Recent evidence suggests that cerebrovascular dysfunction may precede and contribute to amyloid beta-(Aβ)-mediated pathology in Alzheimer’s Disease (AD), particularly promoting endothelial cell (EC) damage and stress, causing the cerebral blood flow (CBF) impairments, cerebral hypoperfusion (CH), and blood brain barrier (BBB) permeability that are pathologically characteristic in AD. Studies have emerged suggesting a link between cardiovascular diseases (CVDs) and AD pathology, showing that cerebrovascular/cardiovascular risk factors (CVRFs), including hyperhomocysteinemia (Hhcy) and hypertension (HTN), and the cerebral consequences of these CVRFs, such as CH, contribute to AD pathology and risk. Despite this, the underlying molecular mechanisms for these associations remain unclear. Previously, our lab has demonstrated that exposure of human cerebral microvascular ECs (HCMECs) to Aβ40-Q22 (vasculotropic Dutch mutant) or Aβ42 resulted in potentiated increases in apoptosis and decreases in barrier integrity and angiogenic capabilities. Previous evidence also reveals that Hhcy, HTN, and hypoperfusion exhibit similar dysfunction within peripheral blood vessels. Therefore, we tested the hypothesis that Hhcy, HTN, and CH exacerbate Aβ-induced cerebral EC apoptosis, BBB dysfunction, and angiogenesis impairment and Cerebral Amyloid Angiopathy (CAA) pathology in vitro and in vivo. Human cerebral microvascular ECs (HCMECs) were challenged with AβQ22 and/or homocysteine (Hcy) or AβQ22/Aβ42, glucose deprivation (GD), or a combination of both under normoxia or hypoxia. Conditions involving oxygen and glucose deprivation (OGD) act to mimic tissue hypoperfusion in vitro. Apoptotic mediator expression, caspase activation, cytochrome c (CytC) release, and DNA fragmentation were measured to assess apoptosis. BBB- modulating protein expression, trans-endothelial electrical resistance (TEER), and inflammatory mediators were measured to assess EC barrier integrity. Angiogenesis inhibition and activation (pVEGFR2 Y1175 expression/localization, VEGF-A ELISA) assays were utilized to measure EC angiogenic capability and wound healing and actin polymerization assays were utilized to measure EC wound healing ability. For the in vivo portion of this study, wild-type (WT) and Tg2576 AD mice (Swedish APP mutation) were fed with a Hhcy-inducing diet, HTN-inducing water (L-NAME), or both starting at 5 months. Mice were sacrificed at 13-14 months and tissue was harvested. Apoptotic mediator expression and vascular caspase-3 activation were measured. BBB-associated tight junction (TJ) protein expression, GFAP/IBA1 expression and morphology (neuroinflammation), and microhemorrhages were measured to assess BBB integrity. Angiogenic mediator expression and CD31+ microvessel density were utilized to determine cerebral angiogenic capability. Additionally, AD pathology and cognitive impairment were assessed in these mice. We found that combined challenge of ECs with AβQ22 and Hcy potentiated increases in TRAIL death receptor (DR)-related apoptotic mediator expression, caspase activation, CytC release, and DNA fragmentation and potentiated decreases in anti-apoptotic mediator expression at differential timepoints. Treatment of ECs with AβQ22 and Hcy additively decreased TEER and, at certain timepoints, potentiated decreases in junction protein expression. Additionally, this combined treatment exacerbated increases in the expression of inflammatory mediators and phosphorylated BBB proteins. Combined treatment of ECs with AβQ22 and Hcy also additively decreased angiogenesis progression, angiogenesis-stimulating cytokine expression, wound healing capability, and actin polymerization levels. From our experiments involving OGD, we found that GD and hypoxia differentially potentiate Aβ-induced activation of cell death mechanisms within HCMECs, with GD potentiating apoptosis levels and hypoxia potentiating necrosis levels. Combined challenge of HCMECs with Aβ and OGD revealed exacerbated decreases in TEER, associated to potentiated dysregulation of BBB-TJ proteins, increases in MMP2 expression, and increases in proinflammatory mediator expression and monocyte migration. Furthermore, treatment of HCMECs with Aβ and OGD demonstrated potentiated decreases in angiogenic and wound healing capabilities. Our in vivo data has shown that Tg2576 mice with CVRFs demonstrate exacerbated increase in pro-apoptotic protein expression and caspase-3 activation within AD vulnerable brain regions. Additionally, Tg2576 mice with CVRFs displayed signs of exacerbated BBB dysfunction, evidenced by decreases in TJ protein expression, increases in active MMP2 expression, increases in neuroinflammation (astrogliosis, increased ICAM1), and increased presence of microhemorrhages and/or microthrombi. Tg2576 mice with CVRFs also demonstrated exacerbated increases in VEGF-A expression and a potentiated decrease in phosphorylated VEGFR2 Y1175, suggesting aberrant VEGF signaling and worsened angiogenic impairment. Finally, Tg2576 CVRF mice trended towards potentiated increases in cerebral soluble Aβ40 and fibrillar Aβ42 and as well as worsened cognitive outcomes on the Barnes Maze memory task. To conclude, this study has revealed specific molecular mechanisms through which amyloidosis and CVRFs additively act to produce cerebrovascular damage and dysfunction and potentiate AD pathology within Tg2576 AD mice, specifically increasing cerebrovascular apoptosis, BBB permeability, and angiogenesis impairments. Identifying common molecular effects that Aβ and CVRFs exert on the cerebral vasculature will reveal novel potential druggable targets and biomarkers that could facilitate earlier disease detection, aid in the prevention of the initial stages of vascular damage within AD, and therapeutically slow or halt disease progression in common mixed AD and vascular dementias. / Biomedical Neuroscience

High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation therapeutic that applies bipolar, high-frequency pulsed electric fields to tumors, triggering the formation of irreversible membrane pores and to induce tumor cell death. H-FIRE has demonstrated pre-clinical and clinical utility as a therapeutic for brain tumors, including gliomas. H-FIRE has been shown to induce precise, uniform ablation within the tumor tissue, as well as local changes to the tumor microenvironment and systemic changes to the immune landscape. Namely, disruption of the peritumoral blood-brain barrier (BBB) following H-FIRE ablation of brain tumors, and infiltration and activation of the innate immune system are clinically observed following H-FIRE tumor ablation. Such effects persist long after death of the treated tumor, and therefore an understanding of the mechanisms underlying these local and systemic changes are critical for the development of H-FIRE. Using in vitro models of glioma and lung carcinoma-derived brain metastases, we investigate the interactions between cancer cells that have been ablated with H-FIRE and the brain tumor microenvironments. Specifically, we demonstrate that H-FIRE-treated cancer cells can recover treatment-induced damage and proliferative capacity after treatment with specific electric field doses, while higher doses inhibit such recovery. This suggests that after H-FIRE ablation of brain tumors, tumor cells can still secrete factors to trigger alterations in their local and systemic environments. We then specifically investigate the role of tumor-derived extracellular vesicles (TDEVs) in mediating these changes, namely pBBB disruption and changes in innate immunity. We find that, following H-FIRE ablation of brain cancer cells, treated cells immediately release TDEVs that disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cerebral endothelial cells in vitro, despite reduced release of TDEVs after H-FIRE. We further demonstrate that H-FIRE significantly alters the proteomic payloads of TDEVs. When TDEVs released by sham- and H-FIRE-treated glioma cells are delivered to healthy rats, only TDEVs released by H-FIRE-ablated cells are retained in the brain, suggesting changes to TDEV organotropism after H-FIRE ablation of glioma. Further, once retained in the brain, these post-H-FIRE TDEVs cluster near cerebral endothelial cells, similarly to in vitro. Although the TDEVs released by H-FIRE ablated glioma cells do not disrupt the BBB in vivo, Iba1+ cells were increased in the brains of rats that received TDEVs released by H-FIRE-ablated glioma cells. Together, these data suggest that H-FIRE immediately alters the secretion and proteome of TDEVs, facilitating changes in TDEV organotropism and cellular tropism and immune cell recruitment to the tumor microenvironment. Together, this research indicates mechanisms by which tumor cells continue to modulate their local and systemic environments via the action of TDEVs, which is critical information for the continued development of H-FIRE and its optimization with adjuvant therapeutics for the treatment of malignant brain tumors. / Doctor of Philosophy / All cells secrete extracellular vesicles, which are packets of information that function as communication highways between cells. In cancer, tumor-derived extracellular vesicles (TDEVs) reprogram local and distant cells to support tumor growth. However, they have also been shown to change local and systemic functions, such as blood vessel function and immune response, after tumors are treated with therapeutics. Therefore, a full understanding of the role of TDEVs in how tumors communicate with the body after cancer treatment is necessary when developing new anti-cancer therapeutics. Here, in developing high-frequency irreversible electroporation (H-FIRE), a novel anti-tumor therapeutic for the treatment of malignant brain tumors, we explore how TDEVs released by brain cancer cells treated with H-FIRE interact with various cell types and structures in the body, and how these interactions may affect the response to treatment. Using a glioma model of primary brain cancer, and a lung carcinoma model of brain metastases, we first explore how tumor cells may be able to recover from damage after treatment with H-FIRE. We discover that brain cancer cells treated with specific doses of H-FIRE recover cell damage and continue to proliferate, but cells treated with higher doses of H-FIRE cannot recover these functions. The fact that tumor cells may be able to recover after H-FIRE suggests that cancer cells may still secrete factors, such as TDEVs, that interact with cells in the microenvironment after tumor treatment. We investigated the role of TDEVs released by brain cancer cells treated with H-FIRE to determine whether they cause changes in surrounding cells and structures in the brain cancer microenvironment. We determined that brain cancer cells treated with H-FIRE release TDEVs that carry proteins different from those carried by TDEVs routinely released by untreated cells. We further found that these TDEVs disrupt the blood-brain barrier (BBB) endothelium in vitro, and are uniquely internalized by cells of the endothelium. When these TDEVs were administered to the brains of healthy rats, they were retained in the brain, clustered near the endothelium, and recruited immune cells from circulation into the brain. Conversely, TDEVs that were routinely released from the brain cancer cells, in the absence of H-FIRE treatment, exhibited none of these functions. Taken together, these results show that H-FIRE changes TDEVs in numerous ways: after H-FIRE, the TDEVs may gravitate toward particular organs and cell types, and recruit immune cells. All of these changes can impact the overall therapeutic response after H-FIRE, and may also be specifically optimized and targeted with additional therapeutics to make H-FIRE more effective for brain cancer.

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.

This thesis examines the utility of USPIO-MRI to provide a tool of tracking macrophage recruitment to sites of neuroinflammation within the CNS. Recruited macrophages and microglia resident in CNS tissue play a key role in the pathophysiology of a number of neuroinflammatory diseases such as neuropathic pain and multiple sclerosis. Under activated conditions, microglia and macrophages will phagocytose invading cells and CNS debris. It has been shown that ultrasmall superparamagnetic particles of iron oxide (USPIO), such as Sinerem, injected systemically, are engulfed by macrophages, which in turn migrate to sites of tissue injury. USPIOs can be visualised as a distinct reduction in signal intensity on T2* weighted MR images. However, there are still some issues regarding the distinction between iron-laden recruited macrophages and the entry of free iron across a permeable blood brain barrier (BBB) in disease cases. Hence, it was shown that intravenously injected Sinerem is cleared from the peripheral circulation within 24 hours, indentifying this as a time point as suitable for MCP-1 injection. Data showed that free USPIO can be visualised in the brain and that there is a linear relationship between Sinerem concentration and T2* signal intensity changes. MCP-1 induces macrophage recruitment to the site of microinjection and causes BBB breakdown at between 3 and 4 hours. In particular it was shown that T2* signal intensity changes are seen, in the presence of an intact BBB, as a result of Sinerem laden macrophages. This finding was verified by the co-localisation of ED-1 positive cells and Prussian blue positive regions. It was demonstrated that there is a strong correlation between T2* signal changes and the number of macrophages. This demonstrates that USPIO-MRI can be used to characterise macrophage infiltration in neuroinflammation in the presence of an intact BBB.

A hipertensão arterial cursa com disfunção autonômica e lesão da barreira hematoencefálica (BHE) em áreas de controle autonômico. Demonstramos recentemente que o treinamento aeróbio corrige a lesão da BHE, e a disfunção autonômica, a qual se encontra correlacionada com a integridade da BHE observada nos hipertensos treinados. O objetivo deste trabalho é avaliar a expressão gênica e proteica de componentes da BHE envolvidos na mediação das respostas cardiovasculares à hipertensão e ao treinamento aeróbio (T). Ratos espontaneamente hipertensos (SHR) e seus controles normotensos (WKY) (250-300g) foram submetidos ao protocolo de T em esteira ou mantido sedentários (S) por 4 semanas. Ao final do T os animais dos grupos experimentais foram canulados para aquisição das variáveis hemodinâmicas. A seguir procedeu-se à infusão intra-arterial de 2 corantes (Rodamina-d, 70 KD, e FITC-d,10 KD) e 20 min após os encéfalos foram coletados para realização de ensaios de fluorescência no Núcleo Paraventricular do Hipotálamo (PVN). Outros ratos dos grupos experimentais foram perfundidos com salina via transcardíaca e realizada a microdissecção do PVN. O mRNA foi extraído e sua concentração de foi analisada pela técnica de RT-PCR. Para investigar os efeitos da hipertensão e do T nos componentes da BHE, foram utilizados os seguintes primers: Occludina, Claudina-5, Zônula Ocludens 1 (proteínas da junção oclusiva), Caveolina-1 (indicador de transporte transcelular), Laminina alfa 1 e Colágeno 4 (componentes da membrana basal), PDGFRβ (marcador de pericitos)e Aquaporina-4 (indicador de podócitos de astrócitos), todos eles normalizados para o HPRT endógeno. Os dados de PCR em tempo real foram quantificados pelo método 2Δ ΔCT. Além disso, outros ratos dos mesmos grupos experimentais foram perfundidos com paraformaldeído 4% para a fixação do encéfalo. O tecido foi crioprotegido e seccionado em criostato, 30 um, os cortes foram incubados em anticorpos primários (Reca-1(marcador endotelial), Claudina-5, Caveolina-1, PDGFRβ e Aquaporina-4) e secundários (Alexa Flúor 488 e 594), e sua quantificação no PVN foi realizada através da densidade integrada. Não foi observada diferença na pressão arterial entre os grupos T e S, porém, houve bradicardia de repouso nos animais T (SHR-T:317±3 e WKY-T:308±2) comparados com os animais S (SHR-S: 344±4 e WKY-S: 323±3). A permeabilidade da BHE foi reduzida e normalizada pelo T nos animais hipertensos (SHR-S: 13,6±1,2% e SHR-T: 3,8±0,4%; WKY-S: 3,9±0,2% e WKY-T: 4,1±0,16%), e análise do RT-PCR não mostrou nenhuma diferença para Claudina, PDGFRβ e Aquaporina-4 entre os T e S. A expressão gênica de Caveolina-1 estava aumentada nos SHR comparado aos WKY, e o T foi capaz de reduzir sua expressão (SHR-T: 1,05±0,1). O que foi confirmado pela expressão proteica no PVN: a Caveolina-1 encontrava-se aumentada significativamente nos SHR-S em relação aos WKY, e o T reduziu sua expressão no PVN dos SHR. Conclusão: Nossos dados sugerem que o aumento da permeabilidade da BHE no PVN de hipertensos é devida ao aumento de transcitose, identificada pela expressão de Caveolina-1 e que o treinamento aeróbio reverte esta permeabilidade ao reduzir o transporte transcelular sem alterar o transporte paracelular. / The arterial hypertension courses with autonomic dysfunction and Blood Brain Barrier (BBB) damage in areas of autonomic control. We recently demonstrated that aerobic training corrects the damage to the BBB, and autonomic dysfunction, which is correlated with the integrity of the BBB observed in trained hypertense subjects. The objective of this work is to evaluate the gene and protein expression of BBB components involved in mediating cardiovascular responses to hypertension and aerobic training (T). Spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) (250-300g) were submitted to treadmill protocol or maintained sedentary (S) for 4 weeks. At the end of the T, the animals of the experimental groups were cannulated to acquire the hemodynamic variables. Intra-arterial infusion of two dyes (Rhodamine-d, 70 KD, and FITC-d, 10 KD) and 20 min after brains were collected for fluorescence assays in the Paraventricular Nucleus of hypothalamus (PVN). Other rats from the experimental groups were perfused with transcardiacally with saline and the PVN was microdissected. The mRNA was extracted and its concentration was analyzed by the RT-PCR technique. To investigate the effects of hypertension and T in the BBB components, the following primers were used: Occludin, Claudin-5, Zonula Ocludens 1 (tight junction proteins), Caveolina-1 (indicator of transcellular transport), Laminin α 1 and Collagen-4 (basement membrane components), PDGFRβ (pericyte marker) and Aquaporin-4 (astrocyte podocyte indicator), all standardized for endogenous HPRT. Real-time PCR data were quantified by the method 2ΔΔCT. In addition, other rats from the same experimental groups were perfused with 4% paraformaldehyde for fixation of the brain. The tissue was cryoprotected and cross-sectioned, 30 m, sections were incubated on primary (Reca-1 (endothelial marker), Claudin-5, Caveolin-1, PDGFRβ and Aquaporin-4) and secondary antibodies (Alexa Fluor 488 and 594), and its quantification in thePVN was performed through the integrated density. There was no difference in blood pressure between the T and S groups, but there was resting bradycardia in the T animals (SHR-T: 317 ± 3 and WKY-T: 308 ± 2) compared to S controls (SHR-S: 344 ± 4 and WKY-S: 323 ± 3). The permeability of BBB was reduced and normalized by T in hypertensive animals (SHR-S: 13.6 ± 1.2% and SHR-T: 3.8 ± 0.4%; WKY-S: 3.9 ± 0, 2% and WKY-T: 4.1 ± 0.16%), and RT-PCR analysis showed no difference for Claudin-5, PDGFRβ and Aquaporin-4 between T and S. Caveolin-1 gene expression was increased in SHR compared to WKY, and T was able to reduce its expression (SHR-T: 1.05 ± 0.1). This was confirmed by protein expression in PVN: Caveolin-1 was significantly increased in SHR-S relative to WKY, and T reduced its expression in the PVN of SHR. Conclusion: Our data suggest that increased permeability of BBB in the PVN of hypertense individuals is due to the increase transcytosis as identified by Caveolin-1 expression and that aerobic training reverses this permeability by reducing transcellular transport without altering the paracellular transport.

A hipertensão cursa com importante déficit autonômico e lesão da barreira hematoencefálica (BHE) enquanto que o treinamento aeróbio (T) de hipertensos reduz acentuadamente a lesão da BHE, mantendo sua integridade no PVN, NTS e RVLM mesmo na persistência de níveis pressóricos elevados. Esta rápida resposta ao T (2 semanas) é condicionada pela redução da disponibilidade de ANGII nas áreas encefálicas, simultâneo aumento da expressão de podócitos dos astrócitos e desativação da microglia, os quais ocorrem simultaneamente à redução do simpático vasomotor (2 semanas) e antes mesmo do aumento da variabilidade da frequência cardíaca, da atividade parassimpática ao coração, da instalação da bradicardia de repouso e queda parcial da pressão arterial, que se instalam a partir da 4ª semana de T. Alterações na permeabilidade da BHE de hipertensos (lesão com prejuízo estrutural/funcional) e treinados (manutenção da integridade estrutural/funcional) são importantes fatores a condicionar respectivamente a disfunção autonômica na hipertensão ou a sua correção pelo treinamento. / The arterial hypertension is accompanied by important autonomic dysfunction and blood-brain barrier (BBB) lesion while aerobic training (T) in hypertension strongly decreases the BBB lesion, maintaining its integrity on the PVN, NTS and RVLM even in the persistence of high blood pressure (BP) levels. This early response to T (2 weeks) is conditioned by the reduction of ANGII availability, increased expression of astrocytic podocytes and deactivation of the microglia in brain areas. These responses occurred simultaneously with the reduction of vasomotor sympathetic activity (2 weeks) and before the increase of both heart rate variability and parasympathetic activity, resting bradycardia and partial BP fall, appearing only at the 4th week. Changes on the BBB permeability in hypertension (lesion with structural/functional damage) and trained (maintenance of the structural/ functional integrity) are important factors to condition the autonomic dysfunction in hypertension or its correction by the training, respectively.

Blood-brain barrier (BBB) is a unique membranous barrier, which segregates brain from the circulating blood. It works as a physical and metabolic barrier between the central nervous system (CNS) and periphery. In mammals, endothelial cells were shown to be of BBB and are characterized by the tight junctions along with efflux system which are responsible for the restriction of movement of molecules within the cells. Efflux system consists of multidrug resistance proteins such as P-glycoprotein (P-gp). P-gp removes substances out back from the brain to the blood before they reach to the brain. So the barrier is impermeable to many compounds such as amino acids, ions, small peptides and proteins, making it the most challenging factor for the development of new drugs for targeting CNS. Curcumin is a bioactive compound that has a number of health promoting benefits such as anti-inflammatory, anticancer, anti-oxidant agent; as well as a role in neurodegenerative diseases, but low oral bioavailability is the major limiting factor. Low water solubility and rapid metabolism are the two important factors responsible for poor bioavailability of curcumin. Galaxolide is a musk compound and previously known for the bioaccumulation of toxic components in the aquatic animals by interference with the activity of multidrug/multixenobiotic resistance efflux transporters (MDR/MXR). The bioavailability of curcumin can be enhanced when administered with galaxolide. This study was carried out to investigate the effect of galaxolide on the permeation of curcumin through the epithelial cell monolayers. MDCKII-MDR1 cell monolayer is used an in vitro blood-brain barrier model while Caco-2 monolayer is used as an in vitro intestinal model, which also expresses the P-glycoprotein. The curcumin and galaxolide were separately solubilised in the DMSO and used in combination to perform permeation study, to determine the effect of galaxolide on curcumin permeation through epithelial cell monolayers. The galaxolide shows an efflux protein inhibition activity and this activity was used to enhance permeation of curcumin through the Caco-2 monolayer. In summary, galaxolide is a novel permeation enhancer molecule, which can be used for the improvement of drug delivery of other bioactive compounds in future.

Brain diseases including neurological disorders and tumors remain undertreated due to the challenge in accessing the brain, and blood-brain barrier (BBB) restricting drug delivery, which also profoundly limits the development of pharmacological treatment. Focused ultrasound (FUS) with acoustic agents including microbubbles and nanodroplets remains as the only method to open the BBB noninvasively, locally, and transiently to assist drug delivery. For an ideal medical system to serve a broad patient population, it requires precise and flexible targeting with simulation to personalize treatment, real-time monitoring to ensure safety and effectiveness, and rapid application, as repetitive pharmacological treatment is often required. Since none of current systems fulfills all the requirements, here we designed a neuronavigation-guided FUS system with protocol assessed in in vivo mice, in vivo non-human primates, and human skulls from in silico preplanning, online FUS treatment and real-time acoustic monitoring and mapping, to post-treatment assessment using MRI. Both sedate and awake non-human primates were evaluated with total treatment time averaging 30 min and 3-mm targeting accuracy in cerebral cortex and subcortical structures. The FUS system developed would enable transcranial FUS in patients with high accuracy and independent of MRI guidance.