Department of Medicine, Division of Gerontology and Geriatric Medicine, University of Washington, Seattle, WA, USA; VA Puget Sound Health Care System, Geriatric Research Education and Clinical Center, Seattle, WA, USA.
Department of Neurosurgery, University of Washington, Seattle, WA, USA.
Microvasc Res. 2022 Mar;140:104282. doi: 10.1016/j.mvr.2021.104282. Epub 2021 Nov 20.
The brain microvasculature is altered in normal aging and in the presence of disease processes, such as neurodegeneration or ischemia, but there are few methods for studying living tissues. We now report that viable microvessels (MV) are readily isolated from brain tissue of subjects enrolled in studies of neurodegenerative diseases who undergo rapid autopsy (performed with <12 h postmortem interval - PMI). We find that these MV retain their morphology and cellular components and are fairly uniform in size. Sufficient MV (~3-5000) are obtained from 3 to 4 g of tissue to allow for studies of cellular composition as well as extracellular matrix (ECM). Using live/dead assays, these MV are viable for up to 5 days in tissue culture media (2D) designed to support endothelial cells and up to 11 days post-isolation in a 3-dimensional (3D) matrix (Low Growth Factor Matrigel™). Assays that measure the reducing potential of live cells \demonstrated that the majority of the MV maintain high levels of metabolic activity for a similar number of days as the live/dead assays. Functional cellular components (such as tight junctions and transporter proteins) and ECM of MV in tissue culture media, and to a lesser extent in 3D matrices, were readily visualized using immunofluorescence techniques. MV in tissue culture media are lysed and protein content analyzed, but MV in 3D matrix first require removal of the supporting matrix, which can confound the analysis of MV ECM. Finally, MV can be preserved in cryoprotective media, whereby over 50% retain their baseline viability upon thawing. In summary, we find that MV isolated from human brains undergoing rapid autopsy are viable in standard tissue culture for up to 5 days and the timeframe for experiments can be extended up to 11 days by use of a supportive 3D matrix. Viable human MV allow for temporal and spatial analysis of relevant cellular and ECM components that have implications for microvascular function in neurodegenerative diseases, vascular brain injury, and neurotrauma.
大脑微血管在正常衰老和存在疾病过程中会发生改变，例如神经退行性变或缺血，但研究活体组织的方法很少。我们现在报告说，从参加神经退行性疾病研究的受试者的脑组织中，可以很容易地分离出有活力的微血管（MV），这些受试者在快速尸检（死后间隔时间 <12 小时）中。我们发现这些 MV 保留了它们的形态和细胞成分，并且大小相当均匀。从 3 到 4 克组织中获得足够的 MV（~3-5000），以允许研究细胞组成以及细胞外基质（ECM）。使用活/死测定法，这些 MV 在组织培养培养基（2D）中在长达 5 天内保持活力，该培养基旨在支持内皮细胞，并且在 3 维（3D）基质（低生长因子基质胶™）中分离后长达 11 天。测量活细胞还原潜力的测定法表明，大多数 MV 在与活/死测定法相似的天数内保持高水平的代谢活性。MV 的功能细胞成分（如紧密连接和转运蛋白）和 ECM 在组织培养培养基中，并且在较小程度上在 3D 基质中，很容易使用免疫荧光技术可视化。MV 在组织培养培养基中被裂解并分析蛋白质含量，但 MV 在 3D 基质中首先需要去除支撑基质，这可能会干扰 MV ECM 的分析。最后，MV 可以保存在冷冻保护介质中，其中超过 50%在解冻时保留其基线活力。总之，我们发现从快速尸检的人脑中分离出的 MV 在标准组织培养中最多可存活 5 天，并且通过使用支持性 3D 基质，实验时间可以延长至 11 天。有活力的人 MV 允许对与神经退行性疾病、血管性脑损伤和神经创伤中的微血管功能相关的相关细胞和 ECM 成分进行时空分析。
