Transgenic Bcl-2 expressed in photoreceptor cells confers both death-sparing and death-inducing effects.

To examine its potential role within the retina as a modulator of cell death and photoreceptor degeneration, bcl-2 expression was targeted to the photoreceptors of transgenic mice by the human IRBP promoter. Three transgenic families were established, with levels of transgene expression between 0.2 and two-fold relative to that of endogenous bcl-2. The effect of bcl-2 expression on genetically programmed photoreceptor degeneration was evaluated by crossing these transgenic mice with mice that develop a rapid degeneration of rod photoreceptors due to expression of a distinct transgene, SV40 T antigen (Tag). Transgenic Bcl-2 was localized to photoreceptor inner segments and was capable of abrogating the activation of caspase activity and the resulting cell death associated with ectopic expression of Tag. However, Bcl-2 itself ultimately caused photoreceptor cell death and retinal degeneration. Several proteins not expressed normally in Tag or other transgenic retinas undergoing photoreceptor degeneration were induced in the Bcl-2 transgenic retinas. Analysis by mass spectroscopy identified one of these proteins as alphaA-crystallin, a member of a protein family that associates with cellular stress. Since Bcl-2 can promote as well as spare cell death in the same photoreceptor population, its potential utility in ameliorating photoreceptor death in human hereditary blinding disorders is compromised.

Age-related changes in the mouse outer retina.

PURPOSE: To define the physiological and structural changes that may accompany aging in the normal mouse retina. METHODS: C57BL/6 mice were maintained under cyclic light for either 2, 6, or 12 months. After rod- and cone-mediated corneal electroretinograms (ERG's) were recorded from anesthetized animals, the retinal structure was quantitatively examined. Photoreceptor cell density was measured within 100-microm regions of the central superior and inferior retina. Cone photoreceptor subtypes were identified by immunocytochemistry. RESULTS: The amplitudes of rod- and cone-mediated ERG's were reduced in older mice, although the overall ERG wave-form did not change appreciably and implicit times were not changed in an age-dependent fashion. In comparison, there was no significant age-related decline in rod or cone photoreceptor density. CONCLUSIONS: The amplitude of the mouse ERG declines with age. This change does not appear to reflect a change in the structural integrity of the photoreceptor cells. In functional studies of murine models of late-onset retinal disorders, it will be important to take these changes into consideration.

Retinal degeneration in the nervous mutant mouse. IV. Inner retinal changes.

We have recently noted that the inner nuclear layer (INL) and the inner plexiform layer (IPL) were significantly thinner in mice homozygous for the nervous defect (nr / nr) than in control (nr /+ or +/+) littermates. Here, we have carried out a series of anatomical studies to further understand these inner retinal changes. At postnatal day (P) 13, there was no difference in the inner retina between nervous and control mice, while a significant difference was observed at P30. Similar changes were not seen in other mouse models of photoreceptor degeneration. There was a significant reduction in the density of cells in the INL that were stained by antibodies against the inhibitory neurotransmitters GABA and glycine. These results indicate that the nervous defect causes a degeneration of one or more sub-types of amacrine cells, in addition to the loss of cerebellar Purkinje cells and retinal photoreceptors that is known to occur in these mutant animals. Finally, evidence is provided that photoreceptors die by an apoptotic pathway in nervous mice.

The relationship between opsin overexpression and photoreceptor degeneration.

PURPOSE: To characterize the process by which overexpression of normal opsin leads to photoreceptor degeneration. METHODS: Three transgenic mouse lines were generated that express different levels of an opsin with three amino acid modifications at the C terminus. These modifications created an epitopic site that can be readily distinguished from the endogenous protein using a bovine opsin-specific antibody. Evidence of degeneration associated with opsin overexpression was provided by anatomic studies and electroretinogram (ERG) recordings. Western blot analysis was used to confirm the production of the transgenic opsin, and an enzyme-linked immunosorbent assay (ELISA) was used to determine the amounts of opsin overexpressed in each line. Immunocytochemistry was used to determine the cellular localization of transgenic opsin. Amounts of 11-cis retinal were determined by extraction and high-performance liquid chromatography (HPLC). RESULTS: Opsin expression levels in the three lines were found to be 123%, 169%, and 222% of the level measured in nontransgenic animals, providing direct correlation between the level of transgene expression and the severity of the degenerative phenotype. In the lower expressing lines, ERG a-wave amplitudes were reduced to less than approximately 30% and 15% of normal values, whereas responses of the highest expressing line were indistinguishable from noise. In the lowest expressor, a 26% elevation in 11-cis retinal was observed, whereas in the medium and the high expressors, 11-cis retinal levels were increased by only 30% to 33%, well below the 69% and 122% increases in opsin levels. CONCLUSIONS: The overexpression of normal opsin induces photoreceptor degeneration that is similar to that seen in many mouse models of retinitis pigmentosa. This degeneration can be induced by opsin levels that exceed by only approximately 23% that of the normal mouse retina. Opsin overexpression has potential implications in retinitis pigmentosa.

Ambient hypoxia reverses retinal vascular attenuation in a transgenic mouse model of autosomal dominant retinitis pigmentosa.

PURPOSE: Loss of retinal capillaries is an inherent component of late stage autosomal dominant retinitis pigmentosa (ADRP). This study examined the hypothetical role of tissue hyperoxia in this vascular attenuation process and tested the potential of ambient hypoxia to reverse it. METHODS: Transgenic mice expressing a mutant opsin gene with a 3-bp deletion of isoleucine at codon 255/256 were used. This model is characterized by early onset of a rapidly progressing retinal degeneration that by postnatal day (P)20 results in the loss of all but one row of photoreceptor nuclei. At P20 some mice were placed in 12% oxygen until they were euthanatized at P26. The remainder were maintained in normoxia and killed at the same age. Retinas were dissected, stained for ADPase, and flat-mounted. RESULTS: Deep plexus capillary density was significantly different in normoxic normals versus transgenics at 20 days of age (P:

Retinoid kinetics in eye tissues of VPP transgenic mice and their normal littermates.

PURPOSE: VPP mice, which possess a mutant transgene for opsin (V20G, P23H, P27L), exhibit a progressive rod degeneration that resembles one form of human autosomal dominant retinitis pigmentosa. In the present study the association of the development of VPP rod degeneration with abnormal operation of the retinoid visual cycle was examined. METHODS: Dark-adapted VPP mice and normal littermates were anesthetized and the pupils dilated. One eye of each animal was illuminated for 2 minutes; the other eye was shielded from the light and served as a control. Each animal was then dark adapted for a defined period (0-300 minutes) and killed. Retinoids contained in the retina, retinal pigment epithelium (RPE), and extracellular medium were recovered by means of formaldehyde-, isopropanol- and ethanol-based extractions and analyzed by high-performance liquid chromatography. RESULTS: Total amounts of retinoid recovered from unilluminated eyes of 2-month-old normal and VPP mice were 425 +/- 90 picomoles per eye and 115 +/- 33 picomoles per eye, respectively (mean +/- SD). Relative distributions of retinoids within normal and VPP eyes were similar. In normal and VPP animals, illumination for 2 minutes produced a similar immediate reduction in the molar percent of total retinoid represented by 11-cis retinal in the retina (average reduction of 34% and 28% in normal and VPP animals, respectively) and a similar transient increase of all-trans retinal in the retina. In both groups the decline of all-trans retinal was accompanied by an increase in total retinyl ester. In normal and VPP animals, a period of approximately 40 minutes or more preceded initiation of the recovery of 11-cis retinal in the retina, and the time course of this recovery was generally similar to that for the decline of retinyl ester. The overall dark-adaptation period required for half-completion of 11-cis retinal recovery was approximately 150 minutes. In neither group did illumination produce a substantial peak of all-trans retinol in the retina. CONCLUSIONS: The evident approximately fourfold reduction of total retinoid in the eyes of 2-month-old VPP mice is consistent with histologic and electroretinographic abnormalities determined in previous studies. Despite this marked abnormality in retinoid content, retinoid cycling in the VPP is remarkably similar to that in normal littermates. The data place constraints on the functional consequences of any abnormality in retinoid processing that may be present at this stage of the VPP rod degeneration.

Opsin localization and rhodopsin photochemistry in a transgenic mouse model of retinitis pigmentosa.

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11-cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

The effect of peripherin/rds haploinsufficiency on rod and cone photoreceptors.

Haploinsufficiency because of a null mutation in the gene encoding peripherin/rds has been thought to be the primary defect associated with the photoreceptor degeneration seen in the retinal degeneration slow (rds) mouse. We have compared the effects of this haploinsufficiency on rod and cone photoreceptors by measuring the levels of rod- and cone-specific gene expression, by determining the relative rates of rod and cone degeneration, and by electroretinography. These analyses were performed at ages before and after the onset of degeneration of the photoreceptor cells. The data were consistent in demonstrating that measures for cone photoreceptors are relatively spared in comparison to comparable measures for rod photoreceptors. Blue cones were retained in higher number than red/green cones for the first 3 months of the degeneration. Our results indicate that the haploinsufficiency present in rds/+ mice has a greater impact on the rod than on the cone photoreceptor, a finding that likely reflects the tight regulation of peripherin/rds and the need for two functional alleles to assemble the structure of the rod outer segment and/or differences between the ultrastructure of the rod and cone outer segments.

Light exposure induces ubiquitin conjugation and degradation activities in the rat retina.

PURPOSE: To evaluate the consequences of light exposure on retinal ubiquitin (Ub) conjugation and degradation. METHODS: Two-month-old Long Evans pigmented rats were exposed to constant light (180 foot-candles) or were left in complete darkness for 18 hours. Rats used for cyclic light and diurnal rhythm experiments were removed from their light cycles at different times (24-hour clock): 0700 (before the light was turned on), 1000 (3 hours into the light cycle), 1000D (continued in the dark cycle), 1900 (before the light was turned off), 2200 (3 hours into the dark cycle), and 2200L (continued in the light cycle). The retinas were examined for Ub conjugation, adenosine triphosphate-Ub-dependent degradation, levels of Ub messenger RNA, and localization of Ub immunocytochemistry. RESULTS: There was a statistically significant increase in Ub conjugation and degradation in retinas isolated from light-exposed animals compared with degradation in retinas of dark-adapted animals. However, no significant differences were observed in the levels of Ub messenger RNA from cyclic light, or light-exposed or dark-adapted retinas, suggesting that light-stress-induced changes do not reflect increased transcriptional activity. The daily variations observed in Ub conjugation and degradation suggest that these processes are probably the result of a circadian rhythm. Results of immunohistochemical studies revealed that Ub and its conjugates were uniformly distributed throughout the retinal cell layers in light- and dark-adapted retinas. However, in light-exposed retinas, a strong positive immunoreactivity was observed in the inner retina, specifically in horizontal and ganglion cells. CONCLUSIONS: These results suggest that light exposure may play a role in inducing Ub-conjugating activity in certain retinal cells. Furthermore, the results support the hypothesis that Ub is a stress protein that plays an important role in protecting cells under stress conditions.

Structural and developmental analysis of the mouse peripherin/rds gene.

Mutations in the peripherin/rds gene have been reported to be associated with different forms of human autosomal dominant retinitis pigmentosa (ADRP) and macular degeneration (MD). To better understand the disruptive role of these mutations, knowledge of the structure-function relationship of the peripherin/rds gene is needed. To facilitate that, genomic clones encoding the mouse gene were isolated using bovine cDNA sequences as probes. Sequence analysis of clone lambda 6-1-1, that contained the entire coding sequence for the mouse peripherin/rds, yielded the exon-intron organization of the gene. The gene is composed of three exons (581, 247, and 213 bp) and two introns with the first and second introns 8.6 kb and 3.7 kb in size, respectively. Two major (1.6 and 2.7 kb) and three minor (4.0, 5.5, 6.5 kb) transcripts were detected on RNA blots. The major transcripts first appeared in the brain at embryonic day 13 and in the retina at postnatal day 1. Transcripts were missing in brain and eye of mice at embryonic day 15. Several transcription start sites were mapped within 26 nucleotides approximately 200 bp upstream from the translation initiation site. However, transcripts varied in the lengths of their 3' untranslated portion as a result of the utilization of different polyadenylation signals.

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.

Defective phototransductive disk membrane morphogenesis in transgenic mice expressing opsin with a mutated N-terminal domain.

Retinitis pigmentosa is a heterogeneous group of inherited retinal disorders in which the photoreceptor cells degenerate. A line of transgenic mice expresses a mutant opsin gene that encodes three missense mutations near the amino terminus, including P23H, which is the basis for a common form of dominant retinitis pigmentosa. By studying the photoreceptor cells of these mice and their normal littermates, we found that: (1) opsin was routed correctly, (2) the concentration of opsin in the disk membranes appeared normal by freeze fracture analysis, (3) the amount of disk membrane shedding was normal, but (4) the basal disks of the outer segments were disorganized, indicating defective disk membrane morphogenesis. Defective disk membrane morphogenesis appears to result in the formation of fewer mature disks, thus accounting for observed gradual shortening of the photoreceptor outer segments with age. We suggest that abnormal disk membrane morphogenesis is the primary cellular defect that leads to blindness, and that it arises from the inability of nascent disk membranes, containing normal and mutant opsin, to interact normally with each other.

Polygenic disease and retinitis pigmentosa: albinism exacerbates photoreceptor degeneration induced by the expression of a mutant opsin in transgenic mice.

Expression of a mouse opsin transgene containing three point mutations (V20G, P23H, and P27L; termed VPP) causes a progressive photoreceptor degeneration that resembles in many important respects that seen in patients with autosomal dominant retinitis pigmentosa caused by a P23H point mutation. We have attempted to determine whether the degree of degeneration induced by expression of the transgene is influenced by albinism, a genetically mediated recessive trait that results in a deficiency in melanin formation in pigmented tissues throughout the body. Litters of albino and pigmented mice (normal as well as transgenic) were reared in either darkness or cyclic light. Retinal structure and function were evaluated by light microscopy, electroretinography (ERG), and retinal densitometry. The data were consistent in demonstrating that at similar ages, the extent of photoreceptor degeneration was greater in transgenic albino animals than in their pigmented counterparts. The albino VPP mice had significantly fewer cell bodies in the outer nuclear layer of the retina, a larger reduction in ERG amplitude, and a lower rhodopsin content in the rod photoreceptors. These structural and functional differences could not be attributed to the greater level of retinal illumination experienced by the albino retina under normal ambient conditions, because they persisted when pigmented and albino mice were reared in darkness from birth. Although the explanation remains unclear, our findings indicate that the rate of photoreceptor degeneration in VPP mice is adversely affected by the existence of the albino phenotype, a factor that may have implications for the counseling of human patients with retinitis pigmentosa and a familial history of other genetic disorders.

Rod phototransduction in transgenic mice expressing a mutant opsin gene.

Rod-mediated electroretinograms (ERG's) were recorded from transgenic mice expressing a mouse opsin gene with three point mutations (V20G, P23H, and P27L; termed VPP mice) and from normal littermates. The leading edge of the alpha wave was analyzed in relation to a computational model of rod phototransduction [J. Physiol. 499, 719 (1992)], in which values for the maximum response (RmP3), transduction gain (S), and transduction delay (td) are derived from alpha-wave data. VPP mice exhibited an age-related decrease in RmP3. This decrease was consistent with reductions in the number of rod photoreceptors and in the length of rod outer segments observed in previous histological studies of the VPP retina. Values of S determined for the VPP mice were within the normal range, consistent with a normal amplification of the visual signal in VPP rods. At high stimulus intensities, both normal and VPP mice exhibited a decrease in S, which may reflect depletion of a phototransduction substrate at these stimulus levels. We examined the recovery of the alpha wave after a bright conditioning flash by measuring the rod alpha-wave response to a probe flash presented at varying times after the conditioning stimulus. In both normal and VPP mice a fourfold (0.6-log-unit) increase in conditioning stimulus intensity increased both T50%, the period required for half-maximal recovery, and tau, the exponential time constant describing recovery. However, the increases in T50% and tau were significantly greater in VPP mice, indicating an abnormally slow recovery of the flash response in VPP rods.

Functional abnormalities in transgenic mice expressing a mutant rhodopsin gene.

PURPOSE: To evaluate the consequences of the expression of a mutant mouse opsin gene on rod- and cone-mediated function. Experimental conditions were chosen to provide a basis of comparison to the results reported for patients with autosomal dominant retinitis pigmentosa (ADRP) in whom the proline at position 23 has been replaced by a histidine (P23H). METHODS: The mutated gene product resulted in three substitutions in the rhodopsin molecule: P23H, glycine for valine at position 20 (V20G), and leucine for proline at position 27 (P27L). Mice positive for the transgene were differentiated from normal littermates by the polymerase chain reaction. Electroretinograms (ERGs) were obtained from anesthetized mice between 1 and 9 months of age. After photically bleaching approximately 18% of the available rhodopsin, the time course of rod dark adaptation was examined by monitoring rod ERG amplitude recovery. Rhodopsin densitometry was used to determine the relative amounts of rhodopsin in the retinae of normal and transgenic mice. RESULTS: ERGs obtained from transgenic mice showed a significant reduction in rod-mediated response amplitude at 1 month of age and a relatively slow progressive decrease thereafter. Cone-mediated ERGs, on the other hand, were nearly normal in amplitude for approximately the first 5 months after birth, but at later ages response amplitudes also underwent a progressive decline. In the normal retina, rod ERG amplitudes returned to prebleach levels within 30 minutes, whereas in transgenic mice response amplitudes did not recover within a 2-hour test period. The age-related decline in rod-mediated electroretinal potentials seen in transgenic mice was paralleled by a concomitant fall in rhodopsin density, and the sensitivity losses obtained electroretinographically could be accounted for solely on the basis of reduced quantal absorption. CONCLUSIONS: The pattern of functional changes seen in the transgenic mice are in good agreement with those reported in patients with ADRP with the P23H mutation in the rhodopsin gene. Particularly noteworthy is the fact that the changes in rhodopsin density and visual sensitivity are associated with a progressive shortening of the rod outer segments; the histologic changes induced by the disease process in patients with ADRP have yet to be determined.

Properties of the mouse cone-mediated electroretinogram during light adaptation.

Cone-mediated electroretinograms (ERGs) were obtained from normal mice during the course of light adaptation to a rod-desensitizing adapting field. Responses obtained during the early minutes of light adaptation were smaller in amplitude, and delayed in implicit time in comparison to responses obtained to the same stimulus presented later during light adaptation. These changes resemble those observed in the human cone ERG obtained under similar stimulus conditions, and indicate that the underlying mechanism may be similar. While the use of an adapting field appears to isolate the mouse cone ERG, these adaptation-induced changes should be considered when evaluating this response.

Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene.

Autosomal dominant retinitis pigmentosa (ADRP), slowly progressing over decades, leads to severe visual impairment and in some cases to complete blindness. More than 40 mutations in the human opsin gene have been linked to some forms of this genetically heterogeneous disease. In photoreceptor cells of ADRP patients with mutations in the opsin gene, normal rhodopsin is thought to be synthesized concomitantly with mutated rhodopsin, which, by an unknown mechanism, causes the slow degeneration of the photoreceptor cells. To establish a transgenic mouse line that carries a mutated mouse opsin gene in addition to the endogenous opsin gene, we introduced a mouse opsin gene containing mutations in exon 1 into the germ line of a normal mouse. The alterations consisted of three amino acid substitutions near the N terminus of rhodopsin, Val-20-->Gly (V20G), Pro-23-->His (P23H), and Pro-27-->Leu (P27L). The P23H mutation is the most prevalent mutation in human ADRP patients. During early postnatal development, mice heterozygous for the mutated opsin gene appear to develop normal photoreceptors, but their light-sensitive outer segments never reach normal length. With advancing age, both rod and cone photoreceptors are reduced progressively in number. The slow degeneration of the transgenic retina is associated with a gradual decrease of light-evoked electroretinogram responses. Our results show that simultaneous expression of mutated and normal opsin genes induces a slow degeneration of both rod and cone photoreceptors and that the course of the retinal degeneration of the mutant mouse retina mimics the course of human ADRP.

Oxygen-induced retinopathy in the rat. Vitamins C and E as potential therapies.

Oxygen-induced retinopathy (OIR) was produced by subjecting newborn albino rats to a 60% oxygen atmosphere for 14 days before they were killed and retinal analysis was done. The extent of OIR was measured by estimating the severity of retinal vasoobliteration in ink-perfused flat-mounted retinas. This was done with the aid of a digitizing camera and an image-analysis system designed to create binary images of the retinal blood vessels. Retinal levels of several antioxidant molecules also were measured. Alpha-tocopherol and ascorbic acid were reduced in oxygen-exposed rats by 34% and 20%, respectively, compared with room air-raised control animals. Retinal glutathione reductase, S-transferase, and peroxidase showed no differences between oxygen-treated and -untreated rats. Attempts to increase the newborn rats' retinal ascorbic acid by administering daily subcutaneous injections (5 g/kg body weight) to the mother rats were unsuccessful. However, the level of retinal alpha-tocopherol of newborn rats could be altered by dietary manipulation of the mothers. The mothers were fed diets containing either 1 g alpha-tocopherol acetate/kg food or none, starting 21-25 days before the birth of their litters and lasting throughout the exposure period. This treatment resulted in three- to fourfold differences in the retinal alpha-tocopherol levels of the pups. The combination of dietary and oxygen treatments also resulted in significant differences in retinal glutathione peroxidase activity, with the vitamin E-deprived, oxygen-exposed group having highest levels. Newborn rats both supplemented with and deprived of alpha-tocopherol had less vasoobliteration than did those nursed by mothers fed rat chow.

Photoreceptor physiology in the rat is governed by the light environment.

Albino rats were born and raised in one of three cyclic (12L: 12D) lighting conditions: (1) 5 lux for 9 weeks, (2) 800 lux for 9 weeks, or (3) 800 lux for 9 weeks, followed by 5 lux for 3 weeks (800:5). After the treatment, the following were determined: (i) retinal function as measured by the electroretinogram (ERG); (ii) retinal morphology, including rod outer segment (ROS) length and outer nuclear layer area; (iii) rhodopsin levels in whole retina and ROS preparations; (iv) fatty acid profile of ROS membranes and (v) retinal antioxidant levels. After 9 weeks, rats raised in 800 lux sustained an irreversible loss of photoreceptors which could not be reversed by then placing them in 5 lux. However, these rats displayed significant alterations in all other parameters measured after the 3 weeks in dim cyclic light. ERGs showed a 60% increase of b-wave maximum amplitude in 800:5 rats at 12 weeks over the value at the time of their change to a dim environment, while a-wave amplitude in 800:5 rats increased more than 2.5 times. This increase can be explained by a combination of increased ROS length and increased ROS membrane concentration of rhodopsin during the three weeks in 5 lux. Polyunsaturated fatty acids predominated in the ROS of 5 lux rats and 800:5 rats, but not in 800 lux animals. Measurements of retinal glutathione enzyme activity and vitamin E and C levels were relatively low in 800:5 rats. Some rats from the 800:5 group were exposed to 2000 lux for 24 hr. Retinas of these rats sustained 50% loss of photoreceptors from this exposure. Comparisons are made to previous studies concerning the effect of cyclic light environments on the retinas of albino rats.

Glutathione-dependent enzymes in intact rod outer segments.

We have compared the specific activities of glutathione-dependent enzymes in rod outer segments (ROS) and in whole retina of rabbits and rats, using three different assays. In the first, glutathione peroxidase (GSH-Px) activity was measured as the combined activities of the Se-dependent and Se-independent forms using cumene hydroxide as a substrate. In the second, Se-dependent GSH-Px alone was measured using hydrogen peroxide (H2O2) as a substrate. In the third, the combined activities of several enzymes, collectively known as GSH-S-transferase, were measured. The latter includes the activity of the Se-independent GSH-Px. GSH-Px activity (Se-dependent and Se-independent combined) in ROS of rat and rabbit were found to be 47.7 +/- 8.2 and 72.9 +/- 11.9 nmol of GSH oxidized min-1 mg-1 soluble protein, respectively. From whole retina, values were 67.3 +/- 6.0 and 128.8 +/- 12.3, respectively. Se-dependent GSH-Px specific activities from the above tissues were 43.5 +/- 2.9 (rat ROS). 70.6 +/- 11.3 (rabbit ROS), 30.6 +/- 9.6 (rat whole retina), and 113.2 +/- 12.2 (rabbit whole retina). GSH-S-transferase activity was negligible in rabbit ROS, whereas, in rat ROS, it was 40.4 +/- 8.0, expressed as nmol of S-2,4-dinitrophenylglutathione produced min-1 mg-1 soluble protein. In contrast, the GSH-S-transferase specific activity in whole rabbit retinas was about eight times that found in the rat retina (101.5 +/- 12.3 for rat retina and 885.3 +/- 60.0 for rabbit retina). These results demonstrate that ROS contain glutathione enzymes which are important in protecting membranes from oxidative stresses by reducing hydrogen peroxide and lipid hydroperoxides at the site of their formation.