PurposeTo 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.MethodsThy1-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.ResultsRetinal 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.ConclusionsRetinal 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.

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.

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 8a2[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 ¼ 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.Keywords: glaucoma, mouse, experimental model, sclera, diffusion, permeability, photobleaching, dextran G laucoma is the second leading cause of world blindness and its principal risk factors include the effect of intraocular pressure (IOP) acting to decrease the number of retinal ganglion cells (RGCs). Intraocular pressure is a mechanical load that generates stress and strain in the sclera, which are magnified at the optic nerve head (ONH). The largest mechanical strains have been measured in in vitro inflation experiments in the peripapillary region, immediately adjacent to the ONH.1 The sclera acts on the ONH, where axons of RGCs pass out of the eye, and suffers damage related to the effects of IOP.2,3 Thus, the behavior of the sclera is highly relevant to glaucoma injury and its study may be useful in improved diagnosis as well as new therapeutic avenues.The sclera comprises three-quarters of the human ocular circumference and is 75% to 90% collagen, with additional elastin fibrils and proteoglycans. 4,5 The scleral proteoglycans include heparin sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan, aggrecan, and several small leucinerich proteoglycans.6,7 These may have important functional significance for the mechanical responses of the sclera. 8 Scleral thickness is greatest at the peripapillary zone, followed by the limbus, and is...