June 2009
Volume 50, Issue 6
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Retinal Cell Biology  |   June 2009
Cone–Rod Dependence in the Rat Retina: Variation with the Rate of Rod Damage
Author Affiliations
  • Vicki Chrysostomou
    From the Research School of Biological Sciences, and the
    ARC Centre of Excellence in Vision Science, The Australian National University, Canberra, Australia; and the
  • Krisztina Valter
    From the Research School of Biological Sciences, and the
    ARC Centre of Excellence in Vision Science, The Australian National University, Canberra, Australia; and the
  • Jonathan Stone
    From the Research School of Biological Sciences, and the
    ARC Centre of Excellence in Vision Science, The Australian National University, Canberra, Australia; and the
    Save Sight Institute and Discipline of Physiology, University of Sydney, Sydney, Australia.
Investigative Ophthalmology & Visual Science June 2009, Vol.50, 3017-3023. doi:10.1167/iovs.08-3004
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      Vicki Chrysostomou, Krisztina Valter, Jonathan Stone; Cone–Rod Dependence in the Rat Retina: Variation with the Rate of Rod Damage. Invest. Ophthalmol. Vis. Sci. 2009;50(6):3017-3023. doi: 10.1167/iovs.08-3004.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To assess the effect of accelerated rod damage on the integrity of cones in the rat retina.

methods. Rhodopsin-mutant P23H-3 and Sprague-Dawley (SD) rats were raised in scotopic ambient conditions (12 hours dark, 12 hours 5 lux) and then exposed to photopic conditions (12 hours dark, 12 hours 300 lux). Rods and cones were assessed for cell death, outer segment (OS) morphology, and electroretinogram (ERG) responses.

results. Cones in the P23H-retina were affected rapidly by photopic exposure. Exposure for 2 days caused 50% reductions in LM- and S-cone OS length and cone ERG responses, associated with and preceded by reductions in rod OS length and ERG responses. Although 2 days’ exposure increased the rate of rod death, outer nuclear layer thinning was minimal, and no evidence of cone death was detected. In the SD retina, the same photopic exposure had no measurable effects on death rates, OS length, or ERG responses in either rods or cones. Longer (7 days) photopic exposure reduced cone and rod OS length and ERG responses in SD, as well as P23H-3 retinas, but less severely than in the P23H-3 strain.

conclusions. Cones are damaged rapidly in the P23H-3 retina when rod damage is accelerated by raised ambient illumination. This close dependence of cone integrity on rod integrity contrasts with the life-long persistence of cone function in the scotopic reared P23H-3 rat. In humans suffering comparable photoreceptor dystrophies, the maintenance of steady, low ambient light may, by minimizing acute rod damage, optimize the function of surviving cones.

The vulnerability of cone photoreceptors when rods degenerate is a clinically important feature of retinal disease. Mutations in proteins specifically expressed in rods, for example rhodopsin, cause the degeneration of rods primarily, and of cones secondarily. 1 The loss of cone vision is devastating for the patient, giving urgency to understanding the mechanisms that make cones vulnerable. 
To date, three mechanisms of cone–rod dependence have been proposed. Evidence has been reported that rods secrete a factor essential for cone survival. 2 3 4 Alternatively, we have noted that tissue oxygen levels in outer retina rise chronically in the photoreceptor-depleted retina, 5 6 7 that oxygen is specifically toxic to photoreceptors, 8 9 10 and that the toxicity involves oxidative damage. 11 We proposed therefore that rod depletion causes oxidative damage to cones (the oxygen toxicity hypothesis). 12 More recently, Ripps 13 has proposed that a toxin generated by dying rods reaches cones by gap junctions and induces their damage and death. None of these mechanisms is exclusive, and more than one may contribute to the vulnerability of cones to rod damage. 
Previous work in the P23H transgenic rat suggests that the rate of rod degeneration may influence the onset of cone dysfunction. The genetic defect in the P23H strain, a point mutation in the rhodopsin gene, causes an autosomal dominant photoreceptor dystrophy characterized by a rod–cone sequence of degeneration; however, the onset of cone dysfunction varies between genetic subtypes. P23H line 1 (P23H-1) animals and P23H homozygotes have a higher level of transgene expression and display faster rod degenerations than do P23H line 3 (P23H-3) animals and heterozygotes. Cone ERG responses in P23H-1 heterozygotes are normal at P28 but are significantly depressed by P56. 14 In P23H-3 heterozygotes they are normal until postnatal day (P)360 and are depressed by P540. 15 In the young adult P23H-1 animal, cone ERG responses are 12% of control values in the homozygote, 16 but remain at 50% of control values in the heterozygote. 14 In all these cases, severe rod loss precedes the onset of cone ERG changes. 
In this study, we investigated the effect of rod damage on cone integrity in the P23H-3 heterozygous strain. In the adult P23H-3 retina, rod loss is continuous but slow and cones are highly stable. 14 15 In this study, we accelerated the rate of rod damage in the P23H-3 strain by a modest increase in ambient illumination. 17 18 19 Evidence is presented that cones are damaged rapidly when rods are damaged rapidly, even in the absence of substantial rod loss. The temporal effects of photopic exposure on cone and rod morphology and function are also described. 
Material and Methods
Animal Strains
All procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and with the requirements of The Australian National University Animal Experimentation Ethics Committee. Observations were made in two strains of rat, the P23H-3 transgenic (Beckman Laboratories, University of California, San Francisco) and Sprague-Dawley (SD) albino, aged P90 to P150. The P23H-3 animals were heterozygotes, the offspring of mating P23H-3 homozygotes with SD rats. 
Ambient Light Protocols
All animals were raised from birth in cyclic light (12 hours dark, 12 hours white light), with the brightness of the light phase set at 5 lux (scotopic conditions). At adulthood, some rats were moved to photopic ambient conditions (12 hours dark, 12 hours 300 lux) for up to 7 days. P23H-3 animals were exposed to photopic cyclic light for 1, 2, or 7 days, whereas SD animals were exposed for 2, 4, or 7 days. 
Tissue Collection and Processing
Animals were euthanatized with an overdose of sodium pentobarbital (>60 mg/kg, intraperitoneal). Eyes were marked at the superior aspect of the limbus for orientation, enucleated, and immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at pH 7.4 for 3 hours. One eye from each animal was processed for cryosectioning, whereas the fellow eye was processed for wholemounting. For cryosectioning, the eyes were rinsed twice in 0.1 M PBS and left in a 15% sucrose solution overnight to provide cryoprotection. They were embedded in OCT compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan) and snap frozen in liquid nitrogen before they were cryosectioned at 12 μm (CM1850 Cryostat; Leica, Wetzlar, Germany). Sections were mounted on gelatin and poly-l-lysine–coated slides and dried overnight at 50°C before they were stored at −20°C. For retinal wholemounts, the retina was dissected from the eye cup, flattened by making radial incisions, gently sandwiched between two glass slides and immersed in 4% paraformaldehyde at 4°C for up to 2 weeks before immunolabeling. 
TUNEL Labeling and Quantification
Retinal cryosections were labeled with the TUNEL technique to identify the fragmentation of DNA characteristic of apoptotic cells, using a previously published protocol. 20 TUNEL-labeled sections were scanned from the superior to inferior edge in 1-mm steps, and the number of TUNEL+ profiles in the ONL was recorded. The frequency of TUNEL+ profiles/mm of ONL was averaged from at least two sections per animal, and a minimum of five animals were analyzed at each time point. To assess the regional distribution of dying cells, the retina was divided into superior and inferior hemispheres (relative to the optic nerve head). 
Immunohistochemistry of Retinal Cryosections
Rod and cone outer segment (OS) morphology was assessed by immunohistochemistry. Retinal cryosections were labeled for rhodopsin, long-medium wavelength–sensitive (LM) opsin, and short wavelength-sensitive (S) opsin, using methods described previously. 19 Briefly, cryosections were incubated overnight at 4°C with an antibody to rhodopsin (mouse monoclonal Rho1D4, 1:1000; Chemicon, Temecula, CA) and LM opsin (rabbit polyclonal, 1:1000; Chemicon) or S opsin (rabbit polyclonal, 1:1000; Chemicon). The next day, the sections were treated with goat anti-mouse Alexa Fluor 594 and goat anti-rabbit Alexa Fluor 488 antibodies (1:1000; Molecular Probes, Eugene, OR) for 24 hours at 4°C. The sections were incubated for 2 minutes with the DNA-specific dye bisbenzamide (1:10,000) before they were coverslipped with a glycerol/gelatin medium. 
Immunohistochemistry of Retinal Wholemounts
Retinal wholemounts were immunolabeled for LM opsin, as described previously. 19 Briefly, flattened retinas were blocked with normal goat serum in 0.01% Triton-PBS before overnight incubation with a rabbit polyclonal antibody to LM opsin (1:1000; Chemicon). After washing with PBS, retinas were incubated for 24 hours with an antibody to rabbit IgG conjugated to Alexa Fluor 488 (1:1000; Molecular Probes) and subsequently mounted and coverslipped on glass slides, with the outer surface up. 
LM opsin-labeled cone OSs were assessed for both density and length. Labeled wholemounts were visualized by fluorescence microscopy, and images of the OS layer were captured with a digital camera (AxioCam MRc; Carl Zeiss Meditec, Oberkochen, Germany). In the digital images, LM opsin-labeled OSs were counted over areas of 0.01 mm2 at 0.5-mm intervals across the entire retinal surface (∼100–250 fields per retina). In each counting field, the average length of cone OSs was also recorded. Counts of OSs per 0.01 mm2 were averaged across the entire retina to give an overall cone density for each sample. 
Quantification of Retinal Thickness and Outer Segment Length
Retinal thickness measurements were made on digital images of bisbenzamide-stained cryosections. Sections were scanned from the superior to inferior edge and the retinal thickness was determined every 500 μm (a total of eight measurements per retina). At each measurement location, the thickness of the outer nuclear layer (ONL) and the thickness of the retina, from inner to outer limiting membrane (ILM–OLM), were recorded. The ratio of the thickness of the ONL to the thickness of the retina (measured from the ILM to the OLM) was used for analysis, to account for obliquely cut sections. 
Measurements of rod and cone OS length were made on digital images of immunolabeled cryosections. Retinal sections were scanned from superior to inferior edge and, at regularly spaced intervals, the length of rhodopsin, LM opsin-labeled, and S opsin-labeled OSs were measured (a total of at least 24 measurements per retina). For measurements of OS length and retinal thickness, results from five animals at each time point were averaged and analyzed. 
Electroretinography
The function of photoreceptors was assessed by the flash-evoked electroretinogram (ERG). Animals were dark adapted overnight and prepared for recording in dim red illumination, as described previously. 17 After previous reports, 21 responses to a standard test flash (44.5 cds/m2) were considered to be mixed with contributions from rods and cones. Responses to the test flash preceded, by 395 ms, by a conditioning flash (12 cds/m2), were considered those of cones. By subtracting the cone response from the mixed response, we isolated the rod response. With this method, we used three measurements of amplitude for analysis: rod a-wave, rod b-wave, and cone b-wave, as described previously (see Fig. 1in Ref. 19 ). 
Statistical Analyses
Paired data from P23H-3 and SD animals were compared by using a two-tailed Student’s t-test. Comparisons within each rat strain were made by an analysis of variance with the Tukey posttest used to compare group means. All data are presented as the mean ± SEM. 
Results
Impact of Increased Ambient Light on Rods
The P23H transgene makes rods vulnerable to damage when ambient light is increased. When the level of ambient light is increased from scotopic to mesopic 17 or photopic 18 19 levels, the death of rods is accelerated, rod ERG responses are reduced, and the OSs of the survivors are damaged and shortened. These effects were confirmed in the current data. Increasing the intensity of the “day” component of cyclic ambient light from 5 to 300 lux for 2 days increased the TUNEL-labeling of P23H-3 photoreceptors by 40-fold (Fig. 1) . A more limited increase was observed in the SD rat, in response to the same increase (Fig. 1B) . Further, the acceleration was more rapid in the P23H-3 strain, the frequency of TUNEL+ profiles reaching its observed maximum at 2 days, as against 4 to 7 days in the SD strain. The spatial distribution of the TUNEL+ photoreceptors was not uniform. As reported previously, 22 23 photoreceptors near the edge of the retina were relatively unaffected, and there was a strong concentration of TUNEL+ cells in the superior midperiphery in both strains (Figs. 1C 1D)
The cell death induced by photopic light exposure did not cause a change in the thickness of the retina in either the P23H-3 or SD retina. ONL thickness was greater in the SD retina (confirming previous reports 6 14 ) by a small margin (the ONL/retina ratio was 0.32 vs. 0. 28), but a significant thinning of the layer during 7 days’ exposure to 300 lux was not apparent in either strain (Fig. 2)
In the P23H-3 strain, photopic light exposure reduced the amplitude of rod ERG responses (Fig. 3A) , and this decrease was accompanied by a shortening of rod OSs (Fig. 3B , red). Slower and less severe reductions were seen in the SD strain in response to the same increase in illumination (Figs. 3C 3D)
Impact of Increased Ambient Light on Cones
We tested whether increased ambient light also causes the death of cones in the P23H-3 retina, by examining the density of LM cone (the dominant cone in the rat retina) OSs in wholemount preparations, before and after 7 days’ exposure to photopic ambient light. Measurements were made in the superior midperipheral retina, the area most affected by the increase in ambient light. 19 Although cone OSs were distinctly shorter after exposure to 300 lux (Figs. 4B 4D) , the density of OSs was not measurably reduced (Fig. 4A) . In addition, we labeled sections from eyes exposed for 2 days for both cell death and cone OSs, after Geller et al., 8 and examined several sections for evidence of TUNEL+ cones, without finding any. 
In both P23H-3 and SD strains, the increase in ambient light from scotopic (5 lux) to photopic (300 lux) levels had a major impact on the function of cones and the length of their OSs. In the P23H-3, the b-wave of the cone ERG response declined (Fig. 3A) , and correspondingly, the length of LM- and S-cone OSs (green and blue in Fig. 3B ) was reduced. A similar trend, but slower and less severe, was apparent in the SD strain (Figs. 3C 3D)
Quantitative Analysis: Time Course of Changes in ERG and OS Length
In the P23H-3 retina, the increase in ambient light to photopic levels caused rod and cone components of the ERG to decline in amplitude to less than 50% of control values within 2 days (Fig. 5A) . The loss was progressive over this period. In the SD retina, rod and cone ERG components maintained amplitude at 2 days but decreased to 60% to 70% of control values at 4 days (Fig. 5B)
Correspondingly, the increase in ambient illumination caused a progressive shortening of rod (Fig. 6A)and cone (Figs. 6B 6C)OSs. In the P23H-3 retina, OSs of rods, LM-cones, and S-cones shortened to 77%, 55%, and 57% of control lengths respectively within 2 days. By 7 days, rod and LM-cone OSs had shortened further to 54% and 34% of control values, whereas the length of S-cones OSs remained unchanged from 2 days. In the SD retina, shortening of both rod and cone OSs was delayed; there was no measurable change at 2 days, but by 4 days, the OSs had shortened to ∼70% of control values. By 7 days, the OSs of rods and LM-cones had shortened further to 45% to 55% of control values, while S-cone OS lengths remained unchanged. 
It was significant that, after 2 days exposure to photopic light, the amplitude of the cone b-wave and cone OS lengths were both maintained at control levels in the SD retina, but were reduced to 50% of initial values in the P23H-3 retina (Figs. 5D 6B 6C) . This point is stressed because it is evidence that cones are not directly damaged by 2 days’ exposure to photopic conditions. The loss of cone b-wave response and cone OS length at this time of exposure is specific to the P23H-3 strain, in which rods are damaged with abnormal speed and severity. 
We saw limited evidence that the changes induced by the increase in ambient illumination occurred first in rods, as anticipated by previous observations that cones are more resistant to light damage. 24 25 Specifically, by the 1-day time point (Fig. 5) , the cone b-wave had lost 18% of its amplitude, the rod a-wave had lost 37% and the rod b-wave 27% of amplitude. Perhaps the most striking feature of these data is how closely the changes in the cones matched the changes in the rods, despite the fact that the transgene that creates this vulnerability of the P23H-3 retina is for a protein expressed specifically in rods. 
Discussion
Effect of Accelerated Rod Damage on Cones
In a recent study, 19 we demonstrated the recovery of cones from the damage induced by a relatively short (1 week) exposure of the retina to photopic light (300 lux). In the present study, we investigated features of the initial damage to cones, tracing its time course, its dependence on preceding damage to rods, the degree of involvement of the major subgroups of cones (S, LM) and the correlation between cone OS length and ERG responsiveness. Three features of the cone damage we report are novel: It is very rapid, being substantial within 24 hours of the onset of the light increment used to cause rod damage, it occurs without measurable rod death, and it does not involve the death of cones. In addition, control experiments show that it is not a direct response of cones to the light increment; the light levels used (300 lux) were well within the photopic range. We have further shown that the rod-induced damage to cones involves S- as well as LM-cones. 
The form of cone–rod dependence that we show is distinctive, and contrasts with previous descriptions of cone–rod dependence in the P23H retina. In earlier studies of P23H line 3 heterozygotes 14 15 and line 1 homozygotes 16 and heterozygotes, 14 cone dysfunction was not detected until there had been a severe loss of rods, amounting to an ONL thinning of approximately 50%. In the present study, cone OS lengths and ERG responses were reduced by half after 2 days’ photopic exposure, when rod loss was insufficient to cause a detectable thinning of the ONL. The present findings also contrast with evidence that cones in rhodopsin-mutant degenerative human retinas maintain their function until rod OSs are reduced to 25% of normal length. 26 In the present paradigm, damage to cone OSs was evident when rod OS length was within 30% of normal. The rapid damage to P23H-3 cones in our study cannot be attributed to the increase in ambient illumination, because cone morphology and function were maintained in the SD retina in response to the same exposure, indicating that cones are not directly damaged by this level of light. The present results suggest that cones are dependent on rod integrity (OS morphology and signaling), even when rod cells survive. These features of cone damage raise questions of mechanism and therapeutic opportunity, which are considered in the following section. 
The Mechanism of Cone–Rod Dependence
Two of the mechanisms previously proposed to explain cone–rod dependence (a rod-derived cone survival factor 3 4 27 and depletion-induced oxygen toxicity 12 ) cannot account for the rate-dependent manner in which cones are damaged by rod degeneration, without the presence of an adaptive mechanism. The preservation of cone integrity when rod loss is slow could be reconciled with these mechanisms if, for example, cones adapt to slow reductions in the levels of a rod-derived trophic factor by amplifying intracellular signaling mechanisms, or to slow rises in oxidative stress by expressing antioxidant protective mechanisms. An alternative explanation for this rate dependence is that damaged rods release a toxin, with a concentration that reaches threshold level only when rod damage is sufficiently rapid. The toxin could reach the cytoplasm of cones via gap junctions (the bystander effect proposed by Ripps 13 ) or it could be released into the extracellular space around the cones, as the rod membranes break down. Examples of substances that could damage cones from the extracellular space are excitatory neurotransmitters such as glutamate, or a purine (reviewed in Ref. 28 ) such as ATP. Although the latter ideas remain speculative, the present results encourage the search for rate-dependent mechanisms of cone–rod dependence. 
Death, Damage, and the Capacity for Recovery
The present results provide insight into the mechanisms of rod–cone dystrophies, the forms of human photoreceptor dystrophy in which cone loss is secondary to rod loss. They suggest that, in some forms of the human disease, cone loss may occur when the affected individual experiences an increase in ambient illumination that occurs suddenly and is maintained for 1 to 2 days. Put more constructively, the findings indicate that in rod-fragile retinas, cone damage may be minimized by maintaining both low and constant ambient light exposure. The idea that restricting ambient light will slow retinal degeneration has considerable support from the study of animal models and more limited support from human trials (for reviews see Refs. 12 29 ). The idea that it is important for cone vision to avoid episodes of high illumination is emphasized by the present study. Further work will be needed to extend these findings, but the technology exists for giving a sufferer real-time measures of ambient light and for adjusting, for example, dark glasses dynamically to counter the changes encountered in daily living. 
A still more encouraging note is that the loss of cone (and rod) function in the acute P23H-3 protocol is largely reversible. Much of the functional (ERG) loss results from a shortening of OSs, rather than photoreceptor death, and will readily reverse in both rods 18 and cones. 19 Light is the stimulus for vision, driving the phototransduction mechanisms of the OS. Light is also a powerful regulator of the cell biology of photoreceptors, and light management seems likely to emerge as an effective tool in the management of photoreceptor stability, whether the instability is caused by genetic mutations or by environmental factors such as bright light, depletion-induced hyperoxia, or age. 
Mission and Nemesis: The Paradox of Light-Induced Damage
Most organisms make some investment in light detection, to optimize their behavioral adaptation and individual survival. In higher primates it is often judged that vision is the most powerful of the senses and has dedicated to it numerous subcortical structures and a large component (the occipital lobe and beyond) of neocortex. Less structurally conspicuous visual pathways control circadian rhythms, eye movements, and pupil size. In plants, light is directly harnessed to create energy-rich molecules. 
It is an interesting paradox that, although good vision is clearly a major factor in the individual’s struggle for survival and quality of life, and photosynthesis is the source of plant life, the absorption of light—whether by 11-cis retinal in animals or by photosystems in plants—is an intrinsically damaging process. Light-experienced photoreceptors 30 31 32 and the photosynthetic organelles of plant cells consistently show evidence of damage and an upregulation of protective factors, 33 34 35 a seemingly unavoidable accompaniment of light absorption. 
The paradox is particularly clear in vertebrate photoreceptors. Light decreases dark current, ion pump activity in inner segments, and oxidative phosphorylation (in response to a decrease in ATP turnover). Thus, light can be expected to decrease the production of reactive oxygen species in photoreceptor mitochondria. Nevertheless, light exposure causes severe damage to OSs. It appears possible that the absorption of light is directly damaging to membranes in which the chromophore is embedded. Winkler 36 has recently argued that the rapid turnover of OS membrane is a substitute for membrane repair, evolving because of the magnitude of the repair task in photoreceptors. 
The point of this argument is that repair of the OSs, by continuous rebuilding of its membranes at the cilial base, is prominent in vertebrate photoreceptors, and can be harnessed to restore vision in human patients where (as in most cases) many photoreceptors survive but are poorly functional. 
Figure 1.
 
Frequency of TUNEL-labeled photoreceptor cells in the P23H-3 and SD rat retina during 7 days’ exposure to cyclic photopic light. Fragmentation of photoreceptor DNA occurred earlier and was several orders of magnitude greater in the P23H-3 retina (A) than in the SD retina (B). TUNEL+ photoreceptors were most frequent in the superior midperipheral region of the retina in both the P23H-3 (C) and SD (D) strains. Data are presented as the mean ± SEM for groups of six animals at each time point. *Data points that are significantly different from 0 day control values, by analysis of variance (P < 0.05).
Figure 1.
 
Frequency of TUNEL-labeled photoreceptor cells in the P23H-3 and SD rat retina during 7 days’ exposure to cyclic photopic light. Fragmentation of photoreceptor DNA occurred earlier and was several orders of magnitude greater in the P23H-3 retina (A) than in the SD retina (B). TUNEL+ photoreceptors were most frequent in the superior midperipheral region of the retina in both the P23H-3 (C) and SD (D) strains. Data are presented as the mean ± SEM for groups of six animals at each time point. *Data points that are significantly different from 0 day control values, by analysis of variance (P < 0.05).
Figure 2.
 
Measurements of retinal thickness in the P23H-3 and SD rat retina after a rise in ambient illumination. The thickness of the ONL was greater in the SD retina, but there was no thinning of the layer in either strain during 7 days of exposure to photopic light.
Figure 2.
 
Measurements of retinal thickness in the P23H-3 and SD rat retina after a rise in ambient illumination. The thickness of the ONL was greater in the SD retina, but there was no thinning of the layer in either strain during 7 days of exposure to photopic light.
Figure 3.
 
Sample waveforms of rod and cone ERG responses matched with immunohistochemical labeling of rod (red), LM-cone (green), and S-cone (blue) OSs. In P23H-3 (A, B) and SD (C, D) retinas, 7 days’ exposure to photopic light reduced the amplitude of rod and cone ERG responses, and was accompanied by a shortening of photoreceptor OSs. This effect was more rapid and severe in the P23H-3. The ERG waveforms in (A) and (C) were elicited to a flash stimulus of 44.5 cds/m2. Vertical arrow: the time of the flash stimulus. Scale bars, 20 μm.
Figure 3.
 
Sample waveforms of rod and cone ERG responses matched with immunohistochemical labeling of rod (red), LM-cone (green), and S-cone (blue) OSs. In P23H-3 (A, B) and SD (C, D) retinas, 7 days’ exposure to photopic light reduced the amplitude of rod and cone ERG responses, and was accompanied by a shortening of photoreceptor OSs. This effect was more rapid and severe in the P23H-3. The ERG waveforms in (A) and (C) were elicited to a flash stimulus of 44.5 cds/m2. Vertical arrow: the time of the flash stimulus. Scale bars, 20 μm.
Figure 4.
 
Analysis of cone survival in the P23H-3 retina after photopic light exposure. The average density of cones (A) and the length of their OSs (B) were quantified from flatmounted retinas immunolabeled for LM opsin. (C, D) Representative images from the superior midperiphery of LM opsin-labeled retinal flatmounts. Flatmounts from three animals were assessed and data are presented as the mean ± SEM. Scale bar, 20 μm.
Figure 4.
 
Analysis of cone survival in the P23H-3 retina after photopic light exposure. The average density of cones (A) and the length of their OSs (B) were quantified from flatmounted retinas immunolabeled for LM opsin. (C, D) Representative images from the superior midperiphery of LM opsin-labeled retinal flatmounts. Flatmounts from three animals were assessed and data are presented as the mean ± SEM. Scale bar, 20 μm.
Figure 5.
 
Time course of loss of rod and cone ERG amplitudes in P23H-3 and SD retinas in response to photopic light exposure. (A) In the P23H-3 retina, rod, and cone components were rapidly reduced in amplitude by an increase in photopic light levels. (B) In the SD retina, rod and cone ERG responses were also reduced by photopic light exposure, but the reductions were delayed and less severe. Direct comparison of rod a-wave (C), cone b-wave (D), and rod b-wave (E) amplitudes between the P23H-3 and SD rat retina during 7 days’ photopic light exposure. The stimulus used for all ERG measures was a flash of 44.5 cd/m2 intensity. Data are presented as the mean ± SEM for eight animals.
Figure 5.
 
Time course of loss of rod and cone ERG amplitudes in P23H-3 and SD retinas in response to photopic light exposure. (A) In the P23H-3 retina, rod, and cone components were rapidly reduced in amplitude by an increase in photopic light levels. (B) In the SD retina, rod and cone ERG responses were also reduced by photopic light exposure, but the reductions were delayed and less severe. Direct comparison of rod a-wave (C), cone b-wave (D), and rod b-wave (E) amplitudes between the P23H-3 and SD rat retina during 7 days’ photopic light exposure. The stimulus used for all ERG measures was a flash of 44.5 cd/m2 intensity. Data are presented as the mean ± SEM for eight animals.
Figure 6.
 
Quantitative analysis of rod and cone OS length changes in P23H-3 and SD retinas in response to photopic light exposure. Exposure significantly reduced the length of rod (A), LM-cone (B) and S-cone (C) OSs in both the P23H-3 and SD retina, and this shortening was more rapid in the P23H-3. Data are presented as the mean ± SEM for groups of six animals at each time point. *Data points that are significantly different from 0 day control values, by analysis of variance (P < 0.05).
Figure 6.
 
Quantitative analysis of rod and cone OS length changes in P23H-3 and SD retinas in response to photopic light exposure. Exposure significantly reduced the length of rod (A), LM-cone (B) and S-cone (C) OSs in both the P23H-3 and SD retina, and this shortening was more rapid in the P23H-3. Data are presented as the mean ± SEM for groups of six animals at each time point. *Data points that are significantly different from 0 day control values, by analysis of variance (P < 0.05).
 
JohnSK, SmithJE, AguirreGD, MilamAH. Loss of cone molecular markers in rhodopsin-mutant human retinas with retinitis pigmentosa. Mol Vis. 2000;6:204–215. [PubMed]
Mohand-SaidS, Deudon-CombeA, HicksD, et al. Normal retina releases a diffusible factor stimulating cone survival in the retinal degeneration mouse. Proc Natl Acad Sci USA. 1998;95:8357–8362. [CrossRef] [PubMed]
HicksD, SahelJ. The implications of rod-dependent cone survival for basic and clinical research. Invest Ophthalmol Vis Sci. 1999;40:3071–3074. [PubMed]
LeveillardT, Mohand-SaidS, LorentzO, et al. Identification and characterization of rod-derived cone viability factor. Nat Genet. 2004;36:755–759. [CrossRef] [PubMed]
YuDY, CringleSJ, SuEN, YuPK. Intraretinal oxygen levels before and after photoreceptor loss in the RCS Rat. Invest Ophthalmol Vis Sci. 2000;41:3999–4006. [PubMed]
YuDY, CringleS, ValterK, WalshN, LeeD, StoneJ. Photoreceptor death, trophic factor expression, retinal oxygen status, and photoreceptor function in the P23H rat. Invest Ophthalmol Vis Sci. 2004;45:2013–2019. [CrossRef] [PubMed]
Padnick-SilverL, Kang DerwentJJ, GiulianoE, NarfstromK, LinsenmeierRA. Retinal oxygenation and oxygen metabolism in Abyssinian cats with a hereditary retinal degeneration. Invest Ophthalmol Vis Sci. 2006;47:3683–3689. [CrossRef] [PubMed]
GellerS, KrowkaR, ValterK, StoneJ. Toxicity of hyperoxia to the retina: evidence from the mouse. Adv Exp Med Biol. 2006;572:425–437. [PubMed]
YamadaH, YamadaE, HackettSF, OzakiH, OkamotoN, CampochiaroPA. Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina. J Cell Physiol. 1999;179:149–156. [CrossRef] [PubMed]
WellardJ, LeeD, ValterK, StoneJ. Photoreceptors in the rat retina are specifically vulnerable to both hypoxia and hyperoxia. Vis Neurosci. 2005;22:501–507. [PubMed]
ShenJ, YangX, DongA, et al. Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa. J Cell Physiol. 2005;203:457–464. [CrossRef] [PubMed]
StoneJ, MaslimJ, Valter-KocsiK, et al. Mechanisms of photoreceptor death and survival in mammalian retina. Prog Retin Eye Res. 1999;18:689–735. [CrossRef] [PubMed]
RippsH. Cell death in retinitis pigmentosa: gap junctions and the ‘bystander’ effect. Exp Eye Res. 2002;74:327–336. [CrossRef] [PubMed]
MachidaS, KondoM, JamisonJA, et al. P23H rhodopsin transgenic rat: correlation of retinal function with histopathology. Invest Ophthalmol Vis Sci. 2000;41:3200–3209. [PubMed]
ChrysostomouV, StoneJ, ValterK. Life history of cones in the rhodopsin-mutant P23H-3 rat: evidence of long term survival. Invest Ophthalmol Vis Sci. .Published online December 30, 2008
PinillaI, LundRD, SauveY. Enhanced cone dysfunction in rats homozygous for the P23H rhodopsin mutation. Neurosci Lett. 2005;382:16–21. [CrossRef] [PubMed]
WalshN, Van DrielD, LeeD, StoneJ. Multiple vulnerability of photoreceptors to mesopic ambient light in the P23H transgenic rat. Brain Res. 2004;1013:197–203.
JozwickC, ValterK, StoneJ. Reversal of functional loss in the P23H-3 rat retina by management of ambient light. Exp Eye Res. 2006;83:1074–1080. [CrossRef] [PubMed]
ChrysostomouV, StoneJ, StoweS, BarnettNL, ValterK. The status of cones in the rhodopsin mutant P23H-3 retina: light-regulated damage and repair in parallel with rods. Invest Ophthalmol Vis Sci. 2008;49:1116–1125. [CrossRef] [PubMed]
MaslimJ, ValterK, EgenspergerR, HollanderH, StoneJ. Tissue oxygen during a critical developmental period controls the death and survival of photoreceptors. Invest Ophthalmol Vis Sci. 1997;38:1667–1677. [PubMed]
NixonPJ, BuiBV, ArmitageJA, VingrysAJ. The contribution of cone responses to rat electroretinograms. Clin Exp Ophthalmol. 2001;29:193–196. [CrossRef]
BowersF, ValterK, ChanS, WalshN, MaslimJ, StoneJ. Effects of Oxygen and bFGF on the vulnerability of photoreceptors to light damage. Invest Ophthalmol Vis Sci. 2001;42:804–815. [PubMed]
StoneJ, MervinK, WalshN, ValterK, ProvisJ, PenfoldP. Photoreceptor stability and degeneration in mammalian retina: lessons from the edge.PenfoldP ProvisJ eds. Macular Degeneration: Science and Medicine in Practice. 2005;149–165.Springer Verlag New York.
CiceroneC. Cones survive rods in the light-damaged eye of the albino rat. Science. 1976;194:1183–1185. [CrossRef] [PubMed]
LaVailMM. Survival of some photoreceptor cells in albino rats following long-term exposure to continuous light. Invest Ophthalmol. 1976;15:64–70. [PubMed]
CideciyanAV, HoodDC, HuangY, et al. Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man. Proc Natl Acad Sci USA. 1998;95:7103–7108. [CrossRef] [PubMed]
Mohand-SaidS, HicksD, SimonuttiM, et al. Photoreceptor transplants increase host cone survival in the retinal degeneration (rd) mouse. Ophthalmic Res. 1997;29:290–297. [CrossRef] [PubMed]
BurnstockG. Purinergic signalling and disorders of the central nervous system. Nat Rev Drug Discov. 2008;7:575–590. [CrossRef] [PubMed]
PaskowitzDM, LaVailMM, DuncanJL. Light and inherited retinal degeneration. Br J Ophthalmol. 2006;90:1060–1066. [CrossRef] [PubMed]
PennJ, AndersonR. Effects of light history on the rat retina. Prog Retin Res. 1991;11:75–98. [CrossRef]
PennJ, NaashM, AndersonR. Effect of light history on retinal antioxidants and light damage susceptibility in the rat. Exp Eye Res. 1987;44:779–788. [CrossRef] [PubMed]
PennJS, AndersonRE. Effect of light history on rod outer-segment membrane composition in the rat. Exp Eye Res. 1987;44:767–778. [CrossRef] [PubMed]
IversonT. Evolution and unique bioenergetic mechanisms in oxygenic photosynthesis. Curr Opin Chem Biol. 2006;10:91–100. [CrossRef] [PubMed]
KruseO. Light-induced short-term adaptation mechanisms under redox control in the PS II-LHCII supercomplex?—LHC II state transitions and PS II repair cycle. Naturwissenschaften. 2001;88:284–292. [CrossRef] [PubMed]
AroE, VirginI, AnderssonBE. Photoinhibition of photosystem. II. Inactivation, protein damage and turnover. Biochim Biophys Acta. 1993;1143:113–134. [CrossRef] [PubMed]
WinklerBS. An hypothesis to account for the renewal of outer segments in rod and cone photoreceptor cells: renewal as a surrogate antioxidant. Invest Ophthalmol Vis Sci. 2008;49:3259–3261. [CrossRef] [PubMed]
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