**2. Preclinical models of AMD**

Animal models have been generated by multiple laboratories by reconstructing specific features of AMD. These models have become integral for providing insight into the pathophysiology of this disease, as well as to develop proof-of-principle studies to support the advancement of new therapies [26]. In general, an optimal animal model is inexpensive and mimics the features of the human disease in a timely manner to allow for efficient studies [17]. In studies focused on AMD, these changes include a thickened BrM, sub-RPE deposits, RPE atrophy and hyperplasia, accumulation of immune cells or complement, photoreceptor atrophy, CNV, and fibrosis [17]. However, when trying to recapitulate AMD, animal models can be challenging because AMD is a complex disease with multiple polymorphisms able to be influenced by environmental and epidemiologic factors [15]. Furthermore, there are inherent differences in the eyes of animals and humans, such that no single model perfectly captures all features of AMD. Although space limits inclusion of all animal models, this review highlights the weaknesses and strengths of specific animal models and how they have been useful for understanding aspects of AMD development, progression, and treatment.

#### **2.1 Introduction to rodent models**

Rodents have been the "go-to" model for retinal disease for decades. There are many advantages to the rodent model. Economically, rodents are small animals that require little space and resources, are easy to breed and handle, have short gestation times while producing many offspring, and have short life spans. Diseases can also progress relatively quickly allowing for efficient studies [17, 27]. Mouse, rat, and human genomes have been sequenced, and each were found to have around 30,000 genes, 95% of which are shared among all three species. Further, advances in molecular genetic techniques allow for ease of genetic manipulation [27]. Anatomically, mice have key retinal structures — RPE, BrM, and choriocapillaris — that are affected in human AMD [15]. The economic, genetic, and anatomic benefits of rodents make them invaluable animal models for studying human disease and testing treatments. Clinicians and scientists alike have been working to recapitulate the human AMD phenotype in mice by taking what is known about the human condition and applying it to mice. This may come in the form of genetic manipulation to induce SNPs in known AMD-associated genes or applying risk factors for AMD to mice such as exposure to cigarette smoke or inducing obesity. Several of these manipulations will be discussed below.

It is important to highlight that there are some structural differences in the retinas of humans and rodents. Unlike humans, rodents do not have a macula, defined anatomically as having at least two layers of ganglion cells with a mixture of rod and cone cells [26]. Rodents also lack an area of the retina with high density of cones similar to the fovea. Moreover, interpretation of findings from early murine AMD models was confounded by a spontaneous point mutation in Crumbs homolog 1

(*Crb1*) that segregated in a sub-strain of the C57B/6 J mouse from the Jackson Laboratories. The *Crb1* mutation affects photoreceptor health and development, but is not relevant in human AMD pathogenesis [28]. This mutation is now screened for prior to use of mouse strains originated from the C57B/6 J mouse strain. Although rodents are not the perfect animal model, they have shed light on many aspects of AMD, of which numerous examples are highlighted in this review.

### **2.2 Rodent models of oxidative stress**

The retina is susceptible to oxidative stress due to its high metabolic demand, lipid oxidation by photoreceptors, and the presence of molecules that form ROS mentioned in section 1.2. Below we present several mouse models that mimic AMD pathology induced by the lack of antioxidants or the addition of oxidative stress.
