Attenuation of c-Jun, Bax and Bid expression in retinas of ET_B receptor-deficient rats following ocular hypertension

Abstract

Purpose: Recent observations from our lab showed that loss of retinal ganglion cells (RGCs) and degeneration of optic nerve axons were greatly attenuated in ET_B receptor-deficient transgenic Wistar Kyoto rats, compared to wild type rats following elevation of intraocular pressure (IOP). In addition, in Brown Norway rats, elevated levels of ET_B receptors in RGCs were associated with increased expression of the immediate early gene c-Jun. The current study aimed at investigating whether there are any changes in c-Jun mRNA and protein levels in RGCs in wild type and ET_B receptor-deficient rats following elevation of IOP using the Morrison’s model of glaucoma. Expression of apoptotic proteins Bax and Bid was also assessed in these rats.

Methods: IOP was elevated in one eye of adult wild type and ET_B receptor-deficient rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. Following IOP elevation, rats were maintained for 2 weeks and sacrificed. Rat eyes were freshly embedded in optimal cutting temperature compound (OCT). Laser capture microdissection (LCM) was used to capture the ganglion cell layer from retinal cryosections. Total RNA was extracted from the ganglion cell layer and cDNA prepared from equal amounts of total RNA from different samples. Real-time PCR was performed using cDNA as template to detect gene expression of c-Jun, bax and bid. Beta-actin was used to normalize for equal loading of the cDNA template. In separate experiments, retinal sections were stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. Retinal sections were also immunostained for βIII-tubulin, which is selectively expressed by RGCs in the retina.<br />

Results: Following 2 weeks of IOP elevation, there was a significant decrease in mRNA and protein levels of c-Jun in RGCs of ET_B receptor-deficient rats, as compared to those of wild type rats. In addition, mRNA levels of bax and bid were significantly reduced in RGCs of the ET_B receptor-deficient rats. Moreover, there was a marked decrease in protein levels of Bax in RGCs in ET_B receptor-deficient rats, compared to wild type rats.

Conclusions: Decreased expression of c-Jun, Bax, and Bid contribute to enhanced RGC survival in ET_B receptor-deficient rats following IOP elevation.