Endothelin B Receptors Contribute to Neurodegeneration in a Rodent Model of Glaucoma

Abstract

Purpose: : ET_B receptors have gained increased attention for their neurodegenerative role in glaucoma. The present study was aimed at investigating changes in the expression of ET_B receptors in vivo in a rodent model of glaucoma and whether neurodegenerative changes following IOP elevation are attenuated in ET_B-deficient transgenic rats.

Methods: : In one set of Brown Norway rats, IOP was elevated in one eye using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the corresponding contralateral eye served as control. After 2 or 4 weeks of IOP elevation, animals were sacrificed. Retinal sections were obtained and stained for ET_B receptor expression by immunohistochemistry. Colocalization of ET_B receptor immunostaining was performed using an antibody to neuritin, a selective marker of RGCs. In the second set of Brown Norway rats, retinal ganglion cells were retrogradely labeled with Fluoro-gold following which IOP was elevated in one eye using Morrison’s method, while the contralateral eye served as control. After 2 weeks of IOP elevation, animals were sacrificed. Retinas were sectioned and stained for ET_B receptor expression by immunohistochemistry. In a separate study, adult wild type and ET_B- deficient transgenic Wistar rats were used for retrograde labeling of retinal ganglion cells (RGCs) with Fluoro-gold. Following labeling, IOP was elevated in one eye using the Morrison’s method, while the contralateral eye served as control. After IOP was elevated, rats were maintained for 4 weeks and sacrificed. Fluoro-gold labeled retinas were isolated, flat-mounted, imaged in a Zeiss LSM-510 confocal microscope, and labeled RGCs were counted. Optic nerves were sectioned and stained with p-phenylenediamine (PPD).

Results: : Immunohistochemical analysis showed that IOP elevation for both 2 and 4 weeks produced increased expression of ET_B receptors in retinal ganglion cells. After retrograde labeling and IOP elevation in Brown Norway rats, increased colocalization of ET_B immunostaining with Fluoro-gold was observed in retinal ganglion cells. IOP elevation for 4 weeks in wild type transgenic rats caused significant loss of RGCs and degeneration of axons in the optic nerve, whereas significant neuroprotection of RGCs and optic nerve axons were observed in ET_B-deficient transgenic rats.

Conclusions: : Increased intraocular pressure caused increased ET_B receptor expression possibly through pressure-related mechanism. ET_B receptors appear to play a causative role in retinal ganglion cell death in the Morrison’s rodent model of glaucoma.

Keywords: ganglion cells • neuroprotection • receptors: pharmacology/physiology