Frances E. Cone

Effect of CNTF on retinal ganglion cell survival in experimental glaucoma.

PURPOSE To assess the neuroprotective effect of virally mediated overexpression of ciliary-derived neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in experimental rat glaucoma. METHODS Laser-induced glaucoma was produced in one eye of 224 Wistar rats after injection of adenoassociated viral vectors (type 2) containing either CNTF, BDNF, or both, with saline-injected eyes and noninjected glaucomatous eyes serving as the control. IOP was measured with a hand-held tonometer, and semiautomated optic nerve axon counts were performed by masked observers. IOP exposure over time was adjusted in multivariate regression analysis to calculate the effect of CNTF and BDNF. RESULTS By multivariate regression, CNTF had a significant protective effect, with 15% less RGC axon death (P < 0.01). Both combined CNTF-BDNF and BDNF overexpression alone had no statistically significant improvement in RGC axon survival. By Western blot, there was a quantitative increase in CNTF and BDNF expression in retinas exposed to single viral vectors carrying each gene, but no increase with sequential injection of both vectors. CONCLUSIONS These data confirm that CNTF can exert a protective effect in experimental glaucoma. The reason for the lack of observed effect in the BDNF overexpression groups is unclear, but it may be a function of the level of neurotrophin expression achieved.

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 <or= 0.02). RGC layer and axon loss was greatest in CD1 mice, significantly more than the other groups (p from 0.04 to <0.0001). Young C57BL/6 eyes elongated more and lost more RGC layer cells than older C57BL/6 mice (p = 0.02 and 0.01, respectively). With this mouse glaucoma model, there was differential susceptibility to ocular elongation and RGC layer and axon damage among mouse strains and by age. Factors that determine sensitivity to RGC injury can be studied using transgenic mouse strains with inducible models.

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.

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.

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.

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.

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.

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.

The Scleral Inflation Response of Mouse Eyes to Increases in Pressure

Glaucoma is one of the leading causes of blindness in the world. Evidence suggests that the stress generated in the eye wall by an elevated intraocular pressure plays a role in damaging the visiontransmitting retinal ganglions cells. However, the relationship between the connective tissue’s mechanical properties and how it affects the cellular function is not understood. The purpose of this study was to measure the inflation response of intact C57/BL6 (control) mouse sclera to increases in intraocular pressure, comparing old (11 month) and young (2 month) animals. Mouse eyes were enucleated, mounted by the cornea to a custom fixture, cannulated and immersed in PBS. An active feedback, pressure-controlled syringe pump inflated the cannulated eyes in a series of load-unload and ramp-hold creep tests. A CCD video camera attached to a microscope imaged the expanding scleral surface at 0.5Hz. Scleral displacement was measured with a digital image correlation program. After testing, fresh tissue thickness measurements were taken on scleral slices at multiple locations. An optimized inverse finite element analysis was performed to fit a non-linear anisotropic material model to the experimental data, and material parameters are compared between groups.