title: Imaging alterations in the hemodynamic response in the SHRSP: A model of cerebral small vessel disease
Creators: name: Morgan, Robert
Publisher: name: University of Glasgow
Year: 2021
Subjects: subject: fMRI, cerebral small vessel disease, SHRSP, BOLD.

description: Cerebral small vessel disease (SVD) is associated with various pathological and neurological processes that affect perforating arterioles, capillaries and venules. The characteristic features of cerebral SVD include small lacunar subcortical infarcts, white matter hyperintensities, microbleeds and cortical microinfarcts. The underlying pathology of cerebral SVD is not fully known, owing to the difficulty of studying the disease in humans. Small lacunar infarcts are rarely fatal and often asymptomatic. In cases where death occurs, autopsy tissue is often difficult to interpret since multiple pathological changes have occurred at this late-stage in disease pathology. Spontaneously hypertensive stroke prone rats (SHRSPs) are an accepted and validated model of cerebral SVD, demonstrating many of the features observed in the human condition. Arteriosclerosis, thickening of the vessel wall, narrowing of the arterial lumen, endothelial dysfunction and a loss of vessel reactivity have all been reported in SHRSPs. Given these pathologic cerebrovascular changes, the hemodynamic response in these animals may be sensitive to such changes and therefore provide a marker of cerebrovascular health and an indicator of therapeutic effect. Blunted BOLD responses have been observed in patients with SVD but no study has assessed whether SHRSPs demonstrate a similar reduction in the stimulus-evoked BOLD response. Therefore, the main aim of this thesis was to evaluate whether differences in the hemodynamic response could be detected in between SHRSPs compared to age-matched WKYs. Prior to performing the study described above, we deemed it necessary to evaluate the ability to non-invasively monitor PaCO2¬, by measuring end-tidal CO2 using side-stream capnography. Baseline and stimulus-evoked cerebral blood flow (CBF) and blood oxygenation level dependent (BOLD) responses are sensitive to changes in the partial pressure of arterial carbon dioxide (PaCO2). Therefore, it is important that PaCO2 is monitored and kept within normal physiological values to avoid introducing confounds into the data. Blood gas analysis remains the gold standard for assessing PaCO2 and other blood gases, however these measurements are invasive and discrete, only providing a measure of PaCO2 for a short time following withdrawal and analysis of the blood sample. The ability to continuously and non-invasively assess PaCO2 using side-stream capnography would be beneficial for preclinical fMRI studies, particularly longitudinal studies, as it could minimise/eliminate any invasive surgery/procedure required to periodically take blood samples for blood gas analysis and provide a continuous measurement. However, the combination of the rat’s small tidal volume, long sample lines, and large sample volumes required by most capnographs complicate the accurate assessment of PaCO2 using ETCO2¬ ¬in these small animals. Therefore, an aim of this thesis was to assess the ability of micro-sampling side stream capnography to non-invasively assess PaCO2 by comparing ETCO2 measurements with PaCO2 measurements obtained by blood gas analysis. Despite the smaller sample volumes require by micro sampling capnographs, our findings indicate that a large variability still exists between ETCO2 and PaCO2 values when long sampling lines are used, as would be required for use in an fMRI experiment. Therefore, side-stream capnography was not implemented in any of the other studies described in this thesis. However, the use of shorter lines for bench top experiments may hold promise with some opimisation. It was also deemed necessary to explore the effects of varying forelimb stimulation parameters on stimulus-evoked BOLD, CBF and neural responses under combined medetomidine-isoflurane anaesthesia. Anesthesia is known to have various effects on CBF and neural activity, depending on the anesthetic being used, which can subsequently affect stimulus-evoked hemodynamic and neural responses. Therefore, there is a general consensus that stimulus parameters should be optimised to the anaesthetic being utilised, especially in cases where a novel anaesthetic is being used or if no stimulus optimisation is currently provided in the literature. Since no study has previously characterised the effects of varying stimulus parameters on the hemodynamic and neural responses under this anaesthetic protocol, the effect of varying the stimulus intensity, stimulus frequency and pulse duration on the stimulus-evoked BOLD, CBF and neural responses was subsequently investigated using fMRI, laser speckle contrast imaging and electrophysiology, respectively. The aim of these experiments was to identify the optimal stimulus parameters that evoked the largest response. Tested parameters were selected based on commonly used values in the literature. We observed that frequency had the largest impact on the resultant hemodynamic or neural measurements, with higher frequencies generally evoking larger peak responses. However, analysis of the SEPs revealed that at higher frequencies there was a periodic loss of the SEP response and the overall amplitude of the SEPs declined over the course of stimulation. Stimulus intensity had a modest effect on the hemodynamic response with larger responses generally being observed &gt;4 mA. Ultimately, the optimal parameters were selected as 6 mA, 0.3 ms and 3 Hz. These parameters were subsequently used for the fMRI study in SHRSPs. The findings in this thesis represent one of the few preclinical studies assessing the hemodynamic response in SHRSPs. Several studies have assessed the hemodynamic response in patients with cerebral SVD and observed blunted BOLD responses to stimulation. However, to our knowledge, this has not been assessed in the SHRSP. Given the role of the endothelium in mediating the hemodynamic response, and that dysfunction of the endothelium has been reported in the SHRSP and patients with cerebral SVD, assessing the hemodynamic response may provide a biomarker of cerebrovascular health and therapeutic effect. Several clinical trials have already assessed cerebrovascular reactivity by evaluating changes in the BOLD response to a hypercapnic challenge as a primary endpoint, suggesting this assessment is clinically relevant. Characterising the hemodynamic response in SHRSPs could further support their use as a model of cerebral SVD and allow consistent assessments to be performed in preclinical and clinical stages, which could aid in drug identification and development. Therefore, the main aim of this study was to characterise any differences between the stimulus-evoked BOLD and CBF responses to forelimb stimulation in the SHRSP, including any differences between young and old animals and aged-matched WKYs. The hemodynamic response was found to differ between older SHRSPs and young and older WKYs, with the stimulus-evoked BOLD and CBF responses being larger in the old SHRSPs. This observation opposes the findings of other studies that assess the hemodynamic response in patients with SVD, but aligns with fMRI findings in the SHR parent strain. It will be important for future studies to characterise this difference of responses between preclinical and clinical populations to confirm whether it is underpinned by a biological and pathologic mechanism or whether it has been artificially created by some of the requirements for preclinical studies i.e. anaesthesia.