Mild traumatic brain injury (mTBI)/ concussion accounts for 70-90% of all reported TBI cases in the United States and can cause long-term neurological outcomes that negatively impact quality of life. Previous studies revealed that increased blood-brain barrier (BBB) leakage is correlated with poor neurological outcomes after mTBI, yet the biological mechanisms linking BBB damage to the onset of neurological deficits after mTBI are not well understood. Previously, we found that astrocytes lose expression of homeostatic proteins after mTBI, characterizing the changes in astrocytic protein expression as an "atypical astrocyte response." Yet, the upstream mechanisms that induce this atypical astrocyte response after mTBI have yet to be elucidated. In models of more severe TBI, exposure to blood-borne factors triggers astrogliosis via upregulation in markers, such as glial fibrillary acidic protein (GFAP), but how exposure to blood-borne factors affects astrocyte protein expression in the context of mTBI is not well understood. Therefore, we hypothesized that mTBI-induced BBB damage causes atypical astrocytes via exposure to blood-borne factors. To test this hypothesis, we use a mTBI mouse model, two-photon microscopy, an endothelial cell-specific genetic ablation model, and serum-free primary astrocyte cultures. Here, we found that mTBI causes BBB damage through the loss of proteins involved in maintaining the BBB's physical and metabolic barriers, and BBB damage is sustained long-term after injury. Also, we demonstrated that leakage of blood-borne factors is sufficient to trigger atypical astrocytes, and plasma exposure triggers a similar response in vitro. Overall, these findings suggest that mTBI induces long-term BBB damage, and exposure to blood-borne factors triggers the loss of key homeostatic astrocytic proteins involved in maintaining healthy neuronal function. / Doctor of Philosophy / Mild traumatic brain injury (mTBI)/ concussion makes up 70-90% of all TBI cases reported in the United States and is commonly observed after car crashes, sports-related tackles, and blast exposure during military combat. People who experience mTBI develop debilitating long-term neurological consequences, such as sleep disturbances, depression, and dementia. Clinical data suggests mTBI causes damage to the barrier between the brain and blood, known as the blood-brain barrier (BBB). This damage has been correlated to the onset of poor neurological deficits, yet how damage to this barrier is causally linked to long-term neurological consequences remains to be fully understood. In our lab, we found that mTBI causes loss of proteins important for maintaining a healthy environment in the brain in specialized cells called astrocytes. However, the biological events that trigger the loss of protein expression in astrocytes after mTBI have yet to be fully investigated. Thus, we hypothesized that mTBI causes loss of these proteins via leakage of blood-borne factors. To test this hypothesis, we used a mTBI mouse model, two-photon microscopy, genetic manipulation, and cell cultures. In our studies, we found that mTBI triggers BBB damage via loss of proteins that make up its protective properties. Also, we demonstrated that leakage of blood-borne factors is sufficient to cause loss of astrocyte-specific proteins both in brain and cell cultures. Altogether, we show that a single mTBI is sufficient to cause loss of astrocyte-specific protein expression via exposure to blood-borne factors. These findings may point to targeting either the blood-borne factor(s) or their corresponding receptor pathways in astrocytes to halt the progression of long-term neurological deficits after mTBI.

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.

Epilepsy affects 3.4 million patients in the USA and is characterized by recurring seizures. The blood-brain barrier is leaky in epilepsy and may contribute to seizure progression but the mechanisms which cause this leakage are not fully understood. We hypothesized that seizures trigger LOX- and COX-mediated blood-brain barrier leakage and that dual LOX/COX inhibition prevents barrier leakage in vivo. To test this hypothesis, we administered either the dual LOX/COX inhibitor licofelone or a combination of the 5-LOX inhibitor zileuton and the COX-2 inhibitor celecoxib to rats that experienced status epilepticus (SE). Serum and brain capillaries were isolated 48 hours after SE and serum S100β levels were measured and Texas Red™ leakage rates were determined. Dual inhibition of 5-LOX and COX prevented serum S100β elevations observed in SE rats in a dose-dependent manner with licofelone. Inhibition of 5-LOX and COX-2 with zileuton and celecoxib completely prevented serum S100β elevation. Texas Red™ leakage rates for SE rats were also reduced in a dose-depended manner with licofelone and reduced to control rates with zileuton and celecoxib. These data support our hypothesis that seizure-induced blood-brain barrier leakage is mediated by LOX and COX, and inhibition of these enzymes prevents barrier leakage.

Thesis (doctoral)--Universitetet i Lund. / Reprints of journal articles inserted in pocket inside back cover.

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).

Thesis (doctoral)--Universitetet i Lund. / Reprints of journal articles inserted in pocket inside back cover.

The initial purpose of this work was to investigate the interaction of methylmercuric chloride (MMC) and X-irradiation given as a co-insult upon the rat blood-brain barrier (BBB). The indicators used to determine BBB alterations were mortality and the in vivo tissue uptake of radioactive sulfate administered as 3 5S-sodium sulfate. The results of the interaction studies indicated a neutralization of effects when MMC and X-irradiation were given together. X-irradiation as a single insult generally caused an increase in sulfate uptake by the brain regions monitored, whereas MC treatment generally resulted in decreased sulfate uptake. The neutralization patterns following co-insult treatments were somewhat varied in the different brain regions, exhibiting cancellation of effects in some cases and overriding by one insult in other eases. From the data obtained by this work and in the literature, it is hypothesized that the P-L organelle system of the perivascular glia serves as a trap for MMC, preventing MMC from reaching the neurons. The system appears to proliferate in response to increased peroxides in the body fluids, thereby increasing tolerance to larger doses of MMC.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains x, 154 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 135-149).

Background: Brain metastasis is a leading cause of cancer mortality and affects 20-40% of all cancer patients. The BBB is responsible for isolation and protection of the brain parenchyma from many diagnostic and therapeutic agents. New molecular agents that target tumour-associated VCAM-1 expression on the brain endothelium show improvements in the early diagnosis of brain metastasis. The vascular endothelium of the CNS plays an important role in the maintenance of the brain microenvironment and possibly aids the extravasation of tumour cells via expression of CAMs. Aims: Using the breast carcinoma-derived 4T1 cell line, syngeneic to BALB/c mice, this work aimed (i) to determine the level of colocalisation between VCAM-1 expression at sites of brain metastasis and the presence of VCAM-MPIO-induced hypointensities in MR datasets; (ii) to describe the normal developmental characteristics of the intracardial BALB/c-4T1 brain metastatic model in the absence of overt inflammation; (iii) to test the effects of an adenovirus-induced systemic inflammatory challenge on metastatic uptake and development in the brain. Results: The level of correspondence of VCAM-MPIO-derived hypointensities with VCAM-1 expression at the tumour site was found to be dependent on the size of metastasis. An improved method for detection of VCAM-MPIO hypointensities using an automated method has been presented. Tumours were found to develop preferentially on venous rather than arteriolar blood vessels, and showed greater and lesser abundance in different anatomical brain regions. Adenovirus injection was found to cause an upregulation of a range of peripheral pro-inflammatory cytokines, and expression of VCAM-1 on cerebral vasculature, preferentially on arteriolar blood vessels. Both pre- and post-treatment with adenovirus caused a two-fold reduction in tumour numbers and altered developmental characteristics of established tumours, although no significant differences were observed in VCAM-MPIO hypointensities in MR datasets. Conclusions: The development of molecular MRI approaches to target VCAM-1 expression at the site of brain metastases has improved the sensitivity of tumour detection. 4T1-GFP metastasis to the brain is specific both to anatomical sites and to regions of the vascular bed, suggesting differences in vascular morphology and/or signalling dynamics in these locations. The changes in tumour number and morphology as a result of systemic inflammation suggest an anti-tumour effect of adenoviral treatment and, given the role of the systemic immune system and its importance in the development of immunotherapies, possible future directions for research.

Vascular, especially cerebrovascular, dysfunction may be a critical factor in ageing and dementia. Cerebrovascular impairment due to risk factors such as ageing, stroke, smoking, diabetes and cerebral hypoperfusion has a deterious impact on the normal supply of basic nutrients such as oxygen and glucose to the brain; their absence leads inevitably to neuronal death. The cerebral white matter lesions found in most forms of dementia are reportedly the result of chronic cerebral hypoperfusion. However the temporal and spatial evolution of damage remains unclear. Furthermore, any decrease in the integrity of the blood-brain barrier (BBB) has been hypothesised to be a precocious attack on white matter. The “milieu interieure” the most protected in the body, namely the extracellular fluid of the brain, is no longer maintained homeostatically. The cumulation of these various pathophysiological processes alters cerebral function and it has been postulated that, in the most extreme instances, the outcome of this cascade of nefarious events leads to dementia. This thesis examines the supposition that chronic cerebral hypoperfusion could be responsible for the time-related development of white and grey matter pathology and investigates the relationships between the disturbances in the integrity of the BBB and white matter pathology. Three studies addressed these aims. In the first, chronic cerebral hypoperfusion, induced in male Wistar rats by bilateral common carotid artery occlusion (BCCAo), was chosen as the model to study changes in axons, myelin, perikarya as well as microglial activation. The groups of rats that underwent BCCAo were examined at three hours as well as three, seven, 14 and 28 days after the induction of chronic cerebral hypoperfusion. The microscopic examination revealed that, after three hours post BCCAo, damage was detected only in axons and myelin. In contrast, no visible pathology to the neuronal perikarya or enhancement of activated microglia (compared to the sham group) was observable. Injury in both white and grey matter and enhancement of activated microglia was observed from three days post BCCAo and increased with time post BCCAo. The most severe damage to the white and grey matter and enhancement of microglial activation was detected at seven days post BCCAo. These results would indicate that white matter damage precedes grey matter pathology and the enhancement of activated microglia. In the second study, the integrity of the BBB at three hours (when only white matter pathology was found according to the results of the first study) and seven days post BCCAo (when more severe damage to the white and grey matter was shown) was assessed by the use of MRI on T1-weighted image acquisitions with gadolinium as a tracer for BBB permeability. White matter integrity was measured by MTR maps from MTI acquisitions in four brain structures (corpus callosum, caudatoputamen, the external and internal capsules). No differences in white matter integrity were detected between the BCCAo and sham group at three hours and seven days. No differences in signal enhancement of gadolinium were detected three hours post BCCAo. However, a significant signal enhancement of gadolinium was detected at seven days post BCCAo in the caudatoputamen and in the external capsule. Furthermore, immunohistochemistry revealed a significant enhancement of activated microglia seven days post BCCAo compared to the sham group. This functional and immunohistochemical finding, when taken together, might indicate that chronic cerebral hypoperfusion is not in itself responsible for BBB permeability. Rather, the damage to the white matter caused by cerebral hypoperfusion may be responsible for the dysfunction of the BBB over time. Another point of interest was the evidence that the enhancement of activated microglia may play a critical role in the increased permeability of the BBB. The final study in this thesis aimed to investigate the possible pathway and proteins potentially implicated in white matter damage and BBB permeability. To address this question, protein levels and the expression of genes involved in the apoptotic and nonapoptotic hypoxic pathways were compared to the sham groups (at three hours and seven days after BCCAo), in three brain structures (cortex, corpus callosum and caudatoputamen). The levels of HIF-1α, MMP-2, Caspase-3 and VEGF were unchanged compared to the sham group after BCCAo. However, VEGF mRNA expression was found to be significantly different to the sham group seven days post BCCAo in all the three structures examined. An overexpression of HIF-1α and a significant level of Caspase-3 would indicate the activation of the apoptotic pathway. However, neither of these criteria were met and these negative results suggest that the apoptotic pathway is not implicated in the mechanisms that lead to white matter pathology after cerebral hypoperfusion. Finally, the significant expression of VEGF mRNA, compared to the sham group seven days post BCCAo, may contribute to the time-relate increased permeability of the BBB. The results presented within this thesis provide a body of evidence to support the hypothesis that chronic cerebral hypoperfusion is - at least – causal to the damage to different components of the white matter which precedes either early ischaemic changes to the perikarya or enhancement of activated microglia following BCCAo. The increased permeability of the BBB, which can be related to the significant over-expression of VEGF mRNA (compared to the sham group seven days post BCCAo), does not appear to be primarily responsible for white matter pathology, because the MRI investigations indicated that BBB integrity was not affected after three hours of BCCAo. The increased permeability of the BBB, observed seven days post BCCAo with MRI, seems to be the consequence of increased brain damage; thereafter, there is a time-dependent relationship between increasing BBB permeability and increasing brain pathology. Overall, the studies reported herein, strengthen the initial working hypothesis. The conclusion – and direction for future studies – would be that minimising white matter pathology and protecting components of the BBB represent potential targets to decrease then incidence of neuropsychological function or to obtund the cerebral dysfunction in patients who suffer from chronic cerebral hypoperfusion.