Tim T. Lam

Expression of a mutant opsin gene increases the susceptibility of the retina to light damage.

The question of whether the expression of mutant opsin predisposes the retina to light damage was addressed using transgenic mice that express rhodopsin with three point mutations near the N-terminus of the molecule. The mutations involve the substitution of histidine for proline at position 23 (P23H), glycine for valine at position 20 (V20G), and leucine for proline at position 27 (P27L). These mice express equal amounts of mutant and wild-type transcripts, and develop a progressive photoreceptor degeneration that is similar to that seen in human retinitis pigmentosa (RP). The P23H mutation is associated with the most frequently occurring form of human autosomal dominant retinitis pigmentosa (ADRP) in the United States. Transgenic and normal littermates were exposed to illuminance of 300 foot-candles (ft-c) for 24 h, then placed in darkness for either 6 h, 6 days, or 14 days. Histological and biochemical techniques were used to evaluate the outer retina in light-exposed and control animals reared on 12-h light/12-h dark cycle. The results indicate that light exposure accelerates the pathological changes associated with the transgene expression. Compared with transgenic animals reared in ambient cyclic light, retinas from light-exposed mice had a reduced rhodopsin content, fewer photoreceptor cell bodies, and less preservation of retinal structure. Data obtained from normal mice did not differ for the lighting regimens used. These findings suggest that the expression of VPP mutations in the opsin gene predisposes the transgenic photoreceptors to be more susceptible to light damage. The data also suggest that reducing photic exposure may be beneficial to any patient with RP mediated by an opsin mutation.

DISTURBANCES IN THE DISTRIBUTION OF NEUROTRANSMITTERS IN THE RAT RETINA AFTER ISCHEMIA

PURPOSE Disturbances in neurotransmitter distribution have been observed in cerebral ischemia in the pathophysiologic process of excitotoxicity. The goal of this study was to examine the effect of pressure-induced retinal ischemia on the distribution of the retinal neurotransmitters glutamate and gamma-aminobutyric acid (GABA) within the rat retina. METHODS Animals were subjected to increased intraocular pressure of 110 mm Hg for 45 min using an intracameral hydrostatic pressure device. The distribution of glutamate and GABA immunoreactivity (IR) was determined at 0, 2, 4, 8 and 24 hrs after reperfusion by immunogold with silver intensification. RESULTS Three phases of neurotransmitter immunoreactivity patterns were discernible following retinal ischemia. Immediately following reperfusion (Phase I), a shift of GABA-IR from inner retinal neurons to the Mueller cells and their processes was noted. In contrast, despite marked decreases in neuronal glutamate-IR, a less pronounced shift of glutamate-IR to the Muller cells was simultaneously noted. This shift of neurotransmitter IR to the Mueller cells was transient with the gradual reappearance of IR within the inner retinal neurons noted 2-8 hrs after reperfusion (Phase II). Phase III began at 8 hrs after reperfusion with progressive loss of GABA-IR noted in the inner retina; by 24 hrs, secondary loss of inner retinal glutamate-IR was evident with corresponding dropout and pyknosis of inner retinal neurons apparent. CONCLUSIONS The distribution of glutamate-IR and GABA-IR was significantly altered following retinal ischemia. The alteration noted in Phase I suggested that the regulation of glutamate by Mueller cells was disrupted by this ischemic insult leading to glutamate excitotoxicity, and delayed neuronal cell degeneration as evidenced by the subsequent loss of inner retinal immunoreactivity in Phase III.

A histopathologic study of retinal lesions inflicted by transscleral iontophoresis

In the present study, retinal lesions were induced by transscleral iontophoresis (1.5 mA) in rabbits. The size and severity of the lesions increased with the duration of application (2–25 min). No lesion was noted after <1 min application. Immediately after 5 min iontophoresis, the edematous retina exhibited necrotic retinal pigment epithelium (RPE), loss of outer segments, and thinning of the inner and outer nuclear layers. At 5 days after iontophoresis, there was a proliferation of RPE cells and macrophages in the subretinal space, with thinning of the inner and outer retinal layers continuing. By day 14, the retina had been reduced to a glial membrane. Immediately after 15 min iontophoresis, the damaged retina appeared in a mummified form containing no cellular elements. By day 5 thereafter, macrophages and actively proliferating RPE cells had been noted in the necrotic retina. By day 14, a glial membrane had formed.

A COMPARISON OF CONTINUOUS VERSUS INTERMITTENT LIGHT EXPOSURE ON APOPTOSIS

PURPOSE We recently found that continuous light exposure at a moderate intensity triggered apoptosis of photoreceptor cells. Since intermittent light exposure is known to cause more severe retinal damage than is continuous light exposure, we sought to determine if intermittent light exposure also triggered apoptosis of photoreceptor cells. METHODS Lewis albino rats were reared, for 2 weeks, in cyclic light and dark adapted for 24 hr before light exposure. Rats were exposed to intermittent light or continuous light for 6 or 9 hr, respectively. Light-exposed rats were killed by lethal injection at three timepoints: immediately after light exposure, after 6 hr of dark recovery following light exposure and after 24 hr of dark recovery following light exposure. Retinal damage after light exposure was evaluated by morphology, morphometry, the terminal transferase-mediated biotin dUTP nick end labeling (TUNEL) technique for identification of nicked/cleaved nuclear DNA and agarose gel electrophoresis of retinal DNA. RESULTS Evaluation of morphology confirmed that intermittent light exposure caused more photoreceptor cell damage than did continuous light exposure of the same duration and intensity. The TUNEL technique showed that photoreceptor nuclei contained nicked or cleaved DNA after either intermittent or continuous light exposure, although more TUNEL-positive nuclei were observed after intermittent exposure. Agarose gel electrophoresis of retinal DNA showed internucleosomal DNA fragmentation, which is associated with apoptosis in samples from intermittent light exposure. CONCLUSIONS These data demonstrated that intermittent light exposure triggered apoptosis in more photoreceptor cells than did continuous light exposure of the same intensity and duration.

Methylprednisolone therapy in laser injury of the retina.

The efficacy of methylprednisolone in argon-laser-induced retinal injury in primates was evaluated by clinical, histopathologic, and morphometric criteria. Methylprednisolone was given with a loading dose of 30 mg/kg followed by 5.4 mg/kg per hour in three different regimens: (1) starting 24 h before laser and continuing for 4 days; (2) starting immediately after laser and continuing for 4 days; and (3) starting immediately after laser and continuing for 8 h. Fundus photography, fluorescein angiography, and histologic examination showed significant beneficial effects of all three treatments compared to controls. Morphometrically, at the center of the lesion, the width of disrupted outer nuclear layer, the width of the affected RPE, and the percentage of residual photoreceptor nuclei confirmed the efficacies of treatment regimens 1 and 2, but not treatment regimen 3.

Dexamethasone ameliorates retinal photic injury in albino rats

The effect of dexamethasone in two regimens on retinal photic injury was studied in Lewis albino rats that were exposed to 24 hr of continuous green fluorescent light. Under regimen 1, dexamethasone was given at a daily dosage of 1 mg kg-1 for 8 days, starting 6 days before light exposure. Under regimen 2, dexamethasone was given at the same daily dosage for 3 days, started 1 day before light exposure. Pathologic study of the light-exposed retina, morphometric evaluation of the photoreceptor cell loss, cell counts of the macrophages in the subretinal space, and measurements of rhodopsin levels were undertaken in the dexamethasone-treated and control retinas at various times. The administration of dexamethasone in both regimens did not produce pathologic changes in the retina before light exposure, but rhodopsin levels were significantly lowered in both treated groups when compared to corresponding vehicle treated control animals. Under regimen 1, at 6 hr after light exposure, both the treated and the control groups showed comparable loss of photoreceptor cells, degeneration of the photoreceptor elements and retinal pigment epithelium, but a significantly lowered level of rhodopsin in the treated group was noted. At 6 days after exposure, the outer nuclear layer thickness, and the outer and inner segments showed significant preservation in the treated group. Also in the treated group, the number of macrophages was significantly reduced and the retinal pigment epithelial (RPE) vacuolation was markedly less. However, there was no difference in rhodopsin levels. At 14 days after exposure, the outer nuclear layer thickness and rhodopsin levels of the treated rats had significantly higher values than the controls. Under regimen 2, however, at 6 days after exposure, an ameliorative effect in the RPE was observed but there were no differences of rhodopsin levels, the outer nuclear thickness and number of macrophages between the treated and control groups. Regimen 1 was associated with a significantly higher retinal level of dexamethasone when compared with regimen 2. The ameliorative effect of dexamethasone on rat retinal photic injury may be through inhibition of lipid peroxidation, in which a high retinal level of the steroid is required.

Effects of glycosaminoglycan-degrading enzymes on bovine trabecular meshwork in organ culture.

We evaluated the effects of three specific glycosaminoglycan-degrading enzymes, chondroitinase ABC, testicular hyaluronidase, and heparinase, on the intraocular pressure (IOP) and the anterior chamber structures of bovine eyes. The anterior segment of freshly enucleated bovine eyes was perfused in a modified organ culture system. After each enzyme solution or its corresponding vehicle was introduced into the anterior chamber, the IOP was monitored for </=6 h. Compared to the control group, the chondroitinase ABC-treated group showed a significant IOP reduction after 1 h of perfusion. Testicular hyaluronidase and heparinase did not show any significant effect. Light microscopy revealed that chondroitinase ABC and testicular hyaluronidase induced detachment of the endothelial cells from trabecular beams in the uveal meshwork. Disruption of the tissues in the juxtacanalicular region and fragmentation of the endothelial cells facing the angular aqueous plexus were observed in the chondroitinase ABC- and heparinase-treated specimens. Electron microscopic study revealed detachment of trabecular meshwork cells from the beams and rupture of the inner wall of the aqueous plexus in all the enzyme-treated eyes. Cellular organelles and architectures were better preserved in chondroitinase ABC-treated eyes than those treated with hyaluronidase and heparinase. These results indicate that chondroitinase ABC has a pronounced effect on the IOP with relatively minor effect on the trabecular meshwork structures.

Correlation of Phospholipid Hydroperoxide Glutathione Peroxidase Activity to the Sensitivity of Rat Retinas to Photic Injury

We hypothesize that the differential susceptibility to photic injury among different strains of rat retinas may depend on the levels of phospholipid hydroperoxide glutathione peroxidase (PHGPX) activity, one of the endogenous antioxidant enzymes in the retina. The retinas of four inbred strains of albino rat (Fischer, Wistar, Buffalo and Lewis) were analyzed for glutathione peroxidase activity using H2O2, cumene hydroperoxide, and phospholipid hydroperoxide as assay substrates. In all four strains of rat, PHGPX was observed only in the high salt extract of the retina, while peroxidases determined by H2O2 or cumene hydroperoxide were observed mainly in the low salt extract. PHGPX was highest (66.7 mU/mg) in the most light-resistant Fischer strain and lowest in the most light-sensitive Lewis strain (31.9 mU/mg), while the activity levels in the moderately light-sensitive Buffalo and Wistar strains were 46.6 and 38.5 mU/mg, respectively. In contrast, there was no significant difference in peroxidases determined by H2O2 or cumene hydroperoxide among the four strains. These observations suggested that, in rat retina, the membrane-associated PHGPX may have an important role in the defense against light-induced free radical damage.

The effect of high-dose methylprednisolone on laser-induced retinal injury in primates: an electron microscopic study

Abstract• Background: Previously we reported an ameliorative effect of high-dose methylprednisolone in laser injury to monkey retinas. The ultrastructural modification by methylprednisolone has not been examined. • Methods: Cynomolgus monkeys were given severe kgrade III) retinal laser burns and treated with an intravenous megadose of methylprednisolone. Pathologic features of the retinal lesions with or without methylprednisolone treatment were evaluated by light and electron microscopy. • Results: Ultrastructurally, the treated lesions showed rapid recanalization of choriocapillaris; proliferation of retinal pigment epithelium to replace the necrotic and damaged cells, resulting in rapid re-establishment of blood retinal barrier; mild macrophagic activity; and rapid reformation of the outer limiting membrane by Mueller cells. • Conclusion: A high dose of methylprednisolone affected the responses of the choriocapillaris, retinal pigment epithelium, photoreceptor cells and Mueller cells to laser injury, showing an overall beneficial effect. These modifications might be ascribed to methylprednisclones anti-inflammatory action, protection of the microcirculation and anti-lipid peroxidation effect. Proprietary interest: none

Clearance of S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913) in rats

Complications of radiotherapy in the treatment of retinal and choroidal neoplastic diseases include cataract formation, radiation retinopathy, neovascular glaucoma, cystoid macular edema, and subretinal neovascularization. These side effects may be minimized by the use of compounds known to have a protective effect on normal ocular tissues without impeding the benefits of therapy. Phosphorothioates, first developed under the Antiradiation Drug Development Program of the U.S. Army Medical Research and Development Command, have been reported to protect normal tissues during radiation therapy in a variety of animal models. One of the phosphorothioates, WR-77913 (S-[3-amino-2-hydroxylpropyl]phosphorothioate) was found to inhibit cataract formation in rats after radiation exposure. To test the efficacy of WR-77913 in the retina, we established a high-pressure liquid chromatography method to measure the levels of dephosphorylated WR-77913 and studied the drugs clearance from the lens, retina, blood, kidney, and liver in rats.