Comparison of stimulus-evoked cerebral hemodynamics in the awake mouse and under a novel anesthetic regime

Neural activity is closely followed by a localised change in cerebral blood flow, a process termed neurovascular coupling. These hemodynamic changes form the basis of contrast in functional magnetic resonance imaging (fMRI) and are used as a correlate for neural activity. Anesthesia is widely employed in animal fMRI and neurovascular studies, however anesthetics are known to profoundly affect neural and vascular physiology, particularly in mice. Therefore, we investigated the efficacy of a novel ‘modular’ anesthesia that combined injectable (fentanyl-fluanisone/midazolam) and volatile (isoflurane) anesthetics in mice. To characterize sensory-evoked cortical hemodynamic responses, we used optical imaging spectroscopy to produce functional maps of changes in tissue oxygenation and blood volume in response to mechanical whisker stimulation. Following fine-tuning of the anesthetic regime, stimulation elicited large and robust hemodynamic responses in the somatosensory cortex, characterized by fast arterial activation, increases in total and oxygenated hemoglobin, and decreases in deoxygenated hemoglobin. Overall, the magnitude and speed of evoked hemodynamic responses under anesthesia resembled those in the awake state, indicating that the novel anesthetic combination significantly minimizes the impact of anesthesia. Our findings have broad implications for both neurovascular research and longitudinal fMRI studies that increasingly require the use of genetically engineered mice

Coupling between gamma-band power and cerebral blood volume during recurrent acute neocortical seizures

Characterization of neural and hemodynamic biomarkers of epileptic activity that can be measured using non-invasive techniques is fundamental to the accurate identification of the epileptogenic zone (EZ) in the clinical setting. Recently, oscillations at gamma-band frequencies and above (>30 Hz) have been suggested to provide valuable localizing information of the EZ and track cortical activation associated with epileptogenic processes. Although a tight coupling between gamma-band activity and hemodynamic-based signals has been consistently demonstrated in non-pathological conditions, very little is known about whether such a relationship is maintained in epilepsy and the laminar etiology of these signals. Confirmation of this relationship may elucidate the underpinnings of perfusion-based signals in epilepsy and the potential value of localizing the EZ using hemodynamic correlates of pathological rhythms. Here, we use concurrent multi-depth electrophysiology and 2-dimensional optical imaging spectroscopy to examine the coupling between multi-band neural activity and cerebral blood volume (CBV) during recurrent acute focal neocortical seizures in the urethane-anesthetized rat. We show a powerful correlation between gamma-band power (25-90 Hz) and CBV across cortical laminae, in particular layer 5, and a close association between gamma measures and multi-unit activity (MUA). Our findings provide insights into the laminar electrophysiological basis of perfusion-based imaging signals in the epileptic state and may have implications for further research using non-invasive multi-modal techniques to localize epileptogenic tissue

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

Extensive and Persistent Disruption of Neurovascular Coupling by a Single Cerebral Microinfarct

Cerebral microinfarcts (CMI), microscopic brain lesions caused by blockade of small blood vessels, have recently emerged as a potential determinant of cognitive decline. Though small in size, our recent work demonstrated that a single, strategically placed CMI was sufficient to disrupt sensory input in a behavioral task. However, the means by which such small lesions disrupt brain function remain poorly understood. We imaged vascular function in awake, head-fixed mice using two-photon microscopy to examine the impact of CMI on neurovascular coupling. CMI were generated in cortex by photothrombotic occlusion of single penetrating vessels through a thinned-skull cranial window. Vibrissa-evoked dilation of individual arteries and arterioles within the primary vibrissa cortex were tracked over four time periods: pre-occlusion, acute (2-3 days post-occlusion), subacute (7-9 days) and chronic (14-21 days). In the acute phase, dilatory responses were markedly attenuated compared to pre-occlusion (2.2 ± 0.5% mean dilation over baseline vs. 11.2 ± 0.8%, p \u3c 0.001). Dilatory responses during the subacute (7.8 ± 1.1%) and chronic (6.5 ± 1.1%) phases partially recovered but remained significantly attenuated in magnitude and time to dilation compared to pre-occlusion (p \u3c 0.01). Critically, vascular dysfunction was observed well beyond the borders of the CMI, as infarcts with an average radius of 0.19 ± 0.05 mm generated deficits in dilation at distances exceeding 1 mm away from the vessel targeted for occlusion. Analysis of dilations in a separate cohort of mice during the hyperacute time period (0-3 hours post-occlusion), revealed that the dilatory deficit is first expressed in the immediate vicinity of the stroke and then propagates outward from the occlusion site. While unresponsive to sensory stimulation, vasodilation could be evoked by isoflurane inhalation, albeit attenuated in the subacute phase (156.7 ± 5.3% of pre-stroke levels vs. 134.6 ± 4.3% subacute, p = 0.02). Expression of c-Fos following an extended period of vibrissa stimulation was reduced in the peri-infarct tissue in the acute time period, with gradual recovery initiating distal to the stroke apparent in subacute and chronic mice. This indicated that loss of sensory-evoked vasodilation is attributed to a combination of altered vascular mechanical properties and a deficit in neural connectivity and/or activity. Thus, CMI disrupt brain function well beyond the regions of overt tissue infarction and this effect, combined with their widespread distribution in the aged brain, may contribute to the pathogenesis of CMI in vascular dementia

Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

Animal models of stroke are used extensively to study the mechanisms involved in the acute and chronic phases of recovery following stroke. A translatable animal model that closely mimics the mechanisms of a human stroke is essential in understanding recovery processes as well as developing therapies that improve functional outcomes. We describe a photothrombosis stroke model that is capable of targeting a single distal pial branch of the middle cerebral artery with minimal damage to the surrounding parenchyma in awake head-fixed mice. Mice are implanted with chronic cranial windows above one hemisphere of the brain that allow optical access to study recovery mechanisms for over a month following occlusion. Additionally, we study the effect of laser spot size used for occlusion and demonstrate that a spot size with small axial and lateral resolution has the advantage of minimizing unwanted photodamage while still monitoring macroscopic changes to cerebral blood flow during photothrombosis. We show that temporally guiding illumination using real-time feedback of blood flow dynamics also minimized unwanted photodamage to the vascular network. Finally, through quantifiable behavior deficits and chronic imaging we show that this model can be used to study recovery mechanisms or the effects of therapeutics longitudinally.R01 EB021018 - NIBIB NIH HHS; R01 MH111359 - NIMH NIH HHS; R01 NS108472 - NINDS NIH HHSPublished versio

Contributions and complexities from the use of in-vivo animal models to improve understanding of human neuroimaging signals.

Many of the major advances in our understanding of how functional brain imaging signals relate to neuronal activity over the previous two decades have arisen from physiological research studies involving experimental animal models. This approach has been successful partly because it provides opportunities to measure both the hemodynamic changes that underpin many human functional brain imaging techniques and the neuronal activity about which we wish to make inferences. Although research into the coupling of neuronal and hemodynamic responses using animal models has provided a general validation of the correspondence of neuroimaging signals to specific types of neuronal activity, it is also highlighting the key complexities and uncertainties in estimating neural signals from hemodynamic markers. This review will detail how research in animal models is contributing to our rapidly evolving understanding of what human neuroimaging techniques tell us about neuronal activity. It will highlight emerging issues in the interpretation of neuroimaging data that arise from in-vivo research studies, for example spatial and temporal constraints to neuroimaging signal interpretation, or the effects of disease and modulatory neurotransmitters upon neurovascular coupling. We will also give critical consideration to the limitations and possible complexities of translating data acquired in the typical animals models used in this area to the arena of human fMRI. These include the commonplace use of anaesthesia in animal research studies and the fact that many neuropsychological questions that are being actively explored in humans have limited homologues within current animal models for neuroimaging research. Finally we will highlighting approaches, both in experimental animals models (e.g. imaging in conscious, behaving animals) and human studies (e.g. combined fMRI-EEG), that mitigate against these challenges

Fluorescence Imaging of Cortical Calcium Dynamics: A Tool for Visualizing Mouse Brain Functions, Connections, and Networks

Hemodynamic-based markers of cortical activity (e.g. functional magnetic resonance imaging (fMRI) and optical intrinsic signal imaging) are an indirect and relatively slow report of neural activity driven by electrical and metabolic activity through neurovascular coupling, which presents significant limiting factors in deducing underlying brain network dynamics. As application of resting state functional connectivity (FC) measures is extended further into topics such as brain development, aging, and disease, the importance of understanding the fundamental basis for FC will grow. In this dissertation, we extend functional analysis from hemodynamic- to calcium-based imaging. Transgenic mice expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketaminze/xylazine) and awake states. Sequential LED illumination (λ=470, 530, 590, 625nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. EEG measurements of the global cortical field potential were also simultaneously acquired. First, we validated the ability of our system to capture GCaMP6 fluorescence emission and hemodynamics by implementing an electrical somatosensory stimulation paradigm. The neural origins of the GCaMP6 fluorescent signal were further confirmed by histology and by comparing the spectral content of imaged GCaMP6 activity to concurrently-acquired EEG. We then constructed seed-based FC and coherence network maps for low (0.009-0.08Hz) and high, delta-band (0.4-4.0Hz) frequency bands using GCaMP6 and hemodynamic contrasts. Homotopic GCaMP6 FC maps using delta-band data in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. We next used whole-brain delay analysis to characterize this correlative feature. This structure is potentially explained by the observed propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas, likely corresponding to propagating delta waves associated with slow wave sleep. During wakefulness, this spatio-temporal structure is largely absent, and a more complex and detailed FC structure is observed. Collectively, functional neuroimaging of calcium dynamics in mice provides evidence that spatiotemporal coherence in cortical activity is not exclusive to hemodynamics and exists over a larger range of frequencies than hemoglobin-based contrasts. Concurrent calcium and hemodynamic imaging enables direct temporal and functional comparison of spontaneous calcium and hemoglobin activity, effectively spanning neurovascular coupling and functional hyperemia. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and provide a framework for subsequent studies of sleep disorders and neurological disease

Volatile anesthetics influence blood-brain barrier integrity by modulation of tight junction protein expression in traumatic brain injury

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema

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