[Effect of elongated-needle penetration intervention on spinal apoptosis and cell signal transduction in acute spinal cord injury rabbits].

Objective: To observe the effect of elongated-needle penetration (ENP) stimulation of "Zhibian" (BL 54), "Shuidao" (ST 28), "Qihai" (CV 6) and "Zhongji" (CV 3) on spinal nerve cell apoptosis and cellular signal transduction in spinal cord injury rabbits, so as to reveal its mechanism underlying improvement of spinal injury.

Methods: A total of 80 adult Newzealand rabbits were randomized to control, model, ENP, ENP + LY 294002 (PI3K antagonist), ENP + PD 98059 (MEK antagonist) groups, with 16 rabbits in each group. The spinal cord injury model was established by using modified Allen's method (Gravity-drop device). Elongated-needle penetration was applied to bilateral BL 54, ST 28, CV 3 and CV 6, once daily for 3 times. For rabbits of the ENP+ LY 294002 and ENP+ PD 98059 groups, LY 294002 (10 microg, 20 microL), PD 98059 (3 microg, 20 microL) were separately given by intrathecal injection. Pathomorphological changes of the injured spinal cord (T13-L1) were observed after H.E. stain. Spinal cell apoptosis was detected by TUNEL,and phosphorylated (p)-Akt and p-ERK1/2 immunoactivity was detected by immunohistochemistry, and the expression levels of p-Akt, p-ERK1/2, cytochrome C (Cyt C) and Caspase-3 proteins were determined by Western blot (WB), and serum TNF-alpha content was assayed by ELISA.

Results: H. E. staining showed apparent structural changes as hemmorrhage, inflammatory cell infiltration, cellular edema and necrosis, and formation of vacuolation in the spinal cord in the model group, which was marked milder in the ENP group. TUNEL assay showed that the rate of apoptotic cells was notably increased in the model group than in the control group (P < 0.05), obviously decreased in the ENP group when compared with the model group (P < 0.05). Immunohistochemistry, WB and ELISA results showed that compared with the control group, spinal p-Akt and p-ERK1/2 protein expression levels in the model group were significantly decreased (P < 0.05), and Cyt C and Caspase-3 expression levels and serum TNF-a content were significantly increased in the model group (P < 0.05). Compared with the model group, the expression levels of p-Akt, p-ERK1/2 were significantly increased in the ENP group (P < 0.05), while Cyt C and Caspase-3 expression levels and TNF-alpha content were significantly down-regulated in the ENP group (P < 0.05). After intrathecal injection of PI3K and MEK antagonists, the effects of ENP were significantly weakened in reducing apoptosis rate, upregulating p-Akt and p-ERK1/2 expression and in down-regulating Cyt C and Caspase-3 expression and TNF-alpha content (P < 0.05), suggesting important roles of ERK1/2 mediated extracellular and PI3K/Akt mediated intracellular apoptotic signal transduction pathways in ENP induced repair of the traumatic tissues.

Conclusion: ENP stimulation can decrease spinal injury and cell apoptosis in spinal injury rabbits, which may be closely related to its effects in up-regulating p-Akt and p-ERK1/2 and down-regulating Cyt C and Caspase-3 expression levels in the spinal cord and serum TNF-alpha content.