【作者】 彭建明；

【导师】 蒋星红；

【作者基本信息】 苏州大学 ， 医学神经生物学， 2017， 博士

【摘要】 第一部分炎症大鼠下丘脑弓状核在脊髓背角神经元伤害性反应和大鼠伤害性感受中的调节作用目的尽管以往的研究已经证明下丘脑弓状核(arcuate nucleus,ARC)是内源性伤害性感受调制系统中一个重要的组成部分,但ARC是如何参与炎症大鼠的伤害性感受调制,特别是对脊髓背角神经元伤害性感受的下行调节,仍然不清楚。本实验旨在观察在持续炎症刺激下,脊髓背角神经元放电活动的变化以及ARC对其伤害感受的调控,以探索ARC对脊髓背角神经元伤害性感受的下行调节机制。方法1)选用雄性SD大鼠,制备完全弗氏佐剂(complete Freund’s adjuvant,CFA)炎症痛模型,用电生理学方法记录大鼠脊髓背角伤害感受性神经元单位【广动力范围型(wide dynamic range,WDR)、伤害感受特异型(nociceptive specific,NS)神经元】的单位自发放电和诱发放电;2)采用新生期大鼠腹腔注射谷氨酸单钠(monosodium glutamate,MSG,2 mg/g﹒d)化学毁损和电解毁损两种方法破坏ARC,电生理学方法记录大鼠脊髓背角WDR以及NS神经元单位自发放电以及伤害性电刺激(双脉冲方波,强度:20 V,波宽:0.5 ms,间隔:10 ms,频率:0.2 Hz)坐骨神经诱发的放电;3)通过ARC核团内微量注射谷氨酸(L-Glutamate,L-Glu,0.2 mM/0.5μl)兴奋ARC细胞,并采用辐射热-缩腿法和von Frey法分别测定核团注射L-Glu后2 min,8 min,14 min时炎症大鼠的热和机械伤害感受阈;4)用电生理学方法观察炎症大鼠兴奋ARC后,脊髓背角神经元单位放电的变化。结果1)电生理学结果显示,与非炎症组大鼠比较,CFA炎症组大鼠脊髓背角WDR和NS神经元的自发放电频率、伤害性电刺激诱发的放电频率均显著升高(P<0.001)。2)MSG毁损ARC后,CFA炎症大鼠脊髓背角WDR和NS神经元自发放电无显著改变(P>0.05),但伤害性电刺激诱发放电频率明显下降(P<0.05);与MSG毁损ARC结果类似,电解毁损ARC后,CFA炎症大鼠脊髓背角WDR和NS神经元诱发放电频率明显下降(P<0.01),但自发放电无显著变化(P>0.05);3)行为学检查显示,ARC内注射L-Glu后2 min、8 min时,炎症大鼠热和机械伤害感受阈值均显著降低(P<0.01),14 min后恢复正常水平;电生理学结果显示,ARC内注射L-Glu后,炎症大鼠的脊髓背角WDR和NS神经元对伤害性电刺激所诱发的放电频率显著升高(P<0.01),12 min后恢复正常水平。结论在外周持续炎症作用下,大鼠脊髓背角神经元的伤害性感受发生了敏感化。MSG毁损或电解毁损ARC后,炎症大鼠脊髓背角神经元对伤害性刺激的反应性降低;ARC内注射L-Glu后,CFA炎症大鼠伤害性感受阈降低,对伤害性刺激的反应性增强,说明下丘脑ARC参与了持续炎症刺激导致的脊髓背角神经元伤害感受的敏感化,起着下行易化的调节作用。第二部分炎症大鼠下丘脑弓状核对脊髓背角神经元伤害性反应和大鼠伤害性感受调节的机制研究目的众所周知,脑内存在着内源性痛觉调制系统,即以中脑导水管周围灰质(PAG)为核心,联结延脑头端腹内侧核(RVM),通过下行通路对脊髓背角神经元感受伤害性刺激的反应能力进行调节。PAG下行调制系统至少包括内源性阿片肽,5-HT和NE三种递质系统。本部分的研究旨在探讨炎症大鼠ARC实现下行易化调节脊髓背角神经元伤害性感受的机制。方法1)观察在炎症大鼠中缝大核(nucleus raphe magnus,NRM)内注射局麻药利多卡因(Lidocaine)对ARC内微量注射L-Glu引起的脊髓背角神经元单位放电的影响;2)在炎症大鼠NRM内注射Lidocaine对ARC微量注射L-Glu引起的热和机械伤害感受阈变化的影响;3)在炎症大鼠NRM内注射非选择性5-HT受体拮抗剂美西麦角(methysergide,MSD)对ARC内微量注射L-Glu引起的脊髓背角神经元的兴奋的影响;4)通过ELISA检测CAF炎症引起的中缝大核和脊髓背角的5-HT、NE、Glu以及GABA递质水平变化,以及ARC在此调节中的作用。结果1)电生理学以及行为学检测结果显示,与在炎症大鼠NRM内注射生理盐水的结果比较,炎症大鼠NRM内注射Lidocaine并不改变伤害性电刺激引起的脊髓背角神经元放电和炎症大鼠对伤害性刺激的反应,但阻断了ARC内微量注射L-Glu引起的脊髓背角神经元对伤害性电刺激反应的增强。与NRM内注射生理盐水的结果相比,伤害性电刺激引起的脊髓背角神经元放电明显减少(P<0.001),炎症大鼠热和机械刺激伤害性感受阈明显升高(P<0.05);2)与在炎症大鼠NRM内注射生理盐水的结果比较,炎症大鼠NRM内注射MSD也并不改变伤害性电刺激诱发的脊髓背角神经元放电,但同样阻断了炎症大鼠ARC微量注射L-Glu引起的脊髓背角神经元对伤害性电刺激反应的增强,表现为伤害性电刺激诱发的脊髓背角神经元发放电减少(P<0.01);3)与正常对照组大鼠相比,炎症组大鼠脊髓背角中的5-HT和Glu浓度升高(P<0.01),NE和GABA浓度降低(P<0.01),而在NRM中的5-HT和GABA浓度升高(P<0.01),Glu浓度降低(P<0.01)。MSG毁损或电毁损ARC明显阻断了这些递质浓度的变化。结论下丘脑ARC参与了对炎症大鼠脊髓背角伤害感受性的下行易化调节。该易化作用有可能是通过调节中缝大核(5-HT和GABA上调,Glu下调)和脊髓背角(5-HT、Glu上调,GABA、NE下调)内的神经递质及神经元的伤害感受性而引起的。更多还原

【Abstract】 Part Ⅰ.The role of hypothalamic arcuate nucleus in regulations of the discharge activity of spinal dorsal horn neurons and the nociceptive response of CFA-imflamed ratsObjective The hypothalamic arcuate nucleus(ARC)is an important component of the endogenous pain modulation system.However,it remains not fully clear how the ARC is involved in the modulation of inflammatory pain.In this part of studies,we aimed to identify the role of ARC in regulations of the neuronal activity in the spinal dorsal horn and the nociceptive responses of rats with persistent inflammation in the left hind paw.Methods Inflammation was induced by sub-plantar injection of complete Freund’s adjuvant(CFA)in rats.The lesion of ARC was produced in rats by electrolytic lesion or by a neonatal intraperitonial(i.p.)injection of monosodium glutamate(MSG,2 mg/g).Extracellular single unit recordings were conducted in the dorsal horn of the spinal cord.Electrical stimulation(frequency: 0.2 Hz,intensity: 20 V two-pulses with 0.5 ms duration and 10 ms interval)of the right sciatic nerves was performed to mimic noxious stimulation.Spontaneous and electrical stimulation-evoked discharges were recorded in wide dynamic range(WDR)neurons,which responded to both innocuous and noxious stimulations,and nociceptive specific(NS)neurons,which responded only to noxious stimulations.Thermal responses of rats were measured by the radiant heat-withdrawalmethod.Mechanical responses were measured by the von Frey method.L-glutamate(0.2mM/0.5 μl)was micro-injected into the ARC to activate ARC neurons.Effects of the ARC injection of L-glutamate on nociceptive neurons in the spinal dorsal horn and on rats’ responses to the heat and mechanical stimulations were recorded respectively at 2min,8 min,14 min after the ARC injection.Results When compared with those in control animals both the spontaneous and noxcious electrical stimulation-evoked discharges of WDR and NS neurons in the spinal dorsal horn were significantly increased(P<0.001)in CFA-inflamed rats Lesions of ARC produced by electrolysis or by a neonatal intraperitonial injection of MSG significantly blocked the electrical stimulation-induced increase in neuronal discharges in both WDR(P<0.05)and NS(P<0.01)neurons of CFA-inflamed rats,while no change was noted in their spontaneous activity.Following microinjection of L-glutamate into the ARC area,noxcious electrical stimulation-induced discharges were significantly increased in the spinal dorsal horn neurons of CFA-inflamed rats(P<0.01).Furthermore,animals’ nociceptive responses to both thermal and mechanical stimulations were also significantly(P<0.01)enhanced.These effects induced by microinjection of L-glutamate lasted about12 min.Conclusions Spinal cord dorsal horn neurons were sensitized following peripheral inflammation.While destroying ARC neurons(by electrolytical lesion or by a neonatal intraperitonial injection of MSG)significantly prevented the increase in neuronal activity induced by inflammation,exciting ARC neurons by microinjection of L-glutamate into the ARC area further increased nociceptive responses in the spinal dorsal horn and the whole animal evoked by inflammation.Thus,we conclude that ARC may act as a descending facilitatory component critically involved in the central sensitization process associated with peripheral inflammation.Part Ⅱ.The mechanism of hypothalamic arcuate nucleus in regulations of the activity of spinal dorsal horn neurons of CFA-imflamed ratsObjective Previous studies have shown that endogenous pain modulation system which including the periaqueductal gray(PAG)and the rostral ventromedial nucleus(RVM)regulate the nociceptive response of spinal dorsal horn neurons by the descending pathway.In this part of studies,we investigated potential pathway(s)and mechanisms by which ARC neurons modulate inflammation-induced nociception.Methods Electrophysiological and behavioral studies were conducted to examine effects of lidocaine or methysergide(MSD)microinjected into the nucleus raphe magnus(NRM)area on the neuronal discharge activity in the spinal dorsal horn and on the behavioural response of CFA-inflamed animals to thermal and mechanical stimulation with or without microinjection of L-glutamate into the ARC area.Through ELISA assay concentrations of 5-hydroxytryptamine(5-HT),norepinephrine(NE),glutamate(Glu)andγ-aminobutyric acid(GABA)in the NRM and spinal dorsal horn of rats were examined.Results Microinjection of either lidocaine or MSD into the NRM did not significantly affect both the neuronal discharge activity in spinal dorsal horn and the animals’ responses to thermal and mechanical stimulation in CFA-inflamed rats.However,these drug injections blocked the facilitative effects induced by the microinjection of L-glutamate into the ARC area.ELISA assay showed that when compared with those in the control group of rats,in the spinal dorsal horn of CFA-inflammatory rats,the concentrations of 5-HT(P<0.01)and Glu(P<0.01)were increased but NE(P<0.01)and GABA(P<0.01)decreased.In the NRM,the concentrations of 5-HT(P<0.01)and GABA(P<0.01)were increased,but Glu(P<0.01)decreased.The ARC-lesion prevented all of these changes in both the spinal dorsal horn and NRM of CFA-inflammatory rats.Conclusions Taken all the findings together,we conclude that NRM is a criticalcomponent,through which ARC play a facilitative role resulting in the central sensitization in rats with peripheral inflammation,and that up-regulating the concentrations of 5-HT,Glu in both the NRM and spinal dorsal horn,as well as down-regulting Glu in the NRM,and GABA and NE in the spinal dorsal horn may be important mechanisms underlying the action of ARC neurons in the modulation of nociception associated with CFA inflammation.更多还原