| Objective:This study was to evaluate the treatment effect of methotrexate on secondary spinal cord injury in rats, including the inhibiting effect to oxidative stress, inflammation, edema and neuron apoptosis, and compare with MP which the classical drug for SCI treatment. Besides, explore the effect of joint of MTX and MP to locomotor function and gene expression profiling in rats, and discuss the molecular mechanism of the joint action. Prospect to offer effective experiment datas for understanding the treatment effect of MTX for SCI, and offer a new idea for obtaining rather treat method in clinical.Methods:1. Adult SD rats were randomly divided into Model group, MTX group, MP group and Sham group, and SCI models were set up by a PinPointTM high-precision three-dimensional contact sensor impactor. MTX(0.5 mg/kg-BW) were administrated subcutaneously at 30min after surgery in MTX Model, and MP(30 mg/kg-BW) were administrated by cauda vein in MP group. Model group were deal with nothing, and Sham Model were only for T10 laminectomy. The content of MDA,8-iso-PGF2a, 4-HNE,3-NT, AOPP, Protein carbonyl and 8-OHdG were detected by ELISA method in 3h,6h,12h and 48h after injury, the expression quantity of AQP4, TNF-a, IL-1β, Bax, Bcl-x and Bcl-2 were detected by realtime fluores-cence quantitative PCR method in 3h,6h,12h and 48h after injury, and observe neuron survival status at injury center and the adjacent area by immunohistochemistry.2. The adult SD rats were trained adaptively for five days on computer-controlled ZH-PT treadmill before surgery, and randomly divided into Model group, MTX group, MP group, MTX+MP group and Sham group. At 30min after surgery, MTX(0.5 mg/kg-BW) were administrated subcutaneously in MTX Model, MP(30 mg/kg-BW) were administrated by cauda vein in MP group, and MTX+MP group were administrated MP(30 mg/kg-1) by cauda vein first, then were administrated MTX(0.3 mg-kg-1) subcutaneously by alternate-day dosing from 1 day to 2 week. Model group were deal with nothing, and Sham Model were only for T10 laminectomy. Application of TreadScan gait analysis system was applicated to descript rat behavior characteristics before surgery and at 2,4,8 week after surgery. Rats were executed at 8 week after surgery, then the spinal cord tissue were proceeded sequencing expression spectrum analysis by solexa.Results:1. Results of ELISA shows that content of seven indicators in MTX group were lower than Model group, and the inhibition effect is a bit worse than MP group only at individual time points. Quantify of PCR shows that mRNA expression quantity of AQP4, TNF-α, IL-1β and Bax were lower than Model group, and mRNA expression quantity of Bcl-x and Bcl-2 were higher, and recovery effect is not as good as MP group only at individual time points. NeuN immunohistochemical staining observed that number of survival neurons increased obviously at 3 mm and 5 mm from the center ocompared with Model group, and was no significant difference with MP group.2. Behavior: ①Results of Model group compared with Sham group showed that swing time, stride time and sinimum longitudinal deviation were rised significantly, and instantrun speed, print area and regularity index were reduced significantly(P<0.05). ②Results of MP group compared with Model group showed all gait parameters significantly improved(P<0.05), But the extend improvement effect no longer increases over time. ②The gait recove of MP+MTX group showed better compared with MP group, and showed significant differences at 3 week or (and) 4 week after injury(P<0.05). Gene expression profile: ①The differences of gene expression show in all aspects of life activities between Sham group and Model group; ②Results of MP group compared with Model group showed that up-regulated genes mainly refers to nerve recovery promoting, anti-inflammatory, antioxidant and anti-apoptosis; Down-regulated genes mainly refers to pro-oxidation and pro-inflammatory; ②Results of MP+MTX group compared with MP group showed that up-regulated genes mainly refers to nerve recovery promoting, antioxidant, anti-inflammatory and anti-apoptosis; Down-regulated genes mainly refers to pro-inflammatory and pro-apoptosis.Conclusion:1. Treating with MTX early in rats SCI can effectively inhibit mechanism occurrence of oxidative stress, inflammation, swelling and the apoptosis, its short-term effect is inferior to MP, but MTX could administrate persistently because its treatment time is flexible, so its effect will be add up after injury.2. MTX joint with MP could effectively improve the function movement of SCI rats’ hind limbs, and has good inhibition effects to oxidative stress, inflammation and apoptosis after the injury. |
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