【作者】 周海宇；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 骨外科学， 2007， 博士

【摘要】 目的:头颈痛临床上很常见,明确分布于颈部组织结构包括硬膜、后纵韧带、小关节囊和椎动脉的神经支配和神经递质的化学状况,对于预防和治疗头颈痛有重要的意义。方法:SD大鼠24只,随机分为A、B二组,每组12只,A组行颈交感神经切除术,B组假手术,两组大鼠存活一天后,再次麻醉、灌注,取C3-C5脊髓段硬膜包括DRG、椎管前后壁软组织层包括后纵韧带、小关节囊、椎动脉等标本,采用二步Envision法进行SP(P物质)和TH(酪氨酸羟化酶)免疫组化染色,光镜下观察SP免疫反应(SP-IR)纤维和TH免疫反应(TH-IR)纤维,测量各区免疫反应纤维阳性区域面积。结果:硬膜、后纵韧带、小关节囊和椎动脉壁均有SP-IR和TH-IR阳性纤维分布,其中硬膜阳性纤维主要分布于根袖区和腹侧区,后纵韧带阳性纤维主要分布于椎间部,椎动脉壁外膜外、外膜层和中膜层以TH-IR纤维为主,颈交感神经切除后各结构TH-IR纤维均显著减少(P <0.01)。结论:颈椎周围的组织结构中有肽类递质SP和经典递质NE共存,表明这些结构和颈源性疼痛症状有关,交感神经系统可能在周围神经初级感觉信息的调制中有重要的意义;硬膜根袖区SP反应纤维密度较高,如受到刺激,可能较早出现向上肢的放散性疼痛;椎动脉表面及及中膜肌层有致密的交感神经分布,如有压迫、炎症等因素,可能引起椎动脉的痉挛并出现椎基底动脉系统血流障碍(VBI)。目的:探讨颈部脊神经节(DRG)和交感神经节(SG)内肾上腺素(NA)能神经和肽能神经的分布,脊神经节内NA能纤维的起源以及脊神经节和交感神经节在疼痛调控中的关系。方法:SD大鼠36只,随机分为A、B、C、D、E、F六组,每组6只,进行如下操作:A组动物灌注、固定后取C3-C5段DRG及同侧交感颈上神经节,进行SP和TH免疫组化染色,观察免疫反应纤维在DRG和SG中分布;B组动物应用HRP神经顺行追踪法,C组动物应用Bb神经逆行追踪法观察DRG和SG的神经纤维联系,D组作为对照;E组动物切除交感神经节及交感干后测定双上肢50%机械缩腿阈值,F组作为对照。结果:颈脊神经节中有大量的SP阳性胞体和纤维,无TH阳性细胞,但有一些TH阳性纤维和终末;交感颈上节中TH阳性胞体和纤维大量存在,环绕着节细胞及其卫星细胞,无SP阳性胞体,但有阳性纤维和终末不均匀地分布;交感颈上节内注射HRP后,可在同侧C1-C8段脊神经节观察到标记细胞,脊神经节内注射Bb后,可在同侧交感颈上节内观察到Bb标记神经元胞体;交感神经切除后上肢50%缩腿阈值二周达到稳定,与对照组相比阈值显著升高(P < 0.05)。结论:交感节后纤维可直接到达DRG并与节细胞发生接触,通过作用于DRG胞体来调节初级感觉信息的传入;另外SG内的SP纤维来自于DRG初级传入神经元的轴突,提示DRG和SG神经元在中枢外存在反射调节机制;交感神经切除提高了大鼠痛阈,使其痛觉敏感性降低。目的:研究颈椎小关节的神经支配来源及其通路,为阐明小关节损伤后弥漫性头颈肩部疼痛的机制及其治疗提供依据。方法: 39只SD雄性大鼠,分为颈交感神经未切除组(A组,18只)、切除组(B组,18只)及对照组(C组,3只),A、B组分别于C1-2、C3-4和C5-6小关节囊注射5%双苯甲亚胺(Bb)0.6μl,C组注射等量生理盐水。8h后检查各组C1-C8脊神经节(DRG)和交感神经节(SG)的荧光标记细胞,并作统计学分析。结果: A组脊神经节和交感神经节内均可见到荧光标记细胞,其中,C1-2和C3-4关节囊内注射组C1-C8 DRG内有Bb(+)细胞,C5-6关节囊内注射组的C3-C8 DRG内可见Bb(+)细胞,B组注射节段和相邻节段Bb(+)细胞无明显减少,但远节段的Bb(+)细胞明显减少;C组未见Bb(+)细胞。结论:颈椎小关节囊神经支配来源于感觉和交感神经系统,脊神经节和交感神经节间有神经纤维联系,阻断脊神经和交感神经在临床上有可能缓解患者的头颈肩痛症状。目的:三叉神经节和上位颈脊髓后根在解剖和功能上有密切的联系,本实验观察了三叉神经节(TG)中P物质(SP)免疫反应物的分布,并探讨C1-3小关节和三叉神经节(TG)间的神经纤维联系及其在颈源性头痛(CeH)中的外周作用机制。方法:40只SD雄性大鼠,随机分为5组,前4组分别于C1-2、C2-3、C3-4、C4-5单侧小关节囊注射5%双苯甲亚胺(Bb)0.6μl,8小时后检查TG荧光标记细胞,第5组观察TG中P物质(SP)免疫反应物的分布。结果作统计学分析。结果: C1-2、C2-3、C3-4组TG中可见到荧光标记细胞,其中C3-4组荧光标记细胞明显减少,C4-5组未见荧光标记细胞;TG中SP免疫阳性细胞不均匀地分布于周围突中,荧光标记细胞和SP免疫阳性细胞均以中小型细胞为主,发出细的薄髓和无髓纤维。结论:C1-3小关节有来源于TG中枢突的侧支或周围突的SP神经纤维分布,寰枕、寰枢或C2-3小关节的不稳定或病变可引起枕颈部的疼痛和三叉神经支配区的疼痛,此种疼痛不同于中枢汇聚机制所导致的牵涉痛,表明CeH存在中枢外的反射机制。目的:NPY及其受体在调控躯体感觉伤害性信息中发挥着重要的作用,周围神经损伤后,NPY及其受体mRNA在脊神经节的表达发生可塑性变化,然而颈神经损伤后DRG中神经肽与其受体之间的确切关系仍不清楚,本研究在大鼠颈神经根性痛模型中观察了DRG中NPY、Y1R和Y2R的表型变化,以期能阐明神经病理性痛的外周发病机制,为未来新药的开发提供靶点。方法:制作大鼠颈神经病理痛模型(SNL模型),手术组、假手术组和正常对照组动物分别于术后7d、14d灌注后取左侧C5和C6脊髓背根神经节,应用免疫荧光染色、聚合酶链反应和Western印记分析技术检测了SNL大鼠不同时间点DRG中NPY、Y1R和Y2R基因和蛋白的表达变化。结果:假手术组和对照组DRG中无NPY免疫反应阳性细胞和神经纤维显示,神经损伤后,DRG内出现NPY阳性细胞,主要位于小型细胞;假手术组和对照组DRG中可见Y1R和Y2R红色荧光标记,神经损伤后,Y1R和Y2R表达增强,其中Y1R主要见于小型细胞,Y2R主要见于大型细胞。NPY和Y1R、Y2R双标神经元主要是大细胞。假手术组和对照组DRG中不表达或极低水平表达NPY mRNA和蛋白,但可见Y1R和Y2R的表达,它们伤后表达水平均显著上调(P<0.01)。结论:Y1R和Y2R在调节痛敏中有重要作用,NPY参与抗伤害作用是通过其受体完成的,Y1R和Y2R是镇痛作用的靶点,并且可使镇痛作用维持。Y2R是神经病理性疼痛发病过程中的关键分子,设计针对其靶点的药物,可对疼痛进行基因治疗。更多还原

【Abstract】 Objective: Headache and pain in the neck are common in clinic, it is important for elucidating the details of the neuroanatomy and neurochemistry of the nerves innervating the posterior longitudinal ligament (PLL), spinal dura, facet joint capsule and vertebral artery(VA) so to provide a basis for prevention and therapy. Materials and Methods: 12 male Sprague-Dawley rats were divided into Sympathectomy rats (A group n=6) and Non-Sympathectomy rats(B group n=6),Bilateral cervical sympathetic chain and ganglia were removed in A group, and the operation in B group is in the same manner with A group but sympathetic chain and ganglia were not removed.one day after operation all the rats were perfused under deep anesthesia and the posterior longitudinal ligament (PLL), the spinal dura mater, the facet joint capsules and the vertebral artery were resected and processed for SP and TH immunohistochemistry. Immunoreactive(IR) fibers were traced using a light microscope and the positive area of the immunoreactive fibers were calculated.Results: tissues around the cervical vertebrae innervated by the SP-IR fibers and TH-IR fibers which comprises the dura mater, posterior longitudinal ligaments, facet joint capsule and vertebral artery. The innervation of the dura mater mainly located in the epiradicular sheath and ventral side and immunoreactive fibers on the PLL are mainly in the intervertebral portion,the majority of nerve fibers distributed on the external membrane and median membrane of VA were sympatheticpost-ganglia fiers. The numbers of TH-IR fibers on all tisues were significant difference after Sympathectomy(p<0.01).Conclusion: Neuropeptide SP was approved to co-localize with NE in the tissues around cervical spinal,it is suggested that these tissues are related to cervicogenic pain, and Sympathetic nerves may play an important role for regulations of primary afferent.A large number of SP-IR fibers were found in the dorsal root ganglion area, and Radicular pain in the upper extremities may be induced by stimulation of the sensory fibers of the epiradicular sheath alone. A majority of sympathetic fibers were distributed in the external and median membrane of VA and mechanical pressure or inflammation of the tissues may cause spasm of VA and vertebrobasilar insufficience(VBI). Objective: To observe the distribution of noradrenergic nerves and peptidergic nerves in spinal ganglion(DRG) and superior cervical ganglion(SG), and to investigate the origin of noradrenergic nerves in spinal ganglion and the interaction in regulating primary sensory afferent between DRG and SG. Materials and Methods: 36 male Sprague-Dawley rats were divided into six groups,6 rats in A group were perfused,DRGs from C3 to C5 and SG were removed for SP and TH immunohistochemical observations;The fiber connection between DRG and SG were observed by the HRP anterotracing technique in B group(6 rats) and Bb retrotracing technique in C group(6 rats);The liminal value of mechanical contractive leg in E group(6 rats) were measured after the bilateral sympathetic chain and ganglia were removed; nonoperated rats in F group(6 rats) were used as normal control subjects. Conclusion: Postsympathetic nerve fibers could directly reach the DRG and contact with DRG neurons,through which primary somatic sensory afferent message could be regulated;Futhermore what SP immunoreactive fibers in the SG came from primary sensory neurons in the DRG suggested that a regulation mechenism between DRG neurons and SG neurons outside the central nervous system exists; cervical sympathectomy raised the threshold of pain sensitivity. Objective: To investigate the pattern of innervation of the cervical facet joint and to determine the pathways from the facet joint to dorsal root ganglions in order to clarify the causes of diffuse neck pain headache and shoulder pain and provide a basis for therapy after facet joint lesions. Method: 39 male Sprague-Dawley rats in three groups were used, including Non-Sympathectomy rats (Group A; n=18), Sympathectomy rats (Group B; n=18) and control rats (Group C; n=3). 5% Bisbenzimide(Bb) were injected into the C1-2, C3-4 and C5-6 facet joints in group A and group B while Bb was replaced with 0.9% saline in group C. Numbers of the labeled neurons in dorsal root ganglions (DRG) from C1 to C8 and sympathetic ganglions(SG) were counted and statistic analysis was made. Result: Neurons labeled with Bb were present in both DRG and SG in group A, which were present from C1-C8 DRG after C1-2 and C3-4 Bb administration and from C3-C8 after C5-6 Bb administration. The number of Bb(+) neurons in group B was not significantly different in injected level from that in group A, but in the other levels Bb(+) neurons was significantly less than that in group A; No labeled neurons was found in group C. Conclusion: The innervation of the cervical facet joint were derived from the both sensory and sympathetic nervous system, DRG were associated with SG through nerve fibers. Blockade of other spinal nerves or sympathetic ganglions is possibly effective for patients with facet disorders. Objectives: Trigeminal ganglia is closely associate with upper spinal dorsal root in anatomy and function.The study explored the fiber connection between C1-3 facet joint and trigeminal ganglia , as well as peripheral mechanism of cervicogenic headache stemming from the C1-3 facet joint. Methods:40 male Sprague-Dawley rats were randomly divided into five groups , 5% Bisbenzimide(Bb) were respectively injected into the C1-2(A group), C2-3(B group) ,C3-4(C group) and C4-5(D group) facet joints on left side , Bb labeled cells were observed after 8 hours, and in E group the distribution of SP-IR(immunoreactive) cells were detected by immunocytochemical technique . Results: neurons labeled with Bb were present in A,B,C and D group , the number of Bb labeled neurons in C group was significantly less than that in A and B group while in D group no Bb labeled neurons was found; and SP-IR neurons were unevenly located in the trigeminal peripheral root ,both Bb labeled neurons and SP-IR neurons were composed primarily of middle and small sized cells contained a higher percentage of thin myelinated and unmyelinated fibers. Conclusions :The innervation of C1-3 facet joint partly stemming from collateral branches of trigeminal sensory root or directly peripheral root,injuries or instability of the atlanto-occipital joint ,the lateral atlanto-axial joint and the C2-3 zygapophysial joint can cause occiput and regions innervated by the trigeminal nerve pain.It indicated that a peripheral mechanism of cervicogenic headache outside central system existed, which is differ from referred pain induced by convergence.. Objective: NPY and its receptors play a modulatory role in processing nociceptive information, Peripheral nerve injury can induce a marked plasticity in the expression of the NPY and its receptor mRNA, However, the exact relations between NPY and its receptors that are regulated in DRG neurons after cervical nerve injury have been studied only to a limited extent. NPY and its receptors phenotype of DRG neurons after nerve injury were observed at present study so as to demonstrate the peripheral mechnisms of neuropathic pains and provide a target for developing new drugs.Material and methods: Neuropathic pains model was established in Sprague-Dawley rats,Rats in SNL group and sham group and control group were perfused and left C5 and C6 DRGs were removed respectively 7 and 14 days after operation,The expression of NPY and Y1 and Y2 receptors in gene and protein level were detected by immunofluorescence and transcription polymerse chain reaction(RT-PCR) and western blotting.Results: In sham and control DRGs ,no NPY-immunoreactive (IR) fibers and cell bodies could be observed while Y1R and Y2R signals were visualized,after nerve injuries strongly labeled NPY-immunoreactive cells were Detected and the expression of Y1R and Y2R showed upregulated, Y1R immunoreactive was prominent in a population of small-sized cells and Y2R was in a large cells.NPY and Y1R or Y2R double-labeled neuron mainly were large cells. In sham and control DRGs,there were no expression or in a low level expression of NPY mRNA and protein, significant differences were noticed after nerve injuries(P<0.01).Conclusions: Y1R and Y2R may play an important role in regulation of hyperalgesia; NPY manage nociceptive pain may act on Y1 and Y2 expressed and its receptors represent an attractive target for analgesics that may retain their effectiveness in neuropathic pain states.Y2R was a key molecule in neuropatic pain at which to design a new drug to aim directly may carry out gene therapy for neuropathic pain.更多还原