Abstract
Purpose: :
Changes in the expression of aquaporins have been reported in a number of diseases. However, such changes and mechanisms remain to be evaluated for retinal injury after optic nerve crush. This study was designed to analyze changes in the expression of AQP-4 (water selective channel) and AQP-9 (water and lactate channel) following optic nerve crush. In addition, AQP9 expression was also studied following stress conditions, such hypoxia, in a rat retinal ganglion cell-line.
Methods: :
The optic nerve of the right eye of Wistar rats was crushed. Retinal Ganglion Cells were retrogradely labeled by applying fluoroGold onto the left superior colliculus one week prior to crushing. Q-PCR was used for measuring changes in AQP4, AQP9, thy-1, caspase-3, Kir4.1, and beta-actin messages. Changes in Bcl-XL and GFAP expression were also followed using western blotting. Immunohistochemistry using a combination of AQP9/cytochrome-c antibodies was used to study localization and expression in RGC-5 cells following hypoxia. Reactive oxygen species were measured using CM-H2DCFDA. Cell proliferation was assessed using the MTT viability assay.
Results: :
The mean number (±SD) of RGCs labeled retrogradely from superior colliculus was 2089.6 ± 85.2/mm2 in rats without any treatment, which decreased to 1090.8 ± 77.6 (52.2%) and 496.6 ± 87.3/mm2 (23.7%) on day 7 and 14, respectively. While AQP4, Kir4.1, thy-1 mRNA levels decreased at 7 and 14 days, caspase-3 mRNA levels were upregulated both at 7 and 14 days. By contrast, AQP9 mRNA levels showed unique changes by decreasing at 7 days while increasing at 14 days. In addition, while GFAP increased at 7 and 14 days, Bcl-XL decreased at both time-points as determined by western blotting. AQP9 was detected in mitochondria from RGC-5 cells, and co-localized with cytochrome c in situ. Hypoxia increased AQP9 as well as cytochrome-c release indicating apoptosis. L-Lactic acid but not D-Lactic acid, dose-dependently inhibited cellular proliferation in RGC-5 cells, a mechanism that involves ROS production.
Conclusions: :
The reduced expression of AQP4 and Kir4.1 in retina following optic nerve crush suggest K+-buffering and water movement and likely impaired retinal function. The increased caspase-3 and decreased Bcl-XL expression suggest commitment to cellular death. The enhanced expression of AQP9 following hypoxia in retina rich in lactic acid may explain increased ROS production and subsequent oxidative damage observed in glaucoma following elevation of IOP.
Keywords: ganglion cells • optic nerve • ischemia