Sparse Cell Labeling Reveals Morphologic Diversity Among Mouse Scleral Fibroblasts

Purpose: The purpose of this study was to investigate mouse scleral cell morphology in healthy and ocular hypertensive eyes. Methods: B6.Cg-Ndor1Tg (UBC-cre/ERT2) 1Ejb/1J (UBC-Cre-ERT2) (Strain # 007001; Jackson Labs, Bar Harbor, ME, USA) were crossed with Gt(ROSA)26 Sor tm1(CAG-Brainbow2.1)Cle /J (R26R-Confetti) (Strain # 017492, Jackson Labs) to obtain hemizygous offsprings (UBC-Confetti). This enables tamoxifen-induced recombination of the Brainbow 2.1 cassette, inducing random expression of fluorescent proteins in all cells. Two weeks after tamoxifen treatment (intraperitoneal injection, n = 7 mice), anterior chamber bead injection raised the intraocular pressure (IOP). Eyes were enucleated 3 days after bead injection. YFP and GFP-labeled peripapillary scleral (PPS) and peripheral scleral cells were imaged, cell volume was measured, and individual cell morphology was highlighted using Imaris (version 9.3; 89 control and 136 glaucomatous cells). Results: Imaging of YFP/GFP-labeled cells allowed visualization of individual scleral cells. Differences in the intensity of the Confetti YFP/GFP signal compared to nuclear and fibrillar actin (FA) labels enabled visualization of FA and nuclear structure within YFP/GFP-labeled cells. Under baseline conditions, cells were morphologically diverse, displayed migratory structures, and extensions into the surrounding extracellular matrix (ECM). Mean PPS cell volume (692 µm3, interquartile range [IQR] = 419.8–1304 µm3, n = 62 cells) was greater than peripheral cell volume (336 µm3, IQR = 217–488 µm3, n = 27 cells, P < 0.0001). Cells in glaucomatous eyes exhibited similar morphologic diversity and had reduced PPS cell volume compared with controls (277 µm3, IQR = 154–532, n = 83 cells, P < 0.0001). Conclusions: Sparse cell labeling allowed visualization of individual cell morphology in the scleral environment. Cells demonstrated diverse morphologies, and PPS cells responded to IOP elevation.