Viable human brain microvessels for the study of aging and neurodegenerative diseases.

ABSTRACT

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.