Combined effect of olfactory ensheathing cell (OEC) transplantation and glial cell line-derived neurotrophic factor (GDNF) intravitreal injection on optic nerve injury in rats

Abstract

Purpose: To investigate the combined effect of olfactory ensheathing cell (OEC) transplantation and recombinant human glial cell line-derived neurotrophic factor (rhGDNF) intravitreal injection on optic nerve functional recovery following incomplete injury in adult rats.

Methods: The optic nerves of adult rats were crushed by forceps and then GDNF was injected into vitreous cavity, OECs transplanted into injured optic nerve, or GDNF vitreous injection combined with OECs transplantation, and balanced salt solution was injected into vitreous cavity of control group rats respectively. Flash visual evoked potential (F-VEP) was performed on the injured eye immediately after injury and at 1, 2, 4, and 8 weeks after injury.

Results: The F-VEP waveforms were almost silent immediately after the optic nerve injury. The latency of the F-VEP (LP1) recovered nearly to the normal value 1 week after injury in the treatment groups. The amplitude recovered more slowly. It recovered more obviously and rapidly in the rhGDNF combined with OEC group. At 8 weeks after injury, the amplitude was restored to 64.5% of the pre-injury level in the control group and to 91.8% in the GDNF+OEC treatment group. Wheat germ agglutinin (WGA) labeling showed retinal ganglion cell (RGC) axon regeneration and prolongation in the combined group, and the regenerated axons extended across the traumatized area and reached the distal end of the injured optic nerve.

Conclusions: The combination of OEC transplantation and rhGDNF intravitreal injection will be more effective in promoting the recovery of visual function after incomplete injury of the optic nerve in adult rats.

Figure 1

Olfactory ensheathing cells culture, purification, and immunofluorescence staining. A: OECs cultured for 8 days, B: OECs cultured for 14 days C: cultured for 8 days, D: cultured for 14 days OECs with immuofluorescence staining. NGFR-p75 staining for OEC (red); fibronectin staining for ONF (green). The scale bar is 20 μm.

Figure 2

Representative waveforms of flash visual evoked potential (F-VEP) in rats. A: Each peak of F-VEP could be distinguished clearly in normal rats, including N1, P1, and N2 waves. B: Immediately after the optic nerve injury, the F-VEP waveform appeared silent and peaks could not be identified. C: Four weeks after OEC+GDNF treatment, F-VEP latency obviously decreased and amplitude increased significantly in rats with injured optic nerves.

Figure 3

Latency (LP1) of flash visual evoked potential (F-VEP) before and after optic nerve injury. The LP1 was obviously delayed immediately after the optic nerve injury and was then rapidly restored 1 week after the injury, especially in the OEC+GDNF group. At 8 weeks, the LP1 of the OEC+GDNF group was almost restored to the normal value, but it was still obviously delayed in the Control, OEC, and GDNF groups. Data were shown as mean±SD (n=5). #: p<0.01 versus OEC+GDNF group; *: p<0.05 versus OEC+GDNF group.

Figure 4

Amplitude (AP1-N2) of flash visual evoked potential (F-VEP) before and after optic nerve injury. The AP1-N2 obviously decreased immediately after the optic nerve injury, and was then restored very slowly. At 2 weeks, the AP1-N2 of the OEC+GDNF group were obviously restored compared with the Control, OEC, and GDNF groups. At 4 weeks and 8 weeks after injury, the AP1-N2 of the OEC+GDNF, OEC, and GDNF groups increased significantly compared to the Control group. #: p<0.01 versus Control group; *: p<0.05 versus Control group. Data were shown as mean±SD (n=5).

Figure 5

The longitudinal sections of RGC axons were labeled with wheat germ agglutinin (WGA) for axonal regeneration. A: After optic nerve injury, the ends of axons became accumulated and twisted. B: One week after injured rich WGA-labeled axons were visualized at the proximal end of the traumatized area, a few axons entered the distal end. C: One week after injury, the ends of the axons in the OEC+GDNF group were more extense and outspread. D: Two weeks after injury, some axons entered the traumatized area and reached the distal end of the traumatized area. E: Four weeks and F, 8 weeks after injury, WGA-labeled nerve fibers in the OEC+GDNF group were obviously prolongated and displayed abundant regenerated axons. At these time points the figure showed regenerated axons tangled and fluorescence accumulated there. G: Eight weeks after injury, a few new fibers regenerated in the OEC group and extended across the traumatized area to reach the distal end; however, the regenerated axons appeared significantly less than in the OEC+GDNF group. H: Negative control for WGA-labeled optic nerve axons. All images: left is the proximal end and right is distal end of optic nerve injured point. The scale bar is 100 μm.