Dataset Information

Microglia display altered spatial morphology and proteome after stroke

ABSTRACT: Microglia are abundantly distributed throughout the central nervous system (CNS) to play critical roles in neural development and homeostasis, and act as immune sentinels to constantly monitor their surrounding neural environment. Given their high reactivity to brain insults, we hypothesised that the cerebral microenvironment altered by ischemic stroke would significantly impact microglial morphology and function in a spatially dependent manner. To investigate this, we examined regional changes in the expression of genes associated with microglial activation and neuroinflammation, microglial morphology using 3D image reconstruction and unbiased proteomics at 24 hours after transient middle cerebral artery occlusion (tMCAO). We found the microenvironment within the ischaemic infarct core has a distinct pro-inflammatory profile is very different to versus that of the sham-operated controls. , and more importantly this region of the brain displays dynamic and diverse inflammatory changes. Moreover, stroke induces region-specific changes to microglia morphology with those closer to the infarct displaying a more ameboid shape and less complex dendritic processes. Additionally, we identified 108 differentially expressed proteins in microglia that were isolated from the ipsilateral ischaemic hemisphere compared to those isolated from thethat of contralateral hemisphere. These differentially expressed proteins are predicted to influence signalling pathways that mediate TNF-α superfamily cytokine production, chemokine activities and leukocyte chemotaxis and migration. These findings support microglia as critical regulators of the inflammatory signalling after stroke. Mice Adult C57Bl/6J and Cx3cr1gfp/+ (both reporter strains are on C57Bl/6J background) male and female mice were obtained from the Monash Animal Research Platform and were housed at Monash Translational Health Precinct Animal Facility (MHTP-AF, Clayton, VIC, Australia) in specific pathogen-free (SPF) conditions with access to food and water ad libitum. Mice were housed in temperature-controlled rooms (22 °C) under a standard 12 h light-dark cycle. Prior to the start of experiments, mice were acclimatised for a minimum of 7 days before use. All procedures were approved by the Monash Medical Centre Animal Ethics Committee (MMCB/2018/002 and MMCB/2022/22, Monash Medical Centre, Clayton, Victoria, Australia). Middle cerebral artery occlusion (MCAO) surgery The mouse model of ischaemic stroke was performed as previously described (27), whereby infarct is induced in the right cerebral hemisphere. Briefly, mice were anaesthetised by intraperitoneal injection of ketamine (150 mg/kg) and xylazine (10 mg/kg). Once anaesthetised, an incision at the top of the head was made to expose the skull, and a laser Doppler holder (PeriFlux System 5000, Stroke model kit 407, Perimed, Järfälla-Stockholm, Sweden) was glued to the right side of the skull to monitor blood flow above the right hemisphere of the brain. The fur at the site of the neck incision was removed and the area was sterilised with 80 % ethanol. A 1–2-cm incision was made along the midline of the throat to expose the trachea. The right common carotid artery, external carotid artery, and internal carotid artery were dissected away from any connective tissue. A vessel clip was used on the right common carotid artery, and a slit was made in the external carotid artery. A silicone-coated monofilament with a diameter of 0.21–0.23 mm (Doccol Corporation) was immediately inserted into the external carotid artery and advanced into the internal carotid artery to occlude blood flow into the MCA. A successful occlusion was determined by >70% reduction in Doppler reading. The external carotid artery was tied off and the vessel clip removed. In this study, we utilised the reperfused model termed transient MCAO or tMCAO, whereby the monofilament was retracted to allow for reperfusion of blood flow into the MCA following 1 h of occlusion. After successful surgery, the neck incision was sutured closed, the doppler probe removed, and the head incision closed. As a control for surgical stress, sham operations were performed whereby similar surgical protocols were executed without the insertion of the monofilament. After surgery, all mice were allowed to recover overnight on a heat pad.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

DISEASE(S): Duchenne Muscular Dystrophy

SUBMITTER: Ralf Schittenhelm

LAB HEAD: Connie Wong

PROVIDER: PXD055588 | Pride | 2025-12-22

REPOSITORIES: Pride

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			Action	DRS
	ANALYSIS_Hossein.zip	Other
	AS1_JS_220324_P22_0561_E5_BrookeW_PQC.raw	Raw
	AS1_JS_220324_P22_0561_E5_BrookeW_PQC_20240324152844.raw	Raw
	AS1_JS_220324_P22_0561_E5_BrookeW_S01.raw	Raw
	AS1_JS_220324_P22_0561_E5_BrookeW_S02.raw	Raw

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Microglia Display Altered Spatial Morphology and Proteome After Stroke.

Wanrooy Brooke J BJ Wilson Jenny L JL Suthya Althea R AR Bourne Joshua H JH Steele Joel R JR Kahrood Hossein Valipour HV Schittenhelm Ralf B RB Ballerin Giulia G Skinner Cameron C Wen Shu Wen SW Wong Connie H Y CHY

Proteomics 20251110 23

Microglia are abundantly distributed throughout the central nervous system (CNS) to play critical roles in neural development and homeostasis, and act as immune sentinels to constantly monitor their surrounding neural environment. Given their high reactivity to brain insults, we hypothesised that the cerebral microenvironment altered by ischaemic stroke would significantly impact microglial morphology and function in a spatially dependent manner. To investigate this, we examined regional gene ex ...[more]

PMID: 41208752

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