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Keywords:

  • agmatine;
  • apoptosis;
  • ion gating;
  • lactate dehydrogenase;
  • P23H rat;
  • retinal degeneration

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague–Dawley (SD) rats and a rat model of retinitis pigmentosa [proline-23-histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24-h light exposure period. Short-term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light-damaged retinas in both SD and the P23H rat retina.

Abbreviations used
AGB

agmatine

LDH

lactate dehydrogenase

ONL

outer nuclear layer

P

post-natal day

P23H

proline-23-histidine

RP

retinitis pigmentosa

SD

Sprague–Dawley

TUNEL

terminal deoxynucleotidyl transferase dUTP nick-end labeling

The mammalian retina is a complex array of neurons and glia that converts and encodes light energy into electrical impulses to begin the visual process. Progressive diseases, such as inherited retinal degenerations, cause a loss or impairment of vision through the degeneration of photoreceptors (Jones et al. 2005). The clinical picture of retinal degeneration is often similar, and despite multiple genetic mutations (http://www.sph.uth.tmc.edu/Retnet/disease.htm), all retinal degenerations have the same outcome: photoreceptor death and functional blindness. The extent and the rate of degeneration is dependent on intrinsic factors such as genetic characteristics (Ablonczy et al. 2000; Grimm et al. 2000; Nir et al. 2001; Vaughan et al. 2003), and extrinsic factors such as duration, intensity, and history of light exposure (Grimm et al. 2001; Li et al. 2001; Nir et al. 2001; Organisciak et al. 2003). This study examined one such factor, intense light exposure, which has long been known to have deleterious effects on the normal retina (Noell et al. 1966; Noell 1980; Penn et al. 1989; Fain and Lisman 1993; Ablonczy et al. 2000; Marc and Jones 2003), and on retinas pre-disposed to degeneration (Nir et al. 2001; Organisciak et al. 2003; Vaughan et al. 2003; Walsh et al. 2004; Cideciyan et al. 2005; Zhang et al. 2005).

Retinitis Pigmentosa (RP) encompasses a group of inherited genetic diseases that cause progressive degeneration of photoreceptors (Illing et al. 2002; Traverso et al. 2002; Kalloniatis and Fletcher 2004). Light exposure leads to photoreceptor degeneration at a faster rate than would normally occur because of the inherited mutation in the transgenic model; the proline-23-histidine (P23H) rat model of RP (Walsh et al. 2004; Jozwick et al. 2006), and other rodent models of RP (LaVail et al. 1987; Naash et al. 1996; Wang et al. 1997; Organisciak et al. 1999). Signs of degeneration are detected by electron microscopy as early as 1 h after light exposure (Kuwabara and Gorn 1968; Grignolo et al. 1969; O’Steen et al. 1972). These structural alterations are also associated with functional losses. For example, after exposure to a constant light of 1700 lux for 24 h, 8-week-old rats showed almost complete abolition of the electroretinogram response (Penn et al. 1989; Li et al. 2001, 2003).

Retinal degeneration induced by continuous light exposure may be caused by prolonged activation of the photoreceptors (Noell et al. 1966; Noell 1980; Lisman and Fain 1995), which may result in altered cGMP levels and distorted cation channel permeability (Lolley et al. 1980). Increased permeability of ligand-gated channels is observed in the rd/rd degenerating retina and in the Royal College of Surgeons rat retina (Kalloniatis et al. 2002; Acosta et al. 2005), where increased photoreceptor permeation to agmatine (AGB), a channel-permeable organic cation, has been shown before apoptotic markers. Abnormal functioning of cationic channels could increase Ca2+ levels in the retina and therefore this event may be triggering apoptosis (Fain and Lisman 1999). Although physiological changes in light levels lead to closure of cyclic nucleotide gated cation channels in the outer segment (Fesenko et al. 1985), entry of cations (i.e. Ca2+) via cyclic nucleotide or through other cation channels, has been thought to be responsible for some forms of degeneration, for example in the rd/rd mice (Farber 1995), or in the GCAP1 and Y99C mutations (Payne et al. 1998; Sokal et al. 1998). Although cation entry may be one mechanism leading to photoreceptor apoptosis after light exposure, other mechanisms include oxidative damage caused by the bleaching of the rhodopsin molecule (Ablonczy et al. 2000; Grimm et al. 2001; Vaughan et al. 2003), and also the activation of microglia and chemokines (Zhang et al. 2005).

Studies in retinal degeneration animal models have shown down-regulation of metabolic activity secondary to photoreceptor degeneration and alteration in lactate dehydrogenase (LDH; EC 1.1.1.27) isoenzymes (Bonavita et al. 1963; Graymore 1963; Bonavita 1965; Acosta et al. 2005). Marked alteration in amino acid levels is observed in the developing Royal College of Surgeons rat retina (Fletcher and Kalloniatis 1996, 1997), and there is also a significant decrease in LDH activity secondary to photoreceptor degeneration (Bonavita et al. 1963; Bonavita 1965). Given that LDH activity increased in response to short-term metabolic insult in hypoglycemic and anoxic rat retinas (Acosta and Kalloniatis 2005), we expect that the initial response to light-induced stress would be an upsurge in energy requirement (increased LDH activity) followed by a decrease in LDH activity as a result of photoreceptor cell death. We also investigated the time course of photoreceptor labeling to an apoptotic marker [terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and developed a protocol for the colocalization with a probe for cation channel gating (AGB labeling) and TUNEL labeling, to determine if cation entry is required to trigger apoptosis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

All procedures were approved by The University of Auckland Animal Ethics Committee and comply with the Association for Research in Vision and Ophthalmology statement for the use of animals in Ophthalmic and Vision research. Both Sprague–Dawley (SD) and P23H rats were born and reared at the Faculty of Medical & Health Sciences Animal breeding facility. The P23H-line 3 homozygous animals were bred with albino SD rats to produce heterozygous rats carrying the P23H mutation in one chromosome. The P23H-line 3 rat undergoes slow rod degeneration and is consistent with clinical findings of RP patients (Machida et al. 2000; Traverso et al. 2002; Ranchon et al. 2003). Moreover, although both homozygous and heterozygous P23H rats undergo a decline in visual response (Pinilla et al. 2005), heterozygous rats were used, to match the prevalent genotype found in human P23H conditions (Machida et al. 2000). A mixture of male and female rats were used in the light experiments with young animals [post-natal day 20 (P20)] and females for the adult experiments (P59). Adult and young SD rats were kept in cycled 12 h white light: 12 h dark per day [mean illuminance was 270 lux with 95% confidence intervals (CI) of (lower and higher CI: 249, 298 lux)], while adult and young P23H rats were reared at 12 h light [60 lux with 95% CI of (lower and higher CI: 34, 90 lux)]: 12 h dark cycle during their lifespan. The CI were derived by using a non-parametric bootstrap approach, a method that does not rely on any underlying assumptions about the data (Efron and Tibshirani 1986). A pseudoreplication of the data set of light readings (total of nine for each condition), was used to estimate the true distribution of the mean value including the lower and upper CI (Foster and Bischof 1987; Darvas et al. 2004; Bui et al. 2005). Although ambient light levels have been shown to lead to altered susceptibility to light damage, e.g. Walsh et al. (2004), Jozwick et al. (2006), our experimental paradigm differed from other studies in that both groups of animals were raised in low photopic light levels rather than very low (scotopic–mesopic) versus photopic levels, e.g. Walsh et al. (2004), Jozwick et al. (2006). The range of light adaptation in the albino rat visual system is at least over a six log unit range, with a distinct rod/cone break in threshold versus intensity (increment threshold) curves (Green 1973; Green and Powers 1982). Although reported light levels are in lux and therefore calibrated for the human visual system, it is possible to estimate the relative light adaptation range (scotopic–mesopic–photopic) based upon these measurements and known adaptation characteristics of the albino rat (Green 1973), and primate (Kalloniatis and Harwerth 1990, 1991), and the assumption that a vertebrate mammalian rod will display quantal efficiency as does the human rod (Hecht et al. 1942). Further, an illuminance of 60 and 270 lux, will lead to a luminance of 9.5 and 43 cd/m2 (for a surface with a reflectance of 0.5). The relative troland value for the two luminance levels would correspond to 269 and 1216 troland for 9.5 and 43 cd/m2 for a 6 mm pupil, placing the primate visual system just into Weber contrast region (within 1–2 log units above cone threshold: Kalloniatis and Harwerth 1990). With the assumption of quantal efficiency for the primate and rodent rod, and the fact that both visual system display a rod/cone break approximately 2–3 log units above absolute rod threshold (Green 1973; Kalloniatis and Harwerth 1990, 1991), and the calculations above, it is likely that both the SD and P23H rat are operating within 1–2 log units of cone adaptation at the low photopic range (within 0.66 log units of each other).

Light-exposure experiments started immediately after the animals were transported to the laboratory (transportation time from 5 to 10 min). All experiments were started at approximately the same time in the morning (between 10 and 11 am), and the analysis was conducted immediately after the relevant light exposure, of either 2 or 24 h light exposure. For experiments when tissue was collected 1 week after light exposure, the animals were anesthetized and their eye removed for analysis at around 10–11 am. A total of 80 rats, including repeated experiments were used in this study.

The different experimental paradigms employed in this study are outlined in Fig. 1 with all experiments using five animals (one eye was used for histology/immunocytochemistry and the second eye for biochemical assays). The illuminance was 2700 lux, created by placing fluorescent white light lamps directly above the cages. Animals were selected at P20, when cell death by apoptosis is visualized in P23H rats (Walsh et al. 2004; Yu et al. 2004), and during the active phase of photoreceptor degeneration in P23H rat at age P59 (Lewin et al. 1998). P23H and SD rats were subjected to a range of light exposure including double (Fig. 1a) or a single (Fig. 1b) 24 h exposure at P20 and collecting the tissue 1 week later. We will be referring to ‘1 week after light exposure’ throughout the manuscript. As Fig. 1 illustrates, the 1 week time frame reflects the ‘total’ time and thus is only 6 days after the intense light exposure was completed, but 7 days from the beginning of the experiment. In one paradigm, we collected the tissue immediately after the 24 h light exposure (Fig. 1c). A similar approach was used for P59 animals using a single 24 h exposure where the tissue was collected immediately after (Fig. 1f) or after 1 week (Fig. 1e). In one experimental series, P59 rats were exposed for 2 h and the tissue collected immediately after the light exposure (Fig. 1d).

image

Figure 1.  Schematic diagram showing the different experimental manipulations employed in this study.

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Dissection, incubation, and sectioning

Rats from each exposure group were deeply anesthetized with medetomidine hydrochloride (1 mg/mL) and ketamine hydrochloride (100 mg/mL) before their eyes were dissected from the orbit. The posterior eyecup was made flat and mounted on filter paper (0.8 μm pore size, Gelman Sciences, Ann Arbor, MI, USA) with the neural retina attached to the filter. The sclera and attached pigmented epithelium were peeled off. The filter paper mounted retinas were incubated for 5 min in a modified physiological medium with 25 mmol/L AGB (Marc et al. 2005; Sun and Kalloniatis 2006). The physiological medium was bubbled in 95% O2 and 5% CO2 at 37°C. AGB is a polyamine that permeates cells via ionic channels, and is a suitable marker of permeability of cationic channels, including those found in the vertebrate retina (Yoshikami 1981; Kuzirian et al. 1986; Marc 1999a; Kalloniatis et al. 2002, 2004; Marc et al. 2005; Sun and Kalloniatis 2006). AGB gating has been previously used as a marker of impending photoreceptor degeneration (Kalloniatis et al. 2002; Acosta et al. 2005), and we have used it in this series of experiments as an indicator of the properties of photoreceptor cation channels after light exposure. Brief incubation of the isolated retinas in physiological buffer containing AGB resulted in AGB entry into cells with open cationic channels. The AGB molecule is subsequently trapped using aldehyde fixation (Marc 1999a,b; Marc et al. 2005; Sun and Kalloniatis 2006). Thus, the technique allows for the assessment of endogenous excitatory drive and determination of the activity of cation channels (Marc et al. 2005).

Immunocytochemistry

Retinas were processed for immunocytochemistry after immersing them in 4% paraformaldehyde and 0.01% glutaraldehyde fixative for 30 min. The tissue was washed in 0.1 mol/L phosphate buffer and then cryoprotected in graded sucrose solutions up to 30%. Retinal sections (16 μm) were cut using a cryostat (Leica CM3050 S, Heidelberg, Germany) and collected onto Superfrost Plus glycerol-coated slides and immunolabeled using a rabbit polyclonal anti-AGB antibody (Marc 1999a,b; Sun and Kalloniatis 2006). The primary antibody was detected using goat anti-rabbit Alexa Fluor A594 or A488 antibodies (Molecular Probes, Eugene, OR, USA). Retinal sections were viewed using a fluorescent microscope with a ×40 objective lens, and digital images for quantification analysis were acquired with a LEICA DC 500 (Leica Microsystems Ltd, Heerbrugg, Germany) camera. AGB-labeled photoreceptors were counted in areas of the retina with no signs of conspicuous AGB labeling or anatomical damage (Marc 1999a). Cell counts were conducted across the whole retina except for the edges within approximately 1.5 mm from the ora serrata. Values were represented as AGB-labeled cells per mm2 of retina.

Apoptosis assay

A fluorescein-based in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) was used to visualize apoptotic cells. Retinal sections were processed for TUNEL following the supplier procedure. Retinas were immunolabeled using AGB antibodies and the TUNEL technique was applied 1 h before completion of the secondary antibody labeling. As a negative control, the retinal sections were processed omitting the incubation step with terminal deoxynucleotidyl transferase during DNA nicked-end labeling. Apoptotic cells were visualized by fluorescence microscopy using FITC filters. Images were acquired using confocal laser scanning microscopy (Leica Microsystems TCS 4D). The number of TUNEL labeled cells were counted per unit area and the number of AGB positive cells colocalizing with TUNEL were normalized against number of AGB cells in unexposed retinas.

Morphological analysis

Retinal sections were stained with toluidine blue and examined and photographed using bright field microscopy. The ratio of the outer nuclear layer (ONL) thickness to the whole retina (inner limiting membrane-nerve fiber layer) was measured using Scion Image software (Scion Corporation, Frederick, MD, USA). One retina per animal (n = 5) was used in these experiments. Values were expressed as the ratio of ONL thickness over total retinal length. Because of the morphological changes induced by the light damage, it was not always possible to determine the orientation of the photoreceptors, and thus to assure that the absolute size of the ONL was not biased by the non-vertical orientation of the tissue, we calculated the ONL/total retinal thickness ratio. The ratio value is not dependent upon photoreceptor orientation and allows for comparison across all experimental conditions.

Biochemical analysis

For the biochemical analysis, individual retinas (n = 5 retinas for each condition), were homogenized in ice-cold 0.9% saline. After centrifugation at 6000 g for 6 min, the supernatant was removed, diluted to 1 : 5 in 0.9% saline and used for the assays. Protein concentration was measured in a colorimetric reaction using a bicinchoninic acid protein assay reagent and detected using a microplate reader at 565 nm wavelength (ELx800 Bio-Tek Instruments Inc., Winooski, VT, USA). LDH activity was determined in each retina (n = 5) for each of the light-exposure experiments, using the Thermo Trace LDH-P reagent to which a known volume of the supernatant was added. Six measurements were taken from each retina to calculate the mean activity for that retina that was subsequently normalized to protein content. The change in NADH absorbance over time (ΔA/min) was measured in a spectrophotometer (Shimadzu UV-2501; Kyoto, Japan) to obtain the activity of LDH, which was expressed in μmoles per minute per milligram of protein.

LDH isoenzyme distribution

Lactate dehydrogenase isoenzymes were separated using a 1.2% Agarose gel (Agarose 1000, Invitrogen, Carlsbad, CA, USA) made in a buffer containing 25 mmol/L Tris and 250 mmol/L glycine, pH 8.6 (Acosta et al. 2005). The running buffer consisted of 5 mmol/L Tris and 40 mmol/L glycine, pH 9.5 and the gel was run for 2 h at 80 V. All retinal samples consisted of 15 μg of retina sample mixed with 10% of loading buffer (0.05% bromphenol blue/8% sucrose). Upon completion of electrophoresis, the gel was placed in 0.01 mol/L Tris buffer, pH 8.6 containing 0.04 mol/L l-(+)-lactic acid, 4 mmol/L NAD+, 9.8 mmol/L nitro blue tetrazolium, and 5.5 mmol/L phenazine methosulfate for 1 h. Following staining, the gel was washed with running tap water for 1–2 h and images were captured with a Kodak DC290 zoom digital camera and processed using the Kodak electrophoresis documentation and analysis system (RADS 290; Eastman Kodak Company, New Haven, CT, USA). To obtain the relative distribution of each of the LDH isoenzymes the intensity of each LDH band was measured using Kodak 1D Image Analysis Software. The relative activity of each isoenzyme was calculated based on the total LDH activity for each sample (n = 5 retinas per condition).

Statistics

For all the assays the mean average value of unexposed (control) and exposed rat retinas were statistically analyzed using a Student’s t-test or one-way anova whenever appropriate. The bootstrap analysis (Efron and Tibshirani 1986) was used in the estimation of the mean light levels.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Effect of light exposure on retinal morphology

Many studies have found a significant decline in the number of cells in the ONL of RP rats as a result of light damage (Abler et al. 1996; Reméet al. 1998; Vaughan et al. 2003). Therefore, as an indicator of morphological damage caused by light exposure, we determined the ratio between the ONL and the whole retinal thickness in adult and young SD rats reared in 12 h dark/light (270 lux) and adult and young P23H rats reared in 12 h dark/light (60 lux). Changes in the number of photoreceptors (outer nuclear thickness), will be reflected by an increase or decrease of this ratio, under the assumption that the inner retina is not modified shortly after light exposure.

Intense light exposure for 2 h had no significant effect (anova, p = 0.06) on the thickness of the ONL of adult SD rats (n = 5), indicating that morphological damage is not triggered immediately after light exposure. In addition, 24 h light exposure showed a non-significant (Student’s t-test, p = 0.06) reduction in the ONL thickness of adult SD rats (n = 5; Fig. 2a). However, at P21, SD rats exposed for 24 h showed a significant reduction (Student’s t-test, p < 0.001) in the ONL. An increase in the ONL thickness at P27 is observed in both unexposed SD and P23H rats, which is due to a developmentally controlled expansion of the photoreceptor layer in the third week of the rat development (Vogel and Möller 1980). However, 24 h light exposure at P20 caused a significant reduction in the ONL thickness of both SD and P23H rats at P27 (Student’s t-test, p < 0.05) (Fig. 2a and b). Adult P23H rats exposed to light for 24 h had a reduction in the ONL thickness, and 1 week later (P66) this ratio decreased significantly compared with unexposed rats (Student’s t-test, p < 0.001). We also analyzed whether further exposure affected the morphology of the P23H retina, and the results in Fig. 2b show that double exposure of young P23H retinas reduced the ratio a further 16.7% at P27 (anova, p < 0.001).

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Figure 2.  The effect of light exposure on the ratio of the outer nuclear layer (ONL) : total retinal thickness at different developmental ages; P21 (immediately after light exposure), P27 (1 week after light exposure), P60 (immediately after light exposure), and P66 (1 week after light exposure). (a) Shows the change in the ONL thickness of unexposed and light-exposed (for 2 and 24 h) female Sprague–Dawley (SD) rats. (b) Shows the relative change in the ONL thickness of unexposed and exposed (2 and 24 h, and double 24 h) P23H rats. When indicated, significant values compared with unexposed retinas at the same age are: Student’s t-test *p < 0.05, **p < 0.001, anova statistical comparison of unexposed and 2 h exposed data was not significant (NS).

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Effect of light exposure on photoreceptor AGB labeling

After light exposure, retinas were incubated in physiological brain buffer containing AGB to assess photoreceptor cation channel permeation (Kalloniatis et al. 2002; Marc et al. 2005; Acosta et al. 2005; Sun and Kalloniatis 2006). Figure 3 show examples of the pattern of AGB labeling immediately or 1 week after a 24 h light exposure of young retinas. In SD retinas, cation channel permeability probed by AGB entry was always greater after light exposure if compared with unexposed retinas (Fig. 3a and b), and decreased 1 week later (Fig. 3c and d). In P23H unexposed retinas, there were several AGB-labeled photoreceptors (Fig. 3e), and after light exposure the number of AGB-labeled photoreceptors increased (Fig. 3f). One week after exposure of the P23H retina, the number of AGB-labeled photoreceptors was still high (Fig. 3g and h). The quantification of AGB labeling is illustrated in Fig. 4 and Table 1. AGB labeling in SD and P23H indicates that the onset of retinal degeneration in P23H rats has an effect on channel permeability (compare control values in Fig. 4a and b), given that unexposed P23H rats have higher AGB-labeled photoreceptors per unit area than unexposed SD rats at all ages analyzed. Cation gating was readily altered after 24 h, as indicated by the significant increase in AGB-labeled photoreceptors in SD and P23H retinas (Fig. 4). The total number of labeled cells was considerably lower for light-exposed young SD reared at 270 lux than P23H rats reared at 60 lux (Table 1). In addition, exposure for 2 h triggered AGB permeation into photoreceptors, in both adult SD and P23H retinas. The results show that photoreceptor cells display a marked increase in cation channel permeability within 2 h of light exposure.

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Figure 3.  Confocal micrographs showing the pattern of agmatine (AGB) immunolabeling immediately (b and f) or 1 week (d and h), after light exposure of post-natal day 20 (P20) rats. Control unexposed retinas correspond to a, c, e, and g. White arrows are indicating photoreceptors that have AGB labeling. (a–d) Shows AGB labeling of Sprague–Dawley (SD) rat retinas. (e–h) Shows AGB labeling of P23H rat retinas. ONL, outer nuclear layer; IPL, inner plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. The white lines on the left-hand side of each image indicate the extension of the INL. Calibration bar is equal to 20 μm.

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Figure 4.  Quantitative analysis of photoreceptors agmatine (AGB) labeling in unexposed and exposed (2 and 24 h) retinas. Counts were taken at ages P21 (immediately after light exposure), P27 (1 week after light exposure), P60 (immediately after light exposure), and P66 (1 week after light exposure). (a) Shows the effect of 2 and 24 h light exposure on Sprague–Dawley (SD) rat retinas. (b) Shows the effect of 2 and 24 h light exposure on P23H retinas. Note the increase in the y-axis in panel (b) for P21 and P27. Significant values compared with unexposed retinas of same age are: Student’s t-test *p < 0.05, **p < 0.001, anova statistical comparison of unexposed and 2 h exposed data was *p < 0.05, **p < 0.001.

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Table 1.   Quantitative data of AGB labeling of photoreceptors in SD and P23H rat retinas. One retina from five animals was used for every experimental condition
 P21P27P60P66
Unexposed24 hUnexposed24 hUnexposed24 h2 hUnexposed24 h
  1. AGB, agmatine; ONL, outer nuclear layer; SD, Sprague–Dawley; P23H, proline-23-histidine.

SD
 Number of AGB positive cells in the ONL64337630701699072209
 Total area of ONL examined (μm2)254375309375308750220000206875239 375120000203125240000
 No. of cells per mm2252110420138342703759359873
P23H
 Number of AGB positive cells in the ONL1419907310827833611274301
 Total area of ONL examined (μm2)21187512812518812517187516000017625070625143125146250
 No. of cells per mm266577483906311492189715985172060

Effect of light exposure on photoreceptor TUNEL labeling and colocalization with AGB labeling

To determine whether increased AGB permeation was related to the initiation of cell death, we analyzed light-exposed retinas with AGB and with a marker of apoptotic cells. Figure 5 shows the colocalization pattern of AGB (red) and the apoptotic marker (green) on adult unexposed and light-exposed P23H retinas. P23H unexposed retinas had very few apoptotic cells at P60 (Fig. 5a–c) and, nevertheless, some of them were colocalized with photoreceptor AGB immunoreactivity. After 2 h of light exposure, an increase in both AGB labeling and apoptotic cells was observed. The number of AGB-labeled cells and apoptotic cells rose dramatically in light-exposed P23H retinas (Fig. 5d–f), and the pattern of cell death was not different from 24 h light-exposed retinas (Fig. 5g–i). Figure 5j shows the proportion of AGB and TUNEL cells in SD rats, normalized against the number of AGB cells per unit area found in unexposed retinas. The analysis indicates that light exposure of SD rats increased the proportion of AGB-labeled cells in the ONL, of which a small proportion corresponded to cells colocalizing with the apoptotic marker. Exposure of the SD rat to light evoked an equal increase in both AGB gating and apoptotic cells after 24 or 2 h light exposure. However, a different pattern in AGB gating and TUNEL labeling was observed in the P23H rat (Fig. 5k). In unexposed P23H retinas, there was an enduring number of AGB-labeled cells and TUNEL positive photoreceptors that are related to the onset of photoreceptor loss because of the P23H mutation. Nevertheless, the proportion of AGB- and TUNEL-labeled cells steadily increased after light exposure. After 2 h light exposure there was almost a 10-fold increase in the number of apoptotic cells and four times more AGB-labeled cells than in unexposed P23H rats. After 24 h light exposure, the proportion of AGB-labeled cells and apoptotic cells did not significantly increase (Student’s t-test, p = 0.055). However, the comparison of the amount of AGB labeling and TUNEL labeling indicated that light exposure of the P23H retina caused more TUNEL labeling than AGB gating, and that the two labeling patterns largely identify different populations of photoreceptor cells. The data indicates that increased AGB gating and TUNEL labeling may be concomitant events after light exposure.

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Figure 5.  Immunocytochemical analysis of the colocalization of agmatine (AGB) labeling and TUNEL labeling in unexposed and light-exposed (2 and 24 h) P23H rat retinas at P60. (a–c) Confocal micrographs of unexposed P23H rat retinas. (d–f) Shows the pattern of colocalization of AGB and TUNEL after 2 h light exposure in P23H rat retina. (g–i) Shows the pattern of colocalization of AGB and TUNEL after 24 h light exposure in P23H rat retina. (j) Shows the proportion of AGB-labeled cells, TUNEL labeled cells, and AGB + TUNEL cells. All values were normalized to the number of AGB cells found in unexposed Sprague–Dawley (SD) retinas (derived from Fig. 4). (k) Shows the proportion of AGB only, TUNEL only, and AGB + TUNEL cells normalized against the number of AGB cells in unexposed P23H retinas (derived from Fig. 4). The abbreviations are as in Fig. 3. The scale bar is equal to 20 μm.

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Effect of light exposure on retinal LDH activity and isoenzyme distribution

We wanted to investigate whether these functional modifications in the light-exposed retina were also associated with retinal metabolic changes. Therefore, we measured LDH activity which is a reliable indicator of changes secondary to retinal metabolic damage or degeneration (Bonavita et al. 1963; Graymore 1963; Acosta and Kalloniatis 2005; Acosta et al. 2005). After 2 h of light exposure a 15% increase in LDH activity was detected in the adult P23H rat (Fig. 6b), although significant changes were not observed in the adult SD retina (Fig. 6a). The effect of 24 h light exposure showed the opposite result: LDH activity decreased for both SD and P23H rats (Fig. 6a and b) at all ages analyzed, and resulted in a 37% reduction in P23H retinas and 26% reduction in SD rat retinas. In addition, when we exposed young P23H rats to 24 h periods of intense light (Fig. 6b), LDH activity was lower than unexposed rats of the same age (41% reduction). However, double exposure of P20 retinas did not further reduce the LDH activity, when compared with the single 24 h light exposure group, suggesting that the LDH activity recorded 24 h after exposure may correspond to the baseline level of retinal metabolism, secondary to photoreceptor dysfunction.

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Figure 6.  The effect of light on total lactate dehydrogenase (LDH) activity in the retina of rats at ages P21 (immediately after light exposure), P27 (1 week after light exposure), P60 (immediately after light exposure), and P66 (1 week after light exposure). (a) Shows the change in LDH activity of unexposed and exposed (2 and 24 h) Sprague–Dawley (SD) rats. (b) Shows the change in LDH activity of unexposed and exposed (2 and 24 h, and double 24 h) P23H rats. Significant values compared with control are: Student’s t-test *p < 0.05, **p < 0.001, anova statistical comparison of unexposed and 2 h exposed data was *p < 0.05; NS, not significant.

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Our previous results point to an up-regulation of LDH in the P23H rat retinas exposed to light for 2 h and down-regulation of LDH if exposed for 24 h. We tested whether this change was reflected by altered LDH isoenzyme distribution by analyzing the relative activity of each LDH isoenzyme (Fig. 7). We determined the relative activity of each isoenzyme by finding the proportion of each band and multiplying it by the total LDH activity value found for that group. In accordance with previous studies, LDH-5 was the predominant LDH isoenzyme in the adult rat retina (Graymore 1963; Acosta and Kalloniatis 2005). In both the SD and P23H retinas the changes in LDH relative activity were largely associated with a reduction of all LDH isoenzymes activity (Fig. 7). For the 2 h exposure condition where total LDH activity increased in the P23H rat retina (Fig. 6b), we found a significant increase in LDH2–4 (LDH1 makes a relatively small contribution to total LDH activity and a significant 1–2% increase was noted: Fig. 7).

image

Figure 7.  The effect of light on lactate dehydrogenase (LDH) isoenzyme distribution at ages P60 (immediately after 2 and 24 h light exposure), and P66 (1 week after 24 h light exposure). (a) Shows LDH relative activity in Sprague–Dawley (SD) rat retinas. (b) Shows LDH relative activity in P23H rat retinas. Significant values compared with control are *p < 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Many studies have shown the impact of light on the functional and morphological state of photoreceptors in both normal rat strains and those with inherited forms of retinal degeneration (Noell et al. 1966; Wang et al. 1997; Grimm et al. 2001; Organisciak et al. 2003). These studies have shown that SD rats have greater ability for neuroprotective mechanisms that reduce retinal damage caused by intense constant illumination (Li et al. 2001), and this resistance is enhanced by raising the rats to be adapted to the physiological 12 h dark/12 h light scheme (Li et al. 2003). Our results support this observation, where the adult SD rats reared under 270 lux appeared to be morphologically unaffected by light damage in the short term and up to 1 week after exposure. However, the altered retinal cation gating, TUNEL labeling, and LDH activity for the rats exposed at P59 suggests that morphological changes were imminent and likely to be evident if a longer time point was examined. The relationship between light exposure and photoreceptor viability is complex (Wenzel et al. 2005). For example, Vaughan et al. (2003) measured the ONL thickness 2 weeks after light exposure and showed that there was a significant reduction of the ONL in both dark-reared and cyclic light-reared adult P23H rats. In the P23H rat, light experience shows a trend of accelerating photoreceptor death but lacks the expected up-regulation of protective mechanisms (Wang et al. 1997; Nir et al. 2001). Nevertheless, our results corroborate with these findings, by demonstrating that photoreceptors from both P23H and SD rats showed exaggerated ion gating when exposed to intense light. Studies of the P23H mice also show this vulnerability evaluated by retinal structure and electroretinography, retinal densitometry, and TUNEL labeling (Naash et al. 1996; Wang et al. 1997), and morphological evidence of photoreceptor and retinal pigmented epithelium hypervulnerability to light-induced damage (Vaughan et al. 2003). The exaggerated photoreceptor apoptosis was also observed when light exposure for 24 h was applied at an adult stage of development supporting other investigations on light-induced damage of adult P23H rats (Vaughan et al. 2003), adult SD rats (Noell 1965; Noell and Albrecht 1971; Grimm et al. 2001), and increased vulnerability of the P23H retina to more severe light-induced damage (Wang et al. 1997).

Agmatine has been described as a useful marker for photoreceptors destined to degeneration before apoptotic markers are evident in the rd/rd mice (Acosta et al. 2005), and in the Royal College of Surgeon rats (Tso et al. 1994; Kalloniatis et al. 2002). The properties of AGB permeation in the P23H rat also allow a better understanding of the mechanisms of cell death in the degenerating retina. Our data suggests that there is a temporal relationship between length of light exposure and increased channel permeability to organic molecules (AGB) and the presence of TUNEL positive cells. Donovan et al. (2001) have demonstrated that exposure to white light causes an early increase in intracellular calcium levels during photoreceptor apoptosis accompanied by mitochondrial membrane depolarization and the potential involvement of cGMP. Our analysis supports a causal relationship between ion gating and apoptosis for a subpopulation of photoreceptors. Also, the photoreceptors with increased cation channels conductance may progress to demonstrate TUNEL labeling. However, a large cohort in both SD and P23H photoreceptors show TUNEL labeling alone. Marc (1999a) has demonstrated that AGB gating in retinal cells is a sensitive marker of cytopathology. It is therefore unlikely that a cell will display cation gating, and subsequently control cation flow and only demonstrate TUNEL labeling. We conclude that cells demonstrating TUNEL labeling alone reflect an apoptotic pathway through as yet an unidentified mechanisms, i.e. apoptosis triggered through a non-cation gating mechanism.

Whether altered energy metabolism leads to degeneration is unclear. However, studies in retinal degeneration models show down-regulation of LDH activity secondary to photoreception degeneration (Bonavita et al. 1963; Graymore 1963; Acosta et al. 2005), and up-regulation before any morphological signs of photoreceptor death (Acosta et al. 2005), suggesting that LDH activity is an early indicator of impending dysfunction and is associated with down-regulation of retinal function. In accordance with these findings our results show that LDH activity is increased after 2 h exposure of the P23H rat, and decreased after 24 h light exposure likely as a result of photoreceptor loss (confirmed by TUNEL labeling). Therefore, our results further support the contention that LDH activity can be used as a marker of retinal photoreceptor degeneration (Acosta and Kalloniatis 2005; Acosta et al. 2005). In addition, investigations into the relationship between LDH levels and metabolic insult showed an increase in LDH activity during short periods (40 min) of hypoglycemia, hypoxia, and anoxia (Acosta and Kalloniatis 2005). Accordingly, LDH is responsible for the short-term shielding of neurons during transient metabolic insult (Schurr et al. 1997; Zeevalk and Nicklas 1997).

Studies involving the use of degenerating retinas have shown that an elevation in LDH activity appears to be consistent with increases in oxygen consumption, glucose utilization, and lactic acid production that occurs in early metabolic dysfunction of the rd/rd mouse (Noell 1965; Acosta et al. 2005), and was observed in this study after a 2 h light exposure period of P23H rats. The significant decrease in LDH levels or isoenzyme distribution that is observed in light-induced photoreceptor degeneration and other forms of retinal degeneration (Graymore 1963; Sweasey et al. 1971; Acosta et al. 2005), appears to be related to altered neuronal activity, as acute exposure of intense light does not seem to have an effect on the activity of glutamine synthetase in Müller cells (Darrow et al. 1997), until light induces hypertrophy of Müller cells (Jones et al. 2003, 2006). Altogether, the results emphasize that the changes that precede light-induced degeneration are only comprised of neuronal cells.

Any attempt to define a general light damage pathway is complicated by the variation in the degree of damage that occurs for different light levels, light pre-conditioning, species or breed of rats (Reméet al. 1998; Walsh et al. 2004). Several light damage mechanisms and molecules associated with light damage have been identified for different light conditions (Hao et al. 2002; Wenzel et al. 2005). Our results show that a similar mechanism involving cation gating is in place early in the photoreceptor degeneration process in both SD and P23H rats. However, a large cohort of photoreceptor cells demonstrates a cation-independent process of apoptosis indicative of the complexity in the underlying mechanism(s) leading to photoreceptor degeneration.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by a professorship funded by the Robert G. Leitl estate (MK) and the University of Auckland Staff Research Fund (MK). Y-LC received support in the form of a summer scholarship from the SA & GJ Ombler Trust and both Y-LC and T-YY, received a summer fellowship from the New Zealand Optometric Vision Research Foundation (NZOVRF). Also supported, in part, by a Health Research Council of New Zealand grant (HRC 05/247) and an Auckland Medical Research Foundation (AMRF) grant. The agmatine antibody was a kind gift of Dr Robert Marc (University of Utah) and we thank Dr Bang Bui for providing the EXCEL programme to undertake the bootstrap estimates of the mean distribution of light levels. We also thank Professor Matt LaVail, Beckman Vision Center, UCSF School of Medicine for providing access to the P23H-3 rat line.

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  6. Acknowledgements
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