Cerebrovascular reactivity measured in awake mice using diffuse correlation spectroscopy.

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.1 Impaired CVR is present in numerous disease states, including cerebrovascular disease,2–8 stroke,9–12 cardiovascular disease,13,14 cardiac arrest,15 traumatic brain injury,16–19 diabetes,20 and sleep apnea.21 Moreover, several studies have suggested that impaired CVR may serve as a prognostic biomarker of functional outcome.22–25 Despite mounting evidence for the relevance of CVR as a potential disease biomarker, mechanisms of cerebrovascular dysregulation are poorly understood, and effective treatment strategies for impaired CVR are lacking. Preclinical murine models provide an excellent platform for interrogating mechanisms underlying vascular dysregulation and determining novel therapeutics that restore impaired CVR. However, quantification of CVR in mice is challenging. Common techniques to assess CVR include intravenous injection of the potent vasodilator acetazolamide (ACZ) and induction of hypercapnia via inhalation of a high-concentration (typically 5% to 6%) carbon dioxide gas mixture. For both of these techniques, cerebral blood flow is monitored before and after intervention, and CVR is defined as the relative percent change in blood flow from preintervention levels normalized to either the dose of ACZ or to the change in arterial partial pressure of carbon dioxide. Although these assessments are routinely used in humans, delivery of the vasoactive stimulus and reliable monitoring of the stimulus response is more challenging in mice. In the case of ACZ, although intravenous tail vein injection is possible, longitudinal assessment of CVR with this approach is challenging because repeated tail vein injections without the use of a catheter cause inconsistent drug delivery and increase risk of infection and vein collapse.26 Alternatively, hypercapnia requires anesthesia for gas delivery in mice, which can induce significant confounding effects on the cerebrovasculature that lead to errors in the estimation of CVR.27–32 Moreover, monitoring the amount of carbon dioxide that has reached the blood stream to accurately estimate CVR during hypercapnia requires either invasive insertion of an arterial catheter to sample blood or intubation to measure end tidal carbon dioxide concentrations. Finally, both interventions to assess CVR (ACZ/hypercapnia) require quantification of cerebral blood flow, which is non-trivial in mice due to their small size. Although a handful of techniques exist to measure CBF in mice, e.g., autoradiography, perfusion magnetic resonance imaging, laser Doppler flowmetry, laser speckle contrast imaging, and microultrasound, these techniques typically involve invasive surgery, anesthesia, and/or manual restraint.33 In sum, challenges in both stimulus delivery and monitoring response to the stimulus make CVR measurements in murine models technically challenging, time consuming, and preclude longitudinal assessment. Herein, we present a novel means of assessing CVR in mice that overcomes several of these technical limitations and enables longitudinal monitoring of CVR in awake, free behaving animals. In this approach, mice are given ACZ intraperitoneally, and the cerebral blood flow response is continuously monitored with diffuse correlation spectroscopy (DCS) using a minimally invasive sensor that is secured to the intact skull. Given the relative ease of both intraperitoneal injections and assessment of blood flow with DCS, multiple longitudinal measurements of CVR can easily be acquired. In this work, we characterize average CVR in healthy mice, and we quantify the spatial and temporal heterogeneity of the CVR response.