Negative impact of AQP-4 channel inhibition on survival of retinal ganglion cells and glutamate metabolism after crushing optic nerve.

The purpose of this study was to determine whether inhibition of aquaporin 4 (AQP4) is neuroprotective or neurodestructive after crushing the optic nerve of rats. The left optic nerves of rats were crushed, and TGN-020 (5.0 mg/kg, crush TGN-020) or its vehicle (DMSO, crush placebo) was injected intraperitoneally just after the crushing. As controls, the left optic nerves were exposed but not touched in other rats (sham controls). The retinal damages were determined by the density of retinal ganglion cells (RGCs) and the ratio of BAX/Bcl-2 on day 7. The glutamate level in the optic nerve on day 1 after the crushing was determined. The expressions of glutamine synthetase, glutamate-aspartate transporter (GLAST), and AQP4 were determined on day 3 by immunoblotting. The effects of AQP4 inhibition on the glutamate-induced changes of AQP4 expression and on the glutamate uptake were determined for optic nerve astrocytes in culture. The results showed that the density of RGCs was 2040 ± 91.3 cells/mm(2) (n = 6) in the sham control, and it was significantly decreased to 1072 ± 134.3 cells/mm(2) after crushing the optic nerve (P < 0.0001, crush placebo, n = 7; Fisher). An intraperitoneal injection of TGN-020 led to a further significant (P = 0.02, Fisher) decrease of the density of RGCs to 743 ± 371 cells/mm(2) (crush TGN-020, n = 7). The mRNA level of BAX/Bcl-2 ratio was 0.37 ± 0.05 in the sham control (n = 6) which was significantly increased to 0.88 ± 0.10 after crushing the optic nerve (placebo crush, n = 7; P = 0.0001, Scheffe). TGN-020 also significantly increased the BAX/Bcl-2 ratio to 1.29 ± 0.4 (n = 6) from the crush placebo group (P = 0.04, Scheffe). Immunoblotting showed similar changes in the protein levels. The glutamate level in the optic nerve was significantly increased to 53.7 ± 6.0 μM/mg/protein on day 1 (n = 4) from the sham control level of 45.9 ± 3.1 μM/mg/protein (n = 4; P = 0.04, t test). TGN-020 significantly (P < 0.05, Scheffe) depressed the expression of glutamate metabolism-related proteins on day 3. Exposure of cultured optic nerve astrocytes to glutamate (1.0 mM, n = 4) significantly increased the expression of AQP4 (P < 0.001, Scheffe) that was depressed by TGN-020 (100 nM, n = 4). In addition, glutamate uptake was inhibited by TGN-020 at 10 nM or higher. These results indicate that an inhibition of AQP4 enhances the loss of RGCs and retinal damages after crushing the optic nerve. Inhibition of AQP4 impairs glutamate metabolism which may account in part for these neurodestructive events.