Temporal remodeling of pial collaterals and functional deficits in a murine model of ischemic stroke

Abstract

Background Leptomeningeal anastomoses play a critical role in regulating reperfusion following cerebrovascular obstruction; however, methods to evaluate their temporospatial remodeling remains under investigation. New method We combined arteriole-specific vessel painting with histological evaluation to assess the density and diameter of inter-collateral vessels between the middle cerebral artery and anterior cerebral artery (MCA-ACA) or posterior cerebral artery (MCA-PCA) in a murine model of permanent middle cerebral artery occlusion (pMCAO). Results While the overall density was not influenced by pMCAO, the size of MCA-ACA and MCA-PCA vessels had significantly increased 2 days post-pMCAO and peaked by 4 days compared to the un-injured hemisphere. Using a combination of vessel painting and immunofluorescence, we uniquely observed an induction of cellular division and a remodeling of the smooth muscle cells within the collateral niche following post-pMCAO on whole mount tissue sections. Vessel painting was also applied to pMCAO-injured Cx3cr1GFP mice, in order to identify the spatial relationship between Cx3cr1-positive peripheral-derived monocyte/macrophages and the vessel painted collaterals. Our histological findings were supplemented with analysis of cerebral blood flow using laser Doppler imaging and behavioral changes following pMCAO. Comparison with existing methods Compared to polyurethane and latex methods for collateral labeling, this new method provides detailed cell-type specific analysis within the collateral niche at the microscopic level, which has previously been unavailable. Conclusions This simple and reproducible combination of techniques is the first to dissect the temporospatial remodeling of pial collateral arterioles. The method will advance investigations into the underlying mechanisms governing the intricate processes of arteriogenesis.

EphA4/Tie2 crosstalk regulates leptomeningeal collateral remodeling following ischemic stroke

“…Evidence of early cellular remodeling in EphA4 fl/fl /Tie2-Cre vessel painted MCA-ACA collaterals. EphA4 suppresses EC proliferation in vitro (27) and the pial collateral niche undergoes active cellular remodeling following pMCAO (28). To address whether the loss of EC-specific EphA4 improved early remodeling of pial vessels by enhancing EC growth properties, we performed immunolabeling for cell division marker, PCNA on wild type and KO cortical tissue whole mounts 1 day after pMCAO and vessel painting.…”

“…For peptide inhibitor studies, 15,000 cells were plated in each well of a 96-well plate containing serum-free media treated with either VTM-EEKK, VTA-EEKK, KYL, or water (vehicle control) at 75, 250, or 500 μM. BrdU (Sigma-Aldrich, 10 μM) was added, and 24 hours later cells were fixed for immunohistochemistry using 1:1000, rat anti-BrdU Surgical procedures and treatments Ischemic stroke was induced by pMCAO as previously described (28). Briefly, 8-to 12-week-old male mice were injected with analgesic Buprenorphine-SR (0.15 mg/kg, ZooPharm) followed by induction of anesthesia using 2% isoflurane-30% oxygen.…”

“…Fixed brains were imaged at ×4 magnification on an upright fluorescence microscope (BX-51, Olympus America) or inverted 880 Zeiss confocal microscope. Images were imported into ImageJ (NIH) for quantification of the number and diameter of inter-collaterals, as described (28). Similarly, images were assessed using ImageJ to quantify the number of PCNA + /VP + cells within the collateral vessel walls from maximum Z-projected images using the cell-counting tool.…”

“…Cortical whole mounts were dissected and placed in 1X PBS overnight as previously described (28). Briefly, whole mounts were blocked in 2% fish gelatin (Sigma-Aldrich) with 0.1% Triton X-100 and incubated overnight in mouse anti-smooth muscle actin (SMA) (1:1000; Abcam, ab7817) at 4°C.…”

Pericyte-Mediated Tissue Repair through PDGFRβ Promotes Peri-Infarct Astrogliosis, Oligodendrogenesis, and Functional Recovery after Acute Ischemic Stroke

“…Mice were euthanized on day 7 by intraperitoneal administration of pentobarbital and transcardially perfused with 20-ml ice-cold saline and 20-ml 4% PFA. For immunostaining, the cortical surface of the brain was dissected (Okyere et al, 2018). Whole mounts were subsequently blocked in 5% skimmed milk for 30 min at room temperature, then incubated at 4°C overnight in primary antibodies: anti-a smooth muscle actin (SMA; 1:100; Abcam #ab7817), anti-CD31 (1:200; BD Biosciences #550274), and anti-F4/80 (1:100; Abcam #ab6640).…”