Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

Chapter 2

ANIMAL MODELS OF CHOROIDAL

NEOVASCULARIZATION

Monika L. Clark,1 Jessica A. Fowler,2 and John S. Penn1,2

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

1.BACKGROUND

There are many ocular conditions in which pathological angiogenesis is a key component. Ocular angiogenesis may occur as preretinal neovascularization, deep retinal neovascularization, or subretinal neovascularization (Figure 1). Subretinal neovascularization occurs in conditions such as age-related macular degeneration (AMD), Sorsby’s fundus dystrophy, Pseudoxanthoma Elasticum, ocular histoplasmosis and multifocal choroiditis. AMD is the leading cause of blindness in individuals 65 years or older in developed countries.1 Although it encompasses a wide range of pathologies, the disease is generally classified into two forms: “dry” and “wet.” Of these

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J.S. Penn (ed.), Retinal and Choroidal Angiogenesis, 41–56.

© Springer Science+Business Media B.V. 2008

two recognized forms, wet AMD is the more debilitating and life-changing in its progression. Fortunately, it is also the less prevalent form, affecting only about 10% of the general AMD population.2 Wet AMD typically becomes manifest with progressive choroidal neovascularization (CNV) characterized by abnormal blood vessel growth of the choriocapillaris through the retinal pigment epithelial (RPE) layer.1 Because the consequences of wet AMD can be so devastating, the vision research community has invested a tremendous amount of time and effort in its attempts to better understand the progression of this form of AMD. A major focus of this effort has involved the development of animal models of CNV, which are essential for identifying early diagnostic markers and for developming of better drug regimens.

Figure 2-1. Schematic diagram of pathological blood vessel growth within the posterior segment. A. Normal vasculature; B. Subretinal neovascularization (eg. age-related macular degeneration); C. Deep retinal neovascularization (eg. retinal angiomatous proliferation,3 Type II idiopathic juxtafoveolar telangiectasia4); D. Pre-retinal neovascularization (eg. retinopathy of prematurity, diabetic retinopathy).

2.ANIMAL MODELS OF LASER-INDUCED CNV

2.1Primate

2.1.1Development of the primate model

The basis for the development of a laser-induced CNV model was the finding that argon laser photocoagulation, used clinically to obliterate neovascularization in the treatment of macular degeneration, could actually induce subretinal neovascularization.5 In 1982, Ryan and colleagues administered argon laser photocoagulation to primate eyes with the intention of inducing CNV rather than treating it, thus using criteria contradictory to what is used clinically.6 Specifically, following sedation and pupillary dilation, high intensity (600-900 mW), short duration (0.1 s) laser burns of a small exposure size (100 μm) were applied through a slit lamp and a Goldmann fundus contact lens to three distinct areas of the fundus of rhesus monkeys. Subretinal neovascularization, assessed by fluorescein leakage during angiography, was observed after three weeks in 39% of laser-induced lesions in the macular region and less than 3% of lesions located nasal to the optic nerve head and in the periphery. The increased incidence of subretinal neovascularization in the lesions of the macular region correlated well with the high predisposition of the human macula to develop CNV, an initial appeal of this new model.

Closer inspection of the morphology of laser burn sites generated in the eyes of cynomolgus monkeys provided more direct evidence that laser photocoagulation can experimentally induce CNV, lending even more promise to the relevance of this model. Morphological assessment of cross sections of the laser lesions one day after laser treatment revealed that the choroid, Bruch’s membrane and RPE cells were disrupted or destroyed. By approximately one week following laser treatment, choroidal vessels had proliferated through the laser-induced breaks in Bruch’s membrane into the subretinal space and were observed overlying proliferating RPE cells.7,8

Histological examination of cross-sectioned lesions confirmed that all lesions exhibiting fluorescein leakage and pooling contained CNV, and interestingly, 80% of non-leaky lesions also contained subretinal vessels that morphologically had the potential to leak fluorescein. That is, like the leaky vessels, the walls of these non-leaky vessels contained diaphragmed fenestrations and intermediate interendothelial cell junctions. Fluorescein leakage was not observed in these vessels due to the absence of a fluid-filled space overlying the subretinal vessels that occurs as the result of serous

retinal detachment.7,9 Thus, the lack of fluorescein leakage is not always an accurate representation of the absence of subretinal neovascularization.

Together, the initial experiments demonstrated that high intensity laserinduced rupture of Bruch’s membrane is a highly effective and reproducible method for inducing CNV.

2.1.2Advantages and disadvantages of the primate model

The primate model of laser-induced CNV has proven to be a valuable tool for investigating the pathogenesis of CNV, especially given that it is similar in many respects to the disease process in humans. As mentioned previously, clinical laser photocoagulation induces CNV in humans when Bruch’s membrane is ruptured, and the features of this photocoagulation-induced neovascularization are the most similar to those produced in the primate model. Laser-induced CNV mimics many features of CNV resulting from AMD as well. In both cases, new choroidal vessels migrate through holes in Bruch’s membrane into the subretinal space, where fluid accumulates. These

new vessels contain fenestrations and interendothelial cell junctions characteristic of choroidal vessels.10,11 Also, in both laser-induced CNV and

AMD, polymorphonuclear leukocytes and macrophages can be observed around the budding endothelial cells, and macrophages are also often found around thinned or ruptured areas of Bruch’s membrane, indicating that an intense inflammatory response is a feature of both forms of CNV.8,12-18

The growth factor profiles in CNV resulting from AMD and laser injury are comparable as well. For example, in both cases, immunohistochemistry has revealed that vascular endothelial growth factor (VEGF) is expressed in the RPE and leukocytes. VEGF receptors, basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β) and tumor necrosis factor- α (TNF-α) are likewise expressed in the same cell types in CNV stimulated by both processes.19

Its similarities to the human condition, as well as its high reproducibility, make the primate model an attractive and highly accepted one in which to study CNV. However, the laser injury model does not perfectly mimic the pathogenesis of CNV in human disease states. Laser-induced CNV is a wounding model, and consequently neovascularization occurs in a manner similar to that which occurs in the process of wound healing. Also, the CNV in the laser lesions regresses with time, or undergoes involution, as demonstrated by decreased vessel leakage in fluorescein angiograms. The

cessation of leakage is due to RPE proliferation and subsequent envelopment of the new vessels.10,20 This aspect of laser-induced CNV contrasts with that

of the human condition, where CNV is more chronic and leakage can

continue for years. Furthermore, the induction of CNV in this model, as well as in other experimental models, occurs in relatively young eyes, whereas AMD in humans occurs in the eyes of the elderly. Therefore, when using the laser-induced CNV as a model to study the pathogenesis of CNV as it occurs in humans, the findings must be interpreted with caution, keeping these differences in mind.

2.1.3Knowledge gained from the primate model

In spite of the discrepancies between the pathogenesis of the CNV induced by laser photocoagulation and by the various human conditions, the primate model has been a valuable tool for increasing our understanding of CNV. From it, a great deal of knowledge has been gained regarding the natural progression of CNV.8-10 It has been used to define the roles of the RPE in

reestablishing the blood-retina barrier, in the scarring process, and in involution of new subretinal vessels.10,20 The laser-induced model in primates

has also been useful in investigating drug treatments and developing other therapeutic strategies to prevent CNV. For example, intravitreal administration of non-steroidal anti-inflammatory drugs has been shown to prevent angiographic leakage in this model for up to eight weeks,21 and photodynamic therapy using verteporfin prevented angiographic leakage for at least four weeks.22 Laser-induced CNV in primates is an excellent model in which to test the long-term effects of potential therapeutic strategies for AMD prior to the onset of clinical trials.

2.2Rat

2.2.1Development of the rat model

The primate model of laser-induced CNV provided the best method of the time for studying subretinal neovascularization. However, expense and availability limited its widespread use, and the need for a rodent model was evident. Pollack and colleagues reported several studies in which laser photocoagulation in rats produced CNV when Bruch’s membrane was breached.23-25 Subsequently, in 1989 a rat model of laser-induced CNV was developed by two different groups, Dobi and colleagues26 and Frank and colleagues,27 by administering krypton laser radiation between the major retinal vessels of the fundus. In this protocol, the animals were anesthetized and pupils dilated, and a handheld coverslip was used as a contact lens for the maintenance of corneal clarity during photocoagulation. The criteria for laser treatment included a small exposure size (100 or 500 μm), a power of

50 to 100 mW, and an exposure duration of 0.02- 0.1s. Two weeks following this procedure, fluorescein leakage indicated the presence of CNV in 30% of laser-induced lesions, while histological examination revealed that CNV actually occurred in 60% of the laser lesions. Again, this discrepancy is due to the lack of fluid overlying the subretinal vessels in some eyes, a feature necessary for the pooling of dye. These lesions exhibited disruption of Bruch’s membrane and degeneration of RPE as well as the photoreceptors, outer nuclear layer, outer plexiform layer, and part of the inner nuclear layer of the retina. The choroidal capillaries proliferated through the break in Bruch’s membrane into these disrupted outer layers of the retina.

Current rat models of laser-induced CNV have evolved from the earliest model. In addition to krypton, argon and diode laser photocoagulators are used, and there are some variations in the specific laser treatment criteria. However, the premise for all of these protocols is the same. A laser beam is focused on Bruch’s membrane with the intention of rupturing it, as evidenced by subretinal bubble formation with or without intraretinal or choroidal hemorrhage at the lesion site.

2.2.2Advantages and disadvantages of the rat model

In comparison to the primate model, the rat model of laser-induced CNV is advantageous because of the high availability, low cost, and ease of maintenance of rats. The rat model is also more practical for investigating the efficacy of therapeutic strategies in prevention or treatment of CNV, where large sample sizes are beneficial. While it shares many of the benefits attributed to the primate model, the rat model also shares the drawback of producing CNV that, unlike in human disease states, regresses after a short time.28 Furthermore, the rat model is somewhat less ideal for studying CNV as it relates to humans since primate eyes are anatomically and functionally more analogous to human eyes. Nevertheless, the advantages conferred by the rat model far outweigh these disadvantages, causing it to be one of the most extensively used methods for studying CNV today.

2.2.3Knowledge gained from the rat model

The rat model of laser-induced CNV has been used to obtain much

information about the temporal and spatial expression patterns of various growth factors, such as VEGF29,30 and bFGF,31 during the progression of

CNV. This knowledge is necessary for increasing our limited understanding of the pathogenesis of CNV. As mentioned previously, the rat model is valuable for investigating the modulation of CNV by various drugs or treatment strategies. It has been utilized to explore the efficacy of anti-

angiogenic agents administered orally,32 by intravitreal injection,33 and by intravitreal implants.34 The rat model is useful for evaluating the effect of such strategies on CNV before testing them in the primate model or human clinical trials. For example, the value of verteporfin photodynamic therapy was evaluated in this model.35

2.3Mouse

2.3.1Development of the mouse model

In 1998, Tobe and colleagues produced a murine model of laser-induced CNV.36 The method for inducing CNV in mice was similar to that which produces CNV in rats. Adult C57BL/6J mice were anesthetized and their pupils dilated. Krypton laser photocoagulation was administered to the posterior retina through a slit lamp using a cover slip as a contact lens. The laser treatment criteria consisted of a spot size of 50 μm, a power of 350-400 mW, and an exposure duration of 0.05 s. As evidenced by bubble formation, Bruch’s membrane was successfully ruptured in 87% of the laser burns. In addition to the disruption of Bruch’s membrane, all layers of the choroid were destroyed within the burn site and ablative damage occurred to the outer retina. Fluorescein leakage and histopathological evidence revealed that over 80% of the lesions contained CNV one week after laser treatment. These new vessels, characterized by large lumens and fenestrations, proliferated into the subretinal space where they were partially enveloped by the RPE.

Presently, laser-induced CNV is produced in mice by methods similar to that published by Tobe et al. The laser treatment criteria might have slight variations, and krypton, argon, or diode laser photocoagulators may be used.

2.3.2Advantages of the mouse model

The mouse model of laser-induced CNV possesses a distinct advantage over the primate and rat models, namely, that manipulation of gene expression is possible. The impact of specific genes on the development of CNV can be evaluated by observing the effects of laser photocoagulation administered to mice overexpressing or underexpressing these genes. The molecular mechanisms underlying the pathogenesis of CNV as well as anti-angiogenic approaches for therapy can thus more readily be explored using the mouse as an experimental animal model in laser-induced CNV.

2.3.3Knowledge gained from the mouse model

Like the rat model, the mouse model of laser-induced CNV has been implemented to further define the role of various growth factors in the development of CNV. For instance, it has been used to show that FGF2 is not necessary for the occurrence of CNV,36 whereas VEGF is a major stimulator.37 The roles of cellular adhesion molecules have been explored in the mouse model,38 as well as the role of complement, since inflammation is thought to be an important part of the pathogenesis of CNV.39 Furthermore, this model has increasingly been used to test the efficacy of various potential anti-angiogenic treatments. The effect of a non-steroidal anti-inflammatory drug administered topically,40 a kinase inhibitor taken orally,41 and subretinal injection of siRNA targeting VEGF42 are a few examples of therapeutic strategies that have successfully inhibited neovascularization in the mouse model.

2.4Evaluation of laser-induced CNV

When each of the experimental animal models of laser-induced CNV described above was first introduced, the primary methods for evaluating the extent of subretinal neovascularization were fluorescein angiography and histological examination of serial cross sections. While these methods are still widely used today, they are not without limitations. Leakage of fluorescein is not easily quantified and cannot always be directly correlated to the amount of CNV. Its use is limited in rodent eyes due to difficulty in performing fundus photography and a poor view of the periphery. Quantifying new vessels in histological sections requires thorough sampling of many sections and can be laborious.

In 2000, Edelman and Castro introduced a new, high-resolution angiographic method to assess experimentally induced CNV using high molecular weight fluorescein isothiocyanate (FITC)-dextran.43 This method had been previously employed to examine neovascularization from oxygeninduced retinopathy in mouse retinal flatmounts.44 FITC-labeled two million molecular weight dextrans are retained in the blood vessels after fixation allowing the entire vasculature to be viewed by microscopy.45 To examine CNV, FITC-dextran is injected into the left ventricle of animals that have undergone laser photocoagulation. RPE-choroid-sclera flatmounts of the eyes must then be obtained. This is done by hemisecting the eye, peeling away the neural retina and making four incisions in the eyecup in order to flatten it on a microscope slide with the RPE on top. The entire choroid can be visualized by a fluorescence microscope, and whole mount images can be

captured and analyzed in order to obtain a precise measurement of the neovascular area (Figure 2).

Figure 2-2. FITC-dextran-perfused choroidal flatmount. A diode pumped solid state laser and slit lamp delivery system were used to deliver laser burns to the eyes of C57BL/6J mice. Laser parameters were 50 μm spot size, 0.10 s exposure time, and 150 mW power. Two weeks following laser treatment FITC-dextran (2 x 106 MW) in PBS solution was injected into the mouse via the tail vein. Eyes were enucleated, and choroidal flatmounts were obtained by removing the cornea, iris, and retina and peeling away the retinal pigment epithelium. CNV at one laser lesion site is shown. The green color demonstrates the extent of fluorescently tagged dextran accumulation within the subretinal space.

3.OTHER ANIMAL MODELS OF CNV

While laser photocoagulation-induced CNV remains a widely utilized model, there are various animal models in which CNV develops spontaneously. These models include transgenic and knockout mice, as well as mice in which the retinas are transfected with relevant growth factors, such as VEGF, FGF, and others. The nature of the animal model depends on the methods used to develop CNV. For example, in several of the transgenic mouse models of CNV, photoreceptor degeneration or a breaching of Bruch’s membrane is necessary for initiation of abnormal vessel growth. Because the progression of CNV is largely dependent on the level of integrity in the barrier between the choroid and the retina, factors that

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contribute to loss of this integrity will have a major impact on the development of CNV.

3.1Correlation between drusen and CNV

When cells of the RPE layer lose their ability to effectively remove waste produced by the photoreceptors during disk membrane turnover, these materials accumulate and ultimately form localized deposits between the basement membrane of the RPE and Bruch’s membrane. These deposits are commonly known as drusen (singular, druse).19 As drusen continue to accumulate in the subretinal spaces, RPE cell death can occur. This can lead to further photoreceptor damage, since the cells of the RPE layer are essential for filtering out debris to ensure healthy and functional photoreceptors.19 The presence of drusen constitutes a landmark feature of AMD, and in fact, there is a strong correlation between the number of drusen present and the rate of CNV progression. Although the presence of drusen correlates with CNV, it is not the only factor involved. Consequently, AMD animal models, such as the rhesus monkey, where age-related druse accumulation is the primary abnormality observed,46 are not adequate models of CNV.

3.1.1Ceruloplasmin (Cp) and Hephaetin (Heph) deficient mice

Hahn and colleagues observed the accumulation of iron deposits in retinas and RPE of mice deficient in ceruloplasmin (Cp) and its homolog hephaestin (Heph).47 There is evidence that implicates these proteins in iron export from cells, explaining the increases in retinal iron in these knockout mice. In retinas of these mice, subretinal neovascularization was observed in areas of RPE hyperplasia and photoreceptor degeneration. The source of this neovascularization was not determined by the investigators. The dominating feature of this model is the accumulation of iron, otherwise considered drusen, which further constrains these animals as a model for drusen deposition rather than for CNV.

3.1.2Ccl-2 and Ccr-2 deficient mice

Ambati and colleagues have recently generated a mouse model that spontaneously develops a clinical syndrome very similar to AMD.48 These mice are deficient in Ccl-2, a monocyte chemoattractant protein-1, and Ccr- 2, its C-C chemokine receptor-2. Because of their role in recruitment and accumulation of monocytes in various diseases, animals that are deficient in Ccl-2 and Ccr-2 are unable to recruit macrophages that subsequently