Gnotobiotic Operations and Assembly for Development of Germ-Free Animal Model of Laser-Induced Choroidal Neovascularization.

PURPOSE

Compelling new evidence reveals a close link between the gut microbiome and the pathogenesis of neovascular age-related macular degeneration (nAMD). Germ-free (GF) animal models are the current gold standard for studying host the microbe interactions in vivo; yet, no GF animal models of nAMD are available today. This protocol describes gnotobiotic operations and assembly for a laser-induced choroidal neovascularization (CNV) model in GF mice to study the gut microbiome in neovascular AMD.

METHODS

We developed a step-wise approach to performing retinal laser photocoagulation in GF C57BL/6J mice that were bred and maintained at the gnotobiotic facility. Following a strict sterility protocol, we administered laser photocoagulation via an Argon 532-nm laser attached to a customized slit-lamp delivery system. Sterility was confirmed by weekly fecal cultures and reverse transcriptase-polymerase chain reaction.

RESULTS

The experiment was repeated twice at different time points using seven mice (14 eyes). Stool cultures and RT-PCR remained negative for 14 days post-procedure in all mice. Lectin immunostaining performed on choroidal flatmounts confirmed the presence of CNV lesions 2 weeks after laser treatment.

CONCLUSIONS

We established a GF mouse model of nAMD with detailed guidelines to deliver retinal laser in GF mice maintaining sterility after the laser procedure.

TRANSLATIONAL RELEVANCE

To our knowledge, this is the first protocol that describes a GF murine model of laser-induced CNV. In addition to nAMD, this animal model can be used to investigate host-microbial interactions in other eye diseases with laser-induced mouse models such as glaucoma and retinal vein occlusion.