June 2005
Volume 46, Issue 6
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Retina  |   June 2005
Activation of the Mitochondrial Apoptotic Pathway in a Rat Model of Central Retinal Artery Occlusion
Author Affiliations
  • Yi Zhang
    From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Chang-Ho Cho
    From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • La-ongsri Atchaneeyasakul
    From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Trevor McFarland
    From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • Binoy Appukuttan
    From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
  • J. Timothy Stout
    From the Casey Eye Institute, Oregon Health and Science University, Portland, Oregon.
Investigative Ophthalmology & Visual Science June 2005, Vol.46, 2133-2139. doi:10.1167/iovs.04-1235
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      Yi Zhang, Chang-Ho Cho, La-ongsri Atchaneeyasakul, Trevor McFarland, Binoy Appukuttan, J. Timothy Stout; Activation of the Mitochondrial Apoptotic Pathway in a Rat Model of Central Retinal Artery Occlusion. Invest. Ophthalmol. Vis. Sci. 2005;46(6):2133-2139. doi: 10.1167/iovs.04-1235.

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

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Abstract

purpose. Apoptosis is known to play a role in cell death in transient retinal ischemia. Little is known about the specific molecular pathways involved. The purpose of the current study was to evaluate a rat model of central retinal artery occlusion (CRAO) that simulates the clinical features of CRAO in humans and to elucidate whether the mitochondrial apoptotic pathway is involved.

methods. CRAO was induced in the central retinal artery by intravenous injection of rose bengal and green laser irradiation of the artery. CRAO was documented at 1, 3, 6, and 24 hours after laser irradiation. Changes in Bax (proapoptotic Bcl-2–associated X protein), cytochrome c, and caspase-9 cleavage in the cytosolic and mitochondrial fractions of neural retinal tissues were measured by Western blot analysis. Apoptosis within the retina was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).

results. Complete CRAO was induced; however, occlusion became incomplete with spontaneous reperfusion of branch arteries, starting at 3 hours after laser irradiation. Only one or two branch arteries remained occluded at the 24-hour time point. Time-dependent, apoptotic changes were observed in inner and outer retinal cell layers. Western blot analysis revealed mitochondrial translocation of Bax from the cytoplasm, starting at 3 hours and peaking at 6 hours after laser irradiation. This translocation was accompanied by cytosolic accumulation of cytochrome c and cleavage of caspase-9.

conclusions. This model is highly relevant to clinical manifestations of CRAO and is an ideal animal model for research. These findings indicate the activation of the mitochondrial pathway in ischemic retina induced by CRAO. The model provides a better understanding of ischemia-induced retinal apoptosis. Antiapoptosis therapy directly targeting the mitochondrial pathway in CRAO or other retinal ischemic diseases may be beneficial.

Central retinal artery occlusion (CRAO) results in severe retinal ischemia, which can cause sudden, irreversible damage to visual function within hours of onset. 1 Patients with CRAO experience an unexpected, rapid loss of vision that is normally painless and usually results in visual acuity of 20/200 or worse. 1 2 The exact molecular events that lead to retinal cell damage and loss of vision from CRAO-induced ischemia are unclear. 
The development of animal models that simulate the clinical features of CRAO is a prerequisite for studying the damage caused by retinal ischemia. Several models of transient retinal ischemia have been reported and include techniques that increase intraocular pressure (IOP) to higher than the systolic arterial blood pressure 3 4 5 or ligate the optic nerve with the central retinal artery (CRA). 6 7 8 Although much information has been gained with these models, they are not without drawbacks. The increased-IOP model has the potential to induce a more global ischemic insult (occlusion of both uveal and retinal circulation), and the ligation model involves occlusion of the posterior ciliary arteries, which results in occlusion of the uveal blood supply that nourishes outer retinal layers, as well as the CRA. In human CRAO, only inner retinal layers are deprived of blood supply. 9 In addition, mechanical damage to neuronal cells due to increased IOP and possible pleiomorphic effects associated with optic nerve strangulation influences the interpretation of results of studies that employ these models. Thus, these models do not accurately represent the clinical exhibition of CRAO in humans. 
A minimally invasive model of transient retinal ischemia was introduced by Daugeliene et al. 10 In this model, photothrombotic CRAO is induced by a combination of intravenous injection of rose bengal and green laser irradiation of the CRA of a rat. Rose bengal is a photosensitive dye that releases active oxygen when irradiated by green light, resulting in vascular endothelial injury, platelet activation, and subsequent formation of an intraluminal thrombus at the irradiated area of the blood vessel. 11 Typically, in previous studies the thrombus was dissolved 1 hour after CRAO by an intravenous injection of tissue-type plasminogen activator, resulting in an artificially time-controlled reperfusion. This CRAO model of transient ischemia was used in a histologic and TUNEL study to determine the time of onset of apoptosis within the retina. 10 In patients, arterial occlusion may persist for a variable period; thus, an animal model that depends on well-controlled reperfusion may yield pathogenic information that is less relevant to the clinical situation. Furthermore, in a study of 178 patients with CRAO, ranging in age from 18 to 89 years, the average time to treatment ranged from 10.8 to >24 hours. 12 Thus, there is improved clinical relevance in a CRAO model of long-term ischemia. In the model used for our studies, the intraluminal thrombus produced in the retinal artery was allowed to resolve naturally and spontaneously to emulate the natural course of CRAO as observed in humans. The primary goal of this study is to employ this model to investigate the onset and progression of apoptosis and to determine whether the mitochondrial pathway is activated. 
Recent studies have revealed that apoptosis is involved in neuronal cell death in animal models of transient retinal ischemia. 3 4 10 Apoptosis, as detected by TUNEL staining, has been observed in the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) of the retina. 10 Whether this process is mediated by the death receptor via the mitochondrial pathway is undetermined. These two major apoptotic pathways are fairly well characterized at the molecular level. 13 14 15 With respect to the intrinsic mitochondrial pathway, the proapoptotic protein Bax (proapoptotic Bcl-2-associated X protein) is activated in response to apoptotic signals and translocates from the cytosol to the outer mitochondrial membrane and forms high-conductance channels. This permits release of cytochrome c into the cytosol, which, in turn, associates with dATP (deoxyadenosine triphosphate), Apaf-1, and procaspase-9 to form the apoptosome. Procaspase-9 can now be cleaved to form caspase-9. Caspase-9 then activates caspase-3 which activates other downstream executioner caspases and ultimately leads to cell death (Fig. 1) . 15 16  
The translocation of Bax from cytosol to mitochondria and the activation of caspase-9, both essential processes for the initiation of the mitochondrial pathway of apoptosis, have yet to be studied in any retinal ischemia model. We used a minimally invasive photothrombotic model, which more closely represents the clinical situation in human CRAO, to study the molecular events of retinal apoptosis induced by ischemia. We present in vivo evidence of activation of caspase-9, Bax translocation into the mitochondria, and cytochrome c release, all indicating that the mitochondrial pathway is involved in this clinically relevant CRAO model. 
Methods
Animals
Adult male Sprague-Dawley rats (weight, 300–350 g; Charles River Laboratory, Wilmington, MA) were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
CRAO Experimental Protocol
Photothrombotic CRAO was induced in the right eye of all test animals; left eyes served as the internal control. Rats were anesthetized with a subcutaneous injection (0.6 mL/kg) of a ketamine, xylazine, and acepromazine cocktail (5 mL ketamine [100 mg/mL], 2.5 mL xylazine [20 mg/mL]), and 1 mL acepromazine [10 mg/mL]). The eyes of all animals were topically anesthetized, and pupils were pharmacologically dilated. Rose bengal (20 mg/mL normal saline; Sigma-Aldrich, St. Louis, MO) was injected into the femoral vein (20 mg/kg). Immediately after injection, the photosensitized CRA was irradiated for 0.4 seconds with a 532-nm argon green laser (Iridex Inc., Mountain View, CA). Laser settings were 75-μm diameter and 0.1 W of power. Forty-four rat eyes were subjected to photothrombosis, and eyes were harvested for study at 1, 3, 6, and 24 hours after laser irradiation. Eleven rats were studied at each time point. 
TUNEL Analysis for Apoptotic Cells
Apoptotic cell death was examined in one rat at each time point by TUNEL staining (ApopTag Peroxidase In Situ Apoptosis Detection Kit; Serologic Corp., Norcross, GA). Rats were humanely euthanatized and both eyes were immediately surgically removed. Enucleated globes were frozen in optimal cutting temperature (OCT) embedding medium (Sakura Finetek, Torrance, CA). Cryosections (8 μm thick) were fixed with 1% paraformaldehyde in PBS and postfixed in precooled ethanol and acetic acid (2:1 solution). After the reaction was quenched in 3.0% hydrogen peroxide and the sections were washed with PBS, the tissue was incubated with equilibration buffer for 10 seconds and then incubated with TdT enzyme at 37°C for 1 hour. Samples were washed with PBS, after which anti-digoxigenin peroxidase conjugate was applied to the specimen at room temperature for 30 minutes followed by four washes in PBS. Peroxidase substrate 3,3′-diaminobenzidine (DAB) was used to stain for apoptotic cells. Methyl green (0.5%) was used as a nuclear stain. TUNEL-positive cells were viewed under a light microscope (DMIRB; Leica, Deerfield, IL) and photographed. 
Subcellular Fractionation
Two neural retinas at each time point were pooled for Western blot analysis. Fractionations were performed at 4°C, according to a modified protocol. 17 In brief, immediately after enucleation, the neural retinas were dissected from the RPE layer and homogenized in 1× M-SHE buffer (0.21 M mannitol, 0.07 M sucrose, 10 mM HEPES-KOH [pH 7.4], and 1 mM EGTA). Complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktail I (Sigma-Aldrich) was added. After incubation on ice (30 minutes), nuclei and unlysed cells were pelleted at 1200g twice and discarded. Supernatants were centrifuged twice at 10,000g for 15 minutes to pellet mitochondria, and the resultant supernatant (cytosolic fraction) was removed. To isolate mitochondrial proteins, pellets were resuspended in buffer C (395 mM sucrose, 0.1 mM EGTA, and 10 mM HEPES), and gently layered between 0.9 mL 26% colloidal silica gradient (Percoll; Sigma-Aldrich) and 1.3 mL 60% of the single-density gradient solution in an 8.5-mL ultracentrifuge tube (Beckman, Palo Alto, CA). After centrifugation at 160,000g at 4°C for 60 minutes (SW-28 rotor, L8-60M ultracentrifuge; Beckman) the sediment was collected and mixed with 100 μL lysis buffer containing 20 mM HEPES (pH 7.4), 400 mM KCl, 5% glycerol, 0.5% Triton X-100, 1× complete protease inhibitor cocktail, 10 μL/mL phosphatase inhibitor cocktail 1, 0.1 mg/mL phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL pepstatin, and 2 mM dithiothreitol (DTT) and then sonicated for 15 seconds. Supernatant containing cytosolic proteins was centrifuged at 100,000g at 4°C for 92 minutes. Supernatant was collected and then centrifuged again at 14,000g at 4°C for 10 minutes, to remove any remaining organelles. Protein concentration was measured using the Bradford assay (Sigma-Aldrich). All isolated fractions were stored at −80°C. 
Western Blot Analysis of Subcellular Fractions
Cytosolic (40 μg) and mitochondrial (30 μg) fractions from each time point were denatured in sample buffer at 95°C for 5 minutes and then separated by 12% Tris-glycine SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane and blocked with 5% dry milk. Antibodies against caspase-9 (specific to the active region of caspase-9), Bax (Cell Signaling Technology, Beverly, MA), and cytochrome c (BD-Pharmingen, San Diego, CA) were used. The membrane was washed and incubated with anti-rabbit or anti-mouse IgG horseradish peroxidase–conjugated antibody and examined (SuperSignal West Femto Maximum Sensitivity Substrate kit; Pierce, Rockford, IL). Antibodies against the cytosolic marker β-actin (Sigma-Aldrich) and mitochondrial marker cytochrome c oxidase subunit IV (COX IV; BD-Pharmingen) were used to control for equal sample loading and detection of contamination. Caspase-9, Bax, and cytochrome c bands were semiquantified by densitometric scanning (BioImage, Advanced quantifier or UVP; LabWorks Software, Ann Arbor, MI) and normalized to β-actin (cytosolic fraction) and COX IV (mitochondrial fraction) controls. 
Statistical Analysis
All data are presented as the mean ± SD. Western blot results of Bax, cytochrome c, and cleaved caspase-9 in cytosolic or mitochondrial fractions of laser-treated eyes were compared with their contralateral eyes, by paired Student’s t-tests. P < 0.05 was considered statistically significant. 
Results
Central Retinal Artery Occlusion
Green laser irradiation of the CRA, in animals photosensitized with rose bengal, resulted in instant occlusion of most of the branch arteries. Occluded arteries became pale and white followed by whitening of the retina (Fig. 2B) . Complete CRAO was evident immediately after laser irradiation, as the retina was pale and edematous in appearance. This appearance was maintained up to 3 hours after occlusion, when one or two branch arteries spontaneously reperfused (Figs. 2C 2D) . Progressive reperfusion of branch arteries ensued, and by 24 hours after irradiation, only one to two arteries remained occluded. Despite reperfusion, the whole retina still appeared pale and edematous (Figs. 2E 2F)at 24 hours. 
Apoptosis in Ischemic Retinal Cells after Laser Irradiation
No TUNEL-positive retinal cells were noted in control eyes at any time points studied, whereas a time-dependent increase of TUNEL-positive cells within different retinal cells layers was observed in the CRAO eyes (Fig. 3) . At the 1-hour time point, no TUNEL-positive cells were observed in the GCL or the INL (Fig. 3B) . TUNEL-positive cells appeared in the GCL and INL at the 3-hour time point, and the number increased markedly at the 6-hour time point (Figs. 3C 3D) ; however, TUNEL-positive cells within the ONL were first noticed only at 6 hours after laser irradiation (Fig. 3D) . The greatest number of TUNEL-positive cells was noted at the 24-hour time point in the GCL, INL, and ONL (Fig. 3E)
Translocation of Bax from the Cytosol into the Mitochondria in CRAO Eyes
We wanted to determine whether translocation of Bax protein occurs after arterial occlusion. Western blot analysis comparing cytosolic and mitochondrial fractions revealed a shift in the amount of Bax protein from the cytoplasm to the mitochondria, as the process of apoptosis proceeded. Within the cytosolic fraction, the Bax protein concentrations were similar between the CRAO and the control eyes at the 1-hour time point, but decreased significantly in the CRAO eyes at the 3-hour time point and continued to decrease until the 6-hour time point, a 2.3-fold decrease over the control (Fig. 4A) . Coinciding with a decrease in Bax in the cytosolic fraction, there was a significant increase in Bax protein within the mitochondrial fractions (Fig. 4B) . This increase, compared with the control eye, was initially identified at the 3-hour time point, peaked at 6 hours (a 2.6-fold difference), and remained significantly higher 24 hours after CRAO (Fig. 4B)
Cytochrome c Release from Mitochondria
Bax-mediated release of cytochrome c from the mitochondria into the cytosol is considered an early and crucial step in the mitochondrial apoptotic pathway. Cytosolic cytochrome c content was greater within the retina of ischemic eyes at the 1-hour time point (P = 0.06) and remained higher than control eyes at the 3- and 6-hour time points, although significance was observed only at the 3-hour time point (Fig. 5A) . At the 24-hour time point, the cytosolic cytochrome c content had declined to a level similar to that observed in the control (Fig. 5A)
Cleavage of Procaspase-9 in CRAO Ischemic Retina
For the mitochondrial apoptotic pathway to proceed, binding of procaspase-9 to the apoptosome leads to cleavage, activation, and release of the initiator caspase-9. Western blot data indicate that procaspase-9 was activated in the retinas of eyes with CRAO (Fig. 6) . Procaspases consist of three domains: a prodomain and large and small catalytic subunits. Activation requires cleavage at specific Asp residues in internal linker sites located between each of the three domains. In the rat, the antibody used in this study detected the prodomain linked to the large catalytic subunit after cleavage at two alternate Asp sites (Asp368, 40 kDa; Asp353, 38 kDa) between the large and small subunits (Fig. 6B)
We demonstrate an increase in cleaved procaspase-9 at the 1-hour time point by Western blot analysis, peaking (2.2-fold increase) at 3 hours after laser irradiation (Fig. 6A) . After the 3-hour time point, the levels of cleaved pro-caspase-9 declined, but were still significantly higher than the nonischemic control retina at the 6-hour time point. 
Discussion
In this study, a photothrombotic CRAO was induced in the rat eye by a combination of intravenous injection of rose bengal and green laser irradiation of the CRA. Intraluminal thrombus was allowed to resolve spontaneously without intervention. Fundus photography revealed complete CRAO at the first hour after laser irradiation. After the 3-hour time point, the occlusion became incomplete, as evidenced by one or two reperfused branch retinal arteries. Eventually, only one or two branch arteries remained occluded 24 hours after laser irradiation. Thus, in our CRAO model, incomplete retinal ischemia was present from the 3- to the 24-hour time points. In other retinal ischemic models, either the high-IOP model or the model involving ligation of the optic nerve, the CRA is obstructed completely 3 4 6 18 ; however, complete obstruction of the CRA is rare in clinical situations. Fluorescein angiography studies indicate that complete obstruction of the retinal arteries probably occurs in fewer than 2% of cases. 19 Therefore, we believe this rose bengal-laser model is more clinically relevant and may be a more accurate representation of the CRAO observed in patients. 
Inner versus Outer Retinal Layer Cell Death
It has been noted that the inner-layer neurons (ganglion cells and amacrine cells) are more susceptible to ischemia than the outer-layer neurons of the retina. 4 6 10 18 20 This selective vulnerability could be attributed to the different exposure and susceptibility of neurons to extracellular transmitters, response to free radicals, and/or extracellular pH and intracellular calcium buffering capabilities. 9 We demonstrated delayed apoptosis within the ONL in addition to the early and profound apoptosis within the GCL and INL at the 3-hour time point, similar to results published by Daugeliene et al. 10 A possible explanation for the observed greater tolerance to ischemia by ONL neurons is that the outer retinal layer is physically closer to the choriocapillary blood supply than the INL or GCL. Other studies have reported an absence of apoptotic cells within the ONL after transient (60 minutes) retinal ischemia, even though uveal ischemia was produced simultaneously in these particular models. 3 4 18 We speculate that ONL survival is due to the relatively short period of retinal ischemia in these models. In our study, apoptotic cells in ONL were observed after ischemic injury, despite the availability of blood supplied by the choriocapillaris. This suggests that the duration of ischemia, as is true of reperfusion time, is one factor that influences the extent of injury to neurons. It has been proposed that ganglion cell loss after retinal ischemia is an ongoing process, for which severity and duration are determined by the ischemic interval. 6  
Hayreh et al. 18 reported a retinal ischemic tolerance time of 97 minutes in old, atherosclerotic, and hypertensive nonhuman primate eyes with complete CRAO; little retinal damage was observed by hematoxylin-and-eosin histopathology and morphologic examination in these animals. However, CRAO lasting longer than 97 minutes resulted in substantial cell loss within the GCL and INL. 18 In humans, clinical histopathology indicates nuclear pyknosis in ganglion cells and the cells of the inner part of the INL after 2 hours of CRAO, with eventual atrophy and loss of these neurons over time. 21 At present, there are no data on the retinal tolerance time in a rat with CRAO. We presumed a retinal tolerance time of between 1 and 3 hours in this CRAO rat model, because we did not observe any biological or histologic changes at the 1-hour time point. 
Activation of the Mitochondrial Apoptotic Pathway
Ischemia defines a condition of lack of blood supply to tissues in which deleterious factors are produced and trigger apoptosis in ischemic cells. Although intracellular Bax translocation, mitochondrial cytochrome c release, and subsequent cytosolic caspase activation have been documented in ischemic injuries of the brain, heart, and liver, insufficient attention has focused on the molecular biological changes in the ischemic retina. 22 23 24 25 The specific signaling pathway triggering retinal ischemic apoptosis has not been reported. Our results suggest that cytosolic Bax translocates into mitochondria, cytochrome c accumulates in cytosol, and cytosolic procaspase-9 is activated in an ischemic retina, thus implying activation of the mitochondrial apoptotic pathway. 
Bax is predominantly localized in the cytosol as a monomer in normal cells. On cell death stimulation, Bax translocates from the cytosol into the outer mitochondrial membrane and permits the release of cytochrome c from the mitochondria out into the cytosol by forming specific channels in the outer mitochondrial membrane. 26 27 28 29 Bax translocation is considered a critical event in neuronal apoptosis. 30 Intracellular Bax translocation followed by release of cytochrome c has been reported in transient cerebral ischemia. 22 In transient retinal ischemia induced by high IOP, increased expression of the Bax gene was observed 6 hours after cessation of ischemia. 4 Intracellular Bax translocation and cytochrome c release have been studied in the retina of a rat model of diabetes. 31 In this CRAO model of retinal ischemia, a significant decrease in cytosolic Bax and substantial accumulation of Bax in mitochondria appeared simultaneously at the 3-hour time point. This transition continued at the 6-hour time point, indicating the involvement of Bax mitochondrial translocation in this retinal disease event. 
It is well established that cytochrome c plays a central role in the mitochondrial apoptosis-signaling pathway. Cytosolic cytochrome c forms the apoptosome with Apaf-1, dATP, and procaspase-9, which in turn activates the downstream effector caspases. 16 The release of cytochrome c ensues as a result of Bax translocation and subsequent mitochondrial outer membrane permeabilization, 22 29 and it is a fast process. Once it is initiated, all mitochondria in the cell lose cytochrome c within a very short period. 32 Our results document a marked increase of cytosolic cytochrome c in the ischemic group at the 3- and 6-hour time points, although only the 3-hour time point is statistically significant compared with the control group. It seems that cytochrome c was released completely sometime before the 3-hour time point. An unexpected, albeit low, level of cytosolic cytochrome c was also measured in control samples. We speculate that the specificity of the cytochrome c primary antibody used for Western blot analysis may have contributed to this result. Cytochrome c is a cytosolic protein initially produced in the form of apocytochrome c. After translocation into the mitochondria, apocytochrome c binds to a heme group and is converted into holocytochrome c, and it is that form that is released into the cytosol during apoptosis. The primary antibody used in our study recognizes both apo- and holocytochrome c; thus, the apo part of cytochrome c can be detected in our samples. The detection of low levels of cytosolic cytochrome c in control samples by Western blot analysis has been described and does not detract from our conclusion. 33  
Bax-mediated release of cytochrome c from mitochondria into the cytosol is considered an early and crucial step in the mitochondrial apoptotic pathway. Cytosolic cytochrome c is capable of binding Apaf-1, which contains a nucleotide-binding site and a conserved motif for a caspase-recruitment domain (CARD)—binding that leads to conformational revelation of CARD for subsequent recruitment of procaspase-9. Caspase-9 is the apical caspase in the mitochondrial apoptotic pathway. Active caspase-9 is the proteolytic product of procaspase-9 and triggers the activation of several downstream caspases, leading to nuclear fragmentation and cell death. 16 Thus, identification of caspase-9 in an apoptotic pathway is key evidence to suggest involvement of the mitochondrial pathway. To our knowledge, this is the first report of caspase-9 involvement in any retinal ischemia model. A 2.2-fold increase in cleaved caspase-9 was noted in our CRAO model at the 3-hour time point and continued to increase to the 6-hour time point, indicating a concerted activation of procaspase-9 within the ischemic retina. Thus, we have observed a series of changes at the 3-hour time point, including Bax translocation from cytosol into the mitochondria and cytochrome c release and procaspase-9 activation within the retina under ischemic conditions induced by CRAO. These biological findings, in addition to the histologic examination at the same time point (TUNEL-positive cells in the GCL and INL), strongly indicate that the mitochondrial apoptotic pathway was activated by 3 hours after CRAO. 
Alternative Pathways Are Not Mutually Exclusive
Although activation of caspase-9 in the mitochondrial apoptotic pathway and activation of the “death receptor-mediated” pathway or the p53 pathway classically are considered separate initiators of the apoptotic process, a review of recent evidence suggests that we cannot rule out the possible involvement of either the death receptors or p53 in this animal model of ischemia. Traditionally, activation of a death receptor (DR) such as Fas leads to activation of caspase-8, which in turn activates caspase-3, allowing this effector caspase to activate other proteins, such as inhibitor of caspase activated DNase (ICAD), that can initiate nuclear DNA breakdown. 13 34 Recent evidence suggests that DR-activated caspase-8 can cleave Bid (BH3 interacting domain death agonist), a protein that can bind Bax. 35 36 Caspase-8-cleaved Bid can enter the mitochondria and initiate the release of cytochrome c, resulting in the formation of the apoptosome and cleavage/activation of caspase-9. 34 35 36 37 The p53 protein can induce apoptosis in response to various stimuli 38 and can directly transcriptionally activate the Bax gene and the apoptosis-associated speck-like protein (ASC) gene. 39 40 ASC binds to Bax in the cytosol and promotes translocation of Bax to the mitochondria and subsequent release of cytochrome c and cleavage/activation of caspases-9 and -3. 40 Future experiments will examine whether activation of Bid or p53 are involved in the molecular events that initiate apoptosis in this rat model of CRAO. 
Western blot analysis suggests that full-length procaspase-9 protein is present at a higher concentration within the retina at 1 hour after laser irradiation when compared with the control (data not shown). Whether this upregulation is accomplished at the transcriptional or translational level is unknown. It is of interest that hypoxia response elements (HREs), that bind the transcription factor hypoxia-inducible factor (HIF)-1, are present within the promoter region of rat and human procaspase-9 genes. 41 Under hypoxic conditions, the HIF-1 protein is markedly increased and induced to translocate into the nucleus to initiate the expression of genes that contain HREs. 42 Future experiments will determine whether in our retinal ischemic model an upregulation of HIF-1 protein leads to an increase in the transcription of the procaspase-9 gene. 
In conclusion, we used a rat model of CRAO that is highly relevant to human disease to conduct a temporal investigation into the molecular events that presage retinal apoptosis. Histologic and biological evidence imply an activation of the mitochondrial apoptotic pathway within 3 hours after CRAO. This suggests that strategies designed to inhibit the mitochondrial apoptotic cascade may be useful for patients with CRAO. The antiapoptotic protein Bcl-2 inhibits apoptosis by preventing the formation of Bax-mediated channels and the release of cytochrome c into the cytosol. efforts to employ this protein to minimize CRAO-dependent apoptosis are under way. 43 44  
 
Figure 1.
 
Schematic of mitochondrial apoptotic pathway. (1) Bax translocates from cytosol to outer mitochondrial membrane and forms high-conductance channels or pores; (2) cytochrome c released from mitochondrion forms the apoptosome with Apaf-1, dATP, and procaspase-9, and then procaspase is activated; (3) caspase-9 activates procaspase-3, which, in turn, activates downstream executioner caspases and leads to cell death.
Figure 1.
 
Schematic of mitochondrial apoptotic pathway. (1) Bax translocates from cytosol to outer mitochondrial membrane and forms high-conductance channels or pores; (2) cytochrome c released from mitochondrion forms the apoptosome with Apaf-1, dATP, and procaspase-9, and then procaspase is activated; (3) caspase-9 activates procaspase-3, which, in turn, activates downstream executioner caspases and leads to cell death.
Figure 2.
 
Rat fundus photographs showing retinal vessels at different time points after argon green laser irradiation to produce CRAO. (A) Normal fundus. (B) Immediately and (C) 1, (D) 3, (E) 6, and (F) 24 hours after argon green laser irradiation. Note the whitening and paleness of the retina and the occluded arteries. Red arrow: an artery; black arrow: vein.
Figure 2.
 
Rat fundus photographs showing retinal vessels at different time points after argon green laser irradiation to produce CRAO. (A) Normal fundus. (B) Immediately and (C) 1, (D) 3, (E) 6, and (F) 24 hours after argon green laser irradiation. Note the whitening and paleness of the retina and the occluded arteries. Red arrow: an artery; black arrow: vein.
Figure 3.
 
Positive TUNEL staining of retinal cell layers at various time points after induction of ischemia. (A) Control eye and treated eyes at (B) 1, (C) 3, (D) 6, and (E) 24 hours. Magnification, ×200.
Figure 3.
 
Positive TUNEL staining of retinal cell layers at various time points after induction of ischemia. (A) Control eye and treated eyes at (B) 1, (C) 3, (D) 6, and (E) 24 hours. Magnification, ×200.
Figure 4.
 
Western blot analysis of Bax (A) in retinal cytosolic fractions and (B) in mitochondrial fractions. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. β-actin served as a protein loading control, to ensure equal total protein concentrations for each lane. Results of semiquantitative densitometry of Bax in subcellular fractions at each time point was calibrated with that of β-actin to obtain the Bax/β-actin ratio. Data were analyzed based on five separate cytosolic fractions (n = 5) or three separate mitochondrial fractions (n = 3). +, laser treated eye; −, contralateral control eye. *P < 0.05 compared with the control at the same time point.
Figure 4.
 
Western blot analysis of Bax (A) in retinal cytosolic fractions and (B) in mitochondrial fractions. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. β-actin served as a protein loading control, to ensure equal total protein concentrations for each lane. Results of semiquantitative densitometry of Bax in subcellular fractions at each time point was calibrated with that of β-actin to obtain the Bax/β-actin ratio. Data were analyzed based on five separate cytosolic fractions (n = 5) or three separate mitochondrial fractions (n = 3). +, laser treated eye; −, contralateral control eye. *P < 0.05 compared with the control at the same time point.
Figure 5.
 
(A) Semiquantitative densitometry results for relative abundance of cytochrome c (cyto-c) in retinal cytosolic fractions, calculated as the cyto-c/β-actin ratio. β-Actin served as a protein-loading control, to ensure equal loading. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blot analyses (n = 5). * P < 0.05 compared with the control at the same time point. (B) Representative Western blots and the cyto-c/β-actin ratio show the changes in the cytosolic fractions at different time points after CRAO; +, laser-treated group; −, control group.
Figure 5.
 
(A) Semiquantitative densitometry results for relative abundance of cytochrome c (cyto-c) in retinal cytosolic fractions, calculated as the cyto-c/β-actin ratio. β-Actin served as a protein-loading control, to ensure equal loading. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blot analyses (n = 5). * P < 0.05 compared with the control at the same time point. (B) Representative Western blots and the cyto-c/β-actin ratio show the changes in the cytosolic fractions at different time points after CRAO; +, laser-treated group; −, control group.
Figure 6.
 
(A) Semiquantitative densitometry results showing relative abundance of cleaved caspase-9 in retinal cytosolic fractions as the caspase-9/β-actin ratio. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blots (n = 5). * P < 0.05 compared with the control at same time point. (B) Representative Western blot analysis and cleaved caspase-9/β-actin ratio indicating changes in the cytosolic fractions at different time points in the laser-induced CRAO (+) and the control (−) group. β-Actin served as a protein loading control.
Figure 6.
 
(A) Semiquantitative densitometry results showing relative abundance of cleaved caspase-9 in retinal cytosolic fractions as the caspase-9/β-actin ratio. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blots (n = 5). * P < 0.05 compared with the control at same time point. (B) Representative Western blot analysis and cleaved caspase-9/β-actin ratio indicating changes in the cytosolic fractions at different time points in the laser-induced CRAO (+) and the control (−) group. β-Actin served as a protein loading control.
The authors thank Genevieve Long for editorial comments and Pat Wallace and the Photography Department at Casey Eye Institute for assistance with fundus photography. 
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Figure 1.
 
Schematic of mitochondrial apoptotic pathway. (1) Bax translocates from cytosol to outer mitochondrial membrane and forms high-conductance channels or pores; (2) cytochrome c released from mitochondrion forms the apoptosome with Apaf-1, dATP, and procaspase-9, and then procaspase is activated; (3) caspase-9 activates procaspase-3, which, in turn, activates downstream executioner caspases and leads to cell death.
Figure 1.
 
Schematic of mitochondrial apoptotic pathway. (1) Bax translocates from cytosol to outer mitochondrial membrane and forms high-conductance channels or pores; (2) cytochrome c released from mitochondrion forms the apoptosome with Apaf-1, dATP, and procaspase-9, and then procaspase is activated; (3) caspase-9 activates procaspase-3, which, in turn, activates downstream executioner caspases and leads to cell death.
Figure 2.
 
Rat fundus photographs showing retinal vessels at different time points after argon green laser irradiation to produce CRAO. (A) Normal fundus. (B) Immediately and (C) 1, (D) 3, (E) 6, and (F) 24 hours after argon green laser irradiation. Note the whitening and paleness of the retina and the occluded arteries. Red arrow: an artery; black arrow: vein.
Figure 2.
 
Rat fundus photographs showing retinal vessels at different time points after argon green laser irradiation to produce CRAO. (A) Normal fundus. (B) Immediately and (C) 1, (D) 3, (E) 6, and (F) 24 hours after argon green laser irradiation. Note the whitening and paleness of the retina and the occluded arteries. Red arrow: an artery; black arrow: vein.
Figure 3.
 
Positive TUNEL staining of retinal cell layers at various time points after induction of ischemia. (A) Control eye and treated eyes at (B) 1, (C) 3, (D) 6, and (E) 24 hours. Magnification, ×200.
Figure 3.
 
Positive TUNEL staining of retinal cell layers at various time points after induction of ischemia. (A) Control eye and treated eyes at (B) 1, (C) 3, (D) 6, and (E) 24 hours. Magnification, ×200.
Figure 4.
 
Western blot analysis of Bax (A) in retinal cytosolic fractions and (B) in mitochondrial fractions. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. β-actin served as a protein loading control, to ensure equal total protein concentrations for each lane. Results of semiquantitative densitometry of Bax in subcellular fractions at each time point was calibrated with that of β-actin to obtain the Bax/β-actin ratio. Data were analyzed based on five separate cytosolic fractions (n = 5) or three separate mitochondrial fractions (n = 3). +, laser treated eye; −, contralateral control eye. *P < 0.05 compared with the control at the same time point.
Figure 4.
 
Western blot analysis of Bax (A) in retinal cytosolic fractions and (B) in mitochondrial fractions. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. β-actin served as a protein loading control, to ensure equal total protein concentrations for each lane. Results of semiquantitative densitometry of Bax in subcellular fractions at each time point was calibrated with that of β-actin to obtain the Bax/β-actin ratio. Data were analyzed based on five separate cytosolic fractions (n = 5) or three separate mitochondrial fractions (n = 3). +, laser treated eye; −, contralateral control eye. *P < 0.05 compared with the control at the same time point.
Figure 5.
 
(A) Semiquantitative densitometry results for relative abundance of cytochrome c (cyto-c) in retinal cytosolic fractions, calculated as the cyto-c/β-actin ratio. β-Actin served as a protein-loading control, to ensure equal loading. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blot analyses (n = 5). * P < 0.05 compared with the control at the same time point. (B) Representative Western blots and the cyto-c/β-actin ratio show the changes in the cytosolic fractions at different time points after CRAO; +, laser-treated group; −, control group.
Figure 5.
 
(A) Semiquantitative densitometry results for relative abundance of cytochrome c (cyto-c) in retinal cytosolic fractions, calculated as the cyto-c/β-actin ratio. β-Actin served as a protein-loading control, to ensure equal loading. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blot analyses (n = 5). * P < 0.05 compared with the control at the same time point. (B) Representative Western blots and the cyto-c/β-actin ratio show the changes in the cytosolic fractions at different time points after CRAO; +, laser-treated group; −, control group.
Figure 6.
 
(A) Semiquantitative densitometry results showing relative abundance of cleaved caspase-9 in retinal cytosolic fractions as the caspase-9/β-actin ratio. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blots (n = 5). * P < 0.05 compared with the control at same time point. (B) Representative Western blot analysis and cleaved caspase-9/β-actin ratio indicating changes in the cytosolic fractions at different time points in the laser-induced CRAO (+) and the control (−) group. β-Actin served as a protein loading control.
Figure 6.
 
(A) Semiquantitative densitometry results showing relative abundance of cleaved caspase-9 in retinal cytosolic fractions as the caspase-9/β-actin ratio. Samples were purified from retinas dissected at 1, 3, 6, and 24 hours after CRAO. Data were analyzed based on five individual Western blots (n = 5). * P < 0.05 compared with the control at same time point. (B) Representative Western blot analysis and cleaved caspase-9/β-actin ratio indicating changes in the cytosolic fractions at different time points in the laser-induced CRAO (+) and the control (−) group. β-Actin served as a protein loading control.
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