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%(P<0.01),2T-PNS和4T-PNS后显著增加到84.0±7.8%和93.2±15.0%(P<0.01)。
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(P<0.05)。
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%(P<0.01)。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(P<0.05)。
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%(P<0.01),2T-PNS和4T-PNS后膀胱容量明显增大92.4±12.0%和107.6±14.7%(P<0.01),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,P<0.01)。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(P<0.01)。
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(P<0.05),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。
Introduction
Overactive bladder (OAB) is defined as the presence of urinary urgency, typically accompanied by frequency and nocturia, with or without urgency incontinence. The etiology and pathophysiology of OAB are still unknown. Conservative treatment for OAB includes behavioral modification and medical therapy such as anti-muscarinics. Though clinically effective, anti-muscarinics still have common side effects including dry mouth, constipation, blurred vision, and cognitive impairment leading to poor compliance and unmet expectations. Neuromodulation is an attractive alternative when conservative treatment fails.
Neuromodulation works through afferent nerve stimulation to the sacral nerve root (S2-4) to modulate the function of bladder and/or urethral sphincters to improve the symptoms of OAB. Sacral neuromodulation (SNM) was the first pathway for clinical neuromodulation for OAB. Common complications of SNM include pain and/or infection at the implantation site, lead migration and battery failure. Therefore, less invasive pathways including tibial nerve and pudendal nerve emerged for neuromodulation. However, the mechanisms underlying neuromodulation of OAB are still uncertain. Most studies focus on two aspects:the anatomical site of action and nerotransmitters involved in neuromodulation.
The lower urinary tract is innervated by an integrated afferent and efferent neuronal complex of peripheral neural circuits involving sympathetic, parasympathetic, and somatic neurons. It is known that reflex bladder activity is controlled by two distinct neural pathways-a spinobulbospinal micturition reflex pathway activated by non-nociceptive Aδ afferents and a spinal micturition reflex pathway activated by nociceptive C-fiber afferents.
The potential sites of neuromodulation action consist of spinal cord and peripheral nerve system, and supraspinal system e.g. brain and pontine. A clinical study found that early SNM implantation in patients with spinal cord injury (SCI) prevented detrusor overactivity and urinary incontinence suggesting that SNM works at spinal cord. Acetic acid (AA) induced nociceptive bladder reflex in cats. Bladder overactivity was inhibited by tibial nerve stimulation (TNS) and pudendal nerve stimulation (PNS) mediated by inhibitory effect of interneurons in spinal cord on parasympathetic efferent pathway.
In cats with chronic spinal cord injury, electrical stimulation of the pudendal nerve on one side at different frequencies induced either inhibitory or excitatory effects on bladder activity. Low frequency (3-10Hz) stimulation elicited an inhibitory effect, while higher frequency (20-40Hz) stimulation produced a facilitatory effect on bladder activity. Excitatory and inhibitory pathways can be activated differentially at the level of the pudendal nerve according to stimulus frequency. The ability to control selectively both continence and micturition with a single electrode on a peripheral nerve is an exciting prospect for neurorehabilitation.
Animal studies have found that various neurotransmitters are involved in neuromodulation for OAB, e.g. inhibitory amino acids y-aminobutyric acid (GABA),5-hydroxytryptamine (5-HT), opioid peptide, and glutamate. It has been reported that AA was used to irritate the bladder and induce nociceptive bladder overactivity, while saline was used to distend the bladder and induce non-nociceptive bladder activity in cats. TNS has an inhibitory effect on both nociceptive and non-nociceptive bladder activities. Furthermore, opioid receptors are involved in nociceptive rather than non-nociceptive bladder reflex. However, opioid receptors are not involved in pudendal inhibition of bladder overactivity but metabotropic glutamate5receptors are partially involved. Therefore, it is speculated that multiple neurotransmitters are involved in pudendal inhibition of nociceptive and/or non-nociceptive bladder reflexes.
GABA which is a major inhibitory neurotransmitter at both spinal and supraspinal synapses has been implicated in the control of micturition in animals. In cats, application of GABA or GABAA receptor agonists by the intrathecal route, iontophoretically to bladder parasympathetic preganglionic neurons in the sacral spinal cord inhibits preganglionic neuron firing and/or reflex bladder activity. Because PNS also elicits inhibitory postsynaptic potentials in bladder parasympathetic preganglionic neurons in the cat, we hypothesize that pudendal neuromodulation might inhibit bladder overactivity by stimulating spinal GABAergic inhibitory mechanisms.
This study consists of two parts:(1) Picrotoxin (a GABAA receptor antagonist) was administered intravenously (i.v.) or intrathecally (i.t.) to examine the role of GABAA receptors in PNS inhibition by using anesthetized cats.(2) The effects of tibial and pudendal nerve stimulation on reflex bladder activity were investigated in cats with acute cord injury at T9-10under a-chloralose anesthesia. Under isovolumetric contraction, electrical stimulation of tibial and pudendal nerve on one side at different frequencies were performed to study bladder activity. Hexamethonium bromide was intravenously administered followed by intrathecal injection lidocaine at L6-7to examine the amplitude changes of bladder contraction. This novel animal model was established to study bladder activity mediated by nociceptive spinal reflex and reveal the mechanism of somatic neuromodulation for OAB.
Part1:Differential Role of Spinal GABAA Receptors in Pudendal Nerve Inhibition of Nociceptive and Nonnociceptive Bladder Reflexes in Cats
Objective
To examine the role of spinal GABAA receptors in PNS inhibition of OAB
Method
1. Surgical procedure
Thirty-three cats were used in this study. After the animals were anesthetized, a tracheotomy was performed and a tube was inserted to keep the airway patent. A catheter was inserted into right carotid artery to monitor systemic blood pressure. Right and left cephalic veins were catheterized for i.v. administration of drugs and fluid. The ureters were isolated and cut for external drainage. A double lumen catheter was inserted through the urethra into the bladder. One lumen was connected to a pump to slowly infuse saline or0.25%AA. The other lumen was attached to a pressure transducer to measure bladder pressure. The right pudendal nerve was isolated and a tripolar cuff electrode was implanted around the pudendal nerve and connected to a stimulator via a constant voltage stimulus isolator, In10cats, a small incision was made to remove the L3spinal process and expose the spinal cord. Then, the spinal dura was pierced and a fine catheter was inserted caudally underneath the dura to position catheter tip at SI sacral spinal cord for i.t. injection of picrotoxin.
2. Stimulation protocol
Uniphasic rectangular pulses (5Hz frequency,0.2ms pulse width) were used to stimulate the pudendal nerve. The stimulation threshold (T) was defined as the minimal intensity for inducing anal sphincter twitching. PNS of multiple intensity thresholds (2T/4T) was used to inhibit bladder activity.
3. Drug administration
In the first group (N=13), repeated cystometrogram (CMGs) were performed with infusion of0.25%AA to irritate the bladder, activate nociceptive bladder afferent C-fibers, and induce bladder overactivity. Picrotoxin administered intravenously or intrathecally to examine the role of GABAA receptors in PNS inhibition. In the second group (N=20), repeated saline CMGs were performed to activate non-nociceptive Aδ afferent initiated micturition reflexes. Picrotoxin was administered intravenously or intrathecally to examine the role of GABAA receptors in PNS inhibition. CMG data was collected for statistical analysis.
4. Data analysis
CMGs were performed by slowly infusing the bladder with saline to determine the bladder capacity that is defined as the bladder volume threshold required to induce a micturition contraction of large amplitude (>30cmH2O) and long duration (>20seconds). For each CMG bladder capacity was normalized to the initial saline control capacity in the same animal, which allowed for comparisons between animals. The bladder capacities were averaged for each condition and reported with standard error of the mean. Student T-test or ANOVA followed by Dunnett or Bonferroni post-tests was used to determine statistical significance (p<0.05).
Result
1. AA irritation significantly (p<0.01) reduced bladder capacity to34.3±7.1%of the saline control capacity; while PNS at2T and4T significantly (p<0.01) increased AA bladder capacity to84.0±7.8%and93.2±15.0%, respectively, of the saline control.
2. Picrotoxin (0.4mg, i.t.) did not change AA bladder capacity but completely removed PNS inhibition of AA-induced bladder overactivity.
3. Picrotoxin (i.v.) only increased AA bladder capacity at a high dose (0.3mg/kg) but significantly (p<0.05) reduced2T PNS inhibition at low doses (0.01-0.1mg/kg).
4. During saline cystometry, PNS significantly (p<0.01) increased bladder capacity to147.0±7.6%at2T and172.7±8.9%at4T of control capacity; and picrotoxin (0.4mg, i.t. or0.03-0.3mg/kg, i.v.) also significantly (p<0.05) increased bladder capacity.
5. Picrotoxin treatment did not alter PNS inhibition during saline infusion.
Conclusion
AA-induce bladder overactivety can be inhibitited by PNS and spinal GABAA receptor is involved in PNS inhibition of nociceptive bladder reflex mediated by C-fiber. PNS also has an inhibitory effect on saline distance induced bladder reflex and spinal GABAA receptor is not involved in PNS inhibition of non-nociceptive bladder reflex mediated by Aδ fiber. Spinal GABAA receptors have a tonic facilitatory role of in the control of normal bladder reflex activity. These results indicate that spinal GABAA receptors have different roles in controlling nociceptive and non-nociceptive reflex bladder activities and in PNS inhibition of these activities.
Part2:Somatic Neuromodulation of Bladder Activity Mediated by Nociceptive Spinal Reflex in Cats
Objective
To establish an animal model to study bladder activity mediated by nociceptive spinal reflex and study the mechanism of somatic neuromodulation for OAB
Method
1. Surgical procedure
Twelve cats were used in this study. After the animals were anesthetized, a tracheotomy was performed and a tube was inserted to keep the airway patent. A catheter was inserted into right carotid artery to monitor systemic blood pressure. Right cephalic vein was catheterized for i.v. administration of drugs and fluid. The ureters were isolated and cut for external drainage. A double lumen catheter was inserted through the urethra into the bladder. One lumen was connected to a pump to slowly infuse saline or0.25%AA. The other lumen was attached to a pressure transducer to measure bladder pressure. The right pudendal nerve and left tibial nerve were isolated and a tripolar and bipolar cuff electrodes were implanted around nerves and connected to a stimulator via a constant voltage stimulus isolator. A small incision was made to remove the T9-10spinal process and expose the spinal cord for complete SCI later. Laminectomy was performed at the level of L6-7spinal cord for i.t. administration of lidocaine.
2. Stimulation protocol
Uniphasic rectangular pulses (5Hz frequency,0.2ms pulse width) were used to stimulate the pudendal and tibial nerve. The stimulation threshold (T) was defined as the minimal intensity for inducing anal sphincter twitching or left toe movement. PNS and TNS of multiple intensity thresholds (2T/4T) were used to inhibit bladder activity.
3. Cystometrogram
Repeated CMGs were performed with infusion of saline to determine bladder control capacity. After SCI,0.25%AA was infused to irritate the bladder and induce bladder overactivity. When the bladder capacity stabilized, four CMGs were performed with AA infusion:(1) control CMG without PNS,(2) CMG during2T-PNS,(3) CMG during4T-PNS, and (4) control CMG without PNS to determine any post-stimulation effect. When the bladder capacity stabilized, four CMGs were performed with AA infusion:(1) control CMG without TNS,(2) CMG during2T-TNS,(3) CMG during4T-TNS, and (4) control CMG without PNS to determine any post-stimulation effect.
4. The effect of PNS and TNS with different frequencies on isovolumetric bladder activity
2T-PNS and4T-TNS (0.2ms pulse width) with different frequencies (0.5,1,5,10,20,40Hz) were performed to deterime the effect of somatic neuromodulation on isovolumetric bladder activity.
5. The effect of hexamethonium bromide (i.v.) and lidocaine (i.t.) on isovolumetric bladder activity
Hexamethonium bromide (10mg/kg) was administered intravenously to examine the changes of bladder pressure. Five minutes later, laminectomy was performed at the level of L6-7spinal cord for i.t. administration of lidocaine to examine the changes of bladder pressure.
6. Data analysis
For each CMG bladder capacity was normalized to the initial saline control capacity in the same animal, which allowed for comparisons between animals. Bladder activity was defined as the ratio between area under curve (AUC) during stimulation and AUC before stimulation to determine the effect of PNS and TNS with different frequencies on isovolumetric bladder activity. The amplitude of bladder contraction was measured to study the effect of hexamethonium bromide (i.v.) and lidocaine (i.t.) on isovolumetric bladder activity. All data was expressed with standard error of the mean. Student T-test or ANOVA followed by Dunnett or Bonferroni post-tests was used to determine statistical significance (p<0.05).
Result
1. After acute SCI, AA irritation significantly (p<0.01) reduced bladder capacity to68.8±6.4%of the saline control capacity. PNS at2T and4T significantly (p<0.01) increased A A bladder capacity to92.4±12.0%and107.6±1.7%, respectively, of the saline control; while TNS at2T and4T did not alter bladder capacity.
2. After intravenous injection of hexamethonium bromide, the isovolumetric bladder activity was significantly decreased from19.3±2.9to8.4±1.9cndH2O (p<0.01). After intrathecal injection of2%lidocaine, the isovolumetric bladder activity was significantly decreased from8.4±1.9to3.9±1.0cmH20(p<0.01).
3. The isovolumetric bladder activity was significantly inhibited by2T-PNS at0.5,1,5and40Hz.2T-PNS at10or20Hz and4T-TNS at0.5,1,5,10,20or40Hz did not alter isovolumetric bladder activity.
Conclusion
Our results demonstrate that AA-induce nociceptive bladder activity after acute SCI can be inhibited by PNS; while TNS has no effect on bladder overactivity. The inhibitory effect of PNS is frequency dependent. The spinal nociceptive bladder activity can be partially blocked by intravenous hexamethonium bromide injection. This novel animal model of nociceptive bladder activity provides a new approach to study the mechanism of somatic neuromodulation for OAB.
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