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

Abstract

 

Purpose

 

Hypothermia has been described as a very effective intervention to prevent or treat brain and retinal damage. Under cold conditions, there is a general reduction in protein expression, but there are 2 proteins whose expression is upregulated by hypothermia: CIRP and RBM3. Both are RNA-binding proteins that nowadays are considered cold sensors, thus providing a molecular mechanism of action for the advantages of hypothermia. These proteins have never been characterized in the retina and here we offer a preliminary description of their behaviour in this organ, both in vitro and in vivo.

 

Methods

 

Retinal cell lines R28 (rat neural retina) and mRPE (monkey retinal pigment epithelium) were exposed to different temperatures in a time-dependent manner and the expression of RBM3 and CIRP was measured through quantitative real time PCR (qRT-PCR) and Western blotting. In addition, Sprague-Dawley albino rats of different ages (newborns and adults) were exposed to a cold environment (8 0C) for different periods of time. Retinas were either snap frozen for molecular analysis (qRT-PCR and Western blotting) or fixed in paraformaldehyde for histological and immunohistochemical analysis with specific antibodies against CIRP and RBM3. Data were analyzed with ANOVA tests.

 

Results

 

In both cell lines, there was a time-dependent significant increase of RBM3 and CIRP expression after exposure to a cold environment. Maximum expression was reached after 6 h at 32 0C. Immunoreactivity (IR) for RBM3 and CIRP was negligible in retinas not exposed to hypothermia. On the other hand, retinas of both neonate and adult rats that had been exposed to hypothermia presented high levels of IR for both proteins with different colocalization patterns (Fig. 1).

 

Conclusions

 

As happens in the central nervous system, cells in the retina can sense cold exposure through elevation of RBM3 and CIRP expression. These proteins are expressed in several cell types and can be responsible for the beneficial effects of hypothermia through the binding of specific molecules of mRNA.  

 

Figure 1. Retinas of newborn rats that were exposed to room temperature (CTL) or to hypothermia (HYP), stained with specific antibodies against RBM3 (green) and CIRP (red). Nuclear contrast was accomplished with DAPI (blue).