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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 μm 3 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., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024. Published by Elsevier Inc.)

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., (Copyright © 2020. Published by Elsevier Inc.)

The 3D organization of cerebral blood vessels determines the overall capacity of the cerebral circulation to meet the metabolic requirements of the brain. Imaging methodologies which combine 3D microvascular structural imaging with blood flow quantification can shed light on the relationship between vascular structure and function, in health and disease. This study applies Arterial Spin Labeling (ASL) MRI with a hypercapnic challenge and ex vivo Serial Two-Photon Tomography (STPT) to examine the relationship between blood flow and vascular architecture following traumatic brain injury (TBI) in a mouse. Mice were exposed to a controlled cortical impact TBI and allowed to recover for either 1 day or 4 weeks. At each time point, ASL MRI was performed to quantify cerebral perfusion and the brain vasculature was imaged in 3D with STPT. Registration of ASL to STPT enabled flow changes to be related to the underlying microvascular structure in each ASL voxel. Hypoperfusion under rest and hypercapnia was observed both 1 day and 4 weeks post-TBI. Vessel density and vascular volume were reduced 1 day post-TBI, recovering by 4 weeks; however, the reorganized vasculature at the latter time point possessed an abnormal radial pattern. Our findings demonstrate functionally significant long-term changes in the vascular architecture following injury and illustrate why metrics beyond traditional measures of vessel density are required to understand the impact of vascular structure on function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 (v RBC ) 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 ± 0.84 mV LFP amplitude and 75 ± 48% increase in v RBC ; diffuse: 1.14 ± 0.75 mV LFP amplitude and 35 ± 23% increase in v RBC ). Compared to diffuse photostimulation, focused photostimulation resulted in a ∼65% increase in the yield of cerebrovascular responses (73 ± 10% for focused and 42 ± 29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9 ± 14.7 for focused and 10.4 ± 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., (Copyright © 2019. Published by Elsevier Inc.)

Brain plasticity following focal cerebral ischaemia has been observed in both stroke survivors and in preclinical models of stroke. Endogenous neurovascular adaptation is at present incompletely understood yet its potentiation may improve long-term functional outcome. We employed longitudinal MRI, intracranial array electrophysiology, Montoya Staircase testing, and immunofluorescence to examine function of brain vessels, neurons, and glia in addition to forelimb skilled reaching during the subacute stage of ischemic injury progression. Focal ischemic stroke (~100mm 3 or ~20% of the total brain volume) was induced in adult Sprague-Dawley rats via direct injection of endothelin-1 (ET-1) into the right sensori-motor cortex, producing sustained impairment in left forelimb reaching ability. Resting perfusion and vascular reactivity to hypercapnia in the peri-lesional cortex were elevated by approximately 60% and 80% respectively seven days following stroke. At the same time, the normal topological pattern of local field potential (LFP) responses to peripheral somatosensory stimulation was abolished and the average power of spontaneous LFP activity attenuated by approximately 50% relative to the contra-lesional cortex, suggesting initial response attenuation within the peri-infarct zone. By 21 days after stroke, perilesional blood flow resolved, but peri-lesional vascular reactivity remained elevated. Concomitantly, the LFP response amplitudes increased with distance from the site of ET-1 injection, suggesting functional remodelling from the core of the lesion to its periphery. This notion was further buttressed by the lateralization of spontaneous neuronal activity: by day 21, the average ipsi-lesional power of spontaneous LFP activity was almost twice that of the contra-lesional cortex. Over the observation period, the peri-lesional cortex exhibited increased vascular density, along with neuronal loss, astrocytic activation, and recruitment and activation of microglia and macrophages, with neuronal loss and inflammation extending beyond the peri-lesional cortex. These findings highlight the complex relationship between neurophysiological state and behaviour and provide evidence of highly dynamic functional changes in the peri-infarct zone weeks following the ischemic insult, suggesting an extended temporal window for therapeutic interventions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Physical exercise, besides improving cognitive and mental health, is known to cause structural changes in the brain. Understanding the structural changes that occur with exercise as well as the neuroanatomical correlates of a predisposition for exercise is important for understanding human health. This study used high-resolution 3D MR imaging, in combination with deformation-based morphometry, to investigate the macroscopic changes in brain structure that occur in healthy adult mice following four weeks of voluntary exercise. We found that exercise induced changes in multiple brain structures that are involved in motor function and learning and memory including the hippocampus, dentate gyrus, stratum granulosum of the dentate gyrus, cingulate cortex, olivary complex, inferior cerebellar peduncle and regions of the cerebellum. In addition, a number of brain structures, including the hippocampus, striatum and pons, when measured on MRI prior to the start of exercise were highly predictive of subsequent exercise activity. Exercise tended to normalize these pre-existing differences between mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Functional neuroimaging in animal models is essential for understanding the principles of neurovascular coupling and the physiological basis of fMRI signals that are widely used to study sensory and cognitive processing in the human brain. While hemodynamic responses to sensory stimuli have been characterized in humans, animal studies are able to combine very high resolution imaging with invasive measurements and pharmacological manipulation. To date, most high-resolution studies of neurovascular coupling in small animals have been carried out in anesthetized rodents. Here we report fMRI experiments in conscious, awake common marmosets (Callithrix jacchus), and compare responses to animals anesthetized with propofol. In conscious marmosets, robust BOLD fMRI responses to somatosensory stimulation of the forearm were found in contralateral and ipsilateral regions of the thalamus, primary (SI) and secondary (SII) somatosensory cortex, and the caudate nucleus. These responses were markedly stronger than those in anesthetized marmosets and showed a monotonic increase in the amplitude of the BOLD response with stimulus frequency. On the other hand, anesthesia significantly attenuated responses in thalamus, SI and SII, and abolished responses in caudate and ipsilateral SI. Moreover, anesthesia influenced several other aspects of the fMRI responses, including the shape of the hemodynamic response function and the interareal (SI-SII) spontaneous functional connectivity. Together, these findings demonstrate the value of the conscious, awake marmoset model for studying physiological responses in the somatosensory pathway, in the absence of anesthesia, so that the data can be compared most directly to fMRI in conscious humans., (Published by Elsevier Inc.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The spatial and temporal response of the cerebral microcirculation to mild hypercapnia was investigated via two-photon laser-scanning microscopy. Cortical vessels, traversing the top 200 microm of somatosensory cortex, were visualized in alpha-chloralose-anesthetized Sprague-Dawley rats equipped with a cranial window. Intraluminal vessel diameters, transit times of fluorescent dextrans and red blood cells (RBC) velocities in individual capillaries were measured under normocapnic (PaCO2= 32.6 +/- 2.6 mm Hg) and slightly hypercapnic (PaCO2= 45 +/- 7 mm Hg) conditions. This gentle increase in PaCO2 was sufficient to produce robust and significant increases in both arterial and venous vessel diameters, concomitant to decreases in transit times of a bolus of dye from artery to venule (14%, P < 0.05) and from artery to vein (27%, P < 0.05). On the whole, capillaries exhibited a significant increase in diameter (16 +/- 33%, P < 0.001, n = 393) and a substantial increase in RBC velocities (75 +/- 114%, P < 0.001, n = 46) with hypercapnia. However, the response of the cerebral microvasculature to modest increases in PaCO2 was spatially heterogeneous. The maximal relative dilatation (range: 5-77%; mean +/- SD: 25 +/- 34%, P < 0.001, n = 271) occurred in the smallest capillaries (1.6 microm-4.0 microm resting diameter), while medium and larger capillaries (4.4 microm-6.8 microm resting diameter) showed no significant changes in diameter (P > 0.08, n = 122). In contrast, on average, RBC velocities increased less in the smaller capillaries (39 +/- 5%, P < 0.002, n = 22) than in the medium and larger capillaries (107 +/- 142%, P < 0.003, n = 24). Thus, the changes in capillary RBC velocities were spatially distinct from the observed volumetric changes and occurred to homogenize cerebral blood flow along capillaries of all diameters.

To investigate the role of cyclooxygenase-2 (COX-2) in the cerebrovascular coupling, hemodynamic and neuronal responses to forepaw stimulation were measured in alpha-chloralose-anesthetized rats (N = 18) before and after intravenous administration of Meloxicam (MEL), a preferential COX-2 inhibitor, and following a bolus of prostaglandin E(2) (PGE(2)), a prominent vasodilatatory product of COX-2 catalyzed metabolism of arachidonic acid. The cerebral blood flow (CBF) and blood-oxygenation-level-dependent (BOLD) response was quantified using continuous arterial spin labeling magnetic resonance imaging. Neuronal activity was measured by recording somatosensory-evoked potentials (SEPs) via intracranial electrodes. Both MEL and PGE(2) had a significant effect on the activation-elicited CBF (P < 10(-6)) and BOLD (P < 10(-6)) responses, without affecting the baseline perfusion. Meloxicam decreased brain COX enzymatic activity by 57 +/- 14% and decreased the stimulation-induced CBF response to 32 +/- 2% and BOLD to 46 +/- 1% of their respective pre-drug amplitudes. In turn, PGE(2) bolus resulted in a partial recovery of functional hyperemia, with the CBF response recovering to 52 +/- 3% and the BOLD response to 56 +/- 2% of their values prior to MEL administration. There was no concomitant decrease in either amplitudes or latencies of SEP components. These findings suggest a modulatory role of COX-2 products in the cerebrovascular coupling and provide evidence for existence of a functional metabolic buffer.

In view of the potential of global resting blood flow level to confound the interpretation of blood oxygenation level-dependent (BOLD) fMRI studies, we investigated the effect of pronounced elevation in baseline cerebral blood flow (CBF) on BOLD and CBF responses to functional activation. Twelve healthy volunteers performed bilateral finger apposition while attending to a radial yellow/blue checkerboard. Three levels of global CBF increase were achieved by inhaling 5, 7.5 or 10% CO2. CBF and BOLD signals were simultaneously quantified using interleaved multi-slice pulsed arterial spin labeling (PASL) and T2*-weighted gradient echo sequences. Increasing basal CBF produced a significant decrease in the activation-induced BOLD response, with the slope of the optimal linear fit of activation versus basal BOLD signal changes of -0.32 +/- 0.01%/% for motor and visual cortex regions of interest (ROIs). While the modulation in basal flow level also produced a statistically significant effect on the activation-induced CBF change, the degree of relative attenuation of the flow response was slight, with a slope of -0.18 +/- 0.02%/% in the motor and -0.13 +/- 0.01%/% in the visual cortex ROI. The current findings describe a strong attenuation of the BOLD response at significantly elevated basal flow levels and call for independent quantification of resting CBF in BOLD fMRI studies that involve subjects and/or conditions with markedly elevated global perfusion.

To investigate the coupling between the hemodynamic and metabolic changes following functional brain activation as well as interictal epileptiform discharges (IEDs), blood oxygenation level dependent (BOLD), perfusion and oxygen consumption responses to a unilateral distal motor task and interictal epileptiform discharges (IEDs) were examined via continuous EEG-fMRI. Seven epilepsy patients performed a periodic (1 Hz) right-hand pinch grip using approximately 8% of their maximum voluntary contraction, a paradigm previously shown to produce contralateral MI neuronal excitation and ipsilateral MI neuronal inhibition. A multi-slice interleaved pulsed arterial spin labeling and T(2)*-weighted gradient echo sequence was employed to quantify cerebral blood flow (CBF) and BOLD changes. EEG was recorded throughout the imaging session and reviewed to identify the IEDs. During the motor task, BOLD, CBF and cerebral metabolic rate of oxygen consumption (CMR(O(2))) signals increased in the contra- and decreased in the ipsilateral primary motor cortex. The relative changes in CMR(O(2)) and CBF were linearly related, with a slope of 0.46 +/- 0.05. The ratio of contra- to ipsilateral CBF changes was smaller in the present group of epilepsy patients than in the healthy subjects examined previously. IEDs produced both increases and decreases in BOLD and CBF signals. In the two case studies for which the estimation criteria were met, the coupling ratio between IED-induced CMR(O(2)) and CBF changes was estimated at 0.48 +/- 0.17. These findings provide evidence for a preserved coupling between hemodynamic and metabolic changes in response to both functional activation and, for the two case studies available, in response to interictal epileptiform activity.

Functional magnetic resonance imaging (fMRI) was used to investigate the changes in blood oxygenation level dependent (BOLD) signal, cerebral blood flow (CBF) and cerebral metabolic rate of oxygen consumption (CMR(O(2))) accompanying neuronal inhibition. Eight healthy volunteers performed a periodic right-hand pinch grip every second using 5% of their maximum voluntary contraction (MVC), a paradigm previously shown to produce robust ipsilateral neuronal inhibition. To simultaneously quantify CBF and BOLD signals, an interleaved multislice pulsed arterial spin labeling (PASL) and T(2)*-weighted gradient echo sequence was employed. The CMR(O(2)) was calculated using the deoxyhemoglobin dilution model, calibrated by data measured during graded hypercapnia. In all subjects, BOLD, CBF and CMR(O(2)) signals increased in the contralateral and decreased in the ipsilateral primary motor (M1) cortex. The relative changes in CMR(O(2)) and CBF were linearly related, with a slope of approximately 0.4. The coupling ratio thus established for both positive and negative CMR(O(2)) and CBF changes is in close agreement with the ones observed by earlier studies investigating M1 perfusion and oxygen consumption increases. These findings characterize the hemodynamic and metabolic downregulation accompanying neuronal inhibition and thereby establish the sustained negative BOLD response as a marker of neuronal deactivation.