Biology subjects

Sutherland, B. A.

Publications and source records attributed to Sutherland, B. A..

7 recordsLinked to original sources

bioRxiv2026

Peptidylglycine α-amidating monooxygenase restores brain microvascular blood flow and improves recovery following ischemic stroke

IntroductionReduced or absent capillary blood flow (termed no-reflow) even after arterial recanalization is associated with poorer neurological outcomes following ischemic stroke. The aim of the current study was to test whether acute administration of peptidylglycine -amidating monooxygenase (PAM) can increase capillary blood flow and improve brain recovery after ischemic stroke. MethodsA 60-minute ischemic stroke was induced using middle cerebral artery occlusion (MCAO) in rats. A modified, long-acting PAM enzyme was administered 30 minutes after induction of ischemia and rats were recovered for either 24 hours or 7 days. In all animals, real-time cerebral blood flow was assessed before, during and after MCAO using trasncranial contrast enhanced ultrasound (tCEU). For rats in the 7-day protocol, a modified neuroscore test was used to assess neurological deficit following MCAO. At the end of each experiment, a transcardiac perfusion was used to generate a fluorescent vascular cast and histology was used to examine capillary diameters and determine infarct volume. ResultsFollowing MCAO and arterial recanalization, untreated rats had reduced cerebral blood flow across brain regions affected by ischemia, indicative of no-reflow. PAM administration led to enhanced cerebral blood flow in affected regions, and this was associated with increased capillary diameters 24 hours after ischemic stroke. Although there was no difference in infarct volume at 24 hours, by day 7, infarct volume was markedly reduced in the PAM group and these animals exhibited improved neurological function compared to the untreated group. ConclusionAdministration of PAM improves capillary blood flow after ischemic stroke leading to enhanced neurological and brain recovery. This work highlights PAM as a novel theraputic approach to improve brain blood flow and recovery after ischemic stroke. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/732201v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@3095ddorg.highwire.dtl.DTLVardef@1bf159borg.highwire.dtl.DTLVardef@239afdorg.highwire.dtl.DTLVardef@19476e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Rapamycin Treatment Reduces Brain Pericyte Constriction in Ischemic Stroke

The contraction and subsequent death of brain pericytes may play a role in microvascular no-reflow following the re-opening of an occluded artery during ischemic stroke. Mammalian target of rapamycin (mTOR) inhibition has been shown to reduce motility/contractility of various cancer cell lines and reduce neuronal cell death in stroke. However, the effects of mTOR inhibition on brain pericyte contraction and death during ischemia have not yet been investigated. Cultured pericytes exposed to simulated ischemia for 12 hours in vitro contracted after less than 1 h, which was about 7h prior to cell death. Rapamycin significantly reduced the rate of pericyte contraction during ischemia, however, it did not have a significant effect on pericyte viability at any time point. Rapamycin appeared to reduce pericyte contraction through a RhoA-dependent pathway, independent of changes in intracellular calcium. Using a mouse model of middle cerebral artery occlusion, rapamycin significantly increased the diameter of capillaries underneath pericytes and increased the number of open capillaries 30 minutes following recanalization. Our findings suggest rapamycin may be a useful adjuvant therapeutic to reduce pericyte contraction and improve cerebral reperfusion post-stroke.

neuroscience↗

bioRxiv2024

Microglia contact cerebral vasculature through gaps between astrocyte endfeet

The close spatial relationship between microglia and cerebral blood vessels implicates microglia in vascular development, homeostasis and disease. In this study we used the publicly available Cortical MM^3 electron microscopy dataset to systematically investigate microglial interactions with the vasculature. Our analysis revealed that approximately 20% of microglia formed direct contacts with blood vessels through gaps between adjacent astrocyte endfeet. We termed these contact points "plugs". Plug-forming microglia exhibited closer proximity to blood vessels than non-plug forming microglia and formed multiple plugs, predominantly near the soma, ranging in surface area from [~]0.01 m2 to [~]15 m2. Plugs were enriched at the venule end of the vascular tree and displayed a preference for contacting endothelial cells over pericytes at a ratio of 3:1. In summary, we provide novel insights into the ultrastructural relationship between microglia and the vasculature, laying a foundation for understanding how these contacts contribute to the functional cross-talk between microglia and cells of the vasculature in health and disease.

neuroscience↗

bioRxiv2023

Pericyte ablation causes hypoactivity and reactive gliosis in adult mice

Pericytes are contractile cells that enwrap capillaries allowing them to control blood flow, maintain the blood-brain barrier and regulate immune cell trafficking in the CNS. Pericytes are lost or become dysfunctional in neurodegenerative diseases such as Alzheimers disease, stroke, and multiple sclerosis, but their role in health and disease is poorly understood. Our aim was to evaluate blood-brain barrier integrity and glial reactivity, and to assess behavioural phenotypes that emerge following pericyte ablation in adult mice. The delivery of tamoxifen to PDGFR{beta}-CreERT2 :: Rosa26-DTA transgenic mice produced a dose-dependent ablation of pericytes. A single low dose of tamoxifen ablated approximately half of all brain pericytes, and two consecutive daily high doses ablated more than 80% of brain pericytes. To determine whether pericyte ablation could induce a behavioural phenotype, we assessed patterns of voluntary movement, as well as balance and coordination using the open field and beam walk tasks. Mice with [~]50% pericyte loss travelled half the distance and spent half as much time moving in the open field as control mice. Mice with more than 80% pericyte ablation also slipped more frequently in the beam walk task than control mice. In brain cryosections from pericyte-ablated mice, blood vessel structure was unchanged, but lumen area was increased. Pericyte-ablated mice also experienced blood-brain barrier leakage, hypoxia and increased microgliosis and astrogliosis compared to control mice. Our results highlight the importance of pericytes for brain health, as pericyte loss can directly drive brain injury and behavioural alterations in mice.

neuroscience↗

bioRxiv2023

Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

BackgroundPericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and PDGF receptor-beta inhibitors impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive meditators. iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

cell biology↗

bioRxiv2022

Microglia associations with brain pericytes and the vasculature are reduced in Alzheimer's disease

Cerebral blood flow is important for the maintenance of brain function and its dysregulation has been implicated in Alzheimers disease (AD). Subpopulations of microglia have well-characterised associations with the vasculature in the central nervous system but the precise relationship between microglia and cells which exist on the vasculature is not yet clear. In this study we explored the relationship between microglia and pericytes, a vessel-resident cell type that has a major role in the regulation of cerebral blood flow and maintenance of the blood brain barrier. Using fixed tissue sections and in vivo live imaging, we discovered a subset of microglia that closely associated with pericytes, termed PEricyte-associated Microglia (PEM). PEM are present throughout all regions of the brain and spinal cord in NG2DsRed x CX3CR1+/GFP mice, and in the human frontal cortex. They reside adjacent to pericytes at all levels of the capillary tree and can maintain their position for at least 28 days. PEM associate with pericytes lacking astroglial endfeet coverage but are segregated from pericytes by capillary basement membranes and capillary vessel width is similarly increased beneath pericytes with or without an associated PEM. Deletion of the microglia fractalkine receptor (CX3CR1) did not disrupt the association between pericytes and PEM, suggesting the association is not reliant on fractalkine signalling. Finally, we found that the proportion of microglia that are capillary-associated and PEM declines in the superior frontal gyrus (SFG) in AD, which is exacerbated by the APOE {varepsilon}3/{varepsilon}4 genotype. In summary, we identify and characterise a subpopulation of microglia that specifically associate with pericytes and find this population is reduced in the SFG in AD. This reduction may be a novel mechanism contributing to vascular dysfunction in diseases such as AD.

neuroscience↗

bioRxiv2021

An automated approach to improve the speed and accuracy of pericyte and microglia quantification in whole mouse brain sections

Whole slide scanning technology has enabled the generation of high-resolution images of complete tissue sections. However, commonly used analysis software is often unable to handle the large data files produced. Here we present a method using the open-source software QuPath to detect, classify and quantify fluorescently-labelled cells (microglia and pericytes) in whole coronal brain tissue sections. Whole brain sections from both male and female NG2DsRed x CX3CR1+/GFP mice were analysed. Small regions of interest were selected and manual counts were compared to counts generated from an automated approach, across a range of detection parameters. The optimal parameters for detecting cells and classifying them as microglia or pericytes in each brain region were determined and applied to annotations corresponding to the entire cortex, hippocampus, thalamus and hypothalamus in each section. 3.71% of all detected cells were classified as pericytes, however this proportion was significantly higher in the thalamus (6.39%) than in other regions. In contrast, microglia (4.45% of total cells) were more abundant in the cortex (5.54%). No differences were detected between male and female mice. In conclusion, QuPath offers a user-friendly, rapid and accurate solution to whole-slide image analysis which could lead to important new discoveries in both health and disease. Significance StatementRapid and accurate quantification of cell numbers and distributions from whole tissue sections represents a difficult challenge in biomedical research. Slide scanning microscopes generate high-resolution images of complete tissue sections but most common image analysis software packages struggle to cope with the large data files they produce. We provide a method for rapidly and accurately quantifying pericyte and microglia cell numbers in whole brain tissue sections using QuPath, a new open-source software designed specifically to overcome this challenging roadblock.

neuroscience↗