| Glaucoma, optic nerve injuries, retinitis pigmentosa which are caused by neural cells degeneration in retina are most-common irreversible ocular diseases. Since the neural cells exhibit limited regeneration ability after injure in adult mammalian retina, these diseases become a big problem to ophthalmologist. There're two parts in this study. For the pathologic condition which the neural axon injures while the neuron still surivive, Part I aims to develop a new type of tissue engineered Schwann cell graft transplanted on the end of transected optic nerve in adult rats to promote axonal regrowth. For the pathologic condition which neural cells died and can't take their function, Part II aims to look for an endogenous protein------ephrinA3 to regulate the proliferation and differentiation of retinal stem cells as a new target to treat retinal neural cells diseases.Purpose:To develop a new type of biodegradable Schwann cell (SC) graft transplanted on the end of transected optic nerve to promote axonal growth in adult rats.Methods:(1) Prepare the CNTF gene-modified Schwann cells (CNTF-SC): GFP-CNTF fusion plasmid was constructed and transferred into SC in vitro by electroporation (EP). The expression of GFP in SC was observed under fluorescence microscope from 3h to 7days after electroporation. The transfection effect was valued by RT-PCR and Immunostaining 24h after transfection. (2) Prepare the tissue engineered SC grafts:80 Poly-glycolic-acid (PGA) filaments were inserted into Poly-glycolic-lacicco-acid (PGLA) conduit, and CNTF-SC or pure SC were injected into this conduit and cultured in 37℃overnight before transplantation. (3)Animal surgery:there're 3 groups, including CNTF-SC filled conduit transplanted group, SC filled conduit transplanted group and empty conduit transplanted group. (4) Evaluate the regerenation of optic nerve:four weeks after transplantation, the GAP-43 staining was used to evaluate the regeneration of optic nerve. The number of regrowed axons in every slice was counted every 625μm from the transected end of optic nerve. Five slices were counted for one animal and six to eight animals were calculated in one group. The statistical difference was anlysized by One Way Anova. Meanwhile, the survival of SC and the ultrastructure of regenerated axons were observed by transmission electron microscope. (5) Evaluate the imflammation and degradation of conduit:the imflammation in the conduit was detected by microglia and macrophage maker-OX42 staining. The degradation of conduit was observed 4weeks and 2 months after transplantation.Results:(1) Succesful preperation of CNTF-SC:the successful construction of GFP-CNTP plasmid was verified by sequencing. Under fluorescence microscope, the expression of GFP was detected since 3 hours after EP, reached the peak at the 24hours after EP and still detectable till 7days. Twenty four hours after SC were transfected, RT-PCR and Immunostaining results showed the effective transfection. (2) The evaluation of regenerated axons:Four weeks after CNTF-SC filled grafts, SC filled grafts and empty conduits were transplanted, there're some GAP-43 labeled regrowed axons in the conduits. At the distance of 1875μm from the optic nerve head, the average number of regenerated axons in SC grafts (14.3±2.94/mm/section) and that in CNTF-SC grafts (16.2±1.56/mm/section) were significant higher than that in empty conduits (5±2.06/mm/section, P＜0.05, One Way Anova, n=6 or 8). It suggested the SC graft had a significant effect to promote optic nerve regeneration. At the distance of 5000μm from the optic nerve head, the average number of regenerated axons in CNTF-SC grafts (4.23±1.18/mm/section) was significantly more than that in SC grafts (1.33±0.58/mm/section, P＜0.05, student's t test, n=6 or 8). At the distance of 5625μm from the optic nerve head, the average number of regenerated axons in CNTF-SC grafts (2.86±1.08/mm/section) was significant more than that in SC grafts (0.3±0.1/mm/section, P＜0.05, student's t test, n=6 or 8). It suggested the CNTF-SC grafts can reach a better effect for promoting the axonal regrowth. (3) the observation by EM:Under transmission electron microscope, the survival of SCs and new unmylined axons were oberserved. (4) The observation of imflammation and degradation of the conduits:the conduits were degraded in 2 months, and no serious imflammation in the conduit.Conclusions:The tissue-engineered SC grafts can provide a feasible environment for optic nerve regeneration and may become an alternative to bridge the damaged nerves and repair nerve defects in the future. Propose:To clarify the role of ephrinA3 in adult retinal stem cell niche to understand the endogenous mechanism of regulating the proliferation and differentiation of adult retinal stem cells.Methods:(1) Detect the expression of ephrinA3 in mouse retina:The expression of ephrinA3 in retina of different ages was checked by western blot. And the distribution of ephrinA3 in adult retina was analyzed by immunostaining. (2) Detect the proliferation of retinal stem cell in adult ephrin knockout (KO) and wildtype mice in vivo:Pulse BrdU labeling was used to label the proliferating adult retinal stem cells in ephrinA3 knockout (KO) and wildtype mice in vivo. And BrdU positive cells were counted and compared. (3) Test the inhibitory role of ephrinA3 to the proliferation of retinal stem cells in vitro:Neurospheres were cultured from ciliary epithelium of ephrinA3 KO mice and wildtype mice. The numbers of primary and secondary neurospheres from ephrinA3 KO mice and wildtype mice were counted and compared. And the BrdU incorporation assay was used to compare the proliferation ability between ephrinA3 KO retinal stem cells and wildtype retinal stem cells. The ephrinA3-clusters were added into the wildtype retinal stem cell culture to detect the changes of BrdU incorporation ratio of retinal stem cells. (4)Detect the stem cell markers expression in neurospheres derived from ephrinA3 KO and wildtype mice: the primers of stem cell markers were designed and generated, the mRNA level of stem cell markers in ephrinA3 KO neurospheres and wildtype neurospheres were analyzed by realtime PCR and RT-PCR. (5)Study the differentiation of retinal stem cells:adult retinal stem cells from neurospheres were induced to differentiate in vitro and was labeled by different cell markers. (6)Study the receptor of ephrinA3 on adult retinal stem cells:RT-PCR was used to detect the expression of EphA4 and EphA7 in neurospheres, immunoprecipitation was used to detect the protein combined to ephrinA3 in ciliary epithelium. And the phosphorylation of EphA4 in ephrinA3 KO and wildtype cilary epithelium was detected by western blot. (7)Study the downstream pathway of ephrinA3 on retinal stem cells:after Wnt3a was added into the retinal stem cell culture, BrdU incorporation assay was used to study the effect of Wnt3a to promote the proliferation of retinal stem cells. And the expression ofβ-catenin and phosphorylatedβ-catenin in ephrinA3 KO and wildtype neurospheres was measured by western blot.Results:(1) EphrinA3 expresses in mouse retina:Western blot result shows the expression of ephrinA3 was detected in mice retina since the postnatal 10 days and increased in adult. In adult mouse eye, the ephrinA3 expressed strongly in the inner plexiform layer and outer plexiform layer and weekly in the ciliary epithelium. (2) EphrinA3 inhibit the proliferation of retinal stem cells in vivo:After 7 days BrdU i.p. injection, the number of proliferating cells labeled by BrdU in ciliary marginal zone (CMZ) in ephrinA3 KO mice (0.0835±0.0478/slice) is significant more than that in wildtype mice (0.00337±0.00337/slice, P＜0.05, Mann-Whitney Rank Sum Test, n= 9). The BrdU positive cells are co-stained by stem cell marker-Chx10, which suggests the proliferatin cells are retinal stem cells. (3) EphrinA3 inhibit the proliferation of retinal stem cells in vitro:in vitro, the number of primary neurosphere formed from ephrinA3 KO adult retinal stem cells is 1.72±0.07 fold of that from wildtype adult retinal stem cells (P<0.01, student's t test, n=3); the number of secondary neurospheres formed from derived from ephrinA3 KO mice is 2.13±0.235 fold of that from wildtype adult retinal stem cells (P<0.01, student's t test, n=3). BrdU incorporation ratio of ephrinA3 KO retinal stem cells (57.5%±2.4%) is significant higher than that of wildtype retinal stem cells (34%±5.2%, P＜0.05, student's t test, n=3), which suggested the ephrinA3 KO retinal stem cells exhibited the increased proliferation ability than that from wildtype mice. Adding the ephrinA3-clusters decreased BrdU incorporation ratio of wildtype adult retinal stem cells from 33.8%±0.8%to 25.6%±0.8%(P<0.05,student's t test, n= 3), which confirmed the inhibitory effect of ephrinA3 to the proliferation of retinal stem cells. (4) Increased expression of stem cell markers in ephrinA3 KO neurospheres:Real time PCR results showed the neurospheres from ephrinA3 KO mice expressed a higher mRNA level of stem cell markers, especially Wnt families, than that from wildtype mice. (5) Increased potential of differentiating to retinal specific neural cells in ephrinA3 KO retinal stem cells:In differentiation assay, cells from neurospheres can differentiate to several kinds of retinal neural cells. The differentiated cells from ephrinA3 KO retinal stem cells expressed a higher level of photoreceptor markers---rhodopsin and recoverin than that from wildtype retinal stem cells, which suggested the ephrinA3 KO retinal stem cells exhibit an increased potential to differentiate to retinal specific cells. (6) EphA4 as the receptor of ephrinA3 in retinal stem cells:the expression of EphA4 is higher than that of EphA7 in neurospheres by RT-PCR. EphA4 was detected in the ciliay epithelial lysate immunopricipitated by ephrinA3 antibody. The phosphorylation level of EphA4 in ephrinA3 KO retinal stem cells is lower than that in wildtype retinal stem cells. (7) Wnt3a/β-catenin as the downstream pathway of ephrinA3 to regulate the proliferation of retinal stem cells: BrdU incorporation assay showed the Wnt3a increased the proliferation of retinal stem cells. Western blot result showed the expression ofβ-catenin and phosphorylatedβ-catenin is higher in ephrinA3 KO neurospheres than that in wildtype neurospheres.Conclusions:ephrinA3 is an inhibitor in adult retinal stem cell niche which inhibits the proliferation and differentiation of retinal stem cells by combining with EphA4 receptor and downregulating the Wnt/β-catenin. The knowledge on the inhibitory effect and molecular mechanism of ephrinA3 will help us to develop new method to modulate the proliferation and differentiation of retinal stem cells for treating retinal neural diseases. In the future, ephrinA3 will be knocked out in genetic retinal degeneration mouse model to see whether the deletion of ephrinA3 can rescue the degenerated retina in vivo.
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