|Molecular Vision 2002;
Received 28 June 2002 | Accepted 27 August 2002 | Published 5 September 2002
Alterations in retinal rod outer segment fatty acids and light-damage susceptibility in P23H rats
Ina Rea Bicknell,1 Ruth
Darrow,1 Linda Barsalou,1 Steven
J. Fliesler,2 Daniel T.
1Petticrew Research Laboratory, Department of Biochemistry and Molecular Biology, School of Medicine, Wright State University, Dayton, OH, USA; 2Departments of Ophthalmology & Pharmacological and Physiological Sciences, Saint Louis University School of Medicine, St. Louis, MO, USA
Correspondence to: Ina Rea Bicknell, PhD, Wright State University, Cox Institute, 3525 Southern Boulevard, Kettering, OH, 45429; Phone: (937) 298-3399 ext. 54725; FAX: (937) 395-8109; email: email@example.com
Purpose: To determine whether dietary-induced alterations in the long-chain polyunsaturated fatty acid content of retinal rod outer segments (ROS) of P23H rats, a transgenic model of retinitis pigmentosa (RP), prolongs photoreceptor cell life.
Methods: Heterozygous P23H and normal Sprague-Dawley rats were fed a standard house diet or a diet deficient in 18:3n-3. Diet-deficient rats were given supplements of either linseed oil (high in 18:3n-3) or fish oil (high in 20:5n-3). ROS fatty acid profiles and serum fatty acids were determined by gas chromatography. Serum cholesterol was evaluated by HPLC. Retinal damage was assessed by measuring whole-retina rhodopsin and DNA content before and after exposure to high-intensity light.
Results: The retinas of 60 day old, cyclic-light-reared, P23H transgenic rats contained 50% of the rhodopsin and 75% of the DNA content found in control Sprague-Dawley rats. Eight hours of intense light had little effect on the rhodopsin or DNA content in the Sprague-Dawley rats, but resulted in rhodopsin and DNA losses of nearly 70%, compared to controls, in P23H animals fed either a standard or an 18:3n-3-deficient diet. Supplementation with linseed oil resulted in small, statistically insignificant, increases in the rhodopsin and DNA losses, which occurred after exposure to intense light, in P23H transgenics. In unexposed animals, supplementation with linseed oil or fish oil had no effect on either rhodopsin or DNA levels in P23H rats or in Sprague-Dawley controls. On standard diet, the ROS 22:6n-3 (DHA) content in P23H rats was lower than that of control animals. DHA decreased in both groups when an 18:3-deficient diet was fed. The reduction was greater in controls than in P23H transgenics, but a concomitant increase in 22:5n-6 was nearly the same in both groups. Supplementation of the 18:3-deficient diet with linseed oil or fish oil in P23H rats resulted in a ROS fatty acid profile comparable to that of Sprague-Dawley rats raised on a standard diet. Serum DHA and 22:5n-6 levels were low in both groups. No significant differences in serum cholesterol were observed as a function of genotype or diet.
Conclusions: Heterozygous P23H rats are capable of forming ROS DHA from dietary fatty acid precursors found in linseed oil (18:3n-3) or fish oil (20:5n-3). Under all dietary conditions, P23H transgenics are highly susceptible to retinal damage from exposure to intense light. Although levels of DHA in the ROS of P23H rats could be altered by dietary manipulation, only small changes in photoreceptor cell survival, as measured by whole-retina rhodopsin and DNA content, were observed. The lower-than-normal levels of ROS DHA may reflect an adaptive, possibly protective, mechanism in the P23H transgenic rat model of RP.
Retinitis pigmentosa (RP), a diverse group of inherited retinal disorders, is characterized by progressive degeneration and functional loss in rod and cone photoreceptors, accompanied by secondary degeneration of tissue underlying the retina [1-3]. The rate of occurrence of RP is generally 1:4000 worldwide, with rates being higher in isolated populations and in populations with frequent intermarriage . Early clinical manifestations of RP are night blindness, loss of peripheral vision, decreased visual acuity [2,4,5], and abnormal electroretinograms [6-9].
Some RP patients have altered levels of n-3 and/or n-6 polyunsaturated fatty acids (PUFAs) in plasma [10-12], red blood cells (RBCs) [10,11,13,14], or sperm . Maude et al.  found reduced levels of 20- and 22-carbon n-3 and n-6 PUFAs in RBCs of individuals with Usher's syndrome Type I, but not in individuals with Usher's Type II. Sperm from Usher's Type II patients were found to have reduced levels of n-3 and n-6 fatty acids compared to non-Usher's RP patients . Notable was the reduction in the amount of the 22:6n-3 PUFA, docosahexaenoic acid (DHA). In addition to alterations in PUFAs, both Usher's Type II and non-Usher's RP sperm had significantly elevated levels of cholesterol, but not of desmosterol, an immediate precursor to cholesterol and a quantitatively significant component of sperm [16,17]. In a large cohort of RP patients, Gong et al.  found lower-than-normal mean plasma levels of 18:3n-3 (α-linolenic acid), 22:3n-3, and DHA. Extensive investigations into animal models of progressive retinal degeneration also have shown decreased levels of n-3 fatty acids, particularly DHA, in the rod outer segment (ROS) and/or plasma of dogs [18,19], cats , and mice .
The importance of n-3 fatty acids, especially DHA and 18:3n-3, for normal retinal function is well documented in humans [22-24] and in animals [25,26]. 18:3n-3 is an "essential" fatty acid; hence, it must be obtained from the diet. Through a series of oxidation (desaturation) and chain elongation reactions, 18:3n-3 is converted to DHA (Figure 1) , the major PUFA in vertebrate rod photoreceptors . Because mammalian ROS have a high turnover rate, maintenance of a normal phospholipid composition in the rods depends to some degree on the availability of fatty acids in the diet. In rats fed diets deficient in n-3 fatty acids, DHA levels are reduced in most tissues, but ROS DHA is reduced by only small amounts [29-33]. Despite decreases in DHA, total 22-carbon PUFAs in ROS remain fairly constant, primarily through compensatory increases in 22:5n-6. Second generation offspring of mothers fed n-3-deficient diets show a marked reduction in retinal DHA and a marked elevation in 22:5n-6 [32,34].
An association between environmental light conditions and DHA content of photoreceptor rods is well established [34-36]. Penn and Anderson  demonstrated that rats raised in bright light were less susceptible to damage from intense light than were rats raised under dim light. In a study in which DHA content in rat ROS was altered by feeding an 18:3n-3-deficient diet, Organisciak et al.  reported decreased ROS DHA and reduced susceptibility to damage from intense light. In a later study, it was concluded that retinal susceptibility to light damage appeared to be related to relative levels of DHA and 22:5n-6 . Koutz et al.  found that ROS DHA was lower in second-generation rats reared in dim light on 18:3n-3-deficient diets than in animals reared in bright cyclic light on a diet high in n-3 fatty acids. However, the photoreceptor cells of the dim-light-reared animals were more susceptible to the damaging effects of intense light. The authors concluded that the increased susceptibility of the dim-light-reared animals reflected differences in antioxidant protective mechanisms, which were possibly related to environmental light conditions, rather than to differences in PUFA levels.
Other associations between DHA levels and visual function have been observed. Bush et al.  noted an elevated rhodopsin content in rats raised on a diet deficient in n-3 fatty acids. Although the level of steady-state rhodopsin bleach was the same, the rate of rhodopsin regeneration in these animals was significantly slower than that in rats raised on a diet containing 18:3n-3. The authors concluded that the capacity for photon absorption by rhodopsin was reduced in n-3-deficient rats and would account for a reduced susceptibility to light damage.
Although animal model studies have not yet elucidated the mechanism underlying the apparent inverse relationship between retinal DHA levels and protection of photoreceptor cells from light-induced damage, it is clear that abnormalities in n-3 fatty acid content are associated with retinal dysfunction. Based upon the results of studies mentioned above, we hypothesized that retinal photoreceptor cell life might be prolonged in an animal model of RP by reducing ROS DHA levels. In this study, we used a P23H transgenic rat model of human RP. P23H denotes a mutation in which a proline residue at position 23 in the N-terminal domain of rhodopsin is replaced with a histidine residue. This mutation represents the most common genetic alteration in autosomal dominant RP and leads to the gradual loss of rod photoreceptor cells both in humans and in animal models [10,11,38-42]. In the present study, dietary restriction and supplementation were used to manipulate the ROS fatty acid profile of P23H and normal Sprague Dawley rats. Animals were subsequently subjected to intense light exposure, and the effect of altered ROS fatty acid composition on photoreceptor survival was assessed. We demonstrate that reduction of ROS DHA levels does not offer significant protection of P23H rats from light-induced retinal damage.
Animals and rearing environment
Albino transgenic rats, heterozygous for the P23H rhodopsin mutation, were bred from a cross between homozygous transgenic P23H rats and normal Sprague Dawley animals (Chrysalis DNX Transgenic Sciences, Princeton, NJ). Line 3 of the P23H mutants, chosen for this study because it exhibits a moderate rate of retinal degeneration, was obtained from Dr. M. LaVail. Based on actual counts of remaining rows of nuclei in the outer nuclear layer (ONL) of the retina, it is estimated that 50% of the photoreceptors in line 3 have degenerated by 60 days of age .
Weanling (21 day old) transgenic rats and Sprague-Dawley (wild type) controls were fed either a standard rat chow diet or a diet deficient in linolenic acid (18:3n-3) for 40 days (Harlan Tekland 8640, Madison, WI). Food and water were available ad libitum. When possible equal numbers of male and female animals were used in all studies. In an attempt to increase DHA levels, rats receiving the 18:3n-3-deficient diet were given weekly supplements by gavage (1 ml) of either linseed oil (53 mol% 18:3n-3) or fish oil (rich in 20:5n-3). Control animals received an equal volume of saline.
Animals were maintained under a dim (20-30 lux) cyclic-light environment (12 h light, 12 h dark) until they were 60 days of age. Some 60 day old rats were then kept in constant darkness for two additional weeks. Prior to euthanasia and rhodopsin, retinal DNA, and lipid analyses, or exposure to intense light, all animals were dark adapted for 16 h. Animals were euthanized under dim red illumination in a chamber saturated with CO2. All procedures involving animals in this study followed the protocols outlined in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Wright State University Laboratory Animal Care and Use Committee.
Exposure to intense light
For intense light exposure, animals were placed into cylindrical green Plexiglas (No. 2092) chambers (Dayton Plastics, Dayton OH), which transmit light between 490 and 580 nm . During exposure, light intensity inside the chamber was 1350 to 1500 lux (~200 μW/cm2 corneal irradiance). The onset of light exposure was at 0900 and was either 4 or 8 h in duration. After intense light treatment, the animals were placed into a dark environment for two weeks. This dark period allows time for repair of damaged retinal tissue and removal of necrotic tissue. At the same time that light-exposed animals were transferred to the dark environment, unexposed rats (0 h intense light) also were transferred to the dark. The dark environment was interrupted by red light (λ = >600 nm) during routine animal care for approximately 30 min daily.
Quantification of rhodopsin and retinal DNA
Methods for measuring whole eye rhodopsin and retinal DNA have been described . In brief, rhodopsin in one eye was extracted into 1.5% detergent (Emulphogene BC-720, Sigma, St. Louis, MO). Quantification was made by comparing absorbance at 500 nm before and after bleaching in white light. The retina from the fellow eye was excised for determination of total DNA. A modified Hoechst dye-binding assay was used for retinal DNA measurements . The proportion of total DNA contributed by photoreceptor cells was determined by subtracting the DNA content of retinas excised from 6-month-old Royal College of Surgeons (RCS) dystrophic rats (which lack photoreceptor cells) from the total retinal DNA of the experimental animals.
ROS isolation and fatty acid determinations
Rod outer segments (ROS) were isolated from the pooled retinas of 5 to 8 animals using sucrose density gradient ultracentrifugation . Band I and II fractions from each isolate were rinsed with Krebs-Ringer phosphate buffer (KRP) and precipitated by centrifugation at 8000 rpm at 4°C for 10 min. Band I contains the purest ROS fraction; Band II is a less pure fraction and includes broken rods and mitochondria. ROS lipids were prepared for analysis as described previously . Briefly, lipids were extracted from the ROS precipitates with chloroform:methanol (2:1 vol/vol). Following four extractions at room temperature, a fifth extraction was done after heating to 55°C for 10 min. Lipid extracts were combined and partitioned with water at 4°C. The lower phase was then washed with Folch's pure upper phase, transferred to a conical transesterification tube, and dried under N2. Lipids were transesterified by adding 14% boron trifluoride in methanol to the dried residue and heating the solution for 20 min at 95°C. Following the addition of ice-cold water, esterified fatty acids were extracted with hexane and subsequently dried in a Biodryer (Virtis, Gardner, NY). Fatty acid methyl esters were redissolved in hexane prior to analysis by gas chromatography, which was performed using a Varian 3700 GLC controlled by a Varian GC Star Workstation (Varian Chromatography Systems, Walnut Creek, CA). The methyl esters were separated on a glass column (6 ft x 1/4 in OD) containing 10% SP-2330 on 100/120 Chromosorb WAW (Supelco, Inc., Bellefonte, PA) with helium as the carrier gas. The gas chromatograph was programmed for a temperature gradient ranging from an initial temperature of 187°C to a final temperature of 207°C. The rate of temperature change was 2°C/min, starting 2 min after sample injection.
Blood serum fatty acid and cholesterol analysis
Blood was obtained by heart puncture. After a 30-minute clotting period, blood serum was separated from cellular elements by centrifugation. Fatty acids in blood serum were determined as described above. Aliquots of serum (50 μl each) from rats were analyzed for cholesterol by reverse-phase HPLC, as described in detail elsewhere . Briefly, serum samples were saponified in methanolic KOH, and the nonsaponifiable lipids were extracted with petroleum ether and analyzed by reverse-phase HPLC, in comparison with an authentic cholesterol standard (Sigma, St. Louis, MO). We employed an IB-SIL 3 C18 BDS (150 x 4.6 mm, 3 μm particle diameter; Phenomenex, Torrance, CA), with a NovaPak C18 guard column (Waters/Millipore, Milford, MA). Isocratic runs were performed at room temperature with HPLC-grade MeOH (Burdick & Jackson, Muskegon, MI) as the mobile phase (flow rate, 1 ml/min) and detection at 205 nm. Each serum sample was "spiked" with an internal standard of [3H] cholesterol (ca. 2x105 dpm; American Radiolabeled Chemicals, St. Louis, MO) to adjust for recovery efficiency and to serve as an internal chromatographic standard for peak assignment. Cholesterol was quantified by integrated peak area analysis in comparison with the empirically determined response factor (integration units per nmol) determined for the authentic cholesterol standard, within the linear range of the assay (ca. 1 to 150 nmol). Replicate analyses of the same sterol-containing sample were within ±3% by this method.
The means of treatment groups were compared for significant differences using Student's two-tailed t-test. Data are reported as group means and standard deviations (SD).
Influence of diet and light/dark conditions on retinal rhodopsin and DNA levels
The effect of diet on retinal rhodopsin and DNA levels in transgenic and normal animals raised under dim cyclic light conditions is shown in the left-hand panels of Figure 2A,B, respectively. Under conditions of standard diet, the rhodopsin content in the eyes of 60 day old P23H transgenic rats was only about one half that found in normal Sprague Dawley rats (0.96 vs. 1.78 nmole/eye). Photoreceptor cell DNA content in the transgenics was about 75% of that observed in the normal animals (142 vs. 188 μg/retina). After an additional two weeks in darkness, the levels of rhodopsin increased in both the transgenic and normal animals (Figure 2A, right panel), but neither P23H rats nor control rats showed any change in DNA levels (Figure 2B, right panel). Thus, ROS length, but not photoreceptor cell number, increased during the dark period. Although it appears that the ROS in the transgenic rats behave functionally as expected, the percent increase in rhodopsin was slightly greater in the transgenic animals (35%) than in the Sprague-Dawley controls (29%).
A diet deficient in 18:3n-3 had no effect on retinal rhodopsin or DNA levels in transgenic rats. Rhodopsin and DNA levels were the same as those under conditions of standard diet, both in a cyclic-light rearing environment (Figure 2A,B, left panel) and after two weeks in darkness (Figure 2A,B, right panel). Previous studies in our laboratory  have shown that rhodopsin and DNA levels in normal Sprague-Dawley rats are the same when fed either an 18:3n-3-deficient or a standard diet, irrespective of rearing environment.
Supplementation of the 18:3n-3-deficient diet with fish oil had little effect on retinal rhodopsin or DNA levels in either normal or transgenic animals under standard light-rearing conditions (Figure 3A,B). Likewise, no major differences were observed in the transgenic animals supplemented with linseed oil. In each case, rhodopsin and DNA levels were the same as in rats given an equal volume of saline. A comparable lack of an effect from linseed oil supplementation was noted in an earlier dietary study of normal Sprague-Dawley rats .
Kinetics of in vitro rhodopsin bleaching in P23H rats
As P23H is an autosomal dominant mutation, both normal and mutated rhodopsin are expressed in the ROS of transgenic P23H rats . To determine if there were alterations in rhodopsin bleaching in P23H rats, the in vitro kinetics were examined and compared to those in normal (age-matched control) animals. Bleaching of the visual pigment extracts from transgenics and normals, both reared on a standard diet, resulted in the same end point after 5 min of light treatment (Figure 4, left panel). When the P23H data were plotted as a percentage of unexposed (0 time) rhodopsin levels (Figure 4, right panel), it is clear that the proline-to-histidine alteration in the primary amino acid sequence of rhodopsin in the transgenic rats did not affect in vitro visual pigment bleaching kinetics.
Influence of diet on susceptibility to damage from intense light
As was noted earlier, under conditions of standard diet, pre-exposure P23H rhodopsin and DNA levels were 50% and 25% lower, respectively, compared to those in normal animals (Figure 5A,B, 0 h). While both P23H and normal rats suffered retinal damage from 8 h of intense light exposure (Figure 5A,B, 8 h), the declines in rhodopsin and DNA content were disproportionately greater in the transgenics. Rhodopsin and DNA levels decreased by less than 10% in the Sprague-Dawley rats after exposure to 8 h of intense light. In contrast, reductions of 70% or more in DNA and rhodopsin were observed in P23H animals, compared to levels observed in unexposed Sprague-Dawley rats. Reduction of the length of exposure to intense light alleviated rhodopsin and DNA losses in the transgenics. When exposure was limited to 4 h (Figure 5A,B, 4 h), DNA and rhodopsin levels declined by 55% and 70%, respectively. Maintenance on an 18:3n-3-deficient diet resulted in higher levels of rhodopsin and DNA, under both 8 and 4 h light treatments, but the increases were not statistically different (α=0.05) from those in P23H animals fed a standard diet. Interestingly, after 4 h of intense light exposure, rhodopsin and DNA levels in transgenics fed a deficient diet supplemented with linseed oil (Figure 5A,B) were even lower than those in transgenics fed either standard (not significant, α=0.05) or deficient diet (significant at 0.02<p<0.05).
Rod outer segment fatty acid composition: Band I
ROS fatty acid content was analyzed to determine if differences observed in normal Sprague-Dawley rats and transgenic rats, with regard to changes in rhodopsin and DNA levels upon light exposure, were related to alterations in fatty acid levels. As shown in Figure 6, DHA content of ROS from normal rats fed a standard rat chow diet was high, with this single PUFA comprising nearly 42% of the total fatty acids. In contrast, DHA content of ROS from P23H heterozygotes fed a standard diet was just 34% of the total Band I fatty acids. Levels of 22:5n-6 were less than 1% of ROS total fatty acids in both normal Sprague-Dawley and P23H transgenic rats. Only minor differences were seen in other fatty acids, including 18:3n-3 and 20:5n-3, both of which were were 0.2% or less of the total fatty acids under all conditions.
With the 18:3n-3-deficient diet, ROS DHA levels were reduced in both normal and transgenic animals. The reduction was small, however, in P23H rats (34% to 31%) compared to normal rats (42% to 28%). A nearly 10-fold increase in ROS 22:5n-6 levels occurred in both normal and transgenic animals.
Supplementation of deficient diet with linseed oil or fish oil elevated ROS DHA levels and decreased 22:5n-6 levels in both Sprague-Dawley and P23H rats. In both groups supplementation produced a fatty acid profile that was strikingly similar to that of normal rats fed a standard diet.
Rod outer segment fatty acid analysis: Band II
To ensure that changes in DHA levels seen in Band I ROS were not due to a loss of material to the denser Band II fraction, the effect of diet on fatty acid content of Band II ROS was also analyzed. DHA levels in Band II ROS decreased when 18:3n-3-deficient diet was fed (data not shown). Thus, the decline in DHA levels was not due to a physical loss of ROS material from Band I into Band II. As was observed in Band I ROS, Band II ROS 22:5n-6 levels increased with the deficient diet and declined with linseed or fish oil supplementation of the deficient diet (data not shown).
Serum fatty acids and serum cholesterol
Serum fatty acid levels were measured in normal Sprague-Dawley rats and P23H heterozygotes fed a standard diet and in P23H rats fed either an 18:3n-3-deficient diet or the deficient diet supplemented with linseed oil. Only small differences in fatty acid composition between Sprague-Dawley and P23H rats were seen when standard rat chow was fed (Table 1). Of particular note are the comparably low levels of DHA and 22:5n-6 in both groups of rats. As expected, relative levels of DHA and 22:5n-6 were changed in P23H rats fed an 18:3n-3-deficient diet. Supplementation with linseed oil produced an elevation of DHA content to levels similar to those obtained with the standard diet; 22:5n-6 levels declined to less than 0.1 mol%. No significant diet-related differences in serum cholesterol were observed. Serum cholesterol values for the P23H transgenics were 92 to 95% of those observed in Sprague-Dawley rats.
Our results demonstrate that it is possible to alter ROS PUFAs in both normal rats and in the P23H rat model of retinitis pigmentosa. Except for the DHA content, under like dietary conditions, ROS fatty acid profiles of P23H mutants and control Sprague-Dawley rats are quite similar. ROS DHA levels were about 8% lower in the mutants, under conditions of standard diet. Although DHA levels were reduced in the P23H ROS, their serum DHA content was comparable to that in the normal animals. Anderson et al.  found similar results in a study of a mouse model of progressive retinal degeneration. Mutant mice had lower levels of ROS DHA compared to wild-type mice, but there were no differences in the DHA content of their serum. These results are compelling because some, but not all, humans suffering from RP are reported to have lower-than-normal plasma DHA levels [11,12]. The results from animal models suggest that blood levels of DHA may not be a reliable indicator of retinal levels.
It has been observed previously that feeding rats a diet deficient in 18:3n-3 induces a decrease in ROS DHA and a concomitant increase in 22:5n-6 [29,30,32,33]. In the present study, we also observed this relationship, but, while the levels of 22:5n-6 were approximately the same in normal and P23H rats fed the deficient diet, the reduction in ROS DHA content in the normal animals was nearly three times greater than the DHA decrease observed in the mutants. Possibly there is a minimal level of DHA that is required for maintenance of a critical 22:5n-6/DHA ratio or that is vital for the survival of the remaining photoreceptor cells.
Both linseed oil and fish oil were used as supplements to the 18:3n-3-deficient diet in this study. Linseed oil is high in linolenic acid (18:3n-3). Fish oil is high in eicosapentaenoic acid (20:5n-3), which is synthesized from 18:3n-3 by the addition of a 2-carbon unit and two desaturations (Figure 1). The rationale for using two precursors is that restoration of DHA levels by supplementation with 20:5n-3, but not with 18:3n-3, would permit a more precise location of a functional deficit in the synthetic pathway. Our data suggest that the enzymatic machinery for DHA synthesis is unaltered in P23H rats.
We found, in normal Sprague-Dawley and P23H rats, that supplementation of a deficient diet with fish oil or linseed oil produced ROS fatty acid profiles very similar to those of normal rats on a standard diet. Reme et al.  also found no differences in ROS DHA content in Sprague-Dawley rats fed semi-purified diets containing either fish oil or soybean oil (which contains 18:3n-3). Our results are different from those reported by Aquirre et al. , in a study of the effects of dietary manipulation in dogs affected with progressive rod-cone degeneration (prcd), an RP animal model. On basal diet, which was very low in n-3 PUFA content, prcd-affected dogs had nearly 20% less ROS DHA than normal dogs. Contrary to the effects of fish oil supplementation in our study, no significant changes in DHA levels were observed with fish oil supplementation in the canine model of RP.
The present study shows that, both under conditions of cyclic light and cyclic light with a subsequent two weeks in darkness, the retinas of 75 day old P23H heterozygotes have only 50% of the rhodopsin and 75% of the DNA found in normal Sprague-Dawley rats. These findings were seen in transgenics maintained on either standard or 18:3n-3-deficient diets. Previous studies in our laboratory  have shown that rhodopsin and DNA levels in normal Sprague-Dawley rats are also the same on either diet in both kinds of rearing environment. Both control and P23H mutant animals showed a comparable increase in the rhodopsin content of the ROS under dark conditions. Thus, the normal mechanism for increasing rod length in the absence of light is functionally intact in the P23H heterozygotes.
It has been suggested that individuals afflicted with RP who work in high-intensity light environments exhibit a greater-than-normal rate of photoreceptor cell loss . One hypothesis for the observed decrease in DHA levels in RP is that the reduction is an adaptive mechanism that affords protection from the potentially damaging effects of light [20,32]. Previously, we found that normal rats fed an 18:3n-3-deficient diet are protected from light damage . The current study demonstrates that ROS DHA is lower in P23H rats fed a standard diet than it is in normal Sprague-Dawleys on the same diet. Yet, it appears that lower ROS DHA levels do not have a significant protective effect on photoreceptor cell survival. In fact, P23H transgenics were shown to be much more sensitive to damage from intense light than normal rats under all dietary conditions. It is intriguing, however, that in the 18:3n-3-deficient transgenics, which had lower ROS DHA levels than P23H rats on a standard diet, there was greater, although not statistically significant, photoreceptor survival (Figure 5). Increasing the ROS DHA level by supplementation with linseed oil, which resulted in a fatty acid profile nearly like that of transgenics on a standard diet, produced a small, statistically significant (0.02<p<0.05), decrease in photoreceptor cell survival compared to that in deficient-diet rats. These observations suggest that even in the P23H animals the retina can be induced, by dietary manipulation, to activate an adaptive mechanism that results in lower DHA and higher 22:5n-6 levels, affording some degree of protection against light damage. Overriding the adaptive mechanism by artificially altering the stoichiometry between DHA and 22:5n-6 induces an increased susceptibility to light damage.
This work was supported by The Ohio Lions Research Foundation, U.S.P.H.S. (NEI/NIH) grants EY01959 (to DTO) and EY07361 (to SJF), and by funds from Mrs. Mary Petticrew, Springfield, OH.
1. Humphries P, Kenna P, Farrar GJ. On the molecular genetics of retinitis pigmentosa. Science 1992; 256:804-8.
2. Merin S. Inherited eye diseases: diagnosis and clinical management. New York: Dekker; 1991.
3. Ryan SJ, editor. Retina. Vol 1. St. Louis: Mosby; 1989.
4. McLeod ML, Wisnicki HJ, Medow NB. Vision impairment in the pediatric population. In: Silverstone B, Lang MA, Rosenthal BP, Faye EE, editors. The Lighthouse handbook on vision impairment and vision rehabilitation. Vol 1. Vision impairment. New York: Oxford University Press; 2000. p. 19-31.
5. Boughman JA, Halloran SL, Cohen MM. Genetic aspects of retinitis pigmentosa. In: LaVail MM, Hollyfield JG, Anderson RE, editors. Retinal degenerations: experimental and clinical studies. New York: Alan R. Liss; 1985. p. 3-24.
6. Heckenlively JR, Yoser SL, Friedman LH, Oversier JJ. Clinical findings and common symptoms in retinitis pigmentosa. Am J Ophthalmol 1988; 105:504-11.
7. Berson EL. Nutrition and retinal degenerations. Int Ophthalmol Clin 2000; 40:93-111.
8. Berson EL. Retinitis pigmentosa: The Friedenwald Lecture. Invest Ophthalmol Vis Sci 1993: 34:1659-76.
9. Dryja TP, McGee TL, Hahn LB, Cowley GS, Olsson JE, Reichel E, Sandberg MA, Berson, EL. Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. N Engl J Med 1990; 323:1302-7.
10. Hoffman DR, DeMar JC, Heird WC, Birch DG, Anderson RE. Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa. J Lipid Res 2001; 42:1395-401.
11. Maude MB, Anderson EO, Anderson RE. Polyunsaturated fatty acids are lower in blood lipids of Usher's type I but not Usher's type II. Invest Ophthalmol Vis Sci 1998; 39:2164-6.
12. Gong J, Rosner B, Rees DG, Berson EL, Weigel-DiFranco CA, Schaefer EJ. Plasma docosahexaenoic acid levels in various genetic forms of retinitis pigmentosa. Invest Ophthalmol Vis Sci 1992; 33:2596-602.
13. Simonelli F, Manna C, Romano N, Nunziata G, Voto O, Rinaldi E. Evaluation of fatty acids in membrane phospholipids of erythrocytes in retinitis pigmentosa patients. Ophthalmic Res 1996; 28:93-8.
14. Hoffman DR, Uauy R, Birch DG. Red blood cell fatty acid levels in patients with autosomal dominant retinitis pigmentosa. Exp Eye Res 1993; 57:359-68.
15. Connor WE, Weleber RG, DeFrancesco C, Lin DS, Wolf DP. Sperm abnormalities in retinitis pigmentosa. Invest Ophthalmol Vis Sci 1997; 38:2619-28.
16. Lin DS, Connor WE, Wolf DP, Neuringer M, Hachey DL. Unique lipids of primate spermatozoa: desmosterol and docosahexaenoic acid. J Lipid Res 1993; 34:491-9.
17. Connor WE, Lin DS, Wolf DP, Alexander M. Uneven distribution of desmosterol and docosahexaenoic acid in the heads and tails of monkey sperm. J Lipid Res 1998; 39:1404-11.
18. Anderson RE, Maude MB, Alvarez RA, Acland GM, Aguirre GD. Plasma lipid abnormalities in the miniature poodle with progressive rod-cone degeneration. Exp Eye Res 1991; 52:349-55.
19. Anderson RE, Maude MB, Acland G, Aguirre GD. Plasma lipid changes in PRCD-affected and normal miniature poodles given oral supplements of linseed oil. Indications for the involvement of n-3 fatty acids in inherited retinal degenerations. Exp Eye Res 1994; 58:129-37.
20. Anderson RE, Maude MB, Nilsson SE, Narfstrom K. Plasma lipid abnormalities in the abyssinian cat with a hereditary rod-cone degeneration. Exp Eye Res 1991; 53:415-7.
21. Anderson RE, Maude MB, Bok D. Low docosahexaenoic acid levels in rod outer segment membranes of mice with rds/peripherin and P216L peripherin mutations. Invest Ophthalmol Vis Sci 2001; 42:1715-20.
22. Neuringer M, Anderson GJ, Connor WE. The essentiality of n-3 fatty acids for the development and function of the retina and brain. Annu Rev Nutr 1988; 8:517-41.
23. Uauy RD, Birch DG, Birch EE, Tyson JE, Hoffman DR. Effect of dietary omega-3 fatty acids on retinal function of very-low-birth-weight neonates. Pediatr Res 1990; 28:485-92.
24. Uauy R, Peirano P, Hoffman D, Mena P, Birch D, Birch E. Role of essential fatty acids in the function of the developing nervous system. Lipids 1996; 31:S167-76.
25. Neuringer M, Connor WE, Van Petten C, Barstad L. Dietary omega-3 fatty acid deficiency and visual loss in infant rhesus monkeys. J Clin Invest 1984; 73:272-6.
26. Neuringer M, Connor WE, Lin DS, Barstad L, Luck S. Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys. Proc Natl Acad Sci U S A 1986; 83:4021-5.
27. Harwood JL. Lipid metabolism. In: Gunstone FD, Harwood JL, Padley FB, editors. The lipid handbook, 2nd ed. London: Chapman and Hall; 1994. p. 605-33.
28. Fliesler SJ, Anderson RE. Chemistry and metabolism of lipids in the vertebrate retina. Prog Lipid Res 1983; 22:79-131.
29. Anderson RE, Maude MB. Lipids of ocular tissues. 8. The effects of essential fatty acid deficiency on the phospholipids of the photoreceptor membranes of rat retina. Arch Biochem Biophys 1972; 151:270-6.
30. Wiegand RD, Koutz CA, Stinson AM, Anderson RE. Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency. J Neurochem 1991; 57:1690-9.
31. Organisciak DT, Wang HM, Noell WK. Aspects of the ascorbate protective mechanism in retinal light damage of rats with normal and reduced ROS docosahexaenoic acid. In: Hollyfield JG, Anderson RE, LaVail MM, editors. Degenerative retinal disorders: clinical and laboratory investigations. Proceedings of the Sendai Symposium on Retinal Degerneration; 1986 Sep 20-24; Sendai, Japan. New York: Liss; 1987. p. 455-68.
32. Bush RA, Malnoe A, Reme CE, Williams TP. Dietary deficiency of N-3 fatty acids alters rhodopsin content and function in the rat retina. Invest Ophthalmol Vis Sci 1994; 35:91-100.
33. Organisciak DT, Darrow RM, Jiang YL, Blanks JC. Retinal light damage in rats with altered levels of rod outer segment docosahexaenoate. Invest Ophthalmol Vis Sci 1996; 37:2243-57.
34. Wiegand RD, Koutz CA, Chen H, Anderson RE. Effect of dietary fat and environmental lighting on the phospholipid molecular species of rat photoreceptor membranes. Exp Eye Res 1995; 60:291-306.
35. Wiegand RD, Giusto NM, Rapp LM, Anderson RE. Evidence of rod outer segment lipid peroxidation following constant illumination of the rat retina. Invest Ophthalmol Vis Sci 1983; 24:1433-5.
36. Penn JS, Anderson RE. Effect of light history on rod outer-segment membrane composition in the rat. Exp Eye Res 1987; 44:767-78.
37. Koutz CA, Wiegand RD, Rapp LM, Anderson RE. Effect of dietary fat on the response of the rat retina to chronic and acute light stress. Exp Eye Res 1995; 60:307-16.
38. Sung CH, Davenport CM, Hennessey JC, Maumenee IH, Jacobson SG, Heckenlively JR, Nowakowski R, Fishman G, Gouras P, Nathans J. Rhodopsin mutations in autosomal dominant retinitis pigmentosa. Pro Natl Acad Sci U S A 1991; 88:6481-5.
39. Naash MI, Hollyfield JG, al-Ubaidi MR, Baehr W. Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene. Proc Natl Acad Sci U S A 1993; 90:5499-503.
40. Olsson JE, Gordon JW, Pawlyk BS, Roof D, Hayes A, Molday RS, Mukai S, Cowley GS, Berson EL, Dryja TP. Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron 1992; 9:815-30.
41. Machida S, Kondo M, Jamison JA, Khan NW, Kononen LT, Sugawara T, Bush RA, Sieving PA. P23H rhodopsin transgenic rat: correlation of retinal function with histopathology. Invest Ophthalmol Vis Sci 2000; 41:3200-9.
42. LaVail MM, Yasumura D, Matthes MT, Drenser KA, Flannery JG, Lewin AS, Hauswirth WW. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy. Proc Natl Acad Sci U S A 2000; 97:11488-93.
43. Noell WK, Albrecht R. Irreversible effects of visible light on the retina: role of vitamin A. Science 1971; 172:76-9.
44. Organisciak DT, Jiang YL, Wang HM, Pickford M, Blanks JC. Retinal light damage in rats exposed to intermittent light. Comparison with continuous light exposure. Invest Ophthalmol Vis Sci 1989; 30:795-805.
45. Noell WK, Organisciak DT, Ando H, Braniecki MA, Durlin C. Ascorbate and dietary protective mechanisms in retinal light damage of rats: electrophysiological, histological and DNA measurements. In: Hollyfield JG, Anderson RE, LaVail MM, editors. Degenerative retinal disorders: clinical and laboratory investigations. Proceedings of the Sendai Symposium on Retinal Degerneration; 1986 Sep 20-24; Sendai, Japan. New York: Liss; 1987. p.469-83.
46. Organisciak DT, Xie A, Wang HM, Jiang YL, Darrow RM, Donoso LA. Adaptive changes in visual cell transduction protein levels: effect of light. Exp Eye Res 1991; 53:773-9.
47. Fliesler SJ, Richards MJ, Miller C, Peachey NS. Marked alteration of sterol metabolism and composition without compromising retinal development or function. Invest Ophthalmol Vis Sci 1999; 40:1792-801.
48. Ablonczy Z, Knapp DR, Darrow R, Organisciak DT, Crouch RK. Mass spectrometric analysis of rhodopsin from light-damaged rats. Mol Vis 2000; 6:109-15 <http://www.molvis.org/molvis/v6/a15/>.
49. Reme CE, Malnoe A, Jung HH, Wei Q, Munz K. Effect of dietary fish oil on acute light-induced photoreceptor damage in the rat retina. Invest Ophthalmol Vis Sci 1994; 35:78-90.
50. Aquirre GD, Acland GM, Maude MB, Anderson RE. Diets enriched in docosahexaenoic acid fail to correct progressive rod-cone degeneration (prcd) phenotype. Invest Ophthalmol Vis Sci 1997; 38:2387-407.
51. Heckenlively JR, Rodriguez JA, Daiger SP. Autosomal dominant sectoral retinitis pigmentosa. Two families with transversion mutation in codon 23 of rhodopsin. Arch Ophthalmol 1991; 109:84-91.
52. Voss A, Reinhart M, Sankarappa S, Sprecher H. The metabolism of 7,10,13,16,19-docosapentaenoic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat liver is independent of a 4-desaturase. J Biol Chem 1991; 266:19995-20000.
53. Mohammed BS, Sankarappa S, Geiger M, Sprecher H. Reevaluation of the pathway for the metabolism of 7,10,13,16-docosatetraenoic acid to 4,7,10,13,16-docosapentaenoic acid in rat liver. Arch Biochem Biophys 1995; 317:179-84.
54. Alvarez RA, Aguirre GD, Acland GM, Anderson RE. Docosapentaenoic acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected miniature poodle dogs. Invest Ophthalmol Vis Sci 1994; 35:402-8.