医用臭氧通过下调神经病理性痛大鼠核因子κB/核因子κB抑制蛋白α/核因子κB抑制蛋白激酶β的表达发挥镇痛效应

目的观察医用臭氧(OZ)对坐骨神经慢性缩窄性损伤(CCI)致神经病理性痛大鼠的镇痛作用及对核因子κB(NF-κB)、核因子κB抑制蛋白α(IκBα)及核因子κB抑制蛋白激酶β(IKKβ)表达水平的影响。方法采用CCI法复制大鼠神经病理性痛动物模型,同时给予不同剂量(0.8、0.4、0.2ml)的OZ予以干预,用Von Frey纤维丝机械刺激触痛仪及冷板测痛仪测定不同剂量OZ对CCI大鼠的机械缩足反射阈值与冷缩足反射阈值的影响;用RT-PCR法和Western blotting法检测不同剂量OZ对CCI大鼠脊髓组织NF-κB p65、IκBα及IKKβmRNA和蛋白表达水平的影响。结果与CCI神经病理性痛模型组比较,OZ 0.8、0.4ml剂量升高CCI大鼠机械缩足反射阈值,降低冷缩足反射阈值(P0.05,P0.01);OZ 0.8、0.4ml剂量可下调CCI大鼠脊髓组织NF-κB p65、IκBα及IKKβmRNA和蛋白表达水平(P0.05,P0.01)。结论 OZ对CCI致神经病理性痛大鼠有镇痛作用,其机制可能与下调NF-κB p65、IκBα及IKKβ的表达有关。     

慢性坐骨神经结扎神经痛大鼠脊髓背角和背根神经节p-JNK表达及意义

目的： 观察磷酸化应激激活蛋白激酶(p-JNK)在神经病理性疼痛大鼠脊髓背角和背根神经节表达的变化,探讨慢性神经痛的发生机制。方法: 采用坐骨神经慢性结扎损伤（CCI）模型,18只SD大鼠随机分为3组（n=6）。对照组（Con组）：不接受任何手术操作;假手术组（sham组）：只分离坐骨神经;CCI组：结扎坐骨神经。于术前2 d和术后1、3、5、7、10、14 d 测定机械痛阈（MWT）和热痛阈（TWL）,术后3、7、14 d取大鼠术侧L4、L5脊髓背角和背根节, 免疫组化法分析脊髓背角和背根节p-JNK动态变化。结果: CCI组较Con组术后各时点TWL和MWL明显降低（P<0.05）。CCI组较Con组各时点脊髓背角和背根神经节p-JNK表达明显增加（P<0.01）。结论: 初级感觉神经元中p-JNK表达可能是神经损伤的因素之一。     

内质网应激特有的caspase-12在神经病理性疼痛大鼠脊髓背角的表达

目的:检测内质网应激特有caspase-12凋亡途径与神经病理性疼痛模型大鼠脊髓背角神经元凋亡之间的关系。方法:SD雄性大鼠随机分为假手术组(sham)和坐骨神经慢性压迫(CCI)模型组。检测大鼠在手术前及术后21 d的热痛敏和机械痛敏;CCI术后21 d,免疫组织化学检测大鼠脊髓背角内葡萄糖调节蛋白78(GRP78)、caspase-12和caspase-3的表达;TUNEL法检测脊髓背角内细胞的凋亡。结果:与假手术组相比,模型组的热痛敏和机械痛敏在术后均明显下降,脊髓背角内GRP78、caspase-12和caspase-3的表达均增高,凋亡染色阳性细胞数目也较多。结论:坐骨神经慢性压迫模型大鼠脊髓背角内神经元凋亡的途径之一是内质网应激特有caspase-12介导的,可能与神经病理性疼痛的发生、发展有关。     

鞘内注射BN50730抑制SNI大鼠痛敏和脊髓TNF-α的表达

目的： 观察鞘内注射血小板活化因子受体拮抗剂BN50730对坐骨神经分支选择损伤（SNI）神经病理痛大鼠痛阈及脊髓肿瘤坏死因子α（TNF-α）表达的影响,探讨脊髓血小板活化因子(PAF)及其受体参与痛觉信号调节的可能机制。方法: 鞘内置管的Sprague-Dawley大鼠24只随机等分为4组：假手术组,SNI组,SNI+DMSO对照组和SNI+BN50730组,建立SNI疼痛模型,手术后1、3、5、7、10和14 d鞘内给药并测痛阈,第14 d取腰段脊髓免疫组化法和ELISA法检测脊髓TNF-α的表达。结果: SNI神经损伤大鼠机械缩爪阈值明显降低(P<0.05),同侧脊髓背角TNF-α表达增强,ELISA检测脊髓TNF-α含量升高(P<0.05);鞘内应用BN50730降低脊髓TNF-α的表达,同时伴有大鼠痛行为的改善。各组大鼠辐射热缩爪潜伏期无明显差异。结论: BN50730对SNI神经病理性疼痛有治疗作用,TNF-α的表达下调可能与其镇痛机制有关。     

移植微囊化嗅鞘细胞对神经病理性疼痛及L4-5段脊髓P2X7受体表达的影响

目的 探讨微囊化嗅鞘细胞移植对坐骨神经损伤引起病理性疼痛及L4-5段脊髓P2X7受体的表达影响。 方法 取1只健康SD大鼠,取其嗅球组织并运用差速贴壁方法,在体外进行嗅鞘细胞培养扩增。取48只健康SD大鼠,随机分成4组,即对照组（control）、慢性压迫性坐骨神经损伤组（CCI）、嗅鞘细胞移植组（OECs）及微囊化嗅鞘细胞移植组（MC-OECs）。术后7 d及14 d,运用行为学方法检测各组大鼠机械缩足反射阈值,运用原位杂交方法检测各组大鼠L4-5段脊髓P2X7受体阳性细胞百分比及平均吸光度表达情况。 结果 术后7 d及14 d,与对照组比较,CCI组大鼠机械缩足反射阈值明显减低,L4-5段脊髓P2X7受体阳性细胞百分比及平均吸光度明显增加（P<0.05）;与CCI组比,OECs组大鼠机械缩足反射阈值明显增高,P2X7受体阳性细胞百分比及平均吸光度明显减低（P<0.05）,与OECs组对比,MC-OECs组大鼠机械缩足反射更高。P2X7受体阳性细胞百分比及平均吸光度更低（P<0.05）。 结论 微囊化嗅鞘细胞移植能更好地缓解神经病理性疼痛,减低L4-5段脊髓内P2X7受体的表达水平,修复坐骨神经损伤。     

鞘内注射M_ERK2-shRNA腺病毒对糖尿病神经痛小鼠的影响

目的 探讨细胞外调节蛋白激酶2 (ERK2）在糖尿病神经病理性疼痛（DNP）小鼠模型中的作用机制。方法40只小鼠随机分4组：对照组,DNP组,siERK2-DNP组（鞘内注射M_ERK2-shRNA腺病毒）,siRNA-DNP组（鞘内注射siRNA腺病毒）。模型制作成功后检测痛阈值;模型制作前、模型制作后2周、 4周及6周检测机械痛阈值;6周后取脊髓组织,利用Western blotting及免疫组织化学法检测脊髓后角ERK2及核因子κB (NFκB)的表达。 结果 与对照组比较,DNP组和siRNA-DNP组痛阈值显著降低, ERK2、NFκB的表达显著上调（P<0.05）,siERK2-DNP组与对照组比较,痛阈值无显著变化, ERK2、NFκB的表达无显著差别（P>0.05）。 结论 下调ERK2可能是治疗糖尿病神经病理性疼痛的脊髓后角神经元损伤的方法之一。     

神经病理性疼痛大鼠脊髓NF-κB、NR2B和iNOS的表达及意义

 目的： 探讨坐骨神经慢性压迫性损伤（CCI）大鼠脊髓核因子κB（NF-κB）、N-甲基D-天冬氨酸受体2B亚基（NR2B）和诱导型一氧化氮合酶（iNOS）的表达及意义。方法: 56只180~220 g雄性SD大鼠随机分为2组：sham组 (n=8)和CCI组（n=48）。于术前1 d、术后1 d、4 d、7 d、14 d和21 d测定机械缩爪阈值（MWT）和热刺激缩爪潜伏期（PWL）后处死,取L4~L6脊髓,采用RT-PCR和Western blotting检测脊髓NF-κB、NR2B和iNOS的表达。结果: CCI组MWT及PWL值较sham组明显降低（P<0.05, n=8）。RT-PCR和Western blotting结果显示CCI组术后脊髓NF-κB、NR2B和iNOS明显高于术前（P<0.05, n=4）。iNOS mRNA与NF-κB mRNA和NR2B mRNA的表达呈显著正相关（r=0.842, P<0.05; r=0.833, P<0.05）。结论: CCI大鼠痛觉过敏的产生和维持可能与脊髓NF-κB和NR2B活化并上调iNOS表达水平有关。     

芒果苷对CCI大鼠神经病理性疼痛的改善作用北大核心CSCD

目的:探究芒果苷对慢性缩窄性损伤(CCI)大鼠神经病理性疼痛的作用及机制。方法:采用成年SpragueDawley大鼠构建CCI模型,以模拟临床神经病理性疼痛。将大鼠随机分为假手术组(Sham组)、CCI组、CCI+Man组[手术后立即用40 mg/(kg·d)芒果苷灌胃,连续治疗17 d],10只/组。术后连续17 d观察缩爪机械阈值(PWMT)和缩爪热潜伏期(PWTL);术后17 d,处死大鼠,ELISA检测脊髓中细胞因子IL-1β、IL-6及趋化因子CCL2、CCR2表达水平;免疫荧光法检测脊髓神经元中Cleaved caspase-3和pJNK蛋白表达水平;免疫组化法和Western blot分析脊髓中c-Fos蛋白表达水平。结果:与Sham组相比,CCI组大鼠术后第1~17天的PWMT和PWTL显著降低,脊髓中促炎因子IL-6、IL-1β,趋化因子CCL2、CCR2及c-Fos蛋白水平明显升高(P<0.001);脊髓神经元细胞中Cleaved caspase-3和pJNK蛋白水平明显升高(P<0.001)。经芒果苷治疗后,CCI组大鼠术后第1~17天的PWMT和PWTL显著升高,脊髓中促炎因子IL-6、IL-1β,趋化因子CCL2、CCR2及c-Fos蛋白水平明显降低(P<0.01);脊髓神经元细胞中Cleaved caspase-3和pJNK蛋白水平明显降低(P<0.01)。结论:芒果苷对CCI大鼠的神经病理性疼痛具有改善作用,其可能与降低脊髓炎症、凋亡、c-Fos及pJNK蛋白表达有关。     

MiR-139-3p通过靶向下调TRPV1缓解大鼠神经病理性疼痛

目的:研究miR-139-3p在大鼠神经病理性疼痛发生发展中的作用及其作用机制。方法:将大鼠随机分为假手术组(sham)、坐骨神经慢性压迫损伤模型组(chronic constriction injury group,CCI)、2 mg/kg miR-139-3p模拟物(mimic)鞘内注射组、4 mg/kg miR-139-3p mimic鞘内注射组。术后的第14 d,Real-time PCR法检测大鼠背根神经节(DRG)中miR-139-3p及TRPV1的表达。于注射前及注射后的第1、3、7、14 d,利用von Frey检测大鼠机械性痛阈及热辐射法检测大鼠热痛阈值。Western Blot法检测DRG中TRPV1的表达;ELISA法检测脊髓L4-L5组织中TNF-α及IL-1β的含量;双荧光素酶报告基因法检测miR-139-3p与TRPV1的靶向关系。结果:(1)与sham组相比较,miR-139-3p在CCI大鼠DRG中的表达显著下降,而TRPV1的表达显著上升(P0.05)。(2)与sham组相比较,CCI组中大鼠机械刺激缩足反射阈值及热刺激缩足反射潜伏期显著下降(P0.05);而与CCI组相比较,鞘内注射miR-139-3p mimic可显著促进大鼠机械刺激缩足反射阈值及热刺激缩足反射潜伏期的上升,抑制TRPV1的表达(P0.05)。(3)与sham组相比较,CCI组大鼠TNF-α及IL-1β含量显著上升(P0.05);而与CCI组相比较,鞘内注射miR-139-3p mimic可显著抑制TNF-α及IL-1β的产生(P0.05)。(4)miR-139-3p mimic转染可显著降低荧光素酶报告基因的荧光强度。结论:miR-139-3p通过靶向下调TRPV1的表达缓解大鼠神经病理性疼痛。     

内质网应激介导的JNK通路在大鼠神经病理性疼痛中的作用

目的:检测内质网应激介导的JNK通路在坐骨神经慢性压迫(CCI)模型大鼠中的作用。方法:SD雄性大鼠随机分为假手术组(sham)和模型组(CCI)。CCI前、后1、3、5、7、14 d测定大鼠的热痛敏和机械痛敏;CCI后14 d,免疫组织化学检测大鼠脊髓背角内葡萄糖调节蛋白78(GRP78)、p-JNK、caspase-3和胶质纤维酸性蛋白(GFAP)的表达;TUNEL法检测脊髓背角内的细胞凋亡。结果:与假手术组相比,模型组的热痛敏和机械痛敏在术后均明显下降,脊髓背角内GRP78、p-JNK、caspase-3和GFAP的表达均增高,凋亡染色阳性细胞数目也较多。结论:内质网应激介导的p-JNK途径参与了神经病理性疼痛模型大鼠脊髓背角内神经元凋亡,可能与星形胶质细胞的活化有关。     

氯胺酮鞘内给药对神经病理性痛大鼠脊髓背角小胶质细胞活化、TNF-α和IL-1β产生的影响

目的:探究鞘内注射氯胺酮(ketamine,KTM)对坐骨神经结扎(CCI)神经病理性疼痛大鼠模型行为、脊髓背角肿瘤坏死因子-α(TNF-α)和白介素1β(IL-1β)以及小胶质细胞标记蛋白OX42表达的影响。方法:(1)建立CCI模型大鼠,鞘内注射KTM或MI(米诺四环素)进行干预,测定大鼠机械缩足阈值(MWT)和热缩足潜伏期(TWL)。(2)运用ELISA法检测TNF-α和IL-1β表达水平。(3)使用Western Blot法检测OX42的表达情况。结果:(1)KTM组和MI组大鼠MWT和TWL值较CCI组显著升高;KTM组大鼠MWT和TWL值高于CCI组(P0.05)。(2)KTM组和MI组TNF-α和IL-1β表达水平低于CCI组(P0.05);其中KTM组TNF-α和IL-1β表达水平显著高于MI组(P0.05)。(3)KTM组和MI组OX42表达水平显著低于CCI组;KTM组OX42表达水平高于MI组(P0.05)。结论:鞘内KTM可以抑制脊髓背角小胶质细胞激活,减少TNF-α和IL-1β表达,显著改善坐骨神经结扎引起的神经病理性疼痛。     

大麻素CB_1受体活化对神经病理性疼痛大鼠脊髓背角嘌呤能P2X_2受体表达的抑制作用

目的:观察脊髓背角大麻素CB_1受体(CB_1R)在坐骨神经缩窄性损伤(CCI)所致的神经病理性疼痛中的作用及其对嘌呤能P2X_2受体表达的调节。方法:7~8周龄SD大鼠分为4组:(1)sham组;(2)CCI组;(3)CP55940+CCI组;(4)AM251+CP55940+CCI组。分别于CCI术前1 d,术后1、3、5、7、10、14 d测定热缩足反射潜伏期(TWL);免疫印迹技术检测各组大鼠损伤侧L_4~L_6段脊髓背角P2X_2受体表达。结果:CCI术后大鼠出现热痛敏,TWL明显缩短;鞘内给予非选择性大麻素受体激动剂CP55940可明显延长CCI大鼠TWL(P0.05);预先鞘内注射CB_1R拮抗剂AM251(0.05 mg/kg)可显著降低CP55940的镇痛效果(P0.05)。免疫印迹实验结果显示:CCI大鼠脊髓背角P2X_2受体在术后7、14 d表达明显增加(P0.05);鞘内给予CP55940可显著降低P2X_2受体表达(P0.05),而预先给予AM251可降低CP55940抑制P2X_2受体表达的效应(P0.05)。结论:脊髓背角CB_1受体激活对CCI所致的神经病理性疼痛具有良好的镇痛作用,其镇痛效应可能与抑制CCI大鼠嘌呤能P2X_2受体表达有关。     

Upregulation of the GABA-transporter GAT-1 in the spinal cord contributes to pain behaviour in experimental neuropathy

Sciatic nerve ligation in rats (chronic constriction injury (CCI)) induces signs and symptoms that mimic human conditions of neuropathy. The central mechanisms that have been implicated in the pathogenesis of neuropathic pain include increased neuronal excitability, possibly a consequence of decreased availability of spinal GABA. GABA availability is regulated by the presence of the GABA-transporters (GATs). This study investigates the dorsal horn expression of the transporter GAT-1 and its functional involvement towards pain behaviour in the CCI model. Male Lewis rats (total n=37) were subjected to CCI or to a sham procedure. A sub-group of animals was treated with the GAT-1 antagonist NO-711. Behavioural testing was performed pre-surgery and at 7 days post-surgery. Testing included evaluation of mechanical allodynia using Von Frey filaments, thermal allodynia with a hot-plate test and observational testing of spontaneous pain behaviour. Subsequently, spinal protein expression of GAT-1 was assessed by Western blotting. Animals were sacrificed 7 days following surgery. CCI markedly increased mechanical and thermal allodynia and spontaneous pain behaviour after 7 days, while the sham procedure did not. GAT-1 was increased in spinal cord homogenates compared contralateral to the ligation side after 7 days. NO-711 treatment significantly reduced all tested pain behaviour. These data provide evidence for possible functional involvement of GAT-1 in the development of experimental neuropathic pain. The latter can be derived from observed analgesic effects of early treatment with NO-711, a selective GAT-1 inhibitor. The obtained insights support the clinical employment of GAT-1 inhibitors to treat neuropathic pain.     

Changes of expression of glial cell line-derived neurotrophic factor and its receptor in dorsal root ganglions and spinal dorsal horn during electroacupuncture treatment in neuropathic pain rats

Injury to the nervous system occasionally leads to intense and persistent neuropathic pain, which is resistant to conventional analgesic methods. It was reported that electroacupuncture (EA) had potent analgesic effect on neuropathic pain by activating various endogenous transmitters such as the opioid peptides. Glial cell line-derived neurotrophic factor (GDNF) has been hypothesized to play an important role in modulation of nociceptive signals especially during neuropathic pain state. Using immunohistochemistry, Western blot, and RT-PCR analysis techniques, the present study observed the effects of EA on the expression of GDNF and GDNF family receptor alpha-1 (GFRalpha-1, the high-affinity receptor of GDNF) in neuropathic pain rats. The results showed that both protein and mRNA levels of GDNF and GFRalpha-1 in the dorsal root ganglions (DRG), as well as GDNF protein in the spinal dorsal horn, were significantly increased after chronic constriction injury (CCI) of the rats' sciatic nerve and could be further enhanced by EA treatment. The present data demonstrated that EA could activate endogenous GDNF and GFRalpha-1 system of neuropathic pain rats and this might underlie the effectiveness of EA in the treatment of neuropathic pain.     

Analgesic effect of intrathecally γ-aminobutyric acid transporter-1 inhibitor NO-711 administrating on neuropathic pain in rats

To investigate the analgesic effect of intrathecally administered γ-aminobutyric acid (GABA) transporter-1 inhibitor NO-711 on the sciatic nerve chronic constriction injury (CCI) rats. 5 days after intrathecal catheter placement, neuropathic pain model was established by CCI of sciatic nerve on rats. Withdrawal thresholds for mechanical allodynia and latency for thermal hyperalgesia were measured in all animals. All rats operated upon for CCI displayed decreased withdrawal thresholds for mechanical allodynia and latency for thermal hyperalgesia, which has significant difference compared with sham groups. After intrathecal NO-711 administration, withdrawal thresholds and latency were significantly increased on CCI rats compared with control group after 1 day. The results show that GABA transporter-1 inhibitor could effectively develop analgesic effect in sciatic nerve CCI rats’ model.     

Remote astrocytic and microglial activation modulates neuronal hyperexcitability and below-level neuropathic pain after spinal injury in rat

In this study, we evaluated whether astrocytic and microglial activation mediates below-level neuropathic pain following spinal cord injury. Male Sprague–Dawley (225–250 g) rats were given low thoracic (T13) spinal transverse hemisection and behavioral, electrophysiological and immunohistochemical methods were used to examine the development and maintenance of below-level neuropathic pain. On postoperation day 28, both hind limbs showed significantly decreased paw withdrawal thresholds and thermal latencies as well as hyperexcitability of lumbar (L4-5) spinal wide dynamic range (WDR) neurons on both sides of spinal dorsal horn compared to sham controls (* P<0.05). Intrathecal treatment with propentofylline (PPF, 10 mM) for 7 consecutive days immediately after spinal injury attenuated the development of mechanical allodynia and thermal hyperalgesia in both hind limbs in a dose-related reduction compared to vehicle treatments (* P<0.05). Intrathecal treatment with single injections of PPF at 28 days after spinal injury, attenuated the existing mechanical allodynia and thermal hyperalgesia in both hind limbs in a dose related reduction (* P<0.05). In electrophysiological studies, topical treatment of 10 mM PPF onto the spinal surface attenuated the neuronal hyperexcitability in response to mechanical stimuli. In immunohistochemical studies, astrocytes and microglia in rats with spinal hemisection showed significantly increased GFAP and OX-42 expression in both superficial and deep dorsal horns in the lumbar spinal dorsal horn compared to sham controls (* P<0.05) that was prevented in a dose-related manner by PPF. In conclusion, our present data support astrocytic and microglial activation that contributes to below-level central neuropathic pain following spinal cord injury.     

Electroacupuncture inhibits cyclooxygenase-2 up-regulation in rat spinal cord after spinal nerve ligation

Electroacupuncture (EA) has long been used to treat pain including neuropathic pain, but its mechanisms remain to be delineated. Since cyclooxygenase-2 (COX-2) has been reported to increase in the spinal dorsal horn following spinal nerve ligation (SNL) and it may play a role in the neuropathic pain, we hereby tested the hypothesis that EA may affect COX-2 expression and hence neuropathic nociception after SNL. The results showed that EA (2 Hz) can significantly reduce mechanical and thermal hypersensitivity following lumbar L5 SNL in rats. Immunostaining demonstrated suppression of COX-2 expression in the spinal L4-L6 dorsal horn after EA. The present results suggest that EA may alleviate neuropathic hypersensitivity by, at least partially, inhibiting COX-2 expression in the spinal cord.