Animal Model Of Choroidal Neovascularization Research Articles

Multimodal imaging of laser-induced choroidal neovascularization in pigmented rabbits

This study aimed to demonstrate longitudinal multimodal imaging of laser photocoagulation-induced choroidal neovascularization (CNV) in pigmented rabbits. Six Dutch Belted pigmented rabbits were treated with 12 laser lesions in each eye at a power of 300 mW with an aerial diameter spot size of 500 μm and pulse duration of 100 ms. CNV progression was monitored over a period of 4 months using different imaging techniques including color fundus photography, fluorescein angiography (FA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT). All treated eyes developed CNV with a success rate of 100%. The margin and morphology of CNV were detected and rendered in three dimensions using PAM and OCT. The CNV was further distinguished from the surrounding melanin and choroidal vessels using FDA-approved indocyanine green dye-enhanced PAM imaging. By obtaining PAM at 700 nm, the location and density of CNV were identified, and the induced PA signal increased up to 59 times. Immunohistochemistry with smooth muscle alpha-actin (αSMA) antibody confirmed the development of CNV. Laser photocoagulation demonstrates a great method to create CNV in pigmented rabbits. The CNV was stable for up to 4 months, and the CNV area was measured from FA images similar to PAM and OCT results. In addition, this study demonstrates that contrast agent-enhanced PAM imaging allows for precise visualization and evaluation of the formation of new blood vessels in a clinically-relevant animal model of CNV. This laser-induced CNV model can provide a unique technique for longitudinal studies of CNV pathogenesis that can be imaged with multimodal imaging.

Fiji-Assisted Automatic Quantitative Volumetric Analysis of Choroidal Neovascularization in a Laser-Induced Choroidal Neovascularization Mouse Model.

The laser-induced choroidal neovascularization (CNV) mouse model is the most frequently used animal model of CNV. To test new therapeutic agents that suppress CNV, CNV measurement in an accurate, precise, and efficient manner is important. We present the utility of Fiji-assisted automatic volumetric quantification of CNV in comparison with two-dimensional CNV analyses. Laser-induced CNV was induced in C57BL/6J mice according to the established protocol. After CNV induction, mice were treated with intravitreal injection of either phosphate-buffered saline solution (PBS) or Aflibercept, an anti- vascular endothelial growth factor agent. One week after intravitreal injection treatment, retina pigment epithelium/choroid flat mounts were stained with rhodamine-conjugated Griffonia simplicifolia lectin B4. Z-stacks of the entire CNV lesion obtained using laser confocal microscopy were converted to binary stacks using Fiji for volumetric analysis. Data from volumetric analysis and multiple area analyses from z-stack projection, the maximum, blindly selected, and mean area were compared using Fiji. Fiji-assisted automatic quantitative volumetric analysis of CNV was useful in detecting experimental outliers in laser-induced CNV genesis and provided accurate and precise measurements of total areas of CNV with a lower coefficient of variance (63%) than in multiple area analyses, including the z-stack projection, maximum, blindly selected, and mean areas (67%, 67%, 76%, and 69%, respectively). A lower coefficient of variance in volumetric analysis than in multiple area analyses resulted in increased statistical significance when comparing CNV lesions in PBS, and Aflibercept-treated groups; P = 0.004 in volumetric analysis versus P value range between 0.03 and 0.05 in multiple area analyses. Fiji-assisted automatic quantitative volumetric analysis can be useful for accurate, precise, and efficient measurements of total areas of CNV. Volumetric measurement for CNV lesions can be advantageous in verifying the efficacy of therapeutic agents in the laser-induced CNV mouse model.

Animal Models of Choroidal Neovascularization: A Systematic Review.

PurposeAnimal models of choroidal neovascularization (CNV) are extensively used to characterize the pathophysiology of chorioretinal diseases with CNV formation and to evaluate novel treatment strategies. This systematic review aims to give a detailed overview of contemporary animal models of CNV.MethodsA systematic search was performed in PubMed and EMBASE from November 20, 2015, to November 20, 2020, for mammalian animal models of CNV. Following inclusion by two investigators, data from the articles were extracted according to a predefined protocol.ResultsA total of 380 full articles, representing 409 independent animal models, were included. Mice were by far the most utilized animal (76%) followed by rats and non-human primates. The median age of rodents was 8 weeks but with a wide range. Male animals were used in 44% of the studies, but 32% did not report the sex. CNV was laser induced in 89% of the studies, but only 44% of these reported sufficiently on standard laser parameters. Surprisingly, 28% of the studies did not report a sample size for quantitative CNV evaluation. Less than half of the studies performed quantitative in vivo evaluation, and 73% evaluated CNV quantitatively ex vivo. Both in vivo and ex vivo evaluations were conducted primarily at day 7 and/or day 14.ConclusionsThe laser-induced mouse model is the predominant model for experimental CNV. The widespread use of young, healthy male animals may complicate clinical translation, and inadequate reporting challenges reproducibility. Definition and implementation of standardized methodologic and reporting guidelines are attractive.

Preclinical SPECT Imaging of Choroidal Neovascularization in Mice Using Integrin-Binding [99mTc]IDA-D-[c(RGDfK)]2.

Integrin ɑvβ3, an adhesion molecule overexpressed in neovascular endothelial cells, is involved in ocular angiogenesis. Integrin ɑvβ3-binding arginine-glycine-aspartic acid (RGD) peptide has been used to target and visualize new vessels. We explored the use of integrin ɑvβ3-targeted RGD peptide ([99mTc]IDA-D-[c(RGDfK)]2) for in vivo molecular imaging of choroidal neovascularization (CNV). To induce CNV in animals, the right eyes of C57BL/6 mice were treated with retinal argon laser photocoagulation. CNV formation was confirmed on immunohistopathological examination of retinal and choroidal tissues. To explore the association of integrin with angiogenesis, integrin mRNA expression in the retinal and choroidal tissues was measured using real-time reverse transcriptase-polymerase chain reaction. For in vivo imaging, mice were intravenously injected with [99mTc]IDA-D-[c(RGDfK)]2 and single-photon emission computed tomography (SPECT) images of [99mTc]IDA-D-[c(RGDfK)]2 were obtained before laser induction (baseline) and at 1, 3, 7, and 14days post-induction. CNV-induced regional alterations were measured using radiotracer uptake count. Immunohistopathological examination revealed that CNV lesions showed intense fluorescein isothiocyanate (FITC)-D-[c(RGDfK)]2 immunofluorescence, in contrast to the normal retina and choroid. Retinal integrin mRNA expression peaked at day 1 following CNV induction. On SPECT images using [99mTc]IDA-D-[c(RGDfK)]2, the radio-uptake count in eyes with CNV was significantly higher than in normal controls on days 1-7 (all p < 0.05), with a peak at day 3 representing the highest angiogenic activity. Our preclinical data demonstrated that [99mTc]IDA-D-[c(RGDfK)]2 can detect CNV and its associated angiogenesis in an animal model of CNV. SPECT imaging using an integrin ɑvβ3-targeted RGD peptide radiotracer may be a useful tool for in vivo functional molecular imaging of CNV.

AMG 386, a Selective Angiopoietin 1/2-Neutralizing Peptibody, Inhibits Angiogenesis in Models of Ocular Neovascular Diseases

To determine whether systemic treatment with AMG 386, a selective angiopoietin 1/2-neutralizing peptibody, inhibits neovascular processes in animal models of ocular disease. AMG 386 was tested in a laser-induced choroidal neovascularization (CNV) model in monkeys using fluorescein angiography. The biodistribution of (125)I-AMG 386 was determined in cynomolgus monkeys by whole-body autoradiography and radioanalysis of ocular tissues. A murine retinopathy of prematurity (ROP) model was used to examine the effect of AMG 386 on established and newly formed retinal vessels, either as a single agent or when combined with VEGF inhibition.AMG 386 pharmacokinetics were evaluated in each model. In the CNV model, AMG 386 significantly decreased fluorescent angiographic leakage and reduced fibroplasia, indicating an impaired healing response consistent with angiogenesis blockade. Radiolabeled AMG 386 was widely distributed across ocular tissues, with highest concentrations in the choroid, cornea, retinal pigmented epithelium, iris/ciliary body, and sclera. In the ROP model, AMG 386 prevented pathologic retinal angiogenesis when administered from P8 to P16 but transiently impeded regression of these abnormal vessels when administered from P17 to P23. Combining AMG 386 with VEGF inhibition led to cooperative prevention of retinal angiogenesis in this model. No AMG 386-related ocular toxicities occurred, and no treatment-related clinical observations were made in any of the studies. In this study, AMG 386 inhibited angiogenesis in animal models of CNV and ROP, supporting investigation of AMG 386 for the treatment of ocular neovascular diseases in the clinical setting.

Relationship between Complement Membrane Attack Complex, Chemokine (C-C Motif) Ligand 2 (CCL2) and Vascular Endothelial Growth Factor in Mouse Model of Laser-induced Choroidal Neovascularization

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

Animal models of choroidal and retinal neovascularization

There have been numerous types of animal models of choroidal neovascularization (CNV) and retinal neovascularization (RNV). Understanding the pathobiology of CNV and RNV is important when evaluating and utilizing these models. Both CNV and RNV are dynamic processes. A break or defect in Bruchs' membrane is necessary for CNV to develop. This may be induced with a laser, mechanically via surgery, or in the setting of transgenic mice. Some of the transgenic mouse models spontaneously develop RNV and/or retinal angiomatous proliferation (RAP)-like lesions. The pathogenesis of RNV is well-known and is generally related to ischemic retinopathy. Models of oxygen-induced retinopathy (OIR) closely resemble retinopathy of prematurity (ROP). The streptozotocin (STZ) rat model develops features similar to diabetic retinopathy. This review summarizes general categories and specific examples of animal models of CNV and RNV. There are no perfect models of CNV or RNV and individual investigators are encouraged to choose the model that best suits their needs.

SiRNA therapeutics for age-related macular degeneration: promises and pitfalls

With the elucidation of the link between age-related macular degeneration and angiogenesis, and the success of therapies targeting VEGF, recent years have seen a great interest in molecular therapeutics for this disease. Another recent scientific discovery, RNA interference, offers the ability to silence specific genes, as well as hope of more powerful and specific therapies with negligible toxicity. In animal models of choroidal neovascularization, several siRNA therapies have shown promising results, including agents targeting VEGF, VEGF receptor 1, and a separate ischemia-induced molecule, RTP801. In this review, preclinical and early clinical data on these agents will be summarized and evaluated in the context of current age-related macular degeneration management guidelines, and the advantages as well as the challenges associated with siRNA therapies will be discussed.

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Choroidal neovascularization (CNV) occurs in a variety of chorioretinal diseases including age-related macular degeneration (AMD), and is the major cause of severe visual loss in patients with AMD. Oxidative stress has been thought to play an important role in the development of CNV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. In this study, we examined the expression of p22phox, an integral subunit in the NADPH oxidase complex, in the mouse eye. We determined that p22phox is expressed in the retinal pigment epithelial (RPE) cells and inner retinal neurons. A small-interfering RNA (siRNA) designed against p22phox efficiently reduced the expression of the protein in the eye when delivered by means of recombinant adeno-associated virus (AAV) vector. Vector treatment inhibited CNV in the mouse when delivered into the subretinal space where RPE cells were transduced. These results suggest that NADPH oxidase-mediated ROS production in RPE cells may play an important role in the pathogenesis of neovascular AMD, and that this pathway may represent a new target for therapeutic intervention in AMD.

Comparing Pegaptanib and Triamcinolone Efficacy in the Rat Choroidal Neovascularization Model

To evaluate the prophylactic effect of intravitreal pegaptanib sodium on choroidal neovascularization membrane (CNVM) development and compare its performance with that of triamcinolone acetonide. In drug-treated and control groups, CNVMs were induced by laser trauma. Immediately after undergoing the laser procedure, animals received intravitreal injections of pegaptanib sodium, 8 or 17 mug; triamcinolone acetonide, 200 mug; or a vehicle solution. After 21 days, fluorescein angiography was performed. The CNVM mean diameters and radial thicknesses were measured histologically. Mean CNVM diameters were 10% to 13% smaller in pegaptanib-treated eyes and 43% smaller in triamcinolone-treated eyes compared with laser-only control eyes. Late-stage fluorescein angiography leakage scores, on a scale of 0 to 3, suggested a statistical difference between triamcinolone- (0.6) and pegaptanib(8 microg)-treated (1.5) groups compared with the laser-only control group (2.0). The CNVM mean thicknesses were greater in the pegaptanib(8 microg)- (79 microm) and pegaptanib(17 microg)-treated (71 microm) groups and significantly smaller in the triamcinolone-treated group (26 microm) compared with the laser-only control group (67 microm). In this animal model of choroidal neovascularization, intravitreal pegaptanib exhibited marginal or no effect on CNVM development; whereas intravitreal triamcinolone evoked robust inhibition of CNVMs. Clinical Relevance Pegaptanib treatment may be insufficient to prevent CNVM formation.

Comparison of Multiple Reduced-dose and Standard Light Application in Photodynamic Therapy in an Animal Model of Choroidal Neovascularization

Current photodynamic therapy (PDT) for choroidal neovascularization (CNV) uses a standard radiant exposure of 50 J/cm2 at an irradiance of 600 mW/cm2. However, low-intensity PDT with verteporfin for neovascular tissue has been shown to be more effective than regular high-intensity PDT in an animal model of healthy choroids and corneal neovascularization. Low-intensity PDT also supposedly induces less retinal damage. In this study, we compared the effect of reduced-dose and standard light application PDT in an animal model of CNV. A laser injury model was used to induce CNV 3 weeks prior to PDT in brown Norway rats. The CNV lesions were then treated with verteporfin PDT with a dose of verteporfin 6.0 mg/m2 and 5 activating doses of light energy (43, 53, 63, 73 and 83 seconds). Closure of CNV was assessed by fundus fluorescein angiography (FAG). Histopathologic study was done after the last FAG. PDT with verteporfin significantly reduced the CNV area compared with control non-treated groups 1 week after PDT (p < 0.05). Only those lesions treated for 63 seconds or longer retained their significantly attenuating effect on CNV up to 3 weeks after PDT. There was no significant difference between the inhibition effects induced by reduced-intensity light application for 63 or 73 seconds compared to the standard radiant exposure (83 seconds). Histopathologic study showed that eyes treated with PDT showed significantly less extent and vascularity of CNV lesion than control lesions. Reduced-intensity PDT with 63-second duration seemed to be as effective as standard dose for CNV suppression. Considering the possible retinal damage following standard PDT, the PDT dose might be adjusted to reduce side effects. Further preclinical study will provide more data on what constitutes appropriate dosimetry for effective and safe PDT in CNV.

A new model of experimental subretinal neovascularization in the rabbit

Existing animal models of choroidal neovascularization (CNV) present several problems: they are hard to reproduce, they are inefficient, and the CNV created is not sustainable. The purpose of this study is to develop a highly efficient, reliable, sustainable rabbit model of CNV to facilitate the study of anti-angiogenic and anti-proliferative therapies for ocular diseases. Twenty-two pigmented rabbits were used in this study. Eleven rabbits received subretinal injections of either 10 μl of Matrigel with 500 ng of vascular endothelial growth factor (VEGF) or 20 μl of Matrigel with 750 ng of VEGF; eight rabbits received subretinal injections of either 10 or 20 μl of Matrigel only; three rabbits used as controls received subretinal injections of 20 μl phosphate-buffered saline (PBS) alone. Fundus photography, fluorescein angiography, optical coherence tomography, and histologic examinations were performed 1, 2, 4, and 9 weeks after injection. All experimental eyes showed angiographic leakage within this localized area 1 week after injection. The amount of leakage usually increased at weeks 2 and 4 and, in most cases, persisted at week 9. Control eyes demonstrated no leakage at any time point. Optical coherence tomography of treated eyes showed subretinal fluid and the presence of a lesion, possibly vascular or fibrotic, at the site of the leakage. Histologic analysis confirmed the presence of new subretinal blood vessels in the areas of Matrigel deposit. In conclusion, this novel method provides a highly reproducible, reliable, and sustainable rabbit model of experimental choroidal neovascularization. Such a model may prove useful for screening new anti-angiogenic therapies in a larger animal eye.

A new animal model of choroidal neovascularization

The purpose of this study was to evaluate the ability of different methods to induce choroidal neovascularization (CNV) in the domestic pig. A total of 26 Danish landrace pigs was used. A sample of 22 eyes in 12 pigs underwent retinal photocoagulation with a xenon lamp, six eyes in four pigs underwent retinal photocoagulation with a diode laser, and mechanical rupture of Bruch's membrane (BM) was induced in 12 pigs following surgical debridement of the retinal pigment epithelium without damage to the neuroretina. All 12 pigs (100%) in the group with mechanical rupture of BM developed CNV. The induced membranes were morphologically similar to CNV membranes in humans. Induced CNV was found in 13 of 22 (54%) xenon lamp-treated animals and in five of six (83%) diode laser-treated animals. The CNV in these groups was small and the morphology of the induced lesions was dominated by retinal gliosis and retinal neovascularization, probably due to a marked destruction of the neuroretina. Surgical debridement of the retinal pigment epithelium followed by mechanical rupture of BM is a reproducible method of producing CNV in the domestic pig, whereas photocoagulation gives rise to glially derived subretinal fibrovascular membranes and primarily retinal neovascularization.

Anastomotic Vessels Remain Viable after Photodynamic Therapy in Primate Models of Choroidal Neovascularization

Anastomotic vessels in exudative age-related macular degeneration (AMD) represent a serious clinical feature that reportedly does not respond well to either photocoagulation or photodynamic therapy (PDT). Anastomoses also occur in various animal models of choroidal neovascularization (CNV). In the present study, anastomotic vessels and their patency were evaluated in two primate CNV laser-trauma models after PDT, by using two novel photosensitizers. In cynomolgus (Macaca fascicularis) and squirrel (Saimiri sciureus) monkey eyes (n = 20), matrix placement of laser photocoagulation sites elicited CNV as a component of the development of fibrovascular tissue (FVT). FVT sites received PDT according to specific drug infusion and laser light treatment parameters. FVTs and anastomoses were evaluated by fundus photography, fluorescein angiography, and histologic examination. Anastomoses averaged approximately 48% of FVT sites, with greatest occurrence in the macaque. Although PDT with each photosensitizer effectively produced FVT closure, both retinal vessels and anastomoses remained patent. Although PDT is effective in closing the choroidal neovascularization in FVT, this technique was ineffective in occluding anastomotic vessels and their associated tributaries within the mid- to proximal retina. Various factors (vascular diameter, composition, blood flow, orientation) may contribute to continued anastomotic patency. By convention, such vessels would typically be defined as chorioretinal anastomoses (CRAs); however, continuing studies suggest the possibility that these neovessels constitute dual-origin hybrids. Regardless of origin, viable anastomoses provide one potential mechanism for revascularization to occur after PDT and may help to explain why CRAs are considered a poor prognostic sign in patients with AMD.

Selective visualization of choroidal neovascular membranes

Purpose. Laser-targeted angiography has unique advantages over conventional angiography of the fundus. Its efficacy in visualizing choroidal neovascular membranes was tested in a rat model and compared to that of fluorescein angiography. Method. Laser-targeted angiography was performed in rats with choroidal neovascularization (CNV) by injecting heat-sensitive carboxyfluorescein liposomes intravenously, locally releasing a bolus of dye in the choroid with a weak laser pulse, and recording advancement of the bolus on a video camera. Conventional fluorescein angiography also was performed. Results. Laser-targeted angiography revealed CNV as an abnormal pattern of brighdy fluorescent vessels. The flow pattern of the bolus and histology, performed in some cases, confirmed the choroidal nature of the vessels. The angiographic visualization was not dependent on dye leakage through the vessels or staining of their walls. Laser-targeted angiography also provided visualization of new vessels that could not be diagnosed by fluorescein angiography. It demonstrated that blood flow was typically more sluggish in CNV than in normal choriocapillaris. Fluorescein angiography failed to demonstrate flow dynamics in all cases of CNV. Conclusions. This study, in an animal model of CNV, shows that laser-targeted angiography demonstrates CNV and its flow dynamics in a manner not provided by conventional fluorescein angiography. It holds clinical promise as a method to delineate CNV considered difficult or impossible to detect by fluorescein angiography. The method also may permit selective photocoagulation of feeding vessels in the choroid, thereby limiting damage to the overlying retina. Invest Ophthalmol Vis Sci. 1996;37:1642-1650. J. he delineation of choroidal neovascularization (CNV) is based on fluorescein angiography, but, in a majority of cases, it suffers from a number of limitations. First, the dye rapidly fills both the retinal and the choroidal vessels; thus, the visualization of small vascular beds, such as CNV, is often obscured by the lack of contrast caused by the bright fluorescence emanating from the large volume of dye present in the underlying choroidal vessels. Second, visualization of

Photodynamic Therapy of Experimental Choroidal Neovascularization Using Lipoprotein-Delivered Benzoporphyrin

Objective: To investigate photodynamic therapy of experimental choroidal neovascularization using benzoporphyrin derivative monoacid (Verteporfin). Methods: Photodynamic therapy using benzoporphyrin derivative monoacid was investigated in cynomolgus monkeys. Following intravenous injection of benzoporphyrin derivative monoacid (1 to 2 mg/kg) complexed with low-density lipoprotein, the eyes were irradiated with 692-nm light at a fluence of 50 to 150 J/cm 2 and irradiance of 150 to 600 mW/cm 2 . Choroidal neovascularization was documented before photodynamic therapy and closure was demonstrated by fundus photography, fluorescein angiography, and light and electron microscopic examination. Results: Following photodynamic therapy, vessels within choroidal neovascularization were occluded, and there was damage to the choroidal neovascularization endothelium and the subjacent choriocapillaris. Damage to the retinal pigment epithelium and photoreceptors was also observed. Conclusion: Photodynamic therapy with lipoprotein-delivered benzoporphyrin derivative monoacid was effective in this animal model of choroidal neovascularization and may be a promising, potentially selective, therapy for choroidal neovascularization.

Photodynamic Therapy of Experimental Choroidal Neovascularization Using Lipoprotein-Delivered Benzoporphyrin

To investigate photodynamic therapy of experimental choroidal neovascularization using benzoporphyrin derivative monoacid (Verteporfin). Photodynamic therapy using benzoporphyrin derivative monoacid was investigated in cynomolgus monkeys. Following intravenous injection of benzoporphyrin derivative monoacid (1 to 2 mg/kg) complexed with low-density lipoprotein, the eyes were irradiated with 692-nm light at a fluence of 50 to 150 J/cm2 and irradiance of 150 to 600 mW/cm2. Choroidal neovascularization was documented before photodynamic therapy and closure was demonstrated by fundus photography, fluorescein angiography, and light and electron microscopic examination. Following photodynamic therapy, vessels within choroidal neovascularization were occluded, and there was damage to the choroidal neovascularization endothelium and the subjacent choriocapillaris. Damage to the retinal pigment epithelium and photoreceptors was also observed. Photodynamic therapy with lipoprotein-delivered benzoporphyrin derivative monoacid was effective in this animal model of choroidal neovascularization and may be a promising, potentially selective, therapy for choroidal neovascularization.