Objective To evaluate the role of nitric oxide (NO) in the spinal cord in the maintenance of diabetic neuropathic pain in rats.Methods Male Sprague-Dawley rats,aged 2 months,weighing 180-200 g,were used in the study.Diabetes mellitus was induced by intraperitoneal streptozotocin (STZ) 60 mg/kg and confirmed by blood glucose ＞ 16.7 mmol/L on day 2 after STZ injection.Twenty diabetic rats were randomly allocated into diabetes mellitus group (DM group,n =10) and L-NAME (non-selective NOS inhibitor) group (LN group,n =10).Another 10 age-matched normal rats served as control group (C group).On 21 days after STZ injection,L-NAME 10 mg/kg was injected intraperitoneally once a day for 7 consecutive days in LN group,whereas the equal volume of normal saline 5 ml/kg was given instead of L-NAME in DM group.Paw withdrawal threshold to yon Frey filament stimulation (PWT) was measured before STZ infection and on 7,14,21 and28 days after STZ injection.The rats were sacrificed after the last measurement of PWT and the lumbar segments of spinal cord were removed for determination of NO content and neuronal nitric oxide synthase (nNOS) expression (by Western blot analysis) in spinal cord tissues.Results Compared with C group,PWT was significantly decreased on 14,21 and 28 days after STZ injection,and the NO content and nNOS expression in spinal cord tissues were increased in DM and LN groups (P ＜ 0.05).Compared with DM group,PWT was significantly increased on 28 days after STZ injection,and the NO content and nNOS expression in spinal cord tissues were decreased in LN group (P ＜ 0.05).Conclusion NO in the spinal cord is involved in the maintenance of diabetic neuropathic pain in rats and the mechanism is related to the enhanced function of nNOS.

Objective To evaluate the role of microRNA9 (miR-9) in spinal dorsal horn neurons in over-expression of calcium homeostasis modulator 1 (CALHM1) in rats with diabetic neuropathic pain (DNP).Methods Ninety-three healthy male Sprague-Dawley rats,aged 2 months,weighing 180-200 g,were used in the study.Diabetes mellitus was induced by intraperitoneal 1％ streptozocin (STZ) 60 mg/kg.The experiment was performed in two parts.Experiment Ⅰ The rats were divided into control group (group C,n=10) and DNP group (n=83) using a random number table.The mechanical paw withdrawal threshold (MWT) was measured before STZ injection and at 1,2,3,4,5 and 6 weeks after STZ injection.The expression of miR-9 in the spinal dorsal horn was determined using the in situ hybridization at 6 weeks after STZ injection.Experiment Ⅱ The rats with DNP were selected at 6 weeks after STZ injection,and the spinal dorsal horn neurons were isolated and cultured.The neurons were seeded in culture plates at the density of 5×106 cells/ml (2 ml/well) and divided into 2 groups (n=18 each) using a random number table:control group (group C) and miR-9 antisense oligonucleotide group (group ASO).The neurons were cultured in normal culture atmosphere in group C.In group ASO,the single nucleotide sequence of miR-9 antisense oligonucleotide sequence 5'-UUCUCCGAACGUGUCACGUTT-3'was added with the final concentration of 100 pmol/L.The expression of miR-9 and CALHM1 mRNA was detected using quantitative real-time polymerase chain reaction at 48 h of incubation.The expression of CALHM1 was detected by Western blot at 72 h of incubation.Results Experiment Ⅰ Compared with group C,the MWT was significantly decreased at 2-6 weeks after STZ injection,and the expression of miR-9 in the spinal dorsal horn was up-regulated in group DNP (P＜0.05).Experiment Ⅱ Compared with group C,the expression of miR-9 and CALHM1 protein and mRNA in spinal dorsal horn neurons was significantly down-regulated in group ASO (P＜0.05).Conclusion miR-9 in spinal dorsal horn neurons probably mediates the over-expression of CALHM1 in rats with DNP.

Objective:To analyze the radiographic improvements after Halo-gravity traction in severe kyphoscoliosis patientswith type III spinal cord on preoperative apex MRI, and to assess the clinical outcomes and surgical safety of Halo-gravity traction in this cohort.Methods:A total of 47 severe thoracic kyphoscoliosis patients with type III spinal cord on preoperative apex MRI who underwent preoperative Halo-gravity traction followed by one-stage posterior spinal fusion from February 2019 to June 2021 in the Nanjing Drum Tower Hospital were retrospectively analyzed. There were 18 males and 29 females with an average age of 22.5±12.8 years (range, 9-60 years). The average duration of traction was 7.4±3.9 weeks (range, 4-16 weeks). Radiographic parameters were measured including the coronal Cobb angle, distance between C 7 plumb line and center sacral vertical line (C 7PL-CSVL), sagittalglobal kyphosis (GK) and sagittal vertical axis (SVA) atpre-traction, post-traction and post-operation, respectively. The traction correction rate was measured as "traction degree before traction-traction degree after traction)/traction degree before traction" and the surgical correction rate was represented as "traction degree before traction-postoperative degree)/ traction degree before traction". The Frankel scoring system was used for the evaluation of neurological status at pre-traction, post-traction and post-operation. Results:All of 47 patients underwent the Halo-gravity traction and posterior spinal correction surgery. The C 7PL-CSVL was 35.7±16.9 mm at initial visit. At post-operation, C 7PL-CSVL was improved to 22.0±13.7 mm ( t=13.75, P<0.001), and the improvement rate was 39.9%±15.5%. The GK was 110.9°±22.1° at initial visit, which was improved to 84.1°±19.9° ( t=8.84, P<0.001) after Halo-gravity traction with an average correction of 23.7%±8.9%. At post-operation, GK was improved to 65.3°±19.3° ( t=10.63, P<0.001), and the improvement rate were 40.1%±20.7%. The SVA was 43.8±19.5 mm at initial visit. At post-operation, SVA was improved to 21.1±14.9 mm ( t=10.32, P<0.001), and the improvement rate were 53.1%±27.0%. A total of 14 patients showed neurological deficits of lower limbs at pre-traction, of which 8 patients had significant neurological improvement after Halo gravity traction; 3 patients had significant neurological improvement after surgery, and the remaining 3 patients had no significant neurological improvement during treatment. No new neurological deficits were observed after Halo-gravity traction or surgery. Conclusion:For severe kyphoscoliosis patients with type III spinal cord on preoperative apex MRI, the Halo-gravity traction could effectively correct the deformity, improve neurological function, enhance the tolerance of spinal cord to surgery and reduce the risk of intraoperative iatrogenic neurological deficit.