Purpose: To directly visualize the live cellularity of the intact human trabecular meshwork (TM) and quantitatively analyze tissue viability in situ.
Methods: Human donor corneoscleral rims were sectioned immediately before intravital dye incubation to label nuclei (Hoechst 33342 & propidium iodide [PI]); cytosol (CellTracker Red CMTPX, calcein AM); and membranes (octadecyl rhodamine B chloride [R18]), followed by 2-photon microscopy. Viability was assessed by counting cells in tissue colabeled with PI and Calcein AM. Some tissues were exposed to Triton X-100 to establish dead tissue controls. Fresh postmortem eyes (within 48 hours of death) represented viable tissue controls. Tissues with live cellularity exceeding 50% were considered viable.
Results: Hoechst nuclear labeling was seen throughout the TM, among the autofluorescent beams, plate-like structures and fibers of the meshwork, and within tissue gaps and pores. CellTracker-labeled live cells were attached to autofluorescent TM structures and filled corneoscleral meshwork pores. R18-labeling revealed the membrane distributions of interconnected cells. Calcein-positive cells were visible in all TM layers, but not in tissues killed by Triton X-100 exposure. Dead control tissues showed PI staining in the absence of Calcein-positive cells. Two-thirds of the standard donor tissues we received possessed viable TM, having a mean live cellularity of 71% (n = 14), comparable with freshly postmortem eyes (76%; n = 2). Mean live cellularity of nonviable tissue was 11% (n = 7).
Conclusions: We have visualized and quantified the live cellularity of the TM in situ. This provided unique perspectives of live cell-matrix organization and a means of assaying tissue viability.
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| http://dx.doi.org/10.1167/iovs.12-10479 | DOI Listing |
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