First Author: C.Kaur SINGAPORE
Co Author(s): G. Rathnasamy 0 0 0 0 0 0 0 0 0
Back to previous
Retinal ganglion cells (RGCs) undergo apoptosis in the developing retina in hypoxic conditions. Increased release of glutamate and production of inflammatory mediators, nitric oxide and free radicals induced by it have been implicated in RGCs death. The role of glutamate in masking neuroprotection rendered by neurotrophic factors such as insulin-like growth factor-1 (IGF1) is unknown. This study was aimed at evaluating the effect of glutamate on IGF-1 pathway in hypoxic retina and in the death of RGCs.
One-day-old wistar rats were subjected to hypoxia (5% oxygen+95% nitrogen) for 2 hours and subsequently sacrificed at different time points ranging from 3h to 14d. In vitro cultures of RGCs were prepared from retinas collected from 6-d-old rats.
The protein expression of IGF-1 in the immature retina was studied using western blotting and double-immunofluorescence, while the tissue concentration of glutamate and IGF-1 was estimated by ELISA. Along with IGF-1, molecules such as phosphorylated phosphoinositide 3-kinase (p-PI3K), insulin receptor substrate-1 (IRS-1) phosphorylated at ser residue (p-IRS-1ser) and phosphorylated protein kinase-A (p-PKA), which are known to be involved in IGF-1 signalling pathway, were studied in the retina and primary cultures of RGCs using western blotting and double-immunofluorescence. Furthermore, in cultured RGCs, while glutamate concentrations were measured by ELISA, apoptosis was evaluated by caspase-3 immunolabelling.
Following hypoxic exposure the concentration of IGF-1 in retina was increased up to 24h in comparison to the controls, which was also evident with double-immunofluorescence and western blotting. In addition, the concentration of glutamate in hypoxic retinal tissues and hypoxic cultured RGCs was found to be significantly increased. In primary cultured RGCs, the expression of proteins p-PI3K, p-IRS-1ser and p-PKA was up-regulated following a hypoxic insult. Moreover, there was an increased caspase-3 labelling of cultured RGCs subjected to hypoxia confirming their apoptosis.
The above results suggest that hypoxia alters the tissue concentration of glutamate and IGF-1. IGF-1 is known to provide neuroprotection by eliciting PI3K activity when bound to IRS1. Hence, the up-regulation of p-PI3K in the cultured RGCs in response to hypoxia suggests an effort of IGF-1 in protecting the RGCs. However, this neuroprotective action of IGF-1 may be masked by excess glutamate produced by RGCs, and this was evident with the increased expression of p-PKA and p-IRS-1ser in the hypoxic cultured RGCs. Phosphorylation of IRS-1 at the Ser307 is enabled by excess glutamate through PKA dependant pathway and may result in reduced binding of IGF-1 to its receptor IRS-1. The uncoupling of IGF-1 from IRS-1 might favour the death of RGCs as shown by caspase-3 labelling. These results implicate that glutamate, by blocking IGF-1 mediated neuroprotection, could cause the apoptosis of RGCs in hypoxic neonatal retina.