As shown in
Figures 8C and
8D, mouse retinas were double-stained using anti-BDNF and anti-GS antibodies. Significantly lower BDNF expression was noted in the inner nuclear layer of microbead-injected eyes (6.0 ± 4.5%) (
Figs. 8Ce–
8Ch,
8D) compared to controls (12.9 ± 4.9%) (
Figs. 8Ca–
8Cd,
8D) (
P < 0.05). Microbead injection with 30-µM RSV increased the expression of BDNF compared with microbead injection alone (18.3 ± 6.0%;
P < 0.001) (
Figs. 8Cm–
8Cp,
8D). There was no significant increase in the expression of BDNF as a result of injections of 30-µM RSV alone compared with the control group (14.6 ± 5.6%;
P = 0.558) (
Figs. 8Cq–
8Ct,
8D). BDNF protein levels of whole retina in western blot analysis showed similar patterns in all groups (
Supplementary Fig. S3). To investigate that this observed change in expression could be from MGCs, we also studied the gene expression of BDNF in the cultured MGCs exposed to RSV at concentrations of 3, 30, 70, and 140 µM in vitro. We investigated the viability of cultured MGCs exposed to each concentration of RSV.
Figure 8E shows that MGC viability increased in response to each of the concentrations of RSV treatment (
P < 0.01), indicating that RSV and the vehicle are not toxic to the cultured MGCs. Gene expression of BDNF significantly increased by 2.9-fold compared to the control (
P < 0.001) (
Fig. 8F) following treatment with RSV at a concentration of 140 µM, but was not affected at concentrations of 3 µM, 30 µM, or 70 µM. Furthermore, we analyzed the expression of TrkB in RGCs (
Figs. 8G,
8H). Mouse retinae were double-stained using anti-Brn-3a and anti-TrkB antibodies. TrkB-immunolabeled areas were observed in the GCL of controls (6.4 ± 2.1%) (
Figs. 8Ga–
8Gd,
8H). TrkB expression was markedly diminished in the microbead injection group compared with the control group (3.1 ± 2.0%;
P < 0.05) (
Figs. 8Ge–
8Gh,
8H). Microbead injection with either 3-µM or 30-µM RSV significantly upregulated the expression of TrkB compared with microbead injection alone (7.3 ± 3.3% and 12.5 ± 4.6%, respectively;
P < 0.01) (
Figs. 8Gi–
8Gp,
8H). Administration of 30-µM RSV alone did not significantly alter the expression of TrkB compared with controls (7.0 ± 4.7%;
P = 0.999) (
Figs. 8Gq–
8Gt,
8H). In western blot analysis, TrkB protein levels of whole retina showed similar patterns in all groups (
Supplementary Fig. S4).