Mary E. Pease

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.

Retinal Ganglion Cell Loss in a Rat Ocular Hypertension Model Is Sectorial and Involves Early Optic Nerve Axon Loss

PURPOSE Previous analyses of the DBA/2J mouse glaucoma model show a sectorial degeneration pattern suggestive of an optic nerve head insult. In addition, there are large numbers of retinal ganglion cells (RGCs) that cannot be retrogradely labeled but maintain RGC gene expression, and many of these have somatic phosphorylated neurofilament labeling. Here the authors further elucidate these features of glaucomatous degeneration in a rat ocular hypertension model. METHODS IOP was elevated in Wistar rats by translimbal laser photocoagulation. Retina whole mounts were analyzed for Sncg mRNA in situ hybridization, fluorogold (FG) retrograde labeling, and immunohistochemistry for phosphorylated neurofilaments (pNF) at 10 and 29 days after IOP increase. A novel automatic method was used to estimate axon numbers in plastic sections of optic nerves. RESULTS Sncg mRNA was confirmed as a specific marker for RGCs in rat. Loss of RGCs after IOP elevation occurred in sectorial patterns. Sectors amid degeneration contained RGCs that were likely disconnected because these had pNF in their somas and dendrites, were not labeled by FG, and were associated with reactive plasticity within the retina. Most of the axon loss within the optic nerve already occurred by 10 days after the onset of IOP elevation. CONCLUSIONS These data demonstrate that the pattern of RGC loss after laser-induced ocular hypertension in rats is similar to that previously reported in DBA/2J mice. The results support the view that in glaucoma RGC axons are damaged at the optic nerve head and degenerate within the optic nerve before there is loss of RGC somas.

Glaucomatous optic nerve injury involves early astrocyte reactivity and late oligodendrocyte loss

Glaucoma, a neurodegenerative disease affecting retinal ganglion cells (RGC), is a leading cause of blindness. Since gliosis is common in neurodegenerative disorders, it is important to describe the changes occurring in various glial populations in glaucoma animal models in relation to axon loss, as only changes that occur early are likely to be useful therapeutic targets. Here, we describe changes occurring in glia within the myelinated portion of the optic nerve (ON) in both DBA/2J mice and in a rat ocular hypertension model. In both glaucoma animal models, we found only a modest loss of oligodendrocytes that occurred after axons had already degenerated. In DBA/2J mice there was proliferation of oligodendrocyte precursor cells (OPCs) and new oligodendrocyte generation. Activation of microglia was detected only in highly degenerated DBA/2J ONs. In contrast, a large increase in astrocyte reactivity occurred early in both animal models. These results are consistent with astrocytes playing a prominent role in regulating axon loss in glaucoma.

Intraocular injection of dibutyryl cyclic AMP promotes axon regeneration in rat optic nerve.

The optic nerve is a CNS pathway containing molecules capable of inhibiting axon elongation. The growth program in embryonic retinal ganglion cell (RGC) neurons enables axons to regenerate in the optic nerve through at least two mechanisms. Namely, high cyclic AMP (cAMP) levels abrogate the ability of CNS molecules to inhibit elongation, and the pattern of gene expression enables axons to undergo rapid, sustained, and lengthy elongation. In adult mammals, recovery of visual function after optic nerve injury is limited by both the death of most RGC neurons and the inability of surviving axons to regenerate. We now report that a single intraocular injection of the membrane-permeable cAMP analogue dibutyryl cAMP (db cAMP) promotes the regeneration of RGC axons in the optic nerves of adult rats, but does not prevent the death of RGC neurons. This regeneration in optic nerves crushed within the orbit (2 mm from the eye) was equally effective either 1 day before or 1 day after db cAMP injection. The number of regenerating axons, which was maximal 14 days after crush, declined with increasing time after injury (i.e., 28, 56, and 112 days) and distance beyond the crush site (i.e., 0.25, 0.5, and 1.0 mm). Thus, db cAMP promotes optic nerve regeneration without increasing the survival of axotomized RGC neurons. Furthermore, since db cAMP does not enable axons to undergo rapid, sustained, and lengthy elongation, strategies that increase survival and promote these changes in elongation may critically complement the ability of db cAMP to promote regeneration.

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.

Manometric calibration and comparison of TonoLab and TonoPen tonometers in rats with experimental glaucoma and in normal mice

PurposeTo compare the TonoPen and TonoLab tonometers to each other and to manometrically set intraocular pressure (IOP) in the eyes of normal mice, normal rats, and rats with chronic IOP elevation. MethodsThe measurement of IOP by the TonoPen and TonoLab tonometers was made in 21 normal rat eyes, 10 normal mouse eyes, and 16 rats that had either 2 or 4-week experimental glaucoma. IOP was varied from 10 to 50 mm Hg in steps of 10 mm Hg under conditions in which the eye was either open or closed to the reservoir controlling IOP. ResultsIn normal rat eyes, TonoPen overestimated manometric IOP at 10 mm Hg and underestimated it by up to 6 mm Hg at higher IOP, whereas the TonoLab matched set IOP within 1 mm Hg. In glaucoma rat eyes, the TonoLab accurately reflected manometric IOP under open stopcock conditions (linear regression: y=0.99x −0.62, R2=0.98), whereas in the closed stopcock condition, IOP measured lower at the higher IOP levels (P=0.0059, paired t test). In uncannulated rat glaucoma eyes, the tonometer used first gave higher IOP [paired t test, P=0.015 (TonoLab first); P=0.005 (TonoPen first)]. In normal mouse eyes under the open stopcock condition, the TonoLab nearly matched manometric IOP (linear regression: y=0.98x+1.57, R2=0.98). ConclusionsIn mouse and rat eyes, including rats with chronic IOP elevation, the TonoLab accurately reflected manometrically set IOP in an efficient manner.

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.

Changes in Optic Disk Characteristics and the Number of Nerve Fibers in Experimental Glaucoma

We compared the change in cup/disk ratio and neuroretinal rim area-to-disk area to estimated change in the number of optic nerve fibers in 12 cynomolgus monkeys with unilateral experimental glaucoma. Changes in the cup/disk ratios and neuroretinal rim area-to-disk area were estimated from stereoscopic optic disk photographs that were obtained before and after the development of increased intraocular pressure. Change in the number of optic nerve fibers in the glaucomatous nerves was estimated by comparing them to their fellow normal nerves. A significant linear correlation was present between change in the cup/disk ratios and neuroretinal rim area-to-disk area, and estimated change in the number of optic nerve fibers (r > or = .85; P < .0002). In optic disks with initial cup/disk ratios of 0.2 to 0.3 and neuroretinal rim area-to-disk area of 0.9, an increase in the cup/disk ratio of 0.1 and a decrease of 0.1 in the neuroretinal rim area-to-disk area is associated with a 10% and 9% loss of optic nerve fibers, respectively.

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.