Scleral fibroblast response to experimental glaucoma in mice.

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.

Time-Lapse Retinal Ganglion Cell Dendritic Field Degeneration Imaged in Organotypic Retinal Explant Culture.

PURPOSE: To develop an ex vivo organotypic retinal explant culture system suitable for multiple time-point imaging of retinal ganglion cell (RGC) dendritic arbors over a period of 1 week, and capable of detecting dendrite neuroprotection conferred by experimental treatments. METHODS: Thy1-YFP mouse retinas were explanted and maintained in organotypic culture. Retinal ganglion cell dendritic arbors were imaged repeatedly using confocal laser scanning microscopy. Maximal projection z-stacks were traced by two masked investigators and dendritic fields were analyzed for characteristics including branch number, size, and complexity. One group of explants was treated with brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) added to the culture media. Changes in individual dendritic fields over time were detected using pair-wise comparison testing. RESULTS: Retinal ganglion cells in mouse retinal explant culture began to degenerate after 3 days with 52.4% surviving at 7 days. Dendritic field parameters showed minimal change over 8 hours in culture. Intra- and interobserver measurements of dendrite characteristics were strongly correlated (Spearman rank correlations consistently > 0.80). Statistically significant (P < 0.001) dendritic tree degeneration was detected following 7 days in culture including: 40% to 50% decreases in number of branch segments, number of junctions, number of terminal branches, and total branch length. Scholl analyses similarly demonstrated a significant decrease in dendritic field complexity. Treatment of explants with BDNF+CNTF significantly attenuated dendritic field degeneration. CONCLUSIONS: Retinal explant culture of Thy1-YFP tissue provides a useful model for time-lapse imaging of RGC dendritic field degeneration over a course of several days, and is capable of detecting neuroprotective amelioration of dendritic pruning within individual RGCs.

Scleral permeability varies by mouse strain and is decreased by chronic experimental glaucoma.

PURPOSE: To determine differences in scleral permeability, as measured by diffusion of macromolecules, by using fluorescence recovery after photobleaching (FRAP), with reference to differences by mouse strain, scleral region, and the effect of experimental glaucoma. METHODS: In three mouse strains (B6, CD1, and B6 mice with mutation in collagen 8α2 [Aca23]), we used FRAP to measure the diffusion of fluorescein isothiocyanate-dextran, molecular weight 40 kDa, into a photobleached zone of sclera. Scleral regions near the optic nerve head (peripapillary) and two successively more anterior regions were compared. Sclera from mouse eyes subjected to chronically elevated intraocular pressure after bead injection into the anterior chamber were compared to fellow eye controls. FRAP data were compared against estimated retinal ganglion cell axon loss in glaucomatous eyes. RESULTS: Diffusion rates of dextran molecules in the sclera were significantly greater in Aca23 and B6 mice than in CD1 mice in a multivariate model adjusted for region and axial length (P < 0.0001). Dextran diffusion significantly decreased in glaucomatous eyes, and the decline increased with greater axon loss (P = 0.0003, multivariable model). Peripapillary scleral permeability was higher in CD1 than B6 and Aca23 mice (P < 0.05, multivariable model, adjusted by Bonferroni). CONCLUSIONS: Measurement of the diffusion rates of dextran molecules in the sclera showed that glaucoma leads to decreased scleral permeability in all three mouse strains tested. Among mouse strains tested, those that were more susceptible to glaucomatous loss of retinal ganglion cells had a lower scleral permeability at baseline.

Susceptibility to glaucoma damage related to age and connective tissue mutations in mice.

The purpose of this research was to study the effects of age and genetic alterations in key connective tissue proteins on susceptibility to experimental glaucoma in mice. We used mice haploinsufficient in the elastin gene (EH) and mice without both alleles of the fibromodulin gene (FM KO) and their wild type (WT) littermates of B6 and CD1 strains, respectively. FM KO mice were tested at two ages: 2 months and 12 months. Intraocular pressure (IOP) was measured by Tonolab tonometer, axial lengths and widths measured by digital caliper post-enucleation, and chronic glaucoma damage was measured using a bead injection model and optic nerve axon counts. IOP in EH mice was not significantly different from WT, but FM KO were slightly lower than their controls (p = 0.04). Loss of retinal ganglion cell (RGC) axons was somewhat, but not significantly greater in young EH and younger or older FM KO strains than in age-matched controls (p = 0.48, 0.34, 0.20, respectively, multivariable regression adjusting for IOP exposure). Older CD1 mice lost significantly more RGC axons than younger CD1 (p = 0.01, multivariable regression). The CD1 mouse strain showed age-dependence of experimental glaucoma damage to RGC in the opposite, and more expected, direction than in B6 mice in which older mice are more resistant to damage. Genetic alteration in two genes that are constituents of sclera, fibromodulin and elastin do not significantly affect RGC loss.

Scleral structural alterations associated with chronic experimental intraocular pressure elevation in mice.

PURPOSE: To study changes in scleral structure induced by chronic experimental intraocular pressure elevation in mice. METHODS: We studied the effect of chronic bead-induced glaucoma on scleral thickness, collagen lamellar structure, and collagen fibril diameter distribution in C57BL/6 (B6) and CD1 mice, and in collagen 8α2 mutant mice (Aca23) and their wild-type littermates (Aca23-WT) using electron and confocal microscopy. RESULTS: In unfixed tissue, the control B6 peripapillary sclera was thicker than in CD1 mice (p<0.001). After 6 weeks of glaucoma, the unfixed CD1 and B6 sclera thinned by 9% and 12%, respectively (p<0.001). The fixed sclera, measured by electron microscopy, was significantly thicker in control Aca23 than in B6 or CD1 mice (p<0.05). The difference between fresh and fixed scleral thickness was nearly 68% in untreated control B6 and CD1 mice, but differed by only 10% or less in fresh/fixed glaucoma scleral comparisons. There were 39.3±9.6 lamellae (mean, standard deviation) in control sclera, categorized as 41% cross-section, 24% cellular, 20% oblique, and 15% longitudinal. After glaucoma, mean peripapillary thickness significantly increased in fixed specimens of all mouse strains by 10.3 ±4.8 µm (p=0.001) and the total number of lamellae increased by 18% (p=0.01). The number of cellular and cross-section lamellae increased in glaucoma eyes. After glaucoma, there were more small and fewer large collagen fibrils (p<0.0001). Second harmonic generation imaging showed that the normal circumferential pattern of collagen fibrils in the peripapillary sclera was altered in significantly damaged glaucomatous eyes. CONCLUSIONS: Dynamic responses of the sclera to experimental mouse glaucoma may be more important than baseline anatomic features in explaining susceptibility to damage. These include decreases in nonfibrillar elements, alterations in lamellar orientation, an increased number of smaller collagen fibrils and fewer larger fibrils, and relative increase in the number of scleral fibroblast layers.