Development of diagnostic and treatment strategies for glaucoma through understanding and modification of scleral and lamina cribrosa connective tissue.

Considerable evidence indicates that the state of ocular connective tissues and their response in glaucomatous disease affect the degree of glaucoma damage. Both experimental and clinical data suggest that improved diagnostic and prognostic information can be derived from the assessment of the mechanical responsiveness of the sclera and lamina cribrosa to intraocular pressure (IOP). Controlled mutagenesis of the sclera has produced a mouse strain that is relatively resistant to increased IOP. Alteration of the baseline scleral state can be accomplished through either increased cross-linking of fibrillar components or their reduction. The sclera is a dynamic structure, altering its structure and behavior in response to IOP change. The biochemical pathways that control these responses are fertile areas for new glaucoma treatments.

Studies of scleral biomechanical behavior related to susceptibility for retinal ganglion cell loss in experimental mouse glaucoma.

PURPOSE: To study anatomical changes and mechanical behavior of the sclera in mice with experimental glaucoma by comparing CD1 to B6 mice. METHODS: Chronic experimental glaucoma for 6 weeks was produced in 2- to 4-month-old CD1 (43 eyes) and B6 mice (42 eyes) using polystyrene bead injection into the anterior chamber with 126 control CD1 and 128 control B6 eyes. Intraocular pressure (IOP) measurements were made with the TonoLab at baseline and after bead injection. Axial length and scleral thickness were measured after sacrifice in the CD1 and B6 animals and compared to length data from 78 eyes of DBA/2J mice. Inflation testing of posterior sclera was conducted, and circumferential and meridional strain components were determined from the displacement response. RESULTS: Experimental glaucoma led to increases in axial length and width by comparison to fellow eyes (6% in CD1 and 10% in B6; all P < 0.03). While the peripapillary sclera became thinner in both mouse types with glaucoma, the remainder of the sclera uniformly thinned in CD1, but thickened in B6. Peripapillary sclera in CD1 controls had significantly greater temporal meridional strain than B6 and had differences in the ratios of meridional to effective circumferential strain from B6 mice. In both CD1 and B6 mice, exposure to chronic IOP elevation resulted in stiffer pressure-strain responses for both the effective circumferential and meridional strains (multivariable regression model, P = 0.01-0.03). CONCLUSIONS: Longer eyes, greater scleral strain in some directions at baseline, and generalized scleral thinning after glaucoma were characteristic of CD1 mice that have greater tendency to retinal ganglion cell damage than B6 mice. Increased scleral stiffness after glaucoma exposure in mice mimics findings in monkey and human glaucoma eyes.

Mice with an induced mutation in collagen 8A2 develop larger eyes and are resistant to retinal ganglion cell damage in an experimental glaucoma model.

PURPOSE: To study susceptibility to glaucoma injury as it may be affected by mutations in ocular connective tissue components. METHODS: Mice homozygous for an N-ethyl-N-nitrosourea induced G257D exchange (Gly to Asp) missense mutation (Aca23) in their collagen 8A2 gene were studied to measure intraocular pressure (IOP), axial length and width, number of retinal ganglion cells (RGC), and inflation responses. Three month old homozygous Aca23 mutant and wild type (WT) mice had 6 weeks exposure to elevated IOP induced by polystyrene microbead injection. Additional Aca23 and matched controls were studied at ages of 10 and 18 months. RESULTS: Aca23 mice had no significant difference from WT in IOP level, and in both strains IOP rose with age. In multivariable models, axial length and width were significantly larger in Aca23 than WT, became larger with age, and were larger after exposure to glaucoma (n=227 mice). From inflation test data, the estimates of scleral stress resultants in Aca23 mice were similar to age-matched and younger WT C57BL/6 (B6) mice, while the strain estimates for Aca23 were significantly less than those for either WT group in the mid-sclera and in some of the more anterior scleral measures (p<0.001; n=29, 22, 20 eyes in Aca23, older WT, younger WT, respectively). With chronic IOP elevation, Aca23 eyes increased 9% in length and 7% in width, compared to untreated fellow eyes (p<0.05, <0.01). With similar elevated IOP exposure, WT eyes enlarged proportionately twice as much as Aca23, increasing in length by 18% and in nasal-temporal width by 13% (both p<0.001, Mann-Whitney test). In 4 month old control optic nerves, mean RGC axon number was not different in Aca23 and WT (46,905±7,592, 43,628±11,162, respectively; p=0.43, Mann-Whitney test, n=37 and 29). With chronic glaucoma, Aca23 mice had a mean axon loss of only 0.57±17%, while WT mice lost 21±31% (median loss: 1% versus 10%, n=37, 29, respectively; p=0.001; multivariable model adjusting for positive integral IOP exposure). CONCLUSIONS: The Aca23 mutation in collagen 8α2 is the first gene defect found to alter susceptibility to experimental glaucoma, reducing RGC loss possibly due to differences in mechanical behavior of the sclera. Detailed study of the specific changes in scleral connective tissue composition and responses to chronic IOP elevation in this strain could produce new therapeutic targets for RGC neuroprotection.

Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma.

PURPOSE: To study sequential changes in retinal ganglion cell (RGC) morphology in mice after optic nerve crush and after induction of experimental glaucoma. METHODS: Nerve crush or experimental glaucoma was induced in mice that selectively express yellow fluorescent protein (YFP) in RGCs. Mice were euthanized 1, 4, and 9 days after crush and 1, 3, and 6 weeks after induction of glaucoma by bead injection. All YFP-RGCs were identified in retinal whole mounts. Then confocal images of randomly selected RGCs were quantified for somal fluorescence brightness, soma size, neurite outgrowth, and dendritic complexity (Sholl analysis). RESULTS: By 9 days after crush, 98% of RGC axons died and YFP-RGCs decreased by 64%. After 6 weeks of glaucoma, 31% of axons died, but there was no loss of YFP-RGC bodies. All crush retinas combined had significant decreases in neurite outgrowth parameters (P ≤ 0.036, generalized estimating equation [GEE] model) and dendritic complexity was lower than controls (P = 0.017, GEE model). There was no change in RGC soma area after crush. In combined glaucoma data, the RGC soma area was larger than control (P = 0.04, GEE model). At 3 weeks, glaucoma RGCs had significantly larger values for dendritic structure and complexity than controls (P = 0.044, GEE model), but no statistical difference was found at 6 weeks. CONCLUSIONS: After nerve crush, RGCs and axons died rapidly, and dendritic structure decreased moderately in remaining RGCs. Glaucoma caused an increase in RGC dendrite structure and soma size at 3 weeks.

The effects of anesthesia, mouse strain and age on intraocular pressure and an improved murine model of experimental glaucoma.

The purpose of this study was to improve a mouse model of chronic intraocular pressure (IOP) elevation utilizing microbead injection in two strains of mice and to assess the effect of age and anesthesia on measured IOP. We compared our previous model with two modified protocols for injecting polystyrene microbeads and viscoelastic material in CD1or C57BL/6 mice. The measured outcomes were degree of IOP elevation and production of axonal loss. The first new protocol was injection of 3 µL of equal volumes of 6 µm and 1 µm diameter beads, followed by 2 µL of viscoelastic (3+2). The second new protocol injected 4 µL of the two bead mixture, then 1 µL of viscoelastic (4+1). Both were compared to injection of 2 µL of 6 µm beads with 3 µL of viscoelastic (2+3). We also compared the effects of age and of two anesthetic regimens (intraperitoneal ketamine/xylazine/acepromazine versus isoflurane gas) on measured IOP in untreated eyes of both strains. IOP was 2mm Hg lower with intraperitoneal than with gas anesthesia in both strains (p=0.003, p<0.0001, t-test). IOP measurements were lower in untreated young (2 months) compared to older (10 months) C57BL/6 mice (p=0.001, t-test). In the experimental glaucoma mouse model, mean IOP and number of elevated IOP measurements were higher in newer protocols. Mean axon loss with the 4+1 protocol (all strains) was twice that of the 2+3 and 3+2 protocols (36% vs. 15% loss, p=0.0026, ANOVA), and mean axon loss in CD1 mice (21%) was greater than in C57BL/6 mice (13%) (p=0.047, ANOVA). Median axon loss in 4+1 protocol treated C57BL/6 mice expressing yellow fluorescent protein in 2% of retinal ganglion cells (RGCs) had greater median axon loss than C57BL/6 4+1 protocol treated mice (26% vs. 10%, p=0.03). The 4+1 protocol provided higher, more consistent IOP elevation and greater axonal loss. The effects of age, strain, and anesthesia on induced IOP elevation and axon damage must be considered in mouse experimental glaucoma research.

Semi-automated, quantitative analysis of retinal ganglion cell morphology in mice selectively expressing yellow fluorescent protein.

The development of transgenic mouse lines that selectively label a subset of neurons provides unique opportunities to study detailed neuronal morphology and morphological changes under experimental conditions. In the present study, a mouse line in which a small number of retinal ganglion cells (RGCs) express yellow fluorescent protein (YFP) under control of the Thy-1 promoter was used (Feng et al., 2000). We characterized the number, distribution by retinal region and eccentricity of YFP-labeled RGCs using fluorescence microscopy and Stereo Investigator software (MicroBrightField, VT, USA). Then, we captured images of 4-6 YFP-expressing RGCs from each of 8 retinal regions by confocal microscopy, producing 3-dimensional and flattened data sets. A new semi-automated method to quantify the soma size, dendritic length and dendritic arbor complexity was developed using MetaMorph software (Molecular Devices, PA, USA). Our results show that YFP is expressed in 0.2% of all RGCs. Expression of YFP was not significantly different in central versus peripheral retina, but there were higher number of YFP-expressing RGCs in the temporal quadrant than in the nasal. By confocal-based analysis, 58% of RGCs expressing YFP did so at a high level, with the remainder distributed in decreasing levels of brightness. Variability in detailed morphometric parameters was as great between two fellow retinas as in retinas from different mice. The analytic methods developed for this selective YFP-expressing RGC model permit quantitative comparisons of parameters relevant to neuronal injury.

Lack of neuroprotection against experimental glaucoma in c-Jun N-terminal kinase 3 knockout mice.

To determine if the absence of c-Jun N-terminal kinase 3 (JNK3) in the mouse retina would reduce retinal ganglion cell (RGC) loss in mice with experimental glaucoma. C57BL/6 mice underwent experimental intraocular pressure (IOP) elevation with a bead/viscoelastic injection into one eye. One-half of the mice were Jnk3 homozygous knockouts (KO) and were compared to wild type (WT) mice. IOP was measured under anesthesia with the TonoLab, axial length was measured post-mortem with calipers after inflation to 15mmHg, and RGC layer counts were performed on retinal whole mount images stained with DAPI, imaged by confocal microscopy, and counted by masked observers in an image analysis system. Axon counts were performed in optic nerve cross-sections by semi-automated image analysis. Both WT and Jnk3(-/-) mice had mean elevations of IOP of more than 50% after bead injection. Both groups underwent the expected axial globe elongation due to chronic IOP elevation. The absence of JNK3 in KO retina was demonstrated by Western blots. RGC layer neuron counts showed modest loss in both WT and Jnk3(-/-) animals; local differences by retinal eccentricity were detected, in each case indicating greater loss in KO animals than in WT. The baseline number of RGC layer cells in KO animals was 10% higher than in WT, but the number of optic nerve axons was identical in KO and WT controls. A slightly greater loss of RGC in Jnk3(-/-) mice compared to controls was detected in experimental mouse glaucoma by RGC layer counting and there was no protective effect shown in axon counts. Counts of RGC layer cells and optic nerve axons indicate that Jnk3(-/-) mice have an increased number of amacrine cells compared to WT controls.

Calibration of the TonoLab tonometer in mice with spontaneous or experimental glaucoma.

PURPOSE: To measure the accuracy of TonoLab (TioLat, Helsinki, Finland) tonometry in mice with spontaneous or induced experimental glaucoma. METHODS: Chronic intraocular pressure (IOP) elevation was induced in one eye of 32 mice by injection of polystyrene beads and viscoelastic material. Three to 6 weeks later, the eyes were cannulated and manometrically set to 10, 20, 30, 40, or 50 mm Hg. The mice were 8-week and 8-month-old C57BL/6, 8-week-old DBA/2J, and 8-week-old CD1. The TonoLab calibration was also tested on five aged DBA/2J mice with spontaneous glaucoma. The relation of the TonoLab reading to manometric IOP was evaluated in multivariate linear regression models with axial length, IOP history, and mouse strain as independent variables. RESULTS: The slope of the relationship between TonoLab and manometric IOP in all the mice was 0.998, with an intercept of 2.3 mm Hg (adjusted R in univariate regression = 0.86). Neither the mice with bead-induced glaucoma nor those with spontaneous glaucoma (older DBA/2J mice) differed significantly from the control animals in having an excellent correlation between TonoLab and manometer IOP. Longer and wider mouse eyes had slightly higher tonometrically measured IOP, whether glaucomatous or control (multivariate regression, adjusted R(2) = 0.90, P < 0.0001). There was no difference in tonometric accuracy among the three mouse strains: CD1, C57BL/6, and DBA/2J, nor between 8-week and 8-month-old C57BL/6 mice (multivariate regression, P = 0.32). CONCLUSIONS: The TonoLab accurately reflects IOP in both normal mice and in eyes of mice with experimental or spontaneous glaucoma, with no detectable effect of age.

The in vitro inflation response of mouse sclera.

The purpose of this research was to develop a reliable and repeatable inflation protocol to measure the scleral inflation response of mouse eyes to elevations in intraocular pressure (IOP), comparing the inflation response exhibited by the sclera of younger and older C57BL/6 mice. Whole, enucleated eyes from younger (2 month) and older (11 month) C57BL/6 mice were mounted by the cornea on a custom fixture and inflated according to a load-unload, ramp-hold pressurization regimen via a cannula connected to a saline-filled programmable syringe pump. First, the tissue was submitted to three load-unload cycles from 6 mmHg to 15 mmHg at a rate of 0.25 mmHg/s with ten minutes of recovery between cycles. Next the tissue was submitted to a series of ramp-hold tests to measure the creep behavior at different pressure levels. For each ramp-hold test, the tissue was loaded from 6 mmHg to the set pressure at a rate of 0.25 mmHg/s and held for 30 min, and then the specimens were unloaded to 6 mmHg for 10 min. This sequence was repeated for set pressures of: 10.5, 15, 22.5, 30, 37.5, and 45 mmHg. Scleral displacement was measured using digital image correlation (DIC), and fresh scleral thickness was measured optically for each specimen after testing. For comparison, scleral thickness was measured on untested fresh tissue and epoxy-fixed tissue from age-matched animals. Comparing the apex displacement of the different aged specimens, the sclera of older animals had a statistically significant stiffer response to pressurization than the sclera of younger animals. The stiffness of the pressure-displacement response of the apex measured in the small-strain (6-15 mmHg) and the large-strain (37.5-45 mmHg) regime, respectively, were 287 ± 100 mmHg/mm and 2381 ± 191 mmHg/mm for the older tissue and 193 ± 40 mmHg/mm and 1454 ± 93 mmHg/mm for the younger tissue (Student t-test, p<0.05). The scleral thickness varied regionally, being thickest in the peripapillary region and thinnest at the equator. Fresh scleral thickness did not differ significantly by age in this group of animals. This study presents a reliable inflation test protocol to measure the mechanical properties of mouse sclera. The inflation methodology was sensitive enough to measure scleral response to changes in IOP elevations between younger and older C57BL/6 mice. Further, the specimen-specific scleral displacement profile and thickness measurements will enable future development of specimen-specific finite element models to analyze the inflation data for material properties.

Differential susceptibility to experimental glaucoma among 3 mouse strains using bead and viscoelastic injection.

The purpose of this experiment was to test the susceptibility to retinal ganglion cell (RGC) axon loss and RGC layer cell loss from experimental glaucoma among 3 mouse strains, and between younger and older mice. We obstructed the mouse aqueous outflow channels by injecting 2 microL of 6 mum diameter, polystyrene beads followed by 3 microL of viscoelastic solution into the anterior chamber with a glass micropipette. We evaluated intraocular pressure (IOP) and damage to RGC as measured by optic nerve axon counts and RGC layer neuron counts in 3 strains of young mice (2 month old C57BL/6, DBA/2J, and CD1) and 10 month C57BL/6 mice. Bead and viscoelastic injection produced IOP elevation at >or=1 time point in 94.1% of eyes (112/119), with mean IOP difference from fellow eyes of 4.4 +/- 3.0 mmHg. By 6-12 weeks, injected eyes were 10.8% longer and 7.6% wider (p < 0.0001). Young DBA/2J and C57BL/6 eyes increased axial length significantly more than young CD1 or older C57BL/6 (all p

The presence and distribution of elastin in the posterior and retrobulbar regions of the mouse eye.

The Presence and distribution of elastin in the posterior and retrobulbar regions of the mouse eye was investigated. Mice of two strains (C57/BL6 and DBA/2J) were studied at 2 months and 8-12 months of age. Light, confocal, and transmission electron microscopy were used to identify elastin, using immunohistochemical techniques and ultrastructural evaluation. Elastin was found in the following ocular structures: conjunctiva, muscle tendons, sclera, choroid, and meninges. The elastin in the sclera was most dense in a ring surrounding the peripapillary optic nerve head, with its presence in the inner sclera declining with greater distance from the nerve head. Elastin fibers were oriented in the sclera along what would be expected to be the principal stress directions generated from the intraocular pressure, though actual biomechanical measurements have not yet been made in the mouse sclera. Elastin comprises a portion of the mouse sclera and its distribution in the peripapillary area is similar to that in human eyes.