Rhodopsin-positive cell production by intravitreal injection of small molecule compounds in mouse models of retinal degeneration

We aimed to verify whether the intravitreal injection of small molecule compounds alone can create photoreceptor cells in mouse models of retinal degeneration. Primary cultured mouse Muller cells were stimulated in vitro with combinations of candidate compounds and the rhodopsin expression was measured on day 7 using polymerase chain reaction and immunostaining. We used 6-week-old N-methyl-N-nitrosourea-treated and 4-week-old rd10 mice as representative in vivo models of retinal degeneration. The optimal combination of compounds selected via in vitro screening was injected into the vitreous and the changes in rhodopsin expression were investigated on day 7 using polymerase chain reaction and immunostaining. The origin of rhodopsin-positive cells was also analyzed via lineage tracing and the recovery of retinal function was assessed using electroretinography. The in vitro mRNA expression of rhodopsin in Muller cells increased 30-fold, and 25% of the Muller cells expressed rhodopsin protein 7 days after stimulation with a combination of 4 compounds: transforming growth factor-beta inhibitor, bone morphogenetic protein inhibitor, glycogen synthase kinase 3 inhibitor, and gamma-secretase inhibitor. The in vivo rhodopsin mRNA expression and the number of rhodopsin-positive cells in the outer retina were significantly increased on day 7 after the intravitreal injection of these 4 compounds in both N-methyl-N-nitrosourea-treated and rd10 mice. Lineage tracing in td-Tomato mice treated with N-methyl-N-nitrosourea suggested that the rhodopsin-positive cells originated from endogenous Muller cells, accompanied with the recovery of the rhodopsin-derived scotopic function. It was suggested that rhodopsin-positive cells generated by compound stimulation contributes to the recovery of retinal function impaired by degeneration.

Automated summary

We successfully generated photoreceptor cells in mouse models of retinal degeneration through intravitreal injection of small molecule compounds alone. The optimal combination of compounds selected through in vitro screening was injected into the vitreous, leading to a significant increase in the number of rod cells and the expression of rhodopsin. Lineage tracing demonstrated that the rhodopsin-positive cells originated from endogenous Muller cells, accompanied by the recovery of rhodopsin-derived scotopic function.