Tetrandrine protects mouse retinal ganglion cells from ischemic injury

Figures & data

Figure 1 Structural formula of Tet and a schematic of intravitreal treatment and partitions of the retina for RGC counting.

Notes: (A) Structural formula of Tet. (B) Tet of 10 μM in 2 μL was injected into the central region of each murine vitreous 24 hours before ischemia. For RGC counting, the central, middle, and peripheral regions of the retinas were divided in each retinal cross section.
Abbreviations: RGC, retinal ganglion cell; Tet, tetrandrine.

Figure 2 Effects of Tet on the survival of RGC-5 cells and SSP-induced neuron-like cells under serum deprivation conditions.

Notes: RGC-5 cells cultured under serum deprivation (A) or with 10% FBS (B) were treated with Tet at doses of 10 nM, 0.1 μM, 0.5 μM, and 1 μM for 24 hours, 72 hours, and 96 hours. (A) Relative numbers of RGC-5 cells were increased at all Tet concentrations at 72 hours. (B) After the 96-hour treatment with 10% FBS, relative cell numbers at Tet concentrations of 0.5 μM and 1 μM were significantly lower than the DMSO control numbers (0.5 μM, P=0.012; 1 μM, P=0.006). (C) The numbers of surviving RGC-5 cells cultured under serum deprivation or with 10% FBS were examined after 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours. (D) At 48 hours after exposure, all of the Tet concentrations tested promoted the survival of 1 μM SSP-induced RGC-5 cells. The cell numbers were determined by WST-8 assay (A–D). Results are expressed as means ± SD. *P<0.05 compared to DMSO control, one-way ANOVA (n≥6).
Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; SD, standard deviation; SSP, staurosporine; Tet, tetrandrine.

Figure 3 Tet protected RGC-5 cells from H₂O₂ injury and L-glutamate excitotoxicity.

Notes: (A) Cell death induced by H₂O₂ could be attenuated by the addition of Tet at a concentration of 100 nM, 6 hours prior to H₂O₂ exposure, as compared with RGC-5 cells treated with Tet 48 hours after exposure to 1 mM H₂O₂, but never reached the control level. (B) Tet pretreatments (6 hours prior to L-glutamate exposure) of 10 nM and greater were able to promote RGC-5 cell survival 12 hours after exposure to 25 mM L-glutamate, and the relative cell numbers increased as the Tet concentration increased. Tet (10 nM −0.5 μM) could increase the relative cell numbers significantly. The relative cell numbers of the 0.5 μM Tet plus L-glutamate group reached the control level, while numbers in the other Tet treated groups were lower than in the control. Results are expressed as the means ± SD. *P<0.05 compared to control; ^# P<0.05 compared to 1 mM H₂O₂ or 25 mM L-glutamate, one-way ANOVA (n≥6).
Abbreviations: ANOVA, analysis of variance; L-glu, L-glutamate; SD, standard deviation; Tet, tetrandrine.

Figure 4 The identification, morphology, and survival curve of primary cultured RGCs.

Notes: (A) Immunofluorescence assay of purified RGCs. (B) Differential interference contrast image of primary ganglion cells isolated from neonatal mouse retinas cultured at a low density (approximately 1,000 cells/cm²) for 4 days. (C) The cells have long processes extending from variably sized cell bodies, both of which are characteristic of primary ganglion cells. The viability of purified RGCs at various time points was expressed as a percentage of viability after the first day of culture. Scale bar =20 μm (A). Images in (A) were taken at 60× magnification. The image in (B) was taken at 20× magnification.
Abbreviations: DAPI, 4, 6-diamidino-2-phenylindole; MAP2, microtubule associated protein 2; RGCs, retinal ganglion cells.

Figure 5 Time-dependent effect of Tet on the viability of primary cultured RGCs.

Notes: The relative cell numbers of primary cultured murine RGCs after 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours exposure to Tet. Relative cell survival compared with DMSO in the 1 nM and 10 nM Tet conditions was 1.36±0.104 (P=0.009) and 1.56±0.105 (P=0.004) after 72 hours, respectively. After 96 hours, the relative cell survival in the 1 nM and 10 nM Tet conditions as compared to DMSO was 1.37±0.155 (P=0.011) and 1.41±0.085 (P=0.006), respectively. The differences between Tet, the control, and DMSO conditions were not significant at the 24 hour, 48 hour, or, 120 hour time points (P>0.05). Results are expressed as means ± SD. * or # P<0.05 as compared to DMSO control, one-way ANOVA (n=4).
Abbreviations: ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; RGCs, retinal ganglion cells; SD, standard deviation; Tet, tetrandrine.

Figure 6 Tet protects RGCs from ischemic injury in vivo.

Notes: Effect of Tet on RGC survival in the central (A, D, G, J, M, and P), middle (B, E, H, K, N, and Q), and peripheral (C, F, I, L, O, and R) regions 1 day after retinal ischemia are shown. Injection of 2 μL of 10 μM Tet (A–C) 1 day before retinal ischemia reperfusion increased RGC survival in comparison to the PBS injection plus ischemia eyes (G–I), but did not reach the levels of Tet (J–L) and PBS (P–R) in nonischemic eyes. The GCL cell numbers in the Tet–I/R group were similar to those in the CNTF–I/R group. The ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) are indicated. Scale bar =50 μm (A–L). All images were taken at 60× magnification.
Abbreviations: CNTF, ciliary neurotrophic factor; GCL, ganglion cell layers; I/R, ischemia/reperfusion; PBS, phosphate-buffered saline; RGCs, retinal ganglion cells; Tet, tetrandrine.

Figure 7 Statistical analysis of RGC survival following Tet application in vivo.

Notes: (A) Percent survival of RGCs in PBS intravitreal-injected (PBS), 10 μM Tet intravitreal-injected (Tet), 12.5 ng/mL CNTF intravitreal-injected (CNTF), PBS intravitreal-injected plus ischemia (PBS–I/R), and 10 μM Tet intravitreal-injected plus ischemia (Tet–I/R) as well as 12.5 ng/mL CNTF intravitreal-injected plus ischemia (CNTF–I/R) eyes were determined by DAPI stain. Intravitreal injection of Tet 1 day before retinal ischemic reperfusion significantly improved ganglion cell survival one day after ischemia in the central, middle, and peripheral retinal regions (P<0.05). Under the Tet and CNTF conditions (B), there were no significant differences in RGC survival 3 days after ischemia in any of the retinal regions (P>0.05). (A and B) Results are expressed as a percent of the PBS intravitreous injection control. (C) The total RGC numbers in PBS, Tet, CNTF, PBS–I/R, Tet–I/R, and CNTF–I/R eyes 1 day and 3 days after ischemia. (C) The numbers of RGCs in the PBS–I/R, Tet–I/R, and CNTF–I/R retinas 1 day and 3 days after ischemia were much lower than the corresponding numbers in the PBS, Tet, and CNTF groups, respectively (P<0.05). A 2 μL volume of Tet, CNTF, or PBS was intravitreally injected 24 hours prior to ischemia. Results are expressed as means ± SD. *P<0.05 as compared to control, one-way ANOVA. ^# P<0.05, one-way ANOVA (n≥3).
Abbreviations: ANOVA, analysis of variance; CNTF, ciliary neurotrophic factor; I/R, ischemia/reperfusion; PBS, phosphate-buffered saline; RGC, retinal ganglion cell; SD, standard deviation; Tet, tetrandrine.

Figure 8 Effects of Tet and CNTF on primary cultured RGCs ΔΨm.

Notes: The percentage of red and/or green fluorescence-labeled RGCs and polarized mitochondria in RGCs exposed to PBS (A), FCCP (B), Tet (C), and CNTF (D) detected by flow cytometry. RGC morphology 24 hours after exposure to PBS (E), FCCP (F), Tet (G), and CNTF (H) for up to 60 minutes. Images (E–H) were taken at 20× magnification.
Abbreviations: ΔΨm, inner mitochondrial membrane potential; CNTF, ciliary neurotrophic factor; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; FITC, fluorescein isothiocyanate; LL, lower left; LR, lower right; PBS, phosphate-buffered saline; PE, P-phycoerythrin; RGCs, retinal ganglion cells; Tet, tetrandrine; UL, upper left; UR, upper right.

Figure 9 Tet and CNTF inhibited the expression of cleaved caspase-3 in the retinas 1 day after I/R insult.

Notes: Effect of Tet (A–C, J–L), CNTF (D–F, M–O) and PBS control (G–I, P–R) on the expression of cleaved caspase-3 1 day after retinal ischemia. Cleaved caspase-3 is green and DAPI-labeled nuclei are blue in the merged images. Red arrows indicate green fluorescence. The ganglion cell layer, inner nuclear layer, and outer nuclear layer are indicated. Scale bar =20 μm (A–R). All images were captured at 40× magnification.
Abbreviations: CNTF, ciliary neurotrophic factor; DAPI, 4, 6-diamidino-2-phenylindole; I/R, ischemia/reperfusion; PBS, phosphate-buffered saline; Tet, tetrandrine; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

Figure 10 Tet and CNTF inhibited Bcl-2 expression 1 day after I/R insult.

Notes: Effect of Tet (A–C, J–L), CNTF (D–F, M–O) and PBS control (G–I, P–R) on the expression of Bcl-2 one day after retinal ischemia. Bcl-2 is green and DAPI-labeled nuclei appear blue in the merged images. Red arrows indicate green fluorescence. The ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL) are indicated. Scale bar =20 μm (A–R). All images were captured at 40× magnification.
Abbreviations: CNTF, ciliary neurotrophic factor; DAPI, 4′, 6-diamidino-2-phenylindole; I/R, ischemia/reperfusion; PBS, phosphate-buffered saline; Tet, tetrandrine.