ABSTRACT
Objective To explore the effect on effect of combined use of Y27632 (ROCK II inhibitor)and TDZD-8(GSK-3β inhibitor)on axonal regeneration of dorsal root ganglion (DRG) neurons in neogenetic rats. Methods All the thoracolumbar DRGs of two neogenetic Sprague-Dawley (SD)rats(<5 days)were harvested under the stereopsis raicrostat,and then the DRG neurons were cultured,purified and indentified.Fifteen adult female SD rats were randomly divided into three groups,ie,complete paraplegia group(5 rats),sham operation control group(5 rats)and normal group(5 rats)respectively.The T8-10 spinal cord extracts (SCEs) were harvested in the complete paraplegia group,sham operation control group and normal group respectively at day 7 after spinal cord injury.The experiment was divided into group A(DRG neurons + PBS),group B(DRG neurons + complete paralysis SCE),group C(DRG neurons + complete paralysis SCE + different concentration Y27632),group D(DRG neurons + complete paralysis SCE + different concentration TDZD-8)and group E(DRG neurons + complete paralysis SCE + proper concentration Y27632 and TDZD-8).The average axonal length and expression intensity of Tubulin βⅢ at distal end of neuronal axons were observed after two days of co-culture respectively in intro. Results (1)The average axonal length and expression intensity of Tubulin βⅢ at axon shaft and growth cone in the group B were significantly shorter and weaker than that in the group A,with statistical difference.(2)In the group C,the average axonal length and expression intensity of Tubulln βⅢ at axon shaft and growth cone in 5-10 μmol/L Y27632 treatment groups were more than that in the group B but lower than that in the group A.The average axonal length and expression intensity of Tubuhn βⅢ at axon shaft and growth cone in 20-50 μmol/L Y27632 treatment group were longer and stronger than that in the group A and the group B,especially the group B.Among different concentration Y27632 treatment groups,there was a longest average axonal length and strongest expression intensity of Tubulin βⅢ in 30 μmol/L treatment group.(3) In the group D,there was a longer average axonal length in 0.5-3 μmol/L TDZD-8 treatment group than that in the group A and the group B,with the longeat average axonal length in l μmool/L TDZD-8 treatment group.In 5-25 μmol/L TDZD-8 treatment groups,the average axonal length showed no difference compared with the group B but wns shorter than that in the group A.In all different concentration TDZD-8 treatment groups,the expression intensity of Tubulin βⅢ at axon shaft and growth cone was significantly stronger than that in the groups A and B.(4) In the group E,although the average axonal length was increased in the group E,there was no statisilcal difference compared with the group A,30 μmol/L Y27632 treatment group and l μmol/L TDZD-8treatment group.There was a significantly longer average axonal length in the group E than it in the group B and the expression intensity of Tubulin βⅢ at axon shaft and growth cone was stronger in the group E compared to the group A,30 μmol/L Y27632 treatment group and l μmol/L TDZD-8 treatment group.Conclusion The complete paralysis SCEs obviously inhibits DRG axonal growth,induces axonal retraction and growth cone collapse.High concentration of Y27632 can more obviously promote the axon growth compared with the low concentration,while the low concentration of TDZD-8 can obviously promote the axon growth.Combined use of appropriate concentration of TDZD-8 and Y27632 can promote the axon growth and induce the axons branching,as facilitates the formation of the axon circuit.