Development Of Rat Models For Choroidal Neovascularisation (CNV)

A Comparison between Laser and Recombinant Adenovirus Induced CNV
  • P. E. Rakoczy
  • W. Y. Shen
  • K. Spilsbury
  • M. Yu
  • M. Lai
  • C. J. Barry
  • I. J. Constable


This project aimed to investigate whether the overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium (RPE), induced by recombinant adenovirus mediated delivery of VEGF164, is sufficient to initiate the development of leaky choroidal blood vessels in a rat model. Eyes of pigmented rats were subjected to krypton laser photocoagulation or subretinal injection with recombinant adenovirus constructs Ad.RSV.VEGF or Ad.CMV.VEGF. Initially the animals were examined with fundus photography followed by fluorescein angiography. Retinal photographs demonstrated that the krypton laser produced moderate burns and that the subretinal injection resulted in the formation of a bleb in the subretinal space. Injection of control recombinant adenovirus Ad. RSV.βgal resulted in the expression of βgal reporter gene in the RPE layer. Of the 141 laser spots delivered into a total of 17 rat eyes, 52.4% developed fluorescein leakage that was detectable by angiography. Of seven eyes injected with Ad.RSV.VEGF, 5 developed leakage at 2 weeks post injection, and of 8 eyes which were injected with Ad.CMV.VEGF, 5 developed leakage at 1 week post injection. However, the development of leaky blood vessels was temporary and both Ad.RSV.VEGF and Ad.CMV.VEGF injected animals showed regression of leaky vessels by 4 and 5 weeks post injection, respectively. These results suggest that the upregulation of VEGF in the RPE layer may be a key factor in the series of events leading to the development of CNV.


Vascular Endothelial Growth Factor Retinal Pigment Epithelium Post Injection Recombinant Adenovirus Choroidal Neovascularisation 


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  1. 1.
    W.R. Green and D.J. Wilson, 1986, Choroidal neovascularization, Ophthalmology 93:1169–1176.PubMedGoogle Scholar
  2. 2.
    S.H. Sarks, 1973, New vessel formation beneath the retinal pigment epithelium in senile eyes, Br J Ophthalmol 57:951–965.PubMedGoogle Scholar
  3. 3.
    W.R. Green, C. Enger, 1993, Age-related macular degeneration histopathologic studies. The 1993 Lorenz E. Zimmerman Lecture. Ophthalmology 100:1519–1535.Google Scholar
  4. 4.
    N.M. Bressler, J.C. Silva, S.B. Bressler, S.L. Fine, and W.R. Green, 1994, Clinicopathologic correlation of drusen and retinal pigment epithelial abnormalities in age-related macular degeneration. Retina 14(2):130–142.PubMedCrossRefGoogle Scholar
  5. 5.
    S.H. Sarks, 1976, Ageing and degeneration in the macular region: a clinico-pathological study, Br J Ophthalmol 60:324–341.PubMedGoogle Scholar
  6. 6.
    S.H. Sarks, D. van Driel, L. Maxwell, and M.C. Killingsworth, 1980, Softening of drusen and subretinal neovascularization, Trans Ophthalmol Soc UK 100:414–422.PubMedGoogle Scholar
  7. 7.
    W.J. Heriot, P. Henkind, R.W. Bellhorn, and M.S. Burns, 1984, Choroidal neovascularization can digest Bruch’s membrane—A prior break is not essential, Ophthalmology 91(12):1603–1608.PubMedGoogle Scholar
  8. 8.
    A. Pollack, W.J. Heriot, and P. Henkind, 1986, Celluar processes causing defects in Bruch’s membrance following krypton laser photocoagulation, Ophthalmology 93:1113–1119.PubMedGoogle Scholar
  9. 9.
    R.N. Frank, A. Das, and M.L. Weber, 1989, A model of subretinal neovascularization in the pigmented rat, Curr Eye Res 8:239–247.PubMedGoogle Scholar
  10. 10.
    X Yi, N Ogata, M. Komada, K Takahashi, K. Omori,and M. Uyama, 1997, Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefe’s Arch Clin Exp Ophthalmol:313–319.Google Scholar
  11. 11.
    T. Ishibashi, Y. Hata, H. Yoshikawa, K. Nakagawa, K. Sueishi, and H. Inomata, 1997, Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Graefe’s Arch Clin Exp Ophthalmol 235:159–167.CrossRefGoogle Scholar
  12. 12.
    E.T. Dobi, C.A. Puliafito, and M. Destro, 1989, A new model of experimental choroidal neovascularization in the rat, Arch Ophthalmol 107:264–269.PubMedGoogle Scholar
  13. 13.
    L. Feeney-Burns, C. Gao, and E.R. Berman, 1988, The fate of immunoreactive opsin following phagocytosis by pigment epithelium in human and monkey retinas, Invest Ophthalmol Vis Sci 29:708–719.PubMedGoogle Scholar
  14. 14.
    W. Shen, M. Yu, C. Barry, I.J. Constable, and P.E. Rakoczy, 1998, Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat, Br J Ophthalmol 82—in pressGoogle Scholar
  15. 15.
    M.C. Killingsworth, S.H. Sarks, 1982, Giant cells in disciform macular degeneration of the human eye, Micron 13(3):359–360.Google Scholar
  16. 16.
    S.H. Sarks, P.L. Penfold, M.C. Killingsworth, and D. van Driel, 1985, Patterns in Macular Degeneration, in: Retinal Diseases, (S.J. Ryan, A.K. Dawson, eds.) pp. 87–93, Grune & Stratton. Inc. NY.Google Scholar
  17. 17.
    P.L. Penfold, M.C. Killingsworth, and S.H. Sarks, 1985, Senile macular degeneration: The involvement of immunocompetent cells, Graefe’s Arch Clin Exp Ophthalmol 223:69–76.CrossRefGoogle Scholar
  18. 18.
    N. Ferrara, T. Davis-Smyth, 1997, The biology of vascular endothelial growth factor, Endocr Rev 18(1):4–25.PubMedCrossRefGoogle Scholar
  19. 19.
    D.W. Leung, G. Cachianes, W. Kuang, D.V. Goeddel, and N. Ferrara, 1989, Vascular endothelial growth factor is a secreted angiogenic mitogen, Science 246:1306–1309.PubMedCrossRefGoogle Scholar
  20. 20.
    H.F. Dvorak, L.F. Brown, M. Detmar, and A.M. Dvorak. 1995, Vascular permeanility factor vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 146:1029–1039.PubMedGoogle Scholar
  21. 21.
    A. Minchenko, T. Bauer, S. Salceda, and J. Caro, 1994, Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo, Lab Invest 71:374–379.PubMedGoogle Scholar
  22. 22.
    R.J. D’ Amato, A.P. Adamis, 1995, Angiogenesis inhibition in age-related macular degeneration, Ophthalmology 102:1261–1262.PubMedGoogle Scholar
  23. 23.
    P.A. D’Amore, 1994, Mechanisms of Retinal and Choroidal neovascularization, Invest Ophthalmol Vis Sci 35:3974–3979.PubMedGoogle Scholar
  24. 24.
    H.P. Heidenkummer, 1991, Age-related macular degeneration: current aspects of pathogenesis and treatment, Eye Science 7:6–20.PubMedGoogle Scholar
  25. 25.
    P.E. Rakoczy, C.M. Lai, M. Baines, S. Di Grandi, J.H. Fitton, and I.J. Constable, 1997, Modulation of cathepsin D activity in retinal pigment epithelial cells, Biochem J 324:935–940.PubMedGoogle Scholar
  26. 26.
    W.R. Green. 1985, Pathology of the macula, in: Ophthalmic Pathology, Volume 2 (W.H. Spencer, ed.) pp. 924–1034, W.B. Saunders Company.Google Scholar
  27. 27.
    S.O. Sykes, N.M. Bressler, M.G. Maguire, and A.P. Schachat, 1994, Detecting recurrent chorodial neovascularization comparison of clinical examination with and without fluorescein angiography, Arch Ophthalmol 112(12):1561–1566.PubMedGoogle Scholar
  28. 28.
    L. da Cruz, P. Rakoczy, M. Perricaudet, and I. Constable, 1996, Dynamics of gene transfer to retinal pigment epithelium, Invest Ophthalmol Vis Sci 37(12):2447–2454.PubMedGoogle Scholar
  29. 29.
    T.C. Becker, R.J. Noel, W.S. Coats, A.M. Gomez-Foix,T. Alam, R.D. Gerard, and C.B. Newgard, 1994, Use of recombinant adenovirus for metabolic engineering of mammalian cells, in: Methods in Cell Biology, Volume 43 pp. 161–189, Academic Press.PubMedCrossRefGoogle Scholar
  30. 30.
    W. Zhang, P.E. Koch, and J.A. Roth, 1995, Detection of wild-type contamination in a recombinant adenoviral preparation by PCR, Biotechniques 18:444–446.PubMedGoogle Scholar
  31. 31.
    A.C. Bird, N.M. Bressler, S.B. Bressler, I.H. Chisholm, G. Coscas, and M.D. David, 1995, An international classification and grading system for age-related maculopathy and age-related macular degeneration. Surv Ophthalmol 39(5):367–374.PubMedCrossRefGoogle Scholar
  32. 32.
    S.J. Ryan. 1989, Subretinal neovascularization, in: Retina, (S.J. Ryan, E.J. Murray, and Patz, eds.) pp. 107–125, Mosby, St Louis.Google Scholar
  33. 33.
    R.N. Frank, R.H. Amin, D. Eliott, J.E. Puklin, and G.W. Abrams, 1996, Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes, Am J Ophthalmol 122:393–403.PubMedGoogle Scholar
  34. 34.
    R.N. Frank, 1997, Growth factors in age-related macular degeneration:pathogenic and therapeutic implications, Ophthalmic Research 29:341–353.PubMedCrossRefGoogle Scholar
  35. 35.
    P.F. Lopez, B.D. Sippy, H.M. Lambert, A.B. Thach, and D.R. Hinton, 1996, Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes, Invest Ophthalmol Vis Sci 37:855–868.PubMedGoogle Scholar
  36. 36.
    A. Kvanta, P.V. Algvere, L. Berglin, and S. Seregard, 1996, Subfoveal fibrovascular membranes in Age-related macular degeneration express vascular endothelial growth factor, Invest Ophthalmol Vis Sci 37(9):1929–1934.PubMedGoogle Scholar
  37. 37.
    G.A. Lutty, D.S. McLeod, C. Merges, A. Diggs, and J. Plouet, 1996, Localization of Vascular Endothelial Growth Factor in Human Retina and Choroid, Arch Ophthalmol 114:971–977.PubMedGoogle Scholar
  38. 38.
    N.C. Tsang, P.L. Penfold, P.J. Snitch, and F. Billson, 1992, Serum levels of antioxidants and age-related macular degeneration. Documenta Ophthalmologica 81(4):387–400.PubMedCrossRefGoogle Scholar
  39. 39.
    B. Martini, S.J. Ryan, 1992, Argon laser lesion of the retina: occurrence and origin of macrophages. Euro J Ophthalmol 2:51–57.Google Scholar
  40. 40.
    X. Yi, K. Takahashi, N. Ogata, and M. Uyama, 1996, Immunohischemical proof of origin of macrophages in laser photocoagulation lesion in the retina. Jpn J Ophthalmol 40:192–201.PubMedGoogle Scholar
  41. 41.
    H. Kimura, T. Sakamoto, C. Spee, and et al. 1995, A new model for experimental subretinal neovascularisation in the rabbit, Invest Ophthalmol Vis Sci 35:3429–3435.Google Scholar
  42. 42.
    N. Ogata, M. Matsushima, Y. Takada, T. Tobe, and et al. 1996, Expression of basic fibroblast growth factor mRNA in developing choroidal neovascularization, Curr Eye Res:1008–1018.Google Scholar
  43. 43.
    H.G.T. Blaauwgeers, G.M. Holtkamp, P. Koolwijk, van Hinsbergh, V.M. Kroon, A. Kijistra, and R. Schlingemann, 1998, Preferential basal secretion of VEGF by RPE in vitro and expression of its receptor KDR on the inner choriocapillaris in vitro suggest a trophic relation in the normal eye, Invest Ophthalmol Vis Sci 39(4):S372Google Scholar
  44. 44.
    L. da Cruz, P. Rakoczy, M, Perricaudet, and I.J. Constable, 1996, The dynamics of gene transfer to retinal pigment epithelium. Invest Opthalmol Vis Sci 37(12):2447–2454.Google Scholar
  45. 45.
    M.B. Reichel, R.R. Ali, A.J. Thrasher, D.M. Hunt, S.S. Bhattacharya, and D. Baker, 1998, Immune responses limit adenovirally medited gene expression in the adult mouse eye, Gene Therapy 5:1038–1046.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic / Plenum Publishers 1999

Authors and Affiliations

  • P. E. Rakoczy
    • 1
  • W. Y. Shen
    • 2
  • K. Spilsbury
    • 2
  • M. Yu
    • 1
  • M. Lai
    • 1
  • C. J. Barry
    • 2
  • I. J. Constable
    • 1
  1. 1.Centre for Ophthalmology and Visual ScienceUniversity of Western AustraliaNedlandsAustralia
  2. 2.Lions Eye InstituteNedlandsAustralia

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