Assessment of Neurovascular Coupling & Cortical Spreading Depression in Mixed Models of Atherosclerosis & Alzheimer’s Disease

Neurovascular coupling is a critical brain mechanism whereby changes to blood flow accompany localised neural activity. The breakdown of neurovascular coupling is linked to the development and progression of several neurological conditions including dementia. In this study, we examined cortical haemodynamics in preparations that modelled Alzheimer’s disease (J20-AD) and atherosclerosis (PCSK9-ATH) between 9-12m of age. We report novel findings with atherosclerosis where neurovascular decline is characterised by significantly reduced blood volume, levels of oxyhaemoglobin & deoxyhaemoglobin, in addition to global neuroinflammation. In the comorbid mixed model (J20-PCSK9-MIX), we report a 3x fold increase in hippocampal amyloid-beta plaques. A key finding was that cortical spreading depression (CSD) due to electrode insertion into the brain was worse in the diseased animals and led to a prolonged period of hypoxia. These findings suggest that systemic atherosclerosis can be detrimental to neurovascular health and that having cardiovascular comorbidities can exacerbate pre-existing Alzheimer’s-related amyloid-plaques.


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Alzheimer's disease (AD) is the most common form of dementia worldwide, with the vast majority of  Rahimi & Kovacs, 2014). A major cardiovascular pathology that affects as many as up to 60% of all 2 individuals after the age of 55 is atherosclerosis. Atherosclerosis is the progressive thickening, 3 hardening and narrowing of major arteries, including those that supply the brain, such as the carotids 4 (Lusis, 2000). Intracranial atherosclerosis does not occur until much later in life, around 75 years and 5 above. As such, Alzheimer's disease that begins around the 8 th decade of life is usually present with 6 other comorbidities such as atherosclerosis (Napoli et al., 1999). There is also evidence that, not only 7 do these often exist as comorbidities, but they may interact pathogenically with vascular disease and 8 neurovascular unit changes contributing to AD (Iadecola, 2017;Kapasi & Schneider, 2016). To date, 9 there are very limited models of comorbidity with respect to preclinical studies, and instead models have 10 been very specific and 'pure', and not reflective of the clinical pathology in humans. Atherosclerosis is 11 known to be a major risk factor for the development of dementia. The progressive atheromatous plaque 12 build-up within cerebral arteries that supply the cortex over time can lead to stenosis producing 13 insufficient oxygen delivery to the brain parenchyma, potentially resulting in neuronal death and  which currently has no disease-modifying cure, are to succeed.

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The breakdown of NVC is thought to be an important and early pathogenic mechanism in the onset and 22 progression of a range of neurological conditions (Zlokovic, 2011). In the present study, we aimed to 23 investigate neurovascular function in mid-aged (9-12m old) mice where atherosclerosis was a 24 comorbidity. We used a novel model of atherosclerosis that utilises a single adeno-associated virus 25 (AAV) i.v. injection of a gain of function mutation (D377Y) to proprotein convertase subtilisin/kexin type 26 9 (rAAV8-mPCSK9-D377Y), combined with a high-fat Western diet to induce atherosclerosis in most  hypothesised that atherosclerosis would exacerbate Alzheimer's disease pathology in the brain and 38 that neurovascular function would be further worsened compared to AD or ATH models alone. We have 39 previously reported no significant alterations to evoked-haemodynamics in the J20-AD model of the 40 3 same age (9-12m); however, under acute imaging sessions where an electrode was inserted into the 1 brain, we found significantly perturbed haemodynamics (Sharp et al., 2019). We hypothesised that 2 electrode insertion causes cortical spreading depression (CSD). Based on recent data linking migraine 3 with aura with cardiovascular disease (Kurth et al., 2020), we hypothesised that experimental CSD 4 might be heightened in all disease models. We report that experimentally induced atherosclerosis in 5 the J20-AD model increased the number of Aβ plaques by 300%. Furthermore, experimental CSD is 6 more severe in all diseased groups compared to WT controls.  We performed chronic imaging of the brain cortex 3-weeks post-surgery, where the thinned cranial

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these stimulations, activation maps of blood volume; total haemoglobin (HbT), can be generated ( Figure   23 1C). Mice were allowed to recover and after 1-week, a final acute imaging session was performed. In 24 this setup, a small burr-hole was drilled through the thinned skull overlying the active region of interest 25 (ROI) as determined from the chronic imaging sessions ( Figure 1D), and a multichannel electrode was 26 inserted into the brain ( Figure 1E) to record neural activity simultaneously. We then imaged and 27 recorded the baseline haemodynamics for a 35-minute period to observe the effect electrode insertion, 28 before commencing the first stimulation. This was also done to record baselines on chronic imaging 29 sessions.

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Chronic Haemodynamic Responses in the Brain are Reduced in PCSK9-ATH Mice 32 Cortical haemodynamics were imaged through a thinned cranial window to determine whether evoked 33 cortical haemodynamics were different between 9-12m old wild-type (WT), atherosclerotic (PCSK9-

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HbT responses; although initially are smaller in J20-PCSK9-MIX mice, recovered to match that of J20-

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AD mice later in the experimental protocol under normoxia ( Figure 2D). Levels of oxyhaemoglobin 40 4 (HbO) were significantly reduced in PCSK9-ATH mice but showed a reduced trend in J20-PCSK9-MIX 1 mice too. The washout of deoxyhaemoglobin (HbR) was significantly reduced in PCSK9-ATH mice 2 compared to WT, but also showed a reduced trend across all diseased groups across all conditions 3 compared to WT mice. All mice displayed stable and robust haemodynamic responses across the 4 experimental protocol ( Figure S2). Finally, vascular reactivity as determined by the response to 10% 5 hypercapnia was not significantly different between any of the diseased groups ( Figure S3).

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CSD is Worse in Diseased Animals and Impacts Haemodynamic Recovery to Baseline 8 1-week after recovery from the chronic imaging protocol, an acute imaging experiment was performed 9 wherein a small-burr hole was drilled into the skull overlying the active region (determined from HbT 10 responses from chronic experiments) and a microelectrode was inserted into the brain to a depth of     Figure 3B). In all disease 24 mouse models, the constriction below baseline was more severe and persisted for a much longer time 25 with a slower haemodynamic recovery. Following CSD, stimulation-evoked haemodynamic changes 26 were not significantly different in any of the diseased groups overall, although they were initially smaller 27 in the first two stimulations for PCSK9-ATH mice ( Figure S1).

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Stimulus-Evoked Neural Activity is Not Significantly Altered in Any Disease Groups Compared to WT   Immunohistochemistry was performed on J20-AD and J20-PCSJK9-MIX mice to assess whether there 6 were any specific differences in AD neuropathology changes. Staining was performed for Aβ plaques 7 and these were quantified within the hippocampus and the cortex. Aβ plaques were significantly 8 increased by 3-fold in the hippocampi of J20-PCSK9-MIX mice compared to J20-AD mice ( Figure 5A/B).

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Within the cortex, there was no significant difference in Aβ plaques between the 2 groups (data not 10 shown). Next, neuroinflammation was assessed by qRT-PCR for 2 key inflammatory markers: 11 interleukin-1β (IL1β) and tumour necrosis factor-α (TNFα) to assess the degree of neuroinflammation 12 present globally within the brain. IL1β mRNA was significantly upregulated in J20-AD and PCSK9-ATH 13 mice ( Figure 5C). TNFα mRNA was significantly upregulated in PCSK9-ATH mice only ( Figure 5D). 14 J20-PCSK9-MIX mice displayed the lowest inflammatory changes in IL1β & TNFα compared to the 15 other diseased groups, though this was still higher than WT mice.

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The present study investigated neurovascular function in a novel experimental model of atherosclerosis

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(PCSK9-ATH) and for the first time, in a comorbid setting whereby atherosclerosis was experimentally 20 induced in a well characterised model of AD; J20-hAPP, to create a mixed comorbid model (J20-21 PCSK9-MIX). These mice were compared to age-matched (9-12m) WT C57BL/6J controls, and J20-

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AD mice. Given that systemic atherosclerosis is a major risk factor for dementia, the mechanisms 23 underpinning the relationship between atherosclerosis, neurovascular decline and dementia are still 24 largely unclear.

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In the first part of the study, we characterised evoked-haemodynamic responses using a chronic skull-

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intact & surgery-recovered mouse preparation. We found that PCSK9-ATH mice displayed significantly 28 reduced evoked blood volume (HbT) responses, in addition to reduced levels of oxyhaemoglobin (HbO) 29 and notably, an impaired washout of deoxyhaemoglobin (HbR) across all stimulations and conditions.

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The J20-PCSK9-MIX mice did not display a significant reduction in HbT, nor in HbO or HbR levels. With 31 respect to J20-AD mice, we did not observe any significant alterations to HbT as previously published  typically occur in healthy brain tissue, however, it is a common neurophysiological occurrence in certain 10 pathological conditions including migraine, epilepsy, brain injury, hyperthermia, hypoxia & ischaemia 11 (Dreier, 2011). In WT mice, the initial constriction wave is small, and a robust haemodynamic recovery 12 occurs which allows for neurovascular coupling to occur to sustain neurons metabolically. This is a 13 marked difference to the diseased animals, which upon electrode insertion to cause a CSD, exhibit    study examined women who suffered from migraines with and without aura and found that those that 37 suffered migraines with aura had a higher incidence rate of cardiovascular disease compared to women 38 without aura or any migraines (Kurth et al., 2020). In addition, another recent study found that migraine 39 history was positively associated with an increased risk of developing both all-cause dementia and AD,

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A question that arises is why the J20-PCSK9-MIX mice HbT responses are not more severely impaired 15 than J20-AD and PCSK9-ATH? There may be redundancies that occur physiologically to compensate 16 for mild hypoxia in the brain, such as the possible angiogenesis within the brain. Angiogenesis is known 17 to be triggered in cerebral microvessels in AD in response to increased Aβ and neuroinflammation and

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There are several notable limitations with the present study. Firstly, all imaging was performed on lightly 27 anaesthetised animals, which is known to compromise neurovascular function (Gao et al., 2017).

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However, previous research from our laboratory has developed an anaesthetic regimen that is 29 comparable to awake imaging in terms of the haemodynamic responses to physiological whisker in which we showed that regardless of the baseline blood volume estimation used, our percentage 1 change was scaled by it (i.e. always the same change). Therefore, the observations in this paper with 2 respect to the different diseased animals are robust.

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In conclusion, we report novel findings of impaired neurovascular function in a novel experimental 5 model of atherosclerosis (PCSK9-ATH) characterised by reduced stimulus-evoked blood volume 6 without any significant alterations to evoked neural activity. We induced atherosclerosis in a mild fAD 7 model (J20-AD) to create a mixed comorbid model (J20-PCSK9-ATH) in which we report a significant 8 increase in the number of hippocampal Aβ plaques, however, without any significant changes to evoked 9 haemodynamic or neural responses compared to WT or J20-AD mice. A key finding from this study 10 was CSD was more severe in diseased animals. This may reflect the global inflammatory state of the 11 brain and could also serve to be an effective preclinical and human clinical biomarker for baseline state

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HbT, HbR and HbO concentration estimates were generated from baseline values in which the 20 concentration of haemoglobin in the tissue was assumed to be 100µM and O2 saturation to be 70%.

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For the stimulation experiments, whiskers were mechanically deflected for a 2s-duration and a 16s-

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duration at 5Hz using a plastic T-shaped stimulator which caused a 1cm deflection of the left-whisker.

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Each individual experiment consisted of 30 stimulation trials (for 2s) and 15 stimulation trials (for 16s)

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of which a mean trial was generated after spectral analysis of 2D-OIS. Stimulations were performed 25 with the mouse breathing in 100% O2 or 21% O2, and a gas transition to medical air (21% O2) as well 26 as an additional 10% CO2-hypercapnia test of vascular reactivity.

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Neural Electrophysiology   Analysis was performed using MATLAB (MathWorks). An automated region of interest (ROI) was 3 selected using the stimulation data from spatial maps generated using 2D-OIS. The threshold for a pixel 4 to be included within the ROI was set at 1.5xSD, therefore the automated ROI for each session per 5 animal represents the area of the cortex with the largest haemodynamic response, as determined by 6 the HbT. For each experiment, the response across all pixels within the ROI was averaged and used 7 to generate a time-series of the haemodynamic response against time.

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Statistical Analysis

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ANOVA was used as they were considered fairly robust against small deviations from normality).