Age related macular degeneration (AMD) is the leading cause of irreversible blindness in adults over 50 in the industrialized world. Approximately 11 million individuals have AMD in the United States (US) alone and is expected to reach 22 million by 2050, while globally is expected to reach 288 million by 2040. Currently, no cure or prevention measures exist. AMD is a complex disease where genetic predispositions play an important role but the disease development appears to be significantly affected by environmental and lifestyle factors. Nutrition, smoking, and obesity have been implicated but the role of high fat diet has not been clearly delineated.

Our preliminary results clearly demonstrate the synergistic effect of high fat diet and genetic predisposition in AMD animal models. The mechanistic pathways by which high fat diet and environmental/lifestyle factors interact with genetic predisposition and influence susceptibility risk towards disease progression remain a mystery and a key missing piece of the puzzle of AMD halting the development of efficacious preventive/curative interventions.

Over the past decade, it has become apparent that gut microbiome plays a critical role in the function of our health and disease through the regulation of our immune system and has been implicated in the pathogenesis of vascular, neurodegenerative diseases and obesity. Gut microbiota is heavily influenced by environmental and lifestyle factors and in particular by Western style diets rich in fats and high in sugars. Our collaborator Dr Eugene Chang's work has shown that changes in gut microbiota by high fat diet seem to be responsible for the development of inflammatory bowel disease in genetically predisposed animals by altering the immune response and promoting low grade inflammation. Minimal data are available about the effect of gut microbiota changes in the eye development and eye diseases.

We hypothesize that gut microbiota could be the common denominator that connects high fat diet and other environmental/lifestyle risk factors, genetic predisposition and AMD development through retinal inflammation upregulation.

AIM 1: To study the effect of high fat diet and gut microbiome on retinal inflammation and stress biomarkers.

AIM 2: To study the effect of high fat diet and gut microbiome on angiogenesis and choroidal neovascularization (CNV) formation in laser-induced mouse model.

In this innovative pilot proposal, we plan to investigate how gut microbiota changes can affect retina inflammation biomarkers as well as abnormal vessel formation in AMD animal models and study how diet types can correlate with different gut microbiota statuses gradually simulating an "AMD risk environment" that both and diet factors interact with different gut microbiome. These "proof of concept" experiments will be led by Dr Dimitra Skondra, MD, PhD retinal surgeon/ scientist, Assistant Professor at The University of Chicago with valuable experience in translational research in laser induced AMD animal models in collaboration with expert microbiome scientist Professor Dr Eugene Chang's team at the Knapp Center of Biomedical Discovery at The University of Chicago. This work could provide a new breakthrough in AMD with gut microbiome as the missing link bridging diet/environment/lifestyle factors, genetic predisposition and AMD development. Gut microbiota can be easily manipulated and altered with targeted probiotics, diet, new agents already in the pipeline for other conditions like food allergies, cancer and this approach could open a new chapter in developing treatment targets and preventative strategies and cures to stop AMD in earlier stages before irreversible vision loss occurs. In summary, this "high risk-high reward" translational project could revolutionize our understanding of AMD and could pave the way into a completely new era of therapeutic approach for preventing/treating the leading cause of blindness by altering gut microbiota and developing treatments tailored for an individual's unique genetic makeup, environment and lifestyle.

AIM 1: To study the effect of high fat diet and gut microbiome on retinal inflammation and stress biomarkers.

A. Young wild-type C57Bl/6J conventional microbiome specific germ-free mice (SPF) will be placed in regular diet (SPF-ND) and high fat diet for 4-6 weeks (SPF-HFD). Biomarkers of inflammation (macrophage infiltration, HMGB-1, IL1b, TNF-a) and retinal stress biomarkers (vimentin, GFAP) will be evaluated with immunostaining of retina/RPE and choroidal layers. Mice will be sacrificed and eyes will be enucleated and cryopreserved. Eyes will be sectioned and cryosections will be used for immunostaining of the retinal/RPE/choroidal after incubation with antibodies for inflammation and retinal stress biomarkers mentioned above. Extraction of bacterial DNA of fecal contents collected by mice cages will be performed and extracted DNA will be sent to Argonne Laboratory where it will be analyzed using 16SrRNA and sequencing protocols to characterize and quantify the microbiome populations. Inflammation biomarkers and gut microbiome in the SPF-HGD group will be compared with SPF-ND group in order to investigate if high fat diet changes gut microbiome composition and upregulates inflammation in retina/RPE and choroid.

B. Germ free (GF) young wild-type C57Bl/6J bred and maintained in plastic isolators will be placed in regular diet (GF-ND) and high fat diet for 4-6 weeks (GF-HFD). Gnotobiotic diets for GF mice will be autoclaved or irradiated and sterility will be tested prior to and after the experiment and these diets will be provided by specialized vendors. Inflammation biomarkers and gut microbiome populations will be evaluated as described above. By using germ free mice on regular and high fat diet we will able to evaluate if eliminating gut microbiome and microbiome changes induced by high fat diet, the inflammation in retina/RPE and choroidal layers can be ameliorated thus implicating gut microbiome and its role upregulating inflammation as a potential mechanism for the effect of high fat diet on promoting AMD pathology in animal models shown in our preliminary data.

AIM 2: To study the effect of high fat diet and gut microbiome on CNV formation in the laser-induced CNV mouse model.

Germ free (GF) in sterile incubators and conventional microbiome SPF mice will be placed on normal diet and high fat diet for 4-6 weeks in 4 separate groups as described in aim 1. CNV will be created using the laser-induced technique and the size of CNV lesions will be evaluated in choroidal flatmounts. For the laser CNV model, mice will be anesthetized and dilated. Four to 6 laser spots will be applied around optic nerve using bubble formation as indicator of Bruch's membrane rupture. One week later, mice will be anesthetized and perfused with FITC high molecular weight dextran to stain vessels and eyes will be enucleated. After anterior segment of eye will be removed and retina will be dissected, RPE, choroid, sclera complex will be flatmounted. Size of angiogenesis (new blood vessel formation)-CNV membranes will be measured in all groups. Retinal inflammation biomarkers within CNV and gut microbiome will be analyzed as described in aim 1. The effect of gut microbiota on CNV formation and inflammation and retinal stress biomarkers within CNV in correlation with high fat diet will be analyzed. The purpose is to evaluate if CNV development and exacerbation caused by high fat diet can be halted and if CNV inflammatory biomarkers can be suppressed by eliminating gut microbiota as the common denominator of the interactions between diet and CNV development through regulation of retinal inflammation.

STATA

Weekly Ophthalmology Grand Rounds and Didactics; Ophthalmology clinics; Research meetings with mentor every week; Ophthalmology Seminars