Abstract
Purpose: :
The c–Jun N–terminal kinase (JNK) has been shown to be involved in various insults that cause apoptotic death of retinal ganglion cells (RGC) in vivo, such as intravitreal injection of NMDA, retinal ischemia, optic nerve transection, and ocular hypertension induced by laser trabeculoplasty. Elevated level of phosphorylated JNK (p–JNK) was also observed in retinal neurons of glaucoma patients. We tested if a prototypical JNK inhibitor can exert a direct protective effect on cultured RGC.
Methods: :
Cytotoxicity was induced in primary cultured adult rat retinal cells by glutamate treatment or by the removal of selective trophic factors. The potential protective effect of various compounds was evaluated by treatment of the cells concurrent with insults. RGC survival was assessed by counting Thy–1–positive cells. Immunocytochemistry was used to assess content of p–JNK.
Results: :
The primary cultured retinal cells used in these studies contained mainly retinal neurons enriched in RGC. After a 3–day treatment, glutamate induced a concentration–dependent loss of RGC with an EC50 of 22 µM. In the presence of 100 µM glutamate, RGC survival was 57.0 ± 1.2 % (mean ± SEM, n = 52) of control (p < 0.001). The glutamate toxicity was completely eliminated by concomitant addition of 100 nM MK801, an NMDA receptor antagonist. Similarly, trophic factor withdrawal for 3 days also induced a loss of RGC to 52.1 ± 1.3 % (n = 57) of control (p < 0.001). These insults caused a corresponding significant (p < 0.05) increase in p–JNK in RGC: control = 100.0 ± 2.1 % (n = 9); glutamate treatment = 206.1 ± 17.6 % (n = 5); trophic factor withdrawal = 206.8 ± 22.9 % (n = 9). The selective JNK inhibitor SP600125 was protective against both insults in a concentration–dependent manner, but with different protective potencies. It was significantly more potent in preventing cell death induced by trophic factor withdrawal (EC50 = 0.3 nM) than glutamate (EC50 = 16 nM). However, at 100 nM it completely prevented cell death for both insults (n = 6; p < 0.001).
Conclusions: :
An adult rat RGC–enriched culture system was established and used successfully to evaluate cytotoxic and cytoprotective agents. In this culture system, both glutamate treatment and trophic factor withdrawal induced RGC loss and activated JNK. The JNK inhibitor SP600125 potently and efficaciously protected against both insults. The neuroprotective effect of JNK inhibitors may represent a novel approach for the potential treatment of various retinopathies.
Keywords: ganglion cells • neuroprotection • pharmacology