Figure 1.
The neuroprotective effects of rapamycin in the COH.
(A): Experimental protocols showing the treatment regimen of rats subjected to the COH receiving intraperitoneal injection of rapamycin (4 mg/kg) daily for 2 d before cauterization and then every other day for 6 weeks. (B): IOP was measured at the indicated time points after challenge in different groups. (C): Representative images of fluorogold-labeled RGCs of retinas in a normal and a COH rat. (D): Quantification of fluorogold labeling of RGCs. Each group includes 8 rats, and experiments were repeated once. The data are either representative or the mean ± SEM of 2 separate experiments. **p<0.01. Abbreviations: COH, chronic ocular hypertension model; RAPA, rapamycin; IOP, intraocular pressure; RGC, retinal ganglion cell.
Figure 2.
Rapamycin treatment inhibited apoptosis signaling and neurotoxic mediator release in COH retinas.
Caspase-3 activity (A) and the expression of active caspase-3 (B) in retinas were detected by ELISA and western blotting, respectively. (C): The nitrotyrosine content in retinas was determined using the OxiSelectTM Nitrotyrosine ELISA Kit. (D): The level of iNOS in retinas was measured by western blotting. TNF-α protein (E) and mRNA expression (F) was determined based on real-time PCR and ELISA. Each group includes 8 rats, and experiments were repeated once. Values are presented as the mean ± SEM of 3 replicates. **P<0.01. Abbreviations: COH, chronic ocular hypertension model; RAPA, rapamycin; iNOS, induced nitric oxide synthase; TNF-α, tumor necrosis factor-alpha.
Figure 3.
Rapamycin inhibited neurotoxic mediator release in microglia by modulating NF-κB signaling.
BV2 microglia were plated in six-well plates for 24 hours and subsequently stimulated with LPS for another 16 hours in the presence or absence of rapamycin. The TNF-α level (A) in the supernatants and the expression of TNF-α mRNA (B) in microglia were determined by ELISA and real-time PCR, respectively. (C): The nitrite content in the supernatants was measured by the Griess reaction. The expression of iNOS (D), NF-κB (E) and IκB-α (F) in microglia was determined based on western blotting. Values are presented as the mean ± SEM of 3 replicates, and experiments were repeated at least 2 times with similar results. **p<0.01. Abbreviations: LPS, lipopolysaccharides; RAPA, rapamycin; TNF-α, tumor necrosis factor-alpha; iNOS, induced nitric oxide synthase; NF-κB, nuclear factor-kappa B; IκB-α, I kappa B-alpha.
Figure 4.
Inhibition of microglial activation by rapamycin is involved in rapamycin–mediated neuroprotection in the COH.
(A): Representative images of Iba1 immunostaining (red) of retinas in a normal rat, a COH rat, and a COH rat after treatment with rapamycin. (B): Quantification of Iba1+ cells in the retinas of different experimental groups. The expression of Iba1 mRNA (C) and protein (D) in the retinas of different experimental groups was determined by real-time PCR and western blotting, respectively. (E): The expression of NF-κB in retinas was determined by western blotting. Each group includes 8 rats, and experiments were repeated once. Values are presented as the mean ± SEM of 3 replicates. **p<0.01. Abbreviations: COH, chronic ocular hypertension model; RAPA, rapamycin; NF-κB, nuclear factor-kappa B; Iba1, ionized calcium-binding adapter molecule 1.
Figure 5.
Rapamycin inhibited apoptosis in glutamate-injured primary RGCs.
(A): Representative images of Thy-1 immunostaining (green) in primary RGCs. (B): The cell viability of primary RGCs was measured by MTT assay. (C, D): The numbers of PI+Annexin+ cells, indicating apoptosis, were detected by flow cytometry. Values are presented as the mean ± SEM of 3 replicates, and experiments were repeated at least 2 times with similar results. **p<0.01. Abbreviations: GLUT, glutamate; RAPA, rapamycin; PI, propidium iodide.
Figure 6.
Inhibition of Akt dephosphorylation is implicated in rapamycin-mediated neuroprotection in primary RGCs and the COH.
Akt phosphorylation at sites Thr308 (A) and Ser473 (B) in primary RGCs was determined by western blotting. (C): The cell viability of primary RGCs was measured by MTT assay. (D): Akt phosphorylation at Thr308 in retinas was measured by western blotting. Values are presented as the mean ± SEM of 3 replicates, and experiments were repeated at least 2 times with similar results. **p<0.01. Abbreviations: GLUT, glutamate; RAPA, rapamycin; Akti-1/2, an Akt inhibitor.