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Retina Effects of Ischemic Preconditioning and Bevacizumab on Apoptosis and Vascular Permeability Following Retinal Ischemia–Reperfusion Injury

Steven F. Abcouwer,1,2,3 Cheng-mao Lin,2 Ellen B. Wolpert,2 Sumathi Shanmugam,2 Eric W. Schaefer,4 Willard M. Freeman,5 Alistair J. Barber,3 and David A. Antonetti2,3

PURPOSE. Using transient ischemia followed by reperfusion (IR) etinal neovascularization and macular edema are associ- to model ischemic retinal disease, this study compares the Rated with vision loss in several retinal diseases, including effects of ischemic preconditioning (IPC) and therapies target- age-related macular degeneration (AMD) and ischemic retinop- ing vascular endothelial growth factor (VEGF) and tumor ne- athies, such as retinal vascular occlusive diseases and diabetic crosis factor (TNF)-␣ on retinal apoptosis, vascular permeabil- retinopathy. Although the vascular components are most ob- ity, and mRNA expression. vious, these retinopathies are also associated with various de- METHODS. Rats were subjected to 30 or 45 minutes of retinal grees of inflammation and neurodegeneration. For example, development of AMD is linked to complement activation and ischemia followed by reperfusion for up to 48 hours. Neuro- 1,2 degeneration was quantified by caspase-3 (DEVDase) activity inflammation. Diabetic retinopathy is associated with micro- and by measuring nucleosomal DNA content (cell death glial activation, increased expression of inflammatory cytokines, adherence of leukocytes to the retinal microvasculature ELISA). Vascular leakage was quantified by the Evans Blue dye 3–6 method. A set of IR-responsive mRNAs was identified by whole- (leukostasis), and apoptotic death of neurons. The func- genome microarray and confirmed by RT-PCR analyses. VEGF tional interactions between inflammatory, neuronal, and vas- protein was measured by Western blot analysis. IPC was ac- cular components of retinopathies are just now beginning to complished with 10 minutes of ischemia 24 hours before IR. be explored. For example, inflammation has been hypothe- VEGF and TNF␣ signaling was inhibited by intravitreal injec- sized to contribute to the development of vascular abnormali- tion of bevacizumab or etanercept, respectively. ties through leukostasis and subsequent microvascular occlusion, production of vasoactive compounds such as VEGF, and RESULTS. IR caused significant retinal cell apoptosis and vascu- subsequent vascular permeability and vascular cell drop out.5,7 lar permeability after 4 and 48 hours. Whereas IR decreased Vascular occlusion and vessel drop out can lead to focal retinal VegfA mRNA, VEGF protein was significantly increased. IPC ischemia, which may be an initiating event for both neurode- effectively inhibited neurodegeneration, bevacizumab effec- generation and neovascularization.8,9 Alternatively, adaptive tively inhibited vascular permeability, and etanercept failed to responses to neurodegeneration may include the expression of affect either outcome. IPC significantly altered the IR re- neurotrophic factors that, in turn, cause vascular permeability sponses of 15 of 33 IR-responsive mRNAs, whereas bevaci- and angiogenesis.10 zumab had no significant effect on these mRNAs. TNF␣ and VEGF have been identified as therapeutic targets CONCLUSIONS. IR provides an acute model of ischemic reti- for treating inflammatory and ischemic retinal diseases.11–13 nopathy that includes neurodegeneration and VEGF- TNF␣-targeted treatments, including etanercept and inflix- dependent vascular permeability and is amenable to rapid imab, have been applied with promising results in ocular in- drug therapy testing. The distinct effects of IPC and bevaci- flammatory disease, AMD, and diabetic macular edema.14–16 In zumab demonstrate that the apoptotic and vascular re- a rat endotoxin-induced uveitis model of inflammatory retinop- sponses to IR may be separated and that therapeutics tar- athy, systemic administration of the TNF␣ inhibitor etanercept geting each pathologic endpoint may be warranted in diminished leukostasis, endothelial and neuronal apoptosis, treating ischemic retinal diseases. (Invest Ophthalmol Vis and vascular permeability.17 Systemic etanercept suppressed Sci. 2010;51:5920–5933) DOI:10.1167/iovs.10-5264 retinal inflammatory markers (ICAM-1, eNOS, and NF-␬B), while reducing leukostasis and vascular dysfunction in rats with short-term diabetes.18 Systemic etanercept inhibited reti-

1 2 nal apoptosis in a rat model of short-term diabetes and retinal From the Departments of Surgery, Cellular and Molecular Phys- 19 3 4 5 vascular cell loss in a mouse model of longer-term diabetes. iology, Ophthalmology, Public Health Science, and Pharmacology, ␣ Penn State College of Medicine, Hershey, Pennsylvania. Intravitreal injection of the TNF inhibitor pegsunercept inhib- Supported by a grant from the Juvenile Diabetes Research Foun- ited retinal microvascular cell death in rat models of both type 20 dation (SFA, DAA, Co-Principal Investigators) that is part of a JDRF 1 and type 2 diabetes. Diabetic Retinopathy Center Grant (Thomas W. Gardner, MD, Direc- VEGF contributes to the vascular angiogenesis and vascular tor). permeability associated with many retinopathies (for a review, Submitted for publication January 25, 2010; revised May 7 and 27, see Ref. 21). Its effect is supported by case studies and small 2010; accepted May 28, 2010. clinical trials demonstrating that treatment with VEGF antago- Disclosure: S.F. Abcouwer, None; C. Lin, None; E.B. Wolpert, nists can alleviate edema and prevent neovascularization in None; S. Shanmugam, None; E.W. Schaefer, None; W.M. Freeman, ischemic and inﬂammatory retinopathies.22,23 However, there None; A.J. Barber, None; D.A. Antonetti, None is concern that inhibition of VEGF function could cause neu- Corresponding author: Steven F. Abcouwer, Cellular and Molecu- 24 lar Physiology and Ophthalmology, Penn State University College of rodegeneration. VEGF acts as a potent neurotrophic factor 25,26 Medicine, Milton S. Hershey Medical Center, C4842, P.O. Box 850, and retinal neurons express VEGF receptors. Blocking Surgery H051, Hershey, PA 17033-0850; [email protected]. VEGF function by repeated application of a soluble VEGF

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receptor protein or neutralizing VEGF antibody caused the loss Francisco, CA), and a pharmaceutical-grade formulation of a dimeric of retinal ganglion cells in mice and rats.25 However, clinical fusion protein of the extracellular portion of the human 75-kDa (p75) trials and other animal experiments suggest that the risks of TNF-␣ receptor (TNFR) linked to the Fc portion of human IgG1, ocular anti-VEGF therapies are minimal.27–29 etanercept (Enbrel, 50 mg/mL solution; Immunex Corp., Thousand The intraocular pressure-induced ischemia–reperfusion (IR) Oaks, CA), were obtained from the Hershey Medical Center Pharmacy. model involves temporary ischemia followed by natural reper- Each drug was injected intravitreally (2 ␮L/eye) with a 32-gauge needle fusion, which causes an inflammatory and neurodegenerative 48 hours before IR or sham treatment. Phosphate-buffered saline (PBS, response in the intact retina. This procedure models the neu- 2 ␮L/eye) was injected in control eyes. ronal damage observed in diseases with transient vessel occlusions. Most IR studies employ periods of ischemia lasting 45 to Ischemia–Reperfusion 120 minutes and examine retinal function and histology after 7 to 10 days of reperfusion (for a review, see Ref. 30). Electro- Deep anesthesia was induced in the rats with intramuscular injection retinogram (ERG) analysis reveals significant decreases in neu- of ketamine and xylazine (66.7 mg/kg and 6.7 mg/kg body weight, ronal function at 1 week after IR, with reduced a- and b-wave respectively). Ischemia was applied to the eye by increasing the in- amplitudes.31 IR also induces the loss of retinal neurons indi- traocular pressure and thus cutting off the blood supply from the cated by decreased thicknesses of retinal layers, including the retinal artery. Increased pressure was achieved by introduction of ganglion cell layer (GCL), inner nuclear layer (INL), and inner sterile saline through a 32-gauge needle that was inserted into the plexiform layer (IPL) as observed by histologic analysis.32,33 anterior chamber of the eye through the cornea. The needle was Terminal deoxynucleotidyl transferase-mediated dUTP nick- attached by Tygon tubing linked to a syringe pump (Braintree Scien- ␮ end labeling (TUNEL) of ischemic retinas demonstrates the tific, Braintree, MA), and the flow rate was set at 40 L/min. The retina presence of apoptotic neurons in all retinal layers. A recent was monitored for blanching, indicating loss of blood flow. Intraocular study also suggested that the IR model replicates vascular pressures were measured with a rebound microtonometer designed abnormalities observed in the diabetic retinopathy.34 Neuronal for use on rodent eyes (TonoLab; Icare, Helsinki, Finland). Published apoptosis and loss of retinal ganglion cells occurs within 2 days comparison of rat intraocular pressure measurements with the micro- of ischemia, whereas the loss of vascular cells occurs after 7 to tonometer and direct micromanometry suggested that the measure- 37 14 days.34 However, little is known regarding the impact of IR ments are approximately 5 mm Hg lower than actual pressures. The Ϯ Ϯ on vascular permeability. Ischemic preconditioning (IPC), in microtonometer showed pressures ranging from 9 1to11 1mm Ϯ Ϯ which a brief (8- to 10-minute) ischemic episode and recovery Hg in sham eyes and from 90 3to94 4 mm Hg in eyes undergoing period precede the IR procedure, effectively prevents subse- ischemia. Unless otherwise stated, the pressure was held for 45 min- quent neurodegeneration.35,36 However, although the effect of utes and then released, allowing the eye to reperfuse naturally for 48 IR on retinal neurodegeneration has been documented and the hours. Sham-treated eyes were treated by briefly inserting a 32-gauge effectiveness of IPC in reducing neural cell damage has been needle into the anterior chamber of the eye through the cornea. IPC shown, this model has seldom been used to address the rela- was accomplished by performing the IR procedure with a 10-minute tionship between neuronal cell death and vascular changes. period of ischemia of and then allowing natural reperfusion for 24 In this study, we compared the effects of IPC and intravit- hours before the 45-minute period of ischemia. real injection of bevacizumab or etanercept on retinal vascular permeability, neurodegeneration, and mRNA expression in rat Caspase-3 Activity retina after intraocular pressure–induced IR. The results dem- Caspase-3 activity was measured in retinal protein homogenates by onstrate that IPC, while effective at preventing IR-induced using a fluorometric assay system (CaspACE; Promega, Madison, WI). neurodegeneration, has little effect on vascular permeability. Immediately after euthanatization, the retina was excised, carefully Bevacizumab prevented IR-induced vascular permeability, cleaned of all vitreous, and bisected, and a half portion was placed in while having no effect on overall caspase activation or inter- 60 ␮L of cold lysis buffer (25 mM HEPES [pH 7.5], 5 mM MgCl ,5mM ␣ 2 nucleosomal DNA cleavage. In contrast, blocking TNF had EDTA, 5 mM DTT, 2 mM PMSF, 10 ␮g/mL leupeptin, and 1% NP40). little effect on either vascular permeability or neurodegenera- The retina was gently sonicated followed by a 30-minute incubation at tion. A set of 33 IR-responsive mRNAs were used to examine the 4°C and a 20-minute centrifugation at 16,000g at 4°C. Protein content effects of the treatments on the gene expression response to IR, in cleared lysate was measured by Bradford protein assay (Bio-Rad, revealing a unique subset of genes that correlated with the pre- Hercules, CA). Caspase-3 activities in the supernatants were measured vention of neurodegeneration but not with vascular permeability. in a 96-well plate format, according to the manufacturer’s protocol, This study demonstrates that neurodegeneration and vascular using 50 ␮g retinal protein and a 60-minute incubation at 37°C. dysfunction in response to retinal IR may be functionally separated and suggests that diseases that include an ischemic retinal response may require combination therapies protective of Apoptotic DNA Cleavage ELISA both vascular and neural function. Apoptotic DNA cleavage was assayed with an ELISA kit (Cell Death Detection; Roche Applied Science, Indianapolis, IN) and normalization METHODS to retinal wet weight. This assay measures cytoplasmic nucleosome- associated DNA fragments. Immediately after euthanatization, the ret- Animal Model ina was excised, carefully cleaned of all vitreous, and placed in a Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, mass-tared microtube containing chilled lysis buffer supplied in the kit MA) weighing 150 to 175 g (unless otherwise noted) were maintained (200 ␮L/retina), which was then weighed to obtain the retinal mass. in specific pathogen-free conditions, monitored by quarterly sentinel Retinal tissue was homogenized with a rotating pestle. The resulting testing, and were treated in accordance with the guidelines of the homogenate was vortexed and incubated for 30 minutes at room Institutional Animal Care and Use Committee of the Penn State Her- temperature with gentle rocking. After centrifugation for 10 minutes at shey College of Medicine and in compliance with the ARVO Statement 10,000g and 4°C, the supernatant was collected and placed on ice. For for the Use of Animals in Ophthalmic and Visual Research. each sample, duplicate 20-␮L aliquots of retinal supernatant, along with positive control (provided) and negative control (lysis buffer), Drugs were subjected to DNA-fragmentation ELISA according to the kit man- A pharmaceutical-grade formulation of humanized VEGF antibody, ufacturer’s instructions. After color reaction to detect captured DNA bevacizumab (25 mg/mL solution; Avastin; Genentech Inc., South San fragments, relative DNA fragmentation was expressed as optical den-

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sity (light absorbance at 405 nm with a 490-nm reference wavelength) Quantitative RT-PCR normalized by retinal mass in each aliquot of retinal supernatant. Quantitative PCR analysis was performed according to a published method39 (7900HT Sequence Detection System, 384-well optical Terminal Deoxynucleotidyl Transferase dUTP plates, with Assay-On-Demand gene-specific primers and probes, and Nick-End Labeling SDS 2.2.2 software; all from ABI). The 2Ϫ⌬⌬Ct analysis method41 was ␤ Eyes were excised, fixed for 20 minutes in 2% paraformaldehyde, and used to quantify relative amounts of mRNAs, with -actin as an endog- ␤ frozen in OCT embedding compound (Tissue Tek; Sakura Finetek USA, enous control. -Actin levels were determined to be unchanged in an Torrance, CA). Two 10-␮m sections were obtained through the plane absolute quantification experiment (data not shown). For a full listing of the optic nerve and were analyzed for DNA strand breaks by the of primer and probe sets see Table 1. TUNEL technique (ApopTag Apoptosis Detection Kit; Millipore, Bel- lerica, MA) and using 3-amino-9-ethylcarbazole (AEC; Sigma-Aldrich, St. Western Blot Analysis Louis, MO) to detect digoxigenin-labeled dNTPs incorporated in the The rat retinas were removed and the vitreous separated before snap DNA strands. Images of noncounterstained tissue were obtained with freezing in liquid nitrogen. Western blot analysis for VEGF was per- differential interference contrast (DIC) microscopy (BH-2 microscope formed as described elsewhere,42 with 100 ␮g protein per sample with 20ϫ objective; Olympus America, Inc. Lake Success, NY). separated on 4% to 12% Bis-Tris gels (NuPAGE; Invitrogen, Carlsbad, CA) and a mouse monoclonal anti-VEGF antibody (clone C-1, sc-7269, Retinal Permeability 1:500; Santa Cruz Biotechnology, Santa Cruz, CA). To control for protein loading, the membranes were probed with a mouse monoclo- Blood–retinal barrier leakage in male Sprague-Dawley rats (200–250 g) nal anti-actin antibody (clone C4, Mab1501, 1:5000; Millipore). After 38 was measured according to the method of Xu et al., with 2 hours of incubation with anti-mouse secondary antibody linked to horseradish circulation time and blood drawn from the inferior vena cava at the peroxidase (GE Health Care, Piscataway, NJ), the signal was detected end of the circulation to obtain the concentration of Evans blue dye in by a Western blot analysis detection reagent (Amersham ECL; GE the plasma. Retinal leakage was calculated on the basis of the accumu- Health Care, Piscataway, NJ), read by a bioimaging system (Geneg- lation of dye in the retinal tissue and the plasma dye concentration and nome; Syngene, Frederick, MD), and quantified (ImageQuant 5.0 soft- was expressed as microliters of plasma equivalent per gram dry retina ware; Molecular Dynamics, Sunnyvale, CA). weight per hour of circulation. Statistical Analysis RNA Isolation A mixed-effects, two-way analysis of variance (ANOVA) model that Total RNA was isolated as described previously39 (Tri-Reagent/BCP; varied both treatment and ischemia status between animals was fit for Molecular Research Center, Cincinnati, OH), followed by a purification the data from all experiments. The fixed effects were the treatment column procedure (RNeasy; Qiagen, Valencia, CA), to further purify status (treatment versus control) of the animal and the ischemia status and remove contaminating organics. Quality and quantity were as- (IR versus sham) of the eye. Individual animal differences were mod- sessed on a bioanalyzer (RNA 6000 Nano LabChip with a 2100 Expert eled by using random effects, which accounted for the positive corre- Bioanalyzer; Agilent, Palo Alto, CA). Only RNA samples with an RNA lation between the eyes of the same animal. The ANOVA model was integrity number (RIN) greater than 8 were used for further analyses completely specified by including model parameters for all main ef- (based on the Agilent RIN software algorithm assigninga1to10 fects and their interaction. Tests for differences between groups (e.g., integrity scale, with 10 being totally intact and 1 being totally de- treatment-IR versus control-IR) were based on t-statistics from the graded). ANOVA model. For the experiments examining mRNA at 4 and 48 hours of reperfusion, a mixed-effects, one-way ANOVA model was fit Microarray Analysis separately for each time point, with ischemia status used as the only fixed effect and a random effect used for individual animal differences. Microarray analysis was performed with microarrays (RatRef12; Illu- Similarly, a t-statistic from the one-way ANOVA model was used to test mina, San Diego, CA) in the Penn State College of Medicine Functional the difference between the sham and ischemia groups. The outcomes Genomics Core Facility, according to standard procedures. These ar- modeled in this framework were permeability, caspase activity, rays contained 22,519 probes corresponding to rat mRNA sequences in DNA fragmentation, and mRNA expression. The mRNA data points the NCBI RefSeq database (provided in the public domain by the were normalized to a reference group (control-sham group) and National Center for Biotechnology Information, Bethesda MD, available were log transformed before statistical analysis (SAS ver. 9.1 soft- at www.ncbi.nlm.nih.gov/locuslink/refseq/). Briefly, 250 ng of RNA ware; SAS, Cary, NC). was transcribed to cDNA according to the manufacturer’s instructions (Ambion-Applied Biosystems, Inc. [ABI], Foster City, CA). After second- strand synthesis and purification, biotin-labeled cRNA was generated RESULTS by in vitro transcription. cRNA from each sample was fragmented, IR-Induced Retinal Apoptosis denatured, and hybridized to microarray slides. The slides were incu- and Vascular Permeability bated with Cy3-labeled streptavidin and washed. Microarrays were scanned (BeadStation scanner; Illumina), and images were imported The neurodegenerative and vascular effects of IR were exam- into the allied software (BeadStudio; Illumina). Initial quality control ined by inducing a relatively mild ischemic insult. The rat for sample preparation, labeling, and hybridization was performed retinas were subjected to a 45-minute period of complete (GenomeStudio software; Illumina) with quality control probes built ischemia induced with elevated intraocular fluid pressure by into the microarray design for positive and negative controls. The data pumping sterile saline into the anterior chamber of the eye were then exported (GeneSpring GX; Agilent Technologies) for sub- followed by natural reperfusion of blood for periods of 4 or 48 sequent statistical analyses. The genes were filtered by expression hours. The effects of IR on retinal cell death were determined level, as described elsewhere,39 to eliminate genes that were below by measuring caspase-3/7 activity (Fig. 1A) and nucleosomal detection or were not expressed in the tissue. Differential expression DNA fragmentation (Fig. 1B). Caspase activity was not signifi- was determined with a combination of P-value (P Ͻ 0.05, two-tailed cantly changed at 4 hours, but was significantly increased t-test) and magnitude change (Ն1.4-fold increase and Յ0.7-fold de- (24%) at 48 hours. Nucleosomal DNA fragmentation was sig- crease), as published39 and in accordance with standards of data nificantly increased (5.8- and 4.1-fold) at 4 and 48 hours of analysis.40 reperfusion, respectively. Vascular leakage of plasma, as mea-

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TABLE 1. IR-Responsive Transcripts Validated by qRT-PCR

Symbol Name Alias AOD No. RefSeq ID

Bbs2 Bardet-Biedl syndrome 2 Rn00586096_m1 NM_053618.1 C1S Complement component 1, s subcomponent r-gsp Rn00594278_m1 NM_138900.1 Carhsp1 Calcium regulated heat stable protein 1 Crhsp24 Rn00596083_m1 NM_152790.2 Cntf Ciliary neurotrophic factor Rn00755092_m1 NM_013166.1 Cx3cl1 Chemokine (C-X3-C motif) ligand 1 Scyd1, Cx3c Rn00593186_m1 NM_134455.1 Dcamkl1 Doublecortin-like kinase 1 DCLK1, Ania4, Cpg16 Rn00584294_m1 NM_053343.2 Ddit3 DNA-damage inducible transcript 3 Chop10, Gadd153, MGC124604 Rn00492098_g1 NM_024134.2 Edn2 Endothelin 2 ET-2 Rn00561135_m1 NM_012549.1 Elovl4 Elongation of very long chain fatty acids-like 4 FEN1/Elo2, SUR4/Elo3 Rn01403757_m1 XM_236476.4 Gbp2 Guanylate binding protein 2 Rn00592467_m1 NM_133624.1 Gfap Glial ﬁbrillary acidic protein Rn00566603_m1 NM_017009.2 Hspb1 Heat shock protein 1 Hsp25, Hsp27 Rn00583001_g1 NM_031970.3 Igf2 Insulin-like growth factor 2 somatomedin A Rn00580426_m1 NM_031511.1 Jak3 Janus kinase 3 Rn00563431_m1 NM_012855.1 Kcne2 Potassium voltage-gated channel, Isk-related family, Mirp1 Rn02094913_s1 NM_133603.2 member 2 Lgals3 Lectin, galactoside-binding, soluble, 3 gal-3 Rn00582910_m1 NM_031832.1 Lgals3bp Lectin, galactoside-binding, soluble, 3 binding protein Ppicap Rn00478303_m1 NM_139096.1 Litaf Lipopolysaccharide-induced TNF factor EET-1, Pig7 Rn01424675_m1 NM_001105735.2 Mct1 Monocarboxylate transporter 1 Slc16a1 Rn00562332_m1 NM_012716.2 Nppa Natriuretic peptide precursor A ANF, ANP, Pnd, RATANF Rn00561661_m1 NM_012612.2

Pcgf1 Polycomb group ring ﬁnger 1 Nspc1 Rn01425394_g1 NM_001007000.1 5923 IR Retinal in Apoptosis and Permeability Prkcb1 protein kinase C, beta Rn00562312_m1 NM_012713.2 Serping1 (C1INH) Serine (or cysteine) peptidase inhibitor, clade G, member 1 C1Inh Rn01485600_m1 NM_199093.1 Slc6a11 (Gat3) Solute carrier family 6 (neurotransmitter transporter, Gabt4, Gat3 Rn00577664_m1 NM_024372.2 GABA), member 11 Spp1 Secreted phosphoprotein 1 Sialoprotein (osteopontin) Rn00681031_m1 NM_012881.2 Stat1 Signal transducer and activator of transcription 1 Rn00583505_m1 NM_032612.3 Stat3 Signal transducer and activator of transcription 3 Rn00562562_m1 NM_012747.2 Syn1 Synapsin I Rn00569468_m1 NM_019133.1 Syp Synaptophysin Syp1 Rn00561986_m1 NM_012664.1 Timp1 Tissue inhibitor of metallopeptidase 1 Timp Rn00587558_m1 NM_053819.1 Tnfrsf12a Tumor necrosis factor receptor superfamily, member 12a Fn14, MGC72653 Rn00710373_m1 NM_181086.2 Vamp2 Vesicle-associated membrane protein 2 Synaptobrevin 2, RATVAMPB, Rn00360268_g1 NM_012663.2 RATVAMPIR, SYB, Syb2 Vegfa Vascular endothelial growth factor A VEGF164 Rn00582935_m1 NM_031836.2

Gene names, aliases, and Applied Biosystems (Foster City, CA) qRT-PCR assay catalogue numbers (AOD No.). 5924 Abcouwer et al. IOVS, November 2010, Vol. 51, No. 11

INL at this time point. No TUNEL-positive cells were observed to be associated with the retinal vasculature. Given that VEGF is necessary for vascular permeability in numerous settings, including retinal diseases, we sought to determine whether IR induces VEGF protein expression. Rat retinas were subjected to 45 minutes of ischemia followed by 4 or 48 hours of reperfusion, at which time whole retinal lysates were obtained and subjected to immunoblot analysis, to compare VEGF protein contents (Fig. 3). A 19- to 20-kDa form of VEGF (presumably VEGF-165) and a band with electro- phoretic mobility corresponding to approximately 60 kDa (presumably a VEGF-165 dimer) were significantly increased by comparable extents in response to IR. Quantification of these two bands suggested that total VEGF-165 protein levels in retinas significantly increased after 4 (ϳ1.7-fold) and 48 (1.3- fold) hours of reperfusion. This result suggests that retinal permeability after IR is mediated by increased VEGF expression. Identification and Validation of mRNAs Responsive to IR Whole-genome microarrays were used to identify alterations in retinal mRNA levels caused by 45 minutes of ischemia followed by 48 hours of reperfusion (Fig. 4). Transcriptomic analysis was performed with RNA from six sham and six IR retinas. After the results were filtered to remove mRNAs that are not appreciably expressed in retina, mRNA corresponding to 7904 genes remained for statistical analysis. According to statistical analysis (P Ͻ 0.05, t-test) and magnitude change (Ն1.4-fold increase or Յ0.7-fold decrease), 1099 probes were identified as differentially expressed in the IR group relative to the sham group (848 decreased and 251 increased). The full set of microarray gene expression data has been deposited in the NIH/NLM Gene Expression Omnibus43 (GEO accession number GSE20521; http:// www.ncbi.nlm.nih.gov/projects/geo/ provided by the National Cen- ter for Biotechnology Information, National Institutes of Health,

FIGURE 1. Retinal IR caused neurodegeneration and vascular leakage. One eye of each animal was subjected to retinal ischemia for 45 minutes and reperfused for a speciﬁc period (4 or 48 hours) before being assayed for (A) caspase-3 activity, (B) DNA fragmentation, and (C) Evans blue dye leakage. The contralateral eye was subjected to needle puncture and served as the sham control. Results shown are the means and SE of means obtained from four to eight animals in each group. Comparisons between sham and IR eyes were calculated with the mixed-effects, two-way ANOVA model: **P Ͻ 0.01, and ***P Ͻ 0.001.

sured by Evans blue dye accumulation in the retina, was sig- FIGURE 2. Retinal IR caused DNA strand breaks in cells of the ONL of nificantly increased (4.9- and 4.6-fold) at 4 and 48 hours after retinas 2 days after ischemia reperfusion. Representative differential ischemia, respectively (Fig. 1C). Thus, biochemical measures interference contrast (DIC) microscopy images of TUNEL-stained sham confirmed that significant apoptosis was caused by 45 minutes (A) and IR (B) retinas. The IR eye was subjected to retinal ischemia for of ischemia. Furthermore, IR caused a rapid and sustained 45 minutes and reperfused for 48 hours before being sectioned and increase in vascular permeability. TUNEL of retinal sections mounted for TUNEL after 48 hours of reperfusion. The contralateral was used to identify the location of apoptotic cells in IR retinas eye was subjected to needle puncture and served as the sham control. Images are oriented with the inner limiting membrane toward the top. after 48 hours of reperfusion. Whereas sham retinas exhibited TUNEL-positive nuclei were detected in groups in the ONL (arrows) virtually no TUNEL-positive cells in any layer, numerous posi- and occasionally in the INL (arrowhead) of the IR retina. Retinal layers tive cells were identified in clusters located predominantly were easily discernable and are labeled as: IPL, inner plexiform layer; within the outer nuclear layer (ONL) of the IR retinas (Fig. 2). INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nu- Isolated TUNEL-positive cells were also observed within the clear layer; OS, photoreceptor outer segments.

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genes (Hsp1, Lgals3, Mct1, Syn, and Tnfrsf12a) were significantly altered by IR after 4 hours of reperfusion. In a second experiment, nine genes were signiﬁcantly altered by IR at this time point (Supplementary Data). It should be noted that VegfA mRNA exhibited a consistent decrease in abundance in response to IR (Tables 2, 3; Supplementary Data), suggesting that the observed increase in VEGF protein content is due to posttranscriptional regulation.

Effect of Ischemic Preconditioning on Neurodegeneration and Permeability The protective effects of IPC were examined by applying a 10-minute ischemic insult followed by 24 hours of reperfusion before IR. IPC diminished caspase activation from a 27% increase to a 20% increase 48 hours after the 45-minute ischemic event (Fig. 5A). However, the difference between mean caspase activity in retinas subjected to IR with and without preconditioning did not reach statistical significance. In contrast IPC, significantly reduced the increase in DNA fragmentation after IR from 3.0- to 1.7-fold, (Fig. 5B). The difference FIGURE 3. Retinal IR caused increased expression of VEGFA protein. between mean DNA fragmentation measures for IR retinas One eye of each animal was subjected to retinal ischemia for 45 with and without IPC was highly significant. To confirm the minutes and reperfused for a specific period (4 or 48 hours) before protective effects on DNA fragmentation, the experiment was being assayed for VEGF protein content by Western blot analysis, repeated with a 30-minute ischemic insult. In this case, IPC ␤ normalized to the -actin protein content. The contralateral eye was completely abrogated the effects of IR on DNA fragmentation subjected to needle puncture and served as the sham control. The (Fig. 5C). IPC diminished the increase in vascular leakage results are the means and SE of means obtained from eight animals in each group. Comparisons between sham and IR eyes were calculated caused by 45 minutes of ischemia from 4.9-fold to 1.9-fold (Fig. by using the mixed-effects, two-way ANOVA model: *P Ͻ 0.05, and 5D). However, this reduction was mainly due to the increased ***P Ͻ 0.001. basal permeability caused by IPC itself, and there was no significant difference in Evans blue dye leakage into IR retinas, with and without IPC. Thus, IPC itself caused some permeabil- Bethesda, MD). The mRNA alterations were examined by using ity, but did not prevent a further increase in permeability in pathway-analysis software (Ingenuity Systems, Redwood CA) response to IR. When the analysis was repeated with a 30- to detect relationships to existing data on disease states, cellu- minute ischemic insult, IPC again increased basal permeability, lar functions, and canonical pathways (see Supplementary but had no significant effect on dye accumulation in IR retinas Data, http://www.iovs.org/cgi/content/full/51/11/5920/DC1). (Fig. 5E). In fact, the mean leakage in IPC-IR retinas was greater An overrepresentation of genes associated with endocrine sys- than that in control-IR retinas. tem disorders (69 genes), metabolic disease (77 genes), and neurologic disease (195 genes) was observed (P Ͻ 0.001, Bevacizumab’s Effect on Permeability Fisher exact test). The three most common cellular functions and Apoptosis of the differentially expressed genes were cell death (207 genes), cell–cell signaling (74 genes), and cellular movement To determine whether VEGF function plays a neuroprotective (106 genes). Phototransduction (14 genes) and complement role and/or contribute to vascular leakage after IR, bevaci- pathways (9 genes) were the two most highly regulated canon- zumab (Avastin; Genentech), was intravitreally injected 48 ical pathways in the database. hours before IR. This treatment had virtually no effect on To validate the microarray findings and examine the tem- caspase activation after 45 minutes of ischemia and 48 hours of poral course of gene expression changes, we performed qRT- reperfusion (Fig. 6A). Likewise, bevacizumab had no signifi- PCR confirmation on retinal RNA samples from sham and IR cant effect on internucleosomal DNA fragmentation after IR experiments, using samples from both 4 and 48 hours of (Fig. 6B). It should be noted that the treatment had no effect, reperfusion. Thirty-three mRNAs (Table 1) were confirmed to ex- positive or negative, on basal or IR-induced retinal cell death. hibit IR-responsive expression by qRT-PCR analysis (Table 2). These In contrast, vascular leakage was significantly decreased by 33 mRNAs included 17 mRNAs increased with IR treatment bevacizumab from a 3.1-fold increase with IR in vehicle-treated (C1S, Carhsp1, Cntf, Edn2, Gbp2, Gfap, Hspb1, Jak3, Lgals3, eyes to 1.9-fold increase with IR in bevacizumab-treated eyes Lgals3bp, Litaf, Serping1 [C1INH], Spp1, Stat1, Stat3, Timp1, (Fig. 6C). and Tnfrsf12a), and 16 mRNAs decreased with IR treatment (Bbs2, Cx3cl1, Dcamkl1, Ddit3, Elovl4, Igf2, Kcne2, Mct1, Etanercept’s Effect on Neurodegeneration Nppa, Pcgf1, Prkcb1, Slac6a11 [Gat3], Syn, Syp, Vamp2, and and Vascular Permeability after IR VegfA). The confirmation by qRT-PCR was performed in the same set of samples used in the microarray analysis and in Similarly, intravitreal injection of etanercept was used to de- samples from additional independent IR experiments employ- termine whether TNF␣ function contributes to the retinal ing 4- and 48-hour reperfusion times. All 33 mRNAs examined response to IR. Etanercept had no significant effects on caspase demonstrated statistically significant changes in retinal con- induction, DNA fragmentation, or vascular permeability (Fig. 7). The tent in at least three of four of the independent experiments increase in retinal permeability caused by IR was slightly with 48 hours of reperfusion (Tables 2, 3; Supplementary greater in etanercept-injected eyes (9.2-fold versus 6.3-fold in Data). These genes exhibited changes that were lesser in vehicle-treated eyes). However, the difference between mean magnitude and less consistent after 4 hours of reperfusion. vascular leakage in vehicle-treated IR retinas and etanercept- For example, in the experiment shown in Table 2, only five treated IR retinas was not significant.

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FIGURE 4. Retinal IR caused numerous alterations of mRNA expression levels. Retinal mRNA expression was analyzed by microarray profiling for differences between sham control and IR retinas (45-minute ischemia followed by 48-hour reperfusion). Results were filtered for genes detected as present, and differential expression was determined as statistically significant (t-test P Ͻ 0.05) with an increase of 1.4-fold or greater or a decrease to 0.7-fold or less. All the 1099 differentially expressed genes are plotted for each of the six animals per group. Each abundance value is scaled to provide a mean sham control value of unity.

Effects of IPC and Bevacizumab on the and bevacizumab significantly affected only the Evan’s blue Expression of IR-Responsive mRNAs leakage, the 33 IR-responsive mRNA set developed seems indicative of the neurodegenerative response and not the vascu- The effects of IPC and bevacizumab on mRNA expression after lar permeability response to IR. IR were examined, to identify transcriptomic responses to IR that may be indicative of neurodegeneration or vascular function (Table 3; Supplementary Data). IPC, which had a marked DISCUSSION effect on neurodegeneration after IR, significantly altered the responses of 15 of the 33 IR-responsive mRNAs (Table 3, TIR In the present study, the retina responded to a relatively brief versus CIR columns). These included the genes Bbs2, Cntf, ischemic event with both neurodegeneration and vascular per- Cx3cl1, Elovl4, Igf2, Lgals3, Pcgf1, Prkcb1, Slc6a11, Spp1, meability. Further, these physiologic outcomes could be Syn, Syp, Tnfrsf12a, Vamp2, and VegfA. The mRNA expres- largely separated according to specific pretreatment. The ef- sion of 11 of these was decreased by IR, with IPC resulting in fects of IR on retinal vascular permeability have been examined a smaller decrease in their expression in response to IR. It is in only one other study. Using magnetic resonance imaging, also noteworthy that IPC alone had no significant effects on the Wilson et al.44 demonstrated that IR produced vascular leakage basal expression of the 33 IR-responsive mRNAs in sham- in rabbit retinas that slowly declined between 1 and 8 days of treated retinas at 48 hours after the ischemia procedure, which reperfusion. To our knowledge, no study has been undertaken corresponds to 72 hours after IPC (Table 3, TS versus CS to examine the effects of preconditioning on retinal vascular columns). Bevacizumab, which inhibited the vascular perme- permeability in IR. In the present study, IPC was effective in ability response without affecting neurodegeneration, did not preventing neurodegeneration, but had little effect on perme- significantly change the effect of IR on expression of any of the ability after IR. Conversely, blocking VEGF function with bev- 33 mRNAs. Although the magnitude of IR effects on Cntf, Igf2, acizumab inhibited the vascular response without affecting Lgal3, and Spp1 mRNAs were altered by bevacizumab, none of measures of apoptosis. Thus, intraocular pressure–induced these changes were statistically significant (Table 3, TIR versus IR can serve as a convenient in vivo model of VEGF-mediated CIR columns). Bevacizumab treatment alone significantly al- retinal vascular permeability. Of importance, these results tered the basal expression of 3 of the 33 IR-responsive mRNAs, demonstrate that the neurodegenerative and vascular re- including Igf2, Kcne2, and Slc6a11 (Table 3, TS versus CS sponses to IR are not functionally linked. In further support column). Thus, IPC had a relatively pronounced effect on the of this conclusion is the observation that IPC and bevaci- expression of these mRNAs in response to IR without signifi- zumab treatment had very different effects on the gene cantly affecting the basal mRNA levels, whereas bevacizumab expression responses to IR. affected the basal expression of a subset of these mRNAs Ocular pressure–induced retinal IR injury can serve as a without significantly interfering with the mRNA responses to useful model of the damage and responses that occur on a IR. Given that IPC significantly diminished apoptosis after IR smaller scale during focal ischemic events caused by vessel

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TABLE 2. Quantitative RT-PCR Validation of IR-Responsive mRNA Expression Changes at 4 and 48 Hours of Reperfusion

4 h Reperfusion 48 h Reperfusion

Gene Sham 4 h (S4) Ischemia 4 h (IR4) IR4 vs. S4 Sham 48 h (S48) Ischemia 48 h (IR48) IR48 vs. S48

Bbs2 1.00 Ϯ 0.05 0.81 Ϯ 0.03 7 1.00 Ϯ 0.08 0.50 Ϯ 0.04 2** C1S 1.00 Ϯ 0.30 0.88 Ϯ 0.14 7 1.00 Ϯ 0.17 1.91 Ϯ 0.19 1* Carhsp1 1.00 Ϯ 0.16 1.49 Ϯ 0.20 1 1.00 Ϯ 0.09 2.94 Ϯ 0.23 11*** Cntf 1.00 Ϯ 0.09 1.15 Ϯ 0.05 7 1.00 Ϯ 0.19 2.75 Ϯ 0.10 11** Cx3cl1 1.00 Ϯ 0.17 0.75 Ϯ 0.07 7 1.00 Ϯ 0.11 0.45 Ϯ 0.04 22** Dcamkl1 1.00 Ϯ 0.08 0.90 Ϯ 0.08 7 1.00 Ϯ 0.16 0.58 Ϯ 0.03 2* Ddit3 1.00 Ϯ 0.27 0.52 Ϯ 0.04 2 1.00 Ϯ 0.15 0.12 Ϯ 0.02 2222*** Edn2 1.00 Ϯ 0.21 1.47 Ϯ 0.31 1 1.00 Ϯ 0.64 3.50 Ϯ 0.67 11* Elovl4 1.00 Ϯ 0.11 0.88 Ϯ 0.09 7 1.00 Ϯ 0.10 0.39 Ϯ 0.03 22*** Gbp2 1.00 Ϯ 0.56 1.50 Ϯ 0.40 1 1.00 Ϯ 0.14 2.48 Ϯ 0.16 11** Gfap 1.00 Ϯ 0.21 0.71 Ϯ 0.09 7 1.00 Ϯ 0.21 5.49 Ϯ 0.34 111** Hspb1 1.00 Ϯ 0.25 2.21 Ϯ 0.31 11* 1.00 Ϯ 0.21 3.42 Ϯ 0.98 11* Igf2 1.00 Ϯ 0.07 0.89 Ϯ 0.03 7 1.00 Ϯ 0.08 0.26 Ϯ 0.06 22*** Jak3 1.00 Ϯ 0.13 1.12 Ϯ 0.17 7 1.00 Ϯ 0.17 3.32 Ϯ 0.22 11** Kcne2 1.00 Ϯ 0.18 1.06 Ϯ 0.15 7 1.00 Ϯ 0.27 0.71 Ϯ 0.12 7 Lgals3 1.00 Ϯ 0.17 2.91 Ϯ 0.56 11** 1.00 Ϯ 0.41 12.79 Ϯ 2.20 1111** Lgals3bp 1.00 Ϯ 0.08 0.64 Ϯ 0.09 2 1.00 Ϯ 0.16 2.14 Ϯ 0.27 11** Litaf 1.00 Ϯ 0.13 1.32 Ϯ 0.26 7 1.00 Ϯ 0.12 3.14 Ϯ 0.26 11*** Mct1 1.00 Ϯ 0.15 0.74 Ϯ 0.04 7** 1.00 Ϯ 0.11 0.49 Ϯ 0.06 22** Nppa 1.00 Ϯ 0.06 0.69 Ϯ 0.04 2 1.00 Ϯ 0.31 0.28 Ϯ 0.03 22* Pcgf1 1.00 Ϯ 0.17 0.83 Ϯ 0.02 7 1.00 Ϯ 0.17 0.51 Ϯ 0.06 2* Prkcb1 1.00 Ϯ 0.30 0.94 Ϯ 0.36 7 1.00 Ϯ 0.28 0.48 Ϯ 0.16 22* Serping1 1.00 Ϯ 0.22 0.63 Ϯ 0.08 2 1.00 Ϯ 0.21 1.86 Ϯ 0.19 1* Slc6a11 1.00 Ϯ 0.14 0.65 Ϯ 0.064 2 1.00 Ϯ 0.22 0.53 Ϯ 0.08 2 Spp1 1.00 Ϯ 0.26 1.09 Ϯ 0.24 7 1.00 Ϯ 0.28 3.02 Ϯ 0.78 11* Stat1 1.00 Ϯ 0.11 0.82 Ϯ 0.12 7 1.00 Ϯ 0.11 1.63 Ϯ 0.19 1* Stat3 1.00 Ϯ 0.08 1.18 Ϯ 0.08 7 1.00 Ϯ 0.20 1.73 Ϯ 0.19 1* Syn 1.00 Ϯ 0.10 0.76 Ϯ 0.05 7* 1.00 Ϯ 0.25 0.39 Ϯ 0.03 22* Syp 1.00 Ϯ 0.11 0.79 Ϯ 0.05 7 1.00 Ϯ 0.06 0.61 Ϯ 0.05 2** Timp1 1.00 Ϯ 0.55 1.25 Ϯ 0.08 7 1.00 Ϯ 0.24 7.82 Ϯ 1.14 111** Tnfrsf12a 1.00 Ϯ 0.44 2.79 Ϯ 0.32 11* 1.00 Ϯ 0.13 5.54 Ϯ 0.80 111** Vamp2 1.00 Ϯ 0.12 0.80 Ϯ 0.04 7 1.00 Ϯ 0.21 0.36 Ϯ 0.03 22** VegfA 1.00 Ϯ 0.16 0.84 Ϯ 0.06 7 1.00 Ϯ 0.10 0.44 Ϯ 0.05 22**

Mean retinal mRNA levels were compared between IR and sham (S) retinas after 4 or 48 hours of reperfusion. Numerical data are expressed as the mean Ϯ SEM. Arrows indicate the direction and magnitude of changes in mRNA level: 7, 0.7 Ͻ IR/S Ͻ 1.4; 1, 1.4 Յ IR/S Ͻ 2.0; 11, 2.0 Յ IR/S Ͻ 4.0; 111, 4.0 Յ IR/S Ͻ 8.0; 1111, 8.0 Յ IR/S Ͻ 16.0; 2, 0.7 Ն IR/S Ͼ 0.5; 22, 0.5 Ն IR/S Ͼ 0.25; 222, 0.25 Ն IR/S Ͼ 0.125; and 2222, 0.125 Ն IR/S Ͼ 0.0625. Statistical comparisons between IR and S eyes were calculated by using mixed-effects, one-way ANOVA model: *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001. Additional data are presented in the Supplementary Data.

drop out or vessel occlusion during diseases such as diabetic Singh et al.45 suggested that this is indeed the case. They retinopathy. The model has been widely used for the study of observed that active caspase-3 staining became evident in the retinal neurodegeneration and the study of IPC.30 Some inves- INL and was extensive in the ONL after IR, whereas early tigators have used ERG and the examination of retinal layer ganglion cell death was associated with active caspase-2, but thinning as endpoints of neuronal damage.31–33 In the present not caspase-3, staining. study, we used caspase-3 activity and DNA fragmentation as A set of 33 mRNAs were identified and validated as being indicators of cell death. Although both increased after IR, these significantly and reproducibly altered by IR. The mRNAs were measures did not correlate perfectly. For example, whereas not chosen as representatives of specific biological processes, DNA fragmentation was similarly increased after both 4 and 48 but rather as being significantly and reproducibly altered by IR. hours of reperfusion, a significant increase in caspase-3 activity Kamphuis et al.46 also used whole genome arrays to examine was not observed at the earlier time point. We observed a very the genetic response to IR in rat retinas, as well as the effects significant increase in caspase-3 activity at 24 hours (data not of IPC on these responses. Their analysis identified several of shown), but the maximum increase in activity seemed to occur the same IR-responsive genes described herein, including C1s, at 48 hours. Thus, although both assays are interpreted as Carhsp1, Dcamkl1, Edn2, Gfap, Gbp2, Hspb1, Lgals3, Litaf, indicators of cell death, the temporal nature or sensitivities of Nppa, Serping1, Spp1, Stat1, Stat3, and VegfA. Hspb1 was also these two endpoints differ. identified as being responsive to IPC in the rat brain.47 In The cell death observed 2 days after IR where largely re- several additional studies Gfap was identified as being upregu- stricted to the ONL. Whereas IR causes the eventual thinning lated during retinal IR48,49 as well as cerebral IR.50 In the of the IPL, TUNEL staining indicated that death occurred retina, Gfap expression is indicative of Mu¨ller cell astroglio- mainly in the ONL at 48 hours. Nishijima et al.25 found that sis.51 death occurs in the GCL and INL soon after IR and then Several previous studies of gene expression changes after IR migrates to the ONL (with a virtually identical staining pattern have focused on inflammatory cytokines.52–54 Approximately as shown in our Fig. 2). It is therefore possible that the death one fourth of the mRNAs identified in the present analysis are of neurons in the inner layers is characterized by DNA frag- closely associated with inflammation, including complement mentation but not caspase-3 activation, whereas appreciable component C1s, the chemokine Cx3cl1 (fractalkine), Lgals3 caspase-3 activity coincides with the death of photoreceptors. (galectin-3), its associated binding protein Lgal3bp, Litaf, and

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TABLE 3. Effect of IPC and Bevacizumab on IR-Responsive mRNA Expression

IPC Bevacizumab

Gene CIR vs. CS TS vs. CS TIR vs. TS TIR vs. CIR CIR vs. CS TS vs. CS TIR vs. TS TIR vs. CIR

Bbs2 2*** 77*** 7** 7*** 77 7 C1S 11*** 711*** 711*** 71* 7 Carhsp1 11*** 711*** 711** 71* 7 Cntf 11*** 711*** 7* 11*** 71 2 Cx3cl1 22*** 72*** 1** 2*** 77* 7 Dcamkl1 2*** 72*** 77* 77 7 Ddit3 22*** 72*** 77** 77 7 Edn2 11*** 711* 7 111*** 11 11* 7 Elovl4 2*** 72** 7* 2** 77 7 Gbp2 11*** 711*** 711** 71* 7 Gfap 111*** 7 111*** 7 111** 7117 Hspb1 11*** 71* 71* 71 7 Igf2 22*** 72*** 11** 22** 2* 71 Jak3 11** 711** 711*** 711** 7 Kcne2 22*** 72*** 72** 2* 77 Lgals3 1111*** 2 111*** 2* 1111** 11 7 22 Lgals3bp 11*** 711*** 71** 71** 7 Litaf 11*** 711** 711*** 711*** 7 Mct1 22*** 72*** 72** 77 7 Nppa 2*** 77*** 72** 77 7 Pcgf1 2*** 72*** 7** 2*** 72** 7 Prkcb1 22*** 72*** 1** 2** 77 7 Serping1 11** 711*** 71** 77 7 Slc6a11 22*** 72*** 1** 2* 2* 27 Spp1 11*** 71 2* 1* 77 2 Stat1 1* 71** 71** 77 7 Stat3 1** 71** 71** 77 7 Syn 2*** 72*** 7* 2*** 72 7 Syp 2*** 77*** 7** 2* 77 7 Timp1 11*** 211*** 71** 211* 7 Tnfrsf12a 11*** 711*** 7* 11*** 711* 7 Vamp2 2*** 72*** 7** 2** 72** 7 VegfA 22*** 72*** 1** 2* 27 7

Mean retinal mRNA levels were compared between groups including control (not pretreated) IR with control sham (CIR vs. CS), treated sham with control sham (TS vs. CS), treated IR with treated sham (TIR vs. TS), and treated IR with control IR (TIR vs. CIR). Arrows indicate the direction and magnitude of the respective ratios of mean values for mRNA levels, and asterisks indicate the signiﬁcance of differences between the mean values in the groups: 7, 0.7 Ͻ ratio Ͻ 1.4; 1, 1.4 Յ ratio Ͻ 2.0; 11, 2.0 Յ ratio Ͻ 4.0; 111, 4.0 Յ ratio Ͻ 8.0; 1111, 8.0 Յ ratio Ͻ 16.0; 2, 0.7 Ն ratio Ͼ 0.5; 22, 0.5 Ն ratio Ͼ 0.25; 222, 0.25 Ն ratio Ͼ 0.125; and 2222, 0.125 Ն ratio Ͼ 0.0625. Statistical comparisons between groups were calculated by using the mixed-effects, two-way ANOVA model: *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001. Numerical data are presented in the Supplementary Data.

members of the JAK/STAT pathway (Jak3, Stat1, and Stat3). In In the present study, TNF␣ mRNA was not identified as a recent proteomics study, the galectin-3 protein was identified being upregulated after IR, and we detected no significant as being upregulated in rat retinas at 2 days after a 2-hour difference in the TNF␣ protein level in whole retinal lysates at ischemic insult.55 Few cytokines traditionally associated with 4 or 24 hours of reperfusion after 45 minutes of ischemia (data inflammation were found in the array analysis or were included not shown). Using immunoblot analysis with antibody to hu- in the present set of 33 mRNAs altered by IR, and expression man IgG, we were able to detect etanercept in retinas at 4 and of Cx3cl1 was actually decreased at both 4 and 48 hours after 48 hours after intravitreal injection (data not shown). The lack ischemia. Because fractalkine is expressed by neurons, the of effect of etanercept in the present study could be due to the reduction in Cx3cl1 may be an indication of neurodegenera- lesser severity of the ischemic insult used, or it may indicate tion rather than an inflammatory condition. In contrast, Zheng that TNF␣ has no direct role in neurodegeneration or vascular et al. 34 demonstrated increased expression of several inflam- permeability during the first 48 hours of reperfusion after matory markers, including TNF␣ mRNA, after 2 and 7 days of ischemia. Three studies have shown that TNF␣ expression is reperfusion in the rat retina. Using GFAP-promoter expression increased after more extended retinal ischemia.34,56,57 Vinores of a dominant-negative I␬B mutant transgene, Dvoriantchikova et al.58 used knockout mice to show that TNF␣ is essential for et al.53 demonstrated that upregulation of TNF␣, as well as that leukostasis during oxygen-induced retinopathy and after intra- of many other inflammatory genes during IR, is dependent on vitreal injection of VEGF, IL-1␤, and platelet-activating factor. NF-␬B activity in glial cells. Furthermore, histologic observa- However, they also found that TNF␣ was not necessary for tion 1 week after ischemia showed that blocking inflammatory vascular permeability in response to these factors. The role of gene expression is highly neuroprotective. However, it should TNF␣ in retinal neurodegeneration may be complex. Berger et be noted that both these groups used periods of ischemia al.57 demonstrated a causal role for TNF␣ in IR injury in mice longer than we used. Furthermore, we did not specifically when they found that TNF receptor knockout alleviated the examine the expression of inflammatory markers and therefore detrimental effects of IR on ERG amplitudes. In contrast, Fon- cannot speculate on their role in neurodegeneration or vascu- taine et al.59 found that TNF␣-knockout mice demonstrated no lar permeability after a relatively mild ischemic insult. significant histologic differences after IR. An interesting finding

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FIGURE 5. IPC diminished neurodegeneration but not vascular leakage in response to IR. For preconditioning, both eyes were subjected to 10 minutes of retinal ischemia. After 24 hours of ischemia, one eye of each animal was subjected to retinal ischemia for 30 or 45 minutes and reperfused for 48 hours before being assayed for (A) caspase-3 activity with 45 minutes ischemia, (B) DNA fragmentation with 45- minute ischemia, (C) DNA fragmentation with 30-minute ischemia, (D) Evans blue dye leakage with 45-minute ischemia, and (E) Evans blue dye leakage with 30-minute ischemia. The contralateral eye was subjected to needle puncture and served as sham control. Results shown are the means and SE of means obtained from eight animals in each group. Comparisons between sham and IR eyes and between control-IR and IPC-IR eyes were calculated using the mixed-effects, two-way ANOVA model: *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001.

in studies of mice deficient in TNFR1 and TNFR2 established mately twofold in guinea pig retinas 7 days after 90 minutes that TNFR1 plays a neuroprotective role, whereas TNFR2 pro- of ischemia,56 and brain VEGF mRNA and protein expres- motes neurodegeneration after IR. Berger et al.57 also found sion are increased after cerebral IR in rats.61,62 However, that intravitreal injection of recombinant TNF␣ protein exac- changes in VEGF may be restricted to focal regions. Using erbated the detrimental effects of IR on ERG amplitudes in rats, immunohistochemical analysis in a rat intraocular pressure– whereas intravitreal injection of neutralizing antibody to TNF␣ induced IR model, Ogata et al.63 showed increased VEGF alleviated this response. They also demonstrated that intravit- antibody binding on vascular cells, despite a lack of overall real injection of a TNF-blocking antibody significantly de- increase in the entire retina. During sustained cerebral isch- creased the number of TUNEL-positive cells in the retina at 24 emia, VEGF protein was confined to microglial cells in the hours after a 45-minute ischemic insult. Thus, although other penumbra of the infarct region.64 Thus, although global studies have identified a role for TNF␣ in IR response, in the VEGF transcription may decrease during IR, focal expression present study no change in TNF␣ expression or effect of TNF␣ of VEGF protein may still contribute an important role in the inhibitor was observed. The reasons for these discrepancies are retinal response. Alternatively, VEGF can be regulated at the not presently known. translational level under ischemic conditions due to internal IR caused significant increases in VEGF protein in whole ribosome entry sites in the mRNA65 and the change in VEGF retinal lysates. Surprisingly, VegfA mRNA was identified as protein observed herein may result from such posttranscrip- an IR-responsive mRNA with decreased expression at 4 and tional regulation. The present demonstration that bevaci- 48 hours of reperfusion. Although VEGF expression is zumab significantly blocked the increase in vascular perme- known to increase in animal models of ischemic retinopa- ability after IR supports a role for VEGF protein in this thy, such as oxygen-induced retinopathy60 and diabetic ret- process. The results also suggest that the IR model may inopathy,4 the expression of VEGF during IR is not well serve as a useful tool for studying VEGF-dependent retinal characterized. VEGF protein levels are increased approxi- edema.

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They also found that intravitreal injection of recombinant VEGF protein or a VEGF receptor agonist prevented neuronal death after IR. Furthermore, the neuroprotective effects of IPC have been attributed to VEGF expression. Blocking VEGF with a soluble VEGF receptor or VEGF-neutralizing antibodies diminishes the protective effects of IPC in this same model.25 Like- wise, adenoassociated virus–mediated expression of VEGF prevents neuronal damage in cerebral IR,68 and antisense targeting of VEGF or VEGF receptor diminishes the protection of IPC in a cerebral IR model.28 The present results show that inhibition

FIGURE 6. Intravitreal bevacizumab injection inhibited vascular leakage but not overall apoptosis in response to IR. Both eyes of each animal in the treatment group were injected with 50 mg bevacizumab, and the eyes of the control animals were injected with the same volume (2 ␮L) of saline. After 48 hours of ischemia, one eye of each animal was subjected to retinal ischemia for 45 minutes and reperfused for 48 hours before assay to determine (A) caspase-3 activity, (B) DNA fragmentation, and (C) Evans blue dye leakage. The contralateral eye was subjected to needle puncture and served as the sham control. Results shown are the means and SE of means obtained from eight animals in each group. Comparisons between sham and IR eyes and between control-IR and treatment-IR eyes were calculated using the mixed effects ANOVA model: *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001. FIGURE 7. Intravitreal etanercept injection had no effects on neurodegeneration and vascular leakage in response to IR. Both eyes of each Surprisingly little is known regarding the role of VEGF in the animal in the treatment group were injected with 100 mg of etaner- physiological response to IR in the retina. Inhibiting VEGF cept, and the eyes of the control animals were injected with the same signaling has been shown to prevent permeability in cerebral volume (2 ␮L) of saline. Forty-eight hours later, one eye of each animal models of IR. Blockade of the KDR VEGF receptor kinase was subjected to retinal ischemia for 45 minutes and reperfused for 48 inhibits edema after cerebral IR,66 and soluble VEGF receptor hours before assay to determine (A) caspase-3 activity, (B) DNA fragmentation, and (C) Evans blue dye leakage. The contralateral eye was reduces edema as well as lesion volume in a mouse model of 67 subjected to needle puncture and served as the sham control. Results cerebral IR. A potential neuroprotective effect of VEGF has are the mean and SE of means obtained from eight animals in each also been observed. Using a rat model of IR induced by optic group. Comparisons between sham and IR eyes and between con- 25 sheath occlusion, Nishijima et al. demonstrated an increase trol-IR and treatment-IR were calculated using the mixed-effects, two- in VEGF protein expression after 3 and 6 hours of reperfusion. way ANOVA model: *P Ͻ 0.05, **P Ͻ 0.01, and ***P Ͻ 0.001.

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of VEGF function during ischemia and after reperfusion does 6. Antonetti DA, Barber AJ, Bronson SK, et al. Diabetic retinopathy: not exacerbate overall retinal cell death. Although this conclu- seeing beyond glucose-induced microvascular disease. Diabetes. sion is consistent with the hypothesis that VEGF does not 2006;55:2401–2411. provide an essential neurotrophic function during IR, it cannot 7. Chibber R, Ben-Mahmud BM, Chibber S, Kohner EM. Leukocytes in be confirmed without specifically examining the death of ret- diabetic retinopathy. Curr Diabetes Rev. 2007;3:3–14. inal neurons. 8. Yeung L, Lima VC, Garcia P, Landa G, Rosen RB. Correlation Anti-VEGF therapies are increasingly used for the treat- between spectral domain optical coherence tomography findings 69,70 and fluorescein angiography patterns in diabetic macular edema. ment of retinopathy. In recent trials, intravitreal ranibi- Ophthalmology. 2009;116:1158–1167. zumab and bevacizumab have each significantly improved 9. Kaur C, Foulds WS, Ling EA. Blood-retinal barrier in hypoxic vision in patients with diabetic macular edema—better than ischaemic conditions: basic concepts, clinical features and man- 71,72 laser treatments. Likewise, preliminary results suggest agement. Prog Retin Eye Res. 2008;27:622–647. that both ranibizumab and bevacizumab can improve mac- 10. Bringmann A, Pannicke T, Grosche J, et al. Muller cells in the ular edema caused by branch or central retinal vein occlu- healthy and diseased retina. Prog Retin Eye Res. 2006;25:397–424. sions.73–77 There is concern that inhibition of VEGF function 11. Rodrigues EB, Farah ME, Maia M, et al. Therapeutic monoclonal could cause neurodegeneration by blocking its neurotrophic antibodies in ophthalmology. Prog Retin Eye Res. 2009;28:117– effects. Chronic application of VEGF antagonists caused the 144. loss of retinal ganglion cells in mice and rats.25 However, 12. Tezel G. TNF-alpha signaling in glaucomatous neurodegeneration. expression of a transgene encoding a soluble VEGF receptor Prog Brain Res. 2008;173:409–421. in the retina blocks VEGF-induced permeability and choroi- 13. Mohamed Q, Wong TY. Emerging drugs for diabetic retinopathy. dal neovascularization, but does not significantly alter ERGs Expert Opin Emerg Drugs. 2008;13:675–694. or retinal ganglion cell loss.29 In another study, the results 14. Theodossiadis PG, Liarakos VS, Sfikakis PP, Vergados IA, Theodos- showed that repeated intravitreal injection of bevacizumab siadis GP. Intravitreal administration of the anti-tumor necrosis 28 factor agent infliximab for neovascular age-related macular degen- has no detrimental effects in rats. In addition, clinical trials eration. Am J Ophthalmol. 2009;147:825–830, 830 e821. of bevacizumab have not revealed any detrimental effects on 27 15. Theodossiadis PG, Markomichelakis NN, Sfikakis PP. Tumor necro- human retinal function. sis factor antagonists: preliminary evidence for an emerging ap- The present results demonstrate that VEGF function con- proach in the treatment of ocular inflammation. Retina. 2007;27: tributes to vascular permeability after retinal IR and thus sug- 399–413. gest that the IR model can serve as a useful tool in the study of 16. Tsilimbaris MK, Panagiotoglou TD, Charisis SK, Anastasakis A, VEGF-induced retinal edema. The observation that bevaci- Krikonis TS, Christodoulakis E. The use of intravitreal etanercept zumab inhibits vascular permeability without increasing over- in diabetic macular oedema. Semin Ophthalmol. 2007;22:75–79. all apoptosis after retinal IR further supports the conclusion 17. Koizumi K, Poulaki V, Doehmen S, et al. Contribution of TNF-alpha that this treatment is not necessarily neurodegenerative. This to leukocyte adhesion, vascular leakage, and apoptotic cell death result is highly relevant to concerns that treatments blocking in endotoxin-induced uveitis in vivo. Invest Ophthalmol Vis Sci. VEGF function may cause neuronal degeneration by blocking 2003;44:2184–2191. neurotrophic effects. Separable effects of IPC and bevacizumab 18. Joussen AM, Poulaki V, Mitsiades N, et al. Nonsteroidal anti-inflam- on neurodegeneration and vascular permeability demonstrate matory drugs prevent early diabetic retinopathy via TNF-alpha suppression. FASEB J. 2002;16:438–440. that these are independent responses to IR. If this independent 19. Joussen AM, Doehmen S, Le ML, et al. TNF-alpha mediated apo- response is true of other retinal diseases that include vascular ptosis plays an important role in the development of early diabetic dysfunction and neuronal death, such as diabetic retinopathy, retinopathy and long-term histopathological alterations. Mol Vis. then complementary therapeutics targeting both permeability 2009;15:1418–1428. and neurodegeneration may be needed to restore normal func- 20. Behl Y, Krothapalli P, Desta T, DiPiazza A, Roy S, Graves DT. tion. The IR model may prove useful for preclinical testing of Diabetes-enhanced tumor necrosis factor-alpha production pro- treatments directed toward each of these pathologic end- motes apoptosis and the loss of retinal microvascular cells in type points. 1 and type 2 models of diabetic retinopathy. Am J Pathol. 2008; 172:1411–1418. Acknowledgments 21. Penn JS, Madan A, Caldwell RB, Bartoli M, Caldwell RW, Hartnett ME. Vascular endothelial growth factor in eye disease. Prog Retin The authors thank Georgina V. Bixler and Robert M. Brucklacher Eye Res. 2008;27:331–371. (Penn State College of Medicine Functional Genomics Core Facility; 22. Jardeleza MS, Miller JW. Review of anti-VEGF therapy in prolifer- Willard M. Freeman, PhD, Director) for excellent technical contri- ative diabetic retinopathy. Semin Ophthalmol. 2009;24:87–92. butions; Wendy Dunton and Melissa Bridi (Penn State Hershey 23. Lott MN, Schiffman JC, Davis JL. Bevacizumab in inflammatory eye Diabetic Animal Models Core Facility; Sarah K. Bronson, PhD, Di- disease. Am J Ophthalmol. 2009;4:4. rector) for aid with animal acquisition and husbandry; and Wade 24. Saint-Geniez M, Maharaj AS, Walshe TE, et al. Endogenous VEGF is Edris for skilled technical assistance with retinal sectioning and required for visual function: evidence for a survival role on muller staining. cells and photoreceptors. PLoS One. 2008;3:e3554. 25. Nishijima K, Ng YS, Zhong L, et al. Vascular endothelial growth References factor-A is a survival factor for retinal neurons and a critical neu- roprotectant during the adaptive response to ischemic injury. Am J 1. Patel N, Adewoyin T, Chong NV. 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