多次鞘内注射CREB反义寡核苷酸对神经病理性痛小鼠脊髓NR2A表达的影响

目的 评价多次鞘内注射cAMP反应元件结合蛋白(CREB)反义寡核苷酸对神经病理性痛小鼠脊髓含2A亚基的NMDA受体(NR2A)表达的影响.方法 鞘内置管成功的C57BL/6雄性小鼠40只,体重20 ～ 25 g,采用随机数字表法,将其随机分为4组(n=10):生理盐水组(NS组)、CREB同义寡核苷酸组(S组)、CREB错义寡核苷酸组(M组)和CREB反义寡核苷酸组(A组).采用结扎坐骨神经的方法制备小鼠神经病理性痛模型,结扎坐骨神经后第1～6天,4组分别鞘内注射生理盐水5μl、同义寡核苷酸5 μg/5μl、错义寡核苷酸5μg/5μl、反义寡核苷酸5μg/5μl,1次/d.于术前1d、造模后1、3、5、7d时测定小鼠结扎侧足底的机械缩足阈值(PWMT)和热辐射刺激潜伏期(PWTL).于结扎坐骨神经后7、14 d时各取5只小鼠断头处死,取L3～5脊髓,采用Western blot和RT-PCR法测定NR2A的表达水平.结果 与基础值相比,A组小鼠结扎坐骨神经1～7d时PWMT和PWTL差异无统计学意义(P＞0.05),NS组、S组和M组PWMT和PWTL降低(P＜0.05).与NS组、S组和M组相比,A组小鼠结扎坐骨神经后7、14 d时脊髓NR2A mRNA和蛋白表达下调(P＜0.05).与结扎坐骨神经后7d时相比,4组结扎坐骨神经后14 d时小鼠脊髓NR2A mRNA和蛋白表达上调(P＜0.05).结论 在神经病理性疼痛形成阶段多次鞘内注射CREB反义寡核苷酸可减轻神经病理性痛,其机制可能与抑制脊髓NR2A表达有关.     

瑞芬太尼和芬太尼对大鼠脊髓背角神经元NMDA受体通道电流的影响

目的 探讨瑞芬太尼和芬太尼对大鼠脊髓背角神经元NMDA受体通道电流的影响.方法 采用全细胞膜片钳技术记录NMDA受体通道电流.原代培养的E14SD大鼠脊髓背角神经元(DH细胞)30个,采用随机数字表法,将其分为3组(n=10):瑞芬太尼组(R组)、芬太尼组(F组)、对照组(C组).4 nmol/L瑞芬太尼(R组)、10 μmol/L芬太尼(F组)灌流DH细胞60 min后洗脱.于给药后即刻(T0)、药物作用15 min(T1)、30 min(T2)、45 min(T3)、60 min(T4)、洗脱后15 min(T5)、30 min(T6)时记录NMDA受体通道电流.结果 与C组比较,F组各时点NMDA受体通道峰电流差异无统计学意义,R组T0、T1时NMDA受体通道峰电流差异无统计学意义(P＞0.05),T2-T6时NMDA受体通道峰电流升高(P＜0.01);与T0时比较,R组T3-T6时NMDA受体通道峰电流升高(P＜0.01);与T5时比较,R组T2-T4时、T6时NMDA峰电流下降(P＜0.01).结论 瑞芬太尼可增强大鼠脊髓背角神经元NMDA受体功能,于洗脱后达峰效应,芬太尼无此作用.     

姜黄素对神经病理性痛大鼠脊髓背角及背根神经节神经元大麻素受体1和NR2B表达的影响

目的 探讨姜黄素对神经病理性痛大鼠脊髓背角及背根神经节(DRG)神经元大麻素受体1 (CBR1)及含NR2B亚基N-甲基-D-天冬氨酸(NMDA)受体表达的影响.方法 雄性SD大鼠72只,体重200～230 g,采用随机数字表法,将其随机分为4组(n=18):假手术组(S组)、慢性压迫性损伤组(CCI组)、姜黄素组(Cur组)和溶媒对照组(SC组).S组仅分离、暴露坐骨神经,其余3组采用慢性压迫性损伤法制备神经病理性痛模型.Cur组术后腹腔注射姜黄素100 mg·kg-1·d-1,连续14 d,SC组给予等容量二甲基亚砜.分别于术前2d、术后1、3、7、10、14 d时测定机械缩足反应阈值(MWT)和热缩足潜伏期(TWL).分别于术后3、7、14 d时处死6只大鼠,取脊髓背角和DRG,采用免疫组化法检测神经元CBR1和NR2B的表达.结果 与S组比较,其他3组术后MWT降低,TWL缩短,脊髓背角和DRG神经元CBR1和NR2B表达上调(P＜0.05);与CCI组比较,Cur组术后MWT升高,TWL延长,脊髓背角和DRG神经元CBR1表达上调,NR2B表达下调(P＜0.05),SC组上述指标差异无统计学意义(P＞0.05).结论 姜黄素可减轻大鼠神经病理性痛,其机制与脊髓背角及DRG神经元CBR1表达上调、NR2B表达下调有关.     

艾芬地尔预先给药对CCI大鼠脊髓NMDA受体NR2B亚基表达的影响

目的探讨艾芬地尔预先给药对坐骨神经慢性压榨性损伤(CCI)大鼠脊髓背角NR2B亚基表达的影响及其对神经病理性疼痛的预防作用。方法雄性SD大鼠50只,体重180～200 g,随机分为3组:假手术组(n=10)、CCI组(n=20)和艾芬地尔组(n=20)。建立CCI动物模型,假手术组大鼠仅分离坐骨神经,但不结扎神经。艾芬地尔组大鼠在术前30 min及术后1、2 d连续3 d腹腔内注射艾芬地尔(10 mg/kg);CCI组大鼠腹腔内注射相同体积的生理盐水;假手术组未给药。各组均于术前,假手术组于假手术后3 d、艾芬地尔组和CCI组均于CCI术后7、14 d分别取10只大鼠,以von Frey纤维测定机械痛阈,随后断头处死,取患侧L4.5脊髓背角组织,采用Western blot方法测定NR2B亚基的蛋白表达。结果各组术前基础机械痛阈比较差异无统计学意义(P>0.05)。与假手术组比较,CCI组术后7、14 d时机械痛阈降低(P<0.05),艾芬地尔组术后7、14 d时差异无统计学意义(P>0.05);与CCI组比较,艾芬地尔组术后7、14 d时机械痛阈升高(P<0.05)。与假手术组相比,艾芬地尔组和CCI组术后7、14 d时脊髓背角NR2B蛋白表达均升高(P<0.05);与CCI组比较,艾芬地尔组术后7、14 d时脊髓背角NR2B蛋白表达降低(P<0.05)。结论大鼠腹腔内预先注射艾芬地尔,通过特异性地拮抗含NR2B亚基NMDA受体,可有效地预防CCI导致的神经病理性疼痛,其机制与降低脊髓背角NR2B亚基表达升高有关。     

鞘内注射DREAM-shRNA对神经病理性痛大鼠脊髓背角p-CREB表达的影响

目的 探讨鞘内注射转录因子下游调控元件拮抗因子-短发夹RNA(DREAM-shRNA)对神经病理性痛大鼠脊髓背角磷酸化环磷酸腺苷反应元件结合蛋白(p-CREB)表达的影响.方法 成年健康雄性SD大鼠,体重280～320 g,采用坐骨神经慢性压迫(CCI)法建立大鼠神经病理性痛模型.于CCI后第3天鞘内置管.取鞘内置管成功的大鼠24只,随机分为4组,每组6只,假手术组(S组):仅暴露坐骨神经,不结扎;神经病理性痛组(NP组):于CCI后第8天鞘内注射生理盐水10 μl;RNA干扰组(RNAi组):于CCI后第8天鞘内注射DREAM-shRNA 5 μl和牛理盐水5 μl;空白载体组(BV组):于CCI后第8天鞘内注射慢病毒空白载体5 μl和生理盐水5μl,各组连续注射7 d.于CCI前1 d(T0,基础状态)、CCI后第7～14天(T1-8)时测定机械痛阈.于CCI后第15天时测定脊髓背角绿色荧光蛋白(GFP)和p-CREB的表达水平.结果 与基础值比较,各组各时点机械痛阈降低(P<0.05或0.01);与T1时比较,NP组和BV组T5-8时机械痛阈降低,RNAi组T8时机械痛阈升高(P<0.05或0.01);与S组比较,NP组和BV组机械痛阈降低,RNAi组T2时机械痛阈降低,T8时升高,NP组、RNAi组和BV组脊髓背角p-CREB表达上调(P<0.05);与NP组比较,RNAi组T6-8时机械痛阈升高,脊髓背角p-CREB表达下调(P<0.05).RNAi组脊髓背角可见大量绿色荧光,即GFP表达阳性,其余3组脊髓背角未见绿色荧光,即GFP无表达.结论 鞘内注射DREAM-shRNA缓解大鼠神经病理性痛的机制可能与抑制脊髓背角p-CREB的表达有关.     

鞘内注射DREAM-shRNA对神经病理性痛大鼠脊髓背角p-CREB表达的影响

Objective To investigate the effects of intrathecal (IT) DREAM-short hairpin RNA (DREAM-shRNA) on expression of phosphorylated cyclic AMP response element binding protein (p-CREB) in the spinal dorsal horn in a rat model of neuropathic pain. Methods Adult male SD rats weighing 280-320 g were anesthetized with intraperitoneal 10% chloral hydrate. Neuropathic pain was induced by chronic constrictive injury (CCI) to sciatic nerve. IT catheters were placed according to the method described by Yaksh on 3rd day after CCI. Twenty-four rots in which IT catheter was successfully implanted were randomly divided into 4 groups (n = 6 each) : group Ⅰ sham operation (group S) ; group Ⅱ neuropathic pain (group NP) ; group Ⅲ RNA interference (group RNAi) and group Ⅳ blank vector (group BV). Lentivius with DREAM-shRNA 5 μl was injected IT in group RNAi, and blank vector 5 μl in group BV, and once a day for 7 days, starting from the day 8 after CCI. The mechanical pain threshold was measured at day 1 before CCI (T0 ,baseline) and day 7-14 after CCI (T1-8). The animals were killed on 15th day after CCI. The L4-6 lumbar segment of the spinal cord was removed for determination of the expression of green fluorescent protein (GFP) and p-CREB by immuno-fluorescent method.Results The mechanical pain threshold was significantly decreased as compared with the baseline at T0 in all 4 groups and returned to the baseline levels at T5-8 in group S and RNAi, but remained low in group NP and BY. The mechanical pain threshold was significantly lower after CCI/sham operation and significanty higher at T8 in group RNAi than in the other 3 groups. The expression of p-CREB in the spinal dorsal horn was up-regulated in group NP, RNAi and BV as compared with group S, and in group NP and BV as compared with group RNAi. The green fluorescence was observed in group RNAi but not in the other 3 groups. Conclusion IT DREAM-shRNA can ameliorate neuropathic pain in rats through inhibiting the expression of p-CREB in the spinal dorsal horn.     

人神经生长因子β基因转染对神经病理性痛大鼠脊髓背角致痛物质含量的影响

目的 探讨人神经生长因子β(hNGFβ)基因转染对神经病理性痛大鼠脊髓背角降钙素基因相关肽(CGRP)和P物质(SP)含量的影响.方法 雄性SD大鼠48只,体重200～250 g,随机分为3组(n=16):假手术组(Ⅰ组)假手术后立即鞘内注射人工脑脊液;Ⅱ组和Ⅲ组制备坐骨神经慢性压迫性损伤(CCI)模型,术后分别立即鞘内注射人工脑脊液或重组腺病毒介导入神经生长因子β(Ad-hNGFβ)基因.于术前1 d、转染后28 d内每4天测定热痛阈、机械痛阈及行为学评分.每组分别于转染后4、7、14及28 d各处死4只大鼠,取脊髓组织,采用免疫组织化学法测定SP和CGRP含量.结果 与Ⅰ组比较,Ⅱ组和Ⅲ组行为学评分升高,机械痛阈及热痛阈均降低(P<0.01);与Ⅱ组比较,Ⅲ组行为学评分及机械痛阈差异无统计学意义(P>0.05),转染后8～24 d热痛阈升高(P<0.05).术后Ⅱ组和Ⅲ组术侧脊髓背角SP及CGRP含量明显高于Ⅰ组,术后7～28 dm组术侧脊髓背角SP及CGRP含量明显低于Ⅱ组(P<0.05或0.01).结论 Ad-hNGFβ基因转染可能通过降低脊髓背角SP及CGRP含量减轻神经病理性痛大鼠的热痛觉过敏.     

鞘内预注射L-NAME对神经性疼痛大鼠脊髓背角降钙素基因相关肽表达的影响

目的 观察鞘内预注射NG-硝基-L-精氨酸-甲基酯(L-NAME)对结扎坐骨神经所致神经性疼痛大鼠脊髓背角降钙素基因相关肽(CGRP)表达的影响。方法 选择鞘内置管后无神经损伤症状的SD雌性大鼠96只,随机分为4组,每组24只。A组:假手术组;B组:坐骨神经结扎组;C组:假手术前15 min鞘内注射L-NAME 10 μl(250 μg);D组:坐骨神经结扎前15 min鞘内注射L-NAME 10 μl(250 μg)。各组分别在术后第1、4、7和14天随机处死6只大鼠。采用免疫组织化学方法观察各组大鼠结扎侧脊髓背角CGRP的表达。结果 与A组比较,B组、D组大鼠结扎侧脊髓背角CGRP表达在术后第4、7和14天明显升高(P<0.05),C组差异无显著性。与C组比较,D组大鼠结扎侧脊髓背角CGRP表达在术后第4、7和14天明显升高(P<0.05)。而D组大鼠结扎侧脊髓背角内的CGRP表达与B组比较,差异无显著性(P>0.05)。结论 鞘内预注射L-NAME不能抑制周围神经损伤所诱导的脊髓背角CGRP表达,提示一氧化氮介导神经性疼痛的作用不是通过促进CGRP释放实现的。     

鞘内注射艾芬地尔对骨癌痛小鼠脊髓NR2B mRNA表达的影响

目的 探讨鞘内注射艾芬地尔对骨癌痛小鼠脊髓N-甲基-D-天冬氨酸(NMDA)受体2B亚基(NR2B)mRNA表达的影响.方法 雄性C3H/HeJ小鼠140只,体重20～25 g,4～6周龄,随机分为5组(n=28):假手术组(S组)、骨癌病组(B组)和艾芬地尔2.5μg、5μg、10μg组(I1-3组).I1-3组和B组于小鼠右侧股骨远端骨髓腔接种NCTC 2472溶骨性纤维肉瘤细胞,建立骨癌痛模型;S组不接种肿瘤细胞.I1～3组于接种肿瘤细胞后14 d分别鞘内注射艾芬地尔2.5、5、10 μg,B组和S组鞘内注射艾芬地尔溶媒.各组于接种肿瘤细胞前1 d、鞘内注射艾芬地尔或溶媒前1 h、注射后2、12和24 h(T1～5)时随机取7只小鼠测定机械痛阈和热痛阈,并于T2～5,时测定后断头处死,取L3～5脊髓,采用RT-PCR法测定脊髓组织NR2B mRNA的表达水平.结果 与S组比较,除I3,组T3时热痛阈差异无统汁学意义(P0.05)外,B组和I1～3组机械痛阈和热痛阈均降低(P<0.05),B组和I1组脊髓组织NR2B mRNA表达上调,I2组该指标表达下调(P<0.05);与B组比较,I2.3组机械痈阈和热痛阈升高,脊髓组织NR2B mRNA表达下调(P<0.05),I1组各时点以上指标差异均无统计学意义(P0.05);与I2组比较,I3组机械痛阈和热痛阈升高,脊髓组织NR2B mRNA表达下调(P<0.05).结论 鞘内注射艾芬地尔可通过阻断含2B亚基的NMDA受体缓解小鼠骨癌痛,并下凋脊髓组织NR2B mRNA的表达抑制痛敏反应.     

产妇鞘内注射舒芬太尼分娩镇痛的效应

目的产妇鞘内注射不同剂量舒芬太尼分娩镇痛的效应。方法 孕足月的初产妇100 例,ASA I或Ⅱ级,随机分为5组(n=20):舒芬太尼1μg组(A组)、舒芬太尼3μg组(B组)、舒芬太尼 5μg组(C组)、舒芬太尼7μg组(D组)和舒芬太尼10μg组(E组)。L2,3行脊椎-硬联合穿刺,鞘内注射 相应剂量舒芬太尼,硬膜外腔置管,接硬膜外自控镇痛(PCEA)泵。记录鞘内给药前、后产妇血压 (BP)、心率(HR)、呼吸频率(RR)、脉搏血氧饱和度(SpO2)。观察各组镇痛起效时间,应用视觉模拟镇 痛评分(VAS)评估疼痛效果,记录PCEA首次给药时间和总用药量,改良Bromage评分评价双下肢运动 阻滞情况。记录产妇出现不良反应和新生儿出生后1、5 min时Apgar评分。结果 各组年龄、身高、 体重、妊娠时间、宫口扩张程度比较差异无统计学意义(P>0．05)。与A组比较,B～E组镇痛起效时 间缩短,首次PCA给药时间延长,最高感觉阻滞平面上移;C～E组PCA药液总量、PCA按压次数减 少;B～E组鞘内给药后5—30min时有效镇痛率升高(P<0．05或0．01)。各组催产素使用率、分娩出 血量、镇痛满意度、新生儿出生后1、5 min时Apgar评分比较差异均无统计学意义(P>0．05)。D、E组 恶心呕吐、瘙痒、下肢麻木的发生率高于A、B组(P<0．01)。结论 产妇鞘内注射舒芬太尼联合 PECA分娩镇痛是安全、有效的,鞘内注射舒芬太尼的剂量以3～5μg为宜。     

神经病理性痛大鼠背根神经节和脊髓背角Nogo-A蛋白表达的变化

目的 评价神经病理性痛大鼠背根神经节和脊髓背角Nogo-A蛋白表达的变化.方法 健康成年雄性SD大鼠72只,体重250～300 g,采用随机数字表法,将大鼠随机分为3组(n=24):对照组(C组)、假手术组(S组)和神经病理性痛组(NP组).C组不做任何处理,NP组采用结扎并剪断胫神经和腓总神经的方法制备大鼠神经病理性痛模型,S组仅切皮暴露坐骨神经但不结扎和剪断神经.于结扎后1、7、14、21 d时采用yon Frey丝测定大鼠机械痛阈.于各时点随机取6只大鼠处死后取损伤侧L5背根神经节和L4,5脊髓,采用免疫荧光标记法测定Nogo-A蛋白的表达(n=3),采用Westernblot法测定Nogo-A蛋白的表达(n=3).结果 与C组和S组比较,NP组大鼠结扎后7、14、21 d时机械痛阈降低,结扎后7、14 d时背根神经节Nogo-A蛋白表达下调,结扎后14、21 d时脊髓背角Nogo-A蛋白表达上调(P＜0.05).结论 背根神经节及脊髓背角Nogo-A蛋白可能在外周神经损伤诱发大鼠神经病理性痛过程中发挥重要作用.

Abstract：

Objective To investigate the changes in the expression of Nogo-A protein in the dorsal root ganglion (DRG) and spinal dorsal horn in a rat model of neuropathic pain (NP) .Methods Seventy-two male SD rats weighing 250-300 g were randomly divided into 3 groups ( n = 24 each) : control group (group C) , sham operation group (group S) and NP group. NP was induced by ligation and severance of tibial and common fibular nerves according to the technique described by Isabelle et al. The mechanical withdrawal threshold (MWT) to von Frey filament stimulation was measured at 1, 7, 14 and 21 days after ligation. Six rats in each group were randomly selected at each time point and sacrificed (3 for determination of Nogo-A protein expression by immunofluorescence, 3 for determination of Nogo-A protein expression by Western blot) . The L5 DRG and L4,5 segment of spinal cord on the injured side were removed for determination of Nogo-A protein expression by immunofluorescence and Western blot. Results Compared with the groups C and S, MWT was significantly decreased at 7, 14 and 21 days after ligation, the expression of Nogo-A protein in the DRG was down-regulated at 7 and 14 days after ligation and the expression of Nogo-A protein in the spinal dorsal horn was up-regulated at 14 and 21 days after ligation ( P ＜0.05) .Conclusion The Nogo-A protein in the DRG and spinal dorsal horn may play an important role in peripheral nerve injury-induced NP in rats.     

脊髓小胶质细胞抑制对神经病理性痛大鼠脊髓背角GABAB受体表达的影响

目的 通过观察鞘内注射特异性小胶质细胞抑制剂米诺环素对神经病理性痛大鼠脊髓背角GABAB受体表达的影响,探讨脊髓小胶质细胞活化介导神经病理性痛发生的作用机制.方法 雄性SD大鼠48只,体重220～260 g,结扎L5神经根制备神经病理性痛模型,随机分为4组(n=12):Ⅰ组仅暴露L5神经根但不结扎,鞘内注射生理盐水10 μl;Ⅱ组暴露并结扎L5神经根,鞘内注射生理盐水10 μl;Ⅲ组仅暴露L5神经根但不结扎,鞘内注射米诺环素50 μg(10μl);Ⅳ组暴露并结扎L5神经根,鞘内注射米诺环素50 μg(10 μl).于术前1 d～术后18 d,每日鞘内注射生理盐水或米诺环素,2次/d.于术前1 d(基础状态)、术后1、2、4、6、8、10、12、14、16、18 d各组取6只大鼠测定机械痛阈,并确定机械痛周最低点,然后在机械痛阈最低点时各组另取6只大鼠测定脊髓背角GABABR2的表达.结果 与Ⅰ组相比,Ⅱ组机械痛阈降低,术侧脊髓背角GABABR2表达下调(P＜0.05或0.01);与Ⅱ组和Ⅲ组比较,Ⅳ组机械痛阈升高,术侧脊髓背角GABABR2表达上调(P＜0.05或0.01).结论 脊髓小胶质细胞活化介导神经病理性痛发生的作用机制可能与抑制GABAB受体的激活有关.     

鞘内注射GDNF对神经病理性痛大鼠脊髓背角p38MAPK蛋白表达的影响

目的 评价鞘内注射胶质细胞源性神经营养因子(GDNF)对神经病理性痛大鼠脊髓背角p38丝裂原活化蛋白激酶(p38MAPK)蛋白表达的影响.方法 取鞘内置管成功的健康雄性SD大鼠120只,周龄6周,体重180～200 g,随机分为4组(n=30):对照组(C组)、假手术组(S组)、神经病理性痛组(P组)和GDNF组.采用结扎L5.6脊神经的方法建立大鼠神经病理性痛模型.C组不给予任何处理;S组只暴露脊神经,但不结扎;P组脊神经结扎后鞘内注射生理盐水10μl,隔日1次,连续14 d;GDNF组脊神经结扎后鞘内注射GDNF 2μg,用生理盐水稀释至10μl,隔日1次,连续14 d.分别于脊神经结扎后3、7和14 d时,取10只大鼠,测定机械痛阈,然后处死,取脊髓背角,分别采用免疫组化法和蛋白质印迹法测定p38MAPK蛋白的表达水平.结果 与S组比较,P组和GDNF组机械痛阈降低,脊髓背角p38MAPK蛋白表达上调(P＜0.05或0.01);与P组比较,GDNF组机械痛阈升高,脊髓背角p38MAPK蛋白表达下调(P＜0.05或0.01).结论 鞘内注射GDNF可通过抑制脊髓背角p38MAPK蛋白的表达减轻大鼠神经病理性痛.     

姜黄素对糖尿病神经病理性痛大鼠脊髓和背根神经节神经细胞凋亡的影响

目的 探讨姜黄素对糖尿病神经病理性痛(DNP)大鼠脊髓和背根神经节(DRG)神经细胞凋亡的影响.方法雄性SD大鼠108只,体重200～230 g,采用腹腔注射链唑霉素70 mg/kg的方法建立大鼠DNP模型.采用随机数字表法,将大鼠随机分为4组(n=27),正常对照组(C组):不制备DNP模型;DNP组;溶剂对照组(SC组)和姜黄素组(Cur组):于腹腔注射链唑霉素后14 d分别腹腔注射玉米油或姜黄素100 mg/kg(25 mg/ml),1次/d,连续2周.于链唑霉素给药前2 d、给药后14 d、姜黄素给药后3、7、14 d时测定机械缩足阈值(MWT)和热缩足潜伏期(TWL);于姜黄素给药后3、7、14 d时分别采用免疫组化法和Western blot法测定脊髓和DRG caspase-3和Bcl-2的表达水平,并测定神经细胞的凋亡率.结果 与C组比较,DNP组、SC组和Cur组MWT降低,TWL缩短,脊髓和DRG神经细胞凋亡率升高,caspase-3表达上调,Bcl-2表达下调(P＜0.05);与DNP组比较,Cur组MWT升高,TWL延长,脊髓和DBG神经细胞凋亡率降低,caspase-3表达下调,Bcl-2表达上调(P＜0.05),SC组差异无统计学意义(P＞0.05).结论 姜黄素可通过抑制脊髓和DRG神经细胞凋亡,从而减轻大鼠DNP,其机制与抑制caspase-3水平、增强Bcl-2水平有关.

Abstract：

Objective To investigate the effect of curcumin on the apoptosis in spinal cord and dorsal root ganglion neurons in a rat model of diabetic neuropathic pain (DNP) . Methods One hundred and eight male SD rats weighing 200-230 g were randomly divided into 4 groups ( n = 27 each): control group (group C), DNP group, solvent control group (group SC) and curcumin group (group Cur) . Diabetes was induced with intraperitoneal streptozocin 70 mg/kg. Successful induction of diabetes was defined as blood glucose ＞ 16.7 mmol/L. Curcumin and com oil 100 mg/kg (23 mg/ml) were given intraperitoneally once a day for 14 consecutive days starting from 14 days after administration of streptozocin in Cur and SC groups respectively. Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured 2 d before and 14 d after streptozocin injection and 3, 7 and 14 d after curcumin injection. The pain threshold measured at 14 d after administration of streptozocin decreased by more than 15% of the baseline in all the rats. The expression of caspase-3 and Bcl-2 in spinal cord and dorsal root ganglion was determined at 3, 7 and 14 d after curcumin injection by immuno-histochemistry and Western blot, and the neuronal apoptosis rate was determined by TUNEL. Results Compared with group C, MWT and Bcl-2 expression were significantly decreased, TWL was significantly shortened, the neurona lapoptosis rate and caspase-3 expression were significantly increased in DNP, SC and Cur groups ( P ＜ 0.05).Compared with group DNP, MWT and Bcl-2 expression were significantly increased, TWL was significantly prolonged, the neuronal apoptosis rate and caspase-3 expression were significantly decreased in Cur group ( P ＜0.05) . There was no significant difference in the parameters mentioned above between DNP and SC groups ( P ＞0.05). Conclusion Curcumin can attenuate DNP by inhibiting the apoptosis in spinal dorsal hom and dorsal root ganglion neurons in rats, and the inhibition of caspase-3 expression and increase in Bcl-2 expression are involved in the mechanism.     

鞘内注射氯胺酮对神经病理性痛大鼠吗啡耐受时脊髓背角突触重塑的影响

目的 探讨鞘内注射氯胺酮对神经病理性痛大鼠吗啡耐受时脊髓背角突触重塑的影响.方法 鞘内置管成功的雄性SD大鼠48只,体重200～250 g,采用随机数字表法,将大鼠随机分为6组(n=8),置管后5 d,神经病理性痛组(NP组)、生理盐水对照组(NS组)、吗啡组(M组)、氯胺酮组(K组)和吗啡+氯胺酮组(MK组)采用背根神经节慢性压迫法制备神经病理性痛模型,假手术组(S组)仅暴露L5椎间孔.背根神经节慢性压迫后1 d NS组鞘内注射0.9%生理盐水20止,M组和K组分别给予吗啡20μg或氯胺酮50μg,MK组给予吗啡20μg+氯胺酮50μg,1次/d,连续14 d.分别于给药前(基础状态)、给药1、3、5、7、9、11、14 d时测定机械缩足阈值(PWT)和热缩足潜伏期(PWL).最后1d给药后立即处死大鼠,取脊髓组织,其中4只采用免疫组织化学方法测定脊髓背角突触数目,另外4只用于测定脊髓背角突触后膜致密物厚度.结果 与S组比较,其余5组PWT降低,PWL缩短,NP组、NS组、M组和K组突触数目和突触后膜致密物厚度增加(P＜0.05);与NP组比较,M组、K组和MK组PWT升高,PWL延长,突触数目和突触后膜致密物厚度降低(P＜0.05);与M组比较,MK组PWT升高,PWL延长,突触数目和突触后膜致密物厚度降低(P＜0.05).结论 鞘内注射氯胺酮可抑制神经病理性痛大鼠吗啡耐受时脊髓背角突触重塑.

Abstract：

Objective To investigate the effects of intrathecal (IT) ketamine on the synapsis remodeling in the spinal dorsal horn during devolopment of morphine tolerance in a rat model of neuropathic pain (NP). Methods Male SD rats weighing 200-250 g were used in this study. IT catheter was placed in the subarachnoid space according to Yaksh. Forty-eight SD rats in which IT catheter was successfully placed were randomly divided into 6groups (n=8 each): group sham operation (group S); group NP; group normal saline 20 μl IT(group NS);group morphine 20 μg IT (group M); group ketamine 50 μg IT (group K) and group morphine 20 μ g + ketamine 50 μg IT (group MK). NP was induced by compression of right L4,5 dorsal root ganglions with steel wire inserted through L4,5 intervertebral foramen in NP,M,K and MK groups. Normal saline or morphine and/or ketamine were injected IT once a day for consecutive 14 days. Paw withdrawal threshold (PWT) to mechanical stimulation and paw withdrawal latency (PWL) to a thermal nociceptive stimulus were measured on 0, 1, 3, 5, 7, 9, 11, 14 days during the consecutive 14 days of administration. The animals were sacrificed after the final IT administration. The lumbar segment of the spinal cord was removed for determination of the number of synapsis in the spinal dorsal horn by immuno-histochemistry in 4 animals in each group and observation of synaptic structure remodeling using electron microscope in another 4 animals in each group. Results Compared with group S, PWT was significantly decreased and PWL was shortened in the other 5 groups, and the number of synapsis was significantly increased and the synaptic structure was thickened in NP, NS, M and K groups (P ＜ 0.05). Compared with group NP,PWT was significantly increased and PWL was prolonged in M, K and MK groups, and the number of synapsis was significantly decreased and the thickness of synaptic structure was significantly reduced in group MK ( P ＜ 0.05).Compared with group M, PWT was significantly increased, PWL was prolonged, the number of synapsis was significantly decreased and the thickness of synaptic structure was significantly reduced in group MK ( P ＜ 0.05). Conclusion IT ketamine can inhibit the synaptic remodeling in the spinal dorsal horn during development of morphine tolerance in a rat model of NP.