Entstehungsmechanismen von Spreading Depolarizations in einem Mausmodell akuter und subakuter kortikaler Blutungen

Background: Spreading depolarizations (SDs) are waves neuronal and glial mass-depolarization that occur spontaneously after brain injury and are associated with detrimental effects in ischemic stroke and subarachnoid hemorrhage. In clinical and experimental intracerebral hemorrhage (ICH), SDs are observed. However, triggers for SD in ICH are poorly understood. In a mouse model, we investigated spatiotemporal characteristics and causes of SD occurrence in acute and subacute stages of ICH. We focused on ischemia, mechanical tissue distortion, and blood constituents or blood breakdown products as potential triggers. Methods: After cannulating the femoral artery to track systemic physiology, ICH was induced by cortical injection of bacterial collagenase VII-S. Immediately, 8h, 24h, or 48h after injection, intrinsic optical signals, laser speckle flowmetry (LSF), and electrocorticography were recorded for 240 min to follow hematoma expansion, cortical blood flow changes, and SD occurrence over time. Subgroups of animals were assigned to normobaric hyperoxia or induced hypertension in the early stages of ICH. In another subset of animals, focal cortical ischemia was induced instead of ICH using the distal Middle Cerebral Artery occlusion model (dMCAo). Brains were collected at the end of the experiment for tissue analysis. Results: During acute stages of ICH (0–4h), 45% of mice developed SDs, that often occurred in couplets and invariably emerged from the hematoma. SD frequency observed in primary hemorrhagic lesions was three-fold lower than in size-matched is-chemic cortical infarcts. Arguing against blood constituents or breakdown products as a trigger for SD in ICH, hematoma size did not correlate with SD occurrence. Further, SDs were only detected 29 to 221 min after ICH induction, whereas not a single SD was recorded at later time points, 8–52h after ICH induction. Likely excluding ischemia as a potential trigger, perihematomal perfusion monitored using LSF did not predict SD occurrence. In line with this, normobaric hyperoxia, known to decrease SD fre-quency by 60% in focal ischemic brains, did not reduce SD occurrence. Instead, SDs always arose during phases of rapid hemorrhage growth, which was doubled immediately preceding an SD, compared with the peak growth recorded in animals that did not develop any SD. Inducing hypertension in a separate cohort of mice yielded severely accelerated hemorrhage growth and increased SD frequency by four-fold com-pared with normotensive controls. Conclusion: Our data provide novel mechanistic insights into the origins of SDs in ICH. They suggest that spontaneous SDs are caused by the mechanical tissue distor-tion of rapidly growing hematomas, with ischemia and blood constituents or breakdown products not contributing to a relevant extent.Hintergrund: Spreading depolarizations (SDs) sind Wellen neuronaler und glialer Massen-Depolarisation, die spontan nach Hirnverletzungen auftreten und mit schädlichen Effekten bei ischämischen Schlaganfällen und Subarachnoidalblutungen in Verbindung gebracht werden. Sie können im Rahmen klinischer und experimenteller int-razerebraler Blutungen (ICH) beobachtet werden. Ihre Auslöser hier sind jedoch unbekannt. In einem Mausmodell analysierten wir das Auftreten und die Ursachen von SDs in akuten und subakuten ICH-Stadien und untersuchten Ischämie, mechanische Gewebsverdrängung und Blutbestandteile oder -abbauprodukte als mögliche Auslöser. Methodik: Nach Kanülierung der Femoralarterie zur Überwachung der systemischen Physiologie wurde durch kortikale Injektion von bakterieller Kollagenase VII-S eine ICH induziert. Unmittelbar, 8h, 24h, oder 48h nach der Injektion wurden intrinsische optische Signale, Laser-Speckle-Flussmessung (LSF) und Elektrokortikographie 240 min lang aufgezeichnet, um die Ausdehnung der Blutung, Veränderungen des kortikalen Blutflusses und das Auftreten von SDs im Zeitverlauf zu beurteilen. Untergruppen von Tieren wurden im Frühstadium der ICH einer normobaren Hyperoxie oder einer induzierten Hypertension unterzogen. In einer anderen Subgruppe wurde anstelle der ICH mit Hilfe des dMCAo Modells eine fokale Ischämie induziert. Am Ende der Experimente wurden die Gehirne zur Gewebsanalyse entnommen. Ergebnisse: In der akuten Phase der ICH (0-4h) entwickelten 45 % der Mäuse SDs, die oft in Paaren auftraten und ausnahmslos von der Blutung ausgingen. Primär hämorrhagische kortikale Läsionen zeigten eine, im Vergleich zu gleich großen kortikalen ischämischen Infarkten, um das dreifach reduzierte SD-Frequenz. SDs traten lediglich 29 bis 221min nach ICH-Induktion auf, nicht jedoch später, 8-52h nach ICH-Induktion, und korrelierten in ihrem Auftreten nicht mit der Blutungsgröße, was gegen Blutbestandteile oder Abbauprodukte als Auslöser der SDs sprach. Weiterhin konnte der peri-hämatomale Blutfluss das Erscheinen von SDs nicht vorhersagen und auch normobare Hyperoxie, die das Vorkommen von SDs in fokal ischämi-schen Gehirnen um 60 % reduziert, beeinflusste die SD-Frequenz nicht. Dies machte eine fokale Ischämie als Auslöser der SDs ebenso unwahrscheinlich. Im Gegensatz dazu zeigte sich, dass SDs immer in Phasen starken Blutungswachstums auftraten, das unmittelbar vor dem Erscheinen einer SD doppelt so hoch war wie das maximale Blutungswachstum in Tieren, die keine SDs entwickelten. Verglichen mit normotensi-ven Tieren führte induzierte Hypertension zu einem stark beschleunigtem Blutungs-wachstum und einer Vervierfachung der SD-Frequenz. Zusammenfassung: Unsere Daten liefern neue pathophysiologische Erkenntnisse zur Entstehung von SDs bei ICH. Sie legen nahe, dass SDs durch mechanischen Druck schnell wachsender Blutungen ausgelöst werden, während Ischämie und Blut-bestandteile, oder -abbauprodukte das Auftreten von SDs nicht relevant beeinflussen

Vascular Response to Spreading Depolarization Predicts Stroke Outcome

Background: Cortical spreading depolarization (CSD) is a massive neuro-glial depolarization wave, which propagates across the cerebral cortex. In stroke, CSD is a necessary and ubiquitous mechanism for the development of neuronal lesions that initiates in the ischemic core and propagates through the penumbra extending the tissue injury. Although CSD propagation induces dramatic changes in cerebral blood flow, the vascular responses in different ischemic regions and their consequences on reperfusion and recovery remain to be defined. Methods: Ischemia was performed using the thrombin model of stroke and reperfusion was induced by r-tPA (recombinant tissue-type plasminogen activator) administration in mice. We used in vivo electrophysiology and laser speckle contrast imaging simultaneously to assess both electrophysiological and hemodynamic characteristics of CSD after ischemia onset. Neurological deficits were assessed on day 1, 3, and 7. Furthermore, infarct sizes were quantified using 2,3,5-triphenyltetrazolium chloride on day 7. Results: After ischemia, CSDs were evidenced by the characteristic propagating DC shift extending far beyond the ischemic area. On the vascular level, we observed 2 types of responses: some mice showed spreading hyperemia confined to the penumbra area (penumbral spreading hyperemia) while other showed spreading hyperemia propagating in the full hemisphere (full hemisphere spreading hyperemia). Penumbral spreading hyperemia was associated with severe stroke-induced damage, while full hemisphere spreading hyperemia indicated beneficial infarct outcome and potential viability of the infarct core. In all animals, thrombolysis with r-tPA modified the shape of the vascular response to CSD and reduced lesion volume. Conclusions: Our results show that different types of spreading hyperemia occur spontaneously after the onset of ischemia. Depending on their shape and distribution, they predict severity of injury and outcome. Furthermore, our data show that modulating the hemodynamic response to CSD may be a promising therapeutic strategy to attenuate stroke outcome

Spectral and Temporal Interrogation of Cerebral Hemodynamics Via High Speed Laser Speckle Contrast Imaging

Laser Speckle Contrast Imaging (LSCI) is a non-scanning wide field-of-view optical imaging technique specifically developed for cerebral blood flow (CBF) monitoring. In this project, a versatile Laser speckle contrast imaging system has been designed and developed to monitor CBF changes and examine the physical properties of cerebral vasculature during functional brain activation experiments. The hardware of the system consists of a high speed CMOS camera, a coherent light source, a trinocular microscope, and a PC that does camera controlling and data storage. The simplicity of the system’s hardware makes it suitable for biological experiments. In controlled flow experiments using a custom made microfluidic channel, the linearity of the CBF estimates was evaluated under high speed imaging settings. Under the camera exposure time setting in the range of tens of micro-seconds, results show a linear relationship between the CBF estimates and the flow rates within the microchannel. This validation permitted LSCI to be used in high frame rate imaging and the method is only limited by the camera speed. In an in vivo experiment, the amount of oxygen intake via breathing by a rat was reduced to 12% to induce the dilation of the vessels. Results demonstrated a positive correlation between the system’s CBF estimates and the pulse wave velocity derived from aortic blood pressure. To exemplify the instantaneous pulsatility flow study acquired at high sampling rate, a pulsatile cerebral blood flow analysis was conducted on two vessels, an arteriole and a venule. The pulsatile waveform results, captured under sampling rate close to 2000 Hz. The pulse of the arteriole rises 13ms faster than the pulse of the venule, and it takes 6ms longer for the pulse of the arteriole to fall below the lower fall-time boundary. By using the second order derivative (accelerated) CBF estimates, the vascular stiffness was evaluated. Results show the arteriole and the venule have increased-vascular-stiffness indices of 0.95 and 0.74. On the other side, the arteriole and the venule have decreased-vascular-stiffness indices of 0.125 and 0.35. Both vascular stiffness indices suggested that the wall of arteriole is more rigid than the venule. The proposed LSCI system can monitor the mean flow over function activation experiment, and the interrogation of blood flow in terms of physiological oscillations. The proposed vascular stiffness metrics for estimating the stroke preliminary symptom, may eventually lead to insights of stroke and its causes

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

Modern laser speckle contrast theory: flaws and consequences

Laser speckle contrast imaging (LSCI) is a non-invasive optical imaging technique for monitoring blood flow in brain, skin, and retina. The simple and cheap instrument makes it a promising technology for both clinical applications and research. Modern LSCI theory takes advantage of the relation between blood flow and the speckle contrast v ~ 1/K^2 to provide an online acquisition of a full-field blood flow image. However, the assumptions about the form of field correlation function, static scattering effect, and the coherence factor make interpretation of the contrast imprecise. Here we examined how the assumptions in modern LSCI theory affect the relative blood flow measurement and utilized Dynamic Laser Speckle Imaging (DLSI) to validate the imprecision of modern LSCI. Most importantly, the contrast models for measuring relative flow in the brain parenchyma and the large vessels were derived. It turns out that modern LSCI underestimates blood flow change and leads to significant error for slow blood flow measurement.2020-06-03T00:00:00

Preclinical models of middle cerebral artery occlusion: new imaging approaches to a classic technique

Stroke remains a major burden on patients, families, and healthcare professionals, despite major advances in prevention, acute treatment, and rehabilitation. Preclinical basic research can help to better define mechanisms contributing to stroke pathology, and identify therapeutic interventions that can decrease ischemic injury and improve outcomes. Animal models play an essential role in this process, and mouse models are particularly well-suited due to their genetic accessibility and relatively low cost. Here, we review the focal cerebral ischemia models with an emphasis on the middle cerebral artery occlusion technique, a “gold standard” in surgical ischemic stroke models. Also, we highlight several histologic, genetic, and in vivo imaging approaches, including mouse stroke MRI techniques, that have the potential to enhance the rigor of preclinical stroke evaluation. Together, these efforts will pave the way for clinical interventions that can mitigate the negative impact of this devastating disease

Optical imaging and spectroscopy for the study of the human brain: status report

This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions

Optical imaging and spectroscopy for the study of the human brain: status report.

This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions

Optical imaging and spectroscopy for the study of the human brain: status report

This report is the second part of a comprehensive two-part series aimed at reviewing an extensive and diverse toolkit of novel methods to explore brain health and function. While the first report focused on neurophotonic tools mostly applicable to animal studies, here, we highlight optical spectroscopy and imaging methods relevant to noninvasive human brain studies. We outline current state-of-the-art technologies and software advances, explore the most recent impact of these technologies on neuroscience and clinical applications, identify the areas where innovation is needed, and provide an outlook for the future directions. Keywords: DCS; NIRS; diffuse optics; functional neuroscience; optical imaging; optical spectroscop