Inhibitory effect of electro-acupuncture on apoptosis after spinal cord injury

BACKGROUND： Acupuncture and electro-acupundura （EA） as complementary and alternative medicine have been applied for neurological recovery after spinal cord injury （SCI）, but the mechanism of treatment is unclear yet. OBJECTIVE： To examine and compare the effects of EA with methylprednisolone （MP） on apoptosis and expression of caspase-3 mRNA and protein in early SCI, in an attempt to provide experimental clues for clinical use of EA in the treatment of early SCI. DESIGN ： A randomized control tria SETTING ： Department of Anatomy, Second Military Medical University of Chinese PLA MATERIALS： Forty-eight adult male Sprague-Dawley （SD） rats aged 12 weeks and weighing （250±20） g were used. The experimental procedures were performed in accordance with the animal care guidelines of the National Institute of Health （NIH）. Equipments and medicine： Methylprednisolone sodium succinate （Pharmacia ＆ Upjohn N.V./S.A., Puurs, Belgium）; sodium pentobarbital （Sigma Chemical Co., St. Louis, MO）; G-6805-2 Multi-Purpose Health Device （Shanghai Medical Instruments High-TECH Co., Shanghai, China）; POD kit （Roche Molecular Biochemicals, Mannheim, Germany）; ISH Detection Kit （Wuhan Boster Biological Technology Ltd., Wuhan, China）; ABC kit （Vector Laboratories, Burlingame, CA）; light microscope （x200, Olympus, Tokyo, Japan）; HPIAS-1000 color High-definition pathological image analysis system （Qianping Imaging Engineering Company of Wuhan Tongji Medical University）. METHODS： The experiment was carried out in Department of Anatomy, Second Military Medical University of Chinese PLA between October 2004 and March 2006. ① The SCI model was established by modified Allen＇s method on T10. The force applied in traumatization was 50 g.cm. ②Forty-eight SD rats were equally randomized into four groups： sham operation （SO） group, in which animals received laminectomy only; model control （MC） group as a negative control, in which animals did not receive any treatment; EA treatment （EA） group, in which animals were treated with electro-acupuncture immediately after SCI, and MP treatment （MP） group, in which animals were treated with 30 mg/kg methylprednisolone sodium succinate through the tail vein immediately after SCI. Electro-acupuncture treatment was performed for EA group at 8 hours and 20 hours after SCI. The needles were inserted at a depth about 5-6 mm into the locus of the Dazhui and Mingmen. Asymmetric waves were used for electro-acupuncture at a stimulating frequency of 1 Hz. The total duration of EA stimulation was 30 minutes. ③ Six rats of each group were sacrificed at 6 hours and 24 hours after SCI. A 1.5 cm segment of the spinal cord encompassing the injury site was removed and prepared for experiment. ④ Cellular apoptosis was observed by terminal deoxynucleotidyl transferase （TDT）-mediated deoxyuridine triphosphate （dUTP）-biotin nick end labeling （TUNEL） method. Expression of caspase-3 mRNA and protein were estimated by ⑤Measurement data among groups were compared with one-way analysis of variance and differences between groups were compared with q test. MAIN OUTCOME MEASURES： The effect of electro-acupuncture on TUNEL-positive cells, the expression of caspase-3 mRNA and protein. RESULTS ： The forty-eight rats were involved in the final experiment. ① TUNEL-positive cells： TUNEL-positive cells in MP group and EA group were 29.00±8.27 and 30.17±6.85, respectively, at 6 hours after SCI; and were 1.00±0.89, 60.17±7.83, 6.67±3.56; 1.17±1.17, 69.50±8.50, 9.67±4.97, respectively at tip, intermediate zone and end of spinal cord at 24 hours after SCI; which were less than those in MC group （47.17±11.12, 9.33±4.76, 92.00±11.14, 22.50±5.43, P〈 0.01）. Cells in MP group were close to those in EA group （P〉 0.05）. ② Expression of positive caspase-3 mRNA： At 6 hours after SCI, numbers of positive caspase-3 mRNA were 133.33±11.84, 117.17±11.70, 120.00±10.73, respectively in MC, MP and EA groups, which were more than those in SO groups （83.50±8.64, P〈 0.01）. At 24 hours after SCI, numbers of positive caspase-3 mRNA were 121.50±11.34, 96.16±8.18, 97.67±9.69, respectively in MC, MP and EA groups, which were more than those in SO groups （83.50±9.64, P〈 0.05-0.01）. Numbers in MP group were close to those in EA group but less than those in MC group （P 〈 0.05-0.01）. ③ Expression of caspase-3 protein： Numbers in MC and MP groups were close to those in EA group （12.83±2.86, 10.00±3.58, 10.50±3.62） at 6 hours after SCI; numbers in MP and EA groups were 11.00±4.43 and 12.17±3.43 at 24 hours after SCI, which were less than those in MC group （17.83±4.92, P〈 0.01, 0.05）. CONCLUSION ： EA may partially inhibit apoptosis and protect nerve after SCI by down-regulating expression of caspase-3 mRNA and protein.

Experimental exploration and research prospect of physical bases and functional characteristics of meridians

The physical basis of meridians and acupoints (acupuncture points) is in a complex system mainly of connective tissue and interwoven with the blood capillaries, nerves, lymph vessels,etc. Elements of Ca, P, K, Fe, Zn, Mn, etc are found concentrated in the deep connective tissue structures in locations corresponding to acupoints. On this physical basis, the specific waveband of high efficiency for transmission of infrared rays exists in the collagenous fibre in a liquid crystal state. The development direction and new idea of researching on acupuncture and meridians are discussed.