce inflammation. To more clearly confirm that OPN has a role in successful synaptic regeneration, we developed a colony of OPN knockout (KO) mice which were used to profile synaptic structure and functional outcome under conditions of UEC-induced synaptogenesis. In Chapter 2, we report that full length OPN responds robustly in the acute (1-2d postinjury) degenerative period following UEC and TBI+BEC. After UEC, time-dependent differences were observed for two alternative, MMP-processed OPN forms, including early increase in a RGD 45 kD, integrin binding fragment (1d), and delayed increase in a C-terminal 32 kD OPN peptide (7d). OPN transcript was also elevated acutely after UEC, a finding which was pronounced in enriched dentate molecular layer (ML) fractions. Parallel immunohistochemistry (IHC) and in situ hybridization localized OPN protein and transcript to reactive glia following UEC. This localization was concentrated within microglia which delineated the border between the intact and deafferented ML, a pattern which was less pronounced in maladaptive TBI+BEC animals. The timing of this glial movement suggests that OPN regulates microglial migration and, potentially, could act as an astrokine to recruit activated astrocytes for influencing subsequent synaptic regeneration. MAP1B staining confirmed dendritic loss during axonal degeneration and dendritic atrophy, with a reemergence during collateral axonal sprouting. However, OPN colocalization with MAP1B was minimal, suggesting a minor role for OPN in reorganization of dendritic/axonal cytoarchitecture in this model of deafferentation. Minocycline reduced acute OPN protein response 2d after UEC, and caused a more random OPN positive glial distribution, similar to that of the maladaptive TBI+BEC. The role of OPN in the inflammation-directed degeneration of terminals is supported by reduced MMP-9 activity, which is temporally correlated with the reduction of MMP-generated OPN lytic fragments (45 kD). Interestingly, this reduction of integrin-binding OPN peptide also matched the impaired removal of presynaptic terminals, evidenced by diminished synapsin 1 clearance in animals which received postinjury minocycline. In Chapter 3, we sought to more precisely evaluate the role of OPN following deafferentation, utilizing wild type (WT) C57BL/6 and OPN KO mice subjected to UEC, comparing the spatio-temporal injury response between WT and KO. To do this we profiled several outcome measures which assessed OPN role in different aspects of recovery: 1) expression of select proteins important in various stages of synaptic recovery, 2) glial response, 3) cognitive recovery, and 4) MMP enzymatic activity. Compared to WT mice, OPN KO mice did not show significant differences in the acute injury-induced alteration of proteins important to cytoarchitectural reorganization (MAP1B) or stabilization of the synaptic junction (N-cadherin). However, both Western blot and IHC analyses showed OPN KO mice had impaired presynaptic terminal clearance, supported by attenuated synapsin 1 breakdown, a result quite similar to that of the minocycline-treated rats with OPN reduction in Chapter 2. This impaired degeneration in OPN KO mice at 2d postinjury correlated with IHC evidence for altered microglial morphology, and hippocampal function assessed by the novel object recognition (NOR) task. Our NOR results confirmed cognitive dysfunction in OPN KO mice during the 4-21d period of synapse reorganization after UEC. In addition, OPN KO decreased MMP-9 activity, an effect associated with reduced MMP-9 bound lipocalin 2 (LCN2), a persistently activated form of that MMP. These latter findings further support the hypothesis that MMP processing of OPN contributes to effective regenerative response after injury. Collectively, the studies presented in the two chapters of this dissertation provide evidence that OPN is a critical element in the acute immune response following injury-induced CNS deafferentation. They suggest that the cytokine can be produced by reactive microglia, may mediate cell migration and acute degenerative clearance, potentially serves as an astrokine to recruit those glia to sites of synaptic repair, and that these processes are disrupted when OPN is either reduced or ablated. Interestingly, this OPN role in synaptogenesis appears to involve ECM interaction with MMP-9, possibly regulated by LCN2. Most importantly, OPN involvement seems to affect the time-dependent progression of synaptic repair, an effect which can be measured by efficacy of functional outcome

In addition to the acute pathology produced by traumatic brain injury, there are chronic alterations that occur after the trauma, including a depressed state of neuronal activity (Feeney, 1991). This study included a preclinical testing of a novel treatment strategy focusing on increasing neuronal activity during the chronic hypofunctional posttraumatic stage. The present investigation tested the effects of repeated post-injury aniracetam administration on cognitive performance in the Morris water maze (MWM) and on the GluR2 - immunoreactivity and protein expression by Western blot analysis in the hippocampus. The first study examined the optimal dose of aniracetam in the MWM task. Animals received aniracetam (25 mg/kg, 50 mg/kg) or vehicle once daily for fifteen days and on days 11-15 were tested in the MWM. The results indicated that injured aniracetam-treated rats had a significant improvement in MWM performance compared to injured saline-treated animals. When the drug was delayed for 11 days post-injury in the second experiment, its beneficial effects were still present, as injured aniracetam-treated rats performed significantly better that injured saline treated rats on the MWM task. In the third experiment, chronic daily aniracetam administration was terminated after 15 days immediately before MWM testing on days 16-20. The results indicated that termination of aniracetam did not enhance MWM performance as injured terminated aniracetam-treated rats did not have significant improvement over injured saline-treated rats. In the fourth study we investigated the mechanism of aniracetam's effects by examining the expression of the AMPA receptor GluR2 subunit, the only AMPA receptor subunit that is Ca++ impermeable. Using a monoclonal antibody selective for the GluR2 subunit, immunohistochemical results indicated that injured rats treated with aniracetam (50mg/kg for 15 days post-injury) had a slight reduction in the GluR2- IR. The fifth study investigated a change in the GluR2 protein expression in the hippocampus with a Western blot analysis. The results were consistent with the immunohistochemical study outcome as the injured vehicle and injured aniracetam treated animals showed a reduced protein expression in the hippocampus. The changes were not significantly different from the controls. The results of these experiments suggested that chronic aniracetam treatment significantly attenuated injury induced spatial memory deficits when administered continually during the hypofunctional posttraumatic stage and when the treatment was delayed for 11 days, but not when the treatment was terminated before the MWM testing. These effects suggest that the compound does not induce chronic receptor changes and has to be biologically active in an organism for it to exert its beneficial properties. Results from the present studies suggest that aniracetam may become a potential treatment option for brain injury induced cognitive deficits.

Catecholaminergic neurotransmission is regionally altered following injury, and drugs aimed at these systems offer promising avenues for post-TBI pharmacotherapies. Atomoxetine is a selective norepinephrine transporter (NET) inhibitor currently indicated for treatment of attention-deficit hyperactivity disorder (ADHD). The studies in this dissertation were designed to test the efficacy of atomoxetine for treating cognitive deficits following experimental TBI and the potential mechanism for any beneficial effect. The first part of the study focused on behavioral recovery following atomoxetine treatment. Several important questions of dose, therapeutic window, and duration of treatment were addressed in these studies. Sprague-Dawley rats were subjected to lateral fluid-percussion injury (L-FPI) of moderate severity (2.08 atm +/- .05). Four experiments were performed. In the first study, atomoxetine (.3 mg/kg, 1mg/kg, 3 mg/kg, or 9 mg/kg) or vehicle was administered daily on post injury days (PID) 1-15. Cognitive assessment was performed using the Morris water maze on PID 11-15. L-FPI resulted in significant cognitive impairment when compared to Sham-Injury. Treatment with lower doses of atomoxetine (.3mg/kg, 1mg/kg, and 3mg/kg) significantly attenuated the cognitive deficits in injured animals. Treatment with the higher dosage (9mg/kg) of atomoxetine resulted in animals that were not significantly different than injured-vehicle treated animals. The optimal response was achieved using 1 mg/kg atomoxetine. In the second study, treatment with atomoxetine (1mg/kg) or vehicle was delayed for 11 days post-injury. Rats were administered atomoxetine daily for 15 days and cognitive assessment was performed on PID 25-29. In this study, treatment with atomoxetine (1 mg/kg) did not result in improved cognitive performance. In the next study atomoxetine was given daily on PID 1-7 and then treatment was terminated. The animals were tested in the MWM on PID 11-15. We found that atomoxetine treatment for 7 days post-injury provides an enhancement of cognitive deficits that is not significantly different from sham animals. We then investigated whether a single treatment of atomoxetine 24 h after brain injury could influence behavioral outcome on days 11-15. From this study, we found a single dose of atomoxetine is not as effective as chronic treatment. Finally, we investigated changes in the protein expression of brain-derived neurotrophic factor, growth-associated protein-43, and synaptophysin on day 7 PID to investigate what effect atomoxetine may have on brain plasticity and regeneration. We found that atomoxetine can enhance both GAP-43 and BDNF, but not synaptophysin at this time point. In conclusion, this is the first study to show that low doses of atomoxetine initiated early after experimental traumatic brain injury results in improved cognition. Furthermore, we show that enhancement of catecholamines via atomoxetine treatment during periods of postinjury-induced plasticity can provide long-term functional and structural benefits.

Traumatic axonal injury (TAI) is a consistent feature of (TBI) and is responsible for much of its associated morbidity. TAI is now recognized to result from progressive/secondary axonal injury, though much remains unknown in regards to the pathobiology and the long-term consequences of axonal injury. TAI has been described in the perisomatic domain, located within the neocortex following mild TBI, and within this domain has been linked to neuronal recovery, not neuronal cell death in the acute setting. Due to technical limitations, our understanding of the long-term fate of this neuronal population and the mechanisms responsible for permitting neuronal survival, recovery and axon regeneration following injury are unknown. The studies presented in this thesis are centered upon the hypothesis that injury within the perisomatic domain is unique, and may allow for enhanced neuronal recovery and axonal regeneration. To address many of these questions, we have utilized a novel model of diffuse brain injury in mice, allowing for the use of transgenic mice to overcome previous limitations in the study of TAI. To address this hypothesis, we first assessed the impact of genetic deletion of cyclophilin D (CypD), a regulator of the mitochondrial permeability transition pore (mPTP), upon TAI within the perisomatic domain. Via this approach it was determined that CypD deletion reduced the number of injured axons by ~50%, indicating that CypD and mPTP formation contribute to TAI in the perisomatic domain. Next, using a fluorescent-based approach, we assessed the temporospatial events associated with TAI, acutely. Here it was determined that the axon initial segment (AIS) is uniquely susceptible to TAI following mild TBI (mTBI) and injury within this domain progresses rapidly to axon disconnection. Last we assessed the long-term fate of axotomized neurons and their associated axonal processes. We report that over a chronic time frame, TAI induces no overt cell death, instead results in significant neuronal atrophy with the simultaneous activation of a somatic program of axon regeneration and recovery of the remaining axonal processes. Taken together, the findings of this work reveal that TAI results in a unique axonal injury that results in a persistent axon regenerative attempt.

Excitotoxic calcium influx and activation of calcium-regulated systems is a common event in several types of neuronal injury. This mechanism has been the focus of intense research, with the hope that a more complete understanding of how neuronal injury affects calcium-regulated systems will provide effective treatment options. This study examines one such calcium-stimulated enzyme, calcineurin, in the context of two common neurological pathologies, status epilepticus and traumatic brain injury.Status epilepticus was induced by pilocarpine injection. NMDA-dependent increases in calcineurin activity were observed in cortical and hippocampal homogenates. Upon closer examination, the most profound increases in activity were found to be present in crude synaptoplasmic membrane fractions isolated from cortex and hippocampus. A concurrent status epilepticus-induced increase in calcineurin concentration was observed in membrane fractions from cortex and hippocampus. Immunohistochemical analysis revealed an increase in calcineurin immunoreactivity in apical dendrites of hippocampal pyramidal neurons. We examined a cellular effect of increased dendritic calcineurin activity by characterizing a calcineurin-dependent loss of dendritic spines. Increased dendritic calcineurin led to increased dephosphorylation and activation of cofilin, an actin-depolymerizing factor. Calcineurin-activated cofilin induced an increase in actin depolymerization, a mechanism shown to cause spine loss in other models. Finally, via Golgi impregnation, we demonstrated that status epilepticus-induced spine loss is blocked by calcineurin inhibitors.To demonstrate that the increase in dendritic calcineurin activity was not model-specific, we examined a moderate fluid-percussion model of brain injury. Calcineurin activity was significantly increased in hippocampal and cortical homogenates. This increased activity persisted for several weeks post-injury, and may be involved in injury-induced neuronal pathologies. Also similar to the SE model, calcineurin immunoreactivity was dramatically increased in synaptoplasmic membrane fractions from cortex and hippocampus, and immunohistochemistry revealed increased calcineurin content in dendrites of hippocampal CA1-3 pyramidal neurons. These changes in calcineurin distribution also persisted for several weeks post-injury.These studies demonstrate a novel, cellular mechanism of calcium-mediated pathology in two models of neuronal injury. Elucidation of cellular events involved in the acute and chronic effects of brain trauma is essential for the development of more effective treatment options.

Traumatic brain injury (TBI) is a serious health concern, causing cognitive, motor, and sensory deficits, including olfactory dysfunction. This dissertation explores the effects of TBI on synaptic plasticity within the olfactory system, seeking to define mechanisms guiding postinjury sensory reinnervation. Physical forces induced by TBI can axotomize olfactory receptor neurons (ORNs), which innervate olfactory bulb (OB). These axons regenerate OB projections after injury, a process involving growth through a complex extracellular matrix (ECM). As such, we investigated a potential molecular mechanism capable of modifying local OB ECM to support postinjury synaptogenesis. Since matrix metalloproteinases (MMPs) and their ECM substrates are recognized for TBI therapeutic potential, we explored the role of MMP9 and its substrate osteopontin (OPN) in promoting ORN reinnervation of the OB after mild fluid percussion injury (FPI). First, we confirmed that FPI deafferented the mouse OB. In Chapter 2, we showed concurrent activation of neuroglia, elevated spectrin proteolysis and reduction in ORN-specific olfactory marker protein (OMP). As OMP normalized during regeneration, growth associated protein-43kD (GAP-43) peaked, marking OB entry of ORN growth cones. Ultrastructural analysis revealed ongoing ORN axon shrinkage and degeneration, glial phagocytosis of cellular debris, and a reorganization of synaptic structure. To explore ECM role in mediating postinjury OB reinnervation, we defined the time course of MMP9 activity and several downstream targets. Chapter 3 reports biphasic MMP9 activity increase during acute/subacute degeneration, accompanied by robust generation of 48kD OPN cell signaling peptide. OPN receptor CD44 also increased during the acute/subacute interval, suggesting potential interaction of the two proteins. Finally, we utilized MMP9 knockout (MMP9KO) mice to confirm MMP9 role in OB synaptogenesis. In Chapter 4, MMP9KO reversed FPI-induced lysis of 49kD OPN and altered postinjury expression of ORN axon degeneration marker OMP. Additional ultrastructural analysis verified delayed recovery of OB synaptic features within the injured MMP9KO. Overall, we demonstrated that mild FPI elicits ORN axotomy to induce OB reactive synaptogenesis, and that MMP9 supports reinnervation by processing OPN for activation of local glia, cells which reorganize the ECM for synapse regeneration.

It is estimated that there are 12.5 million people in England living with neurological disorders (Neurological Alliance, 2014). People with neurological disorders as a result of acquired brain injury (ABI) are living with short and long-term disabilities. These include cognitive impairment, and physical and emotional distress. One of the most common complaints by individuals who have ABI is attention impairment. Attention difficulties can have serious ramifications for daily functioning. Although studies have explored the effects of evidence-based interventions such as mindfulness-based therapy on attention abilities, and have found that it improves individuals' attention skills (Moore et al, 2012), thus far research has been conducted mainly with non-clinical populations. This study set out to investigate whether a mindfulness-based intervention could prove beneficial for people with neurological disorders, particularly whether it could positively impact on attention impairment. The study employed a one group pre-test post-test design. The intervention was adapted from the MBSR programme developed by Kabat-Zinn. Twenty-two participants with ABI were recruited. The Conners Continuous Performance Test 3rd Edition (CPT-3), Mindful Attention Awareness Scale (MAAS), Attention Process Training-II Attention Questionnaire (APT-II AQ) and Clinical Outcome in Routine Evaluation-Outcome Measure (CORE-OM) were utilised to measure outcomes. The results revealed that there was a clinical improvement in self reported measures of mindfulness (MAAS) (Cohen d=0.28), attention (APT-II AQ) (Cohen d=0.33), and psychological distress (CORE-OM) (Cohen d=0.72). This was not observed using the neuropsychological test of attention (CPT-3) for overall group scores, but further evaluation showed some individuals' scores improved. The study is promising as it indicates that mindfulness based treatment can be effective with attentional problems as well as in reducing psychological distress for individuals with ABI. This could be valuable in terms of providing treatment for this client group and adds to the expanding research base on mindfulness-based intervention with this population.