Journal of Neurochemistry, 2001, 77, 466±475
Cell-speci®c caspase expression by different neuronal phenotypes in transient retinal ischemia Manjeet Singh,* Sean I. Savitz,* Romy Hoque,* Gaurav Gupta,* Steven Roth,² Pearl S. Rosenbaum³,§ and Daniel M. Rosenbaum*,³,¶ *Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, USA ²Department of Anesthesia and Critical Care, University of Chicago, Chicago, Illinois, USA ³Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, New York, USA §Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA ¶Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA
Abstract Emerging evidence supports an important role for caspases in neuronal death following ischemia-reperfusion injury. This study assessed whether cell speci®c caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double-labeling with cell-speci®c markers identi®ed which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also
Transient global retinal ischemia and transient global cerebral ischemia share many similarities. Both cause delayed neuronal death which occurs in part by apoptosis (Buchi 1992; Nitatori et al. 1995; Bhat et al. 1996; Rosenbaum et al. 1997; Rosenbaum et al. 1998a). In addition, there is selective vulnerability of speci®c subpopulations of neurons in both types of injury. Pyramidal neurons in the CA-1 zone of the hippocampus are selectively vulnerable to transient cerebral ischemic injury (Kirino 1982; Pulsinelli et al. 1982). Similarly, neurons in the inner nuclear layer of the retina show signi®cantly enhanced susceptibility to transient retinal ischemia
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peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc-aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to signi®cant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y-vad fmk. These observations suggest that ischemiareperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia-reperfusion. These results in the retina may shed light on differential caspase speci®city in global cerebral ischemia. Keywords: apoptosis, ischemia, neuronal death, protease, retina. J. Neurochem. (2001) 77, 466±475.
Received September 12, 2000; revised manuscript received December 21, 2000; accepted December 22, 2000. Address correspondence and reprint requests to Daniel M. Rosenbaum, Departments of Neurology, Neuroscience and Ophthalmology, Kennedy Center, Room 341 Albert Einstein College of Medicine 1410 Pelham Parkway South Bronx, NY 10461, USA. E-mail:
[email protected] Abbreviations used: BAF, Boc-aspartyl fmk; DMSO, dimethyl sulfoxide; ERG, electroretinograms; GFAP, glial ®brillary acidic protein; ILM, inner limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; PBS, phosphate-buffered saline; PFA, paraformaldehyde.
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compared with outer layer neurons (Szabo et al. 1991; Rosenbaum et al. 1997). Many of the same molecular pathways mediating apoptosis operate in cerebral and retinal ischemia. Previous reports have supported a role for the bcl-2 family in forebrain ischemia. Expression of Bax is increased in CA-1 neurons dying from ischemic injury while the expression of Bcl-2 and Bcl-x is decreased (Antonawich et al. 1998; Krajewski et al. 1995). Bax up-regulation is also induced by retinal ischemia but no changes have been detected in bcl-2 expression (Kaneda et al. 1999). The expression of the nuclear phosphoprotein, p53, which is a key determinant in tumor suppression and apoptosis, also increases in retinal ischemia (Rosenbaum et al. 1998b) and may increase in global cerebral ischemia (McGahan et al. 1998). Mice heterozygous for the p53 null mutation are protected against focal cerebral and retinal ischemic damage but homozygotes are not similarly protected (Crumrine et al. 1994; Rosenbaum et al. 1998b). Increasing evidence also support a role for the caspase family in neuronal ischemia (Schulz et al. 1999). The 14 known caspases can be divided into three groups based on their different functions. One class primarily participates in in¯ammation (caspases 1, 4 and 5), the second class controls the activation of other caspases (caspases 2, 8, 9 and 10), and the third class carries out the execution phase of apoptosis (caspases 3, 6, 7 and 14). Previous studies have found evidence for caspase 3 mRNA up-regulation and protein activation in models of transient global cerebral ischemia (Chen et al. 1998; Namura et al. 1998; Ni et al. 1998). Caspase 2 gene expression has also been observed to increase in global ischemia (Kinoshita et al. 1997) but there have been no reports of enhanced translation or activation of caspase 2. Focal cerebral ischemia induces caspase 2 and 3 mRNA (Asahi et al. 1997) but knockout mice for caspase 2 are not resistant to focal ischemic injury (Schulz et al. 1999). Caspase 3 may thus serve a more predominant role in transient focal cerebral ischemia. In transient retinal ischemia, there have been con¯icting data about the role of different caspases (Katai and Yoshimura 1999; Lam et al. 1999). The expression of individual caspases in neuronal ischemia may differ in various neuronal populations. Velier et al. (1999) have shown that permanent occlusion of the middle cerebral artery leads to regional differences between caspase 3 and caspase 8 expression in pyramidal neurons of the cortex. It is also possible that cell type determines the expression of speci®c caspases following ischemic injury. Given the similar mechanisms mediating cerebral and retinal ischemic injury, the retina may serve as an appropriate model to study cell-speci®c caspase expression because there are at least ®ve distinct types of neurons in the retina: retinal ganglion cells, bipolar cells, amacrine cells, horizontal cells, and photoreceptors. In the present study, we
evaluated the role of caspases 1, 2 and 3 following retinal ischemia/reperfusion injury and determined the cell speci®city of these caspases in the ischemic retina. Furthermore, we explored the possibility of rescuing neurons from retinal ischemic injury and preserving retinal function by inhibiting caspase activity. Materials and methods Retinal ischemia by transient elevation of intraocular pressure All animals were treated in accordance with the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals). Male Sprague±Dawley rats weighing 150±175 g were brie¯y anesthetized with an intramuscular injection of ketamine (30 mg/kg; Ketaset, Ft Dodge, IA, USA) and intramuscular injection of xylazine (2.5 mg/kg; Rompun, Bayer, Shawnee Mission, KS, USA) as previously described in this model (Li B. et al. 2000; Nonaka et al. 2000). After topical application of 0.5% proparacaine hydrochloride, the anterior chamber of the right eye was cannulated with a 27-gauge needle connected to normal saline container by silastic tubing and a manometer. The corneal puncture site was sealed with cyanoacrylate cement. Intraocular pressure was raised to 120 mmHg by elevating the saline container for 60 min. Animals were kept normothermic at 36.7 ^ 0.58C with a rectal probe and heating pad during the procedure and then until the animal was awake and mobile. Whitening of the iris and loss of the red re¯ex of the retina con®rmed retinal ischemia. After 60 min of ischemia, the needle was withdrawn from the anterior chamber and the intraocular pressure normalized. One drop of gentamicin ophthalmic solution and atropine 1% ophthalmic solution was applied topically to the right eye before and after cannulating the anterior chamber. The animals were then killed at various times, and their eyes were enucleated for morphologic and immunohistochemical studies. Light microscopy The globes were enucleated 1 week after ischemia/reperfusion and were ®xed in Trump's ®xative at 48C for at least 48 h. The globes were sectioned in the vertical meridian and the inferior portion of the eyeball (retina, choroid and sclera) was post®xed by 2% osmium tetraoxide, dehydrated by graded alcohol and propyline oxide, embedded in epoxy resin, sectioned at 1 mm-thick sections, and were stained with 1% toluidine blue. An ophthalmic pathologist (PSR), blinded to the experimental conditions, evaluated the retinal histoarchitecture. The thickness of the individual retinal layers was measured as follows: (1) outer limiting membrane (OLM) to inner limiting membrane (ILM); (2) outer nuclear layer (ONL); (3) outer plexiform layer (OPL); (4) inner nuclear layer (INL); (5) inner plexiform layer (IPL) to ILM. Averages for these measurements taken in four adjacent areas within 1 mm of the optic nerve were calculated. Selection of the same topographic region of the retina for all of these measurements is important in order to protect against possible regional anatomic variations. Electroretinograms (ERGs) Rats were dark-adapted overnight, brie¯y anesthetized with an intramuscular injection of ketamine (30±40 mg/kg) and xylazine
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Fig. 1 (a and b) Representative photomicrographs showing the histological appearance of the non-ischemic, control (A) and ischemic (B) retinas 7 days after 60 min of ischemia. Toluidine blue; original magni®cation, 40. (c) Measurement (mean ^ SD) of the thickness of retinal layers of non-ischemic (n 5) and ischemic (n 5) retinas 7 days after 60 min of ischemia. Ischemic retinas show signi®cant thinning of the inner retinal layers as compared to the non-ischemic controls. *p , 0.05 by Student's t-test.
(2.5 mg/kg). Pupils were dilated with tropicamide and cyclomydril and the body temperature maintained at 37 ^ 0.58C with a rectal probe and a heating pad. A platinum electrode was placed on the topically anesthetized cornea; a reference electrode placed by the ipsilateral mastoid and a ground electrode was placed close to midline on the lower dorsum. Full-®eld stroboscopic ¯ash stimuli were presented at a distance of 15 cm and a rate of 1.0/s. Neuroelectric signals were impedance matched through a unity gain preampli®er and further differentially ampli®ed with appropriate band pair settings (Ostwald et al. 1997). The `a-wave' and `b-wave' were identi®ed and latencies were scored from stimulus onset using computer cursors. ERG studies were performed at baseline (pre-ischemia), 60 min after onset of ischemia and at 7 days after reperfusion. Immunohistochemistry The eyes of all rats were enucleated at 1 (n 3), 3 (n 3), 6 (n 3), 24 (n 3), 72 (n 3) and 168 h (n 3) after 60 min of ischemia and were then ®xed in 4% paraformaldehyde (PFA) for 2 h. After removing the anterior segment of the eye and the lens, the eyes were further ®xed in 4% PFA for another 4 h and then cryoprotected in 25% sucrose solution overnight. The eye cups were washed and frozen in OCT embedding medium over liquid
nitrogen. 10 mm thick cryosections were prepared at 2 208C, ®xed in cold methanol for 15 min, rinsed in 1 phosphate-buffered saline (PBS) for 5 min, and incubated with 5% goat serum for 60 min at room temperature. The following primary antibodies were used: anti-caspase 1 (rabbit IgG; 1 : 150; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-caspase 2 (also known as anti-Nedd-2; rabbit IgG; 1 : 200; courtesy of Dr Carol Troy), anti-caspase 3 (rabbit IgG; 1 : 100; New England Biolabs, Beverly, MA, USA). Anti-caspase 3 antibody only reacts to the active form of caspase 3. Incubation with each antibody was performed overnight at 48C. Sections were then washed with 1 PBS three times and incubated with either ¯uorescein- or rhodamine-conjugated secondary antibody (anti-rabbit IgG; 1 : 100) at room temperature for 2 h. To investigate the cell types expressing Nedd-2 and caspase 3, double labeling with antiHPC (1 : 150; mouse IgG1; Sigma, St Louis, MO, USA), antiThy-1 (1 : 50; mouse IgG1; Pharmingen, San Diego, CA, USA), anti-PKC (1 : 50; mouse IgG2a; Pharmingen), anti-rhodopsin (RHO42D) (1 : 10; mouse IgG, courtesy of Dr Robert Molday), and anti-glial ®brillary acidic protein (GFAP; 1 : 50; mouse IgG2b; Pharmingen) was performed overnight and then labeled with respective ¯uorescein or rhodamine conjugated secondary antibody for 2 h. Sections were mounted with Antifade (Molecular
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Fig. 2 Caspase 2 (a±e) and caspase 3 (f±j) immunohistochemistry cryosections of non-ischemic rat retina (a,f) and of rat retina at 1 h (b and g), 6 h (c and h), 24 h (d and i) and 72 h (e and j) of reperfusion subsequent to a 60-min period of ischemia. No activated caspase 2 or caspase 3 immunoreactivity is present in the
sham-operated control retinas (a and f). In the ischemic retinas, notable immuno¯uorescence is present in the GCL and INL of the inner retina for caspase 2. There is also signi®cant immuno¯uorescence in the INL and ONL for caspase 3. Fluorescent microscopy, original magni®cation, 40.
Probes, Eugene, OR, USA) and were analyzed under ¯uorescent microscopy. Negative corresponding controls were performed by substitution of the primary antibody with 2% normal goat serum in PBS. Caspase 2 and caspase 3 immunoreactive cells were quanti®ed under 40 magni®cation (high power ®eld) at speci®ed time points after reperfusion.
microscopy. Co-localization utilizing confocal microscopy with Nedd-2 and caspase 3 expressing cells was also performed.
TUNEL staining Staining was based on the method described by Gavrielli et al. (1992) but with several modi®cations. Ten-micron cryosections were incubated in methanol for 15 min at room temperature, washed in 1 PBS for 5 min, and then incubated in a dUTP/Tdt mixture (BM) at 378C for one hour followed by three rinses in 1 PBS and mounted with Antifade. Corresponding negative (without bdUTP) and positive control (DNase-I-treated) sections were also prepared. The sections were analyzed under ¯uorescent
Antisense oligonucleotides Antisense Nedd-2 oligonucleotides (A-Nedd) (5 0 -QGCTCGGCGC CGCCATTTCCAGL-3 0 ) were purchased from Operon (Alameda, CA, USA). The oligonucleotides were coupled to penetratin 1 (Oncor, Gaithersburg, MD, USA), a peptide that facilitates the movement of oligonucleotides across cell membranes as described previously (Troy et al. 1997). A scrambled sequence with the same base composition as the antisense oligonucleotide (5 0 -QCC GTAGCGTAGCTCCGCCTGCL-3 0 ), de®ned as S-Nedd, was synthesized and coupled to Penetratin 1 as a control. A-Nedd was injected intravitreally (5 mL of 8.6 mm) 6 h prior to ischemia into the superotemporal quadrant of the right eye, approximately 1 mm posterior to the limbus, with a 32-gauge needle mounted on a Hamilton syringe. Control animals
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retinal thickness measurements (n 5 per group). In addition, the BAF animals were evaluated for caspase 2 and 3 immunostaining at 24 h following ischemia (n 3 per group).
Results Retinal ischemia Elevating the intraocular pressure to 120 mmHg for 60 min resulted in the typical histopathologic features expected subsequent to acute retinal ischemia (Foos 1976). In the ischemic eye, along with widespread neuronal degeneration, the entire retinal thickness was reduced as compared with the untouched control retinas (Figs 1a and b). Speci®cally, there was a 35±40% reduction in the thickness of the ischemic retina (OLM±ILM) as compared with the untouched controls (Fig. 1c), resulting predominantly from marked thinning of the inner retinal layers (INL, IPL±ILM). Mild disorganization of the cells in the outer nuclear layer (ONL) and of the photoreceptor inner and outer segments were also noted (Fig. 1b).
Fig. 3 Temporal pro®les showing the number of caspase 2-positive cells (a) and caspase 3-positive cells (b) in various retinal layers over time. n 3 per time point. (a) *differs from non-ischemic controls by p , 0.05. b differs from 1 and 72 h by p , 0.05. g Differs from 1 and 6 h by p , 0.05. d Differs from 24 and 72 h by p , 0.05. c Differs from 24 and 72 h by p , 0.05. f Differs from 24 h by p , 0.05. (b) *Differs from non-ischemic controls by p , 0.05. d Differs from 6 and 72 h by p , 0.05. r Differs from 6 h by p , 0.05.
received S-Nedd (5 mL of 8.6 mm). Gentamicin ophthalmic ointment (0.3%) was applied topically after the procedure. The eyes were evaluated immunohistochemically (n 5 per group) at 24 h after ischemia for caspase 2 and 3 expression and morphometrically at 7 days by retinal thickness measurements (n 10 per group). Caspase inhibitors Animals received an intravitreal injection of 5 mL of 400 mm Bocaspartyl fmk (BAF) (Enzyme Systems Products, Livermore, CA, USA), a non-speci®c caspase inhibitor or dimethyl sulfoxide (DMSO)/saline vehicle 6 h before ischemia and 5 min after reperfusion. Another set of animals received an intravitreal injection of 5 mL of 400 mm Y-vad-fmk (Bachem, Torrance, CA, USA), a caspase 1 selective inhibitor or vehicle. The effectiveness of these treatments were evaluated morphometrically at 7 days with
Caspase expression Since caspases have been shown to play an important role in apoptosis, we examined the expression of caspases 1, 2 and 3 in all the retinal layers at 1, 3, 6, 24, 72 and 168 h after 60 min of ischemia. No caspase protein immunoreactivity was noted in sham-operated control retinas. In the ischemic retina, there was no signi®cant expression for caspase 1 at any of the time points. However, caspase 2 expression was detected as early as 1 h following ischemiareperfusion, peaked at 24 h, and diminished markedly at 72 h (Figs 2a±e; Fig. 3a). Immunostaining was mainly appreciated in the GCL but was also found in the INL. Similarly, caspase 3 expression was ®rst noted at 6 h after ischemia/reperfusion, peaked at 24 h and disappeared at 72 h (Figs 2f±j; Fig. 3b). Immunostaining was con®ned primarily to the ONL and some cells of the INL. Double labeling with the cell-speci®c markers Thy-1 for ganglion cells and HPC-1 for amacrine cells showed that the majority of the Nedd-2-positive cells were either ganglion or amacrine in origin (Figs 4 a and b). PKC for bipolar cells, GFAP for MuÈller cells, and rhodopsin for photoreceptor cells demonstrated no colocalization with these markers. Double labeling with rhodopsin and HPC-1 showed that most of the caspase 3-positive cells were photoreceptor or amacrine in origin (Figs 4c and d). The remaining cell markers did not colocalize with caspase 3 expression. Tunel TUNEL staining was performed at different time points after 60 min of ischemia. No TUNEL-positive cells were seen in the control retinas (Fig. 5a). TUNEL positivity was seen in the ischemic retina as early as 6 h in the GCL and INL and was most prominent at 24±48 h (Fig. 5b). Double labeling
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Fig. 4 Double-labeling of both markers for caspase 2 or caspase 3 and phenotypic markers for retinal neurons 24 h following 60 min of ischemia. (a) Caspase 2 reactivity (red) and THY-1, a marker for ganglion cells (green), showing colocalization (yellow, arrow) in the GCL. (b) Caspase 2 reactivity (red) and HPC-1, a marker for amacrine cells (green), showing colocalization (yellow, arrow) in the INL. (c) Caspase 3 reactivity (green) and rhodopsin, a marker for photoreceptor cells (red), showing colocalization (yellow, arrow) in the ONL. (d) Caspase 3 reactivity (green) and HPC-1 (red) showing colocalization (yellow, arrow) in the INL. Fluorescent microscopy, original magni®cation, 40.
Fig. 5 TUNEL staining in rat retina 24 h following 60 min of ischemia. (a) In the nonischemic retina, no TUNEL-positive cells are present. (b) In the ischemic retina, TUNEL-positive cells are seen within the GCL and INL. (c) Confocal microscopy of double-labeling of both markers for caspase 2 (red) and TUNEL (green) showing colocalization (arrow) of retinal neurons in the GCL and INL. (d) Double-labeling of both markers for caspase 3 (red) and TUNEL (green) staining showing colocalization (arrow) of retinal neurons in the INL. (n 3 per group). Fluorescent microscopy, original magni®cation, 40 (a and b) and 60 (c and d).
Fig. 6 Cryosections of rat retina treated with BAF 6 h prior to the onset of 60 min of ischemia and 5 min after reperfusion. Caspase 2 immunohistochemistry is noted at 24 h following ischemia in the GCL and INL of the ischemic, vehicle-treated retina (a). Signi®cantly less immuno¯uorescence is seen in these layers in the ischemic, BAFtreated retina (b). Activated caspase 3 immunohistochemistry is noted at 24 h following ischemia in the ONL and INL of the ischemic, vehicle-treated retina (c) while signi®cantly less immuno¯uorescence is present in the ischemic, BAF-treated retina (d). (n 3 per group). Fluorescent microscopy, original magni®cation, 40.
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Fig. 7 Histopathology of the rat retina in BAF-treated animals 7 days after 60 min of ischemia. Measurements (mean ^ SD) of the thickness of retinal layers of non-ischemic (n 5), ischemic vehicletreated (n 5) and ischemic BAF-treated animals (n 5). BAF-treated animals have signi®cantly less retinal thinning as compared to the vehicle-treated controls. *Differs from non-ischemic controls ( p , 0.05) by ANOVA. d Differs from ischemic, vehicle-treated animals ( p , 0.05) by ANOVA.
utilizing confocal microscopy showed colocalization of TUNEL-positive cells with caspase 2 in the GCL and INL and with caspase 3 in the INL (Figs 5c and d).
Effects of caspase inhibitors Animals treated with intravitreal injection of BAF 6 h prior to ischemia showed signi®cant reduction in the expression of both caspase 2 and caspase 3 at 24 h after ischemia compared to the control group (Fig. 6 a±d). Caspase expression is maximal at this time following ischemia. At 7 days after ischemia, BAF-treated eyes showed signi®cant preservation in thickness and architecture of the retina as compared to the control group (Fig. 7). BAF-treated eyes displayed functional improvement as evidenced by the preservation of the ERG a-wave in all the treated animals as compared with an absent ERG a-wave and b-wave in all vehicle-treated, ischemic controls (Fig. 8). Similarly, animals were treated with an intravitreal injection of A-Nedd 6 h prior to ischemia. The control group received 5 mL of a scrambled version of S-Nedd. At 24 h subsequent to ischemia, eyes treated with A-Nedd compared to S-Nedd showed signi®cant reduction in the expression of caspase 2 in the GCL and INL (Figs 9a and b). Caspase 3 expression was still evident at 24 h in the ONL and INL of both of the antisense groups (Figs 9c and d). The A-Nedd-treated eyes showed signi®cant histoarchitectural preservation of the retina (Fig. 10). Furthermore, A-Nedd-treated eyes showed signi®cantly more function as evidenced by the preservation of the ERG a-wave in four out
Fig. 8 Representative electroretinogram tracings (ERGs). Baseline, non-ischemic control (a); 1 week subsequent to 60 min ischemia, placebo-treated (b); 1 week subsequent to 60 min ischemia, BAFtreated (c). Note extinguished ERG response of the placebo-treated retina, while there is marked accentuation of the a-wave in the BAF-treated animal. (n 10 per group)
of 10 treated animals compared to an absent ERG a-wave and b-wave in all 10 of the S-Nedd-treated eyes. Pretreatment with Y-vad fmk afforded no protection as compared to vehicle treated ischemic controls (data not shown).
Discussion This study has demonstrated that retinal ischemia activates the selective expression of caspase 2 or 3 in different retinal
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Fig. 9 Cryosections of rat retina treated with antisense oligonucleotides for caspase 2 (A-Nedd) or a scrambled version (S-Nedd) 6 h prior to the onset of 60 min of ischemia and 5 min after reperfusion. Caspase 2 immunohistochemistry in the GCL and INL at 24 h following ischemia in the ischemic, S-Nedd-treated retina (a). Signi®cantly less immuno¯uorescence is noted in these layers of the ischemic, A-Neddtreated retina (b). Activated caspase 3 immunohistochemistry at 24 h following ischemia in both the ischemic, S-Neddtreated retina (c) and in the ischemic, A-Nedd-treated retina (d). (n 5 per group). Fluorescent microscopy, original magni®cation, 40.
layers depending on the neuronal phenotype. Amacrine neurons were the only cell type found to stain for both caspases following retinal ischemic injury. No studies to date have reported heterogeneous expression of selective caspases in different neuronal phenotypes after cerebral ischemia. It may be that the CA-1 zone of the hippocampus in global ischemia or the penumbra in focal ischemia contain homogeneous neurons with the same active, downstream apoptotic machinery. Another possibility may be that different extrinsic factors may activate
Fig. 10 Histopathology of the rat retina in A-Nedd-treated animals 7 days after 60 min of ischemia. Measurements (mean ^ SD) of the thickness of retinal layers of non-ischemic (n 5), ischemic scrambled-treated (n 5) and ischemic antisense-treated animals (n 5). A-Nedd animals have signi®cantly less inner retinal thinning as compared to the Nedd-2 scrambled-treated controls. *differs from non-ischemic control (p , 0.05) by ANOVA. d differs from ischemic, S-Nedd-treated (p , 0.05) by ANOVA.
selective caspases. It has been shown that different injuries elicit different caspase pathways in cultured neurons. Oxidative stress-induced neuronal death is mediated by caspase 1 whereas neuronal degeneration as a result of growth factor withdrawal or exposure to b-amyloid involves caspase 2 (Troy et al. 1997, 2000). The pathophysiology of neuronal ischemia involves glutamate excitotoxicity (Lee et al. 1999), calcium overload (Kristian and Siesjo 1998), and oxidative stress (Love 1999). One pathophysiologic process may predominate over another in cerebral versus retinal ischemic damage. Glutamate levels, for example, are higher in the brain than in the retina during ischemia (Iijima et al. 2000). Moreover, glutamate-induced apoptosis of cerebrocortical and cerebellar granule neurons has been shown to be mediated by activation of caspase 3 (Du et al. 1997; Tenneti and Lipton 2000). Higher levels of glutamate may therefore account in part for selective caspase 3 expression in cerebral ischemia. Based on our ®ndings in the retina, further work may yet reveal intrinsic differences of the caspases in different neuronal populations after cerebral ischemia. The importance of the caspases in retinal ischemia was further supported by the neuroprotective effects of the pancaspase inhibitor, BAF and an antisense inhibitor of caspase 2. The lack of protection with Y-Vad, a caspase 1 selective inhibitor was consistent with the absence of caspase 1 immunoreactivity in the ischemic retina. Caspase 1 participates in the in¯ammatory response, which may not be as important in this model of global retinal ischemia. Caspase 2, on the other hand, may play a more pivotal role. Neuroprotection by A-Nedd is at least in part attributed to the down-regulation of caspase 2 since A-Nedd attenuated the expression of caspase 2 but had no effect on the immunostaining for caspase 3. Caspases 2 and 3 may thus act in parallel to one another in amacrine cell death.
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Expression for caspase 2 occured in the inner layers of the retina, the area most susceptible to ischemic injury. TUNEL analysis demonstrated that caspase 2 staining cells are dying by presumptive apoptosis, further supporting this protease as an important mediator of delayed neuronal death in the inner retina. Given the similarities between transient retinal and transient global cerebral ischemia, future investigations may discover an important role for caspase 2 in global cerebral ischemia. Li H. et al. (2000) have shown that the general caspase inhibitor, Z-vad-fmk, and the caspase 3 selective inhibitor, DEVD, do not protect neurons from global cerebral ischemic damage. This ®nding argues against a deleterious role for caspases but speci®c inhibition of caspase 2 was not performed. The expression of caspase 3 in this study was found in the ONL of the retina, a layer which shows more resistance to ischemic damage in this model. It may be that other factors inhibiting caspase 3 predominate and prevent this protease from destroying neurons in this layer. Such factors might include the neuronal inhibitory proteins. Alternatively, the mere expression of caspase 3 may simply be an epiphenomenon. This protease may be more important in the INL where expression was observed in amacrine neurons. TUNEL staining suggested that caspase 3-labeled neurons are dying by apoptosis in the INL. Furthermore, previous studies have shown that preteatment with DEVD is neuroprotective in the INL (Katai and Yoshimura 1999), supporting a deleterious role for caspase 3 in this layer. The results of our study and others (Robertson et al. 2000) suggest that novel therapies should be directed at targeting apoptotic pathways to protect against neuronal ischemia. Down-regulating caspase 2 may be one important intervention. In fact, brain-derived neurotrophic factor has been shown to down-regulate caspase 2 in the retina and rescue retinal ganglion cells from ischemic damage (Kurokawa et al. 1999). However, the pattern of active caspase expression in this study suggests that various selective inhibitors of speci®c caspases may be required to ameliorate the loss of distinct neuronal classes. More importantly, an essential issue is whether antiapoptotic therapy will lead to functional improvement. Pretreatment with A-Nedd led to the preservation of the ERG a-wave, which demonstrates that blocking caspase-mediated death reduces functional de®cits after retinal ischemia. The lack of preservation of the b-wave, however, indicates that bipolar cells are not rescued by down-regulating caspase 2 (Block and Schwarz 1998). It is possible that bipolar cells do not undergo apoptosis in this model of retinal ischemia as evidenced by a lack of TUNEL staining in these neurons. Another study has also shown that horizontal neurons undergo necrosis, not apoptosis, after retinal ischemia (Kuroiwa et al. 1999). Anti-apoptotic therapy therefore may not be suf®cient to fully rescue retinal function. It is likely that multimodal cocktails with anti-apoptotic
inhibitors and antinecrotic agents will be more effective in the treatment of diseases involving neuronal ischemia. In conclusion, retinal ischemia induces different types of cell death and different caspases depending on the neuronal type. These differences may similarly exist in cerebral ischemia. Therefore, multimodal therapy targeting anti-apoptotic and antinecrotic pathways may be more effective in neuronal ischemic diseases. Future studies should investigate caspase differences in degenerative neuronal injury where apoptosis plays a role. Acknowledgements The authors thank Ms Antoinette Barnecott for assistance in the preparation of the manuscript. This study was supported by NIH [EY1253 (DMR) and 10343 (SR)], American Heart Association Grant in-Aid NYC Af®liate (DMR), Research to Prevent Blindness (PSR, DMR), and American Heart Association Student Scholarship in Cerebrovascular Disease (SIS).
References Antonawich F. J., Krajewski S., Reed J. C. and Davis J. N. (1998) Bcl±xL bax interaction after transient global ischemia. J. Cereb. Blood Flow Metab. 18, 882±886. Asahi M., Hoshimaru M., Uemura Y., Tokime T., Kojima M., Ohtsuka T., Matsuura N., Aoki T., Shibahara K. and Kikuchi H. (1997) Expression of interleukin-1 beta converting enzyme gene family and bcl-2 gene family in the rat brain following permanent occlusion of the middle cerebral artery. J. Cereb. Blood Flow Metab. 17, 11±18. Bhat R. V., DiRocco R., Marcy V. R., Flood D. G., Zhu Y., Dobrzanski P., Siman R., Scott R., Contreras P. C. and Miller M. (1996) Increased expression of IL-1beta converting enzyme in hippocampus after ischemia: selective localization in microglia. J. Neurosci. 16, 4146±4154. Block F. and Schwarz M. (1998) The b-wave of the electroretinogram as an index of retinal ischemia. Gen. Pharmac. 30, 281±287. Buchi E. R. (1992) Cell death in the rat retina after a pressure-induced ischaemia-reperfusion insult: an electron microscopic study. I. Ganglion cell layer and inner nuclear layer. Exp. Eye Res. 55, 605±613. Chen J., Nagayama T., Jin K., Stetler R. A., Zhu R. L., Graham S. H. and Simon R. P. (1998) Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J. Neurosci. 18, 4914±4928. Crumrine R. C., Thomas A. L. and Morgan P. F. (1994) Attenuation of p53 expression protects against focal ischemic damage in transgenic mice. J. Cereb. Blood Flow Metab. 14, 887±891. Du Y., Bales K. R., Dodel R. C., Hamilton-Byrd E., Horn J. W., Czilli D. L., Simmons L. K., Ni B. and Paul S. M. (1997) Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons. PNAS 94, 11657±11662. Foos R. Y. (1976) Regional ischemic infarcts of the retina. Albrecht Von Graefes Archiv fuÈr Klinische Experimentelle Ophthalmologie 200, 183±194. Gavrielli Y., Sherman Y. and Ben-Sasson S. A. (1992) Identi®cation of programmed cell death in situ via speci®c labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493±501. Iijima T., Iijima C., Iwao Y. and Sankawa H. (2000) Difference in
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 466±475
Cell-speci®c caspase expression in transient retinal ischemia 475
glutamate release between retina and cerebral cortex following ischemia. Neurochem. Int. 36, 221±224. Kaneda K., Kashii S., Kurosawa T., Kaneko S., Akaike A., Honda Y., Minami M. and Satoh M. (1999) Apoptotic DNA fragmentation and upregulation of Bax induced by transient ischemia of the rat retina. Brain Res. 815, 11±20. Katai N. and Yoshimura N. (1999) Apoptotic retinal neuronal death by ischemia-reperfusion is executed by two distinct caspase family proteases. Invest. Ophthalmol. Vis. Sci. 40, 2697±2705. Kinoshita M., Tomimoto H., Kinoshita A., Kumar S. and Noda M. (1997) Up-regulation of the Nedd2 gene encoding an ICE/Ced-3like cysteine protease in the gerbil brain after transient global ischemia. J. Cereb. Blood Flow Metab. 17, 507±514. Kirino T. (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57±69. Krajewski S., Mai J. K., Krajewksi M., Sikorska M., Mossakowski M. J. and Reed J. C. (1995) Upregulation of bax protein levels in neurons following cerebral ischemia. J. Neurosci. 15, 6364±6376. Kristian T. and Siesjo B. K. (1998) Calcium in ischemic cell death. Stroke 29, 705±718. Kuroiwa S., Katai N. and Yoshimura N. (1999) A possible role for p16INK4 in neuronal cell death after retinal ischemia-reperfusion injury. Invest. Ophthalmol. Vis. Sci. 40, 528±533. Kurokawa T., Katai N., Shibuki H., Kuroiwa S., Kurimoto Y., Nakayama C. and Yoshimura N. (1999) BDNF diminishes caspase-2 but not c-jun immunoreactivity of neurons in retinal ganglion cell layer after transient ischemia. Invest. Ophthalmol. Vis. Sci. 40, 3006±3011. Lam T. T., Abler A. S. and Tso M. O. (1999) Apoptosis and capases after ischemia-reperfusion injury in rat retina. Invest. Ophthalmol. Vis. Sci. 40, 967±975. Lee J. M., Zipfel G. J. and Choi D. W. (1999) The changing landscape of ischaemic brain injury mechanisms. Nature 399, A7±A14. Li B., Yang C., Rosenbaum D. M. and Roth S. (2000) signal transduction mechanism involved in ischemic preconditioning in the rat retina in vivo. Exp. Eye Res. 70, 755±765. Li H., Colbourne F., Sun P., Zhao Z., Buchan A. M. and Iadecola C. (2000) Caspase inhibitors reduce neuronal injury after focal but not global cerebral ischemia in rats. Stroke 31, 176±182. Love S. (1999) Oxidative stress in brain ischemia. Brain Pathol. 9, 119±131. McGahan L., Hakim A. M. and Robertson G. S. (1998) Hippocampal Myc and p53 expression following transient global ischemia. Brain Res. Mol Brain Res. 56, 133±145. Namura S., Zhu J., Fink K., Endres M., Srinivasan A., Tomaselli K. J., Yuan J. and Moskowitz M. A. (1998) Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci. 18, 3659±3668. Ni B., Wu X., Su Y., Stephenson D., Smalstig E. B., Clemens J. and Paul S. M. (1998) Transient global forebrain ischemia induces a prolonged expression of the caspase-3 mRNA in rat hippocampal CA1 pyramidal neurons. J. Cereb. Blood Flow Metab. 18, 248±256.
Nitatori T., Noboru S. and Waguri S. (1995) Delayed neuronal death of the CA-1 pyramidal cell layer of the glial hippocampus following transient ischemia is apoptosis. J. Neurosci. 15, 1001±1011. Nonaka A., Kiryu J., Tsujikawa A., Yamashiro K., Miyamoto K., Nishiwaki H., Mandai M., Honda Y. and Ogura Y. (2000) Administration of 178-estradiol attenuates retinal ischemiareperfusion injury in rats. Invest. Ophthalmol. Vis. Sci. 41, 2689±2696. Ostwald P., Park S. S., Toledano A. Y. and Roth S. (1997) Adenosine receptor blockade and nitric oxide synthase inhibition in the retina: impact upon post-ischemic hyperemia and the electroretinogram. Vision Res. 37, 3453±3461. Pulsinelli W. A., Brierley J. B. and Plum F. (1982) Temporal pro®le of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11, 491±498. Robertson G. S., Crocker S. J., Nicholson D. W. and Schulz J. B. (2000) Neuroprotection by the inhibition of apoptosis. Brain Pathol. 10, 283±292. Rosenbaum D. M., Rosenbaum P. S., Gupta A., Michaelson M. D., Hall D. H. and Kessler J. A. (1997) Retinal ischemia leads to apoptosis which is ameliorated by aurintricarboxylic acid. Vision Res. 37, 3445±3451. Rosenbaum D. M., D'Amore J., Llena J., Rybak S., Balkany A. and Kessler J. A. (1998a) Pretreatment with intraventricular aurintricarboxylic acid decreases infarct size by inhibiting apoptosis following transient global ischemia in gerbils. Ann. Neurol. 43, 654±660. Rosenbaum D. M., Rosenbaum P. S., Gupta H., Singh M., Aggarwal A., Hall D. H., Roth S. and Kessler J. A. (1998b) The role of the p53 protein in the selective vulnerability of the inner retina to transient ischemia. Invest. Ophthalmol. Vis. Sci. 39, 2132±2139. Schulz J. B., Weller M. and Moskowitz M. A. (1999) Caspases as treatment targets in stroke and neurodegenerative diseases. Ann. Neurol. 45, 421±429. Szabo M. E., Droy-Lefaix M. T., Doly M., Carre C. and Braquet P. (1991) Ischemia and reperfusion-induced histologic changes in the rat retina. Demonstration of a free radical-mediated mechanism. Invest. Ophthalmol Vis Sci. 32, 1471±1478. Tenneti L. and Lipton S. A. (2000) Involvement of activated caspase-3like proteases in N-methyl-d-aspartate-induced apoptosis in cerebrocortical neurons. J. Neurochem. 74, 134±142. Troy C. M., Stefanis L., Greene L. A. and Shelanski M. L. (1997) Nedd2 is required for apoptosis after trophic withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J. Neurosci. 17, 1911±1918. Troy C. M., Rabacchi S. A., Friedman W. J., Frappier T. F., Brown K. and Shelanski M. L. (2000) Caspase-2 mediates neuronal cell death induced by beta-amyloid. J. Neurosci. 20, 1386±1392. Velier J. J., Ellison J. A., Kikly K. K., Spera P. A., Barone F. C. and Feuerstein G. Z. (1999) Caspase-8 and caspase-3 are expressed by different populations of cortical neurons undergoing delayed cell death after focal stroke in the rat. J. Neurosci. 19, 5932±5941.
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