Analyzing Live Cellularity in the Human Trabecular Meshwork

Abstract

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 ¼ 14), comparable with freshly postmortem eyes (76%; n ¼ 2). Mean live cellularity of nonviable tissue was 11% (n ¼ 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. (Invest Ophthalmol Vis Sci.

Aqueous outflow - A continuum from trabecular meshwork to episcleral veins

“…The first force occurs through the hinged flap geometry, which provides a means for free movement to permit endothelial cell contractile elements within the walls to actively modulate lumen size (Gonzalez et al, 2013; Gonzalez et al, 2014; Gonzalez et al, 2016; Ko et al, 2016). …”

“…However, the endothelium lining attached to the walls of the SC, the CCE, and the ISCC has perivascular smooth muscle components, which suggest that these regions may possess sufficient contractile components to affect aqueous outflow (Gonzalez et al, 2013; Gonzalez et al, 2014; Gonzalez et al, 2016; Hann et al, 2011; Hann and Fautsch, 2009; Hann et al, 2014; Ko et al, 2016). Such movement is particularly likely given recent evidence that the CCE and ISCC geometry and constituent properties permit free movement in response to pressure changes (de Kater et al, 1992; de Kater et al, 1990; Hariri et al, 2014; Xin et al, 2016a; Xin et al, 2016b).…”

Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

“…TM cell viability was assessed by calcein acetoxymethyl (calcein-AM) and propidium iodide (PI) co-labelling (Gonzalez, Hamm-Alvarez & Tan, 2013). After 180 hours, the anterior segments were collected and washed with PBS three times.…”

A Porcine Ex Vivo Model of Pigmentary Glaucoma

“…The non-fluorescent AM derivative of calcein is transported through the cell membrane into a live cell, whereas PI cannot cross the membrane of a live cell, making it useful to differentiate necrotic, apoptotic, and healthy cells 38 . Viable TM cells can convert nonfluorescent calcein AM to green fluorescent calcein with an intracellular esterase, but do not allow PI to enter or to bind nucleic acids 39 . Pigment granules at a concentration of 1.67x10 7 particles/ml did not increase the percentage of PI-labeled apoptotic or dead cells (0.00±0.00% in the pigment group vs 0.27±0.07% in the normal control, P>0.05).…”