Network response of brain microvasculature to neuronal stimulation.

Neurovascular coupling (NVC), or the adjustment of blood flow in response to local increases in neuronal activity is a hallmark of healthy brain function, and the physiological foundation for functional magnetic resonance imaging (fMRI). However, it remains only partly understood due to the high complexity of the structure and function of the cerebrovascular network. Here we set out to understand NVC at the network level, i.e. map cerebrovascular network reactivity to activation of neighbouring neurons within a 500×500×500 µm3 cortical volume (∼30 high-resolution 3-nL fMRI voxels). Using 3D two-photon fluorescence microscopy data, we quantified blood volume and flow changes in the brain vessels in response to spatially targeted optogenetic activation of cortical pyramidal neurons. We registered the vessels in a series of image stacks acquired before and after stimulations and applied a deep learning pipeline to segment the microvascular network from each time frame acquired. We then performed image analysis to extract the microvascular graphs, and graph analysis to identify the branch order of each vessel in the network, enabling the stratification of vessels by their branch order, designating branches 1-3 as precapillary arterioles and branches 4+ as capillaries. Forty-five percent of all vessels showed significant calibre changes; with 85 % of responses being dilations. The largest absolute CBV change was in the capillaries; the smallest, in the venules. Capillary CBV change was also the largest fraction of the total CBV change, but normalized to the baseline volume, arterioles and precapillary arterioles showed the biggest relative CBV change. From linescans along arteriole-venule microvascular paths, we measured red blood cell velocities and hematocrit, allowing for estimation of pressure and local resistance along these paths. While diameter changes following neuronal activation gradually declined along the paths; the pressure drops from arterioles to venules increased despite decreasing resistance: blood flow thus increased more than local resistance decreases would predict. By leveraging functional volumetric imaging and high throughput deep learning-based analysis, our study revealed distinct hemodynamic responses across the vessel types comprising the microvascular network. Our findings underscore the need for large, dense sampling of brain vessels for characterization of neurovascular coupling at the network level in health and disease.

The effects of voluntary running on cerebrovascular morphology and spatial short-term memory in a mouse model of amyloidosis.

Physical activity has been correlated with a reduced risk of cognitive decline, including that associated with vascular dementia, mild cognitive impairment (MCI) and Alzheimer's disease (AD); recent literature suggests this may in part result from benefits to the cerebrovascular network. Using a transgenic (Tg) mouse model of AD, we evaluated the effect of running on cortical and hippocampal vascular morphology, cerebral amyloid angiopathy, amyloid plaque load, and spatial memory. TgCRND8 mice present with progressive amyloid pathology, advancing from the cortex to the hippocampus in a time-dependent manner. We postulated that the characteristic progression of pathology could lead to differential, time-dependent effects of physical activity on vascular morphology in these brain regions at 6 months of age. We used two-photon fluorescent microscopy and 3D vessel tracking to characterize vascular and amyloid pathology in sedentary TgCRND8 mice compared those who have a history of physical activity (unlimited access to a running wheel, from 3 to 6 months of age). In sedentary TgCRND8 mice, capillary density was found to be lower in the cortex and higher in the hippocampus compared to non-transgenic (nonTg) littermates. Capillary length, vessel branching, and non-capillary vessel tortuosity were also higher in the hippocampus of sedentary TgCRND8 compared to nonTg mice. Three months of voluntary running resulted in normalizing cortical and hippocampal microvascular morphology, with no significant difference between TgCRND8 and nonTg mice. The benefits of physical activity on cortical and hippocampal vasculature in 6-month old TgCRND8 mice were not paralleled by significant changes on parenchymal and cerebral amyloid pathology. Short-term spatial memory- as evaluated by performance in the Y-maze- was significantly improved in running compared to sedentary TgCRND8 mice. These results suggest that long-term voluntary running contributes to the maintenance of vascular morphology and spatial memory in TgCRND8 mice, even in the absence of an effect on amyloid pathology.

In vivo neurovascular response to focused photoactivation of Channelrhodopsin-2.

The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics' potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed ∼40% larger LFP responses (p = 0.05) and twice as large cerebrovascular responses (p = 0.002) under focused vs. diffuse photostimulation (focused: 1.64 ±â€¯0.84 mV LFP amplitude and 75 ±â€¯48% increase in vRBC; diffuse: 1.14 ±â€¯0.75 mV LFP amplitude and 35 ±â€¯23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a ∼65% increase in the yield of cerebrovascular responses (73 ±â€¯10% for focused and 42 ±â€¯29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9 ±â€¯14.7 for focused and 10.4 ±â€¯1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.

Imaging the Effects of ß-Hydroxybutyrate on Peri-Infarct Neurovascular Function and Metabolism.

Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body ß-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Attenuation of functional hyperemia to visual stimulation in mild Alzheimer's disease and its sensitivity to cholinesterase inhibition.

Despite the growing recognition of the significance of cerebrovascular impairment in the etiology and progression of Alzheimer's disease (AD), the early stage brain vascular dysfunction and its sensitivity to pharmacological interventions is still not fully characterized. Due to the early and aggressive treatment of probable AD with cholinesterase inhibitors (ChEI), which in and of themselves have direct effects on brain vasculature, the vast majority of hemodynamic measurements in early AD subjects reported hitherto have consequently been made only after the start of treatment, complicating the disentanglement of disease- vs. treatment-related effects on the cerebral vasculature. To address this gap, we used pseudo continuous arterial spin labeling MRI to measure resting perfusion and visual stimulation elicited changes in cerebral blood flow (CBF) and blood oxygenation dependent (BOLD) fMRI signal in a cohort of mild AD patients immediately prior to, 6months post, and 12months post commencement of open label cholinesterase inhibitor treatment. Although patients exhibited no gray matter atrophy prior to treatment and their resting perfusion was not distinguishable from that in age, education and gender-matched controls, the patients' visual stimulation-elicited changes in BOLD fMRI and blood flow were decreased by 10±4% (BOLD) and 23±2% (CBF), relative to those in controls. Induction of cholinesterase inhibition treatment was associated with a further, 7±2% reduction in patients' CBF response to visual stimulation, but it stabilized, at this new lower level, over the follow-up period. Likewise, MMSE scores remained stable during the treatment; furthermore, higher MMSE scores were associated with higher perfusion responses to visual stimulation. This study represents the initial step in disentangling the effects of AD pathology from those of the first line treatment with cholinesterase inhibitors on cerebral hemodynamics and supports the use of arterial spin labeling MRI for quantitative evaluation of the brain vascular function in mild Alzheimer's disease. The findings provide evidence of a pronounced deficit in the visual cortex hyperemia despite the relative sparing of visual function in early stage AD, its reduction with ChEI treatment induction, and its stabilization in the first year of cholinesterase inhibition treatment. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Venular degeneration leads to vascular dysfunction in a transgenic model of Alzheimer's disease.

Most patients with Alzheimer's disease exhibit accumulation of amyloid-ß peptide on leptomeningeal and cortical arterioles, or cerebral amyloid angiopathy, which is associated with impaired vascular reactivity and accelerated cognitive decline. Despite widespread recognition of the significance of vascular dysfunction in Alzheimer's disease aetiology and progression, much uncertainty still surrounds the mechanism underlying Alzheimer's disease vascular injury. Studies to date have focused on amyloid-ß-induced damage to capillaries and plaque-associated arterioles, without examining effects across the entire vascular bed. In the present study, we investigated the structural and functional impairment of the feeding arteriolar versus draining venular vessels in a transgenic murine Alzheimer's disease model, with a particular focus on the mural cell populations that dictate these vessels' contractility. Although amyloid-ß deposition was restricted to arterioles, we found that vascular impairment extended to the venules, which showed significant depletion of their mural cell coverage by the mid-stage of Alzheimer's disease pathophysiology. These structural abnormalities were accompanied by an abolishment of the normal vascular network flow response to hypercapnia: this functional impairment was so severe as to result in hypercapnia-induced flow decreases in the arterioles. Further pharmacological depletion of mural cells using SU6668, a platelet-derived growth factor receptor-ß antagonist, resulted in profound structural abnormalities of the cortical microvasculature, including vessel coiling and short-range looping, increased tortuosity of the venules but not of the arterioles, increased amyloid-ß deposition on the arterioles, and further alterations of the microvascular network cerebral blood flow response to hypercapnia. Together, this work shows hitherto unrecognized structural alterations in penetrating venules, demonstrates their functional significance and sheds light on the complexity of the relationship between vascular network structure and function in Alzheimer's disease.

A novel 3D mouse embryo atlas based on micro-CT.

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.

Cerebral microvascular network geometry changes in response to functional stimulation.

The cortical microvessels are organized in an intricate, hierarchical, three-dimensional network. Superimposed on this anatomical complexity is the highly complicated signaling that drives the focal blood flow adjustments following a rise in the activity of surrounding neurons. The microvascular response to neuronal activation remains incompletely understood. We developed a custom two photon fluorescence microscopy acquisition and analysis to obtain 3D maps of neuronal activation-induced changes in the geometry of the microvascular network of the primary somatosensory cortex of anesthetized rats. An automated, model-based tracking algorithm was employed to reconstruct the 3D microvascular topology and represent it as a graph. The changes in the geometry of this network were then tracked, over time, in the course of electrical stimulation of the contralateral forepaw. Both dilatory and constrictory responses were observed across the network. Early dilatory and late constrictory responses propagated from deeper to more superficial cortical layers while the response of the vertices that showed initial constriction followed by later dilation spread from cortical surface toward increasing cortical depths. Overall, larger caliber adjustments were observed deeper inside the cortex. This work yields the first characterization of the spatiotemporal pattern of geometric changes on the level of the cortical microvascular network as a whole and provides the basis for bottom-up modeling of the hemodynamically-weighted neuroimaging signals.

Amyloid-ß-dependent compromise of microvascular structure and function in a model of Alzheimer's disease.

The majority of patients with Alzheimer's disease have cerebral amyloid angiopathy, thus showing deposition of amyloid-ß peptides in the walls of leptomeningeal and cortical arterioles. These deposits are believed to result from impaired clearance of parenchymal amyloid-ß peptides. In the current work, we examined the changes in cortical microvascular structure and function in situ in TgCRND8, a transgenic mouse model of Alzheimer's disease. In contrast to venules, cortical arterioles were shown to increase in tortuosity and decrease in calibre with amyloid-ß peptide accumulation. These structural changes were accompanied by progressive functional compromise, reflected in higher dispersion of microvascular network transit times, elongation of the transit times, and impaired microvascular reactivity to hypercapnia in the transgenic mice. Moreover, inhibition of amyloid-ß peptide oligomerization and fibrillization via post-weaning administration of scyllo-inositol, a naturally occurring stereoisomer of myo-inositol, rescued both structural and functional impairment of the cortical microvasculature in this Alzheimer's disease model. These results demonstrate that microvascular impairment is directly correlated with amyloid-ß accumulation and highlight the importance of targeting cerebrovascular amyloid angiopathy clearance for effective diagnosis, monitoring of disease progression and treatment of Alzheimer's disease.

Quantitative estimates of stimulation-induced perfusion response using two-photon fluorescence microscopy of cortical microvascular networks.

Functional hyperemia, or the increase in focal perfusion elicited by neuronal activation, is one of the primary functions of the neurovascular unit and a hallmark of healthy brain functioning. While much is known about the hemodynamics on the millimeter to tenths of millimeter-scale accessible by MRI, there is a paucity of quantitative data on the micrometer-scale changes in perfusion in response to functional stimulation. We present a novel methodology for quantification of perfusion and intravascular flow across the 3D microvascular network in the rat somatosensory cortex using two-photon fluorescence microscopy (2PFM). For approximately 96% of responding microvessels in the forelimb representation of the primary somatosensory cortex, brief (~2s) forepaw stimulation resulted in an increase of perfusion 20±4% (mean±sem). The perfusion levels associated with the remaining 4% of the responding microvessels decreased 10±9% upon stimulation. Vessels irrigating regions of lower vascular density were found to exhibit higher flow (p<0.02), supporting the notion that local vascular morphology and hemodynamics reflect the metabolic needs of the surrounding parenchyma. High dispersion (~77%) in perfusion levels suggests high spatial variation in tissue susceptibility to hypoxia. The current methodology enables quantification of absolute perfusion associated with individual vessels of the cortical microvascular bed and its changes in response to functional stimulation and thereby provides an important tool for studying the cellular mechanisms of functional hyperemia, the spatial specificity of perfusion response to functional stimulation, and, broadly, the micrometer-scale relationship between vascular morphology and function in health and disease.

Quantification of blood flow and volume in arterioles and venules of the rat cerebral cortex using functional micro-ultrasound.

Relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF), and blood flow speed are key parameters that characterize cerebral hemodynamics. We used contrast-enhanced functional micro-ultrasound (fMUS) imaging employing a disruption-replenishment imaging sequence to quantify these hemodynamic parameters in the anesthetized rat brain. The method has a spatial resolution of about 100 µm in-plane and around 600 µm through-plane, which is comparable to fMRI, and it has a superior temporal resolution of 40 ms per frame. We found no significant difference in rCBV of cortical and subcortical gray matter (0.89 ± 0.08 and 0.61 ± 0.09 times the brain-average value, respectively). The rCBV was significantly higher in the vascular regions on the pial surface (3.89 ± 0.71) and in the area of major vessels in the subcortical gray matter (2.02 ± 0.31). Parametric images of rCBV, rCBF, and blood flow speed demonstrate spatial heterogeneity of these parameters on the 100 µm scale. Segmentation of the cortex in arteriolar and venular-dominated regions identified through color Doppler imaging showed that rCBV is higher and flow speed is lower in venules than in arterioles. Finally, we show that the dependence of rCBV on rCBF was significantly different in cortical versus subcortical gray matter: the exponent α in the power law relation rCBV=s·rCBF(α) was 0.37 ± 0.13 in cortical and 0.75 ± 0.16 in subcortical gray matter. This work demonstrates that functional micro-ultrasound imaging affords quantification of hemodynamic parameters in the anesthetized rodent brain. This modality is a promising tool for neuroscientists studying these parameters in rodent models of diseases with a cerebrovascular component, such as stroke, neurodegeneration, and venous collagenosis. It is of particular import for studying conditions that selectively affect arteriolar versus venular compartments.

Functional micro-ultrasound imaging of rodent cerebral hemodynamics.

Healthy cerebral microcirculation is crucial to neuronal functioning. We present a new method to investigate microvascular hemodynamics in living rodent brain through a focal cranial window based on high-frequency ultrasound imaging. The method has a temporal resolution of 40ms, and a 100µm in-plane and 600µm through-plane spatial resolution. We use a commercially available high-frequency ultrasound imaging system to quantify changes in the relative cerebral blood volume (CBV) by measuring the scattered signal intensity from an ultrasound contrast agent circulating in the vasculature. Generalized linear model analysis is then used to produce effect size and significance maps of changes in cerebral blood volume upon electrical stimulation of the forepaw. We observe larger CBV increases in the forelimb representation of the primary somatosensory cortex than in the deep gray matter with stimuli as short as 2s (5.1 ± 1.3% vs. 3.3 ± 0.6%). We also investigate the temporal evolution of the blood volume changes in cortical and subcortical gray matter, pial vessels and subcortical major vessels, and show shorter response onset times in the parenchymal regions than in the neighboring large vessels (1.6 ± 1.0s vs. 2.6 ± 1.3s in the cortex for a 10 second stimulus protocol). This method, which we termed functional micro-ultrasound imaging or fMUS, is a novel, highly accessible, and cost-effective way of imaging rodent brain microvascular topology and hemodynamics in vivo at 100micron resolution over a 1-by-1cm field of view with 10s-100s frames per second that opens up a new set of questions regarding brain function in preclinical models of health and disease.

Covert strokes prior to Alzheimer's disease onset accelerate peri-lesional pathology but not cognitive deficits in an inducible APP mouse model.

It is estimated that up to 1 in 3 healthy middle-aged adults will have had a covert stroke during their lifetime. Furthermore, post-stroke, survivors are more than twice as likely to develop dementia. In the present study, we aimed to model the impact of focal subclinical ischemia prior to the onset of AD pathogenesis in a preclinical model. We utilized endothelin-1 to induce ischemia in an iducible transgenic mouse model of Alzheimer's disease, APPsi:tTA, allowing for temporal control of APP gene expression. We induced the focal subclinical ischemic events in the absence of APP expression, thus prior to AD onset. T2 structural magnetic resonance imaging confirmed the volume and location of focal subclinical ischemic lesions to the medial prefrontal cortex. Following recovery from surgery and 7 weeks of APP expression, we found that two subclinical ischemic lesions resulted in a significant localized increase in amyloid load and in microglial activation proximal to the lesion. However, no differences were found in astrogliosis. A battery of behaviour tests was conducted, in which no significant differences were detected in activities of daily living and cognitive function between stroked and sham cohorts. Overall, our results demonstrated that APP expression was the sole driving force behind behavioural deficits. In conclusion, our results suggest that a history of two subclinical strokes prior to AD onset does not worsen early disease trajectory in a mouse model.

Attenuation of tonic inhibition prevents chronic neurovascular impairments in a Thy1-ChR2 mouse model of repeated, mild traumatic brain injury.

Rationale: Mild traumatic brain injury (mTBI), the most common type of brain trauma, frequently leads to chronic cognitive and neurobehavioral deficits. Intervening effectively is impeded by our poor understanding of its pathophysiological sequelae. Methods: To elucidate the long-term neurovascular sequelae of mTBI, we combined optogenetics, two-photon fluorescence microscopy, and intracortical electrophysiological recordings in mice to selectively stimulate peri-contusional neurons weeks following repeated closed-head injury and probe individual vessel's function and local neuronal reactivity. Results: Compared to sham-operated animals, mTBI mice showed doubled cortical venular speeds (115 ± 25%) and strongly elevated cortical venular reactivity (53 ± 17%). Concomitantly, the pericontusional neurons exhibited attenuated spontaneous activity (-57 ± 79%) and decreased reactivity (-47 ± 28%). Post-mortem immunofluorescence revealed signs of peri-contusional senescence and DNA damage, in the absence of neuronal loss or gliosis. Alteration of neuronal and vascular functioning was largely prevented by chronic, low dose, systemic administration of a GABA-A receptor inverse agonist (L-655,708), commencing 3 days following the third impact. Conclusions: Our findings indicate that repeated mTBI leads to dramatic changes in the neurovascular unit function and that attenuation of tonic inhibition can prevent these alterations. The sustained disruption of the neurovascular function may underlie the concussed brain's long-term susceptibility to injury, and calls for development of better functional assays as well as of neurovascularly targeted interventions.

Cerebrovascular damage after midlife transient hypertension in non-transgenic and Alzheimer's disease rats.

Hypertension, including transient events, is a major risk factor for developing late-onset dementia and Alzheimer's disease (AD). Anti-hypertensive drugs facilitate restoration of normotension without amelioration of increased dementia risk suggesting that transient hypertensive insults cause irreversible damage. This study characterized the contribution of transient hypertension to sustained brain damage as a function of normal aging and AD. To model transient hypertension, we treated F344TgAD and non-transgenic littermate rats with L-NG-Nitroarginine methyl ester (L-NAME) for one month, ceased treatment and allowed for a month of normotensive recovery. We then examined the changes in the structure and function of the cerebrovasculature, integrity of white matter, and progression of AD pathology. As independent factors, both transient hypertension and AD compromised structural and functional integrity across the vascular bed, while combined effects of hypertension and AD yielded the largest deficits. Combined effects of transient hypertension and AD genotype resulted in loss of cortical myelin particularly in the cingulate cortex which is crucial for cognitive function. Increased cerebral amyloid angiopathy, a prominent pathology of AD, was detected after transient hypertension as were up- and down-regulation of proteins associated with cerebrovascular remodeling - osteopontin, ROCK1 and ROCK2, in F344TgAD rats even 30 days after restoration of normotension. In conclusion, transient hypertension caused permanent cerebrovasculature and brain parenchymal damage in both normal aging and AD. Our results corroborate human studies that have found close correlation between transient hypertension in midlife and white matter lesions later in life outlining vascular pathologies as pathological links to increased risk of dementia.

Frequency selective neuronal modulation triggers spreading depolarizations in the rat endothelin-1 model of stroke.

Ischemia is one of the most common causes of acquired brain injury. Central to its noxious sequelae are spreading depolarizations (SDs), waves of persistent depolarizations which start at the location of the flow obstruction and expand outwards leading to excitotoxic damage. The majority of acute stage of stroke studies to date have focused on the phenomenology of SDs and their association with brain damage. In the current work, we investigated the role of peri-injection zone pyramidal neurons in triggering SDs by optogenetic stimulation in an endothelin-1 rat model of focal ischemia. Our concurrent two photon fluorescence microscopy data and local field potential recordings indicated that a ≥ 60% drop in cortical arteriolar red blood cell velocity was associated with SDs at the ET-1 injection site. SDs were also observed in the peri-injection zone, which subsequently exhibited elevated neuronal activity in the low-frequency bands. Critically, SDs were triggered by low- but not high-frequency optogenetic stimulation of peri-injection zone pyramidal neurons. Our findings depict a complex etiology of SDs post focal ischemia and reveal that effects of neuronal modulation exhibit spectral and spatial selectivity.

Refinement of a chronic cranial window implant in the rat for longitudinal in vivo two-photon fluorescence microscopy of neurovascular function.

Longitudinal studies using two-photon fluorescence microscopy (TPFM) are critical for facilitating cellular scale imaging of brain morphology and function. Studies have been conducted in the mouse due to their relatively higher transparency and long term patency of a chronic cranial window. Increasing availability of transgenic rat models, and the range of established behavioural paradigms, necessitates development of a chronic preparation for the rat. However, surgical craniotomies in the rat present challenges due to craniotomy closure by wound healing and diminished image quality due to inflammation, restricting most rat TPFM experiments to acute preparations. Long-term patency is enabled by employing sterile surgical technique, minimization of trauma with precise tissue handling during surgery, judicious selection of the size and placement of the craniotomy, diligent monitoring of animal physiology and support throughout the surgery, and modification of the home cage for long-term preservation of cranial implants. Immunohistochemical analysis employing the glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) showed activation and recruitment of astrocytes and microglia/macrophages directly inferior to the cranial window at one week after surgery, with more diffuse response in deeper cortical layers at two weeks, and amelioration around four weeks post craniotomy. TPFM was conducted up to 14 weeks post craniotomy, reaching cortical depths of 400 µm to 600 µm at most time-points. The rate of signal decay with increasing depth and maximum cortical depth attained had greater variation between individual rats at a single time-point than within a rat across time.

Combinatorial Treatment Using Umbilical Cord Perivascular Cells and Aß Clearance Rescues Vascular Function Following Transient Hypertension in a Rat Model of Alzheimer Disease.

Transient hypertension is a risk factor for Alzheimer disease (AD), but the effects of this interaction on brain vasculature are understudied. Addressing vascular pathology is a promising avenue to potentiate the efficacy of treatments for AD. We used arterial spin labeling magnetic resonance imaging to longitudinally assess brain vascular function and immunohistopathology to examine cerebrovascular remodeling and amyloid load. Hypertension was induced for 1 month by administration of l-NG-nitroarginine-methyl-ester in TgF344-AD rats at the prodromal stage. Following hypertension, nontransgenic rats showed transient cerebrovascular changes, whereas TgF344-AD animals exhibited sustained alterations in cerebrovascular function. Human umbilical cord perivascular cells in combination with scyllo-inositol, an inhibitor of Aß oligomerization, resulted in normalization of hippocampal vascular function and remodeling, in contrast to either treatment alone. Prodromal stage hypertension exacerbates latter AD pathology, and the combination of human umbilical cord perivascular cells with amyloid clearance promotes cerebrovascular functional recovery.

Oophorectomy Reduces Estradiol Levels and Long-Term Spontaneous Neurovascular Recovery in a Female Rat Model of Focal Ischemic Stroke.

Although epidemiological evidence suggests significant sex and gender-based differences in stroke risk and recovery, females have been widely under-represented in preclinical stroke research. The neurovascular sequelae of brain ischemia in females, in particular, are largely uncertain. We set out to address this gap by a multimodal in vivo study of neurovascular recovery from endothelin-1 model of cortical focal-stroke in sham vs. ovariectomized female rats. Three weeks post ischemic insult, sham operated females recapitulated the phenotype previously reported in male rats in this model, of normalized resting perfusion but sustained peri-lesional cerebrovascular hyperreactivity. In contrast, ovariectomized (Ovx) females showed reduced peri-lesional resting blood flow, and elevated cerebrovascular responsivity to hypercapnia in the peri-lesional and contra-lateral cortices. Electrophysiological recordings showed an attenuation of theta to low-gamma phase-amplitude coupling in the peri-lesional tissue of Ovx animals, despite relative preservation of neuronal power. Further, this chronic stage neuronal network dysfunction was inversely correlated with serum estradiol concentration. Our pioneering data demonstrate dramatic differences in spontaneous recovery in the neurovascular unit between Ovx and Sham females in the chronic stage of stroke, underscoring the importance of considering hormonal-dependent aspects of the ischemic sequelae in the development of novel therapeutic approaches and patient recruitment in clinical trials.

Neurogliovascular dysfunction in a model of repeated traumatic brain injury.

Traumatic brain injury (TBI) research has focused on moderate to severe injuries as their outcomes are significantly worse than those of a mild TBI (mTBI). However, recent epidemiological evidence has indicated that a series of even mild TBIs greatly increases the risk of neurodegenerative and psychiatric disorders. Neuropathological studies of repeated TBI have identified changes in neuronal ionic concentrations, axonal injury, and cytoskeletal damage as important determinants of later life neurological and mood compromise; yet, there is a paucity of data on the contribution of neurogliovascular dysfunction to the progression of repeated TBI and alterations of brain function in the intervening period. Methods: Here, we established a mouse model of repeated TBI induced via three electromagnetically actuated impacts delivered to the intact skull at three-day intervals and determined the long-term deficits in neurogliovascular functioning in Thy1-ChR2 mice. Two weeks post the third impact, cerebral blood flow and cerebrovascular reactivity were measured with arterial spin labelling magnetic resonance imaging. Neuronal function was investigated through bilateral intracranial electrophysiological responses to optogenetic photostimulation. Vascular density of the site of impacts was measured with in vivo two photon fluorescence microscopy. Pathological analysis of neuronal survival and astrogliosis was performed via NeuN and GFAP immunofluorescence. Results: Cerebral blood flow and cerebrovascular reactivity were decreased by 50±16% and 70±20%, respectively, in the TBI cohort relative to sham-treated animals. Concomitantly, electrophysiological recordings revealed a 97±1% attenuation in peri-contusional neuronal reactivity relative to sham. Peri-contusional vascular volume was increased by 33±2% relative to sham-treated mice. Pathological analysis of the peri-contusional cortex demonstrated astrogliosis, but no changes in neuronal survival. Conclusion: This work provides the first in-situ characterization of the long-term deficits of the neurogliovascular unit following repeated TBI. The findings will help guide the development of diagnostic markers as well as therapeutics targeting neurogliovascular dysfunction.