Introduction:
Neurovascular unit (NVU) dysfunction plays a key role in cerebral small vessel disease (cSVD) pathogenesis. Spontaneously Hypertensive Rats - Stroke Prone (SHRSP) is a relevant model for cSVD where prominent impairment of the NVU has been identified. Previous studies of SHRSP and Wistar-Kyoto control rats (WKY) were performed at tissue level failing to capture the NVU cellular components where the early pathological events occur. Therefore, we developed a novel method for selective endothelial cell (EC) and pericytes capture using Laser Capture Microdissection (LCM).
Methods:
10 SHRSP and 10 WKY male rats were studied at 16 weeks of age. Prior to brain collection, lectin was administered using intracardiac injection for vessel identification. Rapid frozen immunohistochemistry (IHC) staining protocol was optimized by selecting a panel of antibodies that showed first specific staining pattern for EC and pericytes in a routine overnight IHC protocol. LCM collection was subsequently optimized to achieve collection of 1.5-2 million μm
2
of tissue in 30 minutes to ensure RNA stability. RNA was isolated from the LCM samples using PicoPure RNA isolation kit (ThermoFisher Scientific, Inc.) and RNA quantity was measured using NanoDrop technology. qRT-PCR we performed to measure the expression of smooth muscle actin (ACTA2) (pericytes), von Willebrand Factor (vWF) (EC).
Results:
Following optimization of overnight and rapid staining protocols, platelet-derived growth factor (PDGRF) beta (ABCAM, Inc.) (1:10 concentration) showed best results for perdicytes identification while RCA lectin (Vector labs, Inc.) (1:20 concentration) showed best results for EC. Co-staining with CD31 antibody was performed to confirm the specificity of RCA lectin binding to EC. RNA isolation protocol achieved good RNA collection quantity (4-8) ng/μl. qRT-PCR showed higher expression of ACTA2 (9.5-fold increase) in PDGRF+ elements, 2.8-fold increase of vWF (lectin+ elements) compared to (PDGRF-, lectin -) elements confirming the enrichment of these elements by EC and pericytes.
Conclusion:
LCM is feasible in achieving good quality collection of EC and pericyte in SHRSP and WKY allowing for investigation of the proteomic and transcriptomic changes that result in cSVD.
Laser Capture Microdissection of Cells from Plant Tissues