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(PI3Kantagonist)

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(Gravitydrop device).Elongated-needle penetration was applied to bilateral BL 54

ST 28

CV 3and CV 6

once daily for 3times.For rabbits of the ENP+LY 294002 and ENP+PD 98059 groups

LY 294002(10μg

20μL)

PD 98059(3μg

20μL)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-ERK1/2immunoactivity was detected by immunohistochemistry

and the expression levels of p-Akt

p-ERK1/2

cytochrome C(Cyt C)and Caspase-3proteins were determined by Western blot(WB)

and serum TNF-αcontent 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-ERK1/2protein expression levels in the model group were significantly decreased(P<0.05)

and Cyt C and Caspase-3expression levels and serum TNF-αcontent were significantly increased in the model group(P<0.05).Compared with the model group

the expression levels of p-Akt

p-ERK1/2were significantly increased in the ENP group(P<0.05)

while Cyt C and Caspase-3expression levels and TNF-αcontent were significantly down-regulated in the ENP group(P<0.05).After intrathecal injection of PI3 Kand 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-α content(P<0.05)

suggesting important roles of ERK1/2mediated 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-ERK1/2and down-regulating Cyt C and Caspase-3expression levels in the spinal cord and serum TNF-αcontent.