July 1999
Volume 40, Issue 8
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Retinal Cell Biology  |   July 1999
Caspaselike Proteases Activated in Apoptotic Photoreceptors of Royal College of Surgeons Rats
Author Affiliations
  • Naomichi Katai
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
  • Takanobu Kikuchi
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
  • Hiroto Shibuki
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
  • Sachiko Kuroiwa
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
  • Jun Arai
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
  • Toru Kurokawa
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
  • Nagahisa Yoshimura
    From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
Investigative Ophthalmology & Visual Science July 1999, Vol.40, 1802-1886. doi:
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      Naomichi Katai, Takanobu Kikuchi, Hiroto Shibuki, Sachiko Kuroiwa, Jun Arai, Toru Kurokawa, Nagahisa Yoshimura; Caspaselike Proteases Activated in Apoptotic Photoreceptors of Royal College of Surgeons Rats. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1802-1886.

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

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Abstract

purpose. To study the role of caspase-like proteases, especially roles of more extensively characterized caspase-1 and caspase-2, in apoptotic photoreceptor cell degeneration in Royal College of Surgeons (RCS) rats.

methods. Both RCS and Sprague—Dawley rats were used. Cryosections of the retinas at various postnatal times were immunostained with antibodies against caspase-1 (interleukin-1β–converting enzyme, ICE) and caspase-2 (Nedd2/Ich-1). Double staining with TdT-dUTP terminal nick–end labeling (TUNEL), propidium iodide, and the antibodies was also performed. To evaluate the time course of protein expression, western blot analysis was carried out. The temporal profile of caspase-like protease activity was studied using a fluorogenic tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid α (4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA). Intravitreal injection of a caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartic-aldehyde (Ac-YVAD-CHO), at postnatal days 21 (P21) and P26 was performed to see if this caused a decrease in apoptotic cell number at P28.

results. TUNEL-positive photoreceptors of RCS rats stained strongly with antibodies against caspase-1 and caspase-2. Double staining studies revealed that caspase-1 and caspase-2 were coexpressed in apoptotic cells. Western blot analysis showed that active forms of caspase-1–like and caspase-2–like proteases were upregulated at P28, concurrent with the peak in TUNEL-positive cells. The enzymatic activity of caspase-1–like protease was elevated in RCS rat retinas at P28, and the inhibitor of caspase-1 transiently reduced the number of the apoptotic photoreceptors.

conclusions. Activation of caspase-like proteases plays an important role in photoreceptor apoptosis of RCS rats.

In Royal College of Surgeons (RCS) rats, photoreceptor degeneration caused by impaired phagocytosis of rod outer segments of the retinal pigment epithelial cells begins at about postnatal day 20 (P20), 1 and almost all photoreceptors die out by P60. Recently, this retinal degeneration has been shown to be caused by apoptotic cell death, based on the finding that cells in the outer nuclear layer (ONL) are stained by TdT-dUTP terminal nick—end labeling (TUNEL) and that agarose gel electrophoresis of DNA extracted from the retina shows a typical ladder pattern. 2  
Apoptosis is a phenomenon underlying normal development and many pathologic conditions and is achieved through activation of a cascade called a “death program.” 3 4 In general, the apoptotic process is divided schematically into five steps: activation, propagation, commitment, execution, and, finally, cell death. 3 Interleukin-1β–converting enzyme (ICE)/caspase family proteins, identified by their homology with the nematode death gene ced-3, are executors of the apoptotic program in some vertebrate cells. 4 Although the final step of apoptosis is invariably cell death, various signals contribute to the final common pathway, and intermediate steps differ depending on the etiology of the apoptosis and cell type. 4 If the apoptotic process has not proceeded beyond the execution step, a cell can be rescued in some instances by blocking expression of apoptosis-related genes or by overexpression of bcl-2. 4  
Apoptosis plays a major role in the pathogenesis of many diseases in which photoreceptors degenerate, such as retinitis pigmentosa, retinal detachment, light injury, ischemic injury, and age-related macular degeneration. 5 6 Therefore, it is important to know which genes are specifically expressed during apoptosis of photoreceptor cells. Herein, we will show that members of the caspase family may play a critical role in the apoptosis of photoreceptor cells in the RCS rat. In the present study, we concentrated on caspase-1 and caspase-2 because these enzymes are known to play an important role in neuronal apoptosis of ischemic brain injury and retinal development and apoptosis of rat pheochromocytoma-derived cell line cells (PC12). 7 8 9  
Materials and Methods
Animals
The care and maintenance of rats conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. RCS (Jcl-rdy/rdy) rats were obtained from Japan CLEA, Tokyo, Japan, and Sprague—Dawley (SD) rats from a local breeder. Rats were maintained under 12-hour light/12-hour dark conditions. RCS and SD rats of 14, 21, 25, 28, 33, 35, 45, and 56 days of age were used in this study. 
Antibodies
Antibodies used in this study were obtained from various sources: Goat anti–caspase-1 (ICE) polyclonal antibody (M-19) and goat anti–caspase-2 (Nedd2/Ich-1) polyclonal antibody (N-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); fluorescein isothiocyanate (FITC)– and rhodamine-conjugated goat anti-rabbit IgG from DAKO (Glostrup, Denmark); and FITC- and rhodamine-conjugated rabbit anti-goat IgG from Jackson ImmunoResearch Laboratories (West Grove, PA). These caspase antibodies recognize both proenzyme and active forms of protease. Anti-phosphotyrosine antibody from Upstate Biotechnology was used to distinguish photoreceptors from microglial cells and peripheral macrophages. 
TUNEL and Propidium Iodide Staining
DNA nick end-labeling was performed according to a slightly modified method of Gavrieli et al. 10 After rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg), they were perfused transcardially with 100 ml of normal saline supplemented with 1 U/ml heparin followed by 200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes were enucleated and placed in 4% paraformaldehyde for 24 hours at 4oC. They were rinsed with phosphate-buffered saline (PBS) and then transferred to 10%, 15%, and 20% sucrose in 0.1 M PBS for 3 hours at room temperature. Samples were then snap-frozen in Tissue–Tek (Miles Laboratories, Elkhart, IN) on dry ice in hexane and stored at −80oC. Cryostated sections (10–15 μm) were thaw-mounted onto glass slides coated with poly-L-lysine and air-dried for 2 hours at room temperature. After cryosections were rinsed three times in 0.02 M PBS (pH 7.4), sections were incubated with biotinylated terminal dUTP transferase and biotinylated dUTP in TdT buffer (30 mM Tris, pH 7.2, 140 mM sodium cacodylate, and 1 mM cobalt chloride) for 60 minutes at 37oC with terminal dUTP in the moisture chamber. After rinsing, the sections were reacted with avidin–FITC and examined with a Zeiss scanning laser confocal microscope (LSM 410 inverted Laser Microscope; Zeiss, Oberkochen, Germany). Positive controls were generated using DNase I in TdT buffer (1 μg/ml) before incubation with terminal transferase and biotinylated nucleotides. Propidium iodide (PI) staining was performed by incubating the sections with 50μ l PI (20 μg/ml) for 10 minutes at room temperature. TUNEL staining of cells, compacted nuclear staining, nuclear fragmentation, and loss of nuclear staining by PI were considered to be signs of apoptosis. 
Immunohistochemistry
After rinsing with PBS, cryosections were incubated with 2% normal goat or rabbit serum for 60 minutes at room temperature. Incubation with the primary antibody (anti–caspase-1, 10 μg/ml, and anti–caspase-2, 10 μg/ml) was carried out in a moisture chamber at 4oC overnight. FITC- or rhodamine-conjugated secondary antibody was reacted at room temperature for 2 hours. 
Quantitative Analysis of TUNEL and Immunohistochemistry
At each point of the time course study for TUNEL and immunohistochemical staining, the number of TUNEL-positive- and immunostained cells in the ONL was determined in 5 meridian sections through the optic nerve. The numbers of cells were counted in 0.4-mm lengths of the 1 mm from the optic nerve head on the superior and inferior hemisphere using the Zeiss confocal microscope with the“ measure” function. Results are expressed as mean ± SEM. 
Analysis of Western Blots
Samples (n = 5 at each time) were homogenized in buffer containing Tris–HCl (pH 8.0), 250 mM NaCl, 0.5% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 20 μg/ml leupeptin, and 10 μg/ml aprotinin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out as described by Laemmli. 11 A 50-μg sample of protein was loaded on each lane, electrophoresed with 10% acrylamide gel, and then transferred to nitrocellulose membranes. After being rinsed with Tris-buffered saline (pH 7.4) containing 0.25% Tween-20 (TBST), the membrane was incubated with the primary antibodies (0.5μ g/ml) in TBST for 1 hour at room temperature. After three washings, the membrane was incubated for 1 hour at room temperature with horseradish peroxidase–conjugated secondary antibody (Amersham, Buckinghamshire, UK) diluted 1:500 in TBST. The membrane was washed again and developed with the chemiluminescence ECL western blotting system (Amersham). 
Intravitreal Administration of Caspase-1 Inhibitor
A specific inhibitor of caspase-1, acetyl-tyrosyl-valyl-alanyl-aspartate-aldehyde (Ac-YVAD-CHO), was obtained from the Peptide Institute (Osaka, Japan). A 20-mM solution of the Ac-YVAD in dimethyl sulfoxide was prepared, and 1 μl of the solution was injected into the vitreous cavity of the RCS rats at P21 and P26 by means of a 30-gauge needle. As a control, 1 μl dimethyl sulfoxide (vehicle) was injected in the same way. At P28, the eyes were enucleated, and the TUNEL staining was performed as described above. 
Assay of Caspase Activity
Caspase-1–like protease activity was assayed as described by Nicholson et al. 12 with slight modification. 13 Briefly, resected retinas were homogenized in 10 mM Hepes–KOH, pH 7.2, 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 20 μg/ml leupeptin, and 10 μg/ml aprotinin. Homogenates were centrifuged at 15,000g for 30 minutes, after which the supernatants were transferred to new Eppendorf tubes. Aliquots of extracts (300 μg protein in 100 μl extraction buffer) were preincubated at 37°C for 30 minutes and then mixed with 5 μl of 10 mM tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acidα (4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA; (Peptide Institute). Free aminomethylcoumarin (AMC) accumulation, which resulted from cleavage of the aspartate-AMC bond, was monitored in each sample at 37°C over 180 minutes using a spectrophotometer (Ultraspec III; Pharmacia, Cambridge, England). The absorbance of the each sample at 370 nm was plotted against time. Linear regression analysis of the velocity of each curve yielded the activity for each sample. Data were expressed as a percentage of the caspase-1–like activities in the samples compared to those in the P28 SD rat retina. 
Statistical Analysis
The data were analyzed statistically by one-way ANOVA followed by Fisher’s post-hoc test. Probability values less than 0.05 were determined to be statistically significant. 
Results
TUNEL Staining
In both RCS and SD rats, TUNEL-positive cells were observed in the ONL at P14. In SD rats, these cells were no longer observed at P21. In contrast, TUNEL-positive cells increased in number in the ONL of RCS rats at P21 to P45, with a peak at P28 (Figs. 1 A, 1B). At P28, the number of TUNEL-positive cells was approximately 250/0.4-mm section, and about one third of the total cell number in the ONL at P28 was stained by TUNEL (Fig. 1B)
Immunohistochemistry
Specific staining of cells in the ONL of RCS rats was obtained using antibodies against caspase-1 and caspase-2 (Figs. 2 A, 2B). In contrast, no specific staining was detected by any of the antibodies in the ONL of P28 SD rat retinas (graphic data not shown). 
Diffuse cytoplasmic staining with anti–caspase-1 and anti–caspase-2 antibodies was seen in apoptotic cells that had condensed nuclei. In cells with weak or no PI staining, caspase-1–like and caspase-2–like immunoreactivities were observed in the nucleus and cytoplasm. Immunopositive cell species could not be identified because apoptotic cells had lost their original appearance. However, we concluded that the immunostained cells were apoptotic photoreceptors because of their location in the ONL and because they were not immunolabeled with anti-phosphotyrosine antibody (graphic data not shown). 
TUNEL-Positive and Immunostained Cells
In RCS rats, quantitative analyses showed that the numbers of cells stained with antibodies against caspase-1 and caspase-2 were very similar during the follow-up period (Fig. 2C) . Also, the time course of TUNEL-positive cells showed a similar pattern of immunostaining. The numbers of immunostained cells were almost equal to that of TUNEL-positive cells at P14 and P21 (less than 6 cells/0.4-mm tissue section), but there were approximately 10 times more TUNEL-positive cells than immunostained cells at P28 (243.3 ± 8.0 TUNEL-positive cells/0.4-mm tissue section, 18.2 ± 0.4 caspase-1–positive cells and 13.7 ± 0.4 caspase-2–positive cells). 
Double staining studies to identify TUNEL-positive cells and immunostained cells using anti–caspase-1 and anti–caspase-2 antibodies showed that these signals were colocalized in the same cell (Figs. 2A 2B) . Caspase-1 and caspase-2 were also double-stained in the same cell (graphic data not shown). However, all the anti–caspase-1–positive cells were not always stained with anti–caspase-2 antibodies. The results of double staining with the antibodies and TUNEL staining showed that expression of caspase-1 and caspase-2 was correlated with photoreceptor apoptosis in RCS rats. Anti-phosphotyrosine antibody–labeled cells did not show coexpression of caspase-1 or caspase-2 (graphic data not shown). 
Immunoblot Analysis of Caspase-1– and Caspase-2–Like Proteases
As shown in Figure 3 A, anti–caspase-1 antibody depicted both 37.5-kDa and 20-kDa bands by western blot analysis. The apparent molecular weight of the bands agrees with that of caspase-1–like protease. Figure 3B shows that caspase-2–like protease is expressed in RCS rat retinas, based on the finding that anti–caspase-2 antibody blotted at both 40.5- and 33-kDa bands. The active form of caspase-1 and caspase-2 consists of long and short fragments that are derived from proteolytic processing of the proenzyme during apoptosis. Antibodies used in this study reacted with the active form of the 20-kDa subunit of caspase-1 and the 33-kDa subunit of caspase-2. The level of expression of the 37.5 kDa caspase-1 subunit was constant, but that of the 20-kDa subunit, the active form, was upregulated at P21, P28, and P35 RCS rat. On the other hand, expression of the 40.5-kDa proenzyme was constant, whereas that of the 33-kDa active form of caspase-2 was upregulated at P14, P21, P28, and P35 in RCS rat retinas. 
Assay of Caspase-1–Like Protease Enzymatic Activity
Activity of caspase-1–like protease was assayed using the specific tetrapeptide substrate. As shown in Figure 4 , caspase-1–like protease activity was elevated in the extract from P28 RCS rat retinas. The levels of the activity were twofold those of P14 rats at P21 and sevenfold at P28. The time course of caspase activity changes was similar to those of immunohistochemical analysis and protein expression level of caspase-1 active form as shown in western blot analysis. 
Effect of the Caspase-1 Inhibitor
Figure 5 indicates that the number of TUNEL-positive cells in ONL was significantly reduced in P28 RCS rat retinas injected with the inhibitor Ac-YVAD-CHO at P26. However, the number of TUNEL-positive cells of P28 rat retinas injected with the tetrapeptide at P21 was not decreased. These results indicate that the caspase-1 inhibitor can suppress, at least transiently, apoptosis of photoreceptor cells. 
Discussion
In this study, we demonstrate that caspase-1– and caspase-2–like proteases play an important role in photoreceptor apoptosis during retinal degeneration in the RCS rat. Activation of caspase-1–like protease contributes to photoreceptor apoptosis in the RCS rat. 
Caspase-1 was first identified as the mammalian homologue of the ced-3 gene, initially identified in Caenorhabditis elegans. Caspase-1 is known to promote apoptosis during development of the nematode, and, currently, 10 Ced-3–related cysteine proteases, termed caspase, have been cloned. 3 4 These family members participate in one of two distinct signaling pathways: activation of proinflammatory cytokines and activation of apoptotic cell death. 4 All the known caspase family proteases are synthesized as inactive proenzymes, which require cleavage to liberate one large and one small subunit to form the active enzyme. 4 Activation of one caspase can lead to cleavage and activation of another molecule of the same caspase, another caspase, or both, leading to an amplified apoptotic cascade. 4 Indeed, in the Fas-mediated apoptosis of lymphoid cells, activation of caspase-3 via specific cleavage of the proenzyme by caspase-1 has been demonstrated. 4 A number of experimental studies suggest that activation of the caspase family plays a critical role in the execution of apoptotic events. 3 7 8 9 Caspase-1 was implicated in death caused by superoxide dismutase downregulation in PC12 cells but not in withdrawal of trophic factor support. 9 Conversely, the antisense constructed to downregulate caspase-2 in PC12 cells inhibited cell death by withdrawal of trophic support but not oxidative stress. 7 The activation of distinct caspases in the same cells thus can promote apoptosis initiated by the various stimuli. 7  
In the present study, immunohistochemical analysis showed that caspase-1 and caspase-2 were colocalized in the same cell. Furthermore, on immunoblot analysis and measurement of enzymatic activities (Figs. 3 4) , the active forms of caspase-1– and caspase-2–like proteases were shown to be upregulated in P28 RCS rat retinas. These results suggest that caspase-1 and caspase-2 are correlated with the photoreceptor apoptotic process in RCS rats. However, it remains to be determined how these two caspases are activated in photoreceptor degeneration. To estimate the potential role of caspase-1–like protease in photoreceptor apoptosis more directly, we examined in vivo effects of a specific inhibitor of caspase-1 on the number of apoptotic photoreceptors. When administrated into the vitreous, Ac-YVAD-CHO reduced the number of apoptotic cells (Fig. 5) . This suggests that caspase-1–like protease induced photoreceptor apoptosis in the RCS rat and further suggests that an inhibitor of such caspases could inhibit apoptosis in other photoreceptor degenerative diseases. In fact, inhibitors of caspase-1 and caspase-3 can inhibit neuronal apoptosis in brain injury induced by ischemia and by trauma. 14  
Compared with the number of TUNEL-positive cells, immunopositive cells were fewer in this study. It is difficult to give satisfactory explanation to this apparent discrepancy. However, there are four possible explanations or speculations for this. First, low level expression of apoptosis-related proteins may not be detectable by the immunohistochemical methods used in this study. Second, photoreceptor apoptosis proceeds very slowly in RCS rats. TUNEL-positive cells accounted for as many as one third of the total cell number in the ONL at P28; if these TUNEL-positive cells were digested rapidly by surrounding cells, all the photoreceptors would disappear within several days. However, because photoreceptors survive until P60, 2 dying cells may accumulate and remain over a long period in the ONL. Thus, only a few cells that have recently entered the apoptotic process and that overexpress the caspase-like proteases may be present in any individual tissue sections. A third possibility is that numerous other mechanisms may be involved in the apoptotic process in RCS rats, and we may be aware of only a small portion of this apoptotic process. For example, caspases other than caspase-1 and caspase-2 may play a more important role in the apoptotic process of the RCS rat retina. Finally, it is likely that the persistence of apoptotic nuclei for long periods reflects a failure of phagocytosis. 
To our knowledge, this is the first report of a correlation between photoreceptor apoptosis and the caspase family. Although this study suggests an important role for caspase-1 and caspase-2 in the process of photoreceptor apoptosis in the RCS rat retina, further studies are necessary to clarify the molecular mechanism underlying retinal degeneration in these animals. 
 
Figure 1.
 
(A) The time course of TUNEL staining in the ONL of the RCS rat. The images represent digital overlay of a black-and-white image using Nomarski differential interference contrast and a green color image of TUNEL staining using a confocal microscope. (B) The number of TUNEL-positive nuclei in the ONL of the RCS rats. TUNEL-stained cells are counted in 0.4-mm cryosections in the ONL of each retina using a confocal microscope. Data are expressed as the mean ± SEM (n = 4).
Figure 1.
 
(A) The time course of TUNEL staining in the ONL of the RCS rat. The images represent digital overlay of a black-and-white image using Nomarski differential interference contrast and a green color image of TUNEL staining using a confocal microscope. (B) The number of TUNEL-positive nuclei in the ONL of the RCS rats. TUNEL-stained cells are counted in 0.4-mm cryosections in the ONL of each retina using a confocal microscope. Data are expressed as the mean ± SEM (n = 4).
Figure 2.
 
(A, B) Confocal microscopic images of TUNEL and immunostaining of RCS rat retina. TUNEL staining (a) and immunostaining (b) with rhodamine-conjugated secondary antibodies to anti–caspase-1 (A) and anti–caspase-2 (B) antibody, Nomarski differential interference contrast (c), and digital overlay (d) of TUNEL and immunostaining. TUNEL-positive cells were colabeled with antibodies against caspase-1 (A) and caspase-2 (B) in the ONL of P28 RCS rats. (C) Time course of immunopositive cells in the ONL of RCS rats. The temporal profile of immunostaining was similar to that of TUNEL staining. Caspase-1–positive cells were seen more frequently than were caspase-2–positive cells. Data are represented as findings per 4-mm tissue section and are expressed as mean ± SEM (n = 4). Scale bar, 50 μm.
Figure 2.
 
(A, B) Confocal microscopic images of TUNEL and immunostaining of RCS rat retina. TUNEL staining (a) and immunostaining (b) with rhodamine-conjugated secondary antibodies to anti–caspase-1 (A) and anti–caspase-2 (B) antibody, Nomarski differential interference contrast (c), and digital overlay (d) of TUNEL and immunostaining. TUNEL-positive cells were colabeled with antibodies against caspase-1 (A) and caspase-2 (B) in the ONL of P28 RCS rats. (C) Time course of immunopositive cells in the ONL of RCS rats. The temporal profile of immunostaining was similar to that of TUNEL staining. Caspase-1–positive cells were seen more frequently than were caspase-2–positive cells. Data are represented as findings per 4-mm tissue section and are expressed as mean ± SEM (n = 4). Scale bar, 50 μm.
Figure 3.
 
Western blot analysis of caspase-1–like (A) and caspase-2–like (B) proteins in the RCS rat. (A) Bands of 20 and 37.5 kDa were depicted with the anti–caspase-1 antibody. Expression of the 37.5-kDa subunit was at a constant level, but that of the 20-kDa subunit was upregulated at P28 and P35. (B) Anti–caspase-2 antibody showed 40.5- and 33-kDa bands. Both subunits were upregulated at P28. Data are representative of five separate experiments with similar results.
Figure 3.
 
Western blot analysis of caspase-1–like (A) and caspase-2–like (B) proteins in the RCS rat. (A) Bands of 20 and 37.5 kDa were depicted with the anti–caspase-1 antibody. Expression of the 37.5-kDa subunit was at a constant level, but that of the 20-kDa subunit was upregulated at P28 and P35. (B) Anti–caspase-2 antibody showed 40.5- and 33-kDa bands. Both subunits were upregulated at P28. Data are representative of five separate experiments with similar results.
Figure 4.
 
Caspase-1–like enzymatic activities in the retinal extracts of RCS rats. Protease activity is expressed as a percentage of that in P28 SD rat retina. Data are expressed as the mean ± SEM (n = 4). The enzymatic activities at P28 are significantly elevated relative to those of SD at P28.* P < 0.01.
Figure 4.
 
Caspase-1–like enzymatic activities in the retinal extracts of RCS rats. Protease activity is expressed as a percentage of that in P28 SD rat retina. Data are expressed as the mean ± SEM (n = 4). The enzymatic activities at P28 are significantly elevated relative to those of SD at P28.* P < 0.01.
Figure 5.
 
TUNEL-positive cells in the ONL of P28 RCS rats after intravitreal injection of the caspase-1 inhibitor Ac-YVAD-CHO. Data are expressed as mean ± SEM (n = 4). Photoreceptor apoptosis was transiently blocked by the caspase-1 inhibitor.* P < 0.01.
Figure 5.
 
TUNEL-positive cells in the ONL of P28 RCS rats after intravitreal injection of the caspase-1 inhibitor Ac-YVAD-CHO. Data are expressed as mean ± SEM (n = 4). Photoreceptor apoptosis was transiently blocked by the caspase-1 inhibitor.* P < 0.01.
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Figure 1.
 
(A) The time course of TUNEL staining in the ONL of the RCS rat. The images represent digital overlay of a black-and-white image using Nomarski differential interference contrast and a green color image of TUNEL staining using a confocal microscope. (B) The number of TUNEL-positive nuclei in the ONL of the RCS rats. TUNEL-stained cells are counted in 0.4-mm cryosections in the ONL of each retina using a confocal microscope. Data are expressed as the mean ± SEM (n = 4).
Figure 1.
 
(A) The time course of TUNEL staining in the ONL of the RCS rat. The images represent digital overlay of a black-and-white image using Nomarski differential interference contrast and a green color image of TUNEL staining using a confocal microscope. (B) The number of TUNEL-positive nuclei in the ONL of the RCS rats. TUNEL-stained cells are counted in 0.4-mm cryosections in the ONL of each retina using a confocal microscope. Data are expressed as the mean ± SEM (n = 4).
Figure 2.
 
(A, B) Confocal microscopic images of TUNEL and immunostaining of RCS rat retina. TUNEL staining (a) and immunostaining (b) with rhodamine-conjugated secondary antibodies to anti–caspase-1 (A) and anti–caspase-2 (B) antibody, Nomarski differential interference contrast (c), and digital overlay (d) of TUNEL and immunostaining. TUNEL-positive cells were colabeled with antibodies against caspase-1 (A) and caspase-2 (B) in the ONL of P28 RCS rats. (C) Time course of immunopositive cells in the ONL of RCS rats. The temporal profile of immunostaining was similar to that of TUNEL staining. Caspase-1–positive cells were seen more frequently than were caspase-2–positive cells. Data are represented as findings per 4-mm tissue section and are expressed as mean ± SEM (n = 4). Scale bar, 50 μm.
Figure 2.
 
(A, B) Confocal microscopic images of TUNEL and immunostaining of RCS rat retina. TUNEL staining (a) and immunostaining (b) with rhodamine-conjugated secondary antibodies to anti–caspase-1 (A) and anti–caspase-2 (B) antibody, Nomarski differential interference contrast (c), and digital overlay (d) of TUNEL and immunostaining. TUNEL-positive cells were colabeled with antibodies against caspase-1 (A) and caspase-2 (B) in the ONL of P28 RCS rats. (C) Time course of immunopositive cells in the ONL of RCS rats. The temporal profile of immunostaining was similar to that of TUNEL staining. Caspase-1–positive cells were seen more frequently than were caspase-2–positive cells. Data are represented as findings per 4-mm tissue section and are expressed as mean ± SEM (n = 4). Scale bar, 50 μm.
Figure 3.
 
Western blot analysis of caspase-1–like (A) and caspase-2–like (B) proteins in the RCS rat. (A) Bands of 20 and 37.5 kDa were depicted with the anti–caspase-1 antibody. Expression of the 37.5-kDa subunit was at a constant level, but that of the 20-kDa subunit was upregulated at P28 and P35. (B) Anti–caspase-2 antibody showed 40.5- and 33-kDa bands. Both subunits were upregulated at P28. Data are representative of five separate experiments with similar results.
Figure 3.
 
Western blot analysis of caspase-1–like (A) and caspase-2–like (B) proteins in the RCS rat. (A) Bands of 20 and 37.5 kDa were depicted with the anti–caspase-1 antibody. Expression of the 37.5-kDa subunit was at a constant level, but that of the 20-kDa subunit was upregulated at P28 and P35. (B) Anti–caspase-2 antibody showed 40.5- and 33-kDa bands. Both subunits were upregulated at P28. Data are representative of five separate experiments with similar results.
Figure 4.
 
Caspase-1–like enzymatic activities in the retinal extracts of RCS rats. Protease activity is expressed as a percentage of that in P28 SD rat retina. Data are expressed as the mean ± SEM (n = 4). The enzymatic activities at P28 are significantly elevated relative to those of SD at P28.* P < 0.01.
Figure 4.
 
Caspase-1–like enzymatic activities in the retinal extracts of RCS rats. Protease activity is expressed as a percentage of that in P28 SD rat retina. Data are expressed as the mean ± SEM (n = 4). The enzymatic activities at P28 are significantly elevated relative to those of SD at P28.* P < 0.01.
Figure 5.
 
TUNEL-positive cells in the ONL of P28 RCS rats after intravitreal injection of the caspase-1 inhibitor Ac-YVAD-CHO. Data are expressed as mean ± SEM (n = 4). Photoreceptor apoptosis was transiently blocked by the caspase-1 inhibitor.* P < 0.01.
Figure 5.
 
TUNEL-positive cells in the ONL of P28 RCS rats after intravitreal injection of the caspase-1 inhibitor Ac-YVAD-CHO. Data are expressed as mean ± SEM (n = 4). Photoreceptor apoptosis was transiently blocked by the caspase-1 inhibitor.* P < 0.01.
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