慢性痛大鼠模型前扣带皮层内小胶质细胞活化与细胞因子表达

目的:观察慢性炎症性疼痛大鼠前扣带皮层(ACC)中小胶质细胞标记物(CD14,IBA1)及促炎症细胞因子(IL-1β,TNF-α)的表达变化。方法:单侧足底注射完全弗氏佐剂(CFA)诱导大鼠慢性炎症性疼痛,用RT-PCR检测ACC中CD14、IL-1β、TNF-αmRNA的表达,用免疫组织化学染色观察ACC中IBA1免疫阳性小胶质细胞的变化。结果:足底注射CFA后4 h、3 d双侧ACC中CD14 mRNA表达较生理盐水(NS)组有显著增加(P<0.001);CFA后4 h、3 d、14 d双侧ACC中IL-1βmRNA表达较NS组有显著增加(P<0.001);CFA后3 d、14 d双侧ACC中TNF-αmRNA表达较NS组有显著增加(P<0.001)。CFA 3 d组双侧ACC中IBA1免疫阳性小胶质细胞胞体变大、突起变粗。结论:ACC中小胶质细胞的活化以及IL-1β和TNF-α的表达增加可能在慢性炎症性疼痛的产生和维持中发挥作用。     

炎性疼痛致大鼠海马CREB结合蛋白的表达变化

目的　探讨完全弗氏佐剂（Complete Freund’s Adjuvant，CFA）致大鼠炎性疼痛后海马区CREB结合蛋白（CREB Binding Protein，CBP)的表达变化及意义。 方法　大鼠随机分为正常组和实验组，实验组大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100 μl，建立炎性疼痛模型，分为注射CFA后6 h、1 d、3 d、7 d和14 d组。采用Von Frey纤维观察不同时间点大鼠机械缩足阈值（Paw withdrawal
threshold, PWT）的变化；采用免疫组化法检测海马CBP的表达变化。 结果　足底注射CFA后1 h PWT即下降，并在6 h后达到最低值，3 d后逐渐上调，至14 d仍低于基础值。正常组大鼠海马各区均可见CBP大量表达。实验组大鼠两侧海马各区尤其是CA1区和齿状回（Dentate Gyrus，DG）区域CBP的表达随时间点变化呈现出先降低后升高再降低的双相表达趋势，即注射CFA后6 h、1 d，CBP表达下降，至注射CFA后3 d，CBP表达明显上调；7 d时CBP表达再次出现下调，持续至14 d。 结论　 CFA能诱导大鼠产生为期2周以上的炎性痛病程；炎性疼痛时海马区CBP的表达呈现出双相性表达趋势，提示海马区的基因表达变化在炎性疼痛的产生和持续过程中起着重要的作用。     

前扣带回TRPC1/4/5通道在小鼠慢性炎性痛所致焦虑样行为中的作用

目的:探讨前扣带回(ACC)中瞬时感受器电位通道1/4/5(TRPC1/4/5)在小鼠慢性炎性痛诱发焦虑样行为过程中的表达变化。方法:将雄性C57/BL6小鼠(n=30)随机分为3组:正常对照组(Control组)、CFA1 d组和CFA 7 d组,小鼠左后肢足底皮下注射完全弗氏佐剂(CFA)建立慢性炎性痛模型。采用小鼠机械痛和热痛敏方法检测痛阈变化,旷场和高架十字迷宫法检测小鼠焦虑样行为,real time RT-PCR和Western Blot方法检测TRPC1/4/5通道表达水平变化。结果:小鼠左后肢足底注射CFA后出现明显的机械性痛敏和热痛敏现象。旷场和高架十字迷宫实验显示CFA诱发炎性痛敏7 d后,小鼠在高架十字迷宫开臂进入次数和时间、旷场中央区活动距离均明显降低,提示慢性炎性痛后小鼠出现显著的焦虑样行为。采用real time RT-PCR和Western Blot实验,观察到炎性痛诱发焦虑样小鼠ACC脑区中TRPC1/4/5通道的mRNA和蛋白表达水平较正常组均出现显著上调。结论:CFA诱发的慢性炎性痛模型中,小鼠出现明显的焦虑样症状,同时ACC脑区的TRPC1/4/5通道表达水平显著升高,提示慢性炎性痛诱发的焦虑样行为可能与痛觉神经通路中ACC脑区的TRPC1/4/5通道蛋白表达有密切关系。     

炎性痛致大鼠脊髓后角ProBDNF及其受体的表达变化

目的:探讨完全弗氏佐剂(complete Freund’s Adjuvant,CFA)致炎性疼痛后大鼠脊髓后角内ProBDNF及受体P75NTR和Sortilin的表达变化及意义。方法:大鼠随机分为正常组和实验组,实验组大鼠左侧足底皮下注射CFA和生理盐水混合溶剂100μl,建立炎性疼痛模型,实验组包括注射CFA后6 h、1、3、7和14 d组。采用Von Frey纤维测定不同时间点大鼠机械缩足阈值(pawwithdrawal threshold,PWT)的变化;采用免疫组织化学方法检测ProBDNF及P75NTR、Sortilin在脊髓后角的表达变化。结果:足底注射CFA后1 h PWT即下降,并在6 h后达到最低值,3 d后逐渐上调,至14 d仍低于基础值。ProBDNF在正常脊髓后角可见阳性表达;注射CFA后1 d注射侧脊髓后角较对侧其ProBDNF的表达明显上调,上调持续到7 d左右,至14 d逐渐恢复。正常大鼠脊髓后角P75NTR有较弱表达,主要集中在后角I、II层纤维;注射CFA后6 h开始,注射侧脊髓后角内P75NTR的表达明显上调,III-V层的阳性产物也逐渐增加,这种上调持续到7 d左右达高峰,至14 d逐渐下降。Sortilin在正常脊髓后角浅层仅有较弱阳性产物表达,注射CFA后不同时间点脊髓后角Sortilin的表达无明显差异。结论:CFA能诱导大鼠产生为期2周以上的炎性痛病程;脊髓后角ProBDNF和P75NTR的表达上调可能与炎性疼痛中外周痛觉信号的传导和中枢敏化有关。     

外周注射福尔马林诱导前扣带皮层中胶质细胞激活和细胞因子表达增高

为了观察外周注射福尔马林对大鼠前扣带皮层(ACC)中星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)和S100B,小胶质细胞标记物CD14以及与胶质细胞密切相关的炎症前细胞因子白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)表达的影响,本研究结合行为学检测和RT-PCR方法,观察了大鼠单侧足底皮下注射福尔马林后的行为学变化,并检测了在不同时间点ACC中GFAP、S100B、CD14、IL-1β和TNF-α在基因水平的表达变化。结果显示:福尔马林急性疼痛刺激的大鼠出现典型的双时相自发性疼痛反应。ACC中GFAP和S100B mRNAs表达水平在刺激后30min、1h增高,2h恢复正常;CD14 mRNA表达水平在15min开始增高,1h达到高峰,6h恢复正常;IL-1β和TNF-αmRNAs表达水平在刺激后30min、1h、2h增高,6h恢复正常。上述结果表明,福尔马林外周急性伤害性刺激能够诱导ACC内短暂性的星形胶质细胞、小胶质细胞激活及IL-1β、TNF-α表达增高,提示激活的胶质细胞及表达上调的炎症前细胞因子可能参与伤害性信息的调制。     

慢性炎性痛诱致小鼠焦虑样行为和前扣带回TRPC3和TRPC6通道的表达上调

目的:探讨慢性炎性痛诱致小鼠焦虑样行为情况下前扣带回(anterior cingulate cortex,ACC)TRPC 3/6通道的表达改变。方法:采用小鼠痛行为检测方法、旷场行为检测方法和免疫印迹实验(Western Blot)。结果:小鼠后肢足底皮下注射完全弗氏佐剂(complete freund’s adjuvant,CFA)建立慢性炎性痛模型。痛行为检测结果显示足底皮下注射CFA可以诱致小鼠产生长时程的机械性和热痛觉过敏现象。旷场实验可见CFA致炎致痛后的小鼠较正常小鼠在旷场中央区活动距离明显降低,停留时间明显减少,提示慢性炎性痛小鼠表现为显著的焦虑样行为。进一步采用Western Blot实验发现炎性痛焦虑样小鼠前扣带回脑区的TRPC3和TRPC6通道蛋白表达水平较对照组动物显著上调。结论:CFA引发的慢性炎性痛模型中,小鼠产生焦虑样情绪,TRPC3和TRPC6通道蛋白在ACC部位的蛋白表达水平明显升高。提示这些焦虑样痛情绪的发生与疼痛痛觉通路中ACC脑区的TRPC3及TRPC6表达密切相关。     

雷公藤内酯醇(T10)通过抑制脊髓内胶质细胞的激活改善大鼠CFA诱导的炎性痛的机制研究

目的:观察腹腔注射雷公藤内酯醇(Triptolide,T10)对完全弗氏佐剂(complete Freund’s adjuvant,CFA)诱导的大鼠慢性炎性痛行为的改善作用并探讨其机制。方法:SD大鼠随机分为四组,即Saline+Veh组、Saline+T10组、CFA+Veh组和CFA+T10组。后两组大鼠于右侧足底注射CFA制备大鼠慢性炎性痛模型,前两组则在相同部位注射生理盐水。第二组和第四组大鼠分别于造模前1 h给予T10腹腔注射,随后每12 h给予腹腔注射药物1次,持续用药7 d;第一组和第三组则以相同方式给予100 ml/kg的生理盐水作为对照。采用辐射热法和机械刺激法连续观察给药后大鼠的痛行为;应用免疫组织化学染色和Western Blot方法观察连续给予T10 3d和7 d后大鼠腰膨大平面脊髓背角内小胶质细胞和星形胶质细胞的活化程度;应用实时定量PCR(RT-PCR)方法观察连续给药7 d后大鼠腰5背根神经节内炎性因子,如白细胞介素-6(interleukin-6,IL-6)、白细胞介素-1β(interleukin-1 beta,IL-1β)和肿瘤坏死因子-α(tumour necrosis factor-α,TNF-α)的表达变化。结果:(1)与CFA+Veh组相比,CFA+T10组大鼠机械缩足阈值及辐射热痛潜伏期显著下降(P0.05),并持续到造模成功后7 d,表明腹腔注射T10可以明显改善CFA诱导的大鼠机械痛敏和辐射热痛敏。(2)免疫组织化学染色和Western Blot结果显示:造模后3 d和7 d后CFA+Veh组大鼠脊髓背角出现大量CFA诱导活化的小胶质细胞和星形胶质细胞;小胶质细胞标记物IBa-1和星形胶质细胞标记物GFAP的表达量分别在CFA造模后3 d和7 d显著增高(P0.05),而给予T10 3 d和7 d后炎性痛大鼠腰膨大平面IBa-1和GFAP的活化程度均显著减轻(P0.05)。(3)RT-PCR结果显示:与Saline+Veh组相比,CFA造模7 d后腰5背根神经节内炎性因子IL-1β、IL-6 mRNA和TNF-αmRNA的表达显著增高(P0.05),腹腔注射T10 7 d后上述炎性因子的表达则显著降低(P0.05)。结论:腹腔注射T10可以显著改善CFA诱导的大鼠慢性炎性痛,其机制可能与抑制脊髓背角小胶质细胞和星形胶质细胞的活化以及炎性因子IL-1β、IL-6和TNF-α的合成有关。     

大鼠背根神经节中的Nogo-A蛋白促进微管聚合和炎症热痛觉敏化的发生

目的 探讨背根神经节（DRG）神经元中的Nogo-A在炎症性热痛觉敏化过程中对微管的调控作用及其下游分子通路。 方法 对野生型大鼠(n=12)和Nogo-A敲除大鼠(n=14)足底注射完全弗式佐剂（CFA）制造炎症痛大鼠模型，取两组大鼠DRG组织，采用Western blotting、免疫荧光染色等方法，研究DRG中Nogo-A在炎症热痛觉敏化过程中对于微管的调节作用。 结果 Nogo-A基因敲除具有缓解CFA诱导的炎症性热痛觉敏化作用，Nogo-A基因敲除大鼠DRG中的微管聚合减少，微管上游信号分子磷酸化脑衰反应调节蛋白2（p-CRMP2）表达增加。 结论 Nogo-A通过增强CRMP2促进微管聚合，促进了炎症热痛觉敏化的发生。     

右美托咪定上调海马脑源性神经营养因子-酪氨酸激酶受体B的表达及改善完全费氏佐剂所致小鼠焦虑样和抑郁样行为

目的 探讨α2-肾上腺素受体激动剂右美托咪定(DEX)对完全弗氏佐剂(CFA)诱导疼痛模型小鼠焦虑样和抑郁样行为的影响及其可能的机制。方法 36只ICR雌鼠随机分成生理盐水(NS)组、CFA组和DEX+CFA组，每组n=12。向小鼠右后肢足底皮下注射10μl CFA建立慢性炎性疼痛模型，DEX+CFA组小鼠在痛阈检测前30 min腹腔注射0.025 mg/kg DEX,1次/1 d,持续7 d。实验采用von-frey纤维丝评价3组小鼠机械性痛阈值；采用旷场实验检测小鼠焦虑样行为；采用糖水偏好、悬尾实验和强迫游泳检测3组小鼠抑郁样行为；采用Western blotting检测3组小鼠海马肾上腺素能受体β₂(ADRB2)、脑源性神经营养因子(BDNF)、酪氨酸激酶B受体(TrkB)及突触相关蛋白谷氨酸受体1(GluR1)和GluR2的表达，每组n=8;采用免疫组织化学染色方法检测各组小鼠海马新生神经元标记物双肾上腺皮质激素(DCX)的表达情况，每组n=4。结果 痛阈检测结果显示，与NS组相比，CFA注射后的第1天、3天、7天小鼠机械痛阈值均显著降低(P<0.0...     

小檗碱激活AMPK抑制3T3-L1脂肪细胞分化

目的 探讨小檗碱对3T3-L1脂肪分化的作用是否与激活腺苷酸活化蛋白激酶（AMPK）有关.方法 在3T3-L脂肪细胞分化全程加入小檗碱,以油红O染色检测3T3-L1脂肪细胞胞浆中脂肪的堆积,实时定量PCR检测过氧化物酶体增殖物激活受体γ2（PPARγ2）、CCAAT增强子结合蛋白α（CEBPα）和AMPK的mRNA表达,以Western印迹法检测AMPK和乙酰辅酶A羧化酶（ACC）的磷酸化水平.结果 小檗碱剂量依赖性地抑制3T3-L1脂肪细胞分化,10 μmol/L小檗碱几乎完全抑制胞浆中脂肪的堆积.5 μmol/L小檗碱在脂肪细胞诱导分化1、3、5、7d后均显著降低CEBPα mRNA表达（P〈0.05或P〈0.01）,诱导分化3、5、7d时显著降低PPARγ2的mRNA表达（P〈0.05或P〈0.01）.AMPK的mRNA水平在分化过程中未受小檗碱的明显影响,而小檗碱明显增加其蛋白磷酸化水平,其下游靶基因ACC磷酸化水平也明显增加.结论 小檗碱抑制3T3-L1脂肪细胞的分化可能与其激活AMPK有关.     

Analgesic effect of electroacupuncture on complete Freund's adjuvant-induced inflammatory pain in mice: a model of antipain treatment by acupuncture in mice

Electroacupuncture (EA) was applied bilaterally to the acupoints of Zu-san-li (ST-36) and Kun-lun (BL-60) in the hindlimbs of mice. The therapeutic effect of EA on inflammatory pain induced by an ipsilateral injection of complete Freund's adjuvant (CFA) into the right paw of the mouse was investigated in this study. The time of paw-withdrawal latency (PWL) was used as an indicator for judging the intensity of the pain induced by the CFA injection. The EA effects were divided into immediate (PWL tests within 2 h after EA treatment) and cumulative (PWL tests during and after repetitive EA treatments for 3 weeks) effects. As immediate effects, PWL was significantly shortened in the CFA-injected paw, but was again prolonged 20 min after an EA treatment and lasted until 30 min after. As cumulative effects, PWL was significantly shortened in the CFA-injected paw, but recovered from the 2nd to the 8th day during repetitive EA treatments. No such effects could be observed after sham EA treatment, which resulted in behavior similar to that in untreated animals. These results demonstrate that the CFA-induced inflammatory pain in mice is an ideal model system for the investigation of EA effects and may serve as a valuable reference for the clinical treatment of inflammatory pain in human beings. Furthermore, the mouse pain model opens the possibility to apply the investigation also to transgenic mice.     

Up-regulation of substance P and NMDA receptor mRNA in dorsal horn and preganglionic sympathetic neurons during adjuvant-induced noxious stimulation in rats.

Substance P (SP) and glutamate-containing terminals are found in the dorsal horn and preganglionic sympathetic neurons (PSNs) in the intermedio-lateral nucleus of the spinal cord. SP receptor (SPR) and N-methyl-D-aspartate type glutamate receptor (NMDAR) were also recognized in portions of the dorsal horn and PSNs. Primary sensory nerve fibers containing SP and glutamate terminated around PSNs, or partly on PSNs directly as well as on dorsal horn neurons (DHNs). The present study was performed to investigate the changes in SPR and NMDAR mRNA expressions during nociception in rats. Upon the injection of complete Freund's adjuvant (CFA) into the front paw, edema and hyperalgesia occurred immediately, with the difference in latency score between injected and non-injected paws continuing to day 10. The up-regulation of SPR and NMDAR mRNAs in DHNs and PSNs was recognized using in situ hybridization and northern blot techniques. CFA injection increased SPR mRNA expression in PSNs at days 1 and 4, and NMDAR mRNA expression at days 1, 4 and 7. At day 14, the mRNA expression of both receptors decreased to the control level. These changes in the amount of receptor mRNAs in DHNs and PSNs may cause hyperalgesia and sympathetically mediated pain.     

Transcutaneous Electrical Nerve Stimulation on Yongquan Acupoint Reduces CFA‐Induced Thermal Hyperalgesia of Rats via Down‐Regulation of ERK2 Phosphorylation and c‐Fos Expression

Activation of extracellular signal‐regulated kinase‐1/2 (ERK1/2) and its involvement in regulating gene expression in spinal dorsal horn, cortical and subcortical neurons by peripheral noxious stimulation contribute to pain hypersensitivity. Transcutaneous electrical nerve stimulation (TENS) is a treatment used in physiotherapy practice to promote analgesia in acute and chronic inflammatory conditions. In this study, a total number of 114 rats were used for three experiments. Effects of complete Freund's adjuvant (CFA)‐induced inflammatory pain hypersensitivity and TENS analgesia on ERK1/2 phosphorylation and c‐Fos protein expression were examined by using behavioral test, Western blot, and immunostaining methods. We found that CFA injection caused an area of localized swelling, erythema, hypersensitivity to thermal stimuli, the decreased response time of hind paw licking (HPL), as well as upregulation of c‐Fos protein expression and ERK2 phosphorylation in the ipsilateral spinal dorsal horn and the contralateral primary somatosensory area of cortex and the amygdala of rats. TENS on Yongquan acupoint for 20 min produced obvious analgesic effects as demonstrated with increased HPL to thermal stimuli of CFA‐treated rats. In addition, TENS application suppressed the CFA‐induced ERK2 activation and c‐Fos protein expression. These results suggest that down‐regulation of ERK2 phosphorylation and c‐Fos expression were involved in TENS inhibition on CFA‐induced thermal hyperalgesia of rats. Anat Rec 293:1207–1213, 2010. © 2010 Wiley‐Liss, Inc.     

Distinct roles of the anterior cingulate cortex in spinal and supraspinal bee venom-induced pain behaviors

A wide variety of human and animal experiments suggest that the anterior cingulate cortex (ACC) is one of the key brain substrates subserving higher order processing of noxious information. However, no sufficient data are now available regarding the mediation by ACC of different levels of pain processing as well as its potential descending modulation of spinal nociception. Using the well-developed rat bee venom (BV) model, the present study evaluated the effect of lesions of bilateral ACC on two levels of spontaneous nociceptive behaviors (spinally-processed persistent paw flinching reflex and supraspinally-processed paw lifting/licking) and heat or mechanical hypersensitivity under the inflammatory pain state. In contrast to the sham lesion group (saline microinjection into the ACC), bilateral complete ACC chemical lesions (kainic acid microinjection into the ACC) significantly decreased the BV-induced paw lifting and licking behavior (less time spent by the animal in paw lifting/licking) but produced no influence upon spinally-processed spontaneous paw flinching reflex (no change in number of paw flinches following subcutaneous BV injection). Moreover, the bilateral ACC lesions relieved the BV-evoked primary thermal or mechanical hypersensitivity compared with the sham control group. However, incomplete lesions of bilateral ACC failed to affect the abovementioned pain-related behaviors. No effects were seen on basal pain sensitivity in either group of rats. Motor coordination, as measured by Rota-Rod treadmill test, was not impaired by bilateral ACC lesions. These results implicate that the ACC area of the brain plays differential roles in the mediation of different levels of spontaneous pain-related behaviors. The present study also provides additional evidence for the ACC-mediated descending facilitation of primary hyperalgesia (pain hypersensitivity) identified in the injured area under inflammatory pain state.     

Characteristic features of actinomycin D-induced paw inflammation of the rat.

Features of paw edema induced by subplantar injection of actinomycin D (act D) were investigated in rats. The paw edema was produced as early as the 1st day and reached a maximal level on the 3rd or the 4th day. Thereafter, it began to subside progressively and was considerably reduced by the 16th day following act D (20 μgm) injection. A direct dose response relationship between the amount of act D injected and the intensity of the paw edema was obtained. No difference in β-glucuronidase and acid phosphatase activity was found between saline and act D-injected paws on the 2nd day. This was followed by an increase in the activity of both enzymes on the 4th, 8th, and 16th days after injection. The histamine content of the saline and act D-injected paws remained unchanged during the early phase of inflammation. A marked increase in the histamine content was noted during the late phase in the drug-injected paw. The effects of act D treatment on capillary permeability to Evans blue dye (EBD) and the edema formation of the paw revealed that a maximal increase in vascular permeability to EBD occurred on the 1st day and was maintained until the 8th day. In contrast to permeability, the paw edema on the 1st day was minimal and increased progressively until the 3rd or 4th day. Thereafter, both the permeability and the paw edema began to diminish and were considerably reduced on the 16th day. Aspirin and hydrocortisone treatment were ineffective in suppressing the act D-induced paw inflammation. Indomethacin produced a somewhat dose-related anti-inflammatory effect against the inflammation caused by this drug.