Effects of ischemic preconditioning and bevacizumab on apoptosis and vascular permeability following retinal ischemia-reperfusion injury.
OBJECTIVE
Using transient ischemia followed by reperfusion (IR) to model ischemic retinal disease, this study compares the effects of ischemic preconditioning (IPC) and therapies targeting vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-α on retinal apoptosis, vascular permeability, and mRNA expression.


METHODS
Rats were subjected to 30 or 45 minutes of retinal ischemia followed by reperfusion for up to 48 hours. Neurodegeneration was quantified by caspase-3 (DEVDase) activity and by measuring nucleosomal DNA content (cell death ELISA). Vascular leakage was quantified by the Evans Blue dye method. A set of IR-responsive mRNAs was identified by whole-genome microarray and confirmed by RT-PCR analyses. VEGF protein was measured by Western blot analysis. IPC was accomplished with 10 minutes of ischemia 24 hours before IR. VEGF and TNFα signaling was inhibited by intravitreal injection of bevacizumab or etanercept, respectively.


RESULTS
IR caused significant retinal cell apoptosis and vascular permeability after 4 and 48 hours. Whereas IR decreased VegfA mRNA, VEGF protein was significantly increased. IPC effectively inhibited neurodegeneration, bevacizumab effectively inhibited vascular permeability, and etanercept failed to affect either outcome. IPC significantly altered the IR responses of 15 of 33 IR-responsive mRNAs, whereas bevacizumab had no significant effect on these mRNAs.


CONCLUSIONS
IR provides an acute model of ischemic retinopathy that includes neurodegeneration and VEGF-dependent vascular permeability and is amenable to rapid drug therapy testing. The distinct effects of IPC and bevacizumab demonstrate that the apoptotic and vascular responses to IR may be separated and that therapeutics targeting each pathologic endpoint may be warranted in treating ischemic retinal diseases.
Abcouwer
Steven F
SF
Department of Surgery, Penn State College of Medicine, Hershey, Milton S Hershey Medical Center, Hershey, PA 17033-0850, USA. sabcouwer@psu.edu
Lin
Cheng-mao
CM
Wolpert
Ellen B
EB
Shanmugam
Sumathi
S
Schaefer
Eric W
EW
Freeman
Willard M
WM
Barber
Alistair J
AJ
Antonetti
David A
DA
D020533:Angiogenesis Inhibitors / Q000008:administration & dosage; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D061067:Antibodies, Monoclonal, Humanized; D017209:Apoptosis; D000068258:Bevacizumab; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D004249:DNA Damage; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D058449:Intravitreal Injections; D019194:Ischemic Preconditioning; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D012164:Retinal Diseases / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D012171:Retinal Vessels / Q000378:metabolism / Q000473:pathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; D053148:Caspase 3