Involvement of Caspase-3 in Photoreceptor Cell Apoptosis Induced by In Vivo Blue Light Exposure

Abstract

purpose. To investigate the potential role of caspase-3 in blue light–induced apoptosis in photoreceptor cells.

methods. Cyclic light–raised Sprague-Dawley rats were exposed to 400 to 480 nm light for 6 hours at an irradiance of 0.64 W/m². The rats were then kept in darkness for recovery and killed at scheduled time points from 0 hours up to 24 hours after exposure to light. The unexposed rats were used as the control. Apoptosis was marked with in situ terminal dUTP nick end labeling (TUNEL). Caspase-3 expression was explored using immunohistochemistry, Western blot analysis, real-time RT-PCR, and enzyme activity assay.

results. Exposure to blue light resulted in photoreceptor cell apoptosis, mostly in the superior temporal area of the retina. TUNEL-positive cells were highest during 8 to 16 hours of recovery in darkness. Procaspase-3 protein was constitutively expressed in the rat retina and was apparently upregulated after exposure to light, with expression peaking at 8 to 16 hours and subsiding at 24 hours. The upregulation was further supported by enhanced transcription of caspase-3 in the postexposure retina. Meanwhile, increased cleavage of caspase-3 into its active fragments of 17 and 19 kDa was detected after exposure to light, peaking at 16 hours. Activation of caspase-3 was subsequently located in the photoreceptor cells and predominantly in the superior part of the temporal quadrant. This coincided with elevated caspase-3–like activity at 16 hours after exposure to light.

conclusions. Procaspase-3 protein is temporally upregulated in the retina after in vivo exposure to blue light, and the upregulation is coupled to activation of caspase-3 and concomitant induction of apoptosis in the photoreceptor cells.

Exposure to light has been implicated in the pathogenesis and enhancement of several photoreceptor degenerative diseases. ^{1 2 3 4 5 6} It has been established that the retina can be injured by a photochemical mechanism. ⁷ The retina’s sensitivity to light can be influenced by such factors as light history, genetic background, age, and temperature. ⁸ The manifestation of damage in the phenotype varies with the spectral component of the light under otherwise similar conditions. ⁹ Across the visible spectrum blue light is most detrimental to the retina. ^{10 11} Consensus indicates that the outer layer of the retina is the major target and that photoreceptor cells die primarily by apoptosis. ^{12 13}  

Apoptosis is a genetically regulated form of cell death. The death process is characterized by selective proteolysis of cytoplasmic and nuclear substrates, which disables homeostatic and repair processes and mediates structural disassembly and morphologic changes, thus marking the dying cell for engulfment and disposal. Apoptosis-related proteolysis is largely achieved by a gene family of cysteinyl aspartate- specific proteinases (caspases), containing at least 14 mammalian members. Caspases are synthesized as catalytically dormant tripartite proenzymes, containing prodomain, large subunit, and small subunit. Both the large and small subunits make up the active form of the enzyme as a consequence of cleavage after receiving death signal(s). ¹⁴  

Caspases are important mediators of neuronal apoptosis. They play a pivotal role in developmental and pathologic death in the nervous system. ¹⁵ Laboratory studies have revealed that apoptosis contributes to the pathogenesis of retinal cell death in retinitis pigmentosa, age-related macular degeneration, glaucoma, retinal detachment, and diabetic retinopathy, as well as pathologic myopia. ^{16 17 18} Caspase activation has been demonstrated in the retina in a number of degenerative models of animals in which apoptosis was caused by ischemia, axotomy, excitotoxicity, and gene mutations. ^{19 20 21 22 23}  

We have previously established that photoreceptor cell degeneration is preceded by apoptosis after the cells are injured in vivo by low-intensity blue light. ¹³ It was therefore of great interest to investigate the potential role of caspases in blue light–induced apoptosis. In this study, we explored whether this form of photoreceptor death involves caspase-3, which is one of the most commonly activated caspases in apoptosis. 

Material and Methods

Exposure to Light

Female albino Sprague-Dawley rats were maintained in a 12-hour light–dark cycle in 110 lux. They were fed ad libitum with commercial pellets and had free access to water. The rats (180–190 g body weight) were exposed to 400 to 480 nm light for 6 hours after 20-hour dark adaptation. The blue light was emitted continuously from 16 tubular, low-pressure, mercury vapor, fluorescent lamps (Phillips, Eindhoven, The Netherlands) at a constant rate of 0.64 w/m². Each lamp was incorporated into a spectrum tube (Sierra Polymer Co., Reno, NV) to filter out the possible emission of UV radiation. 

The exposure device has been detailed elsewhere. ^{24 25} Briefly, it was composed of the light source and a rotating cage. The lamps were mounted vertically round the cage that was driven by an electric motor to rotate around a horizontal axis at a speed of 0.25 rev/min to keep the rat’s eyes open during the exposure session. The rats were returned to the darkness after exposure until death. The treatment of the animals was in conformity with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

TUNEL Analysis

Three rats were killed at each time point with an overdose of pentobarbital, and two rats unexposed to light were used as the control. The eyes were removed immediately and fixed in 4% neutral buffered formaldehyde for 20 to 22 hours. Paraffin-embedded sections of 4 μm were cut in the sagittal plane and stained with TUNEL with a kit (ApopTag; Oncor, Gaithersburg, MD). Briefly, the sections were deparaffinized and incubated step-wise in 20 μg/mL proteinase K, 2.0% H₂O₂, and terminal deoxynucleotidyl transferase (TDT) with digoxigenin-dUTP. After the reaction was terminated with buffer (Stop/Wash; Oncor), the sections were incubated with anti-digoxigenin peroxidase. Staining was developed in 3-amino-9-ethylcarbazole (AEC). The sections were counterstained with Mayer hematoxylin. The negative controls were run by substituting an equal volume of distilled water for TDT enzyme. The sections located between 1.5 to 2.0 mm from the temporal side of the scleral surface were chosen for positive staining comparison among different individuals at different points. ²⁵  

Because all sections were collected from the equivalent area, the counting of TUNEL-positive cells was performed on every whole section after the slide labels were masked. 

Immunohistochemistry

The eyes of three exposed rats and two unexposed at each time point were embedded in mounting medium (OCT; Sakura Fintek, Torrance, CA) and frozen immediately on dry ice after enucleation. Cryosections of 14 μm in thickness were made in the sagittal plane and kept at −80°C until analysis. 

The sections were fixed with 2% paraformaldehyde in PBS at room temperature for 20 minutes, followed by incubation with 1% H₂O₂, 2% sodium azide, 0.1% saponin, 10 mM HEPES in Earle’s balanced salt solution (EBSS-saponin) for 1 hour at room temperature in the dark. The sections were washed and incubated in a humid chamber overnight at room temperature with primary rabbit antibody directed against cleaved caspase-3 (1:100; Cell Signaling Technology, Beverly, MA) in EBSS-saponin. According to the manufacturer, this antibody detects only the large fragment of activated caspase-3 (17–20 kDa) which results from cleavage after Asp 175. It does not recognize the full-length caspase-3 and other caspases. The sections were rinsed and incubated with peroxidase-conjugated secondary antibody (1:200, Amersham, Buckinghamshire, UK) for 60 minutes at room temperature. Staining was visualized by adding a diaminobenzidine-H₂O₂ substrate solution (Vector Laboratories, Burlingame, CA), and sections were counterstained with hematoxylin. As the control, sections were stained without exposure to primary antibody or with normal rabbit serum. The sections located between 1.8 to 2.0 mm from the temporal side of the scleral surface were selected for positive staining comparison among the retinas. ²⁵  

The same quantification method as TUNEL analysis was applied in the immunohistochemistry. 

Western Blot Analysis

Four rats were used at each time point. The retinas were dissected, minced and homogenized in ice-cold buffer containing 20 mM HEPES (pH 7.5), 350 mM NaCl, 1 mM MgCl₂, 0.5 mM EDTA, 0.1 mM EGTA, 1% NP-40, 0.5 mM dithiothreitol (DTT), 0.1% phenylmethylsulfonyl fluoride (PMSF), and 1% aprotinin. The suspensions were then centrifuged at 20,000g for 20 minutes at 4°C. The supernatants were collected, and the protein concentrations were determined with a protein assay kit (Bio-Rad, Richmond, CA) with bovine serum albumin as the standard. 

The protein mixture (80 μg) was then mixed with Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, resolved on 12% SDS-PAGE gels, and electrophoretically transferred to polyvinylidene difluoride membranes at 100 V. The membrane was blocked in 50 mM Tris (pH 7.5) with 500 mM NaCl, 1% bovine serum albumin and 5% nonfat dried milk. It was then probed with either polyclonal rabbit antibody against the p17 fragment of caspase-3 (1:1000; gift of Donald W. Nicholson, Merck Frosst Center, Montreal, Quebec, Canada) or polyclonal rabbit antibody recognizing only cleaved caspase-3, as described in immunohistochemistry. This was followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:10,000; Pierce, Rockford, IL or 1:2000; Amersham) and then visualized with an enhanced chemiluminescence (ECL) Western blot detection kit (Amersham). Quantification was performed by densitometric scanning of bands on the developed film. 

Real-Time Quantitative RT-PCR

To quantify the mRNA content of caspase-3 in the retina after exposure to light, four temporal-half retinas were dissected at individual time points and stored at −80°C for further use. Total RNA was extracted after the manufacturer’s protocol by using a kit (RNeasy Mini Kit; Qiagen, Valencia, CA) and treated with deoxyribonuclease I to remove contaminating genomic DNA. The cDNA synthesis was performed by incubation of 1 μg of each RNA sample in hexanucleotide (pdN6; Pharmacia Biotech AB, Stocknolm, Sweden) at 70°C for 3 minutes, followed by the incubation with 0.5 mM each dNTP, 5 mM DTT, and 60 U/40 μL RNasin (Promega Corp., Madison, WI), 400 U/40 μL reverse transcriptase (Superscript II; Life Technologies, Gaithersburg, MD) in RT buffer at 25°C for 10 minutes, 42°C for 50 minutes, and 94°C for 4 minutes. 

PCR with Taq polymerase (Taqman; Applied Biosystems, Foster City, CA) on the cDNA samples was performed on a sequence detector (Prism 7700; Applied Biosystems, Foster City, CA) Equal amounts of cDNA were used in duplicate and amplified with the PCR mix (Taqman Master Mix; Applied Biosystems). The PCR conditions were set at 50°C for 2 minutes, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Additional reactions were performed on known dilutions of rat cDNA as a PCR template to construct a standard curve relating threshold cycle to template copy number. Amplification efficiencies were validated and normalized against the housekeeping gene glyceraldehyde-3-phyosphate dehydrogenase (GAPDH). 

Taq polymerase primer and probe sets for caspase-3 and GAPDH were designed from sequences in the GenBank database on computer (Oligo ver. 5 software; National Bioscience, Plymouth, MN; and Primer Express; Applied Biosystems. GenBank is provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD, and is available at http://www.ncbi.nlm.nih.gov/Genbank). Primer and probe sequences (forward primer, reverse primer, Taq polymerase probe): caspase-3 (accession number U49930): 5′-AATTCAAGGGACGGGTCATG, 5′-GCTTGTGCGCGTACAGTTTC, 5′-TTCATCCAGTCACTTTGCGCCATG; and GAPDH: (accession number U75401): 5′-GAACATCATCCCTGCATCCA, 5′-CCAGTGAGCTTCCCGTTCA, 5′-CTTGCCCACAGCCTTGGCAGC. 

Caspase Activity Assay

The temporal-half retinas were dissected from the enucleated eyes, snap frozen on dry ice, and stored at −80°C for further assay. The retinas were homogenized in a buffer containing 10 mM HEPES (pH 7.2), 5 mM EGTA, 0.1% 3-([3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPS), 1.5 mM MgCl₂, 5 mM DTT, 10 μg/mL pepstatin, 20 μg/mL leupeptin, 10 μg/mL aprotinin, and 0.5 mM PMSF. The samples were centrifuged at 20,000g for 20 minutes at 4°C, and usually 130 μg protein was transferred to an individual well of a 96-well plate and incubated with 50 μM of the synthetic substrate Acetyl-Asp-Glu-Val-Asp-4-methylcoumaryl-7-amide (Ac-DEVD-MCA; Peptide Institute, Osaka, Japan) in a solution containing 100 mM HEPES (pH 7.25), 10% sucrose, 0.1% CHAPS, 5 mM DTT, and 0.1% NP-40. The DEVD-MCA cleavage activity at 37°C was monitored by measuring excitation at 355 nm and emission at 460 nm on a plate reader (Fluoroscan Imaging Systems, Bedford, MA) over time. Fluorescent units were converted to picomoles of MCA released per minute, using a standard curve generated with free MCA. 

Statistics

The activity and mRNA level of caspase-3 were analyzed by the nonparametric Wilcoxon’s test. The significant level was set at P < 0.05. The correlation coefficient was calculated to evaluate the correlation of the temporal profile of the TUNEL and activated caspase-3 labeling. 

Results

Apoptosis in the Retina after Exposure to Blue Light

The photoreceptor cells, mostly situated in the superior region of the temporal retina, were injured by the blue light and stained positively by TUNEL in a sporadic pattern or in small clusters (Figs. 1B 1C 1D 1E) . Increased labeling was seen with time in darkness after exposure to light, with the labeling reaching peak levels at 8 and 16 hours (Figs. 1C 1D) . The labeling was dramatically reduced at 24 hours of recovery (Fig. 1E) . The temporal quantification of TUNEL staining is illustrated in Figure 2A . No TUNEL-positive nuclei were seen in the unexposed and negative control. These results confirmed our previous finding that apoptosis in photoreceptor cells was seen early after the retina is damaged by the blue light. ¹³  

Caspase-3 Expression in the Retina after Exposure to Blue Light

Expression of caspase-3 protein in the retina was investigated immediately, 8, 16, and 24 hours after exposure to light. Western blot analysis demonstrated the constitutive presence of the 32-kDa procaspase-3 protein in rat retina (Fig. 3) . Of interest is the finding that procaspase-3 protein level in the retina was gradually increased during the period of darkness after exposure to light. This postexposure increase peaked at 8 to 16 hours and subsided at 24 hours (Fig. 3) . The result was shown to be reproducible in another different set of samples. 

Furthermore, caspase-3 mRNA expression was evaluated with real-time quantitative RT-PCR. The content of caspase-3 mRNA in the retina was shown to increase after exposure to light (Fig. 4) . The increase was significant from 10 to 20 hours in comparison with the unexposed control (P = 0.027). Evidently, caspase-3 is temporally upregulated in the retina in response to exposure to blue light. 

Caspase-3–like Activity in the Retina after Exposure to Blue Light

Cleavage of the peptide-based substrate DEVD-MCA was used as an indicator of caspase-3–like activity. Cytosolic extracts of the retinas from rats exposed to the blue light were found to contain increasing levels of DEVD-MCA cleavage activity. The elevation of caspase-3-like activity was statistically significant (P = 0.03) at 16 hours after exposure to light compared with the unexposed controls in the current assay (Fig. 5) . 

Activation of Caspase-3 in the Retina after Exposure to Blue Light

To examine whether the increased caspase-3–like activity is due to an increased cleavage of the 32-kDa procaspase-3 protein into the active fragments, activation of caspase-3 was tested by using an antibody that binds specifically to cleaved caspase-3 subunit. Western blot results revealed slight labeling of 17-kDa and 19-kDa subunits in the unexposed control (Fig. 3) . Both bands increased in density during the 24 hours after exposure to light with the highest at 16 hours (Fig. 3) . 

Immunohistochemistry was further performed with the same antibody. Specific staining of cells was obtained exclusively in the outer nuclear layer (ONL) exposed to blue light irradiation (Fig. 1G 1H 1I 1J) . The positive staining was found predominantly at the superior temporal side of the retina. The number of positive cells was maximal at 8 to 16 hours after exposure to light (Figs. 1H 1I 2B) . This staining pattern was confirmed by at least three separate trials. Although there was nonspecific binding along the outer border of the ONL in both exposed and unexposed retinas, no cellular staining was seen in the unexposed retinas and negative control. 

Discussion

Light-induced photoreceptor apoptosis has served as a convenient model to study retinal degeneration. ²⁶ We have reported that exposure of albino rats to broadband blue light for several hours caused a moderate loss of photoreceptor cells in the superior temporal quadrant of the retina. ²⁵ The photoreceptor cells died by apoptosis early after exposure to light, which was revealed by electron microscopy, TUNEL, and DNA laddering. ¹³ The wave of apoptosis reached its maximum at 8 to 16 hours and declined at 24 hours after exposure to light, which was confirmed in the present study. 

Herein, we demonstrate that a regulation of caspase-3 expression occurred in the retina after exposure to light. The level of caspase-3 protein peaked at 8 to 16 hours followed by a decline at 24 hours. There was a simultaneous increase in the mRNA levels of caspase-3 in the postexposure retina, indicating an enhanced transcription of caspase-3. Meanwhile, DEVD-MCA cleavage activity was found to increase significantly at 16 hours after exposure to light. Therefore, the progression of blue light–induced apoptosis in the photoreceptor cells was correlated temporally to caspase-3 upregulation and the elevation of caspase-3–like activity. 

Furthermore, we performed Western blot analysis and immunohistochemistry to characterize the involvement of caspase-3 in the retina in response to exposure to light. There was a slight expression of p17 and p19 subunits in the unexposed retinas, which has also been reported previously. ²⁰ As expected, the retinas contained increased levels of both subunits of active enzyme after exposure to light. The cellular labeling of cleaved caspase-3 subunits was visualized distinctly in the ONL irradiated by the blue light. Thus, evidence is given that the increased cleavage of procaspase-3 contributes to the increasing caspase-3–like activity and that caspase-3 itself is activated in the photoreceptor cells after exposure to light. 

It should be noted that the cellular labeling in immunohistochemistry was not distributed randomly and uniformly across the retina. Positive cells were confined to the area that is targeted preferentially by the blue light ²⁵ and that subsequently contained apoptotic photoreceptor cells that were marked by TUNEL. Moreover, the number of cleaved caspase-3–positive cells also reached its climax when the highest amount of TUNEL positive nuclei was counted. Given the correlation of spatial and temporal profile between immunostaining and TUNEL labeling in the ONL, we conclude that caspase-3 is involved in blue light–induced photoreceptor cell apoptosis. 

However, there is an apparent difference between the number of immunostain- and TUNEL-positive cells. We cannot, therefore, exclude that other mechanisms may contribute to light-induced apoptosis in the retina. It is possible that in some cells the level of cleaved caspase is too low to be detected with the current method, although it may be sufficient to execute apoptosis. This may be supported by the fact that maximal immunopositive cells were obtained in the ONL when the procaspase-3 level was upregulated to its peak level in the retina. Finally, it may be due to the difference in lifespan between DNA breaks and cleaved protein. 

In addition, we were not able to test the possible protection of photoreceptor cells against light-induced damage by using caspase-3 inhibitor in this model, because intravitreal injection of the inhibitor caused an immediate vitreous opacity. 

The change in gene expression of caspase-3 that was displayed in the present study is in line with the finding that caspase transcription is altered in the process of apoptosis in retinal cells. ^{19 21 27 28} Caspase-3 was activated in the photoreceptor cell during apoptosis caused by administration of N-methyl-N-nitrosourea, rhodopsin mutation and ischemia-reperfusion. ^{19 22 23 27} Caspase-3 can execute apoptosis in the inner nuclear layer and/or the ganglion cells in the case of ischemia-reperfusion, excitotoxicity, and axotomy ^{19 20 22 29 30 31} . Recently, the lipofuscin fluorophore A2E has been shown to mediate in vitro blue light–induced damage to the retinal pigment epithelium through caspase-3–dependent apoptosis. ³² In the present study, caspase-3 was activated and upregulated in the retina during the course of photoreceptor apoptosis after in vivo exposure to blue light. All evidence indicates that the retinal cells share some common pathways in their death programs, despite the diversity of death signals, and caspase-3 appears to be the key executioner of cells in the retina by apoptosis. Indeed, an in vitro study revealed activation of caspase-3 and polyadenosine diphosphate-ribose-polymerase (PARP) cleavage in cultured retinal cells during apoptosis induced by different stimuli. ³³  

To sum up, this study demonstrated for the first time that the level of procaspase-3 protein is upregulated in the retina after in vivo exposure to blue light, and the upregulation is coupled to activation of caspase-3 and concomitant induction of apoptosis in the photoreceptor cells. Further studies should be directed toward a better understanding of the apoptotic mechanism in light-induced retinal degeneration to develop therapeutic strategies for light-related retinal diseases. 

 Supported by Karolinska Institutet Research Funds, Edwin Jordan Ophthalmology Research Foundation, Carmen and Bertil Regner Eye Diseases Research Foundation, Crown Princess Margareta Foundation for Vision Injury, Vision Improvement Research Foundation, and European Commission.

 Submitted for publication January 14, 2002; revised May 1, 2002; accepted May 20, 2002.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Figure 1.

 

TUNEL analysis (B–E) and immunohistochemical staining of cleaved caspase-3 (G–J) in the retinas continuously exposed to blue light for 6 hours. The sections were collected just after exposure to light (B, G) and 8 (C, H), 16 (D, I), and 24 hours (E, J) after withdrawal of the light. Specific staining was located in the ONL. No staining was observed in unexposed control retinas (A, F).

Figure 2.

 

The average counting of TUNEL-positive nuclei (A; n = 3) and of activated caspase-3–positive cells (B; n = 3) per section. The correlation coefficient of the temporal profile of both stainings is 0.987.

Figure 3.

 

Western blot analysis of caspase-3 expression in the retinas after exposure to blue light. The retinas were collected immediately, 8 hours, 16 hours, and 24 hours after recovery in darkness, as well as from unexposed controls (Ctr). Increased cleavage of caspase-3 into 17 kDa and 19 kDa was detected with the maximal density at 16 hours (A). The 32-kDa proenzyme was upregulated to peak level during 8 to 16 hours (C). The 32-kDa band density at 16 hours is set arbitrarily at 1 in the histogram. β-Actin was then labeled as an index for equivalent protein loading (B, D).

Figure 4.

 

The real-time quantitative RT-PCR analysis of caspase-3 expression in the retinas after exposure to blue light. The retinas were collected from unexposed controls (ctrl), 0, 10, and 20 hours after exposure to light. Data represent the median and the range (bar) for each time point. The transcription of caspase-3 was significantly elevated at 10 and 20 hours (*P = 0.027; n = 4), compared with the control.

Figure 5.

 

Caspase-3–like activity assay by measuring the cleavage of the peptide-based substrate DEVD-MCA. Data are expressed as the median and range (bar) for each time point. The enzyme activity was significantly increased in the retinas at 16 hours (*P = 0.03; n = 5) after exposure to light compared with the unexposed control (n = 4).

 

The authors thank Roshan Tofighi for excellent technical assistance in the caspase activity assay.