The influence of voxelotor on cerebral blood flow and oxygen extraction in pediatric sickle cell disease.

ABSTRACT: Voxelotor is an inhibitor of sickle hemoglobin polymerization that is used to treat sickle cell disease. Although voxelotor has been shown to improve anemia, the clinical benefit on the brain remains to be determined. This study quantified the cerebral hemodynamic effects of voxelotor in children with sickle cell anemia (SCA) using noninvasive diffuse optical spectroscopies. Specifically, frequency-domain near-infrared spectroscopy combined with diffuse correlation spectroscopy were used to noninvasively assess regional oxygen extraction fraction (OEF), cerebral blood volume, and an index of cerebral blood flow (CBFi). Estimates of CBFi were first validated against arterial spin-labeled magnetic resonance imaging (ASL-MRI) in 8 children with SCA aged 8 to 18 years. CBFi was significantly positively correlated with ASL-MRI-measured blood flow (R2 = 0.651; P = .015). Next, a single-center, open-label pilot study was completed in 8 children with SCA aged 4 to 17 years on voxelotor, monitored before treatment initiation and at 4, 8, and 12 weeks (NCT05018728). By 4 weeks, both OEF and CBFi significantly decreased, and these decreases persisted to 12 weeks (both P < .05). Decreases in CBFi were significantly correlated with increases in blood hemoglobin (Hb) concentration (P = .025), whereas the correlation between decreases in OEF and increases in Hb trended toward significance (P = .12). Given that previous work has shown that oxygen extraction and blood flow are elevated in pediatric SCA compared with controls, these results suggest that voxelotor may reduce cerebral hemodynamic impairments. This trial was registered at www.ClinicalTrials.gov as #NCT05018728.

DCS blood flow index underestimates skeletal muscle perfusion in vivo: rationale and early evidence for the NIRS-DCS perfusion index.

Significance: Diffuse correlation spectroscopy (DCS) permits non-invasive assessment of skeletal muscle blood flow but may misestimate changes in muscle perfusion. Aim: We aimed to highlight recent evidence that DCS blood flow index (BFI) misestimates changes in muscle blood flow during physiological perturbation and to introduce a novel approach that adjusts BFI for estimated changes in vasodilation. Approach: We measured changes in muscle BFI during quadriceps and forearm exercises using DCS, the latter of which were adjusted for estimated changes in microvascular flow area and then compared to Doppler ultrasound in the brachial artery. Then, we compared adjusted BFI- and arterial spin labeling (ASL) MRI measures of gastrocnemius blood flow during reactive hyperemia and plantar flexion exercise. Results: We observed little-to-no change in quadriceps BFI during maximal-effort exercise. Similarly, forearm BFI was modestly increased during handgrip exercise, but the magnitude was significantly lower than measured by Doppler ultrasound in the brachial artery. However, this difference was ameliorated after adjusting BFI for estimated changes in microvascular flow area. Similar observations were also observed in the gastrocnemius when directly comparing the adjusted BFI values to ASL-MRI. Conclusions: Adjusting BFI for estimated changes in microvascular flow area may improve DCS estimates of muscle blood flow, but further study is needed to validate these methods moving forward.

Comparison of diffuse correlation spectroscopy analytical models for measuring cerebral blood flow in adults.

Significance: Although multilayer analytical models have been proposed to enhance brain sensitivity of diffuse correlation spectroscopy (DCS) measurements of cerebral blood flow, the traditional homogeneous model remains dominant in clinical applications. Rigorous in vivo comparison of these analytical models is lacking. Aim: We compare the performance of different analytical models to estimate a cerebral blood flow index (CBFi) with DCS in adults. Approach: Resting-state data were obtained on a cohort of 20 adult patients with subarachnoid hemorrhage. Data at 1 and 2.5 cm source-detector separations were analyzed with the homogenous, two-layer, and three-layer models to estimate scalp blood flow index and CBFi. The performance of each model was quantified via fitting convergence, fit stability, brain-to-scalp flow ratio (BSR), and correlation with transcranial Doppler ultrasound (TCD) measurements of cerebral blood flow velocity in the middle cerebral artery (MCA). Results: The homogeneous model has the highest pass rate (100%), lowest coefficient of variation (CV) at rest (median [IQR] at 1 Hz of 0.18 [0.13, 0.22]), and most significant correlation with MCA blood flow velocities (Rs=0.59, p=0.010) compared with both the two- and three-layer models. The multilayer model pass rate was significantly correlated with extracerebral layer thicknesses. Discarding datasets with non-physiological BSRs increased the correlation between DCS measured CBFi and TCD measured MCA velocities for all models. Conclusions: We found that the homogeneous model has the highest pass rate, lowest CV at rest, and most significant correlation with MCA blood flow velocities. Results from the multilayer models should be taken with caution because they suffer from lower pass rates and higher coefficients of variation at rest and can converge to non-physiological values for CBFi. Future work is needed to validate these models in vivo, and novel approaches are merited to improve the performance of the multimodel models.

Impaired cerebrovascular reactivity in pediatric sickle cell disease using diffuse correlation spectroscopy.

Cerebrovascular reactivity (CVR), defined as the ability of cerebral vasculature to dilate in response to a vasodilatory stimulus, is an integral mechanism in brain homeostasis that is thought to be impaired in sickle cell disease (SCD). This study used diffuse correlation spectroscopy and a simple breath-hold stimulus to quantify CVR non-invasively in a cohort of 12 children with SCD and 14 controls. Median [interquartile range] CVR was significantly decreased in SCD compared to controls (2.03 [1.31, 2.44] versus 3.49 [3.00, 4.11] %/mmHg, p = 0.028). These results suggest DCS may provide a feasible means to routinely monitor CVR impairments in pediatric SCD.

Cerebrospinal Fluid Pharmacokinetics of Nicardipine Following Intrathecal Administration in Subarachnoid Hemorrhage Patients.

Subarachnoid hemorrhage (SAH) is a devastating type of stroke, leading to high mortality and morbidity rates. Cerebral vasospasm and delayed cerebral ischemia (DCI) are common complications following SAH and contribute significantly to the poor outcomes observed in these patients. Intrathecal (IT) nicardipine delivered via an existing external ventricular drain has been shown to be correlated with reduced DCI and improved patient outcomes. The current study aims to characterize population pharmacokinetic (popPK) properties of intermittent IT nicardipine. Following informed consent, serial cerebrospinal fluid (CSF) samples were obtained from 16 SAH patients (50.4 ± 9.3 years old; 12 females) treated with IT nicardipine every 6 hours (n=8) or every 8 hours (n=8), which were subject to high-performance liquid chromatography for measurement of its CSF concentration. Our popPK analysis showed that the CSF PK of IT nicardipine in the cohort was adequately described by a two-compartment model with a lag time, with reliable parameter estimates (relative standard error < 50%). The intracranial pressure influenced both the total clearance and the central volume. Calculated PK parameters were similar between q6h and q8h dosing regimens. Despite a small cohort of SAH patients, we successfully developed a popPK model to describe the nicardipine disposition kinetics in the CSF following IT administration. These findings may help inform future clinical trials designed to examine the optimal dosing of IT nicardipine.

Profiling the neuroimmune cascade in 3xTg mice exposed to successive mild traumatic brain injuries.

Repetitive mild traumatic brain injuries (rmTBI) sustained within a window of vulnerability can result in long term cognitive deficits, depression, and eventual neurodegeneration associated with tau pathology, amyloid beta (Aß) plaques, gliosis, and neuronal and functional loss. However, we have limited understanding of how successive injuries acutely affect the brain to result in these devastating long-term consequences. In the current study, we addressed the question of how repeated injuries affect the brain in the acute phase of injury (<24hr) by exposing the 3xTg-AD mouse model of tau and Aß pathology to successive (1x, 3x, 5x) once-daily weight drop closed-head injuries and quantifying immune markers, pathological markers, and transcriptional profiles at 30min, 4hr, and 24hr after each injury. We used young adult mice (2-4 months old) to model the effects of rmTBI relevant to young adult athletes, and in the absence of significant tau and Aß pathology. Importantly, we identified pronounced sexual dimorphism, with females eliciting more differentially expressed proteins after injury compared to males. Specifically, females showed: 1) a single injury caused a decrease in neuron-enriched genes inversely correlated with inflammatory protein expression as well as an increase in AD-related genes within 24hr, 2) each injury significantly increased expression of a group of cortical cytokines (IL-1α, IL-1ß, IL-2, IL-9, IL-13, IL-17, KC) and MAPK phospho-proteins (phospho-Atf2, phospho-Mek1), several of which were co-labeled with neurons and correlated with phospho-tau, and 3) repetitive injury caused increased expression of genes associated with astrocyte reactivity and immune function. Collectively our data suggest that neurons respond to a single injury within 24h, while other cell types including astrocytes transition to inflammatory phenotypes within days of repetitive injury.

Normative cerebral microvascular blood flow waveform morphology assessed with diffuse correlation spectroscopy.

Microvascular cerebral blood flow exhibits pulsatility at the cardiac frequency that carries valuable information about cerebrovascular health. This study used diffuse correlation spectroscopy to quantify normative features of these waveforms in a cohort of thirty healthy adults. We demonstrate they are sensitive to changes in vascular tone, as indicated by pronounced morphological changes with hypercapnia. Further, we observe significant sex-based differences in waveform morphology, with females exhibiting higher flow, greater area-under-the-curve, and lower pulsatility. Finally, we quantify normative values for cerebral critical closing pressure, i.e., the minimum pressure required to maintain flow in a given vascular region.

Agreement in cerebrovascular reactivity assessed with diffuse correlation spectroscopy across experimental paradigms improves with short separation regression.

Significance: Cerebrovascular reactivity (CVR), i.e., the ability of cerebral vasculature to dilate or constrict in response to vasoactive stimuli, is a biomarker of vascular health. Exogenous administration of inhaled carbon dioxide, i.e., hypercapnia (HC), remains the "gold-standard" intervention to assess CVR. More tolerable paradigms that enable CVR quantification when HC is difficult/contraindicated have been proposed. However, because these paradigms feature mechanistic differences in action, an assessment of agreement of these more tolerable paradigms to HC is needed. Aim: We aim to determine the agreement of CVR assessed during HC, breath-hold (BH), and resting state (RS) paradigms. Approach: Healthy adults were subject to HC, BH, and RS paradigms. End tidal carbon dioxide (EtCO2) and cerebral blood flow (CBF, assessed with diffuse correlation spectroscopy) were monitored continuously. CVR (%/mmHg) was quantified via linear regression of CBF versus EtCO2 or via a general linear model (GLM) that was used to minimize the influence of systemic and extracerebral signal contributions. Results: Strong agreement ( CCC ≥ 0.69 ; R ≥ 0.76 ) among CVR paradigms was demonstrated when utilizing a GLM to regress out systemic/extracerebral signal contributions. Linear regression alone showed poor agreement across paradigms ( CCC ≤ 0.35 ; R ≤ 0.45 ). Conclusions: More tolerable experimental paradigms coupled with regression of systemic/extracerebral signal contributions may offer a viable alternative to HC for assessing CVR.

Influence of oversimplifying the head anatomy on cerebral blood flow measurements with diffuse correlation spectroscopy.

Significance: Diffuse correlation spectroscopy (DCS) is an emerging optical modality for non-invasive assessment of an index of regional cerebral blood flow. By the nature of this noninvasive measurement, light must pass through extracerebral layers (i.e., skull, scalp, and cerebral spinal fluid) before detection at the tissue surface. To minimize the contribution of these extracerebral layers to the measured signal, an analytical model has been developed that treats the head as a series of three parallel and infinitely extending slabs (mimicking scalp, skull, and brain). The three-layer model has been shown to provide a significant improvement in cerebral blood flow estimation over the typically used model that treats the head as a bulk homogenous medium. However, the three-layer model is still a gross oversimplification of the head geometry that ignores head curvature, the presence of cerebrospinal fluid (CSF), and heterogeneity in layer thickness. Aim: Determine the influence of oversimplifying the head geometry on cerebral blood flow estimated with the three-layer model. Approach: Data were simulated with Monte Carlo in a four-layer slab medium and a three-layer sphere medium to isolate the influence of CSF and curvature, respectively. Additionally, simulations were performed on magnetic resonance imaging (MRI) head templates spanning a wide-range of ages. Simulated data were fit to both the homogenous and three-layer model for CBF. Finally, to mitigate the errors in potential CBF estimation due to the difficulty in defining layer thickness, we investigated an approach to identify an equivalent, "optimized" thickness via a pressure modulation. Results: Both head curvature and failing to account for CSF lead to significant errors in the estimation of CBF. However, the effect of curvature and CSF on relative changes in CBF is minimal. Further, we found that CBF was underestimated in all MRI-templates, although the magnitude of these underestimations was highly influenced by small variations in the source and detector optode positioning. The optimized thickness obtained from pressure modulation did not improve estimation accuracy of CBF, although it did significantly improve the estimation accuracy of relative changes in CBF. Conclusions: In sum, these findings suggest that the three-layer model holds promise for improving estimation of relative changes in cerebral blood flow; however, estimations of absolute cerebral blood flow with the approach should be viewed with caution given that it is difficult to account for appreciable sources of error, such as curvature and CSF.

Microvascular cerebral blood flow response to intrathecal nicardipine is associated with delayed cerebral ischemia.

One of the common complications of non-traumatic subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (DCI). Intrathecal (IT) administration of nicardipine, a calcium channel blocker (CCB), upon detection of large-artery cerebral vasospasm holds promise as a treatment that reduces the incidence of DCI. In this observational study, we prospectively employed a non-invasive optical modality called diffuse correlation spectroscopy (DCS) to quantify the acute microvascular cerebral blood flow (CBF) response to IT nicardipine (up to 90 min) in 20 patients with medium-high grade non-traumatic SAH. On average, CBF increased significantly with time post-administration. However, the CBF response was heterogeneous across subjects. A latent class mixture model was able to classify 19 out of 20 patients into two distinct classes of CBF response: patients in Class 1 (n = 6) showed no significant change in CBF, while patients in Class 2 (n = 13) showed a pronounced increase in CBF in response to nicardipine. The incidence of DCI was 5 out of 6 in Class 1 and 1 out of 13 in Class 2 (p < 0.001). These results suggest that the acute (<90 min) DCS-measured CBF response to IT nicardipine is associated with intermediate-term (up to 3 weeks) development of DCI.

Rebellion, curiosity, and perseverance: a conversation with Maria Angela Franceschini.

Neurophotonics Associate Editor Erin M. Buckley (Georgia Institute of Technology and Emory University) interviewed her colleague Maria Angela Franceschini, professor at the Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, and recognized leader in the field of diffuse optical imaging in both neuroscience and clinical neuro-monitoring applications.

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.

Quantifying the Cerebral Hemometabolic Response to Blood Transfusion in Pediatric Sickle Cell Disease With Diffuse Optical Spectroscopies.

Red blood cell transfusions are common in patients with sickle cell disease who are at increased risk of stroke. Unfortunately, transfusion thresholds needed to sufficiently dilute sickle red blood cells and adequately restore oxygen delivery to the brain are not well defined. Previous work has shown that transfusion is associated with a reduction in oxygen extraction fraction and cerebral blood flow, both of which are abnormally increased in sickle patients. These reductions are thought to alleviate hemometabolic stress by improving the brain's ability to respond to increased metabolic demand, thereby reducing susceptibility to ischemic injury. Monitoring the cerebral hemometabolic response to transfusion may enable individualized management of transfusion thresholds. Diffuse optical spectroscopies may present a low-cost, non-invasive means to monitor this response. In this study, children with SCD undergoing chronic transfusion therapy were recruited. Diffuse optical spectroscopies (namely, diffuse correlation spectroscopy combined with frequency domain near-infrared spectroscopy) were used to quantify oxygen extraction fraction (OEF), cerebral blood volume (CBV), an index of cerebral blood flow (CBFi), and an index of cerebral oxygen metabolism (CMRO2i) in the frontal cortex immediately before and after transfusion. A subset of patients receiving regular monthly transfusions were measured during a subsequent transfusion. Data was captured from 35 transfusions in 23 patients. Transfusion increased median blood hemoglobin levels (Hb) from 9.1 to 11.7 g/dL (p < 0.001) and decreased median sickle hemoglobin (HbS) from 30.9 to 21.7% (p < 0.001). Transfusion decreased OEF by median 5.9% (p < 0.001), CBFi by median 21.2% (p = 0.020), and CBV by median 18.2% (p < 0.001). CMRO2i did not statistically change from pre-transfusion levels (p > 0.05). Multivariable analysis revealed varying degrees of associations between outcomes (i.e., OEF, CBFi, CBV, and CMRO2i), Hb, and demographics. OEF, CBFi, and CBV were all negatively associated with Hb, while CMRO2i was only associated with age. These results demonstrate that diffuse optical spectroscopies are sensitive to the expected decreases of oxygen extraction, blood flow, and blood volume after transfusion. Diffuse optical spectroscopies may be a promising bedside tool for real-time monitoring and goal-directed therapy to reduce stroke risk for sickle cell disease.

Influence of source-detector separation on diffuse correlation spectroscopy measurements of cerebral blood flow with a multilayered analytical model.

Significance: Diffuse correlation spectroscopy (DCS) is an emerging noninvasive optical technology for bedside monitoring of cerebral blood flow. However, extracerebral hemodynamics can significantly influence DCS estimations of cerebral perfusion. Advanced analytical models can be used to remove the contribution of extracerebral hemodynamics; however, these models are highly sensitive to measurement noise. There is a need for an empirical determination of the optimal source-detector separation(s) (SDS) that improves the accuracy and reduces sensitivity to noise in the estimation of cerebral blood flow with these models. Aim: To determine the influence of SDS on solution uniqueness, measurement accuracy, and sensitivity to inaccuracies in model parameters when using the three-layer model to estimate cerebral blood flow with DCS. Approach: We performed a series of in silico simulations on samples spanning a wide range of physiologically-relevant layer optical properties, thicknesses, and flow. Data were simulated at SDS ranging from 0.5 to 3.0 cm using the three-layer solution to the correlation diffusion equation (with and without noise added) and using three-layer slab Monte Carlo simulations. We quantified the influence of SDS on uniqueness, accuracy, and sensitivity to inaccuracies in model parameters using the three-layer inverse model. Results: Two SDS are required to ensure a unique solution of cerebral blood flow index (CBFi). Combinations of 0.5/1.0/1.5 and 2.5 cm provide the optimal choice for balancing the depth penetration with signal-to-noise ratio to minimize the error in CBFi across a wide range of samples with varying optical properties, thicknesses, and dynamics. Conclusions: These results suggest that the choice of SDS is critical for minimizing the estimated error of cerebral blood flow when using the three-layer model to analyze DCS data.

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NFκB pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.

Upcoming Neurophotonics Status Report.

Forthcoming status report articles provide updates on microscopy and on diffuse optical imaging in neurophotonics.

Accuracy of diffuse correlation spectroscopy measurements of cerebral blood flow when using a three-layer analytical model.

Diffuse correlation spectroscopy (DCS) is a non-invasive optical technology for the assessment of an index of cerebral blood flow (CBFi). Analytical methods that model the head as a three-layered medium (i.e., scalp, skull, brain) are becoming more commonly used to minimize the contribution of extracerebral layers to the measured DCS signal in adult cerebral blood flow studies. However, these models rely on a priori knowledge of layer optical properties and thicknesses. Errors in these values can lead to errors in the estimation of CBFi, although the magnitude of this influence has not been rigorously characterized. Herein, we investigate the accuracy of measuring cerebral blood flow with a three-layer model when errors in layer optical properties or thicknesses are present. Through a series of in silico experiments, we demonstrate that CBFi is highly sensitive to errors in brain optical properties and skull and scalp thicknesses. Relative changes in CBFi are less sensitive to optical properties but are influenced by errors in layer thickness. Thus, when using the three-layer model, accurate estimation of scalp and skull thickness are required for reliable results.

Diffuse Optical Spectroscopy Assessment of Resting Oxygen Metabolism in the Leg Musculature.

We lack reliable methods to continuously assess localized, resting-state muscle activity that are comparable across individuals. Near-infrared spectroscopy (NIRS) provides a low-cost, non-invasive means to assess localized, resting-state muscle oxygen metabolism during venous or arterial occlusions (VO2VO and VO2AO, respectively). However, this technique is not suitable for continuous monitoring, and its utility is limited to those who can tolerate occlusions. Combining NIRS with diffuse correlated spectroscopy (DCS) enables continuous measurement of an index of muscle oxygen metabolism (VO2i). Despite the lack of previous validation, VO2i is employed as a measure of oxygen metabolism in the muscle. Here we characterized measurement repeatability and compared VO2i with VO2VO and VO2AO in the medial gastrocnemius (MG) in 9 healthy adults. Intra-participant repeatability of VO2i, VO2VO, and VO2AO were excellent. VO2i was not significantly correlated with VO2AO (p = 0.15) nor VO2VO (p = 0.55). This lack of correlation suggests that the variability in the calibration coefficient between VO2i and VO2AO/VO2VO in the MG is substantial across participants. Thus, it is preferable to calibrate VO2i prior to every monitoring session. Important future work is needed to compare VO2i against gold standard modalities such as positron emission tomography or magnetic resonance imaging.

Cerebrovascular reactivity measured in awake mice using diffuse correlation spectroscopy.

Significance: Cerebrovascular reactivity (CVR), defined as the ability of the cerebral vasculature to dilate or constrict in response to a vasoactive stimulus, is an important indicator of the brain's vascular health. However, mechanisms of cerebrovascular dysregulation are poorly understood, and no effective treatment strategies for impaired CVR exist. Preclinical murine models provide an excellent platform for interrogating mechanisms underlying CVR dysregulation and determining novel therapeutics that restore impaired CVR. However, quantification of CVR in mice is challenging. Aim: We present means of assessing CVR in awake mice using intraperitoneal injection of acetazolamide (ACZ) combined with continuous monitoring of cerebral blood flow. Approach: Measurements of cerebral blood flow were made with a minimally invasive diffuse correlation spectroscopy sensor that was secured to an optical window glued to the intact skull. Two source-detector separations (3 and 4.5 mm) per hemisphere were used to probe different depths. CVR was quantified as the relative increase in blood flow due to ACZ. CVR was assessed once daily for 5 days in 5 mice. Results: We found that CVR and the response half-time were remarkably similar across hemispheres and across 3- versus 4.5-mm separations, suggesting a homogenous, whole brain response to ACZ. Mean(std) intra- and intermouse coefficients of variations were 15(9)% and 19(10)%, respectively, for global CVR and 24(15)% and 27(11)%, respectively, for global response half-time. Conclusion: In sum, we report a repeatable method of measuring CVR in free-behaving mice which can be used to screen for impairments with disease and to track changes in CVR with therapeutic interventions.