We determined the function of ARMS2 and HtrA1 in the choroid and retina using transgenic (Tg) mice and evaluated the effects of mainstream cigarette smoke on these mice. The chicken actin promoter (CAG) was used to drive mouse HtrA1, human ARMS2, and ARMS2 (A69S) expression in the entire body of a mouse for one year. Fundus observations were performed with a Spectralis HRA+ optical coherence tomograph (OCT). Eyes were sectioned, stained with hematoxylin and eosin (H&E), and analyzed with immunohistochemistry. Mice were exposed to cigarette smoke for 30 min/d, 5 d/wk for 12 weeks using a mainstream smoking chamber (INH06-CIGR02A, MIPS). After 12 weeks, fundus observations and pathological analyses were performed. Approximately 18.2% of 12-month-old HtrA1 Tg mice exhibited choroidal neovascularization (CNV) by OCT and positive immunostaining with anti-CD31 and anti-fibronectin antibodies. Furthermore, elastic van Gieson (EVG) staining showed Bruch's membrane damage in HtrA1 Tg mice. No retinal changes were observed in ARMS2 and ARMS2 (A69S) Tg mice. A total of 12 weeks of exposure to mainstream cigarette smoke led to CNV rates of 7.7% for wild type (Wt) mice and 20% for HtrA1 Tg mice, but had no effect on ARMS2 Tg mice. In addition, abnormal deposits were observed between photoreceptor cells and the RPE in an HtrA1 Tg mouse exposed to mainstream cigarette smoke. The HtrA1 overexpression and mainstream cigarette smoke can independently lead to CNV. The HtrA1 gene is a strong risk factor for wet AMD, but not all of the HtrA1 Tg mice developed CNV, suggesting that CNV development depends on multiple risk factors.

Nakayama M, Iejima D, Akahori M, Kamei J, Goto A, Iwata T.

Invest Ophthalmol Vis Sci. 2014 Sep 9;55(10):6514-23. doi: 10.1167/iovs.14-14453.

Primate is a member of the biological order that contains prosimians (lemurs, lorises, galagos, and tarsiers) and simians (monkeys and apes). Simians are divided into two groups, the platyrrhines (new world monkeys) of South and Central America and the catarrhine monkeys of Africa and Southeastern Asia. The new world monkeys include the capuchin, howler, and squirrel monkeys, and the catarrhines include the old world monkeys (baboons and macaques) and the apes. The non-human primates preferred for vision research are the macaque monkeys
(Macaca mulatta, Macaca fascicularis, and Macaca fuscata). Macaques rely on three-color (red, green, and blue) stereoscopic vision, the dominant sensory system, with larger brains compared to other mammals for accurate information processing. Macaques have slower rates of development than other similarly sized mammals, and reach maturity later but have longer lifespans. These characteristics mimic the human development of age-related eye diseases.

When working with macula related diseases, animal model with well-defined fovea is preferred. A search for monkey line affected with macular degeneration has been persistent for decades. A monkey with macular degeneration was first described by Stafford et al. in 1974. More than 6% of the elderly monkeys they examined showed pigmentary disorders and drusen-like spots. El-Mofty et al. also reported that the incidence of maculopathy was high as 50% in a colony of rhesus monkeys at the Caribbean Primate Research Center of the University of Puerto Rico.

Dawson et al. examined 272 eyes in 136 rhesus monkeys during 1986-1988 in the closed Cayo Santiago colony of the Caribbean Primate Research Center of the University of Puerto Rico. The fundi were examined and photographed for all eyes and fluorescein angiography was also performed in some eyes. Selected cases were evaluated for 'acuity' loss by recording of pattern-evoked retinal and cortical signals. Light and electron microscopy were used to evaluate the pigment epithelium of some animals. Among examined eye 38% of all eyes had posterior pole drusen. Incidence was highly age-related. When late-stage lesions were found, no CNV was observed, but late hyperfluorescence was consistent with degenerative scarring and atrophy. Electrophysiology demonstrated moderately reduced acuity in the presence of numerous macular drusen. The electrooculograms were below normal and the histopathology showed changes identical to those reported in human AMD.

At the Tsukuba Primate Research Center (TPRC) in Japan, a single cynomolgus monkey (Macaca fascicularis) with a large number of small drusen around the macular region was found. This single affected monkey has mated to a large pedigree of more than 100 affect and 300 unaffected monkeys. Drusen were observed in the macular region as early as one year after birth, and the numbers increased and spread toward the peripheral retina throughout life. No histological abnormalities have been found in the retina, retinal vessels, or choroidal vasculatures of the eyes with drusen. Another sporadic aged cynomolgus monkey with drusen was found in Simian Conservation Breeding and Research Center (SICONBREC) in the Philippines. More than 10% of the elderly monkeys had soft drusen. Immunohistochemical and proteomic analyses of the drusen from these monkeys showed similar to human. These drusen contained protein molecules that mediate inflammatory and immune processes, which included immunoglobulins, components of complement pathway, modulators for complement activation (e.g., vitronectin, clusterin, membrane cofactor protein, and complement receptor-1), molecules involved in the acute-phase response to inflammation (e.g., amyloid P component, α1-antitrypsin, and apolipoprotein E), major histocompatibility complex class II antigens, and HLA-DR antigens. Cellular components have also been identified in drusen, including RPE debris, lipofuscin, and melanin, as well as processes of choroidal dendritic cells, which are felt to contribute to the inflammatory response. In addition to immune components, a number of other proteins were found in drusen. These appear to be vitronectin, clusterin, TIMP-3, serum amyloid P component, apolipoprotein E, IgG, Factor X, crystallins, EEFMP1, and amyloid-beta. The presence of immunoreactive proteins and oxidative modified proteins implicated that both oxidation and immune functions in the pathogenesis of AMD.

The affected monkeys from TPRC were further used to test new drug, which suppress complement activation in the retina, to delay/cure AMD. To test the effect of long term suppression of complement activation in the retina, an cyclic analogue (Ac-I[CV(1MeW)QDWGAHRC]T-NH2) of the small cyclic synthetic peptide compstatin was intravitreally injected into 8 affected monkey intravitreally injected at different dose and intervals. Four affected monkeys were injected at 50 μg dose at one week interval. Fifty microgram dose were dissolved in 100 μl of saline solution, filtrated and intravitreally injected using 30G needles. After 6 month of injection, diffusion of drusen in the macula and by 9 month partial disappearance of drusen was observed in all 4 monkeys. This preliminary experiment has shown reversal of drusen formation by suppression of complement activation. The information should benefit for development of improved drug and therapy for future AMD prevention.

The macaque genome project, which was completed in 2007 generated large amount genome information including the microsatellite markers for linkage study. Currently 240 loci of the cynomolgus monkey are being investigated to try to identify the disease causing gene and to understand the biological pathways leading to complement activation.

The eyes of monkey are structurally similar to human eyes which make them extremely valuable for macular degeneration studies. However, there are limitations in using this species over other laboratory animals. Monkeys have a relatively longer life span, have a longer gestation period, have a lower birth numbers resulting in a slower rate of expanding the pedigree, more difficult to genetically manipulate, and the cost of maintenance is significantly higher. In the other laboratory animals, the differences in the eye structure have been considered to be a disadvantage for using them as AMD models. However, they are easier to maintain and less expensive. This has made the development of a mouse model of AMD very attractive, and a number of mouse AMD models have been reported recently.

Chi Z-L, Yoshida T, Lambris JD, and Iwata T. Suppression of drusen formation by compstatin, a peptide inhibitor of complement C3 activation, on Cynomolgus monkey with early-onset macular degeneration.

Currrent Topics on Complement and Eye Disease, Advances in Experimental Medicine and Biology 703:127-135 (2010)

Umeda S, Suzuki MT , Okamoto H, Ono F, Mizota A, Terao K, Yoshikawa Y, Tanaka Y, and Iwata T. Molecular composition of drusen and possible involvement of anti-retinal autoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca fascicularis).

FASEB Journal 19:1683-1685 (2005)

Umeda S, Ayyagari R, Allikmets R, Suzuki MT, Karoukis AJ, Ambasudhan R, Zernant J, Okamoto H, Ono F, Terao K, Atsushi M, Yoshikawa Y, Tanaka Y, and Iwata T. Early onset macular degeneration with drusen in a cynomolgus monkey (Macaca fascicularis) pedigree caused by a novel gene mutation.

Investive Ophthalmology and Visual Science 46:683-691 (2005)

Umeda S, Suzuki MT, Yoshikawa Y, Iwata F, Fujiki K, Kanai A, Sanuki N, Tanaka Y, and Iwata T. Cloning and Characterization of ELVLO4 Gene in Cynomolgus (Macaca fascicularis) Monkey.

Experimental Animal 52:(2) (2003)