Am-treated group, while 4 weeks of treatment with hypotensive eye drops (i.e., with Ti/Tr, Ti/D, or Ti/B), which began 4 weeks after IOP elevation, significantly improved RGC survival (**p,0.05) relative to the untreated hypertensive group. Treatment with Ti (0.5 ) alone did not substantially improve RGC survival. doi:10.1371/journal.pone.0049730.gconsisted of 10 ml of TaqMan Universal PCR Master Mix, AmpErase uracil-N-glycosylase (UNG; 26), 1 ml of Assay-onDemand (206), and 1 ml of cDNA in a 20-ml reaction. The PCR conditions for all genes were as follows: UNG activation, 50uC for 2 min; preheating, 95uC for 10 min; then 40 cycles of denaturation (95uC for 15 s) and annealing/elongation (60uC for 1 min). Each sample was run in duplicate. The data were analyzed using SDS 2.2 software (Applied Biosystems). 18S RNA served as the endogenous control against which to normalize the amount of cDNA added to each reaction (DCt), and the mean DCt of control samples was used as the calibrator to calculate DDCt. The comparative Ct method was employed, whereby the relative quantity of the respective target gene mRNA–normalized to the endogenous control and relative to the calibrator–is expressed as the relative change: 2 DCt.with post-hoc analyses using the Tukey HSD test to identify possible differences among the experimental groups. If the distribution was not Gaussian, the Kruskal-Wallis H test was used.Results Pharmacological effects on intraocular pressureThe baseline 1313429 IOP in the normotensive sham-treated group was 15.861.5 mmHg. By 10?2 days after episcleral vein cauterization, the IOP had increased significantly by 1.6-fold to 24.861.7 mmHg (p,0.001). These values are consistent with those obtained by other groups, and are nearly identical to those recorded in humans, rabbits, and anesthetized monkeys [31]. The recordings were sustained for the entire duration of the experimental period if animals remained untreated. If treated hypotensively, IOP was reduced effectively as follows (p,0.05): 1. Ti lowered IOP to 20.0061.65 mmHg (p,0.05). 2. Ti/B reduced IOP to 20.561.4 mmHg (p,0.03). 3. Ti/D and Ti/Tr produced more distinctive 34540-22-2 chemical information reductions in IOP (18.5061.35 and 18.7561.80 mmHg, respectively; p,0.001).Statistical analysisAll data regarding IOP recordings, RGC densities of retinal whole-mounts, and relative protein densities in WBs are presented as mean6SD values. Data were analyzed statistically using the two-independent-samples test (SPPS, Statistica version 7) for Gaussian distributions, with the remaining quantitative data analyzed using two-way analysis of variance (Statistica version 7)Protein Changes in Neurodegeneration5-FG. Topical treatment with the combination compounds Ti/Tr, Ti/D, and Ti/B strongly enhanced RGC survival, preserving 20206548 RGCs/mm2 (p,0.001; n = 3), 20316734 RGCs/mm2 (p,0.004; n = 3), and 19566340 RGCs/mm2 (p,0.001; n = 3), respectively. The RGC densities in the experimental groups are illustrated in Fig. 2.Retinal protein profilingSeveral protein spots were reproducibly detected with 2DE (those for the hypertensive group are shown in Fig. 3A). Landmark protein spots that A 196 site appeared with consistent staining intensities in all experimental groups were first mapped and identified (listed in Table 1). In addition, a conspicuous group of proteins appeared in the middle range of molecular masses (20?0 kDa) at slightly basic pH values (Fig. 3A). This area (within the rectangular frame in Fig. 3A, labeled 3B1) also containe.Am-treated group, while 4 weeks of treatment with hypotensive eye drops (i.e., with Ti/Tr, Ti/D, or Ti/B), which began 4 weeks after IOP elevation, significantly improved RGC survival (**p,0.05) relative to the untreated hypertensive group. Treatment with Ti (0.5 ) alone did not substantially improve RGC survival. doi:10.1371/journal.pone.0049730.gconsisted of 10 ml of TaqMan Universal PCR Master Mix, AmpErase uracil-N-glycosylase (UNG; 26), 1 ml of Assay-onDemand (206), and 1 ml of cDNA in a 20-ml reaction. The PCR conditions for all genes were as follows: UNG activation, 50uC for 2 min; preheating, 95uC for 10 min; then 40 cycles of denaturation (95uC for 15 s) and annealing/elongation (60uC for 1 min). Each sample was run in duplicate. The data were analyzed using SDS 2.2 software (Applied Biosystems). 18S RNA served as the endogenous control against which to normalize the amount of cDNA added to each reaction (DCt), and the mean DCt of control samples was used as the calibrator to calculate DDCt. The comparative Ct method was employed, whereby the relative quantity of the respective target gene mRNA–normalized to the endogenous control and relative to the calibrator–is expressed as the relative change: 2 DCt.with post-hoc analyses using the Tukey HSD test to identify possible differences among the experimental groups. If the distribution was not Gaussian, the Kruskal-Wallis H test was used.Results Pharmacological effects on intraocular pressureThe baseline 1313429 IOP in the normotensive sham-treated group was 15.861.5 mmHg. By 10?2 days after episcleral vein cauterization, the IOP had increased significantly by 1.6-fold to 24.861.7 mmHg (p,0.001). These values are consistent with those obtained by other groups, and are nearly identical to those recorded in humans, rabbits, and anesthetized monkeys [31]. The recordings were sustained for the entire duration of the experimental period if animals remained untreated. If treated hypotensively, IOP was reduced effectively as follows (p,0.05): 1. Ti lowered IOP to 20.0061.65 mmHg (p,0.05). 2. Ti/B reduced IOP to 20.561.4 mmHg (p,0.03). 3. Ti/D and Ti/Tr produced more distinctive reductions in IOP (18.5061.35 and 18.7561.80 mmHg, respectively; p,0.001).Statistical analysisAll data regarding IOP recordings, RGC densities of retinal whole-mounts, and relative protein densities in WBs are presented as mean6SD values. Data were analyzed statistically using the two-independent-samples test (SPPS, Statistica version 7) for Gaussian distributions, with the remaining quantitative data analyzed using two-way analysis of variance (Statistica version 7)Protein Changes in Neurodegeneration5-FG. Topical treatment with the combination compounds Ti/Tr, Ti/D, and Ti/B strongly enhanced RGC survival, preserving 20206548 RGCs/mm2 (p,0.001; n = 3), 20316734 RGCs/mm2 (p,0.004; n = 3), and 19566340 RGCs/mm2 (p,0.001; n = 3), respectively. The RGC densities in the experimental groups are illustrated in Fig. 2.Retinal protein profilingSeveral protein spots were reproducibly detected with 2DE (those for the hypertensive group are shown in Fig. 3A). Landmark protein spots that appeared with consistent staining intensities in all experimental groups were first mapped and identified (listed in Table 1). In addition, a conspicuous group of proteins appeared in the middle range of molecular masses (20?0 kDa) at slightly basic pH values (Fig. 3A). This area (within the rectangular frame in Fig. 3A, labeled 3B1) also containe.
