alpha-Tocopherol in the developing rat retina: a high pressure liquid chromatographic analysis

High pressure liquid chromatography was used to measure alpha-tocopherol in the retinas of rats reared in a cyclic light or dark environment. These measurements were performed on extracts of whole retinas during the developmental period, 18-60 days, and on isolated ROS from adult animals. Similar alpha-tocopherol determinations were performed on retinas and isolated ROS following exposure of rats to intense visible light for 24 hr periods. The results show that alpha-tocopherol is chromatographically separated from the vitamin A derivatives found in the retina and is pure, as judged by mass spectrometry. In the retinas of cyclic light and dark reared rats, alpha-tocopherol accumulates in an age dependent fashion, so that at 60 days the level is nearly double that of animals at 18-20 days of age (P less than 0.001). Because the age dependent accumulation of rhodopsin is greater in dark reared rats, the average molar ratio of rhodopsin to alpha- tocopherol in the retina of dark reared animals is 25% higher than in cyclic light rats. Following exposure of rats to intense visible light for 24 hr periods, alpha-tocopherol concentrations in the retina were unchanged from the levels in control animals. In adult animals the concentration of alpha-tocopherol in ROS is 2.5-3.5 times higher than in whole retina. ROS from adult cyclic light reared rats also contain an average of 43% more alpha-tocopherol per mg protein than ROS from dark maintained animals (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protection by dimethylthiourea against retinal light damage in rats.

The protective effect of dimethylthiourea (DMTU) against retinal light damage was determined in albino rats reared in darkness or in weak cyclic light. Rats maintained under these conditions were treated with DMTU at different concentrations and dosing schedules and then exposed for various times to intense visible light, either intermittently (1 hr light and 2 hr dark) or continuously. The extent of retinal light damage was determined 2 weeks after light exposure by comparing rhodopsin levels in experimental rats with those in unexposed control animals. To determine the effect of DMTU on rod outer segment (ROS) membrane fatty acids, ROS were isolated immediately after intermittent light exposure, and fatty acid compositions were measured. The time course for DMTU uptake and its distribution in serum, retina, and the retinal pigment epithelium (RPE)/choroid complex was determined in other rats not exposed to intense light. After intraperitoneal injection of the drug (500 mg/kg body weight), DMTU appeared rapidly in the serum, retina, and the RPE and choroid. In the ocular tissues, it was distributed 70-80% in the retina and 20-30% in the RPE and choroid. This antioxidant appears to have a long half-life because it was present in these same tissues 72 hr after a second intraperitoneal injection. For rats reared in the weak cyclic light environment, DMTU (two injections) provided complete protection against rhodopsin loss after intense light exposures of up to 16 hr. Only 15% rhodopsin loss was found in cyclic-light DMTU-treated rats after 24 hr of intermittent or continuous light. For rats reared in darkness, DMTU treatment resulted in a rhodopsin loss of less than 20% after 8-16 hr of continuous light and approximately 40% after similar exposure to intermittent light. Irrespective of the type of light exposure, rhodopsin loss in the dark-reared DMTU-treated rats was nearly identical to that found in uninjected cyclic light-reared animals. In rats from both light-rearing environments, DMTU treatment prevented the light-induced loss of docosahexaenoic acid from ROS membranes. As measured by rhodopsin levels 2 weeks later, DMTU was most effective when given as two doses administered 24 hr before and just before intense light exposure. As a single dose given during continuous light exposure, DMTU protected cyclic light-reared rats for at least 4 hr after the start of exposure but was ineffective in dark-reared animals if injected 1 hr after the start of light. It was also ineffective in both types of rats when given after light exposure.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of L-and D-ascorbic acid administration on retinal tissue levels and light damage in rats.

To assess the protective effect of ascorbic acid in retinal light damage of rats, we have determined the uptake and retinal tissue distributions of its L- and D- stereoisomers following interperitoneal or intraocular injections. The effects of intense-intermittent light exposure and darkness on tissue ascorbate were compared by measuring its levels in retina and retinal pigment epithelial tissues at various times after administration. The protective effects of the two forms of ascorbate against retinal light damage were also compared by measuring rhodopsin levels 2 weeks after intense light exposure. After interperitoneal injection, both forms of ascorbic acid were higher in the retinal pigment epithelial-choroid-scleral complex (eye cup) than in the retina. Over a 2 hr post-injection period, L-ascorbate in the eye cup was 2 to 4 fold higher than normal (10-11 nmol); D-ascorbate levels were between 15 and 30 nmol. During the same period retinal L-ascorbate was just above normal (12-14 nmol), whereas less than 5 nmol of D-ascorbate was present. When ascorbate was given by the intraocular route the opposite effect was found. During the 2 hr post-injection period retinal L-ascorbate levels were 2 to 5 fold higher than normal; D-ascorbate was between 25 and 50 nmol/retina. Within 1 hr post-injection, L-ascorbate in the eye cup was near normal and D-ascorbate levels were 10 nmol or less. In uninjected rats perfused with normal saline, the endogenous L-ascorbate was distributed 55% in the retina with 9% and 36%, respectively, in the RPE-choroid and sclera. Ten-thirty min after interperitoneal peritoneal injection about 40% of the L-ascorbate was present in the retina with 17% and 44% in the RPE-choroid and sclera. Total ascorbate (L + D) levels in the same tissues of D- injected rats were similar to those found for rats given L-ascorbate. Following 7 hrs of darkness, tissue ascorbate levels in the injected rats decreased to approximately the same levels present in uninjected animals. For rats exposed to intense light average retinal ascorbate levels decreased further, while RPE-choroid and scleral levels were largely unchanged from the dark control levels. About 50% of the tissue ascorbate was present in the retina 10-30 min after intraocular injection. The RPE-choroid contained between 10 and 14% of the ascorbate, with 35-40% present in the sclera. Retinal ascorbate levels remained high in the injected eyes following 2.5 hrs of darkness, but decreased as a result of intense light treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rod outer segment lipid-opsin ratios in the developing normal and retinal dystrophic rat

The outer segment membrane lipid and opsin contents were determined in photoreceptor cell rods isolated from the eyes of developing normal rats reared in cyclic light or dark environments and dark-reared dystrophic rats. In cyclic light-reared normals rhodopsin/eye increased 49% during the period 20–60 days. Total ROS lipid content, a measure of ROS length, increased 50% while the polyunsaturated fatty acid docosahexaenoate increased from 42–51 mol % during the same period. The phospholipid/opsin ratio of cyclic light reared rat ROS membranes was 67 mol/mol at 20 days and 68 mol/mol at 60 days. In young dark-reared normals the phospholipid/opsin ratio was the same as for cyclic light-reared rats. Although 60 day-old dark-reared normals had 30% more rhodopsin/eye than their cyclic light-reared counterparts, non-significant changes in ROS length (14% longer) and in the phospholipid/opsin ratio (8% lower) were measured in these rats. In addition, light deprivation had no significant effect on the concentrations of polyunsaturated fatty acids or the lipid composition of the isolated ROS. The eyes of dark-reared rats with retinal dystrophy accumulated two times more rhodopsin than dark-reared normals during the 20–60-day period. The phospholipid/opsin ratio of mutant rat ROS was only 7% lower than dark normal at 20 days and 13% lower at 34 days. However, by 60 days of age, the phospholipid/opsin ratio in dystrophic rat ROS was significantly lower than in ROS from either cyclic light-or dark-reared normals. Docosahexaenoic acid in mutant rat ROS lipids averaged 40 mol% during the developmental period. These levels were significantly lower than the levels of docosahexaenoate measured in dark normals at both the 34- and 64-day periods. The glycerophospholipid composition of dystrophic rat ROS was the same as normal at all ages but the cholesterol/phospholipid ratio was higher than in normals.The data show: (1) that the retina accomodates changes in rhodopsin content induced by environmental light, age and genetic differences by alterations in ROS opsin density and length: (2) that the content of ROS membrane polyunsaturated fatty acids (fluidity) increases during development in normals but not in dystrophic rats. The data also suggest that basal membrane synthesis and/or post sythetic membrane modification of ROS lipid are impaired as a function of age in dystrophic rats.     

Adaptive changes in visual cell transduction protein levels: effect of light.

Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protective effect of ascorbate in retinal light damage of rats

Cyclic light and dark-reared rats were exposed to intense visible light for various periods and then rhodopsin-measured following recovery in darkness for up to 14 days. Animals were injected with ascorbic acid or ascorbate derivatives at various doses prior to light exposure in green Plexiglas chambers. The results show that ascorbic acid administration elevates retinal ascorbate and reduces the loss of rhodopsin and photoreceptor cell nuclei resulting from intense light. When given in comparable doses, L-ascorbic acid, sodium ascorbate, and dehydroascorbate were equally effective in preserving rhodopsin. The ascorbate protective effect in the retina is also dose dependent in both cyclic light and dark-reared rats and exhibits a requirement for the L-stereoisomer of the vitamin. Ascorbic acid is effective when administered before, but not after, light exposure, suggesting that protection from light damage in the retina occurs during the light period. In some experiments, rod outer segments were isolated from rats immediately after light exposure, lipids extracted, and fatty acid composition determined. As judged by the preservation of rod outer segment docosahexaenoic acid in rats given ascorbate, the vitamin may act in an antioxidative fashion by inhibiting oxidation of membrane lipids during intense light.     

Light-induced exacerbation of retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome

Potentiation of retinal degeneration by intense light exposure, and its amelioration by an antioxidant, were studied in a rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison with normal (control) Sprague-Dawley rats. The SLOS model is created by treating rats with AY9944, a selective inhibitor of cholesterol synthesis at the level of 3beta-hydroxysterol-Delta7-reductase. A subset of rats was treated with dimethylthiourea (DMTU), a synthetic antioxidant, 24 and 1 hr prior to light exposure. Half of the animals (+/-DMTU) were exposed to intense, constant, green light (24hr, 1700lx, 490-580 nm), while the others were maintained in darkness. Subsequently all animals were returned to dim cyclic light (20-40 lx, 12 hr light-12 hr dark) for 2 weeks, after which electroretinograms were recorded. One eye from each rat was taken for histological and quantitative morphometric analyses; sterol analysis was performed on retinas from contralateral eyes. HPLC analysis confirmed the accumulation of 7-dehydrocholesterol (7DHC) in retinas of AY9944-treated rats; cholesterol represented >99% of the sterol in control retinas. Histology of retinas from unexposed, AY9944-treated rats (6-week-old) was normal. In contrast, age-matched, light-exposed rats exhibited massive photoreceptor cell loss in both the superior and inferior hemispheres, and concomitant rod and cone dysfunction. The severity and geographic extent of the damage was far greater than that observed in normal albino rats exposed to the same conditions. DMTU pre-treatment largely prevented these degenerative changes. These findings indicate that the AY9944-induced rat SLOS model is hypersensitive to intense light-induced retinal damage, relative to normal rats. DMTU protection against light-induced damage implicates free radical-based oxidation in the retinal degeneration process. Furthermore, the use of green light (corresponding to the absorption maxima of rhodopsin) implicates rhodopsin in the initiation of the pathobiological mechanism. We propose that generation of cytotoxic oxysterols (by-products of 7DHC oxidation) is an integral part of retinal cell death in the SLOS rat model, which is exacerbated by intense light. Furthermore, the results predict light-dependent potentiation of retinal degeneration in SLOS patients, and the possible ameliorative effects of antioxidant therapy.     

Inhibition of cytochrome oxidase and blue-light damage in rat retina

The activity of cytochrome oxidase, outer nuclear layer thickness, and edema were quantitatively evaluated in the blue-light exposed rat retina. Dark-adapted or cyclic-light reared rats were exposed to blue light with a retinal dose of 380 kJ/m². Immediately, 1, 2, and 3 day(s) after exposure, the retinas of six rats from each adaptation group were examined. There was no difference between the dark-adapted and cyclic-light reared rats. Immediately after light exposure, cytochrome oxidase activity decreased. The activity in the inner segments remained low at day 1, while severe edema was observed in the inner and outer segments. The outer nuclear layer thickness decreased 1–3 days after exposure. The blue-light exposure inhibited cytochrome oxidase activity and caused retinal injury. Similarity of the injury process in the dark-adapted and cyclic-light reared retinas suggests that rhodopsin was not involved. The inhibition of cytochrome oxidase could be a cause of retinal damage.     

Retinal light damage in rats exposed to intermittent light. Comparison with continuous light exposure

Visible light-induced photoreceptor cell damage resulting from exposure to multiple intermittent light-dark periods was compared with damage resulting from continuous light in albino rats maintained in a weak cyclic-light environment or in darkness before light treatment. The time course of retinal damage was determined by correlative measurements of rhodopsin and visual cell DNA at various times after light exposure, and by histopathological evaluation. The effect of intense light exposures on rhodopsin regeneration and on the level of rod outer segment docosahexaenoic acid was also determined. For rats previously maintained in weak cyclic light, 50% visual cell loss was measured 2 weeks after 12 1 hr light/2 hr dark periods, or following 24 hr of continuous light. A comparable 50% loss of visual cells was found in dark-reared rats after only 5 hr of continuous illumination or 2-3 hr of intermittent light. As judged by histology, cyclic-light-reared rats incurred less retinal pigment epithelial cell damage than dark-reared animals. In both experimental rat models intermittent light exposure resulted in greater visual cell damage than continuous exposure. Visual cell damage from intermittent light was found to depend on the duration of light exposure and on the number of light doses administered. Measurements of rhodopsin and DNA 2 hr and 2 weeks after light exposure of up to 8 hr duration revealed that visual cell loss occurs largely during the 2 week dark period following light treatment. The loss of docosahexaenoic acid from rod outer segments was also greater in rats exposed to intermittent light than in animals treated with continuous light. It is concluded that intermittent light exposure exacerbates Type I light damage in rats (involving the retina and retinal pigment epithelium) and the schedule of intense light exposure is a determinant of visual cell death.     

Lighting conditions and retinal development in goldfish: photoreceptor number and structure

The retinas of 63 goldfish were examined after varying durations of exposure to one of three environmental lighting conditions beginning before hatching: constant light (340 lux), cyclic light (12 hr 320 lux, 12 hr dark) and constant dark. Up to 8 months, no effects of constant light or dark on photoreceptor numbers or structure were apparent. Densities of rod and cone nuclei were normal and all retinal layers appeared normal by light microscopy. Exposure to constant light for 12 months or longer resulted in a reduction in rod density by 37%. Cone numbers were unaffected by constant light, even with exposures of 3 yr, and rod and cone outer segments were normal in length at 11-20 months under all environmental conditions. Due to poor survival, only one animal was available for quantitative examination from the group reared in constant dark 12 months or longer. Photoreceptor size and number in this retina were similar to those in the constant light condition. The results suggest that the formation and maturation of rods and cones in goldfish retina is unaffected by rearing in constant light. However, long-term exposure (greater than or equal to 12 months) may disrupt maintenance of differentiated rods.     

Effect of early and late light exposure on the inherited retinal degeneration in rats

The inherited retinal dystrophy of RCS rats is slowed by maintaining the animals from birth in total darkness. Adult dystrophic rats raised in darkness from birth show nearly four times the ERG amplitude of animals kept in cyclic light conditions. Light deprivation, however, need not begin at birth for the full benefit of the treatment to occur. Rats reared in cyclic light and then transferred to darkness at 15–29 days of age show ERGs in adult-hood equal to animals raised in darkness from birth.Early dark rearing for as long as 45 days does not protect the retina from effects of subsequent light exposure as measured by ERG amplitude.Thus, early rearing conditions neither permanently damage nor permanently protect the dystrophic retina of the RCS rat.     

The protective effect of ascorbic acid in retinal light damage of rats exposed to intermittent light

Retinal light damage in dark-reared rats supplemented with ascorbic acid and exposed to multiple doses of intermittent light was studied and compared with damage in unsupplemented dark-reared and cyclic-light-reared rats. The extent of photoreceptor cell loss from intense light exposure was determined by whole-eye rhodopsin levels and retinal DNA measurements two weeks after light treatment. Two weeks after 3 or 8 hr of intermittent light, ascorbate-supplemented animals had rhodopsin and retinal DNA levels that were two to three times higher than in unsupplemented dark-reared rats. In both types of rats rhodopsin levels were influenced by the number of light doses, the duration of light exposure, and to a lesser extent, by the length of the dark period between exposures. Rhodopsin levels in the dark-reared ascorbate-supplemented rats were significantly higher than in unsupplemented dark-reared rats, and were similar to the levels in unsupplemented cyclic-light-reared animals. Ascorbate treatment had no effect on the rate of rhodopsin bleaching. However, regeneration was greater in supplemented rats after multiple 1-hr light exposures. Intermittent light also resulted in lower ascorbate levels in the retinas of supplemented and unsupplemented rats, with dramatic losses from the retinal pigment epithelium (RPE)-choroid in both types of animals. We conclude that ascorbic acid protects the eye by reducing the irreversible Type I form of light damage in dark-reared rats. Ascorbate appears to shift light damage to the Type II form typical of cyclic-light-reared animals.     

The enzymatic estimation of organic hydroperoxides in the rat retina

An enzymatic procedure for the estimation of organic hydroperoxides has been adapted to biological tissues and applied to the measurement of hydroperoxides in the rat retina. Hydroperoxides are determined from the coupled activities of glutathione peroxidase and glutathione reductase as measured by the loss of NADPH absorbance. To minimize the effects of tissue catalyzed peroxide degradation, incubations were performed in the presence of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); which inhibited the activity of retinal tissue glutathione reductase by 85%. For comparisons to the enzymatic technique, retinal tissue hydroperoxides were also estimated by the absorption of tissue extracts at 232 nm.Using the enzymatic procedure the hydroperoxide concentration in whole retina homogenates was significantly higher in 19-day-old rats than in either 35-day or adult animals. Hydroperoxides in the retina of young rats exposed to light for one hour were significantly lower than in non-exposed controls, while in adult rats, following light, hydroperoxides increased 13%. Fractionation of rat retinas into crude ROS and retina minus ROS components revealed that the ROS fractions contain at least twice the hydroperoxide concentration of the remaining retina. The concentration of hydroperoxides in the ROS fractions from dark-reared rats were significantly lower than in cyclic-light-reared animals. In both types of rats, one hour intense light exposure resulted in an increase in ROS hydroperoxides but the increases were not significant. ROS hydroperoxides were also found to be 85–90% water soluble. Estimates of the retinal hydroperoxide content obtained by absorption at 232 nm gave similar results to the enzymatic technique, but the levels were significantly lower. When retinas were maintained in vitro for one hour before analysis, hydroperoxides determined by either technique were significantly higher than in retinas assayed immediately, but A₂₃₂ hydroperoxides were still significantly lower than hydroperoxides measured by the enzymatic procedure.It is concluded: (1) that the observed retinal hydroperoxide concentration depends upon animal age and the method of measurement; (2) that within the retina the photoreceptor cell contains at least a two-fold higher concentration of hydroperoxides than the remaining retina and that prior light history can affect those hydroperoxide levels (it appears that the photoreceptor cell is also a major site of hydroperoxide formation in the retina); (3) that during intense light exposure of short duration significant levels of hydroperoxides do not accumulate in the retinas of rats.     

Rhodopsin content and rod outer segment length in albino rat eyes: Modification by dark adaptation

Whole eye rhodopsin content and rod outer segment length have been determined in albino rat eyes after different periods of dark adaptation, dark-rearing or cyclic light maintenance. The rhodopsin content in the eyes of dark-reared albino rats is approximately 50% higher than that in the eyes of littermates reared in cyclic light (in-cage illumination less than 15 ft-cd). The same increase in rhodopsin can be obtained in albino rats after only a 10-day dark adaptation period. Furthermore, the increased level of rhodopsin is the same as that in the eyes of pigmented rats reared in cyclic light. The increase in rhodopsin content in the albino rat eyes is due at least in part to an increase in rod outer segment length. The increase in length with dark adaptation and dark-rearing was somewhat variable, with an average increase of approximately 25%. No apparent increase was found in rod outer segment diameter, rod outer segment disc packing density or eye size. Rod outer segment lengths were consistently longer in the superior hemisphere of the eye than in the inferior hemisphere in both dark-adapted albino rats and pigmented rats maintained in cyclic light, but not in albino rats maintained in cyclic light.     

Photoreceptor cell damage by light in young Royal College of Surgeons rats.

PURPOSE. To determine the effects of genetic background and light rearing conditions on intense-light-mediated retinal degeneration in young RCS rats. MATERIALS AND METHODS. Albino rats, homozygous or heterozygous for the rdy gene were bred and born in dim cyclic light. At P7 they were moved to a dark environment, and maintained there until exposure to intense visible (green) light at P18 or P25. Other rats remained in the dim cyclic light environment. At various times between P11 and P40 rats were killed for determinations of rhodopsin and photoreceptor cell DNA levels, western transblot analysis of retinal S-antigen (arrestin) and alpha-transducin, or northern slot blot analysis of their respective mRNA levels. RESULTS. At P18, unexposed dark maintained homozygous RCS rats and their phenotypically normal heterozygous counterparts have nearly equivalent rhodopsin levels and photoreceptor cell DNA. Intense light exposure at this age, to 8 hours of continuous light or 3 hours of intermittent light, did not lead to a loss of either rhodopsin or retinal DNA when compared with their respective unexposed controls. At P25 rhodopsin levels were higher than at P18, while photoreceptor cell DNA was essentially the same as in the younger rats. However, intense light exposure at P25 resulted in substantial losses of rhodopsin and photorecptor cell DNA and the losses were greater in homozygous rats than in heterozygous animals. Light damage of P25 rats maintained in dim cyclic light was essentially the same as in dark maintained homozygous rats, but no damage was found in the heterozygous animals. By western analysis, alpha-transducin levels in the retina increased with time in darkness, while retinal S-antigen levels either remained the same or decreased during the period P15-P35. For rats in the cyclic light environment S-antigen expression was greater than alpha-transducin at all ages. Slot blot analysis of mRNAs for the two proteins generally followed the patterns seen by western analysis. S-antigen mRNA was expressed at an earlier age and at higher levels than alpha-transducin in both types of rats from both light rearing conditions. Peak expression of S-antigen most often occurred at P18 in both the heterozygous and homozygous rats. CONCLUSIONS. The relative expressions of S-antigen and alpha-transducin in P18 and P25 rats correlates with their relative resistance to retinal light damage at P18 and their enhanced susceptibility at P25. Rats homozygous for the rdy gene also exhibit more damage than heterozygous animals when photoreceptor cell DNA is used to estimate the extent of retinal light damage.     

Increased susceptibility to light damage in an arrestin knockout mouse model of Oguchi disease (stationary night blindness)

PURPOSE: To determine whether constitutive signal flow arising from defective rhodopsin shut-off causes photoreceptor cell death in arrestin knockout mice. METHODS: The retinas of cyclic-light-reared, pigmented arrestin knockout mice and wild-type littermate control mice were examined histologically for photoreceptor cell loss from 100 days to 1 year of age. In separate experiments, to determine whether constant light would accelerate the degeneration in arrestin knockout mice, these animals and wild-type control mice were exposed for 1, 2, or 3 weeks to fluorescent light at an intensity of 115 to 150 fc. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal. RESULTS: In arrestin knockout mice maintained in cyclic light, photoreceptor loss was evident at 100 days of age, and it became progressively more severe, with less than 50% of photoreceptors surviving at 1 year of age. The photoreceptor degeneration appeared to be caused by light, because when these mice were reared in the dark, the retinal structure was indistinguishable from normal. When exposed to constant light, the retinas of wild-type pigmented mice showed no light-induced damage, regardless of exposure duration. By contrast, the retinas of arrestin knockout mice showed rapid degeneration in constant light, with a loss of 30% of photoreceptors after 1 week of exposure and greater than 60% after 3 weeks of exposure. CONCLUSIONS: The results indicate that constitutive signal flow due to arrestin knockout leads to photoreceptor degeneration. Excessive light accelerates the cell death process in pigmented arrestin knockout mice. Human patients with naturally occurring mutations that lead to nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form of stationary night blindness. The present findings suggest that such patients may be at greater risk of the damaging effects of light than those with other forms of retinal degeneration, and they provide an impetus to restrict excessive light exposure as a protective measure in patients with constitutive signal flow in phototransduction.     

Prevention of cataracts in pink-eyed RCS rats by dark rearing

Royal College of Surgeons rats have hereditary retinal degeneration and associated posterior subcapsular opacities (PSO) of the lens, detectable by slitlamp at 7-8 postnatal weeks in both pink- and black-eyed rats. The retinal degeneration is intensified by light, especially in pink-eyed rats. A fourth of pink-eyed rats developed mature cataracts by 9-12 months of age, but black-eyed rats whose retinas are protected from light by pigmented irises and pigment epithelium rarely have mature cataracts (3% or less), indicating light may be a factor in cataractogenesis. Prior work had shown that dark rearing reduced the rate of retinal degeneration in pink- but not black-eyed rats, but cataracts were not studied. In the present work, pregnant pink-eyed females were placed in a darkroom 1 week before parturition. Pups were removed over intervals at 20-85 postnatal days for: (a) microscopic study of fresh lenses and of fixed, stained retina and lens, and (b) counts of cells mm-2 of the web-like vitreous cortex after it had been dissected free. The macrophage-like cells are a quantitative index of immune reaction to retinal damage. At 50-53 postnatal days, in pink-eyed cyclic light reared RCS, the mean number of macrophages was 4.6-fold that in congenic controls, but in those that were dark reared it was only 1.4-fold. This was less than the increase in cyclic light reared black-eyed RCS (2.3-fold that in congenic black-eyed controls). Total absence of light reduced retinal degeneration and the number of macrophages, and prevented PSO detectable microscopically.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of eye pigmentation and light deprivation on inherited retinal dystrophy in the rat.

The influence of eye pigmentation and light deprivation on inherited retinal dystrophy has been studied in Royal College of Surgeons (RCS) rats which are pink-eyed and in two congenic strains, RCS-p/+, which are black-eyed and RCS-c, which are albinos. The congenic animals are genetically similar to inbred RCS rats, differing only in pigmentation genes and other genes closely linked to the pigmentation loci. Progression of the disease has been analyzed in a series of animals cytologically with 1–2 μm plastic sections and biochemically by measurement of whole eye extractions of rhodopsin. When the rats are reared in cyclic light (12 hr light-12 hr dark; cage illumination less than 15 ft-c), the rate of photoreceptor degeneration in black-eyed rats is slowed from the rate in pink-eyed rats by about 10 days in the posterior retina. In the far peripheral retina, the disease is slowed by about 30–35 days in the superior half of the eye, along and above the horizontal meridian. No slowing occurs in the inferior half of the eye along the vertical meridian. When pink-eyed RCS and black-eyed RCS-p/+ rats are dark-reared, the pattern of degeneration in both is the same as in black-eyed rats reared in cyclic light. The rhodopsin content of eyes from black-eyed RCS-p/+ rats reared in cyclic light also is the same as that in pink-eyed rats reared in darkness. No difference was found between pink-eyed RCS and albino RCS-c retinas in the rate of the disease or in rhodopsin content. These findings indicate that (1) intrinsic differences exist in different regions of the retina in the rate of retinal dystrophy, (2) black eye pigment slows the progression of the disease as much as does dark-rearing in pink-eyed rats, (3) the very small amount of eye pigment in pink-eyed RCS rats is ineffectual in slowing the rate of the disease from that in albino RCS-c rats, and (4) dark-rearing does not slow the rate of the disease further in black-eyed rats.Additional features of retinal dystrophy in the RCS rat were observed. Some photoreceptor cells survive in clusters immediately adjacent to the optic nerve head and the ora serrata as late as day 96, long after most photoreceptors have disappeared. The rod outer segment debris (extra lamellar material), which is a characteristic of retinal dystrophy in the rat, shows a loss of basophilia and osmiophilia (“blanching”) beginning at days 32–35 in the apical region of the debris in the posterior retina. The debris becomes progressively more “blanched” until about day 96, when most of the debris has lost its basophilia in all regions of the eye; the “blanching” of the membranes correlates closely with the loss of rhodopsin from the eye. The issue of the source of the extra lamellar material is re-examined, and data are provided that indicate the material probably is formed entirely from the breakdown of rod outer segments.