Biomechanical, ultrastructural, and electrophysiological characterization of the non-human primate experimental glaucoma model.

Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We studied NHPs with ExGl of several years duration. As expected, ExGl eyes exhibited selective reductions of the retinal nerve fiber layer that correlate with electrophysiologic measures documenting a link between morphologic and elctrophysiologic endpoints. Softening of unlasered TM in ExGl eyes compared to untreated controls was observed. The degree of TM softening was consistent, regardless of pre-mortem clinical findings including severity of IOP elevation, retinal nerve fiber layer thinning, or electrodiagnostic findings. Importantly, this softening is contrary to TM stiffening reported in glaucomatous human eyes. Furthermore, microscopic analysis of unlasered TM from eyes with ExGl demonstrated TM thinning with collapse of Schlemm's canal; and proteomic analysis confirmed downregulation of metabolic and structural proteins. These data demonstrate unexpected and compensatory changes involving the TM in the NHP model of ExGl. The data suggest that compensatory mechanisms exist in normal animals and respond to elevated IOP through softening of the meshwork to increase outflow

Toluene inhalation exposure for 13 weeks causes persistent changes in electroretinograms of Long–Evans rats

Studies of humans chronically exposed to volatile organic solvents have reported impaired visual functions, including low contrast sensitivity and reduced color discrimination. These reports, however, lacked confirmation from controlled laboratory experiments. To address this question experimentally, we examined visual function by recording visual evoked potentials (VEP) and/or electroretinograms (ERG) from four sets of rats exposed repeatedly to toluene. In addition, eyes of the rats were examined with an ophthalmoscope and some of the retinal tissues were evaluated for rod and M-cone photoreceptor immunohistochemistry. The first study examined rats following exposure to 0, 10, 100 or 1000 ppm toluene by inhalation (6 hr/d, 5 d/wk) for 13 weeks. One week after the termination of exposure, the rats were implanted with chronically indwelling electrodes and the following week pattern-elicited VEPs were recorded. VEP amplitudes were not significantly changed by toluene exposure. Four to five weeks after completion of exposure, rats were dark-adapted overnight, anesthetized, and several sets of electroretinograms (ERG) were recorded. In dark-adapted ERGs recorded over a 5-log (cd-s/m2) range of flash luminance, b-wave amplitudes were significantly reduced at high stimulus luminance values in rats previously exposed to 1000 ppm toluene. A second set of rats, exposed concurrently with the first set, was tested approximately one year after the termination of 13 weeks of exposure to toluene. Again, dark-adapted ERG b-wave amplitudes were reduced at high stimulus luminance values in rats previously exposed to 1000 ppm toluene. A third set of rats was exposed to the same concentrations of toluene for only 4 weeks, and a fourth set of rats exposed to 0 or 1000 ppm toluene for 4 weeks were tested approximately 1 year after the completion of exposure. No statistically significant reductions of ERG b-wave amplitude were observed in either set of rats exposed for 4 weeks. No significant changes were observed in ERG a-wave amplitude or latency, b-wave latency, UV- or green-flicker ERGs, or in photopic flash ERGs. There were no changes in the density of rod or M-cone photoreceptors. The ERG b-wave reflects the firing patterns of on-bipolar cells. The reductions of b-wave amplitude after 13 weeks of exposure and persisting for 1 year suggest that alterations may have occurred in the inner nuclear layer of the retina, where the bipolar cells reside, or the outer or inner plexiform layers where the bipolar cells make synaptic connections. These data provide experimental evidence that repeated exposure to toluene may lead to subtle persistent changes in visual function. The fact that toluene affected ERGs, but not VEPs, suggests that elements in the rat retina may be more sensitive to organic solvent exposure than the rat visual cortex

Death by color: differential cone loss in the aging mouse retina

Differential cell death is a common feature of aging and age-related disease. In the retina, 30% of rod photoreceptors are lost over life in humans and rodents. However, studies have failed to show age-related cell death in mouse cone photoreceptors, which is surprising because cone physiological function declines with age. Moreover in human, differential loss of short wavelength cone function is an aspect of age-related retinal disease. Here, cones are examined in young (3-month-old) and aged (12-month-old) C57 mice and also in complement factor H knock out mice (CFH-/-) that have been proposed as a murine model of age-related macular degeneration. In vivo imaging showed significant age-related reductions in outer retinal thickness in both groups over this period. Immunostaining for opsins revealed a specific significant decline of >20% for the medium/long (M/L)-wavelength cones but only in the periphery. S cones numbers were not significantly affected by age. This differential cell loss was backed up with quantitative real-time polymerase chain reaction for the 2 opsins, again showing S opsin was unaffected, but that M/L opsin was reduced particularly in CFH-/- mice. These results demonstrate aged cone loss, but surprisingly, in both genotypes, it is only significant in the peripheral ventral retina and focused on the M/L population and not S cones. We speculate that there may be fundamental differences in differential cone loss between human and mouse that may question the validity of mouse models of human outer retinal aging and pathology

Relative Contribution of VEGF and TNF-␣ in the Cynomolgus Laser-Induced CNV Model: Comparing the Efficacy of Bevacizumab, Adalimumab, and ESBA105

PURPOSE. To compare the relative contribution of VEGF and TNF-␣ in the development of laser-induced choroidal neovascularization (CNV) in monkeys and to exploit the feasibility of topical use of suitable antibody fragments for the prevention of experimental CNV. METHODS. To induce experimental CNV, small high-energy laser spots were used to treat several areas of the macula in the retinas of cynomolgus monkeys according to previously published protocols. To prevent abnormalities, bevacizumab (a potent VEGF inhibitor) and adalimumab or ESBA105 (potent TNF-␣ inhibitors) were given by intravitreal injection 1 week before and 1 week and 3 weeks after laser treatment. ESBA105 was also applied topically in a separate group. Control animals were treated with either intravitreal or topical saline. Eyes were monitored by ophthalmic examination, color photography, and fluorescein angiography. RESULTS. Inhibition of VEGF by bevacizumab completely blocked the formation of CNV. Both TNF-␣ inhibitors also significantly reduced laser-induced CNV abnormalities after intravitreal administration. Most important, topical use of the anti-TNF-␣ single-chain antibody fragment ESBA105 also reduced the formation of CNV. CONCLUSIONS. TNF-␣ contributes to laser-induced CNV formation, and its inhibition can be a new therapeutic target for CNV. This study suggests TNF-␣ as another therapeutic target for the prevention and treatment of CNV and adds to the emerging clinical data suggesting the therapeutic value of TNF-␣ inhibitors in age-related macular degeneration (AMD). Further, this study shows that topical therapy with suitable antibody fragments has the potential of being introduced to retinal disease treatment regimens. (Invest Ophthalmol Vis Sci