Scientific Poster
Laser Induced Choroidal Neovascularization (CNV) as a Model of Age-related Macular Degeneration (AMD) in the Minipig - SciPos108


Stricker-Krongrad, A.¹; Leigh, H.¹; Harvey, R.M.¹; Jones, M.R.¹; Tellez, A.3; Stanley, J.3; Wicks, J.3; Bouchard G.F.¹
¹Sinclair Research Center, LLC, Auxvasse, MO, USA; ²Sinclair Bio Resources, LLC, Auxvasse, MO, USA; 3 Alizee Pathology, LLC, Thurmont, MD, USA


Choroidal Neovascularization (CNV) is a known cause of Age-Related Macular Degeneration (AMD). Lucentis® (ranibuzumab) is a marketed treatment for AMD and is hypothesized to function by binding to vascular endothelial growth factors (VEGF) and preventing aberrant vascular growth. Ocular similarities between humans and the minipig make it a useful model for ocular therapies, and this study aimed to validate the minipig model of laser induced CNV.

Materials & Methods

Choroidal Neovascularization was generated in Sinclair minipigs using retinal photocoagulation. Retinas were treated with the IRIS 810 nm diode laser (IRIS Medical, Mountain View, California, USA), using a spot size of approximately 600 μm and 100 mW for 500 msec (Figure 1A). After laser induction of CNV, animals received a single 25 μL intravitreal injection of either Lucentis® (10 mg/mL; n=4), or vehicle (10 mM histidine HCl, 10 % α,α –trehalose dehydrate, 0.01 % polysorbate 20 pH, 5.5; n=4). Eye examinations were performed on all animals prior to CNV induction and treatment, and weekly after induction by a board-certified Veterinary Ophthalmologist. Fluorescein angiography (FA) was performed after CNV induction during week 1 and then at 2, and 6 weeks by a Certified Retinal Angiographer (CRA) to assess hemorrhages, and CNV lesions. CNV Hemorrhage Grading Scale is present in Table 1, and CNV Lesions Grading Scale is present in Table 2. Animals were humanely euthanized at week 6 and eyes were collected and fixed in Davidson’s fixative and transferred to 70% Alcohol 1-3 days post fixation. Lesions were identified grossly and collected for paraffin processing/embedding. 5 μm serial sections were taken and stained with H&E or IHC vascular marker CD31 (following melanin/autofluorescent bleaching method prior to antigen retrieval). Slides were assessed by a board-certified veterinary pathologist, and computerized morphometry was performed to quantify positive CD31 staining in the lesioned areas of Lucentis® and Vehicle groups.


Prior to laser photocoagulation, there were no major ophthalmologic findings in any pigs (mild discharge is common and considered an incidental finding in minipigs). After photocoagulation, weekly examinations showed 3/4 eyes in the vehicle group had significant vitreous opacification between Weeks 2 and 5. At Week 2 retinal or pre-retinal hemorrhage was noted in 2/4 eyes. Additionally, significant anterior segment inflammation (demonstrated by fibrin or synechia formation) was present in 2/4 eyes during Week 3. Lucentis® treated eyes also demonstrated focal retinal hemorrhages in 3/4 eyes during Week 2, and significant vitreous opacities were present in 3/4 eyes at Weeks 3 through 5.

During Week 1, after retinal photocoagulation and intravitreal treatment, hypofluorescent post-laser spots and hemorrhages were present. These continued into week 2 and were absent by week 6 (Table 3). Sparse Grade 1-2 CNV lesions were present during week 1 fluorescein angiography. Lesions progressed to grade 2-4 during week 2. By week 6, clinically significant Grade 4 CNV lesions were present (Table 4), and they were present at a higher prevalence in Vehicle treated versus Lucentis® treated eyes.

Macular degeneration is a significant concern for an aging population. Choroid neovascularization is a major cause of vision loss in age-related macular degeneration. While a large number of rodent animal models for AMD exist, fewer large animal models exist [1]. Minipigs have been used in several ocular models of disease, such as uveitis, retinal detachment, cataracts, glaucoma, and diabetic retinopathy [2]. Anatomic similarities to humans, and relative size makes the pig eye a useful model. Furthermore, the conservation of VEGF orthologs in NHP, dog, pigs, minipigs, and humans [3], demonstrates the minipig provides an additional useful model targeting anti-VEGF treatments such as Lucentis®. This study used retinal photocoagulation to induce CNV lesions in a minipig model. The model presented with hypofluorescent post-laser spots, and hemorrhaging in Weeks 1-2 after treatment. Lower grade CNV lesions were present in Weeks 1-2 in all groups. By Week 6, clinically significant Grade 4 lesions were seen in both Vehicle and Lucentis® treated animals. However, Grade 4 lesions were present in Lucentis® animals at a lower incidence than Vehicle treated animals. Furthermore, total number of vessels and vessel area were decreased in examined lesions from Lucentis® treated groups compared to Vehicle treated animals. Taken together with weekly ophthalmological exams, Vehicle treated animals had more severe ocular findings overall, suggesting some level of efficacy of Lucentis® in minipigs.

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  1. Pennesi, M.E., M. Neuringer, and R.J. Courtney, Animal models of age related macular degeneration. Mol Aspects Med, 2012. 33(4): p. 487-509.
  2. Stricker-Krongrad, A., C.R. Shoemake, and G.F. Bouchard, The Miniature Swine as a Model in Experimental and Translational Medicine. Toxicol Pathol, 2016. 44(4): p. 612-23.
  3. Heckel, T., et al., Functional analysis and transcriptional output of the Gottingen minipig genome. BMC Genomics, 2015. 16: p. 93

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