Effects of decompression joint Governor Ves(2)

EA and decompression

The rats in the sham group, the 12-hour SCI group, and the 48-hour SCI group were not handled again. The rats in the 12-hour EA (SCI + EA) group and the 48-hour EA (SCI+ EA) groups were given the EA treatment 2 hours after decompression. The stainless steel acupuncture needles(0.3 mm in diameter, Jiajian Medical Equipment Co., Ltd.,Wuxi, China) were inserted at the acupointsDazhui(GV14;between the seventh cervical vertebra and the fi rst thoracic vertebra at the middle of the back) andBaihui(GV 20; the center of the parietal bone) to a depth of 3—4 mm (Figure 2).The EA treatment (Hans-200A, Jisheng Medical TechnologyCo., Ltd., Nanjing, China) was administered at 2 Hz for 15 minutes each day for 14 consecutive days. The current intensity ranged from 0.6 mA to 1.0 mA. The 12-hour and 48-hour MP groups received 30 mg/kg MP (Pfizer Pharmaceuticals Ltd., New York, NY, USA)viathe tail vein within 1 hour after decompression, and then at a dose of 5.4 mg/kg per hour once every four hours; all doses were given within 24 rats were sacri fi ced after 14 days of treatment. Afterwards,the damaged spinal cord segments were extracted.

Table 1 BBB scores of the groups compressed for 12 and 48 hoursCompressed for 48 hours:Ftime= 3200.61,P= 0.00 ＜ 0.05;Fgroup= 3656.30,P= 0.00 ＜ 0.05;Finteraction= 416.78,P= 0.00 ＜ 0.05. Compressed for 12 hours:Ftime= 4182.29,P= 0.00 ＜ 0.05;Fgroup= 7752.59,P= 0.00 ＜ 0.05;Finteraction= 524.15,P= 0.00 ＜ 0.05. All data are expressed as the mean± SD (n= 6 per group; repeated measures analysis of variance). Lower BBB scores indicate poorer locomotor function. The sham group was constant and the other three groups had an improved trend at various time points. *P＜ 0.05,vs. sham group, #P＜ 0.05,vs. EA and MP group. EA:Electroacupuncture; MP: methylprednisolone; SCI: spinal cord injury; BBB: Basso, Beattie, and Bresnahan locomotor after SCI 0 hours24 hours3 days7 days14 days Compressed for 48 hours Sham21. EA1.*6.*16.*17.*MP1.*6.*16.*17.*SCI1.*#4.*#9.*#11.*#Compressed for 12 hours Sham21. EA1.*10.*17.*17.*MP1.*10.*17.*18.*SCI1.*#7.*#11.*#17.*

Hind limb locomotor function score

Evaluation of motor ability was graded using a 0-21 point scoring system called the Basso, Beattie, and Bresnahan locomotor scale (BBB) (Bhimani et al., 2017), which assesses a combination of rat hind limb movements, trunk position, stability, stepping, coordination, paw placement, toe clearance,and tail position. The score was recorded at 0 and 24 hours, as well as at 3, 7, and 14 days after decompression independently by two outside researchers, and the results were averaged.

Enzyme linked immunosorbent assay (ELISA) for platelet-activating factor concentration in the damaged spinal cords

The platelet-activating factor concentration of damaged spinal cords was measured using a rat ELISA kit (Huamei Biological Engineering Co., Ltd., Wuhan, China). Optical density (OD)values were measured at 450 nm. Platelet-activating factor concentration was calculated using OD values in each group.

Western blot assay for the expression of caspase-9

After caspase-9 protein extraction, protein quantification,and the adjustment of protein concentration, the proteins were subjected to electrophoresis (Mini P-4 electrophoresis chamber; Kaiyuan Xinrui Instrument Co., Ltd., Beijing, China) and then transferred on the membrane. The membrane was incubated with primary GAPDH mouse monoclonal antibodies (ImmunoWay Biotechnology, Newark, DE, USA)for 10 minutes at 4°C overnight, and with secondary IgG/FITC (H + L) horseradish peroxidase antibodies (1:10,000;Kangwei Century Biotechnology Co., Ltd., Beijing, China),and IgG/TRITC (H + L) horseradish peroxidase antibodies(1:10,000; Kangwei Century Biotechnology Co., Ltd.), with oscillation for 40 minutes at room temperature. Reaction products were visualized using ECL Gel Pro analysis software(Image J, National Institutes of health, Bethesda, MD, USA)to quantify the amount of protein. OD values were normalized to those of β-actin (1:400; Sigma, St. Louis, MO, USA).

Statistical analysis

The data were recorded as the mean ± SD and analyzed using SPSS 20.0 software (IBM, Armonk, NY, USA). The BBB scores among the groups were compared using repeated measures analysis of variance, while the other data were analyzed using one-way analysis of variance followed by the least signi fi cant differencepost hoctest. APvalue of ＜ 0.05 was considered statistically signi fi cant.

Results

Effects of decompression combined with EA on hind limb locomotor function in rats with acute upper cervical SCI

BBB scores were signi fi cantly lower in the SCI groups compared with the sham group. There was no signi fi cant difference in BBB scores among the SCI groups at 0 hours after decompression (P＞ 0.05). For the groups compressed for 12 hours, there were no signi fi cant differences in BBB scores at any time point between the 12-hour EA and 12-hour MP groups. BBB scores were lower in the 12-hour SCI group than in the 12-hour EA and 12-hour MP groups at 24 hours,3 days, and 7 days (P＜ 0.05). Also, BBB scores were similar at 0 and 48 hours between the 12-hour EA and MP groups (P＞ 0.05). For the groups compressed for 48 hours, there were no significant differences in BBB scores at any time point between the 48-hour EA and MP groups. BBB scores at all time points were lower in the 48-hour SCI group than in the 48-hour EA and MP groups (P＜ 0.05; Table 1).

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