No description available.

No description available.

Traumatic brain injury, hereon referred to as TBI, can be simply defined as a disruption to normal brain function as a result of an outside force to the head. TBI contributes to one third of all injury related deaths in the United States, and treatment strategies for TBI are supportive. Although primary and secondary mechanisms of injury have been clearly identified, the heterogeneous and intertwined pathophysiology of TBI is not fully understood. Primary injury results from the impact itself and causes immediate damage. However, secondary mechanisms of injury in TBI, such as oxidative stress and inflammation, are points at which intervention may reduce neuropathology. Trials taking advantage of the antioxidant and anti-inflammatory properties of several agents have had little clinical success, while the use of targeted therapeutics in TBI is relatively unexplored. Evidence suggests that reactive oxygen species (ROS) propagate blood-brain barrier (BBB) hyperpermeability and exacerbate inflammation following TBI. In the studies presented herein, we tested the hypothesis that targeted detoxification of ROS may improve the pathological outcomes using the controlled cortical impact mouse model of TBI. Following TBI, endothelial activation results in a time dependent increase in vascular expression of ICAM-1, an endothelial activation and cell adhesion molecule, as was observed by immunohistochemistry and immunofluorescence staining of isolated cortical microvessels. We conjugated catalase, an antioxidant enzyme, to anti-ICAM-1 antibodies and administered the conjugate intravenously to 8-week-old C57BL/6J mice at 30 minutes after moderate controlled cortical impact TBI. Results indicate that catalase targeted to ICAM-1 reduces markers of oxidative stress including levels of hydrogen peroxide and 3-nitrotyrosine detected in the cortex ipsilateral to the area of injury. Anti-ICAM-1/catalase also preserved BBB permeability based on two assays of barrier permeability to the plasma protein fibrinogen and small fluorescent tracer sodium fluorescein. Following TBI, mice receiving the conjugate exhibited attenuated neuropathological indices for astrocyte and microglia activation as well as cortical neuronal loss compared to controls. For each of these endpoints, anti-ICAM-1/catalase was found to be more effective than anti-ICAM-1 antibodies or catalase administered alone. An extensive study of microglia by two-photon microscopy of ex vivo brain segments from CX3CR1-GFP mice revealed that anti-ICAM-1/catalase prevented the transition of microglia to an activated phenotype after TBI. Finally, anti-ICAM-1/catalase offered functional improvement in Rotarod and elevated zero maze performance compared to controls at acute and chronic time points, respectively. Collectively, these findings demonstrate the use of a targeted antioxidant enzyme to interfere with oxidative stress mechanisms acutely in TBI. The results demonstrate histological and functional benefit of anti-ICAM-1/catalase administration and provide a proof-of-concept approach to improve acute TBI management that may also be applicable to other neuroinflammatory conditions. / Biomedical Sciences

Across almost all types of neurological pathophysiology, inflammation and corresponding breakdown of the Blood Brain Barrier (BBB) are hallmarks of injury/disease progression. In fact, BBB disruption can occur early during neuropathophysiological development, in many cases even before neurological and cognitive impairments become apparent. Whether as an early causative factor, a side effect, or both as it pertains to neurological injury/disease, BBB breakdown and dysfunction represents a novel and under investigated target for therapeutic development, especially for neurological pathologies with unmet therapeutic needs. Toward this goal, the endocannabinoid system (ECS) has emerged as a promising biological target for drug discovery efforts. Particularly, the Cannabinoid 2 Receptor (CB2R) has been proposed as a druggable target due to its anti-inflammatory effects and since it is not associated with the neurological side effect profile representative of Cannabinoid 1 Receptor (CB1R) drugs. Interestingly, neuroinflammatory conditions promote upregulation of CB2R on brain microvascular endothelial cells (BMVECs) suggesting a possible role toward resolution of inflammation in this cell type. Moreover, previous research has shown promising effects of CB2R agonists on cerebrovascular function, although these effects cannot be directly attributed to endothelial CB2R. The central hypothesis of this research is that endothelial CB2R activation confers effects which are vascular protective and that promote BBB repair, (irrespective of the effects of CB2R in other central nervous system (CNS) cell types). To address this hypothesis, endothelial CB2R expression dynamics were assessed following experimental Traumatic Brain Injury (TBI) followed by a series of assays to assess the therapeutic potential of a novel chromenopyrazole based CB2R agonist, PM289. Results of these experiments demonstrated upregulation of CNR2, the gene which encodes CB2R, following in vivo experimental TBI and in vitro cytokine induced inflammation. Moreover, PM289 exhibited robust CB2R-dependent therapeutic potential by partially restoring TNFa-induced physical barrier disruption, attenuating TNFa-induced ICAM1 upregulation, and promoting rapid monolayer repair following electrolytic wound. Mechanistically, these effects may be explained via CB2R-dependent inhibition of NFkB/P65 signaling. Overall, these results are supportive of the notion that CB2R in BMVECs could aid in vascular protection and promote BBB function in the context of neuroinflammation. Future studies are warranted to understand the in vivo therapeutic efficacy of PM289 in a variety of injury/disease models. Additionally, alternative cell signaling mechanisms should be considered including a comprehensive examination of potential interplay between ECS components and candidates that fall under the umbrella of the endocannabionoidome (ECBome). / Biomedical Sciences

The most common and aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of large molecular agents to permeate through the blood-brain barrier (BBB); migration of highly invasive GBM cells beyond the solid tumor margin; and gross, macroscopic intratumor heterogeneity. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. Notably, irreversible electroporation (IRE) has been used for BBB disruption and nonthermal ablation of intracranial tumor tissues. Despite promising results, IRE can cause unintended muscle contractions and is susceptible to electrical heterogeneities. Second generation High-frequency IRE (H-FIRE) utilizes bursts of bipolar pulsed electric fields on the order of the cell charging time constant (~1 μs) to ablate tissue while reducing nerve excitation, muscle contraction, and is far less prone to differences in electrical heterogeneities. Throughout my dissertation, I discuss investigations of H-FIRE for the treatment of malignant gliomas and other intracranial disorders. To advance the versatility, usability, and understanding of H-FIRE for intracranial applications, my PhD thesis focuses on: (1) characterizing H-FIRE-mediated BBB disruption effects in an in vivo healthy rodent model; (2) the creation of a novel, real-time impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as an in situ technique to monitor ablation growth and to determine patient-specific ablation endpoints; (4) conducting a preliminary efficacy study of H-FIRE ablation in an orthotopic F98 rodent glioma model; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a novel, intraoperative impedance spectroscopy technique towards determining patient-specific ablation endpoints for intracranial H-FIRE procedures. / Doctor of Philosophy / The most aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of chemotherapy agents to diffuse into brain tumor tissue as these molecular are unable to pass the so-called blood-brain barrier (BBB). This tumor tissue also presents with cells with the propensity to invade healthy tissue, to the point where diagnostic scans are unable to capture this migration. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. For instance, irreversible electroporation (IRE) has been used to successfully treat tumors in the prostate, liver, kidney, and pancreas. Second generation High-frequency IRE (H-FIRE) may possess even greater antitumor qualities and this is the focus of my dissertation. Throughout my dissertation, I discuss investigations of H-FIRE with applications to treat malignant gliomas and other intracranial disorders. My PhD thesis focuses on: (1) characterizing H-FIRE effects for enhanced drug delivery to the brain; (2) the creation of a new, real-time electrical impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as a technique to determine H-FIRE treatment endpoints; (4) conducting a preliminary efficacy study of H-FIRE to ablate rodent glioma tumors; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a new impedance spectroscopy technique to be used in determining patient-specific ablation endpoints for intracranial H-FIRE procedures.

Traumatic brain injury (TBI) is a growing health concern worldwide that affects a broad range of the population. As TBI is the leading cause of disability and mortality in children, several pre-clinical models have been developed using rodents at a variety of different ages; however, key brain maturation events are overlooked that leave some age groups more or less vulnerable to injury. Thus, there has been a large emphasis on producing relevant animal models to elucidate molecular pathways that could be of therapeutic potential to help limit neuronal injury and improve behavioral outcome. TBI involves a host of different biochemical events, including disruption of the cerebral vasculature and breakdown of the blood brain barrier (BBB) that exacerbate secondary injuries. A better of understanding of the mechanism(s) underlying cerebral vascular regulation will aid in establishing more effective treatment strategies aimed at improving cerebral blood flow restoration and preventing further neuronal loss. Our studies reveal an age-at- injury dependence on the Angiopoetin-Tie2 axis, which mediates neuroprotection in a model of juvenile TBI following cortical controlled impact (CCI) that is not seen in adult mice. The protection observed was mediated, in part, by the microvascular response to CCI injury and prompted further detailed analysis of the larger arteriole network across several mouse strains and models of TBI. Our second study revealed both a model and species dependent effect on a specialized network of arteriole vessels, called collaterals after trauma. We demonstrated that a repetitive mild TBI (rmTBI) can induce collateral remodeling in C57BL/6 but not CD1 mice; however, CCI injury had no effect on collateral changes in either strain. Together, these findings demonstrate an age-dependent and species/model dependent effect on vascular remodeling that highlights the importance of individualized therapeutics to TBI. / Ph. D.

An astonishing number of deaths and related disabilities are attributed to traumatic brain injury (TBI) in the United States per year. Due to the unforeseeable nature of TBI and its association with the sequelae of other neurological comorbidities, research is centered around the secondary responses of brain mechanisms proceeding the initial mechanical injury. Blood brain barrier disruption is a well described driver of this secondary injury response and predictive marker of prognosis following TBI. Although BBB disruption plays a role in subsequent edema, inflammation, and the overall TBI outcome, the molecular mechanisms responsible for its regulation remain to be investigated. A large family of receptor tyrosine kinases, known as Eph receptors, that are important for axon growth and guidance embryonically and early-postnatally have been implicated in brain insults. Previous findings have shown that Eph expression is upregulated at the mRNA and protein level immediately following TBI. Moreover, ablation of Eph receptors on endothelial cells (ECs) revealed improved blood flow to the lesioned cortex in knockout (KO) mice compared to wild type (WT). Based on these results, we hypothesize that Eph receptors negatively regulate BBB permeability leading to neural dysfunction and motor deficits following TBI. To investigate this hypothesis, we characterized the temporal profile of the BBB, evaluated the EC-specific effects of Eph receptors, and used RNA sequencing to assess the cell-specific contributions following TBI in WT compared to KO mice. Our results show that EC-specific loss of Eph expression ameliorated BBB permeability at 6hr, 1-, 4-, and 7-days post injury (dpi) correlating with improved motor function at 7- and 14-dpi. Furthermore, mechanistic studies revealed increased mRNA expression of Tie2, Ang1, and the tight junction proteins Zona Occludens and Occludin in KO mice compared to WT. As well as, connection with neuronal processes. Based off of these findings, we utilized a soluble Tie2 inhibitor to elucidate the influence of Eph receptors on the Tie2/Ang pathway, and their role in mediating the effects seen. Tie2 inhibition of the KO mice revealed similar BBB disruption and lesion volume as WT 1dpi, attenuating the previous protection KO mice demonstrated. Future studies are necessary to understand other pathways that may be implicated in Eph receptor influence on endothelial cells such as inflammatory mediators and neurovascular crosstalk. This data provides evidence that Eph receptors negatively mediate EC response through downstream signaling of the Tie2/Ang pathway and may be a means of therapeutic target in the future. / Ph.D. / Traumatic brain injuries (TBIs) impact millions of individuals each year in the United States, making it a significant cause of death and disability. Furthermore, TBI has been linked to other comorbidities such as Alzheimers Disease, mood disorders, and epilepsy. Since the primary impact of a TBI cannot be predicted or prevented, research focuses on the secondary injury response as a therapeutic target to improve the outcomes following brain insult. Blood brain barrier (BBB) disruption is a well described consequence of TBI and has been correlated to a worse prognosis. The BBB normally provides a barrier between the circulating blood and the brain as protection and to maintain homeostasis. It is understood that decreased BBB integrity leads to subsequent edema, inflammatory response, and glial excitotoxicity, however, the mechanisms regulating this response remain to be investigated. Recent focus has been on a family of receptor tyrosine kinases, Eph receptors, that are unregulated following brain injury. Utilizing a mouse model, we can manipulate the temporal and spatial expression of Eph receptors to understand their role in the secondary injury cascade. Findings indicated that ablation of Eph receptors specifically on endothelial cells (ECs) resulted in preservation of BBB integrity at 1-, 4-, and 7- days following injury. Based on these results, we hypothesize that Eph receptor signaling on ECs negatively mediates BBB function and recovery following TBI. To test this hypothesis, we performed a comparative analysis between wild type (WT) and knockout (KO) mice on the expression of genes integral to BBB integrity, functional motor deficits, and loss of tissue in the lesion site following injury. We discovered significant decreases in lesion volume correlating with improvements in motor function in the KO mice compared to the WT. Moreover, KO mice showed increased expression of genes important for BBB maintenance such as Occludin and Tie2. To further discern the mechanism for these effects, we blocked Tie2 in the KO mice and observed similar negative prognostic indicators as in the WT. Future studies are warranted to understand the downstream signaling of Eph receptors on the Tie2 pathway. This data provides evidence that Eph signaling influences the BBB negatively following TBI through the Tie2 pathway and may be exploited for therapeutic means in the future.

Le glioblastome (GBM) est la tumeur cérébrale primaire la plus fréquente et agressive chez l’adulte. Son traitement, une exérèse chirurgicale maximale suivi d’un traitement adjuvant (radiothérapie et témozolomide [TMZ]), n’offre qu’un bénéfice modeste de survie médiane (14.6 mois vs. 12.1 mois pour radiothérapie post-chirurgie seule) (STUPP et al., 2005). Le TMZ demeure l’agent de choix pour le traitement du GBM. Malgré sa biodisponibilité approchant 100% suivant son administration per os (PO) (Diez et al., 2009), sa pénétration dans le liquide céphalorachidien n’est que de 20% (Ostermann et al., 2004). Ainsi, il se peut que les limites thérapeutiques du TMZ soient reliées aux barrières hémato-encéphalique (BHE) et hémato-tumorale (BHT). Plusieurs stratégies alternatives tentent de contourner ces barrières comme l’administration intra-artérielle (IA) avec une ouverture osmotique de la BHE (OBHE). Cette technique permet une plus grande distribution d’agent thérapeutique au système nerveux central (SNC). L’utilisation de cette stratégie avec le témozolomide n’a jamais été étudiée à ce jour. Nous avons émis l’hypothèse que son utilisation permettra d’augmenter la concentration de TMZ dans le SNC et que, lorsque combiné avec la radiothérapie, permettra de rehausser son activité anti-tumorale. Les objectifs du projet sont : (1) l’évaluation de la sensibilité des cellules F98 au TMZ in vitro; (2) la caractérisation de la neuropharmacocinétique du TMZ in vivo, selon différents modes d’administration; et (3) l’évaluation de l’effet anti-tumoral du TMZ in vivo, selon différents modes d’administration. Les expérimentations in vivo ont été exécutées dans le modèle syngénique Fischer-F98, porteur de tumeur gliale. L’expérimentation in vitro a démontré une résistance importante des cellules F98 au TMZ. La méthodologie développée a permis de démontrer que l’infusion IA avec et sans OBHE augmente la concentration maximale et l’aire sous la courbe du TMZ dans la tumeur cérébrale et dans le parenchyme cérébral ipsilatéral du rat Fischer-F98. Par contre, aucun bénéfice de survie n’a été observé en utilisant ces stratégies alternatives. Au contraire, l’acheminement augmenté du TMZ au SNC semble toxique. Un bénéfice de survie a été mesuré suite à l’ajout d’un traitement de radiothérapie, mais de façon indépendante au mode de livraison de TMZ ou de solution saline normale (groupe contrôle). Enfin, nos résultats témoignent de l’impact du mode d’acheminement sur la distribution d’un agent thérapeutique au SNC. En détournant la BHE, l’utilisation judicieuse d’approches alternatives combinée à un agent thérapeutique approprié a un grand potentiel clinique dans le traitement des GBM. / Abstract : Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Its’ standard treatment, maximal surgical resection followed by an adjuvant treatment (radiotherapy and temozolomide [TMZ]) offers only a modest median survival benefit of 14.6 months (vs. 12.1 months with post-surgery radiotherapy alone) (Stupp et al., 2005). TMZ remains the therapeutic agent of choice for the treatment of GBM. Despite its bioavailability approaching 100% after a per os administration (Diez et al., 2009), its cerebrospinal fluid penetration is only of 20% (Ostermann et al., 2004). Thus, TMZ’s therapeutic limitations could be due to the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Alternative routes of drug delivery attempt to bypass these barriers. For example, intra-arterial (IA) administration with an osmotic blood-brain barrier disruption (OBBBD) allows greater drug distribution to the central nervous system (CNS). Its use with TMZ, with or without radiotherapy, has never been studied. We hypothesized that it will increase TMZ concentration in the CNS and that, when combined to radiotherapy, it will intensify its anti-neoplastic activity. The project was divided in three parts: (1) the evaluation of F98 cells’ in vitro sensitivity to TMZ; (2) the in vivo caracterization of TMZ’s neuropharmacokinetics, following different routes of administration; and (3) the in vivo evaluation of TMZ’s anti-tumoral effect, following different routes of administration. The syngenic glioma Fischer-F98 model was used in all in vivo experiments. Our results showed the F98 cells to be resistant to TMZ in vitro. The methodology developed showed that an IA infusion with and without OBBBD increased TMZ’s peak concentration and area under the curve in the brain tumor and ipsilateral brain parenchyma in the Fischer-F98 rat. All the while limiting systemic exposure. However, no survival benefit was observed with the use of these alternative strategies. More so, TMZ’s enhanced delivery to the CNS seemed toxic. A survival benefit was measured following the addition of radiotherapy. This was independent of the route of delivery of TMZ or normal saline. In summary, our results provide evidence that the method of TMZ administration does impact its CNS delivery. By bypassing the BBB, the judicious use of local delivery approaches combined with the appropriate therapeutic agent can have a great clinical potential in the treatment of glioblastomas.

The blood-brain barrier (BBB) is a dynamic interface, mainly consisting of highly specialized brain microvascular endothelial cells (BMECs) that segregate the central nervous system (CNS) from the peripheral circulation. Impairment of the BBB, due to disruption of tight junction (TJ) proteins and inflammatory responses, may initiate and/or contribute to the progress of CNS disorders, including stroke. Stroke is the second leading cause of death worldwide. It has been shown that aging and environmental pollutants can induce brain endothelium dysfunction, and are considered as risk factors for stroke. Deficiency of telomerase is highly linked with aging-associated vascular diseases. Evidence indicates that patients with shorter telomere length are at higher risk of heart disease or stroke. Results in this dissertation address the influence of telomerase reverse transcriptase (TERT), a key component of telomerase, on the BBB integrity in the context of ischemic stroke induced brain injury. Our results indicate that aging-related BBB alterations aggregate the stroke outcomes by inducing oxidative stress and stimulating proinflammatory responses on the brain microvessels. The ability of the BBB to protect the brain from harmful compounds indicates that the BBB may be targeted by chemical toxicants in the peripheral circulation. Polychlorinated biphenyls (PCBs) are persistent organic pollutants that frequently bind to nanoparticles (NPs) in the environment. Our results demonstrate that binding PCB153, one of the most abundant PCB congeners in the environment, to silica nanoparticles (PCB153-NPs) potentiates cerebrovascular toxicity and stroke outcomes via stimulation of inflammatory responses and disruption of BBB integrity. These events are mediated by activation of toll-like receptor 4 (TLR4), which subsequently recruits tumor necrosis factor-associated factor 6 (TRAF6) and initiates the production of multiple inflammatory mediators. Research presented in this dissertation demonstrates that aging and environmental pollutants play crucial roles in modifying the function of the BBB through alterations of inflammatory responses and TJ protein expression, which further contribute to the progression of stroke-induced cerebral ischemic injury.

Convection-enhanced delivery (CED) is a proven method for targeted drug delivery to the brain that circumvents the blood-brain barrier (BBB). Little study has been conducted in understanding CED in pathological brain states. This is of importance when dealing with chemotherapeutic agent delivery to brain tumors, where vasogenic edema (VE) exists. The current study aims to characterize a model of VE suitable for studying CED.VE was produced in the right hemisphere of the rat brain using multiple infusions of hyperosmotic mannitol (0.25mL/kg/s over 30 seconds) delivered through the right internal carotid artery. Magnetic resonance imaging (MRI) revealed consistent edema formation and high water levels in the ipsilateral gray and white matter within an hour of the first infusion. Evan's Blue (EB) staining verified that VE has formed. However, apparent diffusion coefficient (ADC) and histological examination revealed also that some possible cytotoxic edema formed.This model provides a reproducible technique for generating a large area of edema for CED study. Further studies with lower doses of mannitol, while titrating to changes in ADC and values for fractional water content, may modify this model with a greater component of VE and less cerebral toxicity.