Identification of susceptibility loci for light-induced visual impairment in rats.

Bright light exposure in animals results in the selective degeneration of the outer retina, known as "retinal photic injury" (RPI). The susceptibility to RPI differs among rat strains. WKY rats display susceptibility to RPI with extensive retinal degeneration observed in the sagittal eye specimen, whereas LEW strain rats are resistant to it, showing only slight or no degeneration. In the present study, we first established an ethological screening method using the Morris water maze to discern differential susceptibility among the living rats. WKY and LEW were crossed to produce the first filial generation (F1) offspring. Maze-trained individuals were exposed to bright, white light. The screening test results demonstrated that the susceptibility to light-induced visual impairment in rats is a dominant Mendelian susceptibility trait, as F1 rats were susceptible to visual impairment like WKY rats. Therefore, F1 rats were backcrossed with recessive LEW to produce the first backcross offspring (BC1). Subsequent recurrent backcrossing while selecting for the susceptibility, indicated a segregation ratio of ca. 24% in BC1 and BC2 generations, indicating the involvement of two or more genes in the susceptibility. Further, microsatellite analysis of BC1-to-BC4 individuals using microsatellite markers mapped two susceptibility loci on chromosome segments 5q36 and 19q11-q12, named RPI susceptibility (Rpi)1 and Rpi2, respectively. This study provides an insight into mechanisms underlying differential susceptibility, which could help decipher the mechanism underlying the onset/progression of human age-related macular degeneration.

[Oxidative stress in ocular disease].

The definition of oxidative stress implies increased oxidant production in animal cells characterized by the release of free radicals, resulting in cellular degeneration. The imbalance between excess free radical production and the antioxidant defense causes cellular damage resulting in lipid peroxidation. Oxidative stress is involved in many ocular diseases such as age-related macular degeneration, retinopathy of prematurity, retinal light damage, and cataract. Reactive oxygen species are involved in this process. The pathogenesis of age-related macular degeneration is largely unknown. Excessive light and iron may enhance the progression of this disease. In in vitro study of the ciliary body, gamma irradiation inhibits TPR53BP2 expression associated with apoptotic cell death, and increased BCL2 is evident just after gamma irradiation. Exposure to ultraviolet light has been postulated as a cause of age-related macular degeneration (AMD), perhaps through damage to the retinal pigment epithelium. It seems logical, therefore, to replace the aging, yellowing lens with a blue light-absorbing yellow intraocular lens (IOL) in cataract surgery. The issue of whether cataract surgery is a risk factor for the development or progression of AMD remains controversial. In vivo studies suggest that lipid peroxidation decreases in the vitreous and retina after cataract surgery with or without intraocular lens implantation.

Macular carotenoid levels of normal subjects and age-related maculopathy patients in a Japanese population.

PURPOSE: Macular carotenoid pigments composed of lutein and zeaxanthin are thought to affect the development of age-related maculopathy (ARM). Macular carotenoid levels were measured in normal Japanese subjects and Japanese patients with ARM. DESIGN: Observational case-control series. PARTICIPANTS: One hundred normal eyes of 100 normal subjects and 187 eyes of 97 patients with ARM; all were Japanese. The definitions of early ARM and late ARM (exudative age-related macular degeneration [AMD] and dry AMD) were used according to an accepted international classification system. METHODS: Macular carotenoid levels were measured using resonance Raman spectroscopy. MAIN OUTCOME MEASURE: Raman signal intensity generated from carbon-carbon double bond vibrations of lutein and zeaxanthin. RESULTS: The mean (+/-standard deviation [SD]) macular carotenoid level in normal subjects was 1471+/-540 Raman counts. The macular carotenoid levels in normal subjects declined with age. The mean macular carotenoid level was 620+/-204 (+/-SD) in eyes with early ARM and 427+/-283 (+/-SD) in eyes with late ARM (equal to AMD). The macular carotenoid levels of early ARM and AMD were significantly lower than those in normal subjects older than 60 years (1100+/-340 [+/-SD]). No difference was revealed in carotenoid levels by the severity for ARM, type of AMD (exudative, atrophic, and disciform scar), or types of choroidal neovascularization (classic, minimally classic, occult, polypoidal choroidal vasculopathy), although small numbers in some groups weakened statistical power. Macular carotenoid levels were affected by the severity of macular disease in the opposing eye. The average for normal eyes where AMD was found in the opposite eye was significantly lower than that of normal eyes in the absence of AMD in the opposite eye (i.e., healthy volunteers older than 60 years). CONCLUSIONS: Macular carotenoids decreased even in older healthy individuals. The ARM patients showed lower macular carotenoid levels than healthy people. Low macular carotenoid levels may be one of the risk factors of progression in ARM.

In the presence of ferritin, visible light induces lipid peroxidation of the porcine photoreceptor outer segment.

We studied the synergistic effect of visible light and ferritin on the lipid peroxidation on a fraction of porcine photoreceptor outer segment (POS). Reaction mixtures containing the POS fraction and horse spleen ferritin were irradiated under white fluorescent light mainly at 17,000 lx or incubated under dark conditions at 37 degrees C. The lipid peroxidation was evaluated by both the thiobarbituric acid method and the ferrous oxidation/xylenol orange method. The irradiation-induced lipid peroxidation was affected by some experimental factors such as the irradiation dose and acidity of the material. When the irradiation was stopped, the lipid peroxidation was also stopped; thereafter, the re-irradiation induced lipid peroxidation. Moreover, this lipid peroxidation was inhibited by desferrioxamine, an iron chelator, or by dimethylthiourea, a hydroxyl radical scavenger, suggesting that the lipid peroxidation involves hydroxyl radicals generated via the Fenton reaction by iron ion released from ferritin. The lipid peroxidation did not take place under dark conditions or in the absence of ferritin. This study suggested the possibility that the visible light-induced lipid peroxidation of the POS fraction in the presence of ferritin may participate in the etiology of human retinal degenerative diseases as the human retina is exposed to light for life.

Ascorbic acid concentration in rabbit vitreous measured by microdialysis with HPLC-electrochemical detection before and after vitreous surgery.

Microdialysis with high performance liquid chromatography and electrochemical detection (HPLC-ECD) was used to measure ascorbic acid (AA) concentrations in rabbit vitreous before and after vitrectomy. A cellulose microdialysis probe was implanted in the vitreous humor, and after stabilization, AA measurements were made daily over a 10 day period. The effect of removing two-thirds of the vitreous by vitrectomy was examined. The effect of triamcinolone acetonide (TA) was evaluated in four groups of rabbits: Group 1, sub-tenon TA (20 mg) alone; Group 2, intravitreous TA (4 mg) alone; Group 3, sub-tenon TA (20 mg) after vitrectomy, and Group 4, intravitreous TA (4 mg) after vitrectomy. The results showed that the AA concentration after vitrectomy was significantly lower from days 2 to 10 with a maximum reduction of 49.5% (P < 0.005) on day 7. No significant changes in the AA level was observed in Groups 1 and 2, a mild recovery of AA concentration reduction after vitrectomy was detected in Group 3. The highest recovery of the AA concentration reduction was observed in Group 4. The attenuating effect of TA treatment on the reduction of AA in the vitreous after vitrectomy was significant. This attenuating effect of the TA may be due to prevention of the disruption of the blood-aqueous barrier by its anti-inflammatory action.

Iron release analyses from ferritin by visible light irradiation.

We investigated the iron release from ferritin by irradiation from a white fluorescent light in the absence or presence of ADP. Irradiation of a ferritin solution at 17,000 lx in the absence of ADP slightly induces iron release from ferritin but only at acidic pH conditions (pH 5.0 or pH 6.0). Irradiation in the presence of ADP markedly enhances iron release from ferritin under the same conditions. In the absence of irradiation, the iron release from ferritin was low even in the presence of ADP. The induction of the iron release by irradiation in the presence of ADP was also affected by various factors such as irradiation dose and acidity, but not temperature (4-47 degrees C), oxygen concentration, or free radical generations during the irradiation. The iron release during the irradiation ceased to increase by turning off the light and was found to increase again after additional irradiation. These results suggest that visible light directly induces iron release from ferritin via the photoreduction of iron stored inside ferritin.

Membranes of retinal pigment epithelial cells in vitro are damaged in the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions.

The ferrous ions released from haemoglobin and storage-transferrin ions cause oxidative stress in the eyes. We observed the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions in the retinal pigment epithelial (RPE) cells in vitro, and investigated how the ferrous ions influenced RPE in vitro and the photoreceptor outer segment discs. We obtained isolated photoreceptor outer segment discs using sucrose gradient of specific gravity after homogenizing porcine retinas. After bovine RPE cells were cultured with isolated photoreceptor outer segment discs containing FeCl2 for 5 and 24 h, we incubated the specimens with rhodamine phalloidin, antimouse alpha-tubulin antibody and antimouse Ig G (FITC and rhodamine labelled). We observed the specimens by a laser scanning microscopy, and made the ultrathin sections with or without 2% uranyl acetate and 2% lead acetate for examination by transmission electron microscopy. Actin filaments and microtubules of specialized cells such as RPE cells were actively involved in phagocytosis of the photoreceptor outer segment discs. Microtubules were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions. The peroxidation increased the granular and aggregated autofluorescence of the photoreceptor outer segment discs. The membranes of the disc and the phagosomes, and lysosomes in RPE cells were damaged by ferrous ions and had fine particles with high electron density staining without uranium acetate and lead citrate. The cytoskeletons such as actin filaments and microtubules, and the membranes of the phagosomes and the lysosomes in RPE cells in vitro were damaged during the phagocytotic process of the photoreceptor outer segment discs peroxidized by ferrous ions.