【作者】 杨鑫；

【导师】 郭曲练； 王健；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2022， 博士

【摘要】 背景近年来,抑郁等精神障碍疾病发病率逐年上升。抑郁患者往往存在疼痛耐受性下降,在合并疼痛相关疾病后容易出现痛觉过敏、难治性疼痛等困扰,而疼痛可进一步加重抑郁程度,发展为抑郁-疼痛相互促进的恶性循环。抑郁-疼痛共病可导致患者生活质量下降和工作能力受损,使医疗资源消耗增加,加重社会负担。因此,研究抑郁-疼痛共病的发病机制意义重大。目的本研究的目的是通过构建抑郁合并神经病理性疼痛大鼠模型,使用化学遗传学方法,钙成像等技术,研究脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)/酪氨酸激酶受体B(Tyrosine kinase receptor B,Trk B)信号通路参与前扣带皮层(Anterior cingulate cortex,ACC)-脊髓背角(Spinal dorsal horn,SDH)环路介导的抑郁情绪对神经病理性疼痛易化调节的作用机制,为抑郁-疼痛共病的治疗提供新的思路和潜在的靶点。方法(1)研究对象选取无特定病原体级雄性SD大鼠,5周龄,体重140g至160g。通过慢性不可预知温和应激(Chronic unpredictable mild stress,CUMS)处理构建大鼠抑郁情绪模型,在CUMS 21天合并坐骨神经慢性压迫损伤(Chronic constriction injury of the sciatic nerve,CCI)处理。将大鼠随机分为Sham+Control,CCI+Control,Sham+CUMS以及CCI+CUMS共4个实验组。通过糖水偏爱测试和强迫游泳测试测量大鼠抑郁行为学变化,使用Von Frey法测量缩足机械痛阈(Paw withdrawal mechanical threshold,PWMT),使用Plantar Test足底测痛仪测量缩足热痛阈(Paw withdrawal thermal latency,PWTL),观察大鼠疼痛行为学变化。通过蛋白质免疫印迹(Western Blot,WB)和免疫荧光染色(Immunofluorescence,IF)检测ACC和SDH中c-Fos蛋白表达水平和分布,对抑郁合并神经病理性疼痛大鼠ACC和SDH神经元的激活水平进行定量和定性分析。(2)对CUMS合并CCI处理大鼠,使用Cre重组酶依赖的AAV-DIO-h M4Di和化学遗传学方法,从CCI 0天至CCI 14天,持续选择性抑制ACC下行投射神经元的兴奋性。将大鼠随机分为AAV-h M4Di组和Mock空载病毒对照组共2个实验组。通过WB检测ACC和SDH中c-Fos蛋白表达水平改变。使用钙成像技术,检测ACC神经元活动情况。观察ACC-SDH投射神经元活性被抑制后,抑郁行为学和疼痛行为学的改变。(3)对CUMS合并CCI处理大鼠,使用Cre重组酶依赖的AAV-DIO-sh BDNF对ACC-SDH投射神经元选择性敲减BDNF。大鼠随机分为sh BDNF组和Scramble错序病毒对照组共2个实验组。通过WB、q PCR和IF对ACC和SDH中BDNF进行定量和定位分析。(4)对CUMS合并CCI处理大鼠,脊髓鞘内注射Trk B受体选择性拮抗剂ANA-12,阻断脊髓层面BDNF/Trk B通路。大鼠随机分为ANA-12组和Vehicle溶剂对照组共2个实验组。观察抑郁行为学和疼痛行为学变化。(5)利用转录组测序技术,分析CUMS合并CCI处理大鼠经化学遗传学方法抑制ACC-SDH投射神经元后脊髓层面基因的表达改变,筛选可能的下游分子通路。根据测序结果,通过WB和IF进一步对脊髓Lamb1蛋白进行定量检测和细胞定位分析。通过高尔基染色反映SDH神经元树突生长变化情况,对抑郁易化神经病理性疼痛的下游机制进行探索。结果(1)经过21天CUMS处理成功构建稳定的抑郁情绪大鼠模型,CUMS诱导的抑郁情绪加重CCI所致的痛觉过敏。CCI+CUMS组的机械痛阈从CCI 3天开始显著低于CCI+Control组,在CCI 7天时两组差异达到最大(2.178 vs 6.778,p=0.0001),热痛阈的改变趋势与机械痛阈一致。CUMS 21天(即CCI 0天),Sham+CUMS组ACC中c-Fos蛋白水平显著高于Sham+Control组(p=0.0286),而两组在SDH中c-Fos蛋白水平无差异,该结果提示,在缺少CCI刺激时,CUMS诱导的抑郁情绪可导致ACC神经元激活,但不会直接激活SDH。在CCI 7天,CCI+CUMS组和Sham+CUMS组ACC中c-Fos蛋白水平分别显著高于CCI+Control组(p=0.0103)和Sham+Control组(p=0.0003),CCI+Control组略高于Sham+Control组(p=0.0429),但CCI+CUMS组与Sham+CUMS组比较无明显差异。免疫荧光染色结果与WB趋势一致。CCI 7天SDH中c-Fos蛋白水平的组间差异比较结果显示,CCI+Control组和CCI+CUMS组的c-Fos蛋白水平分别显著高于Sham+Control组(p=0.0223)和Sham+CUMS组(p=0.0003)。CCI+CUMS组c-Fos蛋白水平高于CCI+Control组(p=0.0433),而Sham+CUMS组和Sham+Control组比较无差异。这提示CUMS诱导的抑郁情绪在CCI所致神经病理性疼痛发生后可以增强SDH神经元的激活程度,但对于没有发生神经损伤的Sham组,CUMS诱导的抑郁情绪对于SDH神经元的激活无显著影响。免疫荧光染色结果与WB趋势一致。(2)化学遗传学方法抑制ACC-SDH投射神经元后未对CUMS合并CCI处理大鼠的抑郁样行为学产生影响,但可以改善疼痛行为学。从CCI 3天至CCI 14天,AAV-h M4Di组的机械痛阈均高于Mock组,且在CCI 7天两组差异最大(6.778 vs 3.222,p=0.0012)。热痛阈趋势与机械痛阈基本一致。CCI 7天,AAV-h M4Di组在疼痛刺激后的荧光钙信号强度ΔF/F(%)低于Mock组(p=0.0079),AAV-h M4Di组ACC和SDH中c-Fos蛋白水平均低于Mock组。以上结果提示,化学遗传学方法使ACC中兴奋性的下行投射神经元抑制后,脊髓层面SDH神经元的激活也受到了抑制。(3)CUMS组大鼠ACC中BNDF水平在CCI后随时间递增。CCI 7天CUMS组大鼠SDH中BDNF蛋白水平高于Control组(p=0.0286)。化学遗传学方法抑制ACC-SDH投射神经元后逆转了CUMS合并CCI大鼠ACC和SDH中BDNF的上调。sh BDNF敲减后,CUMS合并CCI大鼠ACC中BDNF转录水平和蛋白水平均下调,SDH中BDNF转录水平不变但蛋白水平下调。免疫荧光染色显示ACC和SDH中BDNF与工具病毒报告荧光共定位。以上结果提示,ACC中下行投射神经元表达的BDNF存在经由ACC-SDH投射纤维传输至脊髓背角的物质基础和结构基础。sh BDNF选择性敲减ACC-SDH投射神经元中BDNF后,CUMS合并CCI处理大鼠的抑郁行为学无改变,但疼痛行为学得到改善,sh BDNF组在CCI后机械痛阈高于Scramble组,且在CCI 7天差异最大(7.111 vs 2.889,p=0.0002),热痛阈的改变趋势与机械痛阈一致。(4)脊髓鞘内注射ANA-12对CUMS合并CCI处理大鼠的抑郁样行为学无影响,但可以改善疼痛行为学。ANA-12组在CCI后机械痛阈高于Vehicle组,且在CCI 7天差异最大(6.889 vs 3.111,p=0.0003),热痛阈的改变趋势与机械痛阈一致。以上结果提示,BDNF在脊髓层面通过BDNF/Trk B通路参与CUMS诱导的抑郁情绪对CCI所致神经病理性疼痛的易化。(5)转录组测序筛选发现AAV-h M4Di组树突相关基因表达上调,作为调控细胞外基质的重要分子Lamb1上调倍数最高。进一步对Lamb1蛋白水平定量检测发现,CUMS合并CCI处理后SDH中Lamb1蛋白水平下调,而在化学遗传学方法抑制ACC-SDH投射神经元后,SDH中Lamb1蛋白水平下调被逆转。脊髓免疫荧光染色双标结果显示,有79.59%Lamb1与Neu N共定位,13.62%Lamb1与GFAP共定位,5.82%Lamb1与Iba1共定位,Lamb1主要表达于神经元中。高尔基染色结果提示CCI+CUMS组树突数量和成熟形态(短粗型与蘑菇型)树突棘密度显著多于CCI+Control组,不成熟形态(瘦长型与丝状伪足型)树突棘密度无显著差异。以上结果提示,CUMS诱导的抑郁情绪可促进CCI后SDH神经元树突生长活跃,成熟形态树突棘密度上调。而AAV-h M4Di组树突数量和成熟形态树突棘密度较Mock组显著减少,不成熟形态树突棘密度无显著差异。提示化学遗传学方法抑制ACC-SDH投射神经元后,逆转了CUMS诱导的抑郁情绪对CCI后SDH神经元树突生长的促进作用。结论CUMS诱导的抑郁情绪可导致大鼠ACC神经元激活,BDNF蛋白表达上调。BDNF经由ACC-SDH投射纤维传输至脊髓背角,在脊髓层面激活BDNF/Trk B通路,促进SDH神经元树突生长,参与痛觉过敏形成。Lamb1通过对SDH神经元突触可塑性的调节,共同参与抑郁对神经病理性疼痛的下行易化调节机制。图32幅,表19个,参考文献85篇。更多还原

【Abstract】 BackgroundIn recent years,the incidence of mental disorders such as depression has increased year by year.Depressed patients often suffer from decreased pain tolerance,and are prone to suffer from hyperalgesia and refractory pain when combined with pain-related diseases.Pain can further aggravate the degree of depression and develop into a vicious cycle of mutual promotion of depression and pain.Depression-pain comorbidity can lead to a decline in the quality of life of patients and impairment of work ability,increase the consumption of medical resources,and increase the social burden.Therefore,it is of great significance to study the pathogenesis of depression-pain comorbidity.ObjectiveThe objective of this study is to investigate the role of brain-derived neurotrophic factor（BDNF）/tyrosine kinase receptor B（Trk B）signaling in the mechanism of depression facilitating neuropathic pain mediated by anterior cingulate cortex（ACC）-spinal dorsal horn（SDH）circuitry,based on a rat model of depression combined with neuropathic pain,using chemogenetic methods,calcium imaging and other techniques.It might provide new ideas and potential targets for the treatment of depression-pain comorbidity.Methods（1）Specific pathogen-free（SPF）male SD rats,5 weeks old,weighing 140g to 160g were selected as the research subjects.A rat model of depression was constructed by chronic unpredictable mild stress（CUMS）treatment,and combined with chronic sciatic nerve compression injury（CCI）treatment on the 21^stday of CUMS.Rats were randomly divided into four experimental groups:Sham+Control,CCI+Control,Sham+CUMS and CCI+CUMS.The depressive behavior were measured by sugar water preference test and forced swimming test.The pain behavior,including Paw withdrawal mechanical threshold（PWMT）and Paw withdrawal thermal latency（PWTL）,were measured by Von Frey and Plantar Test respectively.The expression level and distribution of c-Fos protein in ACC and SDH were detected by western blot（WB）and immunofluorescence staining（IF）.（2）In rats treated with CUMS combined with CCI,Cre recombinase-dependent AAV-DIO-h M4Di and chemogenetic methods were used to selectively inhibit the activity of descending projection neurons of the ACC from day 0 of CCI to day 14 of CCI.Rats were randomly divided into two experimental groups,AAV-h M4Di group and Mock group.Protein expression levels of c-Fos in ACC and SDH were detected by WB.Activity of ACC neurons was detected using calcium imaging techniques.The changes in depression behavior and pain behavior were observed after the activity of ACC-SDH projection neurons was inhibited.（3）In rats treated with CUMS combined with CCI,Cre recombinase-dependent AAV-DIO-sh BDNF was used to selectively knock down BDNF in ACC-SDH projection neurons.Rats were randomly divided into two experimental groups,sh BDNF group and Scramble group.The protein and m RNA level as well as the distribution of BDNF in ACC and SDH were detected by western blot（WB）,q PCR and immunofluorescence staining（IF）.（4）In rats treated with CUMS combined with CCI,the Trk B receptor selective antagonist ANA-12 was intrathecally injected to block the BDNF/Trk B pathway at the spinal cord level.Rats were randomly divided into two experimental groups,ANA-12 group and Vehicle group.The changes of depression behavior and pain behavior were observed.（5）Using RNA-sequencing technology,we analyzed the gene expression changes at the spinal cord level after chemogenetic inhibition of ACC-SDH projection neurons in rats treated with CUMS combined with CCI,and screened possible downstream molecular pathways.Based on the RNA-sequencing results,the Lamb1 protein was further quantitatively detected and cell localization analysis by WB and IF.Golgi staining was used to reflect the dendritic growth changes of SDH neurons,and to explore the downstream mechanism of depression-facilitated neuropathic pain.Results（1）After 21 days of CUMS treatment,a rat model of depression was successfully established.CUMS-induced depression exacerbates CCI-induced hyperalgesia.The PWMT of the CCI+CUMS group was significantly lower than that of the CCI+Control group from the 3rd day of CCI,and the difference between the two groups reached the maximum at the 7th day of CCI（2.178 vs 6.778,p=0.0001）.The change trend of PWTL was consistent with PWMT.On the 21st day of CUMS（ie,CCI day 0）,the c-Fos protein level in the ACC of the Sham+CUMS group was significantly higher than that of the Sham+Control group（p=0.0286）,while there was no difference in the c-Fos protein level in the SDH between the two groups.These results suggest that in the absence of CCI stimulation,CUMS-induced depression can lead to ACC neuron activation,but not SDH directly.On the 7th day of CCI,the c-Fos protein level in ACC of CCI+CUMS group and Sham+CUMS group was significantly higher than that of CCI+Control group（p=0.0103）and Sham+Control group（p=0.0003）,respectively.The CCI+Control group was slightly higher than the Sham+Control group（p=0.0429）,but there was no significant difference between the CCI+CUMS group and the Sham+CUMS group.The results of immunofluorescence staining were consistent with the trend of WB.The comparison of the c-Fos protein levels in SDH on the 7th day of CCI showed that the c-Fos protein levels in the CCI+Control group and CCI+CUMS group were significantly higher than those in the Sham+Control group（p=0.0223）and Sham+CUMS group（p=0.0003）.The c-Fos protein level in the CCI+CUMS group was higher than that in the CCI+Control group（p=0.0433）,but there was no difference between the Sham+CUMS group and the Sham+Control group.This suggests that CUMS-induced depression can enhance the activation of SDH neurons after CCI-induced neuropathic pain,but for the Sham group without neurological damage,CUMS-induced depression has no significant effect on the activation of SDH neurons.The results of immunofluorescence staining were consistent with the trend of WB.（2）Chemogenetic inhibition of ACC-SDH projection neurons did not affect depressive-like behaviors in CCI+CUMS rats,but improved pain behaviors.From CCI 3 days to CCI 14 days,the PWMT of the AAV-h M4Di group was higher than that of the Mock group,and the difference between the two groups reached the maximum at CCI 7 days（6.778 vs3.222,p=0.0012）.The PWTL trend is consistent with the PWMT.On the7th day of CCI,the fluorescence calcium signal intensityΔF/F（%）of AAV-h M4Di group after pain stimulation was lower than that of Mock group（p=0.0079）,and the level of c-Fos protein in ACC and SDH of AAV-h M4Di group was lower.in the Mock group.These results suggest that after chemogenetic inhibition of activation descending projection neurons in the ACC,the activation of SDH neurons at the spinal cord level was also inhibited.（3）BNDF levels in the ACC of CUMS-treated rats increased over time after CCI.The level of BDNF protein in SDH of CUMS-treated rats on day 7 of CCI was higher than that of Control group（p=0.0286）.Chemogenetic inhibition of ACC-SDH projection neurons reversed the upregulation of BDNF in ACC and SDH in CCI+CUMS rats.After sh BDNF knockdown,the transcription and protein levels of BDNF were down-regulated in ACC of CCI+CUMS rats,while the transcription level of BDNF in SDH was unchanged but the protein level was down-regulated.Immunofluorescence staining revealed co-localization of BDNF with tool virus reporter fluorescence in ACC and SDH.The above results suggest that BDNF expressed by descending projection neurons in the ACC has a material and structural basis for transmission to SDH via ACC-SDH projection fibers.After selective knockdown of BDNF in ACC-SDH projection neurons by sh BDNF,depressive behaviors were not changed in CCI+CUMS rats,but pain behaviors were improved.The PWMT in the sh BDNF group was higher than that in the Scramble group after CCI,and the difference between the two groups reached the maximum on the 7th day of CCI（7.111 vs 2.889,p=0.0002）.The changing trend of PWTL is consistent with that of PWMT.（4）Intrathecal injection of ANA-12 had no effect on depression-like behavior in CUMS-CCI-treated rats,but improved pain behavior.The PWMT in the ANA-12 group was higher than that in the Vehicle group,and the difference between the two groups reached the maximum at 7days of CCI（6.889 vs 3.111,p=0.0003）.The change trend of PWTL was consistent with PWMT.The above results suggest that BDNF is involved in the facilitation of CUMS-induced depression on CCI-induced neuropathic pain at the spinal level through the BDNF/Trk B pathway.（5）The results of RNA-sequencing indicated that the expression of dendrite-related genes in the AAV-h M4Di group was up-regulated.As an important molecule regulating extracellular matrix,Lamb1 has the highest up-regulation fold.Further quantitative detection of Lamb1protein level found that Lamb1 protein level was down-regulated in SDH of CCI+CUMS rats.In contrast,after chemogenetic inhibition of ACC-SDH projection neurons,the downregulation of Lamb1 protein levels in SDH was reversed.The results of immunofluorescence staining in the spinal cord showed that 79.59%of Lamb1 co-localized with Neu N,13.62%of Lamb1 and GFAP co-localized,and 5.82%of Lamb1 co-localized with Iba1.Lamb1 is mainly expressed in neurons.The results of Golgi staining showed that the number of dendrites and the density of dendritic spines in the CCI+CUMS group were significantly higher than those in the CCI+Control group,and the number of dendritic spines in the CCI+CUMS group was significantly higher than those in the CCI+Control group.Dendritic spine density was not significantly different.The above results suggest that CUMS-induced depression can promote active dendritic growth in SDH neurons after CCI,and the density of mature dendritic spines is up-regulated.However,the number of dendrites and the density of mature dendritic spines in the AAV-h M4Di group were significantly lower than those in the Mock group,and there was no significant difference in the density of dendritic spines in the immature morphology.It is suggested that inhibition of ACC-SDH projection neurons by chemogenetic methods reversed the effect of CUMS-induced depression on dendritic growth of SDH neurons after CCI.ConclusionCUMS-induced depression can lead to activation of rat ACC neurons and up-regulation of BDNF protein expression.BDNF is transmitted to SDH via ACC-SDH projection fibers,activates the BDNF/Trk B pathway at the spinal cord level,promotes the growth of SDH neuron dendrites,and participates in the mechanism of hyperalgesia.Lamb1 participates in the mechanism of depression facilitating neuropathic pain by regulating synaptic plasticity of SDH neurons.更多还原