Spreading depolarization remarkably exacerbates ischemia-induced tissue acidosis in the young and aged rat brain

Spreading depolarizations (SDs) occur spontaneously in the cerebral cortex of subarachnoid hemorrhage, stroke or traumatic brain injury patients. Accumulating evidence prove that SDs exacerbate focal ischemic injury by converting zones of the viable but non-functional ischemic penumbra to the core region beyond rescue. Yet the SD-related mechanisms to mediate neurodegeneration remain poorly understood. Here we show in the cerebral cortex of isoflurane-anesthetized, young and old laboratory rats, that SDs propagating under ischemic penumbra-like conditions decrease intra and- extracellular tissue pH transiently to levels, which have been recognized to cause tissue damage. Further, tissue pH after the passage of each spontaneous SD event remains acidic for over 10 minutes. Finally, the recovery from SD-related tissue acidosis is hampered further by age. We propose that accumulating acid load is an effective mechanism for SD to cause delayed cell death in the ischemic nervous tissue, particularly in the aged brain

Transient Hypoperfusion to Ischemic/Anoxic Spreading Depolarization is Related to Autoregulatory Failure in the Rat Cerebral Cortex

BACKGROUND: In ischemic stroke, cerebral autoregulation and neurovascular coupling may become impaired. The cerebral blood flow (CBF) response to spreading depolarization (SD) is governed by neurovascular coupling. SDs recur in the ischemic penumbra and reduce neuronal viability by the insufficiency of the CBF response. Autoregulatory failure and SD may coexist in acute brain injury. Here, we set out to explore the interplay between the impairment of cerebrovascular autoregulation, SD occurrence, and the evolution of the SD-coupled CBF response. METHODS: Incomplete global forebrain ischemia was created by bilateral common carotid artery occlusion in isoflurane-anesthetized rats, which induced ischemic SD (iSD). A subsequent SD was initiated 20–40 min later by transient anoxia SD (aSD), achieved by the withdrawal of oxygen from the anesthetic gas mixture for 4–5 min. SD occurrence was confirmed by the recording of direct current potential together with extracellular K(+) concentration by intracortical microelectrodes. Changes in local CBF were acquired with laser Doppler flowmetry. Mean arterial blood pressure (MABP) was continuously measured via a catheter inserted into the left femoral artery. CBF and MABP were used to calculate an index of cerebrovascular autoregulation (rCBFx). In a representative imaging experiment, variation in transmembrane potential was visualized with a voltage-sensitive dye in the exposed parietal cortex, and CBF maps were generated with laser speckle contrast analysis. RESULTS: Ischemia induction and anoxia onset gave rise to iSD and aSD, respectively, albeit aSD occurred at a longer latency, and was superimposed on a gradual elevation of K(+) concentration. iSD and aSD were accompanied by a transient drop of CBF (down to 11.9 ± 2.9 and 7.4 ± 3.6%, iSD and aSD), but distinctive features set the hypoperfusion transients apart. During iSD, rCBFx indicated intact autoregulation (rCBFx  0.3) because CBF followed the decreasing MABP. CBF dropped 15–20 s after iSD, but the onset of hypoperfusion preceded aSD by almost 3 min. Taken together, the CBF response to iSD displayed typical features of spreading ischemia, whereas the transient CBF reduction with aSD appeared to be a passive decrease of CBF following the anoxia-related hypotension, leading to aSD. CONCLUSIONS: We propose that the dysfunction of cerebrovascular autoregulation that occurs simultaneously with hypotension transients poses a substantial risk of SD occurrence and is not a consequence of SD. Under such circumstances, the evolving SD is not accompanied by any recognizable CBF response, which indicates a severely damaged neurovascular coupling

Effects of cathodal transcranial direct current stimulation on cortical spreading depression

The purpose of this study was to examine the effects of cathodal transcranial direct current stimulation (tDCS) on cortical spreading depression (CSD) in the rat cerebral cortex. CSD is a propagating wave of hyperexcitability that occurs in a number of neurological disorders characterized by excess cerebral excitability such as migraine, acute brain injury, or stroke. Since tDCS is a non-invasive method capable of inducing polarity-dependent changes in cortical excitability, we hypothesized that cathodal stimulation would prevent, attenuate, or change the characteristics of CSD. Forty Sprague-Dawley male rats were randomly divided into two stimulation condition groups: sham tDCS and cathodal tDCS. In both experimental groups, CSD was induced by applying potassium chloride onto cortical surface. Electroencephalogram (EEG) data was recorded during each experiment and subjected to analysis. CSD incidence was compared between the sham and cathodal tDCS group. We observed that significantly fewer CSD events were exhibited during cathodal tDCS relative to sham stimulation. Evaluation of CSD wave characteristics between experimental groups revealed no differences in propagation velocity, amplitude, or waveform of CSD, nor in the presence of neuronal silencing. The results of this study lend support for the use of cathodal tDCS as an effective method for reducing cortical excitability and provides the groundwork for future study of the mechanisms of tDCS and its treatment targets in neurological disorders whose symptoms are created or exacerbated by CSD

The effects of anodal transcranial direct current stimulation on cortical spreading depression

Cortical spreading depression (CSD) is a depolarizing wave that travels through the cerebral cortex, and is followed by an inhibition of cortical activity. The propagation of CSD elicits metabolic challenges in tissue that may be irrecoverable in an ischemic brain, and thus has implications in neurological disease. Limiting the incidence of CSD may be instrumental in limiting the extent of neuronal damage following brain injury. Transcranial direct current stimulation (tDCS) is a form of brain stimulation that alters the level of cortical activity. Anodal tDCS, which increases cortical excitability, is used to treat a variety of neurological syndromes but may have the potential to exacerbate certain pathologies. This contention has never been evaluated using in vivo brain recordings. This study seeks to determine the effects of anodal tDCS on CSD, a phenomenon common to many neurological disorders. CSD was induced in the rat cortex by administration of potassium chloride. Animals were subjected to either anodal tDCS or sham stimulation. Cortical electrical activity was monitored using an intracortical multielectrode array, and data was analyzed to measure the effects of anodal tDCS versus sham on CSD incidence, velocity, amplitude, and several other characteristics of the wave. The hypothesis of the study was that anodal tDCS would increase the incidence, velocity, and amplitude of the CSD wave. No significant effects of anodal tDCS on CSD were observed in this study. Results indicate that anodal tDCS does not increase the velocity, amplitude, or frequency of the spreading depression wave, nor does it interrupt the wave. These data have implications for the use of anodal tDCS in the treatment of neurological disorders associated with spreading depression

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

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