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From the Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto, Japan.
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Abstract |
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METHODS. Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. Expression of caspase-1 and caspase-3 was studied at the mRNA and protein levels using immunohistochemical staining, western blot analysis, semiquantitative reverse transcription–polymerase chain reaction, and assay of the enzymatic activities. Apoptotic retinal neurons were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method. To study the roles of the caspases in retinal ischemia–reperfusion injury, an inhibitor of caspase-1, acetyl-tyrosyl-valyl-alanyl-aspart-1-al (Ac-YVAD-CHO; total dose, 10-7 moles) and that of caspase-3, acetyl-aspartyl-glutamyl-valyl-aspart1-al (Ac-DEVD-CHO; total dose, 10-7 moles) was injected intravitreally and the number of TUNEL-positive cells was compared with the number in sections not treated with the inhibitors.
RESULTS. In the inner nuclear layer (INL), caspase-3-like immunoreactivity was predominantly detected, whereas caspase-1-like immunoreactivity was more predominant in the outer nuclear layer (ONL). Expression of caspase-1 and -3 was upregulated at the protein and gene levels 24 hours after reperfusion. Intravitreal injection of Ac-DEVD-CHO decreased the number of TUNEL-positive cells more significantly in the INL than in the ONL (P < 0.01) at 24 hours, whereas, intravitreal injection of Ac-YVAD-CHO was more effective in decreasing the number in the ONL (P < 0.05).
CONCLUSIONS. These findings suggest a possibility that cell-type–specific activation of caspases takes place in retinal ischemia–reperfusion injury, and such caspase may induce retinal neuronal cell death.
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Introduction |
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Apoptosis plays a major role in the pathogenesis of many ocular diseases and animal models including retinitis pigmentosa, retinal detachment, light injury, glaucoma, age-related macular degeneration, and ischemia–reperfusion injury.7 8 9 10 It has been determined that c-fos is involved in a photoreceptor apoptotic pathway induced by light injury, because this light-induced apoptosis is virtually absent in mice without c-fos.11 Thus, it is important to know which genes are specifically expressed during apoptosis of retinal cells, because apoptotic cell death can be rescued by inhibiting the expression of essential genes of apoptosis.
Various gene products play a role in the process,3 9 12 13 14 and among them, several members of the IL-1ß-converting enzyme (ICE/caspase-1) family have been implicated in the cascade of apoptotic death in a variety of cell types.4 15 16 17 18 19 20 ICE was initially identified as a ced-3 gene homologue that is required for programmed cell death in the nematode, Caenorhabditis elegans.16 20 At present, at least 11 ICE homologues named caspase-1 through caspase-11 have been identified and are divided into three groups.21 22 Caspase-1 encodes a cysteine protease that cleaves proteins after aspartic acid,23 24 and other caspases are also translated as inactive proenzymes, activated by proteolysis and can cleave a number of cellular proteins. Sometimes the cleavage results in the activation of the protein and sometimes in inactivation.23 24
Recently, we and other groups showed that neuronal apoptosis and necrosis play a role in the pathologic mechanisms of ischemia–reperfusion injury in the rat retina in both the inner nuclear layer (INL) and outer nuclear layer (ONL).25 26 27 28 Herein, we present evidence that different caspase families probably play an important role in the apoptotic processes in photoreceptors and retinal neurons in the INL.
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Methods |
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Antibodies
Antibodies used in this study were obtained from various sources:
goat anti-caspase-1 antibody (M-19)29
and goat
anti-caspase-3 antibody (L-18) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA); rabbit anti-bovine s-100 antibody from
Immunotech (Marseilles, France); rabbit anti-glial acidic fibrillary
protein (GFAP) antibody from Nichirei (Tokyo, Japan); monoclonal
anti-
-tubulin antibody from Sigma (St. Louis, MO);
rhodamine-conjugated goat anti-rabbit IgG from Dako (Glostrup,
Denmark); and rhodamine-conjugated rabbit anti-goat IgG from Jackson
ImmunoResearch Laboratories (West Grove, PA).
The antibody against caspase-1 (M-19) was raised against a peptide corresponding to amino acids 276–294 mapping to the carboxyl-terminus of the 20-kDa subunit of the caspase-1 precursor of mouse origin. This antibody recognizes the 20-kDa subunit of active caspase-1. Anti-caspase-3 antibody was raised against a peptide corresponding to amino acids 157–174 mapping to the carboxyl terminus of the 20-kDa subunit of the 32-kDa cysteine protease precursor of human origin. This antibody also reacts with the active form of caspase-3. The specificity of the antibodies was verified by the manufacturers, and the antibodies were known to cross-react with rat proteins. These antibodies were used for both immunohistochemical studies and western blot analyses.
TUNEL Staining
DNA TUNEL was performed as described.30
After rats
were anesthetized with intraperitoneal injection of pentobarbital (60
mg/kg), they were perfused transcardially with 100 ml normal saline
supplemented with 1 U/ml heparin followed by 200 ml 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes were
enucleated and placed in 4% paraformaldehyde for 24 hours at 4°C.
They were rinsed with phosphate-buffered saline (PBS) and then
transferred to 10%, 15%, and 20% sucrose in 0.1 M PBS (3 hours for
each step) at room temperature. Samples were then snap frozen in
Tissue-Tek (Miles Laboratories, Elkhart, IN) on dry ice in hexane and
stored at -80°C. Cryostat sections (6–10 µm) were thaw mounted
onto glass slides coated with poly-L-lysine and air dried
for 2 hours at room temperature. After sections were rinsed three times
in 0.02 M PBS (pH 7.4) and reacted with proteinase K, they were
incubated with 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 37°C in a moisture chamber. After
rinsing, the sections were reacted with avidin-fluorescein
isothiocyanate and examined with a scanning laser confocal microscope
(LSM 410 inverted laser microscope; Carl Zeiss, Oberkochen, Germany).
Positive controls were generated using DNase I in TdT buffer (1
µg/ml) before incubation with terminal transferase and biotinylated
nucleotides.
Immunohistochemistry
After rinsing with 0.02 M PBS, cryosections were incubated with
2% normal rabbit or goat serum for 60 minutes at room temperature.
Incubation of the primary antibody was performed overnight in a
moisture chamber at 4°C. Working concentrations of the primary
antibodies (anti-caspase-1, 1:100; anti-caspase-3, 1:100) were
determined after applying various concentrations of the antibodies.
Rhodamine-conjugated secondary antibody was reacted at room temperature
for 2 hours. To investigate whether caspase-1 and caspase-3 are
expressed in apoptotic cells, TUNEL staining was performed after
immunostaining. To exclude the possibility that TUNEL-positive cells
were retinal glial cells and Müller cells, anti-s-100 and
anti-GFAP antibodies were reacted before TUNEL staining. Sections were
observed with the laser scanning confocal microscope in the transmitted
light mode.
Quantitative Analysis of TUNEL and Immunohistochemistry
Four eyes were used to obtain data for each time point to
investigate the time course of TUNEL and immunohistochemical staining.
The number of TUNEL-positive and immunostained cells in the INL and ONL
was determined in five meridian sections through the optic nerve head.
The number of cells was counted in 20 areas of each section at 1 mm
from the optic nerve head on the superior and inferior hemisphere using
the confocal microscope’s area measure and measure functions. Data are
represented as number of cells per square millimeter.25
28
Results are expressed as mean ± SEM.
Western Blot Analysis
Retinas were dissected from enucleated eyes (n = 4
for each time point) with fine forceps under an operating microscope;
the samples were immediately frozen on dry ice and kept at -80°C
until used. Samples were homogenized in buffer containing Tris-HCl (pH
8.0), 250 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10
µg/ml pepstatin, 20 µg/ml leupeptin, and 10 µg/ml aprotinin. The
protein content was determined using the method of
Bradford.31
The mixture was then added to 5x Laemmli
sample buffer containing 125 mM Tris-HCl (pH 6.8), 10% (wt/vol)
glycerol, 3% sodium dodecyl sulfate, 6% urea, 5%
ß-mercaptoethanol, and 0.02% bromphenol blue. Sodium dodecyl
sulfate–polyacrylamide gel electrophoresis was performed as
described32
after samples were boiled for 5 minutes. A
sample of 50 µg protein was loaded on each lane and was
electrophoresed on 10% acrylamide gel and then transferred to
nitrocellulose membranes.33
After rinsing with
Tris-buffered saline (pH 7.4) containing 0.25% Tween-20 (TBST), the
membrane was incubated with primary antibodies (1:500) in TBST for 1
hour at room temperature. After the membrane was washed three times, it
was incubated with the horseradish peroxidase–conjugated secondary
antibody diluted 1:500 in TBST for 1 hour at room temperature, washed
again, and developed with the enhanced chemiluminescence western blot
analysis system (ECL; Amersham, Buckinghamshire, UK). The gel was then
exposed to hyperfilm (ECL; Amersham) for 1 to 3 minutes. Loading of
approximately equivalent amounts of intact protein content was
confirmed by the blot with antibody against -tubulin protein.
Semiquantitative RT-PCR
Total RNA was extracted from the frozen stored retina by the
guanidium thiocyanate method. PolyA+RNA was then
purified from the total RNA by an oligo-dT column. Aliquots of
polyA+RNA (0.1 µg) were used to make the cDNA
with a first-strand cDNA synthesis kit (Pharmacia-Biotech, Uppsala,
Sweden). PCR was performed by the method of Saiki et al.34
with slight modifications. The following conditions were used:
denaturation at 94°C for 45 seconds; annealing at 55°C for 45
seconds; and extension at 72°C for 90 seconds. The reaction was
performed for 33 to 35 cycles. After PCR, 1 µl of PCR product was
cloned directly into a plasmid vector (pCRII; Invitrogen, San Diego,
CA). Nucleotide sequencing of the cloned DNA was performed by the
dideoxynucleotide chain termination method by using a gene analyzer
(ABI Prism 310; Perkin–Elmer, Foster City, CA). The sequence data of
the PCR products were identical with rat sequences found in GenBank.
The optimal PCR cycle number for each primer set was obtained by
varying the number of PCR cycles. Primers used to amplify the target
genes were: Rat caspase-1 cDNA, 5'-TTCAGGCATGCCGTGGAG (sense) and
5'-AATGTCCCGGGAAGAGGT (antisense), 363 bp35
; rat caspase-3
cDNA, 5'-ACATGGAAGCGAATCAATGGACTC (sense) and
5'-AAGGACTCAAATTCTGTTGCCACC (antisense), 698 bp35
; and rat
ß-actin cDNA, 5'-AGCTGAGAGGGAAATCGTGC (sense) and
5'-CCACAGGAT-TCCATACCTGA (antisense), 297 bp.36
Expression of ß-actin was used as the internal standard. Reaction
mixture of 1 picomole/ml caspase-1 primer sets and 0.3 picomoles/ml
ß-actin primer sets in a tube was amplified for 33 cycles.
Thirty-five PCR cycles were performed for caspase-3 cDNA amplification
in a tube including 1 picomole/ml caspase-3 primer sets and 0.2
picomoles/ml ß-actin primer sets. PCR products were electrophoresed
on a 3% agarose gel and visualized with ethidium bromide. The digital
photograph was analyzed (Gel Plotting Macros; NIH Image ver. 1.62;
National Institutes of Health, Bethesda, MD). Levels of individual PCR
products were expressed as the ratio of individual product optical
density to that of the internal standard.
Intravitreal Administration of Caspase Inhibitors
Selective inhibitors of caspase-1,
acetyl-tyrosyl-valyl-alanyl-aspart-1-al (Ac-YVAD-CHO), and caspase-3,
acetyl-aspartyl-glutamyl-valyl-aspart-1-al
(Ac-DEVD-CHO),37
were obtained from the Peptide Institute
(Osaka, Japan). A 20- mM solution of these inhibitors in dimethyl
sulfoxide was prepared, and 2 µl of the solution was injected into
the vitreous cavity with a 30-gauge needle 24 hours before ischemic
insult. Another 3 µl was injected into the vitreous at the time of
reperfusion. As a control, dimethyl sulfoxide (vehicle) was injected in
the same way. Twenty-four hours after reperfusion, eyes were
enucleated, and TUNEL staining was performed as described earlier,
because the time point for peak TUNEL staining was at 24 hours after
reperfusion. Eyes that showed vitreous and subretinal hemorrhage from
the intravitreal injection were omitted from the study.
Assay of Caspase-1 and Caspase-3 Activities
Caspase family protease activities were assayed as
described19
38
with slight modification. Briefly, frozen
stored retinas were homogenized in 10 mM Hepes-KOH (pH 7.2), 2 mM EDTA,
0.1% CHAPS, 5 mM dithiothreitol, 1 mM PMSF, 10 µg/ml pepstatin, 20
µg/ml leupeptin, and 10 µg/ml aprotinin. Homogenates were
centrifuged at 15,000g for 30 minutes. Supernatants were
transferred to new tubes (Eppendorf; Fremont, CA). 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 10 mM tetrapeptide
substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid
-(4-methyl-coumaryl-7-amide; Ac-YVAD-MCA) for caspase-1 or
acetyl-aspartyl-glutamyl-valyl-aspartic acid
-(4-methyl-coumaryl-7-amide; Ac-DEVD-MCA) for caspase-3 obtained
from the Peptide Institute. Free aminomethylcoumarin accumulation,
which resulted from cleavage of the aspartate-aminomethylcoumarin bond,
was monitored in each sample at 37°C for 180 minutes in a
microcubbette using a spectrophotometer (Ultraspec III; Pharmacia,
Cambridge, UK). The absorbance of the each sample at 370 nm was plotted
against time. Linear regression analysis of the velocity of each curve
yielded an activity for each sample.38
Data were expressed
as a percentage of the caspase-family–like activity in the samples
from sham-procedure retina.
Statistical Analysis
The data were analyzed by one-way analysis of variance (ANOVA)
followed by Fisher’s post hoc test. P < 0.05 was
taken to be statistically significant.
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Results |
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Semiquantitative RT-PCR
Semiquantitative RT-PCR analyses showed a significant
(P < 0.01 by ANOVA) upregulation of the mRNAs for
caspase-1 and caspase-3 in the experimental retinas compared with that
in sham-procedure rat retinas at 12 to 48 hours after reperfusion (Fig. 4)
. Such mRNA expression decreased at 96 hours after reperfusion. A peak
time point of the two caspases was 24 hours after reperfusion. The
expression level of caspase-1 and caspase-3 was 1.48 and 2.18 times
higher than that of the sham-procedure controls, respectively.
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Discussion |
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Caspase-1 was identified as a mammalian homologue of the ced-3 gene,20 initially identified in Caenorhabditis elegans. Ced-3 is known to promote apoptosis in the nematode during development, and currently, 11 cysteine proteases,21 23 39 40 termed caspases,22 have been cloned. These family members participate in one of two distinct signaling pathways: activation of proinflammatory cytokines and activation of apoptotic cell death.39 41 All the known caspase family proteases are synthesized as inactive proenzymes that require cleavage to liberate one large and one small subunit to form the active enzyme.39 These families have been divided into three groups based on homology,22 and we examined two distinct family members of caspase-1 and caspase-3.
A number of experimental studies have suggested that activation of the caspase family is important in the execution of apoptotic events in neurons.41 42 43 44 45 46 47 48 49 Our previous studies indicate that neuronal apoptosis and necrosis are the main pathologic mechanism in ischemia–reperfusion injury in the rat retina, based on morphologic (TUNEL staining and ultrastructural studies) and biochemical (DNA fragmentation) evidence,25 26 and other reports have supported our idea.27 50 Furthermore, TUNEL-positive cells in INL were double stained with an amacrine cell marker but not anti-s-100 and anti-GFAP antibodies.25 26 In the present study, the same results were obtained (graphic data not shown). Thus, we conclude TUNEL-positive cells were mainly retinal neurons. Although most of the TUNEL-positive cells were neurons, we have to be careful not to misunderstand that TUNEL positivity directly addresses neuronal death.
In the present experiment, caspase-1 and caspase-3 were colocalized with TUNEL-positive cells in the INL and ONL, and the temporal profiles of TUNEL staining and immunostaining were similar. Western blot analysis showed activation of caspase-1-like and caspase-3-like proteases during the retinal degenerative process during ischemia–reperfusion. Also, enzymatic activities of caspase were upregulated at 12 to 48 hours after reperfusion and downregulated at 96 hours. The level of these caspase mRNAs increased after reperfusion and decreased at 96 hours. Caspase-1 has been implicated in the death caused by superoxide dismutase downregulation in PC12 cells, but not by withdrawal of trophic factor support.46 Conversely, an antisense constructed to downregulate caspase-2 in PC12 cells inhibites cell death by withdrawal of trophic support,47 but does not inhibit oxidative stress.48 Activation of distinct caspases in the same cells can thus promote the apoptosis initiated by different stimuli. Furthermore, an in vivo study shows that caspase-1 and caspase-3 contribute to neuronal cell death in ischemic brain injury induced by occlusion of the middle cerebral artery in mice,42 and caspase-3, but not caspase-1, is associated with neuronal apoptosis after fluid-percussion–induced traumatic brain injury.45 These studies indicate that different pathologic stimuli can activate a distinct caspase family in the same cell to induce apoptosis.
Little is known, however, about whether different proteases in the caspase family are activated in apoptotic neurons of different cell types evoked by the same pathologic condition.49 The retina is an appropriate model to answer this question, because there are at least six distinct types of neurons in the retina: retinal ganglion cells, bipolar cells, amacrine cells, horizontal cells, cone photoreceptors, and rod photoreceptors. In the present study, we tried to determine whether different caspases were expressed in apoptotic neurons of the ONL and the INL induced by the ischemia–reperfusion injury. Immunohistochemically, caspase-1-immunoreactive cells in the ONL were more pronounced than those of caspase-3, but in the INL, caspase-3–reactive cells were more numerous than caspase-1 cells. When the caspase-1 inhibitor was injected into the vitreous, the percentage of apoptotic cells in the ONL was decreased significantly more than those in the INL. Conversely, when the caspase-3 inhibitor was administered, the changes in the number of apoptotic cells in the INL was significantly greater than in the ONL. Thus, apoptotic neuronal death in retina after ischemia–reperfusion is executed by two different caspases. In the INL, caspase-3 is the main executor of apoptotic neurons, and in the photoreceptors caspase-1 is predominantly expressed. We thus conclude that the neuronal death evoked in different cell types by the same cause may be mediated by distinct members of the caspase family proteases. Stereologic examination of multiple retinal layers in this model, if possible, would give more satisfactory data. However, in the present investigation such examination was not possible.
Unfortunately, specific inhibitors of both caspase-1 and caspase-3 have not been discovered, and the inhibitors used in this study were selective rather than specific for caspase-1 and caspase-3. Ac-YVAD-CHO inhibites the enzymatic activity of caspase-4, and also, Ac-DEVD-CHO in high concentrations blocks caspase-7 reactivity.51 Therefore, we cannot discriminate between the relationship of caspase-4 and caspase-7 in retinal ischemia–reperfusion injury. Although we have shown the importance of two distinct caspase family proteases in the INL and ONL by a single insult, we have to be careful in drawing a conclusion. Further experiments using specific rather than selective inhibitors or the use of knockout animals will be needed in the future.
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Footnotes |
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Submitted for publication December 11, 1998; revised April 30, 1999; accepted June 15, 1999.
Commercial relationships policy: N.
Corresponding author: Nagahisa Yoshimura, Department of Ophthalmology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan. E-mail: nagaeye{at}hsp.md.shinshu-u.ac.jp
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