Neuroprotective Effects of Human Adipose-Derived Mesenchymal Stem Cells in Oxygen-Induced Retinopathy

Abstract

This study was designed to provide evidence of the neuroprotective of human adipose-derived mesenchymal stem cells (hADSCs) in oxygen-induced retinopathy (OIR). In vivo, hADSCs were intravitreally injected into OIR mice. Various assessments, including HE (histological evaluation), TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, electroretinogram (ERG) analysis, and retinal flat-mount examination, were performed separately at postnatal days 15 (P15) and 17 (P17) to evaluate neurological damage and functional changes. Western blot analysis of ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) was conducted at P17 to elucidate the neuroprotective mechanism. The P17 OIR group exhibited a significant increase in vascular endothelial cell nuclei and neovascularization that breached the ILM (inner limiting membrane) to the P17 control group. In addition, the retinal nonperfusion areas in the P17 OIR group and the number of apoptotic retinal cells in the P15 OIR group were significantly higher than in the corresponding hADSCs treatment group and control group. There was no significant thickness change in the inner nuclear layer (INL) but the outer nuclear layer (ONL) in the P17 OIR treatment group compared with the P17 OIR group. The cell density in the INL and ONL at P17 in the hADSCs treatment group was not significantly different from the OIR group. The amplitude of a-wave and b-wave in scotopic ERG analysis for the P17 OIR group was significantly lower than in the P17 hADSCs treatment group and the P17 control group. Furthermore, the latency of the a-wave and b-wave in the P17 OIR group was significantly longer than in the P17 hADSCs treatment group and the P17 control group. In addition, the expression levels of CNTF and BDNF in the P17 OIR group were statistically higher than those in the P17 control group, whereas the expression of GDNF was statistically lower in the P17 OIR group, compared with the P17 control group. The expression of CNTF and GDNF in the P17 hADSCs treatment group was statistically higher than in the P17 OIR group. However, the expression of BDNF in the P17 hADSCs treatment group was statistically lower than in the P17 OIR group. This study provides evidence for the neuroprotective effects of hADSCs in OIR.

Keywords: TUNEL; brain-derived neurotrophic factor; ciliary neurotrophic factor; electroretinogram; glial cell line–derived neurotrophic factor; histological evaluation; human adipose–derived mesenchymal stem cells; neuroprotection; oxygen-induced retinopathy.

Figure 1.

Culture of hADSCs and experimental timeline. (A) The third passage of hADSCs displays spindle and polygonal shapes. (B) A schematic diagram illustrating the experimental time points. hADSCs: human adipose–derived mesenchymal stem cells; OIR: oxygen-induced retinopathy; MSC: mesenchymal stem cell.

Figure 2.

The HE and retinal flat mount results. (A) The HE of the P17 control group. No VEC nuclei and neovascularization breached the retina’s ILM. (B) The HE of the P17 OIR group. Numerous VEC nuclei and neovascularization breached the ILM (black arrows). (C) The HE of the P17 hADSCs treatment group. Few VEC nuclei and neovascularization broke through the ILM, and the injected cells were observed in the vitreous cavity and above the retina (green arrows). (D) Quantification of VEC nuclei that breached the retina`s ILM. The P17 OIR group exhibited a significant increase compared with the P17 control group (P < 0.0001) and the P17 hADSCs treatment group (P < 0.0001). (n = 5) (E) Retinal flat mount of the P17 control group. No apparent neovascular fluorescence and nonperfusion areas were observed. (F) Retinal flat mount of the P17 OIR group. Extensive neovascular fluorescence and nonperfusion areas (yellow arrows) were evident. (G) Retinal flat mount of the P17 hADSCs treatment group. Slight neovascular fluorescence and no apparent nonperfusion areas were detected. (H) The extent of retinal nonperfusion areas. The P17 OIR group exhibited a significant increase compared with the P17 control group (P < 0.0001) and a significant decrease compared with the P17 hADSCs treatment group (P < 0.0001). (I, J) INL and ONL thickness. Significant reduction in both INL and ONL thickness in the P17 OIR group compared with the P17 normal group (P = 0.000 and P = 0.030; Fig. 2I, J). There was no significant change in INL thickness, but ONL thickness was reduced in the P17 OIR treatment group compared with the P17 OIR group (P = 0.358 and P = 0.043). (K, L) Cells in INL and ONL. The cell density in the INL at P17 OIR group was significantly lower than in the P17 normal group (P = 0.007), with no significant difference compared with the P17 hADSCs treatment group (P = 0.075). There were no significant differences within the ONL between the P17 control group and the hADSCs treatment group compared with the P17 OIR group (P = 0.062 and P = 0.537; n = 5). HE: Histological evaluation; VEC: vascular endothelial cell; ILM: inner limiting membrane; NOR: normal group; OIR: oxygen-induced retinopathy; hADSCs: human adipose–derived mesenchymal stem cells; INL: inner nuclear layer; ONL: outer nuclear layer; MSC: mesenchymal stem cell; GCL: ganglion cell layer; IPL: inner plexiform layer; OPL: outer plexiform layer; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3.

TUNEL results. (A, B, C) TUNEL results from the P15 control group. No apparent TUNEL-positive cells were observed. (D, E, F) TUNEL results from the P15 OIR group. A substantial number of TUNEL-positive cells were evident (white arrows), primarily localized in the INL. (G, H, I) TUNEL results from the P15 hADSCs treatment group. Some TUNEL-positive cells were observed. OIR: oxygen-induced retinopathy; INL: inner nuclear layer; hADSCs: human adipose–derived mesenchymal stem cells; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 4.

Electroretinogram results. (A, B) Amplitudes of the a-wave and b-wave. The amplitudes of the a-wave and b-wave in the P17 OIR group were significantly lower than in the P17 control group (P: < 0.0001, < 0.0001). The amplitudes of the a-wave and b-wave in the P17 hADSCs treatment group were significantly higher than in the P17 OIR group (P: < 0.0001, = 0.001). (n = 5) (A, C) Latencies of the a-wave and b-wave. The latencies of the a-wave and b-wave in the P17 OIR group were significantly longer than in the P17 control group (P: = 0.003, = 0.001). The latencies of the a-wave and b-wave in the P17 hADSCs treatment group were significantly shorter than in the P17 OIR group (P: =0.047, = 0.001). (n = 5). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NOR: normal group; OIR: oxygen-induced retinopathy; hADSCs: human adipose–derived mesenchymal stem cells; MSC: mesenchymal stem cell.

Figure 5.

Expression of CNTF, GDNF, and BDNF. (A) Western blot analysis of the protein expressions of CNTF, GDNF, and BDNF in the P17 control, OIR, and hADSCs treatment groups. The relative protein expression levels were normalized to β-Actin. (B) Quantification illustrating trends in the expressions of CNTF, GDNF, and BDNF in the P17 control, OIR, and hADSCs treatment group. The expression of CNTF and BDNF in the P17 OIR group was statistically higher than in the P17 control group (P < 0.0001). The expression of GDNF in the P17 OIR group was statistically lower than in the P17 control group (P < 0.0001). The expression of CNTF and GDNF in the P17 hADSCs treatment group was statistically higher than in the P17 OIR group (P < 0.0001). The expression of BDNF in the P17 hADSCs treatment group was statistically lower than in the P17 OIR group (P < 0.0001). CNTF: ciliary neurotrophic factor; GDNF: glial cell line–derived neurotrophic factor; BDNF: brain-derived neurotrophic factor; NOR: normal group; OIR: oxygen-induced retinopathy; hADSCs: human adipose–derived mesenchymal stem cells; MSC: mesenchymal stem cell.