Scleral fibroblast response to experimental glaucoma in mice

Abstract

Purpose: To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma.

Methods: Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using (16)O/(18)O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4',6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry.

Results: Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry.

Conclusions: Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition.

Figure 1

Protein expression changes after 1 week of experimental glaucoma. Immunoblot intensity ratios of experimental glaucoma at 1 week for four selected proteins, with significant elevations in three molecules and all near or greater than twofold increase. Mean ± standard error. Asterisk indicates p value <0.05 for significant increase in glaucoma compared to the control eyes, each referenced to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) value from the blot; t test or Wilcoxon rank-sum test.

Figure 2

Immunohistochemistry of selected molecules in scleral whole mounts. Immunohistochemistry in whole mounts of sclera for vimentin (red) in (A) B6 mouse control and (B) 3-day glaucoma sclera cells. C, D: Control (left) and 3-day glaucoma (right) labeling for α-SMA (B6 mice). E, F: Control (left) and 3-day glaucoma (right) labeling for cell adhesion molecule, α-actinin (red, CD1 mice). 4’,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue). Scale bars=20 µm.

Figure 3

Scleral expression of thrombospondin and HINT1. Immunostaining for thrombospondin-1 (TSP-1; red) counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue) shows labeling comparable to the mean increase in the masked scleral grading (see text). The sclera is marked with a bracket in each panel. A: Control. B: Increased labeling in the 3-day glaucoma sclera, choroid, and retina (CD1 mice). Scale bar=50 µm. Lower pair shows immunostaining for HINT1 (red), sclera labeling in the (C) CD1 control and increased labeling in the glaucoma tissue (D). DAPI counterstain (blue in A, B; green in C, D). Scale bar=25 µm.