内源性GDNF、NT-4对针刺猫脊髓备用根背根节神经元的作用

为了解部分背根切除和针刺及内源性GDNF和NT-4对体外培养备用背根节(DRG)的作用,本研究对5只成年猫进行双侧备用根手术(切除双侧L1~L5和L7~S2DRG,其中L6DRG作为备用背根)。术后当日开始针刺一侧L6脊神经后肢分布区的两组穴位,即足三里和悬钟、伏兔和三阴交,每天一次,每次30min,连续针刺7d后无菌条件下取出双侧L6DRG进行体外培养,24h后全量换液,并将针刺侧的一部分培养孔的培养液分别用含有200ng/ml抗GDNF和NT-4抗体培养液替换,分别作为抗GDNF和NT-4抗体封闭组。7d后终止培养,于显微镜下用显微测微尺测量神经突起的长度;并用抗NSE抗体行免疫细胞化学ABC法染色进行神经元鉴定。结果显示:(1)免疫细胞化学染色可见体外培养的细胞95%以上为NSE阳性细胞,且为典型的体外培养的DRG神经元;(2)体外培养备用根组和抗GDNF抗体组神经突起的平均长度比针刺组的短(P<0.05);(3)而针刺组神经突起的平均长度与抗NT-4抗体组间无差异(P>0.05);两抗体组平均突起长度比备用根组的突起长(P<0.05)。本研究结果提示,针刺可促进体外培养DRG神经元突起的生长,进而可能与脊髓可塑性密切相关;内源性GDNF有促进DRG神经元突起生长的作用;而内源性NT-4在DRG神经元突起生长中发挥的作用却不明显。

THE EFFECT OF ENDOGENOUS GDNF, NT-4 ON NEURONS FROM SPARED ROOT GANGLION FOLLOWING ACUPUNCTURE

The present study aimed to investigate the effect of partial dorsal root rhizotomy, acupuncture, endogenous GDNF and NT-4 on extending of neurons from spared dorsal root ganglion (DRG) in vitro. L_1-L_5, L_7-S_2 DRGs from 5 adult cats were removed bilaterally , and L_6 DRG was spared, then unilateral two sets of acupoints (Zusanli and Xuanzhong; Futu and Sanyinjiao located in the distribution area of spinal nerve L_6) were electro-stimulated alternatively 30 min everyday by electro-needling. 7 days after operating, bilateral L_6 DRGs was taken out on the condition of asepsis and were cultured respectively in vitro. The culture medium of some of the wells plated with neurons obtained from acupuncture treated DRG neurons were replaced with a medium containing 200ng/ml anti-GDNF and NT-4-antibody at the 24~ th hour and terminated after 7 days. The length of the neurite was measured by micro-measured ruler in upside-down light microscope. Then, cultured cells were labeled using specific neuron specific enolase (NSE) antibody, by the immunocytochemical ABC method. The results showed that: (1) Immunocytochemical staining revealed that over 95% cells were NSE positive cells which were the typical neuron of DRG in vitro; (2) on the 7~ th day, the neurite length of the spared DRG group and anti-GDNF antibody group were shorter than that of the acupuncture group(P<0.05); (3) while that of anti-NT-4 antibody group was not shorter than that of the acupuncture group; that of the two-antibody group were longer than that of the spared DRG group(P<0.05). These results indicate that DRG has plasticity and acupuncture probably promote the plasticity, which consequently promote the spinal cord plasticity; endogenous GDNF probably promote extending of neuronal dendrite in DRG, whereas the function of NT-4 is not obvious.