外周神经电刺激治疗膀胱过度活动症的神经调节机制

发布时间：2018-04-09 13:23

  本文选题：膀胱　切入点：外周神经　出处：《山东大学》2014年博士论文

【摘要】：前言 膀胱过度活动症是一种以尿急症状为主的症候群,常伴有尿频和夜尿,可伴有或不伴有急迫性尿失禁。其病因和发病机制尚不明确,首选治疗方案包括行为训练和药物治疗,其中药物治疗以抗胆碱能制剂为主。虽然大量研究证实抗胆碱能药物的有效性,但常见的副作用如口干、便秘、视觉模糊和认知损害以及病人耐受性差等因素影响其临床应用。当保守治疗效果不佳或药物治疗有明显副作用时,可以考虑选择神经电刺激进行治疗。 神经电刺激主要是利用特定参数的电流,对骶髓的2-4节段(S2-4)神经根及其分支进行刺激,干预排尿反射的神经通路从而达到调节膀胱和(或)尿道括约肌的作用。临床最先应用的刺激部位是骶神经,电极植入手术创伤大,存在电极移位、疼痛、感染和电池失效等术后并发症。因此,临床开始应用胫神经和阴部神经等外周神经作为电刺激的部位,手术创伤小,安全有效。神经电刺激的作用机理仍不明确,其研究可从以下两方面入手：一是寻找电刺激发挥作用的解剖部位,二是明确神经调节过程中的神经递质及其药理作用。 膀胱反射的神经调节极为复杂,包括脊髓和外周神经,脊髓以上的大脑、脑桥和小脑等。生理状态下,膀胱逼尿肌的张力感受器通过有髓鞘的A6传入纤维传递膀胱内压力,经脊髓传递到脑桥排尿中枢。通过脊髓下行传导通路,介导非伤害性的脊髓脑脊髓膀胱反射。病理状态下,感染、化学性刺激或冷刺激等激活无髓鞘的C传入纤维,介导伤害性的脊髓膀胱反射。 研究发现,外周神经电刺激可通过中枢或外周神经通路起作用,包括脑、脊髓和C传入神经纤维等。完全性脊髓损伤患者早期行骶神经刺激可预防尿失禁发生,说明骶神经刺激可在脊髓水平起作用。动物研究发现,乙酸灌注膀胱后通过激活C传入神经纤维诱发伤害性脊髓膀胱反射。胫神经电刺激(Tibial nerve stimulation, TNS)和阴部神经电刺激(Pudendal nerve stimulation, PNS)后激活脊髓内的抑制性中间神经元,通过抑制副交感传出通路进而抑制膀胱的过度活动。 对慢性脊髓损伤的猫进行阴部神经电刺激,发现不同频率的电刺激可兴奋或抑制膀胱收缩。低频率的阴部神经电刺激(3-10Hz)抑制膀胱活动,而高频率电刺激(20-40Hz)可兴奋膀胱收缩。采用不同频率电刺激阴部神经后可能激活不同的神经传导通路,从而产生兴奋或抑制膀胱的作用。这一发现具有良好的临床应用前景,通过植入外周神经电刺激器,有可能实现脊髓损伤患者的排尿和储尿双相控制。 已知多种神经递质参与外周神经电刺激对膀胱活动的神经调节,如抑制性氨基酸Y-氨基丁酸(γ-aminobutyric acid, GABA)、五羟色胺(5-hydroxy tryptamine,5-HT)、阿片肽、谷氨酸。动物研究发现,胫神经电刺激可抑制伤害性和非伤害性膀胱反射,阿片受体参与正常膀胱反射以及胫神经刺激对伤害性膀胱反射的抑制过程,代谢型谷氨酸受体参与胫神经调节。在阴部神经电刺激抑制膀胱过度活动的过程中,发现代谢型谷氨酸受体5和5-HT3参与其中,阿片受体不起作用。因此推测,阴部神经的抑制过程需要多种受体,其他抑制性神经递质和受体如GABA受体和甘氨酸可能参与阴部神经电刺激对膀胱过度活动的调节。 GABA是哺乳动物中枢神经系统中重要的抑制性神经递质之一,存在于脊髓和脊髓以上中枢神经系统的突触部位。猫鞘内注射GABA或GABAA受体激动剂,膀胱活动受到抑制,膀胱副交感节前神经元放电减弱。阴部神经电刺激同样可引起膀胱副交感节前神经元产生抑制性突触后电位。因此推测,脊髓GABAA受体可能参与阴部神经电刺激对膀胱过度活动的神经调节机制。 本研究分为两个部分：第一部分通过向猫膀胱内灌注生理盐水和乙酸,诱发非伤害性和伤害性膀胱反射。行膀胱压力容积测定,电刺激阴部神经联合GABAA受体拮抗剂印防己毒素(Picrotoxin, PTX)(静脉注射或鞘内注射)。分析膀胱容量变化,研究脊髓GABAA受体在非伤害性膀胱反射和伤害性膀胱反射以及阴部神经电刺激的膀胱抑制过程中的作用。第二部分通过完全横断猫T9-10脊髓,建立伤害性脊髓膀胱反射的动物模型。向猫膀胱内灌注乙酸,行阴部神经和胫神经电刺激后分析膀胱容量变化,以研究外周神经电刺激对伤害性脊髓膀胱反射的影响。等容量收缩条件下,用不同频率刺激阴部神经和胫神经,研究不同频率的外周神经电刺激对膀胱活动的影响。等容量收缩条件下,先静脉注射神经节阻断剂溴化六甲双铵(Hexamethonium bromide, Hx),膀胱稳定后鞘内注射局部麻醉剂利多卡因,分析膀胱收缩强度的改变,研究伤害性脊髓膀胱反射的神经机制。 第一部分：脊髓GABAA受体在阴部神经电刺激治疗膀胱过度活动中的作用 目的： 研究脊髓GABAA受体在阴部神经电刺激治疗膀胱过度活动中的作用,进一步明确阴部神经电刺激的神经调节机制。 方法： 1、手术过程：本实验有33只猫,麻醉成功后行右侧颈动脉和气管插管,记录动脉血压和保持气道通畅。左、右头静脉插管,建立静脉输液和药物通道。离断双侧输尿管,单侧接导管引流尿液。双腔导管经尿道插入膀胱,一端接灌注泵,分别向膀胱内灌注生理盐水或0.25%乙酸；另一端接膀胱压力传感器,进行膀胱压力容积测定(Cystometrogram, CMG)。分离右侧阴部神经,置入刺激电极。取10只猫,在L3脊髓棘突水平暴露脊髓,打开硬脊膜置入导管达S3脊髓水平,作为印防己毒素鞘内给药通道。 2、阴部神经电刺激阈值强度确定：刺激频率为5Hz,波宽0.2ms。逐渐增加刺激强度,观察到明显的肛门括约肌收缩的最小强度就是阴部神经电刺激的阈值(Threshold, T)。实验中分别用2T和4T进行刺激,以观察阴部神经电刺激对膀胱活动的调节作用。 3、分组和给药：共分为2组,第1组向膀胱灌注乙酸,包括静脉给PTX和鞘内给PTX两个亚组。第2组向膀胱灌注生理盐水,包括静脉给PTX和鞘内给PTX两个亚组。记录各组给予PTX前后阴部神经电刺激对CMG的影响,进行统计学分析。 4、数据分析：实验开始时每只猫膀胱灌注生理盐水,引起超过20秒大于30cmH2O的收缩时的膀胱容量为猫的初始膀胱容量。膀胱容量百分比指膀胱容量与同一只猫的初始膀胱容量的比值。统计数据以平均值加减标准误表示,采用GraphPad Prism4统计软件进行t检验或ANOVA分析。 结果： 1、与初始膀胱容量相比,乙酸组膀胱容量在乙酸刺激后显著减少到34.3±7.1%(P0.01),2T-PNS和4T-PNS后显著增加到84.0±7.8%和93.2±15.0%(P0.01)。 2、PTX (0.4mg,it)未改变乙酸组膀胱容量,阴部神经电刺激对膀胱过度活动的抑制作用完全消失。 3、高剂量PTX (0.3mg/kg iv)增加乙酸组膀胱容量,低剂量PTX(0.01-0.1mg/kg iv)后2T-PNS对膀胱过度活动的抑制作用明显减弱(P0.05)。 4、生理盐水组中,与初始膀胱容量相比2T-PNS和4T-PNS后膀胱容量显著增加147.0±7.6%和172.7±8.9%(P0.01)。鞘内注射PTX (0.4mg)和静脉注射PTX (0.03-0.3mg/kg)后膀胱容量显著增加(P0.05)。 5、生理盐水组中,PTX不改变阴部神经电刺激对膀胱活动抑制作用。 结论： 膀胱内灌注乙酸可诱发伤害性膀胱反射,阴部神经电刺激能够抑制膀胱过度活动,脊髓GABAA受体参与此抑制过程。向膀胱内灌注生理盐水可诱发非伤害性膀胱反射,阴部神经电刺激能够抑制正常膀胱反射。脊髓GABAA受体在正常膀胱反射中起兴奋性作用,不参与阴部神经电刺激对正常膀胱反射的抑制过程。脊髓GABAA受体在伤害性和非伤害性膀胱反射以及阴部神经电刺激的膀胱抑制过程中有不同的作用。 第二部分：外周神经电刺激在伤害性脊髓膀胱反射中的作用 目的： 建立伤害性脊髓膀胱反射的动物模型,研究外周神经电刺激治疗膀胱过度活动症的神经调节机制。 方法： 1、手术过程 本实验有12只猫,麻醉成功后行右侧颈动脉和气管插管,记录动脉血压和保持气道通畅。右头静脉插管,建立静脉输液和药物通道。离断双侧输尿管,单侧接导管引流尿液。双腔导管经尿道插入膀胱,一端接灌注泵,向膀胱内灌注生理盐水或0.25%乙酸；另一端接膀胱压力传感器,进行膀胱压力和容积的测定。分离右侧阴部神经和左侧胫神经,分别置入刺激电极。在T9-10脊髓棘突水平行椎扳切除术,膀胱容量稳定后行脊髓完全横向切断术。在L6-7脊髓棘突水平行椎扳切除术,用作实验中向鞘内注射利多卡因。 2、阴部神经、胫神经电刺激阈值强度确定 刺激频率为5Hz,波宽0.2ms。逐渐增加刺激强度,观察到明显的肛门括约肌收缩或左侧脚趾收缩的最小强度就是阴部神经和胫神经电刺激的阈值(Threshold,T).实验中分别用2T和4T进行刺激,以观察阴部神经和胫神经电刺激对膀胱反射的调节作用。 3、膀胱压力和容积的测定 向膀胱内灌注生理盐水测得初始膀胱容量,在T9-10棘突水平行脊髓完全横向切断术,再向膀胱内灌注0.25%的乙酸诱发膀胱过度活动。待膀胱容量稳定后,进行如下CMG测定：(1)乙酸对照；(2)2T-PNS;(3)4T-PNS；(4)乙酸后对照。重复测定待膀胱容量稳定后,进行如下CMG测定：(1)乙酸对照；(2)2T-TNS；(3)4T-TNS；(4)乙酸后对照。 4、等容量收缩条件下,不同刺激频率的阴部神经和胫神经电刺激对膀胱活动的影响 等容量收缩条件下对阴部神经和胫神经进行电刺激,刺激强度分别为2T和4T,波宽为0.2ms,频率依次为5.40.1.20.0.5.10Hz。 5、等容量收缩条件下Hx和利多卡因对膀胱收缩强度的影响 等容量收缩条件下,静脉注射Hx(10mg/kg)检测膀胱压力。5分钟后在脊柱棘突L6-7水平行椎板切除术,鞘内注射2%利多卡因和去甲肾上腺素混合液1-2ml,以观察膀胱压力的改变。 6、数据分析 膀胱容量与同一动物的初始膀胱容量对比,计算膀胱容量百分比。等容量收缩条件下,不同刺激频率时与刺激前曲线下面积对比,计算膀胱活动百分比以比较不同刺激频率对膀胱活动的影响。等容量收缩条件下,测量膀胱压力平均值以表示膀胱收缩强度。统计数据以平均值加减标准误表示,采用GraphPad Prism4统计软件进行t检验或ANOVA分析。 结果 1、与初始膀胱容量相比,T9-10脊髓完全横向切断术后乙酸灌注使膀胱容量明显减小68.8±6.4%(P0.01),2T-PNS和4T-PNS后膀胱容量明显增大92.4±12.0%和107.6±14.7%(P0.01),2T-TNS和4T-TNS后膀胱容量无明显改变。 2、等容量收缩条件下,静脉注射Hx后膀胱压力明显减小(19.3±2.9vs.8.4±1.9cmH2O,P0.01)。鞘内注射利多卡因后膀胱压力再次减小(3.9±1.0cmH2O),膀胱收缩强度明显减弱(P0.01)。 3、等容量收缩条件下,0.5、1、5、40Hz的2T-PNS抑制膀胱收缩,膀胱活动百分比明显降低(P0.05),10、20Hz的2T-PNS时膀胱活动无明显改变。 4、等容量收缩条件下,0.5、1、5、10、20、40Hz的4T-TNS未能抑制膀胱收缩,膀胱活动百分比无明显改变。 结论： 急性脊髓损伤后膀胱内灌注乙酸可诱发膀胱过度活动,阴部神经电刺激能抑制伤害性脊髓膀胱反射,胫神经电刺激则不能。阴部神经电刺激的膀胱抑制作用与刺激的频率有关。Hx可部分阻断伤害性脊髓膀胱反射。本研究建立一种新的伤害性脊髓膀胱反射的动物模型,可用于进行外周神经电刺激治疗膀胱过度活动症的神经调节机制的研究。
[Abstract]:Preface
Overactive bladder is a urgency symptoms syndrome, usually with frequency and nocturia with or without urinary incontinence. Its etiology and pathogenesis is not clear, the preferred treatment including training and drug treatment, including drug treatment with anti cholinergic agents. Although a large number of the research confirmed the effectiveness of anticholinergic drugs, but common side effects such as dry mouth, constipation, blurred vision and the effect of cognitive impairment and poor patient tolerance factor in its clinical application. When conservative treatment ineffective or drug treatment has obvious side effect, can choose the nerve stimulation treatment.
Nerve stimulation is the main current use of specific parameters of the 2-4 sacral segment (S2-4) and its branches of nerve root stimulation, neural pathway intervention to adjust the micturition reflex bladder and (or) urethral sphincter function. Clinical application is the first site of stimulation of sacral nerve electrode implantation, surgical trauma, existence electrode displacement, pain, infection and battery failure and other postoperative complications. Therefore, clinical application of part of the tibial nerve and peripheral nerve pudendal nerve stimulation, surgical trauma, safe and effective. Nerve stimulation for mechanism is not clear, it can start from the following two aspects: one is to look for play the role of stimulating anatomy, two is to clear the neural regulation of neurotransmitter and its pharmacological effects in the process.
Bladder reflex nerve regulation is very complex, including spinal cord and peripheral nerve, spinal cord or brain, pons and cerebellum. Under physiological condition, tension receptors in bladder detrusor by myelinated afferent fibers transmit A6 bladder pressure, the spinal cord to the pontine micturition in the spinal cord. The pivot descending pathway, spinal cord the brain and spinal cord bladder reflex mediated by non noxious. Under pathological conditions, infection, chemical irritation or cold stimulation to activate unmyelinated afferent fibers C, spinal cord mediated nociceptive reflex bladder.
The study found that peripheral nerve stimulation may play a role, through the central or peripheral nerve pathways including the brain, spinal cord and C afferent nerve fibers. Spinal cord injury patients undergoing early sacral nerve stimulation can prevent urinary incontinence, that sacral nerve stimulation could play a role in the spinal cord. Animal studies found that acetic acid perfusion of bladder after activation by C afferent nerve fibers evoked nociceptive spinal cord bladder reflex. The tibial nerve stimulation (Tibial nerve, stimulation, TNS) and pudendal nerve stimulation (Pudendal nerve, stimulation, PNS) after activation of inhibitory interneurons in the spinal cord, by inhibiting parasympathetic efferent pathway and inhibit overactive bladder.
The chronic spinal cord injury of cat pudendal nerve stimulation, different frequency stimulation can excite or inhibit bladder contraction. The low rate of pudendal nerve stimulation (3-10Hz) inhibits bladder activity, and high frequency stimulation (20-40Hz) can be excited by different frequency of bladder contraction. Electrical stimulation of the pudendal nerve may activate nerve different signalling pathways, resulting in excitatory or inhibitory bladder role. This finding has good prospects for clinical application, through the implantation of peripheral nerve stimulator, possible in patients with spinal cord injury urination and urine storage phase control.
The known neurotransmitters involved in peripheral nerve stimulation on bladder activity such as neural regulation of inhibitory amino acid GABA (gamma Y- -aminobutyric acid, GABA), five hydroxytryptamine (5-hydroxy tryptamine, 5-HT), opioid peptide, glutamic acid. Animal studies found that tibial nerve stimulation can inhibit nociceptive and non nociceptive bladder reflex, opioid receptors in normal bladder reflex and tibial nerve stimulation on nociceptive reflex bladder, metabotropic glutamate receptors are involved in the regulation of the tibial nerve. The process of pudendal nerve stimulation inhibits bladder overactivity in the metabotropic glutamate receptor 5 and 5-HT3 involved in opioid receptor does not work. It was suggested that inhibition of the pudendal nerve need a variety of receptors, other inhibitory neurotransmitters and receptors such as GABA receptor and glycine may participate in pudendal nerve stimulation of overactive bladder. Adjust.
GABA is one of the most important inhibitory neurotransmitter in the mammalian central nervous system, the synapses exist in the spinal cord and spinal cord above the central nervous system. The injection of GABA or GABAA receptor agonist cat intrathecal, bladder activity inhibited bladder parasympathetic preganglionic neurons discharge decreases. Pudendal nerve stimulation can cause bladder parasympathetic preganglionic neurons produce inhibitory postsynaptic potential. So, probably GABAA receptors are involved in spinal cord pudendal nerve stimulation on bladder excessive activity of nerve regulation mechanism.
This study is divided into two parts: the first part through to the infusion of physiological saline and acetic acid induced cat bladder, non noxious and noxious bladder reflex. Cystometry, pudendal nerve stimulation combined with GABAA receptor antagonist picrotoxin (Picrotoxin, PTX) (intravenous or intrathecal) analysis. Study on the changes of bladder capacity, spinal GABAA receptor inhibition function in the process of non noxious and noxious bladder reflex bladder reflex and pudendal nerve stimulation of the bladder. The second part of the spinal cord transected cat T9-10 animal model of spinal cord injury, bladder reflex. The cat to intravesical instillation of acetic acid, analysis of bladder volume changes for pudendal nerve and tibial nerve after electrical stimulation, to study the effect of electrical stimulation on peripheral nerve injury of spinal cord bladder reflex. Isovolumic contraction conditions and pudendal nerve stimulation with different frequency Tibial nerve, peripheral nerve electrical stimulation of different frequency effects on bladder activity. Under the condition of isovolumic contraction, the first intravenous injection of ganglionic blocker hexamethonium bromide (Hexamethonium, bromide, Hx), stable bladder after intrathecal injection of local anesthetic lidocaine, analysis of bladder contraction intensity changes, the neural mechanism of injury spinal cord bladder reflex.
Part 1: the role of GABAA receptor in the treatment of overactivity of bladder in the perineum nerve electrical stimulation
Objective:
To study the role of GABAA receptor in the treatment of overactivity of bladder in the perineum nerve stimulation, and to further clarify the mechanism of nerve regulation of the perineal nerve electrical stimulation.
Method锛，

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