Objectives: Trigeminal neuralgia (TN) is a severe chronic neuropathic pain that mainly affects the distribution area of the trigeminal nerve with limited treating efficacy. There are numerous treatments for TN, but currently the main clinical approach is to suppress pain by carbamazepine (CBZ). Brain-derived neurotrophic factor (BDNF) is closely related to chronic pain. This study aims to determine the effects of CBZ treatment on BDNF expression in both the trigeminal ganglion (TG) and serum of TN via a chronic constriction injury of the infraorbital nerve (ION-CCI) rat model.
Methods: The ION-CCI models were established in male Sprague-Dawley rats and were randomly divided into a sham group, a TN group, a TN+low-dose CBZ treatment group (TN+20 mg/kg CBZ group), a TN+medium-dose CBZ treatment group (TN+40 mg/kg CBZ group), and a TN+high-dose CBZ treatment group (TN+80 mg/kg CBZ group). The mechanical pain threshold in each group of rats was measured regularly before and after surgery. The expressions of BDNF and tyrosine kinase receptor B (TrkB) mRNA in TGs of rats in different groups were determined by real-time PCR, and the expression of BDNF protein on neurons in TGs was observed by immunofluorescence. Western Blotting was used to detect the protein expression of BDNF, TrkB, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) in TGs of rats in different groups. The expression of BDNF in the serum of rats in different groups was detected by enzyme-linked immunosorbent assay (ELISA).
Results: The results of mechanical pain sensitivity showed that there was no significant difference in the mechanical pain threshold in the right facial sensory area of the experimental rats in each group before surgery (all P>0.05). From the 3rd day after operation, the mechanical pain threshold of rats in the TN group was significantly lower than that in the sham group (all P<0.01), and the mechanical pain threshold of rats in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 CBZ mg/kg group was higher than that in the TN group (all P<0.05). The BDNF and TrkB mRNA and protein expressions in TGs of rats in the TN group were higher than those in the sham group (all P<0.05), and those in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than the TN group (all P<0.05). The p-ERK levels in TG of rats in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were significantly decreased compared with the TN group (all P<0.05). The BDNF and neuron-specific nuclear protein (NeuN) were mainly co-expressed in neuron of TGs in the TN group and they were significantly higher than those in the sham group (all P<0.05). The co-labeled expressions of BDNF and NeuN in TGs of the TN+ 80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than those in the TN group (all P<0.05). The results of ELISA showed that the level of BDNF in the serum of the TN group was significantly higher than that in the sham group (P<0.05). The levels of BDNF in the TN+80 mg/kg CBZ group, the TN+40 mg/kg CBZ group, and the TN+20 mg/kg CBZ group were lower than those in the TN group (all P<0.05). Spearman correlation analysis showed that the BDNF level in serum was negatively correlated with mechanical pain threshold (r=-0.650, P<0.01).
Conclusions: CBZ treatment can inhibit the expression of BDNF and TrkB in the TGs of TN rats, reduce the level of BDNF in serum of TN rats and the phosphorylation of ERK signaling pathway, so as to inhibit TN. The serum level of BDNF can be considered as an indicator for the diagnosis and prognosis of TN.
目的: 三叉神经痛(trigeminal neuralgia，TN)是一种严重的慢性神经病理性疼痛，主要影响三叉神经分布区域，临床治疗效果不佳。TN的治疗方法众多，但目前临床上主要是通过服用卡马西平(carbamazepine，CBZ)来抑制疼痛。脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)和慢性痛密切相关。本研究通过慢性压迫性损伤眶下神经(chronic constriction injury of the infraorbital nerve，ION-CCI)大鼠模型观察CBZ处理对TN大鼠三叉神经节(trigeminal ganglion，TG)和血清中BDNF表达的影响。方法: 建立雄性SD大鼠ION-CCI模型，并将其随机分为假手术(sham)组、TN组、TN+低剂量(20 mg/kg)CBZ处理组、TN+中剂量(40 mg/kg)CBZ处理组、TN+高剂量(80 mg/kg)CBZ处理组。在手术前后定时测量各组大鼠的面部机械痛阈(mechanical pain threshold)。使用实时聚合酶链反应技术测定各组大鼠TG中BDNF及酪氨酸激酶受体B(tyrosine kinase receptor B，TrkB)的mRNA含量，免疫荧光技术观察各组大鼠TG中BDNF蛋白质在神经元上的表达情况，蛋白质印迹法检测各组大鼠TG中BDNF、TrkB、细胞外调节蛋白激酶(extracellular regulated protein kinases，ERK)及磷酸化的细胞外调节蛋白激酶(phospho-extracellular regulated protein kinases，p-ERK)的蛋白质表达变化，酶联免疫吸附试验(enzyme-linked immunosorbent assay，ELISA)检测各组大鼠血清中BDNF的表达变化。结果: 行为学检测结果表明：手术前，各组大鼠右侧面部感觉区域的机械痛阈差异均无统计学意义(均P>0.05)；术后第3天开始，TN组大鼠机械痛阈与sham组相比均明显降低(均P<0.01)，TN+80 mg/kg CBZ处理组、TN+40 mg/kg CBZ处理组和TN+20 mg/kg CBZ处理组与TN组相比均升高(均P<0.05)。实时聚合酶链反应和蛋白质印迹法结果显示：TN组大鼠TG中的BDNF、TrkB的 mRNA及蛋白质表达量均较sham组升高(均P<0.05)，TN+20 mg/kg CBZ处理组、TN+40 mg/kg CBZ处理组、TN+80 mg/kg CBZ处理组均较TN组降低(均P<0.05)；与TN组相比，TN+20 mg/kg CBZ处理组、TN+40 mg/kg CBZ处理组、TN+80 mg/kg CBZ处理组大鼠TG中的p-ERK水平均显著降低(均P<0.05)。免疫荧光双标结果表明：TN组TG中的BDNF和神经元特异性核蛋白(neuron-specific nuclear protein，NeuN)主要共表达在神经元上，与sham组比较BDNF和NeuN水平升高(P<0.05)，TN+20 mg/kg CBZ处理组、TN+40 mg/kg CBZ处理组、TN+80 mg/kg CBZ处理组与TN组大鼠比较二者表达均降低(均P<0.05)。ELISA检测结果显示：TN组大鼠血清中BDNF的水平较sham组显著升高(P<0.05)，TN+20 mg/kg CBZ处理组、TN+40 mg/kg CBZ处理组、TN+80 mg/kg CBZ处理组均较TN组大鼠降低(均P<0.05)。Spearman相关分析显示血清中BDNF水平与机械痛阈呈负相关(r=-0.650，P<0.01)。结论: CBZ处理可以抑制TN大鼠TG中BDNF及其受体TrkB的表达，降低TN大鼠血清中BDNF水平及ERK信号通路磷酸化水平，进而抑制TN。可以考虑将血清中BDNF水平作为诊断TN和评估预后的指标。.
Keywords: brain-derived neurotrophic factor; carbamazepine; trigeminal ganglion; trigeminal neuralgia; tyrosine kinase receptor B.