Glutamate-mediated blood-brain barrier opening. implications for neuroprotection and drug delivery

The blood-brain barrier is a highly selective anatomical and functional interface allowing a unique environment for neuro-glia networks. Blood-brain barrier dysfunction is common in most brain disorders and is associated with disease course and delayed complications. However, the mechanisms underlying blood-brain barrier opening are poorly understood. Here we demonstrate the role of the neurotransmitter glutamate in modulating early barrier permeability in vivo Using intravital microscopy, we show that recurrent seizures and the associated excessive glutamate release lead to increased vascular permeability in the rat cerebral cortex, through activation of NMDA receptors. NMDA receptor antagonists reduce barrier permeability in the peri-ischemic brain, whereas neuronal activation using high-intensity magnetic stimulation increases barrier permeability and facilitates drug delivery. Finally, we conducted a double-blind clinical trial in patients with malignant glial tumors, using contrast-enhanced magnetic resonance imaging to quantitatively assess blood-brain barrier permeability. We demonstrate the safety of stimulation that efficiently increased blood-brain barrier permeability in 10 of 15 patients with malignant glial tumors. We suggest a novel mechanism for the bidirectional modulation of brain vascular permeability toward increased drug delivery and prevention of delayed complications in brain disorders. SIGNIFICANCE STATEMENT: In this study, we reveal a new mechanism that governs blood-brain barrier (BBB) function in the rat cerebral cortex, and, by using the discovered mechanism, we demonstrate bidirectional control over brain endothelial permeability. Obviously, the clinical potential of manipulating BBB permeability for neuroprotection and drug delivery is immense, as we show in preclinical and proof-of-concept clinical studies. This study addresses an unmet need to induce transient BBB opening for drug delivery in patients with malignant brain tumors and effectively facilitate BBB closure in neurological disorders

Review of photoacoustic imaging plus X

Photoacoustic imaging (PAI) is a novel modality in biomedical imaging technology that combines the rich optical contrast with the deep penetration of ultrasound. To date, PAI technology has found applications in various biomedical fields. In this review, we present an overview of the emerging research frontiers on PAI plus other advanced technologies, named as PAI plus X, which includes but not limited to PAI plus treatment, PAI plus new circuits design, PAI plus accurate positioning system, PAI plus fast scanning systems, PAI plus novel ultrasound sensors, PAI plus advanced laser sources, PAI plus deep learning, and PAI plus other imaging modalities. We will discuss each technology's current state, technical advantages, and prospects for application, reported mostly in recent three years. Lastly, we discuss and summarize the challenges and potential future work in PAI plus X area

Development of an Awake Behaving model for Laser Doppler Flowmetry in Mice

Bien que le cerveau ne constitue que 2% de la masse du corps chez les humains, il présente l’activité métabolique la plus élevée dans le corps, et en conséquence, constitue un organe hautement vascularisé. En fait, l’approvisionnement en sang dans le cerveau est strictement modulé au niveau régional par un mécanisme fondamental nommé couplage neurovasculaire (CNV), qui associe les besoins métaboliques locaux au flux sanguin cérébral [1, 2]. Notre compréhension du CNV sous des conditions physiologiques et pathologiques a été améliorée par un large éventail d’études menées chez les rongeurs. Néanmoins, ces études ont été réalisées soit sous anesthésie, soit chez la souris éveillée et immobilisée, afin d’éviter le mouvement de la tête pendant l'acquisition de l'image. Les anesthésiques, ainsi que le stress induit par la contention, peuvent altérer l'hémodynamique cérébrale, ce qui pourrait entraver les résultats obtenus. Par conséquent, il est essentiel de contrôler ces facteurs lors de recherches futures menées au sujet de la réponse neurovasculaire. Au cours de l’étude présente, nous avons développé un nouveau dispositif pour l'imagerie optique éveillée, où la tête de la souris est immobilisée, mais son corps est libre de marcher, courir ou se reposer sur une roue inclinée. En outre, nous avons testé plusieurs protocoles d'habituation, selon lesquels la souris a été progressivement entraînée pour tolérer l’immobilisation de tête, afin de minimiser le stress ressenti lors des sessions d'imagerie. Enfin, nous avons, pour la première fois, cherché à valider l'efficacité de ces protocoles d'habituation dans la réduction du stress, en mesurant l'évolution des taux plasmatiques de corticostérone tout au long de notre étude. Nous avons noté que les souris s'étaient rapidement adaptées à la course sur la roue et que les signes visibles de stress (luttes, vocalisations et urination) étaient nettement réduits suite à deux sessions d'habituation. Néanmoins, les taux de corticostérone n'ont pas été significativement réduits chez les souris habituées, par rapport aux souris naïves qui ont été retenues sur la roue sans entraînement préalable (p> 0,05). Ce projet met en évidence la nécessité d'une mesure quantitative du stress, car une réduction des comportements observables tels que l'agitation ou la lutte peut être indicative non pas d'un niveau de stress plus faible, mais plutôt d'un désespoir comportemental. Des recherches supplémentaires sont nécessaires pour déterminer si la fixation de la tête lors de l'imagerie optique chez la souris peut être obtenue avec des niveaux de stress plus faibles, et si le stress induit par la contrainte effectuée avec notre dispositif est associé à des changements de la réponse hémodynamique.Whilst the brain only constitutes 2% of total body weight in humans, it exhibits the highest metabolic activity in the body, and as such is a highly vascularized organ. In fact, regional blood supply within the brain is strictly modulated through a fundamental process termed neurovascular coupling (NVC), which couples local metabolic needs with cerebral blood flow [1, 2]. A wide array of optical imaging studies in rodents has enhanced our understanding of NVC under physiological and pathological conditions. Nevertheless, these studies have been performed either under anesthesia, or in the awake mouse using restraint to prevent head-motion during image acquisition. Both anesthetics and restraint-induced stress have been clearly shown to alter cerebral hemodynamics, thereby potentially interfering with the obtained results [3, 4]. Hence, it is essential to control for these factors during future research which investigates the neurovascular response. In the present study, we have developed a new apparatus for awake optical imaging, where the mouse is head-restraint whilst allowed to walk, run or rest on an inclined wheel. In addition, we have tested several habituation protocols, according to which the mouse was gradually trained to tolerate head-restraint, in order to minimize the stress experienced during imaging sessions. Lastly, we have, for the first time, sought to validate the efficiency of these habituation protocols in reducing stress, by measuring the evolution of plasma corticosterone levels throughout the study. We noted that the mice had quickly adapted to running on the wheel, and that the overt signs of stress (struggling, vocalizations and urination) were clearly reduced within two habituation sessions. Nevertheless, corticosterone levels were not significantly reduced in habituated mice, relative to naïve mice that were restrained on the wheel without prior training (p > 0.05). This project highlights the necessity for a quantitative measure of stress, as a reduction in observable behaviors such as agitation or struggling may be indicative not of lower stress, but rather, of behavioral despair. Further research is needed to determine whether head-fixation during optical imaging in mice can be achieved with lower stress levels, and if restraint-induced stress using our apparatus is associated with changes in the hemodynamic response

DEVELOPEMENT OF WIDEFIELD MULTI-CONTRAST OPTICAL METHODS FOR IN VIVO MICROVASCULAR SCALE IMAGING

Traditional in vivo optical imaging methods rely on a single contrast mechanism, thereby limiting one’s ability to characterize more than one biological variable. However, most biological systems are complex and are comprised of multiple variables. Therefore, optical methods that employ multiple contrast mechanisms and are capable of visualizing multiple biological variables would permit a more comprehensive understanding of biological systems. Multi-contrast optical imaging, therefore, has great potential for both fundamental and applied biomedical research. The goal of this dissertation is to develop optical methods to enable multi-contrast imaging in vivo over a wide field of view while retaining a microvascular scale spatial resolution. We present the integration of three types of optical imaging contrast mechanisms: fluorescence (FL), intrinsic optical signals (IOS) and laser speckle contrast (LSC). Fluorescence enables tracking pre-labelled molecules and cells, IOS allow quantification of blood volume and/or intravascular oxygen saturation, and LSC permits assessment of tissue perfusion. Together, these contrast mechanisms can be harnessed to provide a more complete picture of the underlying physiology at the microvascular spatial scale. We developed two such microvascular resolution optical multi-contrast imaging methods, and demonstrated their utility in multiple biomedical applications. First, we developed a multi-contrast imaging system that can interrogate in vivo both neural activity and its corresponding microvascular scale hemodynamics in the brain of a freely moving rodent. To do this, we miniaturized an entire benchtop optical imaging system that would typically occupy 5 x 5 x 5 feet, into just 5 cm3. Our miniaturized microscope weighs only 9 g. The miniature size and light weight permitted us to mount our microscope on a rodent’s head and image brain activity in vivo with multiple contrast mechanisms. We used our microscope to study the functional activation of the mouse auditory cortex, and to investigate the alteration of brain function during arousal from deep anesthesia. Our miniaturized microscope is the world’s first rodent head-mountable imaging system capable of interrogating both neural and hemodynamic brain activity. We envision our microscope to usher an exciting new era in neuroscience research. Second, we developed an optical imaging system to extensively characterize microvascular scale hemodynamics in vivo in an orthotopic breast tumor model. We specifically designed it as a benchtop based system to allow ample space for surgical preparation and small animal manipulation. Using it, we continuously monitored in vivo microvascular scale changes in tissue perfusion, blood volume and intravascular oxygen saturation of an orthotopic breast tumor microenvironment for multiple hours over a field of view encompassing the entire tumor extent. This unique dataset enabled us for the first time to characterize the temporal relationship between different tumor hemodynamic variables at the scale of individual microvessels. We envision our work to inspire a whole new avenue of experimental cancer research where the role of a tumor’s hemodynamic microenvironment is extensively characterized at its native (i.e. microvascular) spatial scale. In summary, this dissertation describes the design, implementation and demonstration of two microvascular resolution, wide-field, multi-contrast optical imaging systems. We believe these methods to be a new tool for broadening our understanding of biology

MULTI-MODAL OPTICAL NEUROIMAGING AND APPLICATIONS

Optical imaging tools provide superior details than MRI, PET in monitoring the physiological and pathological state of brain in preclinical models. By combining different optical imaging modalities, a variety of physiological parameters (e.g. cerebral hemodynamics, metabolism and neuronal activity) could be detected simultaneously; such simultaneous imaging is expected to profoundly enhance our understanding of normal brain regulation and its disruption from neurovascular disorders. As a part of this thesis, I designed a multi-modal optical imaging system that could perform simultaneous laser speckle contrast imaging, wide-field fluorescence imaging and optical intrinsic signal imaging. The principles, processing methods and applications of these imaging modalities are presented in Chapter 1. The system was first applied to study a new atherothrombotic stroke model and to evaluate the recovery of stroke from different treatment protocols in mice (Chapter 2). Cerebral blood flow changes and thrombus formations were imaged by laser speckle contrast imaging and wide-field fluorescence imaging, respectively. We concluded that the combination treatment of tissue plasminogen activator and cathepsin G inhibitor improved the neurological outcomes of ischemic brain injury from induced atherothrombotic stroke. To investigate brain activity in high-resolution by optical imaging tools, cranial window preparation is an essential procedure to allow optical access to the brain. We also employed the optical imaging system to investigate the effects of cranial windows on monitoring neurovasculature by laser speckle contrast imaging (Chapter 3). Open-skull and thin-skull cranial window procedures were performed in separate experiments, and the neurovasculature underlying the cranial windows were monitored for fourteen days. The differences between two window types were systematically compared by parameters such as contrast-to-noise ratio and microvessel density. Finally, the last part of my thesis was to miniaturize the multi-modal bench-top imaging system to a head-mounted microscope, which allows imaging on awake freely moving animals. The natural physiological state of brain activities can be detected without the confounding effects of anesthetics. The current version of the microscope weighs less than 5 g and is able to perform laser speckle contrast imaging, wide-field fluorescence imaging and optical intrinsic signal imaging simultaneously. We are currently testing the miniaturized microscope to study a brain tumor murine model. Finally, I describe the current progress of miniaturized optical neuroimaging systems on awake moving animals in Chapter 4 of this thesis

Multi-scale mapping along the auditory hierarchy using high-resolution functional UltraSound in the awake ferret

A major challenge in neuroscience is to longitudinally monitor whole brain activity across multiple spatial scales in the same animal. Functional UltraSound (fUS) is an emerging technology that offers images of cerebral blood volume over large brain portions. Here we show for the first time its capability to resolve the functional organization of sensory systems at multiple scales in awake animals, both within small structures by precisely mapping and differentiating sensory responses, and between structures by elucidating the connectivity scheme of top-down projections. We demonstrate that fUS provides stable (over days), yet rapid, highly-resolved 3D tonotopic maps in the auditory pathway of awake ferrets, thus revealing its unprecedented functional resolution (100/300µm). This was performed in four different brain regions, including very small (1–2 mm3 size), deeply situated subcortical (8 mm deep) and previously undescribed structures in the ferret. Furthermore, we used fUS to map long-distance projections from frontal cortex, a key source of sensory response modulation, to auditory cortex

Review

Functional ultrasound (fUS) is a hemodynamic-based functional neuroimaging technique, primarily used in animal models, that combines a high spatiotemporal resolution, a large field of view, and compatibility with behavior. These assets make fUS especially suited to interrogating brain activity at the systems level. In this review, we describe the technical capabilities offered by fUS and discuss how this technique can contribute to the field of functional connectomics. First, fUS can be used to study intrinsic functional connectivity, namely patterns of correlated activity between brain regions. In this area, fUS has made the most impact by following connectivity changes in disease models, across behavioral states, or dynamically. Second, fUS can also be used to map brain-wide pathways associated with an external event. For example, fUS has helped obtain finer descriptions of several sensory systems, and uncover new pathways implicated in specific behaviors. Additionally, combining fUS with direct circuit manipulations such as optogenetics is an attractive way to map the brain-wide connections of defined neuronal populations. Finally, technological improvements and the application of new analytical tools promise to boost fUS capabilities. As brain coverage and the range of behavioral contexts that can be addressed with fUS keep on increasing, we believe that fUS-guided connectomics will only expand in the future. In this regard, we consider the incorporation of fUS into multimodal studies combining diverse techniques and behavioral tasks to be the most promising research avenue

Developing Next-generation Brain Sensing Technologies - A Review.

Advances in sensing technology raise the possibility of creating neural interfaces that can more effectively restore or repair neural function and reveal fundamental properties of neural information processing. To realize the potential of these bioelectronic devices, it is necessary to understand the capabilities of emerging technologies and identify the best strategies to translate these technologies into products and therapies that will improve the lives of patients with neurological and other disorders. Here we discuss emerging technologies for sensing brain activity, anticipated challenges for translation, and perspectives for how to best transition these technologies from academic research labs to useful products for neuroscience researchers and human patients