| Objective To establish a calibrated optic nerve crush injury model in rats for researching treatment of optic nerve injury.Method Fifty-four healthy Sptague-Dawley(SD) rats were randomly divided into three groups. Six rats were in normal control group, another group, model of partial optic nerve crush injury group, partial optic nerve crush injury model was induced in the left eye of another 24 rats by a special designed optic nerve clip with 40-gram holding force at optic nerve 2mm behind the eyeball clipped for thirty seconds to partially block the optic nerve axoplasmic transport.The right eyes served as a control. Another 24 rats served as pseudo-injury control group. According to the sacrificing date of the rats, four subgroups in groups in injury group were further divided for three, seven, fourteen and twenty-eight days later. Therefore, six rats were in each subgroup.5% fluorogold was injected into both superior colliculi bilaterally to each subgroup and normal group. The eyes were enucleated seven days later, flat mounts of the retina from both eyes were prepared on a slide and observed under a fluorescence microscope. Twelve photos with 200×magnification were taken from quadrants of the retina 1mm away from the optic disc. The labeled retinal ganglion cells(RGCs) photos were counted by ourselves. The labeled RGCs rate, a comparison of the labeled RGCs from both eyes in a rat were used for statistical analyzing.(The labeled RGCs rate=RGCs from injured left eye/RGCs from uninjured right eye×100%). The retina was sectioned and colored by hematine-eosine for counting the cells in the ganglionic layer of the optic nerve, quantitating optic nerve lesion and observing change of retina-morph. Leptonomorphological change was observed by coloring retinal blade using hematoxylin-eosine after optic nerve injury.Result1. In injury group, factorial analysis of variance showed significant diferences between the control eye (3d,192.16±32.12,7d,189.26±26.16, 14d,191.76±25.29,28d,186.56±23.76) with the injury eye (3d,152.26±25.12,7d,111.19±20.32,14d,101.23±17.19,28d,94.86±18.26) (P<0.05). There were no significant differences between left eye and right eye in normal group and pseudo-injury group(P>0.05).2. The retinal ganglion cells (3d,152.26±25.12,7d,111.19±20.32, 14d,101.23±17.19,28d,94.86±18.26) of the injury eye showed a tendency to decline gradually along with increases of the time in model of partial optic nerve crush injury group (P<0.05). There were no significant differences between 14d group and 28 group(P>0.05).There was a same tendency along with increase of the time in the labeled RGCs rate (3d,79.35%±8.29%,7d,59.76%±7.79%,14d,53.26%±7.26% 28d,51.29%±3.26%) after optic nerve injury (P<0.05). There were no significant differences between 14d group and 28 group(P>0.05). There were no significant differences between left eye and right eye in normal group and pseudo-injury group(P>0.05).3. Pathological change:After optic nerve injury, there was karyopycnosis on ganglion cell layer of retina, thinningz on each layer of retina, derangement of cell and decrease in retinal ganglion cells. There was different degree of the above change in different time after optic nerve injury. There were the swelling, the hemorrhage, derangement of spongiocyte and the denaturation like vacuole in the spot of optic nerve injury. Moreover, they were aggrava-ting with increases of the time after optic nerve injury. There were no pathological changes in normal group and pseudo-injury group.Conclusion Using a special designed clip with 40-gram holding force constantly pressed for thirty seconds to create an optic nerve crush injury model in rats. And the model may be applied to researching repair of the optic nerve injury. Objective To investigate protection of the retinal ganglion cells(RGCs) in the Spragur-Dawly rats with optic nerve injury after human umbilical cord blood stem cells (hUCBSC) were transplanted.Method Ninety-six adult Sprague-Dawley rats were randomly divided into six groups, therefore 16 rats in each group. Calibrated optic nerve crush injury model was induced in the left eyes in the six groups, the right eyes served as a control. Medicine was injected at seventh day after optic nerve injury. PBS was injected into vitreous of the eyes of Group A rats. The neurotrophic factors were injected into vitreous of the eyes of Group B rats. The hUCBSCs were injected into vitreous of the eyes of Group C rats. The hUCBSCs and the neurotrophic factors were injected into vitreous of the eyes of Group D rats. The hUCBSCs were injected in Group E rats by peribulbar injection. The hUCBSCs were injected in vena caudalis of Group F rats. And then according to the time interval between the optic nerve crush and the sacrifice, every group was divided into four subgroups (day 7,day 14,day 21 and day 28), therefore 6 rats in each subgroup. Seven days before sacrifice,5% fluorogold was injected into superior colliculi bilaterally. The eyes were enucleated after the rat was sacrificed, and flat mounts of the retina from both eyes were prepared on a slide and observed under a fluorescence microscope. The labeled RGCs were counted by a computerized image analyzer. The labeled RGCs rate was used for statistical analysis.Result The retinal ganglion cells of the injury eye were less than the control eye in Group A, B, C and D (P<0.05). The labeled RGCs rate showed a tendency to decline gradually along with increases of the time in four groups (P<0.05), but the trend of decrease of Group B, C and D was evidently slower than that of Group A. The labeled RGCs rate of Group B, C, and D was significantly higher than that of Group A on every time period (P<0.05). The retinal ganglion cells of the injury eye were less than the control eye in Group C, E and F (P<0.05). The labeled RGCs rate also showed a tendency to decline gradually along with increases of the time in three groups (P<0.05), but the trend of decrease of Group C was evidently slower than that of Group E and F.The labeled RGCs rate of Group C was significantly higher than that of Group E and F on every time period (P<0.05). The labeled RGCs rate of Group E was the same with that of Group F.Conclusion The hUCBSC can increase the survival rate of the RGCs and can rescue and/or restore the injujed RGCs after transplanted into the vitreous body of optic nerve crush rat model. The protection of the hUCBSC was the same with that of the neurotrophic factors. Moreover, the protection of the blend of the hUCBSC and the neurotrophic factors was the strongest. Objective To study the mechanisms underlying the neuroprotective effect of human umbilical cord blood stem cells (hUCBSC) on retinal ganglion cells after optic nerve injury.Method Thirty-six Sprague-Dawley rats were randomly divided in Group A (transplantation of hUCBSC group) and Group B (control group), therefore 18 rats in each group. Fifteen rats of each group were into left optic nerve injury model and the other three rats of each group were as the normal control. Cultured hUCBSC were injected into the left right vitreous of optic nerve injury model of Group A. The same dose of PBS were injected into the left vitreous of optic nerve injury model of Group B. Semi-quantitative RT-PCR is employed to detect mRNA expression of BDNF and GDNF in normal retina and the retina of after 3 d,7d,14d,21d and 28d after optic nerve injury in two groups.Result After transplanted into the retina, the dose of BDNF and GDNF secreted by Group A were all higher than Group B in every interval except the expression of BDNF had no difference on two groups on seventh day. The expression of BDNF was constantly increasing in Group A, especially, a tendency of increase was more significant after fouteen days. The expression of GDNF was the most on twenty-first day, and was slightly decreased. Which suggested hUCBSC can continue to secrete neurotrophic factors in vivo of retina.Conlusion The hUCBSC can secrete continuous quantities of BDNF in vivo of retina of optic nerve injury rats, which can recruit the neurotrophic factors decreased by optic nerve injury that are necessary for the survival of RGCs. And this was likely one of the important mechanisms of optic nerve injury repair.
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