Surgery-induced brain injury (SBI) refers to trauma caused by routine neurosurgical procedures that may result in post-operative complications and neurological deficits. Unlike accidental trauma, SBI is potentially subject to preemptive interventions at the time of surgery. SBI can cause bleeding, inflammation and the formation of tissue gaps. Conventional haemostatic techniques, though effective, are not necessarily conducive to healing. Inflammation and the absence of extracellular matrix in tissue gaps also hinder regeneration after SBI. This study investigated the applications of RADA16-I, a type I self-assembling peptide nanofibre scaffold (SAPNS), and mesenchymal stem cells (MSCs) in the treatment of SBI. Using animal SBI models, treatments were applied immediately and locally onto the operative fields, taking advantages of the haemostatic and cell-carrying properties of RADA16-I, the immune- modulatory effects of MSCs, and the earliest available therapeutic window for SBI. There were three objectives. Objective 1 was to compare RADA16-I with conventional haemostatic methods, including electrocautery and fibrin sealant, in their effects on the brain’s acute cellular inflammatory response. The hypothesis was that RADA16-I would cause the same or a lesser degree of inflammation. This study showed that RADA16-I was superior to electrocautery, and was noninferior to conventional topical haemostats. Objective 2 was to study the in vitro expansion of MSCs within RADA16-I in preparation for in vivo transplantation. The hypothesis was that the in vitro survival of MSCs would vary between different RADA16-I concentrations and culturing methods. This study showed that plating MSCs onto pre-buffered RADA16-I would protect the cells against RADA16-I’s intrinsic acidity and result in better initial survival. Subsequent integration with the RADA16-I hydrogel, however, was poor. Mixing the cells directly with RADA16-I caused initial cell loss but allowed better integration. RADA16-I at lower concentrations resulted in better survival but also more fragile hydrogels that were mechanically unfit for transplantation. Mixing MSCs with 0.5% RADA16-I for seven days represented a compromise between these competing factors. Objective 3 was to study the in vivo effects of a MSC-RADA16-I implant on tissue reactions after SBI. The hypothesis was that the combinatorial therapy would result in less cellular inflammatory response than MSC alone or RADA16-I alone. Implants of pre-buffered 0.5% RADA16-I hydrogel, with or without cells, were found to cause less inflammation than control. MSCs in free suspension resulted in significantly more pronounced inflammation than when carried in RADA16-I. Supplementing RADA16-I with MSCs, however, did not confer additional benefit over RADA16-I alone. The present study provided new preclinical evidence to support future clinical testing of RADA16-I as a novel surgical haemostat. It also demonstrated the feasibility of early intracerebral transplantation of RADA16-I hydrogel in the treatment of SBI. Whether RADA16-I and/or transplanted MSCs could modulate the brain’s inflammatory response after SBI require further investigations, which may include the search for the optimal ex vivo expansion technique and specifically tailored nanofibre scaffold. The translational applications of these findings would include the treatment of SBI over critical brain regions where trauma would cause severe functional deficits and where better healing would facilitate patient recovery. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

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published_or_final_version / Anatomy / Master / Master of Philosophy

Blast-induced traumatic brain injury (bTBI) has been a health concern in both military and civilian populations due to recent military and geopolitical conflicts. Military service members are frequently exposed to single and repeated blasts throughout their training and deployment. As a result of blast exposures, military personnel report symptoms of various neurological and neurosensory deficits. Our group has previously reported decreased long term potentiation (LTP) following either single or repeated bTBI in a rat organotypic hippocampal slice culture (OHSC) model. LTP is a neuronal correlate for learning and memory and is a neurological metric that can be used to evaluate blast injury severity and the efficacy of therapeutic interventions. In the first aim of this thesis, we characterized LTP deficits following repeated bTBI to develop tolerance criteria for blast exposures. We did so by varying the blast injury severity, the inter-blast interval between blasts, and the recovery period following blast exposure. We determined that LTP deficits were compounded as a result of repeated mild bTBI. LTP deficits were attenuated with increasing inter-blast intervals and with increasing recovery periods after injury. Even after three repeated mild bTBIs, LTP spontaneously recovered after 6 days. In the second aim, we investigated the pathological changes in OHSCs following repeated blast exposures. Following injury, we observed robust microglial activation, evidenced by increased expression of the pro-inflammatory marker, CD-68, and decreased expression of the anti-inflammatory marker, CD-206. We also observed increased expression of MIP-1α, IL-1β, MCP-1, IP-10, and RANTES and decreased expression of IL-10 in the acute period after both single and repeated bTBI. Following partial depletion of microglia prior to injury, injury induced LTP deficits were significantly reduced. Lastly, treatment with a novel drug, MW-189, immediately after a repeated bTBI prevented LTP deficits. In the third aim, we investigated changes in inflammatory markers like cyclooxygenase (COX) and tested the efficacy of COX or prostaglandin receptor (EP3R) inhibitors in attenuating LTP deficits. We observed that expression of COX-2 increased 48 hours following repeated blast injury; however, COX-1 expression was unchanged. Following repeated bTBI, EP3R expression was upregulated and cyclic adenosine monophosphate (cAMP) concentration was decreased. Treatment of blast injured OHSCs with a COX-1 specific inhibitor, SC-560, a COX-2 specific inhibitor, rofecoxib, a pan-COX inhibitor, ibuprofen, or an EP3R inhibitor, L-798,106 improved LTP deficits. Delayed treatment with L-798,106 and ibuprofen also improved LTP deficits. Our data suggests that bTBI induced neuroinflammation may be partially responsible for the functional deficits that we have observed in blast-injured OHSCs. Additionally, we also conclude that COX and EP3R inhibition may be viable therapeutic strategies to reduce bTBI induced neurophysiological deficits. In the final aim, we investigated bTBI induced changes to the electrophysiological network of OHSCs. Following blast exposure, sham and injured OHSCs were administered increasing concentrations of bicuculline, a GABAA receptor antagonist. Doing so revealed an increase in connectivity and clustering coefficients in sham slices compared to injured slices. This suggested that the underlying neuronal network of injured slices was dysfunctional. Biologically, this dysfunction could be explained by the decreased expression of GABAA receptor α1 and α5 subunits. A loss of GABAA receptor expression or function may explain the electrophysiological network disruptions that we observed. More work will be required to determine how blast exposure decreases the expression of GABAA receptors and how these receptors may contribute to network deficits. This thesis has expanded upon the tolerance criteria for repeated blast exposures. These studies have also further characterized the pathological changes in microglial activation and explored promising therapeutic pathways that could be used to attenuate functional deficits. Lastly, this thesis has also provided novel ways to interrogate neuronal networks following blast injury, revealing subtle deficits that will need to be explored in more detail.

Traumatic brain injury (TBI) is the signature injury of the ongoing military conflicts in the Middle East and Afghanistan, largely due to the use of improvised explosive devices (IEDs), which have affected soldiers and civilians alike. Blast-induced TBI (bTBI) biomechanics are complex and multiphasic. While research has clearly demonstrated the negative effects of penetrative (secondary blast) and inertia-driven (tertiary blast) injury, the effect of shock wave loading (primary blast) on the brain remains unclear. Combined primary-tertiary blast exposure in vivo has been reported previously to alter brain function, specifically hippocampal function; however, it is extremely difficult to deliver primary blast exposure in isolation with an in vivo injury model. The research presented in this thesis utilized a custom-designed in vitro blast injury model to deliver military-relevant shock wave exposures, in isolation, to organotypic hippocampal slice cultures (OHSCs). To contextualize blast-induced pathobiology with previous TBI studies, the first goal of this thesis was to experimentally characterize the deformation profile induced in OHSCs with our blast injury model. Using stereoscopic, high-speed cameras and digital image correlation to calculate strain, we found that our blast model induced low strain magnitudes (<9%) but at high strain rates (25-86s-1), which aligned closely with associated computational simulations of our model. The second aim was to determine if primary blast was capable of altering hippocampal electrophysiological function. We exposed OHSCs to a range of shock intensities and found, using a micro-electrode array system, that long-term potentiation (LTP), a measure of synaptic plasticity, was very sensitive to primary blast exposure; a threshold for disruption of LTP was found between 9 and 39 kPa•ms impulse. Alternative measures of basal electrophysiology were less sensitive than LTP. Blast exposure significantly reduced LTP between 1 and 24 hours post-injury, and this deficit persisted through 6 days post-injury. Depending on shock intensity, LTP spontaneously recovered 10 days post-injury. The third aim was to explore the cellular mechanisms for blast-induced LTP deficits. Using a chemical LTP induction protocol, blast exposure altered key proteins necessary for the induction of LTP by 24 hours post-injury including, postsynaptic density protein-95 (PSD-95), a major scaffolding protein that organizes the postsynaptic density (PSD), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor 1 (AMPA-GluR1), and stargazin, an auxiliary GluR1 protein that binds AMPA-GluR1 to PSD-95. Modulation of the cyclic adenosine monophosphate (cAMP) pathway reversed the observed effects of blast on LTP. We theorized that blast-induced disruption of PSD-95 prevented translocation, and subsequent phosphorylation, of GluR1-containing AMPARs to the postsynaptic membrane, which, in turn, prevented potentiation. The final aim was to investigate the efficacy of phosphodiesterase-4 (PDE4) inhibitors, which block degradation of cAMP, as a therapeutic strategy. When delivered immediately following primary blast injury, multiple PDE4 inhibitors proved efficacious in restoring LTP measured 24 hours post-injury. Roflumilast, a Food and Drug Administration-approved PDE4 inhibitor, was effective when delivered at a clinically relevant concentration (1nM) and at a delayed time point (up to 6 hours). Roflumilast reversed blast-induced changes in expression/phosphorylation of the key LTP protein targets. We hypothesized that maintenance of PSD-95 drove the observed therapeutic effect. Greater work is necessary to determine how blast exposure degrades PSD-95 and how roflumilast prevented these detrimental effects. This thesis has shown that primary blast exposure can negatively alter neurological function, as well as protein expression and phosphorylation. These studies expand the understanding of primary blast injury mechanisms, provide computational models with important tissue-level tolerance criteria, inform protective equipment design, inform clinical care guidelines for bTBI, and present a promising therapeutic candidate for further clinical investigation.

This thesis focuses on the effects of pharmacological and environmental interventions following perinatal prefrontal cortex lesions. Rats given postnatal day 3 medial prefrontal cortex lesions were provided with one of the following treatments: basic fibroblast growth factor (bFGF), complex-housing, tactile stimulation, or a combined treatment of both bFGF and tactile stimulation or bFGF and complex-housing. Rats given postnatal day 3 orbital prefrontal cortex lesions were housed in a complex environment. The findings of these studies suggest that bFGF, complex-housing or tactile stimulation are beneficial after early brain injury. The combined treatment of bFGF with complex-housing provides a synergistic effect, as the combined condition is more advantageous than bFGF alone. In contrast, the combined treatment of bFGF with tactile stimulation produced adverse effects. These results suggest that pharmacological and environmental manipulations change cortical plasticity and therefore functional recovery after neonatal cortical injury. / xv, 177 leaves : ill. (some col.) ; 29 cm.

Traumatic brain injury (TBI) is the signature injury of modern military conflicts due to widespread use of improvised explosive devices (IEDs) and modern body armor. However, the exact biophysical mechanisms of blast-induced traumatic brain injury (bTBI) and its pathological effects on the blood-brain barrier (BBB) – a structure essential for maintaining brain homeostasis – remain poorly understood. The specific aims of this thesis are to: 1) determine a threshold for primary blast-induced BBB dysfunction in vitro; 2) determine the effect of repeated blast on BBB integrity in vitro; 3) improve BBB recovery in vitro as a potential therapeutic strategy for mitigating effects of blast; and 4) quantify the time course and pore-size of BBB opening in vivo. In this work we utilized a shock tube driven by compressed gas to generate operationally relevant, ideal pressure profiles consistent with IEDs. By multiple measures, the barrier function of an in vitro BBB model was disrupted after exposure to a range of blast-loading conditions. Trans-endothelial electrical resistance (TEER) decreased acutely in a dose-dependent manner that was most strongly correlated with impulse, as opposed to peak overpressure or duration. Significantly increased hydraulic conductivity and solute permeability post-injury further confirmed acute alterations in barrier function. Compromised ZO-1 immunostaining identified a structural basis for BBB breakdown. These results are the first to demonstrate acute disruption of an in vitro BBB model after primary blast exposure; defined tolerance criteria may be important for development of novel helmet designs to help mitigate effects of blast on the BBB. After determining that exposure to a single primary blast caused BBB disruption, we hypothesized that exposure to two consecutive blast injuries would result in exacerbated damage to the BBB in vitro. However, contrary to our hypothesis, repeated mild or moderate primary blast delivered within 24 or 72 hours did not significantly exacerbate reductions in TEER across a brain endothelial monolayer compared to sister cultures receiving a single exposure. Single blast exposure significantly reduced immunostaining of ZO-1 and claudin-5 tight junction proteins, but subsequent exposure did not cause additional damage to tight junctions. The second injury delayed recovery of TEER and hydraulic conductivity in BBB cultures. Extending the inter-injury interval to 72 hours, the effects of repeated injury on the BBB were independent given sufficient recovery time between consecutive exposures. Investigation of repeated blast on the BBB will help identify a temporal window of vulnerability to repeated exposure. Restoration of the BBB after blast injury has emerged as a promising therapeutic target. We hypothesized that treatment with dexamethasone (DEX) after primary blast would potentiate recovery of an in vitro BBB model. DEX treatment resulted in complete recovery of TEER and hydraulic conductivity 1 day after injury, compared with 3 days for vehicle-treated injured cultures. Administration of RU486 (mifepristone) inhibited effects of DEX, confirming that barrier restoration was mediated by glucocorticoid receptor signaling. Potentiated recovery with DEX treatment was accompanied by stronger ZO-1 tight junction immunostaining and expression, suggesting that increased ZO-1 expression was a structural correlate to BBB recovery. This is the first study to provide a mechanistic basis for potentiated functional recovery of an in vitro BBB model due to glucocorticoid treatment after blast injury. Using an in vivo bTBI model, systemic administration of sodium fluorescein (NaFl; 376 Da), Evans blue (EB; 69 kDa when bound to serum albumin) and dextrans (3 – 500 kDa) was used to estimate the pore-size of BBB opening and time required for recovery. Exposure to blast resulted in significant acute extravasation of NaFl, 3 kDa dextran, and EB. However, there was no significant acute extravasation of 70 kDa or 500 kDa dextrans, and minimal to no extravasation of NaFl, dextrans, or EB 1 day after exposure. This work is the first to quantify the time course and size of BBB opening after bTBI, suggesting that the BBB recovered 1 day post-injury. This study supports our hypothesis that transient opening of the BBB may permit serum-components to infiltrate the brain parenchyma and contribute to pathological secondary cascades. This research has shown that BBB damage, demonstrated in vitro and in vivo, is a major mechanism contributing to vascular and neuronal pathology of bTBI at exposure levels above a critical threshold. Compared with published studies on blast-induced damage to the BBB, we have developed primary blast injury tolerance criteria by precisely controlling the biomechanical initiators of injury and measuring resulting alterations to the structure and function of an in vitro BBB model by methods not possible in vivo. We have also developed a potential glucocorticoid treatment to rapidly restore the BBB after injury, which may lead to more promising therapeutic strategies to treat TBI-related pathologies. This work will also guide the development of novel armor designs to protect service members and civilians in order to more effectively address the burden to society of bTBI.

With over 50,000 deaths and 80,000 disorders annually in the United States resulting from traumatic brain injury (TBI), there is a demand for improved therapeutic strategies. Cell transplantation offers the potential to treat TBI by targeting multiple mechanisms in a sustained fashion. However, efforts are needed to improve survival and integration of transplanted cells, and ultimately enhance functional recovery. Using tissue engineering strategies, we aimed to mimic key aspects of fetal tissue grafts by combining neural stem cells with a fibronectin or laminin based scaffold that could be delivered to the injured brain in a minimally invasive fashion. We found that the incorporation of extracellular matrix proteins into a cell transplantation paradigm led to improved donor cell survival and restored cognitive ability for treated animals. To begin to examine how fibronectin and laminin mediate these improvements, we first examined the endogenous role of these two proteins in the injured brain. Using a clinically-relevant model of TBI, we found both proteins are increased in the injured brain at acute time points. The spatial localization of fibronectin and laminin with specific support cells in the brain suggests a role for these proteins in repair, warranting further investigation. Using conditional plasma fibronectin knockout animals, we found that fibronectin is neuroprotective to the traumatically injured brain. Specifically, injured fibronectin knockout animals had more severe motor and cognitive deficits, increased cell death, and decreased retention of phagocytic cells compared to injured wild type animals. Thus, we have identified novel therapeutic treatments for TBI which utilize tissue engineered transplants and/or exploit endogenous repair mechanisms for fibronectin.