| The injury of neuron used to result in the irreversible neurocyte death in adult mammalian central nervous system(CNS), the disorders of neural function and recovery are the focal point and nodus in the academic circles. Optic nerve(ON) injury usually leads to the death of retinal ganglion cells(RGCs) and irrevocable vision loss. As one part of CNS, it can not regenerate after birth. Because of the characters of RGCs are similar as neurons of CNS, many studies about CNS have been carried out using the transection of ON as model system. Microtubules are the primary components of the neuronal cytoskeleton,which have a strong influence on both the neurotransmitter transportation, morphology and physiology of neurons. Microtubule dynamics have been shown to contribute to neurite outgrowth, branching and guidance. The dynamic reorganization of the microtubule network is an early and important pathogenic event in the process of neuropsychiatry disorder and neurodegenerative diseases. Stathmin is a robust microtubule-destabilizing factor that is involved in the regulation of microtubule dynamics and plays an essential role in the development of CNS, neurite elongation and synaptic plasticity. Studies have reported that the m RNA levels of Stathmin changes may be related to the development of rat retina and ON recovery. However, the mechanism of Stathmin rescue the injured axotomized RGCs has not yet been elucidated.【Objective】:In the present experiments, the ON axotomized rat model was established and we investigate the expressional changes of Stathmin-related genes at various time points(0, 3, 5, 7, 14, 21 days) following the ON axotomy. Subsequently, the Stathmin-overexpression and Stathmin-silencing recombinant adenovirus were constructed and packaged successfully. We administrated the Ad-Stathmin or Ad-RNAi-Stathmin recombinant adenovirus into the vitreous cavity of the ON axotomized adult rats to see whether Stathmin protect injured retinal RGCs. The aim of this study was to investigate if exogenous overexpression or silencing of Stathmin using recombinant adenovirus can further improve the survival of injured RGCs in vivo and to examine the possible mechanisms of Stathmin in optic neuroprotection.【Methods】:(1) Firstly, using RT-PCR, Western blot analysis we detected the expression of Stathmin and Stathmin-related genes in normal retina of adult SD rats at m RNA and protein levels. And using immunohistochemistry(IHC) we detected the expression and distribution of the Stathmin-related proteins in the normal retina. Secondly, in the present experiments the ON axotomized rat model was established by transect the intraorbital ON at 1.5mm from eyeball. To investigate the expressional changes of Stathmin-related genes following optic nerve axotomy, we sampled neural retina at various time points(0, 3, 5, 7, 14, 21 days) after axotomy. Using RT-PCR, Western blot and IHC analysis we detected the expression and distribution of Stathmin-related genes at m RNA and protein levels.(2) Full-length sequence of Rattus Stathmin gene was amplified by PCR method using SD rat retina genomic DNA as template. Then the PCR products were confirmed by sequencing. Meanwhile, according to the Stathmin c DNA sequence of rattus in Gen Bank, the specific RNAi fragments were designed and synthesized, which were annealed and formed double-stranded oligos. Subsequently, the full-length sequence of Stathmin and the specific RNAi oligos targeting Stathmin were cloned into the adenovirus shuttle vector of p DC315, and the recombinant adenovirus vector of p DC315-Stathmin and p DC315-RNAi-Stathmin were constructed. Then recombinant adenovirus vector p DC315-Stathmin and p DC315-RNAi-Stathmin were cotransfected into HEK293 E cells with adenovirus backbone plasmid p BHGlox△ E1,3Cre by LipofectamineTM 2000 for homologous recombination to get packaged recombinant adenovirus of Ad-Stathmin and Ad-RNAi-Stathmin. According to the same methods, we constructed the Ad-wt and Ad-NC as control recombinant adenovirus respectively.Then the plaque technique was used to harvest, enrich, purify the recombinant adenovirus, after that the titers of viruses were tested using Reed-Muench method and the virus was named Ad-Stathmin and Ad-RNAi-Sta-563/424. Finally, specific Stathmin overexpression and silencing effects was investigated in PC12 cells infected with Ad-Stathmin and Ad-RNAi-Sta-563/424.(3)ON axotomized model was used and RGCs were retrograde-labeled by fluorogold(FG) through at the ocular stump of intraorbitally transected ON. To test Stathmin expression after Ad-Stathmin/Ad-wt and Ad-RNAi-Sta-563/Ad-NC recombinant adenovirus administration, the recombinant adenovirus were intravitreally injected at ON transection and the protein levels of Stathmin was detected at 5 days following ON transection. Further defined the specific Stathmin overexpression and silencing effects in vivo. In the follow-up experiment, the FG-labeled RGCs and retinal microglial cells(RMGs) were counted after the ON transection with Ad-Stathmin/Ad-wt and Ad-RNAi-Sta-563/Ad-NC recombinant adenovirus administration to see whether they protect injured RGCs at 3, 9, 16 days after ON transection.【Results】:(1) RT-PCR and Western blot analysis revealed that in normal rat retina, levels of SCG-10 and Stathmin were higher than those of SCLIP and RB3. Following the ON axotomy, the relative Stathmin-related genes m RNA levels obviously increased at 3 days after optic nerve axotomy(P<0.01)and further increased to 7 days. At 14 days after axotomy, the relative Stathmin m RNA levels were specifically higher(P<0.05)than normal comparing to other Stathmin-related genes. To further investigate the expression changes of Stathmin protein levels after ON axotomy, Western blot analysis was carried out on proteins obtained from retinas after axotomy. As we expected, comparison between the pattern of proteins from control retinas and from retinas after ON axotomy, the changes of Stathmin protein levels consistent with the m RNA levels. Most notably, the protein levels of Stathmin were specifically higher( P<0.05) than normal comparing to other Stathmin-related genes at 14 days after the ON transection. To detect the changes and distributions of Stathmin-related family expression in the normal and axotomized retina, we performed IHC on the normal rat retina and retina obtained 0, 3, 5, 7, 14, 21 days after axotomy. Firstly, in the normal rat retina, immunoreactivity signal of Stathmin and SCG-10 was detected in the whole retina layer, specifically associated with the ganglion cell layer(GCL) and inner nuclear layer(INL).The signal of Stathmin and SCG10 was stronger in the GCL and INL than in the inner plexiform layer(IPL). The immunoreactivity signal of SCLIP and RB3 was weaker in the retina, but specifically in the neuron of GCL. And the Stathmin-related family proteins were expressed in cytoplasm of the neuron of GCL. Secondly, the immunoreactivity of Stathmin was increased in the neurons of GCL at 3 days after ON axotomy, and further increased to 7 days, and then gradually decreased at 14 days as compared with the control retina.(2)The full-length sequence of Rattus Stathmin gene was obtained by PCR method, the sequences and orientation of inserted genes were verified by DNA sequencing and enzyme digestion analysis. The specific RNAi fragments targeting Stathmin were designed and synthesized, which were annealed and formed double-stranded oligos. And the sequences were confirmed by DNA sequencing. And the control recombinant adenoviruses were constructed respectively.Then the Stathmin-overexpression and Stathmin-silencing adenovirus vectors were constructed successfully. Then those recombinant adenovirus vectors were efficiently packaged with high-titer recombinant adenoviruses. The titer of Ad-Stathmin, Ad-RNAi-Sta-563 and Ad-RNAi-Sta-424 recombinant adenovirus was 5.9×1010 TCID50/ml, 6.5×1010 TCID50/ml and 4.3×1010 TCID50/ml respectively. PCR and Western blot results demonstrated that the expression of Stathmin was obviously up-regulated in the PC12 cells after infected with the Ad-Stathmin recombinant virus. Conversely, the expression of Stathmin was obviously down-regulated in the PC12 cells after infected with the Ad-RNAi-Sta-563 and Ad-RNAi-Sta-424 recombinant virus. And the interference efficiency of Ad-RNAi-Sta-563 recombinant virus was superior to Ad-RNAi-Sta-424 recombinant virus.(3) Western blot analysis revealed that the protein levels of Stathmin were increased in the rat retina at 5 days after the ON axotomy combined with Ad-Stathmin recombinant adenovirus intravitreal administration; and the protein levels of Stathmin were decreased in the rat retina at 5 days after the ON axotomy combined with Ad-RNAi-Sta-563 recombinant adenovirus intravitreal administration.(4) Compare to Ad-wt and solvent groups, the density of surviving RGCs in Ad-Stathmin recombinant adenovirus administration groups showed less survival FG-labeled RGCs at 9 days after ON transection(p<0.05). Meaningfully, we can see some activated microglia cells at 3 days following ON transection in the Ad-Stathmin recombinant adenovirus administration groups. And at 9 days after ON transection, Ad-Stathmin recombinant adenovirus administration could remarkably boost the number of activated microglia cells and inhibit the the number of surviving RGCs(p<0.05).(5) Density of surviving RGCs has no statistical differences between Ad-RNAi-Sta-563 recombinant adenovirus administration group and Ad-NC, solvent at 3 days after ON transection. Compare to Ad-NC and solvent groups, Ad-RNAi-Sta-563 recombinant adenovirus administration group showed more survival FG-labeled RGCs at 9 days after ON transection( p<0.01). Amazedly, Ad-RNAi-Sta-563 recombinant adenovirus administration could remarkably inhibit the number of activated microglia cells at 9 days after ON transection(p<0.001). The protective effect of Ad-RNAi-Sta-563 recombinant adenovirus administration on injured retinal RGCs could continue to 16 days. Compare to Ad-NC and solvent group, the density of surviving RGCs in Ad-RNAi-Sta-563 recombinant adenovirus administration group was significantly increased at 16 days after ON transection(p<0.001), and the density of activated RMGs was significantly decreased(p<0.01). The number of amoeba microglia was increased in Ad-RNAi-Sta-563 recombinant adenovirus administration group.【Conclusion】(1) We demonstrated the expression of Stathmin and Stathmin-related molecules in normal rat retina at m RNA and protein level. The levels of SCG-10 were highest and then levels of Stathmin and SCLIP were higher than RB3. Immunoreactivity of Stathmin and SCG-10 was detected in the whole retina layer, specifically associated with the GCL and INL, and the signal of Stathmin and SCG10 was stronger in the GCL and INL than in the IPL. The immunoreactivity signal of SCLIP and RB3 was weaker in the retina, but specifically in the neuron of GCL. The Stathmin-related family proteins were expressed in cytoplasm of the neuron of GCL.(2) The relative Stathmin-related genes m RNA levels markedly increased at 3 days after ON axotomy and further increased to 7 days. At 14 days after axotomy, the relative Stathmin m RNA levels were specifically higher than normal comparing to other Stathmin-related genes. Notably, the protein levels of Stathmin were specifically higher than normal comparing to other Stathmin-related genes at 14 days after the ON transection.(3) The successfully constructed recombinant adenovirus bearing the wild type Rattus Stathmin gene or specific RNAi fragments targeting Stathmin, has significant Stathmin overexpression or silencing effect in vitro and in vivo.(4) Compare to Ad-wt and solvent groups, intravitreal administration of Ad-Stathmin recombinant adenovirus immediately after ON transection could remarkably boost the number of activated microglia cells at 3 days and 9 days following the ON transection. Meanwhile, Ad-Stathmin recombinant adenovirus administration groups showed less survival FG-labeled RGCs at 9 days after ON transection. These datum indicate that intravitreal administration of Ad-Stathmin recombinant adenovirus immediately after ON transection were not conductive to the retinal ganglion cells survival.(5) Compared to the Ad-NC and solvent control groups, intravitreal administration of Ad-RNAi-Sta-563 recombinant adenovirus can play a very significant role in rescuing RGCs not only at 9 days but also have a permanent effect on 16 days after ON transection. And the numbers of activated RMGs were remarkably decreased at 9 days and 16 days in Ad-RNAi-Sta-563 recombinant adenovirus administration groups than Ad-NC and solvent control groups following the ON transection.These results demonstrated that inhibit the expression of Stathmin following the ON axotomy may promote the axotomized retinal ganglion cells survival. |
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