A systematic review of neuroprotective strategies after cardiac arrest: from bench to bedside (Part I - Protection via specific pathways).

Neurocognitive deficits are a major source of morbidity in survivors of cardiac arrest. Treatment options that could be implemented either during cardiopulmonary resuscitation or after return of spontaneous circulation to improve these neurological deficits are limited. We conducted a literature review of treatment protocols designed to evaluate neurologic outcome and survival following cardiac arrest with associated global cerebral ischemia. The search was limited to investigational therapies that were utilized to treat global cerebral ischemia associated with cardiac arrest. In this review we discuss potential mechanisms of neurologic protection following cardiac arrest including actions of several medical gases such as xenon, argon, and nitric oxide. The 3 included mechanisms are: 1. Modulation of neuronal cell death; 2. Alteration of oxygen free radicals; and 3. Improving cerebral hemodynamics. Only a few approaches have been evaluated in limited fashion in cardiac arrest patients and results show inconclusive neuroprotective effects. Future research focusing on combined neuroprotective strategies that target multiple pathways are compelling in the setting of global brain ischemia resulting from cardiac arrest

Cerebral Hemodynamic Disturbances in Motor Neuron Disease

An association between motor neuron disease (MND) and dementia was first realized in the late 1800s, yet substantiating research and a description of dementia as part of the clinical syndrome would not appear until the 1990s. In the last two decades, medical imaging has investigated cerebral blood flow changes in the motor and nonmotor cortex to correlate with motor dysfunction and clinical dementia, respectively. The aim of this thesis is to describe early cerebral hemodynamic disturbances with the goal to determine a marker for cognitive decline in MND. Chapter 2 describes the relationship between changes in cerebral hemodynamics and cognition in primary lateral sclerosis (PLS) patients compared to normal controls. Neuropsychological testing revealed subtle frontotemporal changes characterized by executive dysfunction that were associated with global increases in mean transit time (MTT) in grey and white matter, and increased cerebral blood volume (CBV) in the frontotemporal grey matter. Chapter 3 presents a longitudinal clinical study of early cerebral hemodynamic changes in amyotrophic lateral sclerosis (ALS) patients without evidence of cognitive impairment at study onset. This Chapter characterized the relationship between duration of disease and MTT in the cortical grey matter. MTT was found to be the most sensitive indicator of early cerebral hemodynamic change accompanying disease progression in ALS. Furthermore, these findings corroborate the trend of increased MTT in the absence of cognitive impairment found in PLS patients in Chapter 2, and may further indicate that hemodynamic changes may occur before the onset of cognitive impairment. in The aim of Chapter 4 was to elucidate a biological mechanism for increased MTT described in the previous Chapters 2 and 3. A rabbit model of global hypotension was used to demonstrate that MTT is an indicator of cerebral perfusion pressure (CPP). A spectrum of cognitive dysfunction has now been described in MND. The use of sensitive neuropsychological testing has enabled us to identify patients with mild changes in cognitive function from those who are cognitively intact. With the help of this stratification, we were able to show that changes in MTT was associated with disease progression and cognitive impairment. The experimental data presented in this thesis suggest that vascular factors may contribute to cognitive dysfunction in MND

Cerebral Hemodynamic Disturbances in Motor Neuron Disease

An association between motor neuron disease (MND) and dementia was first realized in the late 1800s, yet substantiating research and a description of dementia as part of the clinical syndrome would not appear until the 1990s. In the last two decades, medical imaging has investigated cerebral blood flow changes in the motor and non­ motor cortex to correlate with motor dysfunction and clinical dementia, respectively. The aim of this thesis is to describe early cerebral hemodynamic disturbances with the goal to determine a marker for cognitive decline in MND. Chapter 2 describes the relationship between changes in cerebral hemodynamics and cognition in primary lateral sclerosis (PLS) patients compared to normal controls. Neuropsychological testing revealed subtle frontotemporal changes characterized by executive dysfunction that were associated with global increases in mean transit time (MTT) in grey and white matter, and increased cerebral blood volume (CBV) in the frontotemporal grey matter. Chapter 3 présents a longitudinal clinical study of early cerebral hemodynamic changes in amyotrophie lateral sclerosis (ALS) patients without evidence of cognitive impairment at study onset. This Chapter characterized the relationship between duration ofdiseaseandMTTinthecorticalgreymatter. MTTwasfoundtobethemostsensitive indicator of early cerebral hemodynamic change accompanying disease progression in ALS. Furthermore,thesefindingscorroboratethetrendofincreasedMTTintheabsence of cognitive impairment found in PLS patients in Chapter 2, and may further indicate that hemodynamic changes may occur before the onset of cognitive impairment. iii The aim of Chapter 4 was to elucidate a biological mechanism for increased MTT described in the previous Chapters 2 and 3. A rabbit model of global hypotension was used to demonstrate that MTT is an indicator of cerebral perfusion pressure (CPP). A spectrum of cognitive dysfunction has now been described in MND. The use of sensitive neuropsychological testing has enabled us to identify patients with mild changes in cognitive function from those who are cognitively intact. With the help of this stratification, we were able to show that changes in MTT was associated with disease progression and cognitive impairment. The experimental data presented in this thesis suggest that vascular factors may contribute to cognitive dysfunction in MND

Role of Astrocytes in Neurovascular Coupling

Neural activity is intimately tied to blood flow in the brain. This coupling is specific enough in space and time that modern imaging methods use local hemodynamics as a measure of brain activity. In this review, we discuss recent evidence indicating that neuronal activity is coupled to local blood flow changes through an intermediary, the astrocyte. We highlight unresolved issues regarding the role of astrocytes and propose ways to address them using novel techniques. Our focus is on cellular level analysis in vivo, but we also relate mechanistic insights gained from ex vivo experiments to native tissue. We also review some strategies to harness advances in optical and genetic methods to study neurovascular coupling in the intact brain

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

Impact of Subarachnoid Hemorrhage on Astrocyte Calcium Signaling: Implications for Impaired Neurovascular Coupling

Deficits within the brain microcirculation contribute to poor patient outcome following aneurysmal subarachnoid hemorrhage (SAH). However, the underlying pathophysiology is not well understood. Intra-cerebral (parenchymal) arterioles are encased by specialized glial processes, called astrocyte endfeet. Ca2+ signals in the endfeet, driven by the ongoing pattern of neuronal activity, regulate parenchymal arteriolar diameter and thereby influence local cerebral blood flow. In the healthy brain, this phenomenon, called neurovascular coupling (NVC), matches focal increases in neuronal activity with local arteriolar dilation. This ensures adequate delivery of oxygen and other nutrients to areas of the brain with increased metabolic demand. Recently, we demonstrated inversion of NVC from vasodilation to vasoconstriction in brain slices obtained from SAH model animals. This pathological change, which would restrict blood flow to active brain regions, was accompanied by an increase in the amplitude of spontaneous Ca2+ events in astrocyte endfeet. It is possible that the emergence of higher amplitude endfoot Ca2+ events shifts the polarity of NVC after SAH by elevating levels of vasoactive agents (e.g. K+ ions) within the perivascular space. In the first aim of this dissertation we tested whether altered endfoot Ca2+ signaling underlies the inversion of NVC after SAH. Brain injury is often associated with increased levels of extracellular purine nucleotides (e.g. ATP). A recent study found that ATP levels in the cerebrospinal fluid of aneurysmal SAH patients were roughly 400-fold higher than that of non-SAH controls. Astrocytes express a variety of purinergic (P2) receptors that, when activated, could trigger a spike in intra-cellular Ca2+. It is possible that enhanced signaling via astrocyte P2 receptors underlies the change in endfoot Ca2+ signaling after SAH. In the second aim of this dissertation we determined the role of purinergic signaling in the generation of high-amplitude spontaneous endfoot Ca2+ events after SAH. Parenchymal arteriolar diameter and endfoot Ca2+ dynamics were recorded simultaneously in fluo-4-loaded rat brain slices using combined infrared-differential interference contrast and multi-photon fluorescence microscopy. We report that SAH led to a time-dependent emergence of spontaneous endfoot high-amplitude Ca2+ signals (eHACSs) that were only present in brain slices exhibiting inversion of NVC. Depletion of intracellular Ca2+ stores abolished spontaneous endfoot Ca2+ signals, including eHACSs, and restored arteriolar dilation in SAH brain slices to two downstream elements in the NVC signaling cascade, (1) increased endfoot Ca2+ and (2) elevated extracellular K+. We next tested the role of purinergic signaling in the generation of SAH-induced eHACSs by recording endfoot activity before and after treatment with the broad-spectrum purinergic receptor antagonist, suramin. Remarkably, suramin selectively abolished eHACSs and restored vasodilatory NVC in SAH brain slices. Desensitization of Ca2+-permeable ionotropic purinergic (P2X) receptors had no effect on eHACSs after SAH. However, eHACSs were selectively blocked using a cocktail of inhibitors targeting Gq-coupled purinergic (P2Y) receptors. Collectively, our results support a model in which SAH leads to an emergence of P2Y receptor-mediated eHACSs that cause inversion of NVC. Further, we identify the FDA-approved drug, suramin, as a potential therapy to be used in the treatment of aneurysmal SAH

Quantitative optical imaging platform for studying neurovascular hemodynamics during ischemic stroke

The measurement of cerebral hemodynamics is vital for the study of physiological and pathophysiological conditions in the brain. Preclinical research examining the mechanisms, outcomes, and efficacies of interventions during ischemic stroke require quantitative imaging technologies. This dissertation presents the development of an optical imaging platform capable of chronic in vivo monitoring of cortical hemodynamics during ischemic stroke and the subsequent recovery. The system combines two different imaging techniques, laser speckle contrast imaging (LSCI) and oxygen-dependent quenching of phosphorescence, to measure cerebral blood flow (CBF) and dissolved oxygen tension (pO2) within cortical vasculature. Phosphorescence signal localization is achieved through the use of a spatial light modulator to selectively pattern excitation light in order to overcome the traditional limitations of the imaging modality. The spatial light modulator is also utilized to perform artery-targeted photothrombosis for the induction of highly-localized, reproducible infarcts as an extension of the photothrombotic model of ischemic stroke. The LSCI hardware implements the multi-exposure speckle imaging (MESI) technique for robust estimates of CBF that facilitate day-to-day and between-subject comparisons. The integration of an awake imaging system eliminates the confounding effects of anesthesia upon cerebral hemodynamics. The capabilities of the complete optical imaging platform are demonstrated by monitoring the acute hemodynamic response during targeted photothrombosis and the chronic recovery of the resulting ischemic infarctBiomedical Engineerin

Inhaled Carbon Monoxide Provides Cerebral Cytoprotection in Pigs

Carbon monoxide (CO) at low concentrations imparts protective effects in numerous preclinical small animal models of brain injury. Evidence of protection in large animal models of cerebral injury, however, has not been tested. Neurologic deficits following open heart surgery are likely related in part to ischemia reperfusion injury that occurs during cardiopulmonary bypass surgery. Using a model of deep hypothermic circulatory arrest (DHCA) in piglets, we evaluated the effects of CO to reduce cerebral injury. DHCA and cardiopulmonary bypass (CPB) induced significant alterations in metabolic demands, including a decrease in the oxygen/glucose index (OGI), an increase in lactate/glucose index (LGI) and a rise in cerebral blood pressure that ultimately resulted in increased cell death in the neocortex and hippocampus that was completely abrogated in piglets preconditioned with a low, safe dose of CO. Moreover CO-treated animals maintained normal, pre-CPB OGI and LGI and corresponding cerebral sinus pressures with no change in systemic hemodynamics or metabolic intermediates. Collectively, our data demonstrate that inhaled CO may be beneficial in preventing cerebral injury resulting from DHCA and offer important therapeutic options in newborns undergoing DHCA for open heart surgery