Resveratrol Inhibits Neuronal Apoptosis and Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Diabetic Mouse Retina
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Figures
- FIG. 1.
Determination of retinal thickness and neuronal cell death in retinas of mice 2 months after induction of diabetes compared with control mice. For morphological analysis, H&E staining was performed on 10-μm retinal cryosections of control (A) and diabetic (B) mice. Retinal thickness was measured as the length (in μm) from the GCL to the tip of the INL, and the results were obtained from comparative analysis of four different images of each retina (C). To assess cell death induced by diabetes in the retinas, Western blotting using an antibody to active caspase-3 was performed for the retinas of both groups (D). An immunoblot with an antibody to active caspase-3 was reprobed with an antibody to pro-caspase-3 to control for differences in loading, and the level of each protein was normalized to that of pro-caspase-3. Four independent tests were repeated in different retinal extracts from each group, and the results are indicated as the fold change (E). To investigate retinal neuron death, the TUNEL assay was performed on retinal sections from control and diabetic mice, followed by counterstaining with cresyl violet (F and G). To confirm the death of RGCs, immunofluorescent staining of γ-Syn, the GC marker, using Alexa Fluor 488 goat anti-rabbit IgG (green) and TUNEL staining were performed consecutively on the same sections from diabetic mice, and the sections were counterstained with the nuclear marker DAPI (H–J). The total number of TUNEL+ RGCs was counted in four different fields of the GCL (∼100 μm) from three different retinas of each group (K). Data are means ± SE (n = 4). *P < 0.05 comparing control and diabetic groups. CTL and DM, control and diabetic mice 2 months after injection of buffer or STZ; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Bars, 12.5 μm. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 2.
Molecular and morphological expression of CaMKII and phospho-CaMKII (Thr286), and CaMKII kinase assay in retinas of mice 2 months after induction of diabetes compared with controls. Protein levels of CaMKII and phospho-CaMKII were assessed by Western blotting using 30 μg total protein from each retina, and representative blots from four independent Western blots are shown in A. The immunoblots were reprobed with α-tubulin to control for differences in loading, and levels of each protein were normalized to that of α-tubulin. The results indicate the fold change in B and C, respectively. Analysis of CaMKII kinase activity was performed in the same retinal homogenates (100 μg) prepared for immunoblotting using the CaMKII Kinase Assay Kit. The results are expressed as picomoles of phosphate incorporated into the CaMKII substrate peptide per minute per milligram total protein, and four independent tests were repeated with different retinal extracts for each group (D). Data are means ± SE (n = 4). *P < 0.05 comparing control and diabetic groups. To confirm the distribution of CaMKII (E and F) and phospho-CaMKII (G and H) in retinal neuronal cells from control and diabetic mice, retinal cryosections were immunostained with each antibody and then counterstained with cresyl violet. Arrows indicate immunopositive signals of CaMKII and phospho-CaMKII in the GCL of the diabetic retina, and arrowheads in each insert show the double-immunofluorescent staining of γ-Syn and CaMKII or phospho-CaMKII, using Alexa Fluor 350 (blue) and 594 (red) goat anti-rabbit and mouse IgGs (F and H). CTL and DM, control and diabetic groups 2 months after injection of buffer or STZ; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Bars, 12.5 μm. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 3.
Co-localization of CaMKII or phospho-CaMKII immunopositive signals and apoptotic RGC cells in retinas of mice 2 months after induction of diabetes compared with controls. To examine the correlation between CaMKII or phospho-CaMKII expression and RGC cell death, double-immunofluorescent staining for CaMKII or phospho-CaMKII and γ-Syn, the ganglion cell marker, using Qdot 525 (green) and Alexa Fluor 350 (blue) goat anti-mouse and rabbit IgGs, and TUNEL staining (red) were sequentially performed on the same sections. Arrows in B and E indicate TUNEL-positive RGC cells that were immunopositive for CaMKII and phospho-CaMKII in the diabetic retinas compared with control (A and D). The total number of triple-labeled cells was counted in the GCL (∼100 μm) in four different fields from three different retinas for each group (C and F). Data are means ± SE (n = 4). **P < 0.001 comparing control and diabetic groups. CTL and DM, control and diabetic mice 2 months after injection of buffer or STZ; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Bars, 12.5 μm. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 4.
Changes in CaMKII and phospho-CaMKII protein levels, kinase activity of CaMKII, and the level of active caspase-3 protein after αCaMKII knockdown using RNA interference in retinas of mice 2 months after induction of diabetes compared with controls. siRNA (2 μl) was injected into the right vitreous of the retinas, and an equal volume of distilled water was injected into the left vitreous as a control. A and B: Dose- and time-independent effects of siRNA on the level of CaMKII protein in the retinas of mice 2 months after the induction of diabetes, 2 days after injection with 0.01, 0.1, or 1 mmol/l siRNA, and 0, 1, 2, and 4 days after injection with 1 mmol/l siRNA using immunoblotting. C–F: Changes in CaMKII and phospho-CaMKII protein levels, CaMKII kinase activity, and active caspase-3 protein level in the retinas of control and diabetic groups, 2 days after the injection of 1 mmol/l αCaMKII siRNA. Each immunoblot was reprobed with α-tubulin, and levels of the other proteins were normalized to that of α-tubulin. The fold changes in protein levels compared with the controls (CTL) are indicated below the blots (A–C) or as a bar graph (F). DM, diabetic subjects. Results of the CaMKII kinase assay indicate the picomoles of phosphate incorporated into the CaMKII substrate peptide per minute per milligram of total protein (D). All data show representative results from four independent tests and are expressed as means ± SE (n = 4). *P < 0.05 and **P < 0.001 in A, comparing the DW-treated group with the others; *P < 0.05 and **P < 0.001 in B, comparing day 0 with the others; *P < 0.05 and **P < 0.001 in C–F, comparing the DW-treated control group with the others; †P < 0.05 in C–F, comparing the DW- and siRNA-treated diabetic groups. C and D, control and diabetic groups at 2 months after injection with STZ or buffer; DW, distilled water.
- FIG. 5.
Changes in CaMKII and phospho-CaMKII protein levels, kinase activity of CaMKII, and active caspase-3 protein level after AIP treatment of retinas of mice 2 months after induction of diabetes compared with controls. AIP (2 μl) was injected into the right vitreous in the retinas, and an equal volume of saline was injected into the left vitreous humor as a control. A: Western blot analysis of the time-independent effect of 500 μmol/l AIP on the CaMKII protein level in the retinas of mice 2 months after induction of diabetes at 0, 1, 2, and 4 days after AIP treatment. B–E: Changes in CaMKII and phospho-CaMKII protein levels, CaMKII kinase activity, and active caspase-3 protein level in the retinas of control and diabetic groups 2 days after AIP treatment. Each immunoblot was reprobed with α-tubulin, and the levels of other proteins were normalized to that of α-tubulin. The fold changes in protein levels compared with the controls (CTL) are indicated below the blots (A and B) or as a bar graph (E). DM, diabetic subjects. Results of the CaMKII kinase assay are indicated as picomoles of phosphate incorporated into the CaMKII substrate peptide per minute per milligram of total protein (C). All data show representative results from four independent tests and are expressed as means ± SE (n = 4). *P < 0.05 in A, comparing day 0 with the others; *P < 0.05 and **P < 0.001 in B–E, comparing the saline-treated control group with the others; †P < 0.05 and ‡P < 0.001 in B–E, comparing the saline- and AIP-treated diabetic groups. C and D, control and diabetic groups 2 months after injection of STZ or buffer.
- FIG. 6.
Effects of resveratrol on CaMKII and phospho-CaMKII protein expression and the kinase activity of CaMKII in retinas of mice 2 months after induction of diabetes compared with controls. Resveratrol (20 mg · kg−1 · day−1) was completely suspended in 0.5% CMC/0.9% saline and was administered by oral gavage once a day for 4 weeks, beginning 1 month from the last day of the fifth injection of STZ or buffer, and 0.5% CMC was used as a control. A shows representative immunoblots of CaMKII and phospho-CaMKII. Each immunoblot was reprobed with α-tubulin. The levels of the other proteins were normalized to that of α-tubulin, and the fold changes are indicated in B and C, respectively. Results of the CaMKII kinase assay are indicated in picomoles of phosphate incorporated into the CaMKII substrate peptide per minute per milligram of total protein (D). To confirm changes caused by resveratrol in CaMKII and phospho-CaMKII in neuronal cells of control and diabetic retinas, cryosections were immunostained with specific antibodies and counterstained with cresyl violet. Arrows and arrowheads indicate RGCs immunopositive for CaMKII (E–G) and phospho-CaMKII (I–K), respectively. The image of the resveratrol-treated control group was omitted from the figure because resveratrol and CMC treatment did not significantly affect control mice. The total number of these cells was counted in the GCL (∼100 μm) in four different fields from three different retinas for each group (H and L). All data show representative results from four independent tests and are means ± SE (n = 4). *P < 0.05 and **P < 0.001, comparing the CMC-treated control group with the others; †P < 0.05 comparing the CMC- and resveratrol-treated diabetic groups. C (CTL) and D (DM), control and diabetic mice 2 months after injection of buffer or STZ; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RES, resveratrol. Bars, 12.5 μm. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 7.
Effects of resveratrol on cell death and RGC cells co-stained for TUNEL and phospho-CaMKII in retinas of mice 2 months after induction of diabetes compared with controls. A shows a representative immunoblot using antibody to active caspase-3. Each immunoblot was reprobed with α-tubulin. The levels of other proteins were normalized to that of α-tubulin, and the fold changes are indicated in B. To confirm the death of cells in conjunction with phospho-CaMKII expression, immunofluorescent staining of phospho-CaMKII using Alexa Fluor 488 goat anti-rabbit IgG (green) and TUNEL staining (red) were performed consecutively in the same sections, and the sections were counterstained with the nuclear marker DAPI (C–E). Arrows indicate TUNEL+ cells that are immunopositive for phospho-CaMKII in the retinas of diabetic mice without or with resveratrol. To examine whether the co-positive cells were RGC cells, we performed sequential double-immunofluorescent staining for γ-Syn, the ganglion cell marker, and phospho-CaMKII, using Alexa Fluor 350 (blue) and 488 (green) goat anti-rabbit and -mouse IgGs before TUNEL staining (red). The arrowheads in the insert show the triple-stained RGC cell in the diabetic retina (D). Image of the resveratrol-treated control group was omitted from the figure because resveratrol and CMC treatment did not significantly affect control mice. The total number of double-stained cells was counted in the GCL (∼100 μm) in four different fields from three different retinas for each group (F). Data are means ± SE (n = 4). *P < 0.05 comparing the CMC-treated control group with the others. †P < 0.05 comparing the CMC- and resveratrol-treated diabetic groups. C (CTL) and D (DM), control and diabetic mice 2 months after injection with buffer or STZ; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RES, resveratrol. Bars, 12.5 μm. (A high-quality digital representation of this figure is available in the online issue.)
Tables
- TABLE 1
Changes in body weight and blood glucose level with resveratrol treatment
Body weight (g) Blood glucose (mmol/l) C-C C-R D-C D-R C-C C-R D-C D-R Week 0 28.3 ± 0.3 27.7 ± 0.5 21.2 ± 0.5 20.4 ± 0.4 8.7 ± 0.1 9.0 ± 0.3 25.4 ± 0.8 27.5 ± 0.9 Week 2 27.6 ± 0.3 26.3 ± 0.4 21.1 ± 0.6 21.1 ± 0.5 9.0 ± 0.2 7.4 ± 0.2 31.8 ± 0.7 29.6 ± 0.8 Week 4 28.5 ± 0.3 27.8 ± 0.4 20.6 ± 0.6 21.2 ± 0.5 8.3 ± 0.2 7.9 ± 0.2 31.2 ± 0.8 27.7 ± 1.2* Data are means ± SE (n = 10). Resveratrol (in 0.5% CMC) at a dose of 20 mg/kg of body weight and 0.5% CMC alone were administered daily to control and diabetic mice, respectively, by oral gavage for 4 weeks. Time points were 0, 2, and 4 weeks after administration of resveratrol and CMC to control and diabetic mice, respectively, 1 month after STZ treatment: C-C and C-R, CMC- and resveratrol-treated control mice, respectively; D-C and D-R, CMC- and resveratrol-treated diabetic mice, respectively.
*P < 0.05 compared with CMC- and resveratrol-treated diabetic groups.
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