Abstract
Purpose :
Localization of molecularly distinct cell types within the trabecular meshwork (TM) and iridocorneal angle will aid in our understanding of TM dysfunction. We sought to histologically characterize computationally defined cell types identified by transcriptomic analysis of the human trabecular meshwork (TM) and surrounding structures.
Methods :
Human ocular tissues were obtained from the Lion’s Eye Bank and through a rapid autopsy program at Mass. General Hospital. Corneoscleral segments were fixed in 4% PFA for 2-24 hr, embedded in tissue freezing medium, and mounted onto slides in 10 µm meridional sections. Molecular markers specific to each of 15 transcriptionally distinct TM cell types identified via high-throughput single-cell RNA sequencing (scRNA-seq) of 5 post-mortem eyes from 4 genetically unrelated individuals (ages 44 to 78) were investigated by immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH) and imaged with confocal microscopy.
Results :
At least three endothelial cell types shared podoplanin (PDPN) as a common marker distinct from other cell types. Antibodies against PDPN demonstrated robust preferential staining of TM cells encircling trabecular beams, which were visualized via autofluorescence of matrix molecules such as elastin; autofluorescence was strongest in excitation/emission channels 405 nm/410-475 nm and 488 nm/499-550 nm, so these were avoided for IHC detection. A fourth distinct endothelial cell cluster was exclusively positive for PECAM-1/CD31and localized to Schlemm canal and other vascular lumen. Various immune cell clusters including LYVE-1+/CD163+ macrophages and CD27+ B-cells were confirmed to be present in the TM body through IHC.
Conclusions :
Podoplanin is a positive marker for TM endothelium.
PECAM-1/CD31 is a positive marker for Schlemm canal endothelium.
Autofluorescence of the sclera and TM beams aids in establishing anatomical landmarks in histological sections during image analysis of IHC and FISH.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.