Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

function in degradation and phagocytosis.49 Ambati’s group obtained histopathological sections from the eyes of mutant mice ranging in age from less than 12 months to greater than 24 and compared them to their agematched wild-type controls. The mutant mice exhibited a high frequency of protein complex deposits and pathologies similar to those found in AMD, with photoreceptor and RPE cell death attributed to the progressive subretinal accumulation of these deposits. Because AMD, and more specifically CNV, correlates with age, mice older than 9 months displayed clinical symptoms strikingly similar to those in AMD patients. However, despite the correlation between drusen and CNV progression, this transgenic Ccl-2/Ccr-2 mouse is more typical of a model of drusen deposition rather than of CNV.

3.2Growth factor driven neovascularization

3.2.1VEGF overexpression in photoreceptors

Vascular endothelial growth factor (VEGF) is produced by a variety of cells in the retina, including the RPE, and is implicated as a driving force in choroidal neovascularization. Its contribution to the development of CNV is supported by data showing an increase in VEGF mRNA levels in rat RPE following laser-induced CNV.29 To further elucidate the role of VEGF in the progression of CNV, transgenic mice have been generated that overexpress the growth factor in the photoreceptors under the control of the rhodopsin promoter.50 While VEGF overexpression results in retinal neovascularization,50 VEGF alone is not adequate for the induction and subsequent progression of CNV. Overexpression of VEGF must be coupled with photoreceptor cell death before CNV is observed in these animals.19 The usefulness of this model is further limited and complicated by the fact that both deep-retinal neovascularization and choroidal (subretinal) neovascularization can occur (Figure 1).

3.2.2VEGF overexpression in RPE

Models of CNV involving VEGF overexpression in the RPE cells have also been developed. Campochiaro’s group created a transgenic mouse model where inducible VEGF overexpression in RPE cells is driven by the VMD2 promoter.51 However, these animals exhibited no signs of CNV unless an adenoviral vector containing an expression construct for angiopoietin-2 (Ang2) was injected into the subretinal space. It may be that this injection perturbed the

RPE, thereby facilitating the occurrence of CNV. In an earlier study by Spilsbury and colleagues, injection of an adenovirus vector expressing VEGF164 cDNA into the subretinal space induced CNV in the rat eye.52 The compromise of the barrier between the retina and the choroid, caused by the needle puncture in this model, may be an important factor in producing CNV in these animals.

3.2.3Subretinal injection of Matrigel

In a recent study by Qiu and colleagues,53 CNV was induced in rabbits via sub-retinal injection of VEGF-enriched Matrigel growth matrix. While CNV developed in the animals treated with VEGF-enriched matrix, it also developed in those injected with Matrigel alone. The Matrigel serves as a slow-release reservoir of growth factors and a scaffold for growth of subretinal neovascularization. In this model, the inflammatory response to the Matrigel plays a key role in development of CNV.

3.2.4Prokineticin-1 expression in the retina

Recently, Tanaka and colleagues have produced transgenic mice that express the mitogen prokineticin-1 in the retina.54 Unlike transgenic animals that overexpress VEGF in the photoreceptors with subsequent retinal neovascularization, this model has the added benefit that the effects observed are specific to fenestrated vessels in the choroid. Because this mitogen is not normally expressed in the retina, the rhodopsin promoter was utilized to target its expression in the retina. The retinal vessels were not affected by this mutation, and the animals exhibited no disruption of Bruch’s membrane by choroidal vessels. In fact, the only pathological feature observed that was characteristic of AMD was a thickening of the choroid. Considering the absence of Bruch’s membrane penetration by the choroidal vessels, this transgenic mouse cannot be considered a successful model for CNV.

4.CONCLUSION

Choroidal neovascularization is a pathological condition in which proliferating choroidal blood vessels grow through Bruch’s membrane, penetrate the RPE, and extend into the subretinal space. There, the blood vessels leak fluid through their fenestrations and interendothelial cell junctions, ultimately leading to serous retinal detachment. CNV associated with the wet form of age-related macular degeneration is the major cause of vision loss in the elderly1 and also plays a major role in other diseases such

as Sorsby’s fundus dystrophy, Pseudoxanthoma Elasticum, ocular histoplasmosis and multifocal choroiditis.2 However, in spite of its prevalence, relatively little is known concerning the pathogenesis of CNV. In order to better understand this disease process and explore therapies to treat it, several experimental animal models of CNV have been developed. The most widely used of these models is laser-induced CNV in primates and rodents, but several knockout and transgenic mouse models exist as well. While none of these models accurately reproduce all clinical aspects of CNV, they have been successfully implemented to vastly increase our knowledge of new subretinal choroidal vessel formation.

REFERENCES

1.R. Klein, B. E. Klein, and K. L. Linton, Prevalence of age-related maculopathy. The Beaver Dam Eye Study, Ophthalmology 99 (6), 933-943 (1992).

2.Subfoveal neovascular lesions in age-related macular degeneration. Guidelines for evaluation and treatment in the macular photocoagulation study, Macular Photocoagulation Study Group, Arch. Ophthalmol. 109 (9), 1242-1257 (1991).

3.L. A. Yannuzzi, S. Negrào, T. Iida, C. Carvalho, H. Rodriguez-Coleman, J. Slakter, K. B. Freund, J. Sorenson, D. Orlok, and N. Borodoker, Retinal angiomatous proliferation in age-related macular degeneration, Retina 21 (5), 416-434 (2001).

4.J. D. Gass, Chorioretinal anastomosis probably occurs infrequently in type 2A idiopathic juxtafoveolar retinal telangiectasis, Arch. Opthtalmol. 121 (9), 1345-1346 (2003).

5.J. Francois, J. J. De Laey, E. Cambie, M. Hanssens, and V. Victoria-Troncoso, Neovascularization after argon laser photocoagulation of macular lesions, Am. J. Ophthalmol. 79 (2), 206-210 (1975).

6.S. J. Ryan, Subretinal neovascularization. Natural history of an experimental model, Arch. Ophthalmol. 100 (11), 1804-1809 (1982).

7.H. Miller, B. Miller, and S. J. Ryan, Correlation of choroidal subretinal neovascularization with fluorescein angiography, Am. J. Ophthalmol. 99 (3), 263-271 (1985).

8.T. Ishibashi, H. Miller, G. Orr, N. Sorgente, and S. J. Ryan, Morphologic observations on experimental subretinal neovascularization in the monkey, Invest. Ophthalmol. Vis. Sci. 28 (7), 1116-1130 (1987).

9.H. Miller, B. Miller, and S. J. Ryan, Newly-formed subretinal vessels. Fine structure and fluorescein leakage, Invest. Ophthalmol. Vis. Sci. 27 (2), 204-213 (1986).

10.H. Miller, B. Miller, T. Ishibashi, and S. J. Ryan, Pathogenesis of laser-induced choroidal subretinal neovascularization, Invest. Ophthalmol. Vis. Sci. 31 (5), 899-908 (1990).

11.S. H. Sarks, D. Van Driel, L. Maxwell, and M. Killingsworth, Softening of drusen and subretinal neovascularization, Trans. Ophthalmol. Soc. U.K. 100 (3), 414-422 (1980).

12.M. C. Killingsworth, J. P. Sarks, and S. H. Sarks, Macrophages related to Bruch’s membrane in age-related macular degeneration, Eye 4 (Pt 4), 613-621 (1990).

13.T. L. van der Schaft, C. M. Mooy, W. C. de Bruijn, and P. T. de Jong, Early stages of age-related macular degeneration: an immunofluorescence and electron microscopy study, Br. J. Ophthalmol. 77 (10), 657-661 (1993).

14.H. E. Grossniklaus, K. A. Cingle, Y. D. Yoon, N. Ketkar, N. L’Hernault, and S. Brown, Correlation of histologic 2-dimensional reconstruction and confocal scanning laser

microscopic imaging of choroidal neovascularization in eyes with age-related maculopathy, Arch. Ophthalmol. 118 (5), 625-629 (2000).

15.T. Nishimura, R. Goodnight, R. A. Prendergast, and S. J. Ryan, Activated macrophages in experimental subretinal neovascularization, Ophthalmologica 200 (1), 39-44 (1990).

16.H. Oh, H. Takagi, C. Takagi, K. Suzuma, A. Otani, K. Ishida, M. Matsumura, Y. Ogura, and Y. Honda, The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes, Invest. Ophthalmol. Vis. Sci. 40 (9), 1891-1898 (1999).

17.P. L. Penfold, J. M. Provis, and F. A. Billson, Age-related macular degeneration: ultrastructural studies of the relationship of leucocytes to angiogenesis, Graefes. Arch. Clin. Exp. Ophthalmol. 225 (1), 70-76 (1987).

18.J. P. Sarks, S. H. Sarks, and M. C. Killingsworth, Morphology of early choroidal neovascularisation in age-related macular degeneration: correlation with activity, Eye 11 (Pt 4), 515-522 (1997).

19.J. Ambati, B. K. Ambati, S. H. Yoo, S. Ianchulev, and A. P. Adamis, Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies, Surv. Ophthalmol. 48 (3), 257-293 (2003).

20.H. Miller, B. Miller, and S. J. Ryan, The role of retinal pigment epithelium in the involution of subretinal neovascularization, Invest. Ophthalmol. Vis. Sci. 27 (11), 1644-1652 (1986).

21T. Sakamoto, D. Soriano, J. Nassaralla, T. L. Murphy, A. Oganesian, C. Spee, D. R. Hinton, and S. J. Ryan, Effect of intravitreal administration of indomethacin on experimental subretinal neovascularization in the subhuman primate, Arch. Ophthalmol. 113 (2),

222-226 (1995).

22.D. Husain, M. Kramer, A. G. Kenny, N. Michaud, T. J. Flotte, E. S. Gragoudas, and J. W. Miller, Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment,

Invest. Ophthalmol. Vis. Sci. 40 (10), 2322-2331 (1999).

23.A. Pollack, G. E. Korte, A. L. Weitzner, and P. Henkind, Ultrastructure of Bruch’s membrane after krypton laser photocoagulation. I. Breakdown of Bruch’s membrane, Arch. Ophthalmol. 104 (9), 1372-1376 (1986).

24.A. Pollack, G. E. Korte, W. J. Heriot, and P. Henkind, Ultrastructure of Bruch’s membrane after krypton laser photocoagulation. II. Repair of Bruch’s membrane and the role of macrophages, Arch. Ophthalmol. 104 (9), 1377-1382 (1986).

25.A. Pollack, W. J. Heriot, Fraco, Fracs, and P. Henkind, Cellular processes causing defects in Bruch’s membrane following krypton laser photocoagulation, Ophthalmology 93 (8), 1113-1119 (1986).

26.E. T. Dobi, C. A. Puliafito, and M. Destro, A new model of experimental choroidal neovascularization in the rat, Arch. Ophthalmol. 107 (2), 264-269 (1989).

27.R. N. Frank, A. Das, and M. L. Weber, A model of subretinal neovascularization in the pigmented rat, Curr. Eye. Res. 8 (3), 239-247 (1989).

28.A. Pollack and G. E. Korte, Repair of retinal pigment epithelium and its relationship with capillary endothelium after krypton laser photocoagulation, Invest. Ophthalmol. Vis. Sci. 31 (5), 890-898 (1990).

29.X. Yi, N. Ogata, M. Komada, C. Yamamoto, K. Takahashi, K. Omori, and M. Uyama, Vascular endothelial growth factor expression in choroidal neovascularization in rats,

Graefes. Arch. Clin. Exp. Ophthalmol. 235 (5), 313-319 (1997).

30.M. Wada, N. Ogata, T. Otsuji, and M. Uyama, Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization, Curr. Eye Res. 18 (3), 203-213 (1999).

31.N. L. Zhang, E. E. Samadani, and R. N. Frank, Mitogenesis and retinal pigment epithelial cell antigen expression in the rat after krypton laser photocoagulation, Invest. Ophthalmol. Vis. Sci. 34 (8), 2412-2424 (1993).

32.F. Kinose, G. Roscilli, S. Lamartina, K. D. Anderson, F. Bonelli, S. G. Spence,

G.Ciliberto, T. F. Vogt, D. J. Holder, C. Toniatti, and C. J. Thut, Inhibition of retinal and choroidal neovascularization by a novel KDR kinase inhibitor, Mol. Vis. 11, 366-373 (2005).

33.M. El Bradey, L. Cheng, D. U. Bartsch, K. Appelt, N. Rodanant, G. Bergeron-Lynn, and

W.R. Freeman, Preventive versus treatment effect of AG3340, a potent matrix metalloproteinase inhibitor in a rat model of choroidal neovascularization, J. Ocul. Pharmacol. Ther. 20 (3), 217-236 (2004).

34.M. R. Robinson, J. Baffi, P. Yuan, C. Sung, G. Byrnes, T. A. Cox, and K. G. Csaky, Safety and pharmacokinetics of intravitreal 2-methoxyestradiol implants in normal rabbit and pharmacodynamics in a rat model of choroidal neovascularization, Exp. Eye Res. 74 (2), 309-317 (2002).

35.D. N. Zacks, E. Ezra, Y. Terada, N. Michaud, E. Connolly, E. S. Gragoudas, and

J.W. Miller, Verteporfin photodynamic therapy in the rat model of choroidal neovascularization: angiographic and histologic characterization, Invest. Ophthalmol. Vis. Sci. 43 (7), 2384-2391 (2002).

36.T. Tobe, S. Ortega, J. D. Luna, H. Ozaki, N. Okamoto, N. L. Derevjanik, S. A. Vinores,

C.Basilico, and P. A. Campochiaro, Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model, Am. J. Pathol. 153 (5), 1641-1646 (1998).

37.N. Kwak, N. Okamoto, J. M. Wood, and P. A. Campochiaro, VEGF is major stimulator in model of choroidal neovascularization, Invest. Ophthalmol. Vis. Sci. 41 (10), 3158-3164 (2000).

38.E. Sakurai, H. Taguchi, A. Anand, B. K. Ambati, E. S. Gragoudas, J. W. Miller, A. P. Adamis, and J. Ambati, Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization, Invest. Ophthalmol. Vis. Sci. 44 (6), 2743-2749 (2003).

39.P. S. Bora, J. H. Sohn, J. M. Cruz, P. Jha, H. Nishihori, Y. Wang, S. Kaliappan, H. J. Kaplan, and N. S. Bora, Role of complement and complement membrane attack complex in laserinduced choroidal neovascularization, J. Immunol. 174 (1), 491-497 (2005).

40.K. Takahashi, Y. Saishin, Y. Saishin, K. Mori, A. Ando, S. Yamamoto, Y. Oshima, H. Nambu, M. B. Melia, D. P. Bingaman, and P. A. Campochiaro, Topical nepafenac inhibits ocular neovascularization, Invest. Ophthalmol. Vis. Sci. 44 (1), 409-415 (2003).

41.M. S. Seo, N. Kwak, H. Ozaki, H. Yamada, N. Okamoto, E. Yamada, D. Fabbro, F. Hofmann, J. M. Wood, and P. A. Campochiaro, Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor, Am. J. Pathol. 154 (6), 1743-53 (1999).

42.S. J. Reich, J. Fosnot, A. Kuroki, W. Tang, X. Yang, A. M. Maguire, J. Bennett, and

M.J. Tolentino, Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model, Mol. Vis. 9, 210-216 (2003).

43.J. L. Edelman and M. R. Castro, Quantitative image analysis of laser-induced choroidal neovascularization in rat, Exp. Eye Res. 71 (5), 523-533 (2000).

44.L. E. Smith, E. Wesolowski, A. McLellan, S. K. Kostyk, R. D’Amato, R. Sullivan, and

P.A. D’Amore, Oxygen-induced retinopathy in the mouse, Invest. Ophthalmol. Vis. Sci. 35 (1), 101-111 (1994).

45.R. D’Amato, E. Wesolowski, and L. E. Smith, Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse, Microvasc. Res. 46 (2), 135-142 (1993).

46.R. J. Ulshafer, H. M. Engel, W. W. Dawson, C. B. Allen, and M. J. Kessler, Macular degeneration in a community of rhesus monkeys. Ultrastructural observations, Retina 7 (3), 198-203 (1987).

47.P. Hahn, Y. Qian, T. Dentchev, L. Chen, J. Beard, Z. L. Harris, and J. L. Dunaief, Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration, Proc. Natl. Acad. Sci. U. S. A. 101 (38), 13850-13855 (2004).

48.J. Ambati, A. Anand, S. Fernandez, E. Sakurai, B. C. Lynn, W. A. Kuziel, B. J. Rollins, and B. K. Ambati, An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice, Nat. Med. 9 (11), 1390-1397 (2003).

49.B. Sar, K. Oishi, A. Wada, T. Hirayama, K. Matsushima, and T. Nagatake, Induction of monocyte chemoattractant protein-1 (MCP-1) production by Pseudomonas nitrite reductase in human pulmonary type II epithelial-like cells, Microb. Pathog. 28 (1), 17-23 (2000).

50.N. Okamoto, T. Tobe, S. F. Hackett, H. Ozaki, M. A. Vinores, W. LaRochelle, D. J. Zacks, and P. A. Campochiaro, Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization, Am. J. Pathol. 151 (1), 281-291 (1997).

51.Y. Oshima, S. Oshima, H. Nambu, S. Kachi, S. F. Hackett, M. Melia, M. Kaleko,

S.Connelly, N. Esumi, D. J. Zack, and P. A. Campochiaro, Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization, J. Cell Physiol. 201 (3), 393-400 (2004).

52.K. Spilsbury, K. L. Garrett, W. Y. Shen, I. J. Constable, and P. E. Rakoczy, Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to development of choroidal neovascularization, Am. J. Pathol. 157 (1), 135-144 (2000).

53.G. Qiu, J. M. Stewart, S. Sadda, R. Freda, S. Lee, D. Guven, E. de Juan, Jr., and S. E. Varner,

Anew model of experimental subretinal neovascularization in the rabbit, Exp. Eye Res. 83 (1), 141-152 (2006).

54.N. Tanaka, M. Ikawa, N. L. Mata, and I. M. Verma, Choroidal neovascularization in transgenic mice expressing prokineticin 1: an animal model for age-related macular degeneration, Mol. Ther. 13 (3), 609-616 (2006).

Chapter 3

RODENT MODELS OF OXYGEN-INDUCED RETINOPATHY

Susan E. Yanni,1 Gary W. McCollum,2 and John S. Penn1,2

Departments of 1Cell & Developmental Biology and 2Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nasvhille, Tennessee

1.BACKGROUND

In 1942, Terry first described ROP as a disease of prematurity, characterized by retinal neovascularization.1 An epidemic of ROP occurred during the 1950’s, exposing the need for research focused on the identification and characterization of the pathogenesis of ROP. In 1951, Campbell proposed that the incidence of ROP was linked to the supplemental oxygen administered to premature infants with under-developed pulmonary function.2 During the 1950’s, several convincing studies correlated the use of supplemental oxygen with the incidence and progression of ROP.3-7 This led to the rigorous monitoring of the oxygen being given to premature infants. Consequently, the percentage of blindness attributed to ROP dropped from 50% in 1950 to just 4% in 1965.8 The 1970’s and 1980’s saw

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an increased incidence of ROP,9 presumably from the increased survival of very low-birth-weight premature infants.

According to the most recent estimates of the National Eye Institute, each year approximately 14,000-16,000 premature infants (classified as those weighing 1250 grams or less, and being born prior to 31 weeks’ gestation) develop some stage of ROP. Of these infants, 400-600 will suffer from ROPinduced blindness. ROP is the leading cause of childhood blindness in the developed world.10 For this reason, among others, research focused on understanding physiological and pathological retinal neovascularization is highly significant.

Several animal models have been developed that approximate human ROP. To emphasize the differences between human ROP and experimentally induced retinopathy in animals, the term oxygen-induced retinopathy (OIR) is often used. Rodent models of OIR are widely used to study the cellular and molecular aspects of physiological and pathological retinal neovascularization.

1.1Normal human retinal vascularization

The retina is one of the last organ systems of the developing fetus to undergo vascularization, beginning at approximately 16 weeks’ gestation. At this time, vasculogenesis (the de novo formation of blood vessels from mesodermal precursor cells) occurs, beginning in the most posterior region of the superficial retina (the optic disk) and proceeding to the periphery. At 25 weeks’ gestation, angiogenesis (the development of new capillaries from pre-existing vessels) begins, proceeding also from the optic disk in a peripheral wave, resulting in the development of a deeper (more sclerad) vessel network. It is believed that the hypoxic uterine environment (30 mm Hg) drives retinal vascularization during normal gestation. In utero, retinal hypoxia induces pro-angiogenic growth factors that stimulate the growth of retinal blood vessels. These blood vessels satisfy the increasing demands of the developing fetal retina for oxygen. Complete vascularization is attained at approximately 36-40 weeks’ gestation, and the relatively hyperoxic (55-80 mm Hg) postnatal environment effectively prevents further vasoproliferation.11,12

1.2The pathogenesis of ROP

The pathogenesis of ROP is biphasic. The first phase of ROP results in the vasoattenuation and pruning of the existing vasculature. This is followed by the second, proliferative phase, characterized by retinal neo- vascularization.7,13-15 Vasoattenuation is a cessation of the retinal vascularization

process, which occurs after the infant has been placed on supplemental oxygen therapy. At this time, the oxygen tension within the retina sufficiently inhibits the hypoxia-induced production and secretion of vascular growth factors. Diminished growth factor production results in an incompletely vascularized retinal periphery, a hallmark of ROP. Vasoattenuation results in retinal avascularity. Retinal avascularity results in retinal ischemia when oxygen supplementation to the infant is discontinued because the development and maturation of the neural components within the retina demand more oxygen than they are receiving. At this time, retinal hypoxia ensues. Retinal hypoxia induces the onset of the second, vasoproliferative phase of ROP. Vasoproliferation is best described as deregulated angiogenesis, resulting in the production of fragile, non-patent vascular structures that grow through the inner limiting membrane of the retina into the vitreous cavity. These abnormal vascular structures are often referred to as preretinal neovascular tufts, and they predispose affected infants to intravitreal hemorrhages, retinal detachment, and subsequent vision loss.

The severity of ROP is inversely proportional to gestational age.16 Because retinal vascularization is completed at, or near, the time of birth, premature infants demonstrate an increased area of retinal avascularity. Placing these infants in a post-natal hyperoxic environment leads to vasoattenuation of the already sparse vasculature. Returning the infants to a hypoxic room air environment leads to retinal hypoxia and the subsequent development of ROP. The larger the avascular area at the time of birth, the more severe the retinal hypoxia upon return to room air, and hence, the more severe the ROP.

Roughly half the infants that develop ROP do so while receiving supplemental oxygen therapy. Hypoxia or variable oxygen, therefore, is not the sole determinant in the pathogenesis of ROP. Developmental timing may regulate the responses of the immature retina to oxygen. ROP involves a complex sequence of pathological events.

2.RODENT MODELS OF ROP

2.1Mouse

2.1.1Mouse vascular development

Unlike the human, whose retinal vasculature derives from spindle-shaped mesenchymal precursor cells of the hyaloid artery in a vasculogenic process, research has provided evidence that the retinal vasculature of the mouse

derives from immature retinal astrocytes in an angiogenic process.17 The contributions of vasculogenesis and angiogenesis to retinal vascularization may be species-specific.18 Regardless, the retinal vasculature of a newborn mouse is comparable to that of an infant at 25 weeks’ gestation who is at risk for developing ROP.19 For this reason, the retinal vasculature of the newborn mouse pup is an attractive model of the premature infant’s retinal vasculature.

2.1.2Earliest mouse model

After the initial identification of ROP, experiments were conducted in both laboratory and clinical settings to ascertain the effect of oxygen therapy on retinal angiogenesis. In 1954, Gyllensten and Hellstrom exposed newborn mouse pups to 100% oxygen for 1-3 weeks. Ocular examination after oxygen withdrawal revealed that approximately one-third of the animals experienced hemorrhages in both the vitreous and the anterior chamber. It was further demonstrated that exposing the pups to 100% oxygen and subsequently removing the pups to room air for 5 days induced vasoproliferation of the retinal vessels, a hallmark of ROP.19 It should be noted that removal to room air was required for induction of the ROP-like vasoproliferative changes.20

2.1.3Current mouse model

Gyllensten and Hellstrom provided the research community with a means to explore ROP in greater detail. Early studies were inconclusive, yielding highly varied results. One of the confounding factors in the early attempts to model ROP was the fact that hyperoxic exposure of newborn mice, followed by removal to room air, resulted in the proliferation and engorgement of the hyaloid.21,22 Reasoning that the hyaloidopathy might explain the observed variability in the early attempts to model ROP, Smith and colleagues proposed a novel method for inducing retinopathy in the mouse, a model that sought to minimize any hyaloidopathy.23

The Smith model allows a consistent and reliable reproduction of ROP. The widely used method involves exposing mice at postnatal day 7 (P7) to 75% oxygen for 5 days to induce vaso-attenuation and atrophy of the centralized portion of the retinal vascular bed (Figure 1). Removal of the mice to room air for variable lengths of time induces retinal vasoproliferation and revascularization of the central retina. At P17-P21, the eyes of the mice are analyzed for the presence of neovascularization (Figure 2).