Hypothermic treatment induces the expression of cold sensing proteins CIRP and RBM3 in the retina, both in vitro and in vivo

ALFREDO MARTINEZ; MANUEL REY-FUNES; DANIELA S CONTARTESE; VERÓNICA B DORFMAN; FEDERICO ROLÓN; ANIBAL SAROTTO; FABIÁN LOIDL; IGNACIO LARRAYOZ

Congreso; ARVO Annual Meeting; 2015

PurposeHypothermia has been described as a very e􀃗ective intervention to prevent or treat brainand retinal damage. Under cold conditions, there is a general reduction in proteinexpression, but there are 2 proteins whose expression is upregulated by hypothermia:CIRP and RBM3. Both are RNA-binding proteins that nowadays are considered coldsensors, thus providing a molecular mechanism of action for the advantages ofhypothermia. These proteins have never been characterized in the retina and here weo􀃗er a preliminary description of their behaviour in this organ, both in vitro and in vivo.MethodsRetinal cell lines R28 (rat neural retina) and mRPE (monkey retinal pigment epithelium)were exposed to di􀃗erent temperatures in a time-dependent manner and the expressionof RBM3 and CIRP was measured through quantitative real time PCR (qRT-PCR) andWestern blotting. In addition, Sprague-Dawley albino rats of di􀃗erent ages (newborns andadults) were exposed to a cold environment (8 0C) for di􀃗erent periods of time. Retinaswere either snap frozen for molecular analysis (qRT-PCR and Western blotting) or 􀃒xed inparaformaldehyde for histological and immunohistochemical analysis with speci􀃒cantibodies against CIRP and RBM3. Data were analyzed with ANOVA tests.ResultsResultsIn both cell lines, there was a time-dependent signi􀃒cant increase of RBM3 and CIRPexpression after exposure to a cold environment. Maximum expression was reached after6 h at 32 0C. Immunoreactivity (IR) for RBM3 and CIRP was negligible in retinas notexposed to hypothermia. On the other hand, retinas of both neonate and adult rats thathad been exposed to hypothermia presented high levels of IR for both proteins withdi􀃗erent colocalization patterns (Fig. 1).ConclusionsAs happens in the central nervous system, cells in the retina can sense cold exposurethrough elevation of RBM3 and CIRP expression. These proteins are expressed in severalcell types and can be responsible for the bene􀃒cial e􀃗ects of hypothermia through thebinding of speci􀃒c molecules of mRNA.