Abstract
Purpose :
Hundreds of different mutations in genes with diverse functions are responsible for Retinitis Pigmentosa. Reliable animal models are necessary to understand the disease biology and implement therapeutic approaches. However, various rodent models of RP are developmental and do not mimic the natural time course of human RP. Purpose of this study is to provide the complete characterization of an inducible and rapid model of RP obtained by intravitreal injections of Cytotoxic Necrotizing Factor 1 (CNF1) in adult rats. CNF1 is produced by pathological E. coli and causes constitutive activation of RhoA, Rac1 and Cdc42 GTPases. These key regulators of actin cytoskeleton are highly expressed in photoreceptors. In mouse rods, Rac1 is part of the NADPH oxidase complex and is a molecular switch of oxidative stress.
Methods :
A single intravitreal injection of 2nM CNF1 (C. Fiorentini, ISS Rome) was performed in the right eyes of Long Evans hooded rats aged 2-4 months. Left eyes were injected with vehicle or inactivated toxin. Dihydroethidium (DHE) histochemical assay was performed on fresh-frozen retinal sections obtained 24 hrs post injection. 2, 7 and 14 days post injection, ICCH with cell type-specific antibodies and staining with Phalloidin (a polymerized actin marker) were performed on retinal vertical sections, then analyzed by confocal microscopy. We also studied retinas from eyes injected with CNFY (G. Schmidt, Freiburg University), a toxin that constitutively activates RhoA only. Additional CNF1 injected rats were used for scotopic and photopic ERG recordings.
Results :
Morpho-functional analysis of CNF1 treated retinas at all time points showed a degeneration pattern similar to genetic RP models: a primary rod-to-cone degeneration with a center to periphery gradient paralleled by a decline of the ERG amplitude and followed by inner retinal remodeling. Phalloidin staining showed early actin rearrangement of the outer limiting membrane. DHE staining demonstrated the occurrence of oxidative stress 24 hrs post injection, before morphological signs of degeneration. Control retinas were normal. CNFY injections did not cause retinal abnormalities, excluding RhoA from the degeneration process.
Conclusions :
CNF1 intraocular injections induce a retinal degeneration recapitulating the main features of RP. This is mainly due to Rac1 activation, triggering both NADPH oxidase complex activation and actin polymerization.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.