Abstract P54: Endothelial Cells and Pericytes Collection Using Laser Capture Microdissection in Spontaneously Hypertensive Stroke Prone Rats

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Jordan T Moore ◽  
Mohamed Ewees ◽  
Jay L Zweier ◽  
Daniel Gallego-perez ◽  
Yousef Hannawi
Keyword(s):  
Laser Capture Microdissection ◽  
Small Vessel Disease ◽  
Lectin Binding ◽  
Neurovascular Unit ◽  
Rna Isolation ◽  
Fold Increase ◽  
Male Rats ◽  
Spontaneously Hypertensive ◽  
Qrt Pcr ◽  
Laser Capture

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 Archival Kidney Tissue for qRT-PCR

2015 ◽  
pp. 251-265 ◽  
Author(s):  
Tim D. Hewitson ◽  
Michael Christie ◽  
Edward R. Smith
Keyword(s):  
Laser Capture Microdissection ◽  
Kidney Tissue ◽  
Qrt Pcr ◽  
Laser Capture

213 ANALYSIS OF STEROID HORMONE RECEPTOR GENE EXPRESSION IN THE BOVINE OVARY AFTER LASER-CAPTURE MICRODISSECTION

2010 ◽  
Vol 22 (1) ◽  
pp. 264
Author(s):  
R. Kenngott ◽  
G. Palma ◽  
M.J. Wendl ◽  
M. Vermehren ◽  
F. Sinowatz
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Real Time ◽  
Laser Capture Microdissection ◽  
Real Time Pcr ◽  
Quality Indicator ◽  
Estrogen Receptor Alpha ◽  
Qrt Pcr ◽  
Theca Interna ◽  
Laser Capture

Developmental processes in complex organs like the ovary are difficult to study in terms of a biochemical and molecular biological analysis. Laser-assisted microdissection allows the efficient and precise capture of single cells or groups of cells of an organ within the context of time and space and permits their subsequent molecular characterization. Together with real-time PCR techniques, it is now feasible to study gene expression in defined cell populations of complex tissues, but it is essential to create standards optimized for fixation, preparation, and isolation of RNA, reverse transcription reaction, and real-time PCR protocol for every tissue of interest. The aim of our study was to develop protocols for a precise analysis of estrogen receptor alpha (ER-α) and progesterone receptor (PR) in defined compartments of the ovary (granulosa cells, theca interna cells, zona vasculosa, and zona parenchymatosa of the stroma). Additionally, the receptor proteins were localized by immunohistochemistry. A special focus was put on the question of how formalin fixation and paraffin embedding influences the quality of the isolated RNA from microdissected material, which was used for quantitative reverse transcription-PCR (qRT-PCR). Quality and quantity of total RNA extracted from formalin-fixed, paraffin-embedded (FFPE) sections and from material immersed in RNAlater® (Ambion, Foster City, CA, USA) was checked using an Experion automated electrophoresis system (Bio-Rad, Munich, Germany). The RNA quality indicator for microdissected material was between 6 and 7, and for RNAlater® material was 9 or better. Online qRT-PCR using the iCycler SYBR GreenTM protocol (Bio-Rad) was performed in a 96-well plate. Primer pairs were chosen to generate PCR products between 100 bp (ER-α) and 140 bp (PR), as RNA recovered from FFPE-laser microdissected material was expected to be considerably fragmented. Using GenEx software (BioEPS, Freisling, Germany), we showed that the expression of mRNA for PR was much stronger in the theca interna than in the 3 other compartments. Estrogen receptor alpha, on the other hand, was nearly exclusively expressed in the zona parenchymatosa and zona vasculosa of the stroma. Our results show that cells obtained after laser microdissection from FFPE ovarian material can be successfully used for subsequent real-time PCR, despite the fact the RNA quality indicator number of the isolated RNA was usually comparatively low. The data of our immunohistochemical analysis support the expression data of our RNA studies. In conclusion, laser-capture microdissection in combination with quantitative PCR is a reproducible and reliable technique for quantification of a small number of cells from FFPE material. We gratefully acknowledge the continuous support by the DFG-Graduiertenkolleg 1029 and the BMBF (ARG 08/013).

scholarly journals Effect of mechanical stretch on HIF-1α and MMP-2 expression in capillaries isolated from overloaded skeletal muscles: laser capture microdissection study

2005 ◽  
Vol 289 (3) ◽  
pp. H1315-H1320 ◽  
Author(s):  
Malgorzata Milkiewicz ◽  
Tara L. Haas
Keyword(s):  
Endothelial Cells ◽  
Laser Capture Microdissection ◽  
Skeletal Muscles ◽  
Hypoxia Inducible Factor ◽  
Fold Increase ◽  
Mechanical Stretch ◽  
Specific Gene ◽  
Capillary Endothelial Cells ◽  
Laser Capture

Under physiological nonhypoxic conditions, angiogenesis can be driven by mechanical forces. However, because of the limitations of the specific gene expression analysis of microvessels from in vivo experiments, the mechanisms regulating the coordinated expression of angiogenic factors implicated in the process remain intangible. In this study, the technique of laser capture microdissection (LCM) was adapted for the study of angiogenesis in skeletal muscles. With a combination of LCM and real-time quantitative PCR it was demonstrated that capillary endothelial cells produce matrix metalloproteinase (MMP)-2 and that mechanical stretch of capillaries within muscle tissue markedly increases MMP-2 mRNA (2.5-fold increase vs. control; P < 0.05). In addition, we showed that transcription factor hypoxia-inducible factor (HIF)-1α expression was 13.5-fold higher in capillaries subjected to stretch compared with controls ( P < 0.05). These findings demonstrate the feasibility of this approach to study angiogenic gene regulation and provide novel evidence of HIF-1α induction in stretched capillary endothelial cells.

scholarly journals Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Aisha M. Basudan ◽  
Yanqi Yang
Keyword(s):  
Laser Capture Microdissection ◽  
Genetic Data ◽  
Male Rats ◽  
Cell Populations ◽  
Specific Cell ◽  
Condylar Cartilage ◽  
Rna Integrity ◽  
Tissue Sections ◽  
Mandibular Condylar Cartilage ◽  
Laser Capture

Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.

Improved Method of RNA Isolation from Laser Capture Microdissection (LCM)-Derived Plant Tissues

2019 ◽  
pp. 89-98
Author(s):  
Vibhav Gautam ◽  
Archita Singh ◽  
Sharmila Singh ◽  
Swati Verma ◽  
Ananda K. Sarkar
Keyword(s):  
Laser Capture Microdissection ◽  
Rna Isolation ◽  
Plant Tissues ◽  
Improved Method ◽  
Laser Capture

Analysis of Human Osteoarthritic Connective Tissue by Laser Capture Microdissection and QRT-PCR

2007 ◽  
Vol 48 (6) ◽  
pp. 316-323 ◽  
Author(s):  
Thomas Scharschmidt ◽  
Robin Jacquet ◽  
Jovan Laskovski ◽  
Elizabeth Lowder ◽  
Scott Weiner ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Laser Capture Microdissection ◽  
Qrt Pcr ◽  
Laser Capture

Abstract P52: Temporal Progression of Cerebral Small Vessel Disease Lesions on Brain Magnetic Resonance Imaging in Spontaneously Hypertensive Stroke Prone Rats

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Yousef Hannawi ◽  
Kimerly A Powell ◽  
Anna Bratasz ◽  
Mohamed G Ewees ◽  
Jay L Zweier
Keyword(s):  
Small Vessel Disease ◽  
Brain Mri ◽  
Brain Magnetic Resonance Imaging ◽  
Volumetric Analysis ◽  
Cerebral Small Vessel Disease ◽  
Male Rats ◽  
Small Vessel ◽  
Spontaneously Hypertensive ◽  
Vessel Disease ◽  
Hemosiderin Deposition

Introduction: Spontaneously Hypertensive Rats- Stroke Prone (SHRSP) are a relevant model for human cerebral small vessel disease (cSVD). However, data are still lacking regarding the neuroimaging correlates of cSVD lesions in SHRSP and their temporal evolution in relationship to histological findings. Methods: 40 SHRSP and 40 Wistar Kyoto control (WKY) male rats were divided into 4 groups (10 WKY and 10 SHRSP) per group. Systolic blood pressure (SBP) was measured using tail-cuff device, weekly. Select animals of each group had brain MRI at 9.4T machine at 7, 16, 24, and 32 weeks of age. Volumetric analysis was completed using manual segmentation of the total brain, ventricles and bilateral hippocampi. Following MRI, brain histology was completed at the same time points. Results: At 6 weeks, SBP was similar (WKY 123.8±0.1 vs SHRSP 130.7±3.2, P=0.09). SHRSP developed hypertension between 9-11 weeks of age and maintained it throughout the experiment (SBP at 31 weeks: WKY 138.1±6 vs SHRSP 169.1±6.7, p=0.0006). At 7 weeks, brain MRI was normal in SHRSP and WKY. Histology was largely unremarkable except for few areas of red blood cell extravasation in couple of SHRSP. At 16 weeks, MRI was normal in WKY and it showed small subcortical hyperintensity on T2 sequences in one SHRSP while histology showed microbleeds (MBs) in 85% of SHRSP. At 24 weeks, brain MRI consistently identified subcortical hyperintensities in SHRSP and H&E showed MBs in all SHRSP in addition to hemosiderin deposition and arteriosclerosis. LFB staining showed areas of demyelination in the corpus callosum. At 32 weeks, SHRSP had hydrocephalus and H&E showed widespread hemosiderin deposition. Volumetric analysis showed larger ventricles in SHRSP (SHRSP 42.3±18.1 ml vs WKY 28.4±7.2 ml, p=0.013) and ventricle size significantly increased with age in SHRSP. Hippocampal and brain volumes were similar in both groups (hippocampal volume: WKY 84.5±8.1 ml vs SHRSP 81.6±2.6 ml, p=0.39; brain volume WKY 2103.4±100.4 ml vs SHRSP 2169.6 ±164.2 ml, p=0.2). Conclusions: SHRSP develop cSVD histological changes early in life and brain MRI showed consistent abnormalities at a later time point. These results have implications in defining cSVD phenotypes in SHRSP in future mechanistic studies.

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