A sonotrombólise pela combinação de ultrassom (US) e microbolhas com medicamento trombolítico tem indicado grande eficácia na quebra de coágulos in vitro, devido a efeitos de cavitação. Contudo, estudos in vivo sobre drug delivery demonstram que a cavitação de microbolhas é também capaz de abrir local e transientemente a barreira hematoencefálica (BHE) - estrutura de permeabilidade seletiva que protege o Sistema Nervoso Central. Um estudo clínico sobre sonotrombólise foi interrompido precocemente devido a evolução de casos de Acidente Vascular Cerebral isquêmicos para hemorrágicos associados a danos na BHE e formação de ondas estacionárias. Nesta tese, foram realizados estudos in vitro e in vivo sobre técnicas de US multifrequencial para trombólise. Além disso, verificou-se os limiares para a abertura da BHE e efeitos de neuromodulação ambos causados pelo ultrassom transcraniano. Foi demonstrado que o duplo feixe de US e a variação temporal de frequências (excitação codificada) são capazes de reduzir a formação de ondas estacionárias e gerar regiões focais mais confinadas do que feixes focalizados monofrequenciais. O duplo feixe foi incapaz de gerar ondas de baixa frequência para trombólise (menor ou igual a 1 Pa para feixes primários de 1,58 MPa). Exames histológicos e por imagens de ressonância magnética mostraram que a cavitação de microbolhas pode causar danos ao tecido cerebral para níveis de pressão de mesma ordem necessários para se observar efeito trombolítico. Além disso, foi observado que o US é capaz de disparar atividade neuronal causando respostas motoras e indícios de respostas associadas a modulação de atividades cognitivas. A focalização de feixes por excitação multifrequencial é um grande avanço para sonotrombólise. Contudo, a potencialização do efeito trombolítico do US por cavitação e medicamento é limitada devido a danos a BHE e critérios de exclusão do medicamento. / Sonothrombolysis by combining ultrasound (US) and microbubbles with thrombolytic drugs has been demonstrated capable of breaking blood clots in in vitro studies, due to cavitation effects. However, in vivo drug delivery studies have demonstrated that cavitation of microbubbles is also capable of opening locally and transiently the blood-brain barrier (BBB) - structure with selective permeability that protects the Central Nervous System. A sonothrombolysis clinical study was interrupted prematurely because of the occurrence of intracerebral hemorrhages after treatment associated with damages in the BBB and standing waves formation. In this dissertation, in vitro and in vivo studies evaluated techniques of multifrequency US for thrombolysis. Furthermore, the ultrasound pressure threshold to obtain the BBB opening and neuromodulation effects were explored during transcranial insonation. It has been demonstrated that the double US beam and the time variation of frequencies (coded excitation) are capable of reducing standing wave formation and generating more confined focus zones than monofrequency focused beams. The double US beam was not capable of generating low frequency waves for thrombolysis (less than or equal to 1 Pa obtained from primary beams with 1.58 MPa). Histological exams and magnetic resonance images demonstrated that microbubbles cavitation can damage the brain tissue with acoustic pressures of the same level necessary to observe thrombolytic effects. Furthermore, it was observed motor responses and other responses associated with cognitive activity triggered by US. The capability of multifrequency excitation in focusing US beams is an important advance for sonothrombolysis. However, the enhancement of fibrinolytic effect of US by microbubbles cavitation and with thrombolytic drugs is limited by associated damages to the BBB and by exclusion criteria for the use of the thrombolytic drugs.

Compte tenu de l'absence de traitement pharmacologique efficace contre la maladie d'Alzheimer (MA), le développement d'approches thérapeutiques alternatives telles que le régime cétogène (« ketogenic diet » : KD) pourrait être envisagé. Le KD est un régime riche en graisses, basé sur la production de corps cétoniques (« ketone nodies » : KB) dans le sang. En raison des effets bénéfiques du KD sur le système nerveux central et de l'absence de données publiées sur la barrière hémato-encéphalique (BHE), nous avons utilisé une approche in vivo / in vitro pour étudier l'effet du KD et des KB sur la BHE. Pour l'étude in vivo, le sang de souris 129Sv a été récolté afin d’effectuer le dosage du beta-hydroxybutyrate et du glucose. Les capillaires cérébraux ont été isolés de cortex des souris, et des RT-qPCR ont été effectuées pour évaluer l'expression de l'ARNm des transporteurs / récepteurs impliqués dans la synthèse et le transport de KB, de glucose et du peptide bêta amyloïde. Les analyses transcriptionnelles ont été réalisées également dans un modèle in vitro de BHE, composé de cellules endothéliales dérivées de cellules souches hématopoïétiques (BLECs) en état de cétose. Après confirmation de l'intégrité des jonctions cellulaires des BLECs, Enfin, des expériences de transport de peptides beta amyloïde fluorescents après traitement avec les KBs ont été réalisées in vitro. Nos résultats montrent que les KBs modulent la physiologie de la BBB et l'expression de certains transporteurs et récepteurs du peptide bêta amyloïde, renforcent ainsi notre motivation à décrypter les mécanismes moléculaires et cellulaires au niveau vasculaire et plus précisément au niveau de la BHE. / Given the current absence of an effective pharmacologic treatment for Alzheimer’s disease (AD), the development of alternative therapeutic approaches (such as the ketogenic diet, KD) might be considered. The KD is a low-carbohydrate, high-fat diet based on the production of ketone bodies (KBs) in the blood. In view of the KD’s beneficial effects on the central nervous system and the lack of published data on the blood brain barrier (BBB), we used an in vivo/in vitro approach to investigate the effect of the KD and KBs on the BBB. For the in vivo study, blood from 129Sv mice was assayed for beta-hydroxybutyrate and glucose dosage. Brain capillaries were isolated from mouse cortices, and RT-qPCR assays were used to evaluate the mRNA expression of transporters/receptors involved in the synthesis and transport of KBs, glucose and beta-amyloid peptide. The mRNA assays were also performed in an in vitro BBB model, based on brain-like endothelial cells (BLECs). After a ketotic state had been established and the BLECs’ integrity had been confirmed, we evaluated the mRNA expression of KB-, glucose- and amyloid-beta-related genes. Lastly, the transport of fluorescently labelled beta-amyloid peptide across the BBB was studied after treatment with KBs. Our results showed that KBs modulate the physiology of the BBB by regulating the expression of certain beta-amyloid peptide transporters/receptors and amyloid peptide-synthesizing enzymes. These data suggest that it is possible to modulate key molecular players in beta-amyloid peptide transport and synthesis at the BBB, and thus open up new perspectives for studying KB-related therapeutic approaches.

The blood-brain barrier (BBB) is physiologically essential for brain homeostasis. While it protects the brain from noxious agents, it prevents almost all currently available drugs from crossing to the parenchyma. This greatly hinders drug delivery for the treatment of neurological diseases and disorders such as Parkinson’s, Alzheimer’s and Huntington’s, as well as the development of drugs for the treatment of such diseases. Current drug delivery techniques to the brain are either invasive and target specific, or non-invasive with low special specificity. Neither group of techniques are optimal for long term treatment of patients with neurological diseases or disorders. Focused ultrasound coupled with intravenous administration of microbubbles (FUS) has been proven as an effective technique to selectively and noninvasively open the BBB in multiple in vivo models including non-human primates (NHP). Although this technique has promising potential for clinical outpatient procedures, as well as a powerful tool in the lab, the safety and potential neurological effects of this technique need to be further investigated. This thesis focuses on validating the safety and efficacy of using the FUS technique to open the BBB in NHP as well as the ability of the technique to facility drug delivery. First, a longitudinal study of repeatedly applying the FUS technique targeting the basal ganglia region in four NHP was conducted to determine any potential long-term adverse side effects over a duration of 4-20 months. The safety of the technique was evaluated using both MRI as well as behavioral testing. Results demonstrated that repeated application of the FUS technique to the basal ganglia in NHP did not generate permanent side effects, nor did it induce a permanent opening of the BBB in the targeted region. The second study investigated the potential of the FUS technique as a method to deliver drugs, such as a low dose of haloperidol, to the basal ganglia in NHP and mice to elicit pharmacodynamical effects on responses to behavioral tasks. After opening the BBB in the basal ganglia of mice and NHP, a low dose of haloperidol was successfully delivered generating significant changes in their baseline motor responses to behavioral tasks. Domperidone was also successfully delivered to the caudate of NHP after opening the BBB and induced transient hemilateral neglect. In the final section of this thesis, the safety and efficacy of the FUS technique was evaluated in fully alert NHP. The FUS technique was successful in generating BBB opening volumes larger on average to that of the BBB opening volumes in anesthetized experiments. Safety results through MRI verification as well as behavioral testing during application of the technique demonstrated that the FUS technique did not generate adverse neurological effects. Conversely, the FUS technique was found to induce slight positive effects on the response of the NHP to the behavioral task. Collectively, the work presented in this thesis demonstrates the safety and effectiveness of the FUS technique to open the BBB and deliver neuroactive drugs in the NHP.

O glioblastoma multiforme (GBM) é uma doença grave e sem tratamento eficaz, especialmente pelos agentes terapêuticos disponíveis causarem reações adversas importantes nas doses terapêuticas. O metotrexato (MTX) é um fármaco citotóxico utilizado para tratar diversas neoplasias, no entanto, sua utilização é limitada pela baixa biodisponibilidade e reações adversas. A nanotecnologia tem sido utilizada para aumentar a eficácia dos medicamentos antitumorais, com o intuito de direcioná-los para o sítio de ação e reduzir os efeitos adversos. Nesse sentido, realizamos ensaios com nanocápsulas lipídicas de MTX (LNC MTX) para avaliar os mecanismos de captação em linhagens celulares de glioblastoma e micróglia, além investigar a eficácia terapêutica da LNC MTX em ensaios in vitro e in vivo. Inicialmente, ensaios de microscopia de fluorescência, empregando bloqueadores farmacológicos específicos para transportes de membrana, mostraram que as LNC MTX marcadas com Rodamina B penetram em células tumorais GL261 por endocitose, dependente de caveolinas, e em células de micróglia da linhagem BV2 por fagocitose e macropinocitose. Os tratamentos com LNC MTX ou solução de MTX (em concentrações correspondentes) em células GL261 inibiram a proliferação; aumentaram a fragmentação de DNA, mas, somente as LNC induziram a morte celular por necrose e diminuíram o número de células na fase G1/G0 do ciclo celular. Na linhagem celular BV2, os tratamentos com LNC MTX ou solução de MTX inibiram a proliferação, reduziram a quantidade de células na fase G1/G0 do ciclo celular, aumentaram a fragmentação de DNA e induziram morte celular por apoptose e apoptose tardia. Os ensaios in vivo de microscopia intravital mostraram que a LNC MTX atravessa a barreira Hematoencefálica (BHE) de camundongos fêmea C57Bl/6 após administração intravenosa ou oral, sem danificar a sua estrutura. O tamanho do glioblastoma in vivo foi reduzido em animais tratados com LNC MTX por via oral em relação aos animais tratados com salina. Esta redução não foi detectada em animais tratados com solução de MTX. Em conjunto, os dados obtidos mostram que a LNC MTX penetram em células de glioma e da glia e causam toxicidade, atravessam a BHE in vivo e sugerem que a nanoencapsulação do MTX pode ser uma estratégia importante para o tratamento do glioblastoma. / Glioblastoma multiforme (GBM) is a serious disease and no effective treatment is availabe, especially because the drugs cause significant adverse reactions in therapeutic doses. Methotrexate (MTX) is a cytotoxic drug used to treat many neoplasms, however, their use is limited by the low bioavailability and adverse reactions. Nanotechnology has been used to increase the effectiveness of antitumor drugs in order to direct them to the site of action and to reduce adverse effects. Accordingly, we carried out an experimental approach with MTX lipid nanocapsules (MTX LNC) to evaluate the uptake mechanisms in glioblastoma and microglia cell lines, and the therapeutic efficacy of MTX LNC in vitro and in vivo systems. Initially, fluorescence microscopy assays employing specific pharmacological blockers for membrane transport showed that the MTX LNC stained with Rhodamine B penetrated into GL261 tumor cells by caveolae-mediated endocytosis, and in BV2 microglia cells by phagocytosis and macropinocytosis. Treatment with MTX solution or MTX LNC (at corresponding concentrations) on GL261 cells inhibited the proliferation; increased DNA fragmentation, but only the LNC induced cell death by necrosis and decreased the number of cells in the G1/G0 phase of the cell cycle. In BV2 cells, treatment with MTX solution or MTX LNC inhibited proliferation, reduced number of cells in the G1/G0 phase of the cell cycle, increased DNA fragmentation and cell death, induced by apoptosis and late apoptosis. Intravital microscopy study showed that the MTX LNC across the Blood-Brain Barrier (BBB) of C57BL/6 female mice after intravenous or oral administrations, without damaging its structure. The area of glioblastoma in vivo was reduced in animals oral treated with MTX LNC comparing to saline treated mice. This reduction was not observed in animals treated with MTX solution. Together, the data herein obtained show that MTX LNC penetrate the cell membrane and cause cell toxicity on glioma and neurons lineage, cross the BBB and suggest that the nanoencapsulation of MTX can be an important strategy for the treatment of glioblastoma.

Small vessel disease (SVD) is an important cause of stroke, cognitive decline, and age-related disability. The cause of SVD is unknown, increasing evidence from neuropathology and neuroimaging suggests that failure of the blood-brain barrier (BBB) precipitates or worsens cerebral SVD progression and its failure is associated with SVD features such as white matter hyperintensities (WMH), perivascular spaces (PVS) and lacunar infarcts. The BBB change mechanism may also contribute to other common disorders of ageing such as Alzheimer's disease (AD). Magnetic resonance imaging (MRI) has revolutionised our understanding of SVD features. The MRI contributes to better understanding of the SVD pathophysiology and their clinical correlates. The purpose of this project was to better understand the pathogenesis of SVD, which involves improved understanding of BBB structures and pathophysiology and accurate measurement of cerebral SVD imaging characteristics on MRI scans. We aimed to assess (1) structures related to the BBB and factors that affect the BBB; (2) efficient and consistent WMH measurement method; (3) effect of stroke lesions on WMH and cerebral atrophy progression; (4) development and optimisation of computational PVS measurement method; (5) the relationships between PVS and SVD, blood markers, and BBB permeability. Section one describes structures and pathophysiology of the BBB. I reviewed the BBB structural and functional components from the view of neurovascular unit, PVS, and junctional proteins. The PVS part was done in a systematic search. I also reviewed some common stimuli for BBB permeability including inflammation and ischemia. Ischemic triggers for the BBB permeability were summarized systematically. Based on the literatures above, I summarized changes in junctional proteins in ischemia, inflammatory pain and AD models. Section two describes accurate measurement of WMH progression and atrophy. I used data from 100 patients who participated in a stroke study about BBB permeability changes in lacunar versus cortical stroke. To find a most efficient and consistent WMH measurement method, we tested several computational methods and effect of common processing steps including bias field correction and intensity adjustment. To avoid the effect of artefacts, I did a systematic search about artefacts and tested methods of image segmentation to avoid WMH artefacts as much as possible. To investigate the effect of stroke lesions on WMH and atrophy progression, I did the WMH, atrophy segmentation and stroke lesion measurements in a subgroup of 46 patients with follow-up scans, and showed that stroke lesions distorted measurement of WMH and atrophy progression and should be excluded. Section three describes development and optimization of a computational PVS measurement method, which measures the count and volume for PVS based on a threshold method using AnalyzeTM software. We tested the observer variability and validated it by comparison with visual rating scores. We investigated the associations between PVS results with other SVD features (WMH, atrophy), risk factors (hypertension, smoking and diabetes), blood markers, and BBB permeability. In conclusion, MRI is a valuable tool for the investigation of cerebral SVD features and BBB permeability. Exclusions of artefacts and stroke lesions are important in accurate measurement of WMH. PVS are important features of BBB abnormalities, and they correlate and share risk factors with other SVD features, and they should be considered as a marker of SVD and BBB permeability. Further systematic histological and ultrastructural studies of BBB are desirable in understanding the BBB regarding to the different parts of the cerebral vascular tree.

Cerebral small vessel disease (SVD) is a vascular neurodegenerative disease which is the leading cause of vascular dementia and causes 20% of strokes. 20-30% of those over 80 show signs of the disease as white matter hyperintensities on MRI scans, doubling their risk of stroke and trebling their risk of dementia. Sporadic SVD is thought to be caused by hypertension but 30% of sufferers are normotensive and an alternative hypothesis implicates loss of integrity of the blood brain barrier (BBB). To investigate this, I studied brains from normotensive people with early stage SVD and found reduced capillary endothelial claudin-5 (a BBB tight junction protein), more oligodendrocyte precursor cells (OPCs; the precursors to myelinating oligodendrocytes), and more microglia/macrophages compared to controls. Furthermore, in a relevant rat model of spontaneous SVD, the Stroke Prone Spontaneously Hypertensive Rat (SHRSP; disease model; DM) I found that reduced endothelial claudin-5 was the earliest change, appearing at 3 weeks of age, followed by OPC proliferation, appearing at 4 weeks, and then increased number of microglia/macrophages, appearing at 5 weeks. Importantly, all these changes occurred at a young age (< 5 weeks), before any measurable hypertension. These changes were confirmed in an ex vivo slice culture model (i.e. removing blood flow), ruling out direct damage by leakage of blood components through an impaired BBB and suggesting an inherent endothelial cell dysfunction as the primary cause, with secondary BBB defects. This hypothesis of endothelial dysfunction is supported by increased endothelial cell proliferation in both human SVD tissue and the DM rats, and lower levels of endothelial nitric oxide synthase (eNOS) in brains of DM rats. To study this further I isolated primary brain microvascular endothelial cells (BMECs) from DM and control rats and found that those from DM rats formed less mature tight junctions (less membranous claudin-5) than control BMECs. I also found that conditioned media (CM) from DM BMECs causes OPCs in culture to proliferate more and mature less. This indicates that the endothelial dysfunction is inherent to the endothelial cells, rather than induced by other cell types, and through secreted factors causes OPC changes mirroring what is seen in vivo. Using an antibody array, I identified HSP90α as a candidate secreted factor and showed that it is necessary (by blocking the protein in CM) and sufficient (by adding recombinant HSP90α) to induce the maturation phenotype in OPCs, but not the proliferation phenotype. The idea that endothelial dysfunction causes SVD begs the question of what causes endothelial dysfunction, especially in our inbred DM rat strain. To establish this, I reanalysed sequencing data of the DM and control rats from a previously published study, searching for mutations which lead to truncated proteins in genes expressed in brain endothelial cells. We confirmed the candidate gene Atp11b, a phospholipid flippase, was mutated as predicted. I found that knocking down Atp11b using siRNA in a control endothelial cell line caused endothelial dysfunction and a loss of tight junction maturity, and that CM from these cells causes OPCs to proliferate more and mature less, mirroring what we see in primary DM BMECs and suggesting that Atp11b has a key function in promoting normal endothelial function. Furthermore, I showed that knocking down Atp11b causes cells to secrete increased levels of HSP90α. I propose a mechanism whereby ATP11B regulates the retention of HSP90α within endothelial cells, which in turns regulates eNOS levels and activity, as has been shown previously. In summary, this work shows that there are many pre-symptomatic changes which occur in the brain in the development of SVD in DM rats, and that these are ultimately caused by endothelial dysfunction. As these changes are similar to those found in spontaneous human SVD, I propose that endothelial dysfunction is a key mechanism of human SVD, which may in the future lead to new therapies.

Sepse e suas complicações (sepse grave e choque séptico) ainda são a principal causa de morte nas unidades de terapia intensiva em todo o mundo. Estudos clínicos e experimentais têm demonstrado que na fase inicial da sepse a concentração plasmática de arginina vasopressina (AVP) está elevada. No entanto, durante o processo fisiopatológico os níveis plasmáticos da mesma permanecem inadequadamente baixos, apesar de haver hipotensão persistente. Uma das hipóteses sugeridas para essa deficiência relativa de AVP é a apoptose de neurônios vasopressinérgicos. Nosso objetivo foi identificar elementos envolvidos na morte celular hipotalâmica, além de avaliar o comportamento de células gliais e da barreira hematoencefálica (BHE) durante a sepse. Ratos Wistar foram submetidos à sepse por ligadura e punção cecal (CLP) ou não manipulados (naive) como controle e então divididos em dois grupos. No primeiro, foram perfundidos e os cérebros coletados para imunohistoquímica. Outro grupo foi decapitado para a retirada de sangue para dosagem de interferon- gama (IFN-?) e encéfalo para análise da expressão de proteínas no hipotálamo ou nos núcleos supraópticos (SON) e paraventriculares (PVN). Um terceiro foi separado para investigação da permeabilidade da BHE. Apesar de aumento da imunomarcação de CD8 e MHC-I no SON dos animais sépticos, não encontramos indícios de morte celular mediada por células imunes. No SON e PVN de animais sépticos, a expressão de fatores envolvidos na ativação da via extrínseca de apoptose (tBID, caspase-8 clivada) se manteve inalterada, enquanto fatores anti-apoptóticos relacionados à via intrínseca (BCL-2, BCL-xL) estavam diminuídos no hipotálamo. No SON destes animais a micróglia assumiu uma morfologia associada à sua ativação, concomitante com o aumento plasmático de IFN-?. Houve rompimento transitório da BHE no hipotálamo após 6 horas do CLP. Os resultados indicam que a via intrínseca de apoptose parece ser a responsável pela morte celular que é observada nos núcleos vasopressinérgicos e essa condição está temporalmente associada à ativação microglial e rompimento da BHE / Sepsis and its complications (severe sepsis and septic shock) remain as the main cause of death in intensive care units worldwide. Clinical and experimental studies have shown that in the early phase of sepsis the plasma concentration of arginine vasopressin (AVP) is increased. However, during the pathophysiological process the plasma levels remain inadequately low, despite of persistent hypotension. One of the hypothesis suggested for this relative deficiency is the apoptosis of vasopressinergic neurons. Our objective was to identify elements involved in the hypothalamic cellular death and evaluate the modifications of glial cells and blood-brain-barrier (BBB) during sepsis. Wistar rats were submitted to sepsis by cecal ligation and puncture (CLP) or non-manipulated (naïve), as control and then divided in two groups. In the first one, they were perfused and brains were collected for immunohistochemistry. In another one they were decapitated for blood collection and further plasma interferongama (IFN-?) analysis by ELISA. Brain was also collected for apoptosis-related proteins expression analysis in the hypothalamus or in the supraoptic (SON) and paraventricular (PVN) nuclei. A third set was separated for the investigation of BBB permeability. Despite of increased immunostaining for CD8 and MHC-I in the SON of septic animals, we did not find evidence of cell death mediated by immune cells. In the SON and PVN of septic animals, the expression of proteins involved in the activation of the extrinsic apoptosis pathway (tBID, cleaved caspase-8) was not altered, whereas anti-apoptotic factors related to the intrinsic pathway (BCL-2, BCLxL) were decreased. In the SON of these animals, microglia assumed a morphology related to its activation, associated with the increase of plasma IFN-?. There was a transitory breakdown of BBB in hypothalamus after 6 hours following CLP. The results indicate that the intrinsic apoptosis pathway seems to be responsible for the cell death observed in vasopressinergic nuclei and this condition is temporally associated with microglial activation and BBB leaking

Devant l'impact des maladies neurodégénératives sur la société, les thérapies par ultrasons focalisés apparaissent comme des techniques prometteuses combinant non invasivité, précision spatiale millimétrique et capacité d'atteindre les structures profondes du cerveau. Cependant, des travaux sont encore nécessaires pour renforcer les effets de la neuromodulation, comprendre les mécanismes sous-jacents et contrôler la sûreté de la technique avant d'entreprendre des essais cliniques. Dans cette thèse, la propagation des ultrasons dans le cerveau de rongeurs et de singes a été étudiée numériquement afin d'estimer l'intensité acoustique dans le cerveau, la répartition spatiale des ondes dans la boîte crânienne et l'élévation de température. Afin d'évaluer physiologiquement les effets des ultrasons à l'échelle cellulaire, l'activité de neurones uniques a été mesurée sur des macaques éveillés pendant une neuromodulation ultrasonore. Puis, la durée de l’effet de modulation a été augmentée grâce à une prolongation du tir sur des singes exécutant une tâche visuelle. L'imagerie fonctionnelle par IRM a ensuite permis de faire ressortir des changements de connectivité entre l'aire stimulée et des régions du cerveau éloignées.Enfin, les avantages de la neurostimulation par ultrasons ont été combinés avec l'efficacité d'un agent neuroactif. En utilisant des microbulles conjointement aux ultrasons, la barrière hémato-encéphalique a été ouverte localement et réversiblement dans le cortex visuel de macaques anesthésiés pour permettre le passage d'un neurotransmetteur inhibiteur dans le cerveau. La baisse d'amplitude des réponses EEG du cortex visuel à des stimuli démontre la faisabilité de la délivrance locale et non invasive de neuromodulateurs dans le cerveau. Ainsi, les paramètres ultrasonores ont été optimisés grâce aux simulations numériques et à des expériences in vivo pour renforcer les effets de neuromodulation tout en contrôlant les effets indésirables, avec l'objectif de se diriger vers des applications thérapeutiques et proposer de nouveaux outils pour des études de connectivité cérébrale / Considering the extent of neurodegenerative diseases consequences on the society, focused ultrasound appears as a promising technique combining non-invasiveness, millimetric spatial accuracy and ability to reach deep brain structures. However, efforts still need to be made to amplify the effects of focused ultrasound neuromodulation, understand its mechanism and control the safety of the technique before moving towards human trials.The ultrasound propagation inside the brain of rodents and monkeys was first studied numerically to estimate the maximum intensity in the brain, the pressure distribution in the skull cavity and the thermal rise. To evaluate physiologically the ultrasound effects at the cellular level, the activity of individual neurons was measured on awake macaques during ultrasonic neuromodulation. To further increase the duration of the modulation, a longer sonication was tested successfully on macaques performing a visual task. Functional MRI was then used to highlight the connectivity changes between the stimulated area and distant cerebral regions. Finally, the advantages of ultrasound neurostimulation were combined with the efficiency of a neuroactive agent (GABA). Using microbubbles and ultrasound, the blood brain barrier was opened locally and reversibly in the visual cortex of anesthetized macaques to deliver an inhibitory neurotransmitter in the brain. The amplitude of the EEG response of the visual cortex to stimuli decreased after GABA injection, demonstrating the feasibility of delivering neuroactive drugs non-invasively and locally to any brain region.Overall, ultrasound parameters were optimized with both numerical tools and in vivo experiments to amplify neuromodulation effects while controlling the safety. This work opens the way to the development of novel therapeutic applications and new tools for connectivity studies

Stroke is the 5th leading cause of death and the leading cause of disability in the United States, but there are only two available therapies, tissue plasminogen activator and endovascular thrombectomy. As both therapies focus on removal of the clot, the subsequent pathologic processes, i.e. inflammation, cerebrovascular breakdown, ATP depletion, etc. are left untreated, contributing to worsened patient outcome. Many clinical trials have unsuccessfully attempted to address these mechanisms. The blood-brain barrier (BBB), a system of non-fenestrated endothelial cells, extracellular matrix, and astrocytic endfeet, is significantly impacted after ischemic stroke in its role of preventing the free movement of proteins from the blood into the brain. In fact, BBB dysfunction is viewed as one of the major facilitators of damage following ischemic stroke, leading to increased infarct volumes and worsened patient outcomes. Interestingly, a family of endothelial integrins, the b1 integrins, have been shown to regulate tight junction proteins preventing the free movement of molecules. When expression of the tight junctions are decreased, this results in increased BBB permeability. To test this concept, our laboratory has previously shown the knockout of the particular β1 integrin, α5β1, is neuroprotective following ischemic stroke through BBB stabilization. To determine if therapeutically targeting integrin a5b1 was feasible, we first determined if brain integrin a5b1 expression increases after experimental mouse ischemic stroke model, specifically tandem/transient common carotid artery/middle cerebral artery occlusion. We found that integrin a5b1 does increase acutely, by post-stroke day (PSD)2, and continued in an exponential fashion through PSD4. Next, we determined if integrin a5b1 was therapeutically accessible by systemic treatment (i.e. intraperitoneal or intravenous) by being located on the inside (luminal surface) of vasculature. We found that location of integrin a5b1 was dependent on the area relative to the stroke injury. The core, or area of direct impact, demonstrated expression of integrin a5b1 on the outside vasculature (abluminal surface), while per-infarct expression was localized to the lumen. Lastly, to determine the activity of integrin a5b1 following ischemic stroke, we showed that the potential ligands (binding partners), plasma fibronectin, fibrinogen, and amyloid-b, do not bind integrin a5b1 after ischemic stroke. Next, we determined the therapeutic potential of targeting integrin a5b1 with the small peptide, ATN-161. ATN-161 has undergone clinical trials in solid tumors, with limited side effects reported. First, we determined that intraperitoneal (IP) injection of ATN-161 was safe after ischemic stroke, showing no changes in heart rate, pulse distention (blood pressure), or body temperature. Next, we found that IP administration of ATN-161 after experimental ischemic stroke reduced infarct volumes, edema, and functional deficit. Furthermore, these results were due to reduction of BBB permeability and anti-inflammatory effects. Interestingly, ATN-161 reduced cytokine production, prevented leukocyte infiltration, and leukocyte recruitment. Collectively, these results suggest that targeting integrin a5b1 with ATN-161 is 1) feasible, 2) safe and 3) effective, suggesting that ATN-161 may be a novel therapeutic treatment for ischemic stroke.

Human immunodeficiency (HIV) infection can result in neurocognitive deficits in about one-half of infected individuals. Despite excellent systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting HIV infection within the central nervous system (CNS). Drug abuse exacerbates cognitive impairment and pathologic CNS changes in HIV-infected individuals. This work investigates the effects of the HIV-1 protein, Tat, and drugs of abuse on factors affecting drug penetration into the brain. Firstly, an in vitro model of the blood-brain barrier was built to study effects of HIV-1 Tat and methamphetamine (Meth) on integrity and function of the BBB, in turn how HIV-1 Tat and meth will affect antiretroviral penetration into the brain. We found that co-exposure HIV-1 Tat and Meth results in inhibition or impairment of P-glycoprotein activity at the BBB. Also, simultaneous inhibition of P-glycoprotein (P-gp) and Multidrug Resistant Protein -1 (MRP-1), by verapamil and MK-571 causes an increase in accumulation of atazanavir inside the primary human brain endothelial cells. Secondly, we developed and validated the method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell extracts of CNS cells. This method was used to study how HIV-1 Tat and/or morphine affects antiretroviral penetration in CNS cells like human brain microvascular endothelial cells, human astrocytes, human microglia, and human pericytes. We found that in untreated cells, accumulation of antiretroviral drugs was higher in hCMEC/D3 cells compared to other CNS cell types. Also, HIV-1 Tat and/or morphine had no significant effect on antiretroviral penetration amongst these cell types. Overall, the rank order of intracellular accumulation observed in treated and untreated cells was dolutegravir > emtricitabine > tenofovir.