| Objective: To explore the effect of DRGNs axonal growth and extension with co-application of Rho-ROCKII and GSK-3βinhibitors in vitro after complete spinal cord injury in rats.Methods:⑴15 adult female Sprague-Dawley (SD) rats were randomly divided into three groups and 5 were subjected to weight-drop impact causing complete paraplegia, 5 were in sham operation control group and normal group repectively. The T8-10 spinal cord extracts (SCE) were harvested in complete paraplegia group, sham operation control group and normal group repectively after 7 days.⑵All thoracic-lumbar DRGs (20~25 DRGs per rat) of neogenesis SD rats (<5d) were harvested under the stereopsis microstat, and then DRNGs were harvested, cultured, purified and indentified.⑶The first experiment was divided into 4 groups, group A was DRGNs + PBS, group B was DRGNs + normal SCE, group C was DRGNs + sham operation SCE and group D was DRGNs + complete paralysis SCE.⑷The second experiment was divided into 5 groups, group A was DRGNs + PBS, group B was DRGNs + complete paralysis SCE, group C was DRGNs + complete paralysis SCE + Y27632, group D was DRGNs + complete paralysis SCE + TDZD-8, group E was DRGNs + complete paralysis SCE + Y27632 + TDZD-8.⑸The axonal length, positive expression of TubulinβIII and mean fluorescence density at axonal distal end of DRGNs were observed with phase-contrast microscope and immunofluorescence after co-culture during 1-2 days, and the result was analyzed with one-way ANOVA in SPSS 13.0 statistics software. Results:⑴The average lengths of neuronal axon were 144±18um in group A, 134±11um in group B, 125±30um in group C and 47±5um in group D respectively at 1days and were 211±21um in group A, 182±15um in group B, 156±13um in group C, 80±8um in group D respectively at 2 days after co-culture in the first experiment. The axonal length of the neogenesis DRGNs in group D was significant shorter compared to group A, B and C respectively , it in group C was shorter compared to group A and group B respectively and there was no difference between group A and B in axonal length at 1, 2 days after co-culture in the first experiment. The mean fluorescence densities at axonal distal end were 200.4±4.0 AFU/um in group A, 199.2±6.6 AFU/um in group B, 194.2±7.0 AFU/um in group C and 75.1±4.1 AFU/um in group D respecviely at 2 days after co-culture. The expression of TubulinβIII neuronal axon shaft and growth cone showed obviously in group A and B, while it was weak in group C and weaker in group D compared with group C, especially at the distal end of axon and growth cone at 2 days after co-culture in the first experiment. The mean fluorescence densities of axonal distal end in group D was significant stronger compared to group A, B and C respectively and there was no difference between group A, B and C in the mean fluorescence densities of axonal distal end at 2 days after co-culture in the first experiment.⑵The average lengths of neuronal axon were 402±33um in group A, 125±18um in group B, 404±14um in group C, 398±41um in group D, 405±12um in group E at 2 days after co-culture in the second experiment. The axonal length of the neogenesis DRGNs in group B was significant shorter compared to other groups respectively and there was no difference among group C, D and E in axonal length at 2 days after co-culture in the second experiment. The mean fluorescence densities at axonal distal end were 208.2±5.6 AFU/um in group A, 67.1±4.2 AFU/um in group B, 362.6±5.6AFU/um in group C, 365.6±39.3 AFU/um in group D and 379.8±23.6AFU/um in group E respectively at 2 days after co-culture.The expression of TubulinβIII neuronal axon shaft and growth cone showed obviously in group A, C, D and E, while it was weaker in group B compared with group A, especially at the distal end of axon and growth cone at 2 days after co-culture in the second experiment. The mean fluorescence densities of axonal distal end in group B was significant stronger compared to other groups and it in group E was stronger than group A , C and D at 2 days after co-culture. Conclusion:⑴The complete paralysis SCEs could obviously inhibit DRGNS axon growth and induce growth cone collapse. Sham operation SCEs have also a little effect on the axon growth, but could not induce growth cone collapse. Normal SCEs could not effect axon growth.⑵30uM Y27632 could obviously promote axon growth and induce steamlined growth cone.⑶1uM TDZD-8 could promote axon growth and induce thick growth cone, induce regeneration of many axons, axiramificate and thick growth cone.⑷Co-application of moderate dose TDZD-8 and Y27632 could promote axon extend, induce regeneration of many axons and axiramificate.The effect of co-application is more heavy than single application of inhibitory. |
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