June 5, 2020 Conference


An Eye for Detail: Examining the role of corneal Schwann cells

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Gwendolyn Schultz, UConn School of Medicine (Presenter)
Paola Bargagna-Mohan, Neuroscience, UConn Health
Bruce Rheaume, Neuroscience, UConn Health
Dr. Paul Robson, The Jackson Laboratory for Genomic Medicine
Royce Mohan, Neuroscience, UConn Health
Purpose: The cornea is densely innervated by an intricate network of nerve axons. Injury to the central cornea, whether by surgical procedure or by accident, can disrupt these axons and lead to a loss of sensitivity. However little is known about corneal Schwann cells (cSCs), the resident glial cells which protect and support corneal axons. Specifically, we were interested in the contributions of cSCs to axon regeneration as this has never before been explored. To study this unique cell population, we used single cell RNA-sequence (scRNA-seq) analysis and validated our findings using antibody staining in whole corneal tissue. Methods: We employed rabbit corneas to derive a single cell preparation that was subjected to droplet-based scRNA-seq (10X Genomics) generating data on 7,555 individual cells. The entire procedure was replicated from a different batch of corneas generating data on another 10,057 individual cells. The gene expression matrix output from CellRanger (10X Genomics) of the aggregated data was subjected to unsupervised clustering and dimensionality reduction. Specifically, the 1500 most highly variable genes were used for neighborhood graph generation (using 20 nearest-neighbors) and dimensionality reduction with UMAP. A specific SC cluster represented by conserved SC-genes was obtained. Antibody staining for the cSC proteins L1CAM, SCN7a, and SOX10 was done using corneas from wild-type adult C57/Bl6 mice and whole mount stained tissue was subjected to confocal microscopy. To model corneal injury, wild-type adult C57Bl6 male and female mice were subjected to a penetrating stab injury under systemic and ocular anesthesia that produced a focal lesion through the epithelium, basement membrane and a significant part of the corneal stroma. Eyes were enucleated at 4 and 7 days post injury and whole corneas were stained using antibodies. B3-tubulin was used for marking axons. Results: The scRNA seq analysis of rabbit corneas produced 4 cell clusters, including those representing keratocytes, epithelial cells, inflammatory cells, and Schwann cells. The corneal SC cell cluster revealed that Scn7A, Plp1, Gfra3, Sox10, and L1cam transcripts were highly expressed, identifying for the first time targets for investigation. In undisturbed central corneas, cSC markers showed distinct staining around axon processes co-stained for ?3-tubulin. SOX10 staining was distinctly nuclear in SCs. Following injury to the cornea in which axons are severed, cSCs remained at the injury site. In injured corneas, we also found co-staining for alpha-smooth muscle actin co-staining with cSC markers revealing some cSCs had differentiated into myofibroblastic cells. Conclusions: cSCs are closely associated with nerve axons in the central cornea. Upon injury, these cells remain at the injury site. Our characterization of these cells will allow us to study how these glial cells respond to corneal injury, and how their role can inform clinical practice.
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