Neovascular Models of the Rabbit Eye Induced By Hydroperoxide

  • Toshihiko Ueda
  • Takako Nakanishi
  • Kazushi Tamai
  • Shinichi Iwai
  • Donald ArmstrongEmail author
Part of the Oxidative Stress in Applied Basic Research and Clinical Practice book series (OXISTRESS)


The eye is rich in oxidizable substrates which often increase in ocular disease. In vivo rabbit models described in this chapter provide valuable clinical and biomolecular data, whereas in vitro models are used to define physiological observations such as endothelial cell proliferation, migration and vascular tube ­formation. Antioxidant supplementation significantly inhibits these phenomena and is facilitated when multiple antioxidants are given simultaneously. The 13-­isomer of linoleic acid hydroperoxide (HpODE) stimulates neovascularization when injected into the corneal stroma, the vitreous, or subretinal to create appropriate animal ­models. During this process, nuclear factors, metalloproteinase and angiogenic cytokines are ­upregulated within 3–6 h to initiate the neovascular response. Fluorescein angiography­ provides direct visualization of new vessel growth. A variety of oxidative stress biomarkers can be measured in plasma, ocular homogenates and tissue culture cells, or media. Histology confirms location of injected HpODE, presence or absence of an inflammatory response, hemorrhage and illustrate features that resemble human disease. New vessels grow at the rate of 0.3 mm/day and plateau at 3–4 weeks as the HpODE stimulus is metabolized. Repeat injections result in an accelerated growth rate. Vessel length in diabetic rabbits is double that observed in normal animals. In contrast to surgical or laser-induced neovascularization ­models, the HpODE model uses a natural substrate and is considered a ­non-traumatic ­procedure. All models are valuable to validate antioxidant intervention studies in clinical trials.


Angiogenic cytokines Antioxidants Choroid Cornea Fluorescein angiography Hyperglycemia Light and electron microscopy Lipid hydroperoxides Matrix metalloproteinase Neovascularization Nuclear factors Retina Tissue culture 



The authors thank the following scientists who assisted in these various studies: Akira Higa, Shohei Fukuda, Eichi Nishimura, Richard Browne, Ahmad Aljada, Kathleen Dorey, Ram Sasisekharan, Tadahisa Hiramitsu, Shigehiro Iwabushi, Yuichiro Ogura, Alihisa Matsubara, Mary E. Hartnett, Kaori Watanabe, Miho Chida, Maki Kamegawa, Richard Spaide, Wang Ho-Spaide, Maria Grant and E. Ann Ellis. We also thank Professors Ryohei Koide and Hajimi Yasuhara for department and institutional support and technical assistance and Oxidative Stress Associates for administrative financial aid.


  1. 1.
    Armstrong, D. et al., 1998. Dose dependent mechanisms of lipid hydroperoxide induce retinal pathology. In, Yagi, K, ed, Pathophysiology of Lipid Peroxides and Related Radicals, Japan Sci. Soc. Press, Tokyo/S. Karger, Basel, pp 57–76.Google Scholar
  2. 2.
    Ueda, T. et al., 1997. Lipid hydroperoxide-induced tumor necrosis factor (TNF) -a,vascular endothelial growth factor and neovascularization in the rabbit cornea: effect of TNF inhibition. Angiogenesis 1: 174–184.Google Scholar
  3. 3.
    Armstrong, D, et al., 1998. Lipid hydroperoxide stimulates retinal neovascularization in rabbit retina through expression of tumor necrosis factor-a, vascular endothelial growth factor and platelet-derived growth factor. Angiogensis 2: 93–104.Google Scholar
  4. 4.
    Tamai, K et al., 2002. Lipid hydroperoxide stimulates subretinal choroidal neovascularization in the rabbit. Exp Eye Res 74: 301–308.Google Scholar
  5. 5.
    Higa, A., et al., 2002. Lipid hydroperoxide induced corneal neovascularization hyperglycemic rabbits. Curr Eye Res 25: 49–53.Google Scholar
  6. 6.
    Iwai, S., et al., 2006. Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina by lipid hydroperoxides. Curr Eye Res 31: 337–346.Google Scholar
  7. 7.
    Spaide, RF, et al., 1999. Characterization of peroxidized lipids in Bruch’s membrane. Retina 19: 141–147.Google Scholar
  8. 8.
    Watanabe, K et al., 2000. Quantitative analysis of rabbit corneal neovascularization induced by lipid hydroperoxide. Atarashi Ganka (J of Eye) 17: 710–714.Google Scholar
  9. 9.
    Kamegawa, M et al., 2007. Effect of lipid hydroperoxide-induced oxidative stress on vitamin E, ascorbate and glutathione in the rabbit retina. Ophthalmic Res 39: 49–54.Google Scholar
  10. 10.
    Ogura, M et al., 2007. Effect of dihydrobenzofuran derivatives on lipid hydroperoxide-induced rabbit corneal neovascularization. J Pharmacol Sci 103: 234–240.Google Scholar
  11. 11.
    Ueda, T, et al., 1996. Preventive effect of natural and synthetic antioxidants on lipid peroxidation in the mammalian eye. Ophthalmic Res 28: 184–192.Google Scholar
  12. 12.
    Chida, M, et, al, 1999. In vitro testing of antioxidants and biochemical end-points in bovine retinal tissue. Ophthalmic Res 31: 407–415.Google Scholar
  13. 13.
    Nishimura, E, et al., 2001. Upregulation of vascular endothelial growth factor in cultured rabbit corneal cells during lipid hydroperoxide oxidative stress. Showa Univ J Med Sci 13: 35–41.Google Scholar
  14. 14.
    Yamada, Y et al., 1998. Angiogenic effect of lipid hydroperoxide on bovine aortic endothelial cells. J Clin Biochem Nutr 25: 121–130.Google Scholar
  15. 15.
    Yamamoto, YM, et al., 2000. UV-B and lipid hydroperoxides promote conjunctival epithelial cell migration. Pathophysiology 6: 225–230.Google Scholar
  16. 16.
    Framme, C et al., 2008. Clinical evaluation of experimentally induced choroidal neovascularizations in pigmented rabbits by subretinal injection of lipid hydroperoxide and consecutive preliminary photdynamic treatment with Tookad. Ophthalmologica 222: 254–264.Google Scholar
  17. 17.
    Baba, T., et al., 2009. A rat model for choroidal neovascularization using subretinal injection of lipid hydroperoxide. ARVO Abstract 2055: 140.Google Scholar
  18. 18.
    Armstrong, D and Browne, R, 1998. Synthesis of lipid and cholesterol hydroperoxide standards. Meth Mol Biol 108: 139–145.Google Scholar
  19. 19.
    Armstrong, D and Browne, R 1994. The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress as applied to the clinical chemistry laboratory. In, Armstrong, (ed), Free Radicals in Diagnostic Medicine, Adv Exp Med Biol,vol 366, pp 43–58.Google Scholar
  20. 20.
    Ellis, EA, 2008. Correlative transmission microscopy: cytochemical localization and immunocytochemical localization in studies of oxidative and nitrosative stress. Meth Mol Biol, Adv Protocols 1, 477: 41–48.Google Scholar
  21. 21.
    Armstrong, D, et al., 1982. Studies on experimentally induced retinal degeneration.1. Effect of lipid peroxides on electroretinographic activity in the albino rabbit. Exp Eye Res 35: 157–171.Google Scholar
  22. 22.
    Fernandez-Robredo, P et al., 2005. Vitamins C and E reduce retinal oxidative stress and nitric oxide metabolites and prevent ultrastructural alterations in porcine hypercholesterolemia. Invest Ophthalmol Vis Sci 46: 1140–1146.Google Scholar
  23. 23.
    Moreira, EF, et al., 2009. 7-Ketocholesterol is present in lipid deposits in the primate retina: potential implications in the induction of VEGF and CNV formation. Invest Ophthalmol Vis Sci 50: 523–532.Google Scholar
  24. 24.
    Wang, L. et al., 2009. Lipoprotein particles of intraocular origin in human Bruch membrane: an unusual lipid profile. Invest Ophthalmol Vis Sci 50: 870–877.Google Scholar
  25. 25.
    Dong, A, et al., 2009. Oxidative stress promotes ocular neovascularization. J Cell Physiol 219: 544–552.Google Scholar
  26. 26.
    Xu, AI, et al., 2009. Subtoxic oxidative stress induces senescence in retinal pigment epithelial cells via TGF-beta release. Invest Ophthalmol vis Sci 50: 926–935.Google Scholar
  27. 27.
    Garcia-Layana, A et al., 2009. Development of laser-induced choroidal neovascularization in rats after retinal damage by sodium iodate injection. Ophthal Res 42: 205–212.Google Scholar
  28. 28.
    Niedowicz, DM and Daleke, DL, 2005. The role of oxidative stress in diabetic complications. Cell Biochem Biophys 43: 289–330.Google Scholar
  29. 29.
    Ogarte, M, 2009. Animal models of diabetic retinopathy: how are they helping us? Arch Soc Esp Oftalmol 84: 277–279.Google Scholar
  30. 30.
    Tamai, K, et al, 2002. Lipid hydroperoxide stimulates leukocyte-endothelium interaction in the retinal microcirculation. Exp Eye Res 75: 69–75.Google Scholar
  31. 31.
    Matsubara, A et al., 2005. Protective effect of polyethylene glycol-superoxide dismutase on leukocyte dynamics in rat retinal microcirculation under lipid hydroperoxide-induced oxidative stress. Exp Eye Res, 81: 193–199.Google Scholar
  32. 32.
    Pemp, B, et al., 2009. Effects of antioxidants (AREDS medication) on ocular blood flow and endothelial function in an endotoxin-induced model of oxidative stress in humans. Invest Ophthalmol Vis Sci Aug 13 (Epub ahead of print).Google Scholar
  33. 33.
    Chiu, CJ, et al., 2009. Dietary compound score and risk of age-related macular degeneration in the age-related eye study. Ophthalmology 116: 939–946.Google Scholar
  34. 34.
    Rehak, MN et al., 2008. Lutein and antioxidants in the prevention of age-related macular degeneration. Ophthalmologe 105: 37–38, 40–45.Google Scholar
  35. 35.
    Neelam, K, et al., 2008. Carotenoids and co-loclization in age-related maculopathy: design and methods. Ophthalmic Epidemiol 15: 389–40.Google Scholar
  36. 36.
    Michikawa, T, et al., 2009. Serum antioxidants and age-related macular degeneration among older Japanese. Asia Pac J Clin Nutr 18: 1–7.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Toshihiko Ueda
  • Takako Nakanishi
  • Kazushi Tamai
  • Shinichi Iwai
  • Donald Armstrong
    • 1
    Email author
  1. 1.Department of OphthalmologyUniversity of Florida College of MedicineGainesvilleUSA

Personalised recommendations