Functional and Anatomical Characterization of Atrophic Age-Related Macular Degeneration in an Aged Mouse Model

Experimental Ophthalmology, University of Geneva, Geneva, Switzerland. Department of Ophthalmology, University Hospitals of Geneva, Geneva, Switzerland. Department of Fundamental Neurosciences, University of Lausanne, Switzerland. Pharmaceutical Biochemistry/Chemistry group, School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland. Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland .


Introduction
In industrialized countries excellent health care and progress in medicine have increased life expectancy and the number of elderly people [1], with concomitant increase of age-related diseases such as age-related macular degeneration (AMD) [2]. It is estimated that the number of patients suffering from early and late AMD will increase from 15 and 2.7 Mio, respectively, to 21.5 and 4.8 Mio respectively by 2040 [3]; approximately 80% of these patients will suffer from atrophic AMD (aAMD). No current approved therapies are available to treat aAMD. Even though several promising treatment approaches are being considered, which focus on visual cycle modulation, neuroprotection, suppression of inflammation and complement inhibition [4], progress is hampered by the absence of an appropriate animal model. A significant cause for age-related diseases, including aAMD, is cellular oxidative stress with the resultant accumulation of reactive oxygen species, which damages and causes cell death [2]. Several animal models of aAMD have been developed; however, animal models that develop choroidal neovascularization (CNV) [5][6][7], which is not a feature of aAMD are time-consuming [6,7] or cause rapid retinal degeneration [7,8] are not suited for the analysis of therapeutic approaches that endeavor to interfere with the development of aAMD at an early stage of the disease.

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docosahexaenoic acid and is elevated in the vitreous and plasma of aAMD patients [9,10]. Here, we report a modification of the model of aAMD that Holyfield developed in young mice [9,10] by modifying the immunization protocol and using old mice establishing a model of aAMD that better recapitulates to the human disease without accompanying adverse events and thus, permits the generation of results highly transferable to human patients, which promotes the development of new aAMD therapies.

Synthesis of CEP-conjugated MSA
All reagents for the synthesis were purchased from

Synthesis of CEP-conjugated MSA
The strategy for grafting the CEP entity onto MSA has been inspired by the synthesis work published by Salomon et al. [12] and Lu et al. [13]. Briefly, the synthesis consists of five steps ( Figure 1).

Toluidine staining and electron microscopy
Tissue processing and electron microscopy were done according to Dosso et al. [16].

H&E and immunofluorescent staining
For histology, globes were fixed in 4% PFA at RT.

Statistics
Statistical analysis was performed using GraphPad   The score has been created to include all information of the micrographs in the analysis but excluding false positives by background or autofluorescence (e.g. erythrocytes).

Preparation of CEP-conjugated protein
Protein modifications in which the -amino group of lysyl residues is combined into a 2-CEP using the Consequently, Salomon et al. [12] and Lu et al. [13] reported an efficient synthesis of CEP derivatives, which is specific and efficient for proteins. Their key finding was that the protected DOHA 9fluorenylmethyl ester reacts with primary amines to  [12] and Lu et al. [13] and used for the synthesis of the CEP-MSA in this work is summarized in Figure 1. followed are taken from the previous papers [12,13].

Synthesis of DOHA-Fm
The key intermediates have been characterized by 1 H, 13 C and LRMS to assess their identity and are shown in the text here (Figures 11-15).

Knoor synthesis from DOHA-Fm
The last step of the synthesis is the insertion of the

Determination of OKT and VPT
To p=0.0207) (Figure 8a, b). Similar trends were obtained for the expression of C3; however, the differences are not statistically significant (Figure 8c, d).

Toluidine staining and electron microscopy:
Toluidine-stained semi-thin sections and uranyl acetate and lead citrate-stained thin sections of retinas from one CEP-treated and one age-matched control mouse at 3 months post-immunization were analyzed for CEP-induced alterations by light and electron microscopy, respectively. Figure 9a

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The Muller cells of AgM and CEP mice. d) Similar levels of RPE65 (red) was expressed in both groups of animals.
The micrographs in the figure are from retinas of AgM and CEP mice without reference to age, since no age-dependent differences were observed. Micrographs are representative of n=8-10 animals/group (Magnification of 200x).

Discussion
Recent studies have shown the importance of chronic oxidative damage, inflammation, immune dysregulation, and lipid metabolism in aAMD pathophysiology [4]. that are not found in the human disease. The SOD2 KD model, which is complex to generate and responsible for early animal death [22], causes increased CEP levels, which are known to be elevated in the plasma and vitreous of aAMD patients [25,26]. CEP, an oxidation adducts produced from the docosahexaenoic acid, is generated following dysfunctions of the Nrf2 pathway [22].
The orginal "CEP model" by Hollyfield [9,10] [7,9,10]. The CEP-old-mouse model reproduces the pre-atrophic stages of aAMD including the influence of age. It is also less traumatic for the animal injecting CEP-MSA into the hocks [11] instead of the foot pad Similarly, we did not observe apoptotic cell death ( Figure 6) as reported in other models [23,29].