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 cyto- kines, 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 occlu- sion, 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 Investigative Ophthalmology & Visual Science, November 2010, Vol. 51, No. 11 5920 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/30/2021 IOVS, November 2010, Vol. 51, No. 11 Permeability and Apoptosis in Retinal IR 5921 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 occlu- sions. 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