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Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis

Abstract

The mouse model of oxygen-induced retinopathy (OIR) has been widely used in studies related to retinopathy of prematurity, proliferative diabetic retinopathy and in studies evaluating the efficacy of antiangiogenic compounds. In this model, 7-d-old (P7) mouse pups with nursing mothers are subjected to hyperoxia (75% oxygen) for 5 d, which inhibits retinal vessel growth and causes significant vessel loss. On P12, mice are returned to room air and the hypoxic avascular retina triggers both normal vessel regrowth and retinal neovascularization (NV), which is maximal at P17. Neovascularization spontaneously regresses between P17 and P25. Although the OIR model has been the cornerstone of studies investigating proliferative retinopathies, there is currently no harmonized protocol to assess aspects of angiogenesis and treatment outcome. In this protocol we describe standards for mouse size, sample size, retinal preparation, quantification of vascular loss, vascular regrowth, NV and neovascular regression.

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Figure 1: Cartoon schematic of the mouse OIR model.
Figure 2: Vasculature and quantification of P12H vaso-obliteration.
Figure 3: Quantification of vaso-obliteration and neovascularization (NV) at P17H.
Figure 4: Quantification of neovascularization (NV) during regression: P18H-P25H.

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References

  1. Chen, J., Connor, K.M., Aderman, C.M. & Smith, L.E. Erythropoietin deficiency decreases vascular stability in mice. J. Clin. Invest. 118, 526–533 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Smith, L.E. et al. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat. Med. 5, 1390–1395 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Smith, L.E. et al. Essential role of growth hormone in ischemia-induced retinal neovascularization. Science 276, 1706–1709 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Connor, K.M. et al. Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat. Med. 13, 868–873 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Palmer, E.A. et al. Incidence and early course of retinopathy of prematurity. The Cryotherapy for Retinopathy of Prematurity Cooperative Group. Ophthalmology 98, 1628–1640 (1991).

    Article  CAS  PubMed  Google Scholar 

  6. Madan, A. & Penn, J.S. Animal models of oxygen-induced retinopathy. Front Biosci. 8, d1030–d1043 (2003).

    Article  CAS  PubMed  Google Scholar 

  7. Smith, L.E. et al. Oxygen-induced retinopathy in the mouse. Invest. Ophthalmol. Vis. Sci. 35, 101–111 (1994).

    CAS  PubMed  Google Scholar 

  8. Ashton, N. Animal experiments in retrolental fibroplasia. Trans. Am. Acad. Ophthalmol. Otolaryngol. 58, 51–53 discussion, 53–54 (1954).

    CAS  PubMed  Google Scholar 

  9. Ashton, N., Ward, B. & Serpell, G. Role of oxygen in the genesis of retrolental fibroplasia; a preliminary report. Br. J. Ophthalmol. 37, 513–520 (1953).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Penn, J.S., Tolman, B.L. & Henry, M.M. Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization. Invest. Ophthalmol. Vis. Sci. 35, 3429–3435 (1994).

    CAS  PubMed  Google Scholar 

  11. Flower, R.W. Perinatal ocular physiology and ROP in the experimental animal model. Doc. Ophthalmol. 74, 153–162 (1990).

    Article  CAS  PubMed  Google Scholar 

  12. Flower, R.W., McLeod, D.S., Lutty, G.A., Goldberg, B. & Wajer, S.D. Postnatal retinal vascular development of the puppy. Invest. Ophthalmol. Vis. Sci. 26, 957–968 (1985).

    CAS  PubMed  Google Scholar 

  13. Mammoto, A. et al. A mechanosensitive transcriptional mechanism that controls angiogenesis. Nature 457, 1103–1108 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hellstrom, M. et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007).

    Article  PubMed  Google Scholar 

  15. Kubota, Y., Hirashima, M., Kishi, K., Stewart, C.L. & Suda, T. Leukemia inhibitory factor regulates microvessel density by modulating oxygen-dependent VEGF expression in mice. J. Clin. Invest. 118, 2393–2403 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Checchin, D. et al. Hypercapnia prevents neovascularization via nitrative stress. Free Radic. Biol. Med. 40, 543–553 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Brault, S. et al. Selective neuromicrovascular endothelial cell death by 8-Iso-prostaglandin F2alpha: possible role in ischemic brain injury. Stroke 34, 776–782 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Kanaan, A., Farahani, R., Douglas, R.M., Lamanna, J.C. & Haddad, G.G. Effect of chronic continuous or intermittent hypoxia and reoxygenation on cerebral capillary density and myelination. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R1105–R1114 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Chopp, M., Zhang, Z.G. & Jiang, Q. Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke. Stroke 38, 827–831 (2007).

    Article  PubMed  Google Scholar 

  20. Gardiner, T.A. et al. Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy. Am. J. Pathol. 166, 637–644 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen, J. et al. Suppression of retinal neovascularization by erythropoietin siRNA in a mouse model of proliferative retinopathy. Invest. Ophthalmol. Vis. Sci. 50, 1329–1335 (2009).

    Article  PubMed  Google Scholar 

  22. Aiello, L.P. et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. USA 92, 10457–10461 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Higgins, R.D. et al. Diltiazem reduces retinal neovascularization in a mouse model of oxygen induced retinopathy. Curr. Eye Res. 18, 20–27 (1999).

    Article  CAS  PubMed  Google Scholar 

  24. Lange, C. et al. Intravitreal injection of the heparin analog 5-amino-2-naphthalenesulfonate reduces retinal neovascularization in mice. Exp. Eye Res. 85, 323–327 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Ritter, M.R. et al. Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J. Clin. Invest. 116, 3266–3276 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Davies, M.H., Stempel, A.J. & Powers, M.R. MCP-1 deficiency delays regression of pathologic retinal neovascularization in a model of ischemic retinopathy. Invest. Ophthalmol. Vis. Sci. 49, 4195–4202 (2008).

    Article  PubMed  Google Scholar 

  27. Dorrell, M.I., Aguilar, E., Scheppke, L., Barnett, F.H. & Friedlander, M. Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis. Proc. Natl. Acad. Sci. USA 104, 967–972 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lofqvist, C. et al. IGFBP3 suppresses retinopathy through suppression of oxygen-induced vessel loss and promotion of vascular regrowth. Proc. Natl. Acad. Sci. USA 104, 10589–10594 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chan, C.K. et al. Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization. Lab. Invest. 85, 721–733 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Vanhaesebrouck, S. et al. Oxygen-induced retinopathy in mice: amplification by neonatal IGF-I deficit and attenuation by IGF-I administration. Pediatr. Res. 65, 307–310 (2009).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This research was generously supported by the V. Kann Rasmussen Foundation, the US National Institutes of Health (EY008670, EY017017, EY14811 (L.E.H.S.); 5 T32 EY07145, 1 F32 EY017789-01 (K.M.C.)), William Randolph Hearst Fund (K.M.C.) and Children's Hospital Boston Mental Retardation and Developmental Disabilities Research Center, P01 HD18655 (L.E.H.S.). Support from the Research to Prevent Blindness Lew Wasserman Merit Award (L.E.H.S). Juvenile Diabetes Research Foundation International (3-2006-278, 10-2008-603 (J.C.). The sponsors had no role in the design or conduct of the study, in the collection, analysis and interpretation of data and in the preparation, review or approval of the manuscript.

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Authors

Contributions

K.M.C. wrote the manuscript with N.M.K., R.J.D. and L.E.H.S. with input from C.M.A., J.C., K.G., P.S., A.S. and K.L.W. All authors have worked on and modernized this protocol.

Corresponding author

Correspondence to Lois E H Smith.

Supplementary information

Supplementary Video 1

Retinal Dissection Movie (MOV 156087 kb)

Supplementary Video 2

Movie of retinal flatmount (MOV 87767 kb)

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Connor, K., Krah, N., Dennison, R. et al. Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis. Nat Protoc 4, 1565–1573 (2009). https://doi.org/10.1038/nprot.2009.187

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