| Objective: Survival cells of retinal ganglion decrease significantly after optic nerve injured. Formation of glial scar and various molecules inhibit regeneration by releasing molecules that hinder the axons regenerating through the damaged area. Nerve regeneration is difficult. In perspective of neural tissue engineering, amniotic epithelial cells were seeded into collagen sponge to build amniotic epithelial cells / collagen sponge complex in vitro. Then it was implanted into the damaged parts of the optic nerve in rats to preliminarily observe and discuss repair effects of complex on the injured optic nerve and related mechanisms.Methods: (1)Amniotic tissue was taken from late pregnancy SD rats. After sequential digestion, amniotic epithelial cells were culture primarily. Subcultured cells were stained by immunofluorescence and quantified by PCR. (2)Amniotic epithelial cells were seeded into the collagen sponge, in vitro cultured for one week. Using immunofluorescence staining, quantitative PCR to reassure cells in the collagen sponge. (3)Using H&E staining, scanning electron microscopy, fluorescent dye to label cells, identifying cell growth, and CCK-8 to detect cell proliferation in vitro. (4)The amniotic epithelial cells / collagen sponge composition adult male SD rats were transplanted into the optic nerve injury model. The animals were divided into 4 groups: normal control group, injured group. Rats in the injuried group got complete optic nerve transected by using1ml syringe with a vertical incision of epineurium at 2 mm retrobulbar, cutting off about 0.5 mm of the nerve. But epineurium and vessels intact should be kept complete. Injecting 10μl serum-free culture medium into the injured part. Stent control group was based on the injured group, collagen sponge which was cultured for 1 week was implanted into injured part of the nerve. Experimental group was based on the injured group. 10μl The amniotic epithelial cells / collagen sponge composition (5×10~6 /ml) were injected into the injured part of the nerve. (5) For some of the animal models, utilizing CM-Dil as a membrane marker before the cell implantation. Rats were fixed by cardiac perfusion on 4weeks and 8 weeks after surgery. The optic nerve were harvested for frozen sectioning to observe distribution and numbers of the labeled cells. (6)CTB was injected into vitreous body to label RGCs in some of the animals 48 h before being sacrificed. 2 days later, rats were fixed by cardiac perfusion, and eyeballs were taken for retina flattening to count CTB labeled cells. Some of the animals were sacrificed 4 weeks after surgery ,whose eyeballs were taken for retina flattening and Nissl staining to count RGCs. Some of the animals were sacrificed 4,8 weeks after surgery, the optic nerves were collected for HE staining to observe the changes of tissue structure and cell concentration, and for SABC immunohistochemistry staining to show the expression of GAP-43.Results:(1)Rat AECs were successfully cultured. Immunocytochemical staining showed the rat AECs could express epithelial-specific markers CK-19, neural stem cell marker Nestin, as well as pluripotent cells marker Oct-4, Nanog, and so on. (2)Quantitative RT-PCR showed the same genes were expressed by AECs after grew on the collagen sponge scaffold, and upregulated the mRNA of Nestin. (3)By using H&E staining, scanning electron microscopy, immunofluorescence staining and cck-8, amniotic epithelial cells grew well on the collagen sponge scaffold, collagen sponge can promote the proliferation of amniotic epithelial cells.(4)After CM-Dil labeled cell complex were transplanted into the model in which the optic nerve was cut, frozen section of optic nerve showed great amount of labeled cells survived in the injury area 4 and 8 week after surgery. And these cells could spread to the proximal segment and distal segment of damaged optic nerve, and migration along the nerve membrane.(5)At 4 weeks after injury, CTB and NISSL staining of retinal ganglion cells counts showed that ganglion cell density in each group was significantly lower than normal. In the cell complex group, cell density of ganglion was higher than that in injured group, stent control group; there was no significant difference between the injured group and stent control group. (6)HE staining of optic nerve showed the Collagen sponge transplanted into body can integrated with the injured nerve tissue. it degraded after 8 weeks. There were great amount of cells, forming irregular streak and spreading towards both directions. Shape of the nuclear in the distal part of the optic nerve became smaller, but seemed much similar with normal ones in the cell transplanted groups. By counting the cells within the distal part of the optic nerve in 4 and 8 weeks, we learned that the number of stent control group higher than injury group,the number of complex cells was significantly higher than injury group and stent control group.(7)Immunohistochemistry staining of GAP-43 in the injured group and control stent group, only small amount of GAP-43 expressed in the injury area, and none in the distal part. The expression of GAP-43 in amniotic epithelial cell / collagen sponge graft injury area significantly increased, showing a small amount of GAP-43 positive, similar to the regeneration of axon-like structure stretching from the injured area to the distal segment. The structure transplanted 8 weeks reach farther than to it was transplanted 4 weeks .Conclusions: (1)Amniotic epithelial cells and collagen sponge have good histocompatibility. Collagen cultured in vitro can promote the proliferation of amniotic epithelial cells and enhanced amniotic epithelial cells differentiating towards neural stem cells, the complex have better vitality.(2)After transplanting the complex, part of cells in collagen sponge can survive at least 8 weeks in damaged optic nerve and could migrate to both sides of the damaged zone(.3)After transplanting the complex, it could protect retinal ganglion cells and optic nerve glial cells and improve the growth ability of the regenerating axons, and could facilitate some of the axons to pass injury area and grow into the distal part of the optic nerve. These results indicate that the complex could improve the optic nerve regeneration microenvironment and promote axon regeneration. |
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