Decrease of cone opsin mRNA in experimental ocular hypertension.

PURPOSE: This study was designed to test the hypothesis that photoreceptors are adversely affected in glaucoma. As a measure of this effect, we examined the levels of rod opsin, and red/green and blue cone opsin mRNAs in monkeys with experimental ocular hypertension and glaucoma and in human eyes from donors with diagnosed glaucoma. METHODS: Experimental ocular hypertension was induced in one eye of 19 cynomolgous and 2 rhesus monkeys by laser ablation of the trabecular meshwork. In 15 monkeys, the elevated IOP was reduced by trabeculectomy. When the animals had experienced prolonged elevations of IOP (128 to 260 days), they were killed and the eyes enucleated. Fresh retinal tissue from the macula, inferotemporal retina (mid-peripheral), and far peripheral regions were harvested from some animals using a 3 mm trephine. The remaining retinas from these monkeys, and whole retinas from other animals were fixed. RNA isolated from each trephined sample was used for RNase Protection Analysis or real time PCR analysis to quantify opsin mRNA levels from different photoreceptor cell types. Fixed tissue was used for in situ hybridization studies. Human donor eyes (7 glaucoma and 4 control) were obtained from eye banks. All human specimens were used for in situ hybridization studies. RESULTS: Quantitative mRNA analysis and in situ hybridization studies both showed a reduction in the expression of red/green and blue cone opsin mRNAs in 6 monkey eyes with chronic ocular hypertension, relative to the contralateral eye. No loss of rod opsin mRNA was observed. The principal reduction occurred in cells of the mid-peripheral retina, a region of retina that often shows early and progressive damage in humans with glaucoma. In monkeys with ocular hypertension followed by trabeculectomy, there was a similar decrease in cone opsin mRNAs, but only in six out of fifteen (40%) of the monkeys. The decrease in these animals was correlated with a significantly elevated IOP at some time during the 2 weeks prior to euthanization and not with the extent of glaucomatous damage. Of the 7 human eyes with diagnosed glaucoma that were examined, 5 showed a decrease of cone opsin mRNA in the mid-peripheral retina, whereas none of the 4 normal eyes examined showed a decrease. CONCLUSIONS: Ocular hypertension leading to glaucoma also affects the outer retina, particularly the cone photoreceptors. We speculate that these cells become stressed leading to a disruption in the expression of normal genes, such as that encoding opsin. There is some evidence that this effect is reversible, when IOP levels are reduced.

p53 regulates apoptotic retinal ganglion cell death induced by N-methyl-D-aspartate.

PURPOSE: The tumor suppressor protein p53 plays a central role in regulating apoptosis in a variety of neuronal cell types. Previous studies have indicated that retinal ganglion cell (RGC) death induced by ischemia follows a p53-dependent pathway. Ischemia causes wide-spread damage to the retina, eliciting multiple different damaging pathways. We conducted experiments to specifically investigate the role of p53 in RGC death activated by overstimulation of the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate dependent calcium channel normally involved in glutamate neurotransduction. METHODS: RGC death was induced in both wild-type (CB6F1 or 129/Sv) and p53-deficient (129/Sv background) mice by a single intravitreal injection of either 40 or 160 nmol of NMDA into one eye leaving the other eye as an untreated control. Cell loss was quantified by comparing the number of surviving cells in the retinas from experimental eyes relative to the control eyes of the same animals. The accumulation of p53 mRNA in retinas was monitored by reverse-transcription PCR (RT-PCR) of retinal total RNA isolated from mice injected with 40 nmol of NMDA. The functional requirement for p53 was monitored in p53-deficient mice after intravitreal injection of 160 nmol of NMDA. Immunohistochemistry for cleaved poly(ADP-ribose) polymerase (PARP) was performed on p53-deficient mice after intravitreal injection of 160 nmol of NMDA. RESULTS: In wild-type CB6F1 mice, p53 mRNA levels are elevated within 3 h after NMDA injection. This accumulation correlates with the onset of changes in RGC nuclear morphology that precedes pyknosis, which occurs by 6 h. Mice (129/Sv) deficient for one or both alleles of p53 show no developmental change in RGC number, compared to wild-type animals (Mann-Whitney test, p=0.824), suggesting that p53 is not required for developmental programmed cell death of RGCs. In adult mice, however, p53-dependent changes in the rate of RGC death after exposure to 160 nmol of NMDA were observed. Four days after injection, p53+/+ and p53-/- mice exhibit statistically equivalent amounts of cell loss (p>0.1), while p53+/- mice have significantly attenuated cell loss (p<0.002), relative to the other groups. RGCs from NMDA-treated p53+/+ and p53-/- mice were analyzed further using immunohistochemistry to identify the cleavage products of poly(ADP-ribose) polymerase (PARP), a known substrate for caspases. Cleaved PARP was found in p53+/+ and p53+/- eyes, but not in p53-/- mice. CONCLUSIONS: Developmental RGC programmed cell death does not require p53. Selective overstimulation of the glutamate-dependent NMDA-receptor in adult mice activates a p53-dependent pathway of death in RGCs. The requirement for p53 is not absolute, however, because mice lacking this gene are able to execute an alternative pathway of cell death. Examination of the cleavage of PARP, which is a substrate for caspases, suggests that the p53-dependent pathway utilizes these proteases, but the p53-independent pathway does not.

Adenovirus-mediated gene therapy using human p21WAF-1/Cip-1 to prevent wound healing in a rabbit model of glaucoma filtration surgery.

OBJECTIVE: To determine if adenovirus-mediated p21(WAF-1/Cip-1) (p21) gene therapy can prevent fibroproliferation and wound healing in a rabbit model of glaucoma filtration surgery. METHODS: In vitro studies were performed using rabbit Tenon fibroblasts harvested from fresh tissue. In vivo studies were conducted in New Zealand white rabbits. A full-thickness sclerotomy was performed under a limbal-based conjunctival flap. Reagents tested included a replication-deficient recombinant adenovirus containing the human p21 gene (rAd.p21); the nonspecific marker gene for green fluorescent protein or beta-galactosidase; mitomycin, 0.5 mg/mL; and balanced saline solution. Each treatment was applied episclerally for 5 minutes before the sclerotomy using a soaked cellulose sponge placed under the surgically created conjunctival flap. Independent experiments were conducted to (1) monitor changes in intraocular pressure during a 30-day period after treatment and examine surgical site histological features, (2) examine changes in bleb morphologic features over 30 days, (3) determine outflow facility 14 days after treatment, and (4) examine the localization and persistence of rAd.p21 expression between 3 and 60 days after treatment. RESULTS: Treatment of Tenon fibroblasts with rAd.p21 resulted in a dose-dependent inhibition of DNA synthesis and cell growth in vitro. In vivo, rAd.p21 inhibited wound healing and fibroproliferation after filtration surgery, comparably to mitomycin. Mitomycin caused notable thinning of the bleb wall. In addition, 2 of the 5 mitomycin-treated eyes exhibited an abscess with hypopyon and hyalitis 30 days after surgery, which was not observed in any of the rAd.p21-treated eyes. None of the treatments resulted in a significantly sustained decrease in intraocular pressure during the 30-day period, although mitomycin treatment resulted in a significant (P =.02) increase in outflow facility 2 weeks after surgery in separate animals. Mitomycin- and rAd.p21-treated eyes had functioning blebs at the end of the experiment based on slitlamp examination. CONCLUSIONS: Mitomycin and rAd.p21 were effective in preventing fibroproliferation and wound healing in a rabbit model of glaucoma surgery. Mitomycin treatment increased outflow facility in normal-pressure eyes. CLINICAL RELEVANCE: Gene therapy with rAd.p21 may provide an effective antiproliferative for glaucoma filtration surgery, without the complications associated with mitomycin.