Carotid intima-media thickness (IMT) is a measure of atherosclerosis. A large carotid IMT may be indicative of an impaired blood-brain barrier, providing a pathway for infections and inflammation to more readily enter the brain to contribute to neuronal damage as proposed by the ‘microbial hypothesis’. However, no consensus exists regarding the effects of carotid IMT and infections on cognitive decline. Therefore, the goal of my dissertation was to investigate the potential mechanisms proposed by the ‘microbial hypothesis’ that may result in the outcome of cognitive decline. This goal was accomplished through investigating three specific aims. First, I conducted a scoping review to examine and synthesize existing literature assessing the effects of either carotid IMT or infections on the outcome of change in cognition over at least two years. Secondly, I empirically assessed the association between mid-life carotid IMT and late-life cognitive function at baseline and over time in a well characterized cohort. Finally, in the same cohort, I examined the association between mid- to late-life hospitalized infections and late-life cognitive function at baseline and change over time. Studies included in the scoping review moderately supported an association between carotid IMT and decline in global cognition, though evidence for an association between infections and cognitive decline is lacking. Infections most commonly were identified using antibodies, which are representative of past infections and may explain null findings. When examining carotid IMT empirically, I found no association between mid-life carotid IMT on 6-year change in late-life global cognitive function, however there was a significant association between greater carotid IMT and decreasing executive function scores over time. In a secondary analysis, I was able to expand follow-up time for global cognition up to 21 years, beginning in mid-life immediately following last carotid IMT measurement and found a significant association with change over time with this approach. This may support the need for interventions for reducing or preventing atherosclerosis earlier in the life course to prevent cognitive decline and potentially halt progression to dementia. In the same cohort, I found no significant associations between mid- to late-life hospitalized infections and 6-year change in late-life cognition for global cognition or domains. However, a history of having a hospitalized infection was significantly associated with baseline global cognition and baseline scores for language and executive function, but not memory. This may support theories that infections may result in faster rates of decline immediately following infections while then returning to normal rates in later life, however further examination of trajectories immediately after infections is needed. Results from this dissertation do not directly support the ‘microbial hypothesis’, however, they do provide insight that may be applicable to future studies examining these relationships.

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.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 165 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 131-163).

Persistent cerebrovascular dysfunction has been postulated as one mechanism that may contribute to divergent functional trajectories after ischemic stroke. However, how brain endothelial cells (BECs) acutely respond to ischemia and what endogenous signals subsequently regulate vascular normalization remain poorly understood. To spatiotemporally interrogate neuronal activity and hemodynamics in the acute period after ischemic brain injury we used wide-field imaging. Local ischemia consistently provoked a large-amplitude cortical spreading depolarization (CSD) accompanied by strong vasoconstriction, followed by subsequent diverse CSDs with varying hemodynamic responses. Small CSDs with slow depolarization induced vasodilation in well oxygenated cortical tissue. CSDs of larger amplitude with non-sustained depolarization induced biphasic vascular responses. CSDs of large amplitude, characterized by rapid and prolonged depolarization, drove vasoconstriction in deoxygenated cortical tissue with sustained neuronal depolarization. These observations support a model in which vascular responses after acute brain injury are dependent upon the local relationship between CSD features (i.e. slope, duration, and amplitude of depolarization) and the underlying cortical state (i.e. neuronal activity, perfusion, oxygenation). After this acute period, the ischemic brain is characterized by profound changes in immune cell composition and function. To understand how distinct immune signaling pathways regulate blood-brain barrier (BBB) repair and vascular remodeling after ischemic brain injury, I investigated a unique post-ischemic BEC type one interferon (IFN1) signature. Functional assays and single-cell transcriptomic analyses in IFN1 receptor (Ifnar1) inducible EC knockout (iECKO) mice revealed that loss of BEC IFN1 signaling exacerbated post-stroke barrier disruption and resulted in an expansion of BECs enriched in genes involved in angiogenic processes. Conversely, acute administration of exogenous IFNI ameliorated post stroke BBB disruption. In vitro assays supported that IFNI signaling modulates BEC junctional protein stabilization and vascular endothelial growth factor (VEGF) signaling to enhance BEC barrier properties and suppress angiogenic features, respectively. These findings suggest that endogenous BEC IFN signaling after ischemic brain injury restricts angiogenesis to potentially promote acute barrier function. These studies, which span from the systems to molecular level, demonstrate that brain ischemia and post-ischemic sequelae have a profound impact on cerebrovascular dysfunction and recovery. Furthermore, each study introduces a novel framework to investigate how differences in acute BEC responses may contribute to variable vascular trajectories and longitudinal brain function after ischemic insult.