Ischemic Preconditioning Attenuates Apoptotic Cell Death in the Rat Retina

Abstract

purpose. Ischemic preconditioning (IPC) protects the rat retina against the injury that ordinarily follows prolonged ischemia. It has been shown that release of adenosine, de novo protein synthesis, and mediators, such as protein kinase C and K_ATP channels, is required for IPC protection. However, the molecular mechanisms of neuroprotection by IPC are unknown. Retinal cells die after ischemia by necrosis and apoptosis. This study was undertaken to investigate the effect of IPC on apoptosis after ischemia and some of the key proteins involved in the apoptotic cascade.

methods. Retinal ischemia or IPC was produced in anesthetized Sprague-Dawley rats by increasing intraocular pressure above systolic arterial pressure. Retinal ischemia was induced 24 hours after either IPC or sham IPC. TUNEL staining was used to quantitate the number of cells with DNA fragmentation. The authors examined expression of cleaved forms of caspases-2 and -3, bax, and poly-adenosine diphosphate-ribose-polymerase (PARP) by Western blot analysis for evidence of apoptosis-related gene expression. To examine possible mechanisms of apoptosis after ischemia, the authors studied the expression of mitogen-activated protein kinases (MAP kinases). Functional recovery after ischemia was measured using electroretinography, and retinal histology was examined and quantitated by light microscopy.

results. Positive TUNEL staining, increases in caspase-2 and -3 cleavage, expression of bax and PARP, and activation of MAP kinases were found with ischemia. IPC attenuated these changes, but paradoxically, IPC itself triggered increased expression of MAP kinases.

conclusions. IPC protects against ischemic injury, in part, by diminishing apoptosis-related gene expression and by altering protein phosphorylation.

Earlier reports have shown that ischemic preconditioning (IPC) ^{1 2 3} is remarkably effective in protecting the retina functionally and histologically against ischemic injury and attenuates the severe hypoperfusion that follows ischemia. ⁴ We and others have shown that IPC protection is mediated by a number of mechanisms, including binding of adenosine to its A1 and A2a receptors, ² the opening of K_ATP channels, ^{3 5} activation of protein kinase C, ³ and de novo protein synthesis. However, the molecular mechanisms of retinal IPC remain unknown. 

Emerging evidence suggests that cells die after retinal ischemia by both apoptotic and necrotic mechanisms. The relative contribution of these mechanisms to cell death has not yet been determined, but a number of mediators are involved in retinal apoptosis, including endonucleases, ⁶ Bax/Bcl-2, ⁷ p53, ^{8 9} and caspases. ^{10 11 12} Apoptotic cell signaling is tightly coupled in many cells to other cellular transduction systems, including those involved in proliferation and differentiation (e.g., Ras, and mitogen-activated kinases [MAPKs]) such as extracellular signal-regulated kinase [ERK], p38, and c-Jun N-terminal kinase (JNK). ¹³ Tyrosine phosphorylation of proteins after retinal ischemia may also influence cell survival. ^{14 15} The trigger for cells to undergo apoptosis may also be influenced by protective factors such as heat-shock proteins. ¹⁶ The mechanisms responsible for apoptosis after ischemia in the retina are just beginning to be elucidated. 

Recent studies suggest that IPC limits infarct size and decreases DNA fragmentation in rat hearts in vivo. ^{17 18} In the preconditioned heart, alterations have been found in the bcl-2 family of apoptosis-related genes. ¹⁹ In cerebral ischemia, changes in this family of genes also appear after IPC. ²⁰ The attenuation of IPC neuroprotection or its mimicking by adenosine after blockade of protein synthesis indicates that a change in gene expression is responsible. ^{1 3} In addition, adenosine, which is an essential component of IPC neuroprotection, is known to stimulate protein kinases and phospholipases and may alter the expression of other genes. ²¹ Preconditioning rat retina by light exposure before photic injury stimulates the production of ERK, fibroblast growth factor, and ciliary neurotrophic factor. ²² It therefore seems logical to hypothesize that IPC may be protective by interrupting pathways that stimulate apoptosis, thereby shifting the balance in retinal cells toward survival. To study this possible protective mechanism, we used a well-described model in which retinal cell death after ischemia has the characteristics of apoptosis. ^{6 8 11} To examine apoptosis-related gene expression, we measured levels of caspases-2 and -3, bax, and poly-adenosine diphosphate-ribose-polymerase (PARP), which correlate with the presence of apoptosis in neuronal cells. ²³ The expression of MAPKs was examined to provide insight into mechanisms of cell death after retinal ischemia, and conversely, enhancement of cell survival by IPC. 

Materials and Methods

Ischemia Methodology

Our procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee at the University of Chicago. We studied Sprague-Dawley rats (200–250 g) purchased from Harlan (Indianapolis, IN). Animals were maintained on a 12-hour on/off light cycle and were dark adapted for at least 2 hours before experiments. Before ischemia was induced, animals were anesthetized with chloral hydrate (450 mg/kg). For baseline and postischemia follow-up electroretinograms, rats were injected intraperitoneally with ketamine (35 mg/kg; Parke-Davis, Morris Plains, NJ), and xylazine (5 mg/kg; Miles, Shawnee Mission, KS). Corneal analgesia was achieved using 1 to 2 drops of 0.5% proparacaine (Allergan, Humacao, Puerto Rico). Pupillary dilatation was maintained with 0.5% tropicamide (Alcon, Humacao, Puerto Rico) and 0.2% cyclopentolate HCl-1% phenylephrine HCl (Cyclomydril; Alcon, Fort Worth, TX). Body temperature was maintained at 36.5°C to 37.0°C with a servo-controlled heating blanket (Harvard Apparatus, Natick, MA). 

For preconditioning, the intraocular pressure (IOP) was increased to 160 mm Hg for 8 minutes with a pressurized 1000-mL plastic container of sterile normal saline (Baxter, North Chicago, IL), connected to a 27-gauge needle placed in the anterior chamber of the eye. For ischemia, the IOP was increased to 110 mm Hg by elevating the saline reservoir to sufficient height above the eye for 45 minutes. In preliminary experiments, we directly measured systolic arterial blood pressure in a cannulated femoral artery and found that increasing IOP to 110 mm Hg or above consistently exceeded arterial blood pressure. In sham-treated, nonpreconditioned animals, the IOP was maintained at 10 to 15 mm Hg. The opposite eye of each animal served as the nonischemic control. 

TUNEL Staining

Enucleated eyes were immediately fixed in 4% paraformaldehyde (Sigma, St. Louis, MO) for 2 hours. The cornea and lens were removed, and the remaining eye cup was placed in the same fixative for 4 hours, before cryoprotection in 25% sucrose overnight at 4°C. Tissues, collected 24 hours after ischemia, at which time retinal DNA fragmentation reaches its peak, ^{6 9 11} were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA), and sagittal sections were cut through the optic nerve. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining was performed on 7-μm frozen retinal sections with a kit (TdT-FragEL DNA Fragmentation Detection Kit; Oncogene, Boston, MA), with procedures modified from Gavrieli et al. ²⁴ The sections were incubated with 20 μg/mL proteinase K and then with Tdt/dUTP reaction mixture for 1 hour at 37°C for DNA fragment labeling, followed by three rinses in phosphate-buffered saline. The positive-labeling cell nuclei were made visible by their brown color after exposure to 3,3′-diaminobenzidine (DAB; Dako, Carpinteria, CA). Positive control experiments demonstrating DNA fragmentation were performed by exposing sections to DNAase, whereas negative controls omitted the TdT/dUTP labeling mixture, resulting in no staining. TUNEL-positive cells were considered apoptotic if cellular shrinkage and chromatin condensation were visible under light microscopy. ^{6 9 11} Digital images were then obtained (Axioskop with Axiocam camera; Carl Zeiss, Thornwood, NY). TUNEL-positive cells in the inner retina were counted in four to five adjacent high-powered (40×) fields starting within 50 μm of the optic nerve head, advancing progressively toward the periphery. 

Immunoblot Analysis

After the death of the animals and enucleation of the eyes, retinas were rapidly dissected and frozen in liquid N₂, then crushed using a tissue pulverizer (Beckman, Fullerton, CA) on dry ice. The retinas were solubilized in 9 M urea, 4% Nonidet P-40, and 2% 2-mercaptoethanol (pH 9.5). Protease inhibitor cocktail (P8340, Sigma) consisting of 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, bestatin, leupeptin, E-64, and aprotinin was added to prevent protease activity. Samples were centrifuged 10 minutes at 14,000g. The supernatant was used for SDS-PAGE and the pellet discarded. Protein concentration was determined with a modified Bradford assay (Bio-Rad, Hercules, CA). 

Equal amounts of retinal protein per lane (40 μg) were diluted with SDS sample buffer and loaded onto gels for SDS-PAGE (4%–20% gradient; Invitrogen, San Diego, CA). Proteins were electroblotted to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) and the efficiency of transfer was confirmed by staining the membrane with ponceau S red (Sigma). Gel retention was assessed by staining with Coomassie blue (Pierce, Rockford, IL). Nonspecific binding was blocked with 5% nonfat dry milk in Tween-Tris-buffered saline (TTBS). Membranes were incubated overnight at 4°C with antibodies to caspase-2 (rabbit polyclonal IgG, 1:1000; Stressgen, Victoria, BC, Canada), caspase-3 (rabbit polyclonal IgG, clone CM1, 1:3000; BD-Pharmingen, San Diego, CA), bax (rabbit polyclonal IgG, 1:1000, BD-Pharmingen), and PARP (mouse monoclonal IgG, clone C-2-10, 1:500; Biomol, Plymouth Meeting, PA). Membranes were incubated similarly with anti-phospho-p44/p42 MAPK (P-ERK, rabbit polyclonal IgG, Thr202/Tyr204, 1:2000, Cell Signaling, Beverly, MA), anti-diphosphorylated p38 (rabbit polyclonal IgG, 1:2000, Promega, Madison, WI), and anti-diphosphorylated JNK (rabbit polyclonal IgG, 1:1000, Cell Signaling). All the antibodies were prepared in 5% nonfat dry milk solution in TTBS. 

Actin was used to verify equal loading of protein. The anti-actin antibody (mouse monoclonal IgM, clone JLA 20, 1:200) developed by J. J.-C. Lin, PhD, was obtained from the National Institute of Child Health and Human Development (NICHD)–supported Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA). Appropriate horseradish (HRP)–conjugated secondary antibodies, which were anti-rabbit (goat IgG; Jackson ImmunoResearch, West Grove, PA), or anti-mouse (sheep IgG, Amersham, Arlington Heights, IL) were applied at 1:20,000. Negative controls were performed without primary antibody. Chemiluminescence was developed with a kit (Super Signal West Pico; Pierce). Protein bands were visualized digitally with an imaging system (CCDBIO 16SC; Hitachi Genetic Systems/MiraiBio, Alameda, CA), and quantitated by densitometry (Gene Tools and Gene Snap software; Hitachi). 

Electroretinography

Procedures used in our laboratory have been described in detail previously. ^{1 2 4} In brief, corneal electrical responses to 10-μs white light flashes delivered by a Ganzfeld (Nicolet, Madison, WI) were recorded on a commercial system (Spirit 486 System; Nicolet). Data at each time point were collected by averaging the results of three flashes delivered at least 2 minutes apart. The ERG wave amplitudes at each time point after ischemia (1, 3, and 7 days later) were measured and reported as a percentage of the baseline, nonischemic wave amplitude. 

Histopathology

The eyes were enucleated on the seventh day after ischemia and immediately placed in Trump fixative. ^{1 6 9} A needle was inserted into the anterior chamber to mark the superior limbus. After fixation for 24 hours, the eyes were divided into superior and inferior halves. The inferior half of the eye was further divided into two sectors, and inferior strips of retinal specimens were embedded in epoxy resin. One-micrometer semithin sections were stained with 1% toluidine blue, examined by light microscopy, and quantitated as described by us earlier. ^{1 12}  

Studies

Ischemia for 8 minutes was used as a preconditioning stimulus. ^{1 2 4} Forty-five minutes of ischemia followed 24 hours after IPC. The timing was based on our earlier findings that a 24 or 72 hours separation between IPC and ischemia provided equally efficacious protection from ischemic damage. ¹ Retinal tissues for Western blot analysis were obtained in the following groups: normal eyes; IPC alone (48 hours later); sham IPC followed by ischemia 24 hours later, with eyes removed 24 hours later (sham IPC+ischemia); and IPC followed by ischemia 24 hours later, with eyes removed 24 hours later (IPC+ischemia). Thus, retinas were examined at comparable time points by removing eyes in each case 48 hours after the initial IPC stimulus and 24 hours after the more prolonged ischemia. Moreover, retinas for Western blot analysis were obtained at the same time points as those for TUNEL staining. 

Statistics

TUNEL staining was compared in sham IPC+ischemia, IPC+ischemia, IPC alone, or sham-normal samples using the Kruskal-Wallis test. ERG, histologic, and Western blot data were analyzed as previously described, with ANOVA and post hoc t-test performed by computer (Stata ver. 6.0; Stata Corporation, College Station, TX). ^{1 2 4} Data are expressed as mean ± SEM. 

Results

DNA Fragmentation

TUNEL-positive staining (Fig. 1) , particularly in the inner nuclear, ganglion cell, and outer nuclear layers, was evident 24 hours after ischemia, consistent with results from previous studies. ^{9 11} No TUNEL-positive cells were present in normal retina or after IPC alone (Fig. 1) . Preconditioning significantly decreased the number of TUNEL positive-staining cells after ischemia from 20 ± 3 to 2 ± 0.5 cells per high-power field, in the sham+ischemia and the IPC+ischemia group, respectively (n = 7 per group, P < 0.002). 

Caspases, Bax, and PARP

Figure 2 shows the results of Western blot analyses for caspases-2 and -3 and for bax. For caspase-2, bands were visible at 54 and at 32 kDa. The pro form of rat caspase-2 has been described with molecular masses of 51 or 41 kDa. ^{25 26} The 32-kDa form is a cleavage product indicating activation of caspase-2, described previously with molecular mass of 33 kDa in the retina of RCS rats. ²⁵ Compared with normal, the intensity of the 32-kDa band tended to increase (169% ± 24%, P < 0.06) after ischemia, and this change was attenuated in the preconditioned retina (94% ± 23%). There were no significant changes in the intensity of the 54-kDa pro form, except for a decrease (64% ± 6%) with IPC alone (P < 0.01). The CM1 antibody is specific for the 18-kDa cleaved caspase-3. ²⁷ With ischemia alone, cleaved caspase-3 increased to 226% ± 28% (P < 0.04 versus normal), and the increase was blunted by prior IPC (83% ± 10%, P < 0.02 versus ischemia without IPC). Bax protein increased significantly (239% ± 23%, P < 0.009) with ischemia. This increase was partially attenuated by prior IPC (168% ± 31%). The 116-kDa PARP (Fig. 2) increased to 195% ± 19% (P < 0.03 versus normal) after ischemia, and this increase was prevented by prior IPC (73% ± 7%, P < 0.01 versus ischemia without prior IPC). 

Protein Phosphorylation

Immunoblotanalysis for phosphorylated ERK (Fig. 3 ; MAP42/44 kinase, P-ERK) revealed significant and almost identical increases in the 42- and 44-kDa bands with IPC alone, with ischemia, and with ischemia preceded by IPC. Although IPC tended to decrease MAP42/44 expression after ischemia (290% ± 61% vs. 227% ± 32%, e.g., for p44), this difference was not statistically significant. Changes in phosphorylated JNK consisted of increases in both a 46- and a 54-kDa band (Fig. 3) . The 46-kDa band increased to 286% ± 67% with ischemia (P < 0.03 versus normal), and this increase was attenuated by prior IPC (144% ± 44%). The 56-kDa band increased to 204% ± 42% with ischemia (P < 0.04), and the increase was attenuated with prior IPC (139% ± 45%). Increases in p38 (Fig. 3) were also evident after both IPC or ischemia as two bands, 43 and 48 kDa. Both were increased significantly with sham IPC+ischemia (292% ± 68% and 271% ± 41%, P < 0.04 and P < 0.009, respectively). Prior IPC significantly attenuated the increase in the 43-kDa band (117% ± 19%, P < 0.05), and to a lesser extent, the increase in the 48-kDa band (190% ± 46%). Actin control in Figure 2 demonstrates equal loading of protein in the gel lanes. 

Functional and Histologic Protection by IPC

The recovery of the b-wave after ischemia (Fig. 4) was significantly enhanced at day 7 after ischemia by IPC (88% ± 8% vs. 42% ± 6% recovery of baseline amplitude, for IPC+ischemia versus sham IPC+ischemia, respectively, P < 0.005). Mean thickness of the inner retinal layers was decreased by 22% (P < 0.005). IPC significantly attenuated the decrease in inner retinal thickness (P < 0.005, Fig. 5 ). 

Discussion

Our results show that ischemic preconditioning decreased DNA fragmentation associated with apoptosis after ischemia and that attenuation of caspase activation, bax, and PARP expression and changes in protein phosphorylation are involved in this neuroprotection. Acute and sustained increases in intraocular pressure result in two morphologically distinguishable patterns of ischemic injury in the rat retina. A rapid pattern of necrosis occurs within hours in the ganglion cell and inner nuclear layers, and involves activation of N-methyl-d-aspartate (NMDA) receptors. A late phase starts at approximately 12 hours, remains evident for at least 72 hours, and is notable for features of apoptosis that include condensation of nuclear chromatin and shrinkage of the cell body. ^{6 8 11} Apoptosis after retinal ischemia is associated with increased expression of p53, cyclin D1, endonucleases, bax/bcl-2, caspases, and PARP. ^{6 7 8 9 10 11 12 28}  

An activator of other caspases, caspase-2, is essential for apoptosis, ²⁶ and increased expression has been described after cerebral and retinal ischemia. ^{12 29} Blocking caspase-2 expression attenuates damage after retinal ischemia. ¹² Caspase-3 is a major mediator of apoptosis in Parkinson and Alzheimer diseases, cerebral ischemia, and during development. ³⁰ Caspase-3 activates PARP. Moreover, caspase-3 colocalized in TUNEL-positive rat retinal ganglion cells after axotomy ³¹ and in the inner nuclear layer after retinal ischemia. ¹² Blockade of caspase-3 decreased the damage after both cerebral and retinal ischemia. ^{12 32}  

We confirmed the hypothesis that ischemic preconditioning decreased the activation of caspases-2 and -3 after ischemia. The presence of caspase cleavage molecules indicates caspase activation. ³³ Our results show that IPC attenuated ischemia-induced activation of caspases-2 and -3. The caspase-2 antibody used for Western blot analysis recognizes a full, uncleaved molecule, as well as a smaller activated cleavage product. ²⁵ The caspase-3 antibody is specific for the active 18-kDa fragment. ²⁷ In an unexpected and interesting finding, expression of the caspase-2 proform decreased with IPC alone, but no significant change was seen in levels of the smaller cleaved molecule. This suggests that IPC itself is unassociated with significant activation of caspase-2. 

Bax is a major effector of apoptotic cell death in the brain ³⁴ and in the retina after ischemia, excitotoxicity, axotomy, and in retinal degeneration. ^{35 36 37} The bax gene codes potentially for three proteins, but the 21-kDa product we detected most probably is Bax-α. ^{35 36 37} Blockade of increased bax expression by IPC indicates suppression of yet another critical death-inducing protein after ischemia. 

Caspases and PARP activation, both associated with DNA fragmentation, are hallmarks of apoptosis. ³⁸ Increased expression of PARP, with attenuation of these changes by IPC, indicates that IPC prevents apoptosis after ischemia. Excessive activation of PARP may cause cell death by energy substrate depletion. ³⁹ The exact role of PARP remains controversial, but most studies in brain and retina have shown that inhibition of PARP is protective against ischemic injury. ^{38 40}  

Because caspase and PARP activation are not the initial events in the induction of apoptosis and cell death, the question arises as to exactly how IPC interrupts apoptosis in the retina after ischemia. Mechanisms may include preservation of cellular adenosine triphosphate (ATP); direct blockade of caspase, bax, and PARP expression; inhibition of protein phosphorylation; modification of specific signaling proteins, stimulation, and/or blockade of growth factors, among others. A full elucidation of the mechanisms is not yet available, but our study provides some possible clues. IPC may shift cells into an energy-conserving mode that prevents depletion of ATP, ⁴¹ mitochondrial dysfunction, and the consequent activation of caspases and PARP. ²³ We have demonstrated that retinal blood flow after ischemia is preserved by prior IPC, ⁴ supporting a theory of preservation of energy levels. Another critical event determining cell survival is protein phosphorylation. ⁴² Protein tyrosine phosphorylation of growth factors, tyrosine kinases, and other proteins has been described in cerebral and retinal ischemia and retinal vein occlusion. ^{14 43 44} Moreover, pharmacologic blockade of tyrosine kinase decreased retinal damage after ischemia in a rat model. ¹⁵ Of considerable interest is that tyrosine phosphorylation after ischemia was attenuated in a rat model of cerebral ischemic tolerance, ⁴⁵ similar to our present findings. The inhibition of phosphorylation of several proteins by IPC that we showed, taken together with findings that tyrosine kinase inhibition decreases injury after ischemia, suggests that protein phosphorylation may be a critical event in determining ischemic tolerance and survival in the retina. 

Because of the profound implications of MAPKs in cell survival, they are clearly candidate downstream proteins in the induction of endogenous neuroprotection. This prediction has been borne out by both in vitro and in vivo studies of IPC and ischemia in myocardium and brain. ⁴⁶ In cerebral cortical cells in vitro, activation of ERK by growth factors blocks NMDA-induced cell death, ⁴⁷ and IPC protection against O₂-glucose deprivation required Ras, Raf, MEK, and ERK. ⁴⁸ JNK, ERK, and p38 expression increase after focal and global cerebral ischemia in rats and gerbils. ^{49 50 51} Inhibition of p38 is neuroprotective after global ischemia, ⁵¹ while blocking ERK expression is protective after focal, ⁵² but not global ischemia. ⁵¹ Several studies have shown activation of ERK and p38 after myocardial IPC. ^{53 54} It is intriguing that, although p38 was increased by both IPC and ischemia in rat myocytes, prior IPC abolished the ischemia-induced increase. ⁵⁵ JNK also increased after IPC in myocardium. ⁵⁶ A recent report showed that IPC decreased functional injury and activation of JNK and p38 but not ERK after kidney ischemia. ⁵⁷  

Our results showing increases in ERK, JNK, and p38 with IPC, and attenuation of ischemia-induced increases in p38 and JNK by IPC suggest a paradox. Under conditions of brief ischemia (i.e., IPC) these enzymes may result in the transcription of proteins that are neuroprotective. With prolonged ischemia, the net effect of stimulating MAPKs is an increase in proteins causing cell death. If this hypothesis is correct, IPC prevents cell death by shifting the balance toward expression of proteins favoring cell survival. 

In summary, we provide new data to support a hypothesis that ischemic preconditioning decreases apoptotic injury in the retina. The sites of inhibition of apoptosis by IPC involve caspases, bax, and PARP, as well as MAPKs, but the precise upstream and downstream molecular events whereby IPC is protective remain to be described. 

 Supported by National Institutes of Health Grants EY10343 (SR), EY11253 (DMR), and GM08140, Bethesda, Maryland; The Glaucoma Research Foundation (SR), San Francisco, CA; Research to Prevent Blindness, Inc. (PSR).

 Submitted for publication November 2, 2001; revised March 21, 2002; accepted April 9, 2002.

 Commercial relationships policy: N.

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

 

Figure 1.

 

TUNEL staining (dark cells) 24 hours after ischemia (C) was substantially decreased by IPC (D). (A) Normal; (B) IPC alone; (C) sham IPC+ischemia; (D) IPC+ischemia. Seven-micrometer frozen retinal sections digitally imaged at 20×.

Figure 2.

 

Expression of caspases, bax, and PARP with IPC and ischemia shown by Western blot analysis in whole retinal homogenates. Membranes were incubated overnight at 4°C with anti-caspase-2 (1:1000), anti-caspase-3 (1:3000), anti-bax (1:1000), or anti-PARP (1:500). Images were developed using enhanced chemiluminescence. IPC attenuated caspase cleavage, the expression of bax and of PARP. Also shown are results for anti-actin (1:200), demonstrating equal loading of proteins. Data show expression of caspases, bax, and PARP (n = 4) in the four test groups, expressed as the mean ± SEM. *P < 0.05 versus normal; #P < 0.01 versus normal.

Figure 3.

 

Western blot analysis of whole retinal homogenates showing phosphorylated ERK (42 and 44 kDa, 1:2000), phosphorylated JNK (46 and 57 kDa, 1:1000), and phosphorylated p38 (43 and 48 kDa, 1:2000) with IPC and ischemia. Data show expression of phosphorylated ERK (n = 7), JNK (n = 6), p38 (n = 5) in the four test groups, expressed as the mean ± SEM. *P < 0.05 versus normal; #P < 0.009 versus normal.

Figure 4.

 

IPC improves recovery of the ERG a- and b-waves after ischemia. IPC was produced by increasing intraocular pressure above systolic arterial pressure for 8 minutes. Sham indicates no preconditioning. Prolonged ischemia followed 24 hours later. The ERG a- and b-waves were normalized to the preischemia baseline and corrected for day-to-day variation with the data from the nonischemic control eye. Recovery as a percentage of baseline is plotted on the y-axis; the x-axis shows time after ischemia. Data are expressed as the mean ± SEM, with n = 10 to 13 rats per group. *P < 0.05 versus sham IPC+ischemia; **P < 0.005 versus sham IPC+ischemia.

Figure 5.

 

IPC prevented decreases in mean inner retinal thickness (MTIRL, μm) at 7 days after ischemia. Data are expressed as the mean ± SEM, with n = 5 rats per group. §P < 0.005 versus normal, ‡P < 0.05 versus sham IPC+ischemia.

The authors thank the Digital Light Microscopy Facility, University of Chicago Cancer Research Center, for the use of the facility to perform the digital imaging.