Intravitreal transplantation of human umbilical cord blood stem cells protects rats from traumatic optic neuropathy

Abstract

Objectives: To treat traumatic optic neuropathy (TON) with transplantation of human umbilical cord blood stem cells (hUCBSC) and explore how transplanted stem cells participate in the neuron repairing process.

Methods: A total of 195 Sprague-Dawley rats were randomly assigned to three groups: sham-surgery, optic nerve injury, and stem cell transplant group. Optic nerve injury was established in rats by directly clamping the optic nerve for 30 seconds. hUCBSC was microinjected into the vitreous cavity of injured rats. Optic nerve function was evaluated by flash visual evoked potentials (F-VEP). Apoptosis in retina tissues was detected by TUNEL staining. GRP78 and CHOP gene expression was measured by RT-PCR.

Results: After injury, transplantation of hUCBSC significantly blunted a reduction in optic nerve function indicated by smaller decreases in amplitude and smaller increases in peak latency of F-VEP waveform compared to the injury alone group. Also, significant more in retinal ganglion cell (RGC) count and less in RGC apoptosis were detected after transplantation compared to injured rats. The protective effect correlated with upregulated GRP78 and downregulated CHOP mRNA expression.

Conclusion: Intravitreal transplantation of hUCBSCs significantly blunted a reduction in optic nerve function through increasing RGC survival and decreasing retinal cell apoptosis. The protective role of transplantation was associated with upregulation of GRP78 expression and downregulation of CHOP expression in retinal cells.

Figure 1. Measurement of optic nerve function by F-VEP.

A) F-VEP in normal eye. B) Representative F-VEP in eye injured for 1 hr, 21 days (C), and 28 days (D). E) Representative F-VEP in the hUCBSC-transplanted eyes for 1 hr, 21 days (F), and 28 days (G). H) The amplitude of F-VEP at different time points in injury and hUCBSC transplantation groups. I) Latency of F-VEP at different time points in injury and hUCBSC transplantation groups. N = 5.

Figure 2. Detection of retinal ganglion cells.

A) Normal eye staining. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B–E) Staining for injured (Inj) eyes at 3-day, 1-week, 2-week, and 4-week post injury, respectively. F–I) Staining for hUCBSC transplanted (Inj+SC) eyes at 3-day, 1-week, 2-week and 4-week post surgery, respectively. J) Comparison of RGC number. ^* p<0.05, ^** p<0.001 vs. injury group. N = 5. A scale bar measures 100 microns.

Figure 3. Detection of TUNEL-positive cells.

A) Representative TUNEL staining of normal retina, injured retina 7 days after injury, and hUCBSC-transplanted retinas 7 days after surgery. TUNEL-positive cells were seen in inner nuclear layer and ganglion cell layer. Arrows indicated the TUNEL-positive cells (Brown). ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B) TUNEL-positive cells counting in retina in RGC layer at different time points in injury and hUCBSC transplanted rats. N = 5. ^* p<0.05, ^** p<0.01 vs. 3 hrs. ^# p<0.05, ^## p<0.01 vs. injury group.

Figure 4. Detection of GRP78 mRNA expression.

A) Representative RT-PCR of GRP78 expression. GRP78 mRNA expression was detected 3 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs and 7 days post surgery in the injury (Inj) and hUCBSC transplantation (Inj+SC) groups. B) Comparison of the band-pixel values of GRP78 mRNA in injury and hUCBSC transplantation rats. ^* p<0.001 vs. injury group. N = 5.

Figure 5. Detection of CHOP mRNA expression.

A) Representative RT-PCR of CHOP expression. CHOP mRNA expression was detected 3 hrs, 12 hrs, 24 hrs, 48 hrs and 7 days post surgery in the injury (Inj) and hUCBSC transplantation (Inj+SC) groups. B) Comparison of the band-pixel values of CHOP mRNA in injury and hUCBSC transplantation rats. ^* p<0.001 vs. hUCBSC transplantation. N = 5.