氯胺酮相关性膀胱炎发病机制的研究
|
摘要
研究背景和目的
自2007年以来,我们在临床工作中发现了一些长期吸食新型毒品K粉的患者会出现严重的尿频、尿急、尿痛、血尿、排尿困难或急迫性尿失禁等下尿路症状(LUTS)。我们称之为氯胺酮相关性膀胱炎(ketamine cystitis, KC)。氯胺酮相关性膀胱炎的病人的临床表现、实验室检查及病理检查中所发现的特征均与间质性膀胱炎(interstitial cystitis, IC)的病人相类似,2009年的亚洲间质性膀胱炎指南也认为氯胺酮相关性泌尿系损害与IC有同样的发病机制。然而,对于氯胺酮相关性膀胱炎发病机制的研究仍处在起步阶段,还存在以下三点主要的疑问：1.下尿路症状和氯胺酮吸食是否确实有直接的因果关系?2.氯胺酮造成膀胱损伤的机制是什么?3.对于氯胺酮导致的下尿路病人的有效的合适的治疗方案是什么?为了回答这三个问题,我们设计建立了氯胺酮对人膀胱上皮细胞的细胞毒性作用模型(第二章),氯胺酮滥用大鼠膀胱损害的模型(第三章)以及氯胺酮病人症状评分及尿钾水平测定的模型(第四章)分别进行研究。从细胞、动物及临床水平分别探氯胺酮相关性膀胱炎的发病机制。
方法
体外实验进行人膀胱永生化上皮细胞SV-HUC-1细胞的培养,细胞生长到对数生长期时,将细胞暴露于100nM、1μM、10μM、100μM浓度氯胺酮培养基中,利用MTT比色法检测氯胺酮对人膀胱永生化上皮细胞增殖的影响,利用Amplex-Red荧光法检测氯胺酮作用于人膀胱永生化上皮细胞时所产生的自由基水平。
动物实验取雄性Sprague-Dawley (SD)大鼠24只,体重180-200g,随机分为空白组(control),生理盐水组(NS),低剂量实验组(LK,5mg.kg-1)和高剂量实验组(HK,50mg.kg-1),每组6只。实验组大鼠每日早晨9点腹腔注射盐酸氯胺酮溶液,溶解于500ul生理盐水。生理盐水组大鼠同时注射等量生理盐水,空白组不予药物处理。大鼠单独饲养与自制的代谢笼中,利用自制的尿液染色试纸记录大鼠排尿频率。大鼠排尿测量实验在第一次给药前连续测3天,之后再第4,8,16周的给药后连续测3天。氯胺酮注射对大鼠尿液的短期影响通过分析尿液中抗增殖因子APF和一氧化氮NO水平完成。首先收集第一次注射氯胺酮前大鼠的6小时尿液作为基准对照,跟着收集注射后尿液36小时,每6小时收集一次。氯胺酮注射对大鼠尿液的长期影响通过分析尿液中糖蛋白GP-51和尿钾离子水平完成,在注射前,注射后4、8和16周分别取24小时尿液。氧化氮NO水平通过Griess Reaction试剂盒完成,APF及GP-51的尿液含量通过ELISA试剂盒法测量,尿钾离子水平通过多功能生化分析仪取得。大鼠予氯胺酮注射16周后,断头处死并称重,取膀胱组织行HE染色光镜下读片拍照。利用免疫组化法分析膀胱上皮组织中的紧密链接蛋白ZO-1及Occludin,和诱导性一氧化氮合酶iNOS的表达。利用免疫蛋白印迹法验证ZO-1,Occludin和iNOS在各组大鼠膀胱组织中的表达。
临床中收集43例氯胺酮相关性膀胱炎的患者,分成两组,A组为32例有严重的下尿路症状但能自行排尿的患者,B组为11例由于严重的下尿路症状而不能或不愿自行排尿在入院后留置尿管的患者。入院时,所有患者除接受常规检查外,均在入院时行盆腔疼痛与尿急/尿频(PUF)评分,并留置24h尿,检测尿钾、尿钠和尿肌酐浓度；同时取我院健康成年人30名,男女各15名,平均年龄22-30岁,在正常饮食情况下,连续测定3天的24h尿钾、尿钠、尿肌酐浓度作为空白对照。所有患者均被要求戒断K粉在,试验性给予静脉注射抗生素、口服肾上腺素能受体阻滞剂、胆碱能受体阻滞剂、戊聚硫钠；透明质酸钠膀胱内灌注及止痛等对症支持治疗方法。平均治疗疗程为12天。出院前复查24小时尿钾和PUF评分。
实验数据以均数±标准差表示,第二章采用SPSS13.0统计软件进行统计分析,在各个时间点不同浓度组间对细胞作用采用One-way ANOVA分析,两两比较采用LSD法检验(方差齐时)。方差不齐时分别采用Welch和Dunnett's T3法,显著性标准为P<0.05。第三章采用Prism version5.0(Graphpad Software, San Diego, CA, USA)统计软件分析处理,连续性数据用One-Way ANOVA检验。组间比较采用Dunnett's correction法,显著性标准为P<0.05。第四章采用SPSS13.0对数据进行处理。对治疗前后各组间的尿钾、尿钠、尿肌酐浓度分析采用One-Way ANOVA。治疗前、后组间的PUF评分比较均采用两独立样本t检验,组内治疗前后的PUF评分比较采用配对样本t检验,P<0.05有统计学意义。治疗前后PUF与对应的24h尿钾浓度的统计学分析采用计量资料的Pearson积差相关分析,P<0.05有统计学意义。
结果
当人膀胱上皮永生化细胞生长至对数期后,以不同剂量的氯胺酮100nM、1μM、10μM、100μM分别处理细胞24小时及48小时后,利用MTT法测量可见氯胺酮1μM、10μM、100μM处理后细胞活性与对照组相比具有统计学差异,但是未观察到药物对细胞有显著生长抑制作用,说明氯胺酮在100nM～100μM的剂量范围内对人膀胱上皮永生化细胞的细胞毒作用无生物学意义。当人膀胱上皮永生化细胞生长至完全融合后,各种浓度的氯胺酮被分别加入到含有细胞的和无细胞的96孔细胞培养板中以AmplexRed法来测试其化学荧光的底色。结果发现,测量60分钟之内,氯胺酮各浓度组在有细胞及无细胞的培养板中所记录的荧光改变数据均呈自然氧化的平缓线性表现,有细胞及无细胞组之间的比较均无统计学差异,说明氯胺酮无法刺激人膀胱永生化上皮细胞产生自由基。
大鼠在注射50mg-1kg氯胺酮溶液1分钟后出现全身僵硬症状,不能活动,随后10分钟出现共济失调,头尾不停摇摆晃动,向左右跌倒,接着开始保持平静不动1小时直到复原。在低剂量组,大鼠注射5mg-1kg氯胺酮溶液后,15分钟内相比生理盐水组合空白组的大鼠出现轻微的兴奋和活跃状态,之后恢复正常。在注射氯胺酮溶液16周后,高剂量组(HK)大鼠体重的增加与生理盐水组(NS)相比显著地减慢(15.6±4.4g vs53.1±4.0g,P<0.05；n=6)。3/6只大鼠在注射高剂量氯胺酮溶液16周后出现了明显的血尿。注射氯胺酮溶液12周后,高剂量组大鼠相比生理盐水组大鼠出现显著的尿频增多(24-hour frequency20.4±0.9vs16.4±0.6,P<0.01)。注射氯胺酮溶液16周后,高剂量组(HK)和低剂量组(LK)与生理盐水组相比均出现了显著的尿频增多(24-hour frequency21.0±0.7vs16.4±0.8,P<0.05；28.6±1.4vs16.4±0.8,P<0.001； n=6)。
高剂量组和低剂量组的时间-抗增殖因子曲线自首次氯胺酮注射后均开始出现增长,高剂量组在开始时产生了较快的反应,12小时后两组APF的反应基本一致,在注射后第30小时时达到最高峰,30小时后两组的曲线开始出现下降,而生理盐水和空白组的曲线保持水平。抗增殖因子APF分泌的量是由各时间段的量除以基础数值取百分比求得,高剂量组在第6小时APF增加的百分数较生理盐水组显著增多(HK113%±3%vs.NS99%±3%,P<0.05,n=6)。在第30小时,高剂量组和低剂量组APF增加的百分数均较生理盐水组显著增多(HK127%±4%vs.NS105%±3%P<0.001；LK124%±8%vs.NS105%±3%P<0.001,n=6)。在注射前APF在各组的平均数是22.3±0.13pg.ml-1(均数±方差,n=24)。
注射后6小时内尿液中一氧化氮的水平计算为基础一氧化氮水平的百分比,首次注射氯胺酮溶液6小时内尿液中一氧化氮的水平在高剂量组和低剂量组均较生理盐水组出现了显著的增多(HK277%±6%vs.NS119%±5%: P<0.001,LK173%±9%vs.NS119%±5%,P<0.05,n=6)。生理盐水和空白对照组之间相比没有统计学差异。一氧化氮水平在首次注射氯胺酮6小时之后的各时间段在各组间相比没有统计学差异。
24小时尿液标本中的尿钾结果除以尿肌酐标准化以提高准确性。随着时间的进展,氯胺酮高剂量和低剂量组的尿钾尿肌酐比(K+/Cr)显著降低(高剂量组：at0weeks46±0.8vs.8weeks36±0.6and16weeks31.5±1.4P<0.05, P<0.001:低剂量组：at0weeks:44.7±0.7vs16weeks36.8±0.8, P<0.05,11=6).生理盐水和空白对照组的尿钾和肌酐的比值K+/Cr随时间进展无显著统计学差异。随着时间的进展,氯胺酮高剂量和低剂量组的尿液GP-51水平显著降低(高剂量组：initial38.98±0.35pg/ml vs.8weeks32.83±0.33pg/ml and16weeks15.74±1.22pg/ml P<0.05,P<0.001；低剂量组：initial37.35±0.49pg/ml vs.8weeks33.68±0.32pg/ml and16weeks34.40±0.30pg/ml P<0.01,P<0.05,n=6)。生理盐水和空白对照组的尿液GP-51水平随时间进展无显著统计学差异。
HE染色结果显示：生理盐水组大鼠膀胱上皮较空白对照组未发现明显的结构改变。高剂量组的大鼠膀胱上皮层较空白对照组显得更厚和更致密。在高剂量组里可以观察到膀胱粘膜下层明显的单核粒细胞浸润,结缔组织纤维化和胶原蛋白沉积。而粘膜下层原有正常的疏松结缔组织在高剂量组明显消失。低剂量组的膀胱病理改变程度鉴于高剂量组和生理盐水组之间。
免疫组化结果显示：诱导型一氧化氮合酶(iNOS)和ZO-1均主要表达在膀胱上皮层,而另一个紧密连接蛋白Occludin主要表达在膀胱粘膜下层的血管内皮细胞中。通过Image-Pro Plus v6.0软件处理量化分析可见,iNOS高剂量和低剂量组的表达较生理盐水组显著增高(平均光密度±方差：LK0.432±0.006vs. NS0.215±0.005P<0.001；HK0.450±0.006vs.NS0.215±0.005P<0.001.),生理盐水组较空白对照组也出现了显著的增高(平均光密度±方差：control0.270±0.005vs.NS0.317±0.006P<0.01,n=6)。Occludin的染色密度在低剂量组和高剂量组均较生理盐水组出现了显著降低(平均光密度±方差：LK0.404±0.005vs.NS0-317±0.006P<0.05；HK0.452±0.008vs.NS0.317±0.006P<0.001,n=6),ZO-1在高剂量组和低剂量组的染色密度则较生理盐水组出现显著增高(平均光密度±方差：LK0.340±0.006vs.NS0.453±0.011P<0.05；HK0.255±0.008vs.NS0.453±0.011P<0.001,n=6)。各组大鼠膀胱中NOS、ZO-1和Occludin的表达由免疫蛋白印迹方法检验。免疫蛋白印迹的量化柱状图结果与免疫组化的量化结果完全一致。低剂量和高剂量组中NOS的相对蛋白表达比率较生理盐水组显著增多(平均比率±方差：LK0.83±0.003vs.NS0.70±0.004P<0.001；HK1.09±0.006vs.NS0.70±0.004P<0.001,n=6,),生理盐水组中NOS的表达较空白组也显著增加(平均比率±方差：control0.63±0.008vs.NS0.70±0.004P<0.01,n=6).低剂量和高剂量组中ZO-1的相对蛋白表达比率较生理盐水组显著减少(平均比率±方差：LK1.08±0.008vs.NS1.55±0.003P<0.001；HK0.73±0.005vs.NS1.55±0.003P<0.001,n=6).低剂量和高剂量组中Occludin的相对蛋白表达比率较生理盐水组显著增加(平均比率±方差：LK0.89±0.003vs.NS0.70±0.005P<0.01； HK1.02±0.01vs.NS0.70±0.005P<0.001,n=6)。
临床中氯胺酮相关性膀胱炎患者治疗前24h尿液样本中的尿钠和尿钾的变化：所有数值均以24小时尿肌酐作为内参进行标准化。经标准化后的A组尿钾浓度分别与对照组、B组比较,比较有统计学差异。(A组1.80vs.B组6.22K+/Cr,P<0.01；A组1.80vs.对照组6.47K+/Cr,P<0.01)。各组尿钠浓度没有统计学差异(P>0.05)。所有患者(A、B组)在接受治疗后,下尿路症状均有明显改善。B组所有患者在拔除导尿管后能自行排尿。标准化后的组间尿钾浓度没有显著统计学差异(P>0.05)。治疗前、后组间PUF评分均无统计学差异(P>0.05),治疗前后组内PUF评分有统计学差异(A组：治疗前23.19±3.64VS治疗后18.31±2.19,P<0.05；B组：治疗前21.95±3.86VS治疗后17.18±2.68,P<0.05)。治疗前A组尿钾浓度与PUF评分呈负相关关系,(r=-0.697,P<0.01)；治疗前B组尿钾浓度与PUF评分没有明显相关关系(r=0.012,P=0.973)；治疗后A组(r=-0.847,p<0.01),治疗后B组(r=-0.881,p=<0.01),尿钾浓度与PUF评分均呈明显负相关关系。
讨论
氯胺酮在100nM～100μM的剂量范围内对人膀胱上皮永生化细胞的细胞毒作用无生物学意义,并且也不能诱导膀胱上皮细胞产生氧化应激反应。因此我们认为是氯胺酮的代谢产物而不是氯胺酮本身对泌尿系统产生了损害。
经过动物实验的结果得出,长期滥用氯胺酮的大鼠,会出现明显的尿频和血尿。我们推测其机制可能是氯胺酮的代谢产物在尿液中对膀胱上皮细胞产生了直接的毒性作用,使膀胱上皮细胞产生了一氧化氮和APF。这种蓄积的毒性作用和APF的持续释放,扰乱破坏了膀胱上皮细胞的正常增殖和自我修复。因此膀胱上皮屏障的完整性被破坏,表现为氨基葡聚糖中的糖蛋白GP-51和细胞紧密连接蛋白ZO-1的表达下降。然后,膀胱上皮的渗透性增加。尿液中的成分如尿酸、尿钾等可以渗入膀胱的间质层和肌肉层,导致炎症细胞的浸润,间质层纤维化和血管增生。尤其是尿钾的渗入,促使膀胱的神经和肌肉去极化,产生尿频、血尿和尿痛的IC样症状。
临床中,新发的、未经治疗的氯胺酮相关性膀胱炎患者中尿液钾离子浓度与正常对照组和留置尿管的重症患者相比显著减低,这也可能是由于膀胱黏膜上皮的缺损,尿液中的钾离子渗透进入膀胱内间隙所致。尿钾浓度的测定对于氯胺酮相关性膀胱炎患者疾病严重程度和治疗效果的判定等均具有良好的指导意义。并且治疗后的两组PUF评分及所有患者的PUF总体分值均较治疗前下降,差异有统计学意义。PUF评分与治疗后24h尿钾存在负相关,PUF评分越低,24h尿钾浓度越高。说明我们采用的综合治疗不仅有效减少了尿钾离子在膀胱壁的重吸收且有助于缓解尿频、尿痛等症状。
Background and Objection
Since2007it has been known that long-term ketamine abuse can affect the urinary system, resulting in lower urinary tract syndrome, frequency, nocturia, urgency, dysuria, suprapubic discomfort and occasional hematuria. Cystoscopy findings and histological changes in bladder biopsies from ketamine abusers are similar to those patients with interstitial cystitis. Intravenous urography and urodynamic studies in a small group (n=6) of chronic recreational ketamine users identified various changes in bladder function and appearance. These subjects had narrowing of both ureters, contracted bladder syndrome with mild bilateral hydronephrosis, detrusor instability and urinary leakage. It remains unclear how ketamine abuse leads to these interstitial cystitis-like, functional and histological changes. It has been postulated that the accumulation of ketamine and/or its metabolites in the urine might cause direct toxic effects on the urinary tract. However, apart from the pathological changes caused by long-term ketamine abuse that are seen clinically and in animal models, no direct evidence has been found that gives a causal relationship between ketamine toxicity and the development of cystitis. In addition, there are currently no studies describing the effects of the urinary metabolites of ketamine on bladder function. The aim of this study was to fully explore the pathogenesis of ketamine cystitis in vivo, in vitro and in clinic.
Methods and materials
Sv-HUC-1cells was cultivated in vitro to the logarithmic growth phase. Then, Sv-HUC-1cells was exposed to100nM、1μM、10μM、100μM dose of ketamine respectively. MTT assay was used to measure the proliferative activity of the urothelial cells with the effect of ketamine. Amplex Red assay was used to measure the generation of free radicals produced by the urothelial cells when exposed to ketamine.
Twenty-four2-month-old male Sprague-Dawley (SD) rats weighing180-200g had free access to standard laboratory rodent chow and water. Groups of six rats were randomly assigned to control, normal saline (NS), low-dose (5mg.kg-1) and high-dose (50mg.kg-1) ketamine groups. The two experimental groups received a single intraperitoneal (i.p.) injection of ketamine hydrochloride dissolved in500μL saline at09:00h each day. The NS group received an i.p. injection of vehicle the same time:rats in the control group were untreated. All rats were weighed weekly to adjust the quantity of ketamine administrated.
Urinary frequency was determined by housing the rats individually in modified metabolic cages that had a fine stainless steel mesh, which retained feces but allowed urine to pass, through its base. Paper, impregnated with saturated copper sulfate solution (CuSO45H2O) and dehydrated at200℃for1h, was placed under the base of the cage. The urine falling onto this paper, rehydrated the anhydrous CuSO4, turning it blue. The number of urinations was determined by counting the number of blue spots observed. Baseline urinary frequency was measured3days before ketamine administration, and then at consecutive3day periods after4,8and16weeks of treatment.
Urine samples were collected from the metabolic cage, but with the CuSO4paper removed. The short-term effect of ketamine was assessed by analyzing urine samples for APF and NO. A6h baseline urine sample was collected immediately before the first ketamine injection and subsequent urine samples were collected at6h intervals for30h. The longer term effects of ketamine were monitored using Gp-51and potassium measurements at baseline and after4,8,16weeks of treatment. Urine was collected for24h, immediately before daily ketamine administration and thereafter24-h urinary volume was recorded daily. Aliquots of urine were stored at-80℃and were thawed once before analysis. The levels of APF and Gp51in urine were measured using an ELISA assay. Levels of NO were estimated via the Griess reaction. Urinary potassium concentration was determined using an ion selective electrode technique in an automated chemistry analyzer.
Rats were killed by spine dislocation, and the bladders were excised. Some of the bladder specimens were stained with hematoxylin and eosin (H&E), and examined under a light microscope. Immunohistochemical studies were performed using the DAKO ChemMateTM EnVisionTM Detection Kit. The following primary antibodies were used:anti-iNOS, anti-occludin antibody and anti-ZO-1. The integrated optical density of cytoplasm and cell membranes containing brown-yellow granules was detected by using Image-Pro Plus v6.0software. Western blot analysis was used to examinate the expression of iNOS, Occludin and ZO-1in the bladder. Protein signals were quantified by scanning densitometry using a FluorChem Q system. The results of western blotting were quantified by Quantity One4.4.0software.
43ketamine-associated cystitis patients (male29cases, female14cases) were analyzed.32patients without indwelling urinary catheter were categorized as group A, while the other11patients with indwelling urinary catheter were in group B. The therapy regimes consisted of anti-inflammatory, antioxidant, relieving spasm and pain, improving the microcirculation and repairing the bladder epithelium barrier.30healthy adults were selected as the controls. Urinary potassium(K), sodium (Na) and creatinine (Cr) were determined in24h urine samples from all groups before and after treatments.24h urinary Cr was used as the internal standard.24h urinary K and Na in the individual patients were calculated as concentrations relative to the Cr concentrations. The pelvic pain and urgency/frequency patient symptom (PUF) were used for evaluation before and after the treatments. The differences of urinary K were compared with in each group and between groups before and after treatments. In addition, relationship of urinary K and PUF were assessed by statistics.
In part two, statistical analysis was performed using SPSS13.0software. One-way ANOVA, with LSD test were used to test for differences between groups. In part three, statistical analysis was performed using Prism version5.0. Repeated measures ANOVA, with Dunnett's correction, was used to test for differences from the control values. In part four, statistical analysis was performed using SPSS13.0software. The data were analyzed by using t test-way ANOVA and Pearson product-moment correlation analysis.
Results
Sv-HUC-1cells was cultivated to the logarithmic growth phase. After treated with100mM、1μM、10μM、100μM of ketamine respectively for24h and48h, MTT assay was used to measure the proliferative activity of the Sv-HUC-1cells between ketamine treated groups and control group. Although the results had statistical difference, no obvious inhibitory effects of ketamine on the proliferation of cells were observed. It means that ketamine was not cytoxic to Sv-HUC-1cells. Amplex Red assay was used to measure the generation of free radicals produced by the urothelial cells when exposed to ketamine. The results showed that, in60mins duration, with cells and no cells plates had similar increased fluorescent rates with time. There were no significant difference between with cells and no cells groups, which indicated that ketamine had no effect on the generation of free radicals in Sv-HUC-1cells.
Rats presented with cataleptic immobility within1min of administration of50mg.kg-1ketamine i.p. This was followed by ataxia (head and body swaying) after about10minutes, and falling over and staying still for approximately1h until recovery. Rats in the LK group became mildly excited and active within15min of ketamine injection. Three of six rats in the HK group developed hematuria after16weeks of ketamine treatment. At Week12, high dose ketamine significantly increased micturition frequency compared with that in the NS group (24-hour frequency20.4±0.9vs16.4±0.6, P<0.01), and both low and high-dose ketamine groups showed a significant increase in frequency at Week16(24-hour frequency21.0±0.7vs16.4±0.8, P<0.05;28.6±1.4vs16.4±0.8, P<0.001; n=6). Weight gain at Week16was significantly slower in the HK group than in the NS group (15.6±4.4g vs53.1±4.0g,P<0.05;n=6).
Urinary nitric oxide levels were measured in triplicate samples immediately before and6h after ketamine administration. The fractional change in NO in relative to un-injected controls was1.19±0.05in the NS group, LK1.73±0.09in the LK group and2.77±0.06in the HK group. There was no significant difference in NO levels between control and NS groups. Rat urinary APF was measured at6h intervals for42h. The mean APF concentration at the initial collection point before injection was22.3±0.13pg.mL-1, with no differences being seen between groups. Neither the control nor the NS injected animals deviated from this over the42h collection period, whereas, both low and high dose ketamine increased APF concentrations, with the high dose producing a more rapid response. After12h a similar magnitude of response was seen with both doses of ketamine and the response lasted until the end of the collection period.
Urinary potassium:creatinine ratios significantly decreased with time in both the HK and LK groups (HK:at0weeks46±0.8vs.8weeks36±0.6and16weeks31.5±1.4P<0.05, P<0.001; NK:at0weeks:44.7±0.7vs16weeks36.8±0.8, P<0.05,n=6). There were no changes in the ratios in either the control or NS groups. Urinary GP-51levels were significantly reduced from baseline levels after8and16weeks of treatment with low and high dose ketamine.(HK:initial38.98±0.35pg/ml vs.8weeks32.83±0.33pg/ml and16weeks15.74±1.22pg/ml P<0.05, P<0.001; LK:initial37.35±0.49pg/ml vs.8weeks33.68±0.32pg/ml and16weeks34.40±0.30pg/ml P<0.01, P<0.05, n=6). There were no significant changes over time in GP-51levels in the control or NS groups.
Rats receiving high dose ketamine had a thicker and more compact bladder epithelial layer compared to control rats. Inflammatory cell infiltration and collagen deposition (fibrosis) was present in the bladder submucosa in the HK group. The normal loose connective tissue within the submucosal layer was diminished in the HK group. In both the LK and HK groups there was increased staining for iNOS in the bladder epithelial layer as shown by quantified immunohistochemistry and Western blot densitometry. NS rats also presented increased iNOS expression compared with control rats. The tight junction protein ZO-1was chiefly confined to the bladder epithelial layer, but the staining density and the relative protein expression of ZO-1was decreased in the LK and HK groups compared to the NS group. Expression of occludin mainly occurred in the endothelial cells of the bladder submucosa. The staining density and the relative protein expression of occludin were significantly increased in both LK and HK rats compared to NS rats.
Conclusion
Ketamine, in the dose between100nM to100μM, had not cytoxic effect on Sv-HUC-1cells. Moreover, it can not induce oxidative stress reaction on the cells. Therefore, we thought it was its metabolites rather than ketamine itself affect the urinary system.
Due the results of the rat model of ketamine cystitis, we hypothesized that metabolites of ketamine in urine had a direct toxic effect on bladder epithelial cells, generating nitric oxide and APF. The cumulative toxicity and sustained release of APF impaired the integrity of the bladder epithelial barrier resulting in decreased expression of glycoprotein GP-51and ZO-1within the bladder epithelial layer. The resulting increase in permeability enabled urine constituents, such as urea, and potassium to penetrate the bladder interstitium and muscle layers, resulting in mononuclear cell infiltration, fibrosis, angiogenesis and hypervascularity. Potassium diffusion depolarized nerves and muscles causing symptoms, such as frequency, hematuria and pain. With further development our rat model of ketamine induced cystitic symptoms may help to uncover the etiology and evaluate new methods for treating IC.
In clinic, urinary potassium measurement had a role in evaluating the disease status and efficacy of treatments of patients who suffered from ketamine-associated cystitis.
自2007年以来,我们在临床工作中发现了一些长期吸食新型毒品K粉的患者会出现严重的尿频、尿急、尿痛、血尿、排尿困难或急迫性尿失禁等下尿路症状(LUTS)。我们称之为氯胺酮相关性膀胱炎(ketamine cystitis, KC)。氯胺酮相关性膀胱炎的病人的临床表现、实验室检查及病理检查中所发现的特征均与间质性膀胱炎(interstitial cystitis, IC)的病人相类似,2009年的亚洲间质性膀胱炎指南也认为氯胺酮相关性泌尿系损害与IC有同样的发病机制。然而,对于氯胺酮相关性膀胱炎发病机制的研究仍处在起步阶段,还存在以下三点主要的疑问：1.下尿路症状和氯胺酮吸食是否确实有直接的因果关系?2.氯胺酮造成膀胱损伤的机制是什么?3.对于氯胺酮导致的下尿路病人的有效的合适的治疗方案是什么?为了回答这三个问题,我们设计建立了氯胺酮对人膀胱上皮细胞的细胞毒性作用模型(第二章),氯胺酮滥用大鼠膀胱损害的模型(第三章)以及氯胺酮病人症状评分及尿钾水平测定的模型(第四章)分别进行研究。从细胞、动物及临床水平分别探氯胺酮相关性膀胱炎的发病机制。
方法
体外实验进行人膀胱永生化上皮细胞SV-HUC-1细胞的培养,细胞生长到对数生长期时,将细胞暴露于100nM、1μM、10μM、100μM浓度氯胺酮培养基中,利用MTT比色法检测氯胺酮对人膀胱永生化上皮细胞增殖的影响,利用Amplex-Red荧光法检测氯胺酮作用于人膀胱永生化上皮细胞时所产生的自由基水平。
动物实验取雄性Sprague-Dawley (SD)大鼠24只,体重180-200g,随机分为空白组(control),生理盐水组(NS),低剂量实验组(LK,5mg.kg-1)和高剂量实验组(HK,50mg.kg-1),每组6只。实验组大鼠每日早晨9点腹腔注射盐酸氯胺酮溶液,溶解于500ul生理盐水。生理盐水组大鼠同时注射等量生理盐水,空白组不予药物处理。大鼠单独饲养与自制的代谢笼中,利用自制的尿液染色试纸记录大鼠排尿频率。大鼠排尿测量实验在第一次给药前连续测3天,之后再第4,8,16周的给药后连续测3天。氯胺酮注射对大鼠尿液的短期影响通过分析尿液中抗增殖因子APF和一氧化氮NO水平完成。首先收集第一次注射氯胺酮前大鼠的6小时尿液作为基准对照,跟着收集注射后尿液36小时,每6小时收集一次。氯胺酮注射对大鼠尿液的长期影响通过分析尿液中糖蛋白GP-51和尿钾离子水平完成,在注射前,注射后4、8和16周分别取24小时尿液。氧化氮NO水平通过Griess Reaction试剂盒完成,APF及GP-51的尿液含量通过ELISA试剂盒法测量,尿钾离子水平通过多功能生化分析仪取得。大鼠予氯胺酮注射16周后,断头处死并称重,取膀胱组织行HE染色光镜下读片拍照。利用免疫组化法分析膀胱上皮组织中的紧密链接蛋白ZO-1及Occludin,和诱导性一氧化氮合酶iNOS的表达。利用免疫蛋白印迹法验证ZO-1,Occludin和iNOS在各组大鼠膀胱组织中的表达。
临床中收集43例氯胺酮相关性膀胱炎的患者,分成两组,A组为32例有严重的下尿路症状但能自行排尿的患者,B组为11例由于严重的下尿路症状而不能或不愿自行排尿在入院后留置尿管的患者。入院时,所有患者除接受常规检查外,均在入院时行盆腔疼痛与尿急/尿频(PUF)评分,并留置24h尿,检测尿钾、尿钠和尿肌酐浓度；同时取我院健康成年人30名,男女各15名,平均年龄22-30岁,在正常饮食情况下,连续测定3天的24h尿钾、尿钠、尿肌酐浓度作为空白对照。所有患者均被要求戒断K粉在,试验性给予静脉注射抗生素、口服肾上腺素能受体阻滞剂、胆碱能受体阻滞剂、戊聚硫钠；透明质酸钠膀胱内灌注及止痛等对症支持治疗方法。平均治疗疗程为12天。出院前复查24小时尿钾和PUF评分。
实验数据以均数±标准差表示,第二章采用SPSS13.0统计软件进行统计分析,在各个时间点不同浓度组间对细胞作用采用One-way ANOVA分析,两两比较采用LSD法检验(方差齐时)。方差不齐时分别采用Welch和Dunnett's T3法,显著性标准为P<0.05。第三章采用Prism version5.0(Graphpad Software, San Diego, CA, USA)统计软件分析处理,连续性数据用One-Way ANOVA检验。组间比较采用Dunnett's correction法,显著性标准为P<0.05。第四章采用SPSS13.0对数据进行处理。对治疗前后各组间的尿钾、尿钠、尿肌酐浓度分析采用One-Way ANOVA。治疗前、后组间的PUF评分比较均采用两独立样本t检验,组内治疗前后的PUF评分比较采用配对样本t检验,P<0.05有统计学意义。治疗前后PUF与对应的24h尿钾浓度的统计学分析采用计量资料的Pearson积差相关分析,P<0.05有统计学意义。
结果
当人膀胱上皮永生化细胞生长至对数期后,以不同剂量的氯胺酮100nM、1μM、10μM、100μM分别处理细胞24小时及48小时后,利用MTT法测量可见氯胺酮1μM、10μM、100μM处理后细胞活性与对照组相比具有统计学差异,但是未观察到药物对细胞有显著生长抑制作用,说明氯胺酮在100nM～100μM的剂量范围内对人膀胱上皮永生化细胞的细胞毒作用无生物学意义。当人膀胱上皮永生化细胞生长至完全融合后,各种浓度的氯胺酮被分别加入到含有细胞的和无细胞的96孔细胞培养板中以AmplexRed法来测试其化学荧光的底色。结果发现,测量60分钟之内,氯胺酮各浓度组在有细胞及无细胞的培养板中所记录的荧光改变数据均呈自然氧化的平缓线性表现,有细胞及无细胞组之间的比较均无统计学差异,说明氯胺酮无法刺激人膀胱永生化上皮细胞产生自由基。
大鼠在注射50mg-1kg氯胺酮溶液1分钟后出现全身僵硬症状,不能活动,随后10分钟出现共济失调,头尾不停摇摆晃动,向左右跌倒,接着开始保持平静不动1小时直到复原。在低剂量组,大鼠注射5mg-1kg氯胺酮溶液后,15分钟内相比生理盐水组合空白组的大鼠出现轻微的兴奋和活跃状态,之后恢复正常。在注射氯胺酮溶液16周后,高剂量组(HK)大鼠体重的增加与生理盐水组(NS)相比显著地减慢(15.6±4.4g vs53.1±4.0g,P<0.05；n=6)。3/6只大鼠在注射高剂量氯胺酮溶液16周后出现了明显的血尿。注射氯胺酮溶液12周后,高剂量组大鼠相比生理盐水组大鼠出现显著的尿频增多(24-hour frequency20.4±0.9vs16.4±0.6,P<0.01)。注射氯胺酮溶液16周后,高剂量组(HK)和低剂量组(LK)与生理盐水组相比均出现了显著的尿频增多(24-hour frequency21.0±0.7vs16.4±0.8,P<0.05；28.6±1.4vs16.4±0.8,P<0.001； n=6)。
高剂量组和低剂量组的时间-抗增殖因子曲线自首次氯胺酮注射后均开始出现增长,高剂量组在开始时产生了较快的反应,12小时后两组APF的反应基本一致,在注射后第30小时时达到最高峰,30小时后两组的曲线开始出现下降,而生理盐水和空白组的曲线保持水平。抗增殖因子APF分泌的量是由各时间段的量除以基础数值取百分比求得,高剂量组在第6小时APF增加的百分数较生理盐水组显著增多(HK113%±3%vs.NS99%±3%,P<0.05,n=6)。在第30小时,高剂量组和低剂量组APF增加的百分数均较生理盐水组显著增多(HK127%±4%vs.NS105%±3%P<0.001；LK124%±8%vs.NS105%±3%P<0.001,n=6)。在注射前APF在各组的平均数是22.3±0.13pg.ml-1(均数±方差,n=24)。
注射后6小时内尿液中一氧化氮的水平计算为基础一氧化氮水平的百分比,首次注射氯胺酮溶液6小时内尿液中一氧化氮的水平在高剂量组和低剂量组均较生理盐水组出现了显著的增多(HK277%±6%vs.NS119%±5%: P<0.001,LK173%±9%vs.NS119%±5%,P<0.05,n=6)。生理盐水和空白对照组之间相比没有统计学差异。一氧化氮水平在首次注射氯胺酮6小时之后的各时间段在各组间相比没有统计学差异。
24小时尿液标本中的尿钾结果除以尿肌酐标准化以提高准确性。随着时间的进展,氯胺酮高剂量和低剂量组的尿钾尿肌酐比(K+/Cr)显著降低(高剂量组：at0weeks46±0.8vs.8weeks36±0.6and16weeks31.5±1.4P<0.05, P<0.001:低剂量组：at0weeks:44.7±0.7vs16weeks36.8±0.8, P<0.05,11=6).生理盐水和空白对照组的尿钾和肌酐的比值K+/Cr随时间进展无显著统计学差异。随着时间的进展,氯胺酮高剂量和低剂量组的尿液GP-51水平显著降低(高剂量组：initial38.98±0.35pg/ml vs.8weeks32.83±0.33pg/ml and16weeks15.74±1.22pg/ml P<0.05,P<0.001；低剂量组：initial37.35±0.49pg/ml vs.8weeks33.68±0.32pg/ml and16weeks34.40±0.30pg/ml P<0.01,P<0.05,n=6)。生理盐水和空白对照组的尿液GP-51水平随时间进展无显著统计学差异。
HE染色结果显示：生理盐水组大鼠膀胱上皮较空白对照组未发现明显的结构改变。高剂量组的大鼠膀胱上皮层较空白对照组显得更厚和更致密。在高剂量组里可以观察到膀胱粘膜下层明显的单核粒细胞浸润,结缔组织纤维化和胶原蛋白沉积。而粘膜下层原有正常的疏松结缔组织在高剂量组明显消失。低剂量组的膀胱病理改变程度鉴于高剂量组和生理盐水组之间。
免疫组化结果显示：诱导型一氧化氮合酶(iNOS)和ZO-1均主要表达在膀胱上皮层,而另一个紧密连接蛋白Occludin主要表达在膀胱粘膜下层的血管内皮细胞中。通过Image-Pro Plus v6.0软件处理量化分析可见,iNOS高剂量和低剂量组的表达较生理盐水组显著增高(平均光密度±方差：LK0.432±0.006vs. NS0.215±0.005P<0.001；HK0.450±0.006vs.NS0.215±0.005P<0.001.),生理盐水组较空白对照组也出现了显著的增高(平均光密度±方差：control0.270±0.005vs.NS0.317±0.006P<0.01,n=6)。Occludin的染色密度在低剂量组和高剂量组均较生理盐水组出现了显著降低(平均光密度±方差：LK0.404±0.005vs.NS0-317±0.006P<0.05；HK0.452±0.008vs.NS0.317±0.006P<0.001,n=6),ZO-1在高剂量组和低剂量组的染色密度则较生理盐水组出现显著增高(平均光密度±方差：LK0.340±0.006vs.NS0.453±0.011P<0.05；HK0.255±0.008vs.NS0.453±0.011P<0.001,n=6)。各组大鼠膀胱中NOS、ZO-1和Occludin的表达由免疫蛋白印迹方法检验。免疫蛋白印迹的量化柱状图结果与免疫组化的量化结果完全一致。低剂量和高剂量组中NOS的相对蛋白表达比率较生理盐水组显著增多(平均比率±方差：LK0.83±0.003vs.NS0.70±0.004P<0.001；HK1.09±0.006vs.NS0.70±0.004P<0.001,n=6,),生理盐水组中NOS的表达较空白组也显著增加(平均比率±方差：control0.63±0.008vs.NS0.70±0.004P<0.01,n=6).低剂量和高剂量组中ZO-1的相对蛋白表达比率较生理盐水组显著减少(平均比率±方差：LK1.08±0.008vs.NS1.55±0.003P<0.001；HK0.73±0.005vs.NS1.55±0.003P<0.001,n=6).低剂量和高剂量组中Occludin的相对蛋白表达比率较生理盐水组显著增加(平均比率±方差：LK0.89±0.003vs.NS0.70±0.005P<0.01； HK1.02±0.01vs.NS0.70±0.005P<0.001,n=6)。
临床中氯胺酮相关性膀胱炎患者治疗前24h尿液样本中的尿钠和尿钾的变化：所有数值均以24小时尿肌酐作为内参进行标准化。经标准化后的A组尿钾浓度分别与对照组、B组比较,比较有统计学差异。(A组1.80vs.B组6.22K+/Cr,P<0.01；A组1.80vs.对照组6.47K+/Cr,P<0.01)。各组尿钠浓度没有统计学差异(P>0.05)。所有患者(A、B组)在接受治疗后,下尿路症状均有明显改善。B组所有患者在拔除导尿管后能自行排尿。标准化后的组间尿钾浓度没有显著统计学差异(P>0.05)。治疗前、后组间PUF评分均无统计学差异(P>0.05),治疗前后组内PUF评分有统计学差异(A组：治疗前23.19±3.64VS治疗后18.31±2.19,P<0.05；B组：治疗前21.95±3.86VS治疗后17.18±2.68,P<0.05)。治疗前A组尿钾浓度与PUF评分呈负相关关系,(r=-0.697,P<0.01)；治疗前B组尿钾浓度与PUF评分没有明显相关关系(r=0.012,P=0.973)；治疗后A组(r=-0.847,p<0.01),治疗后B组(r=-0.881,p=<0.01),尿钾浓度与PUF评分均呈明显负相关关系。
讨论
氯胺酮在100nM～100μM的剂量范围内对人膀胱上皮永生化细胞的细胞毒作用无生物学意义,并且也不能诱导膀胱上皮细胞产生氧化应激反应。因此我们认为是氯胺酮的代谢产物而不是氯胺酮本身对泌尿系统产生了损害。
经过动物实验的结果得出,长期滥用氯胺酮的大鼠,会出现明显的尿频和血尿。我们推测其机制可能是氯胺酮的代谢产物在尿液中对膀胱上皮细胞产生了直接的毒性作用,使膀胱上皮细胞产生了一氧化氮和APF。这种蓄积的毒性作用和APF的持续释放,扰乱破坏了膀胱上皮细胞的正常增殖和自我修复。因此膀胱上皮屏障的完整性被破坏,表现为氨基葡聚糖中的糖蛋白GP-51和细胞紧密连接蛋白ZO-1的表达下降。然后,膀胱上皮的渗透性增加。尿液中的成分如尿酸、尿钾等可以渗入膀胱的间质层和肌肉层,导致炎症细胞的浸润,间质层纤维化和血管增生。尤其是尿钾的渗入,促使膀胱的神经和肌肉去极化,产生尿频、血尿和尿痛的IC样症状。
临床中,新发的、未经治疗的氯胺酮相关性膀胱炎患者中尿液钾离子浓度与正常对照组和留置尿管的重症患者相比显著减低,这也可能是由于膀胱黏膜上皮的缺损,尿液中的钾离子渗透进入膀胱内间隙所致。尿钾浓度的测定对于氯胺酮相关性膀胱炎患者疾病严重程度和治疗效果的判定等均具有良好的指导意义。并且治疗后的两组PUF评分及所有患者的PUF总体分值均较治疗前下降,差异有统计学意义。PUF评分与治疗后24h尿钾存在负相关,PUF评分越低,24h尿钾浓度越高。说明我们采用的综合治疗不仅有效减少了尿钾离子在膀胱壁的重吸收且有助于缓解尿频、尿痛等症状。
Background and Objection
Since2007it has been known that long-term ketamine abuse can affect the urinary system, resulting in lower urinary tract syndrome, frequency, nocturia, urgency, dysuria, suprapubic discomfort and occasional hematuria. Cystoscopy findings and histological changes in bladder biopsies from ketamine abusers are similar to those patients with interstitial cystitis. Intravenous urography and urodynamic studies in a small group (n=6) of chronic recreational ketamine users identified various changes in bladder function and appearance. These subjects had narrowing of both ureters, contracted bladder syndrome with mild bilateral hydronephrosis, detrusor instability and urinary leakage. It remains unclear how ketamine abuse leads to these interstitial cystitis-like, functional and histological changes. It has been postulated that the accumulation of ketamine and/or its metabolites in the urine might cause direct toxic effects on the urinary tract. However, apart from the pathological changes caused by long-term ketamine abuse that are seen clinically and in animal models, no direct evidence has been found that gives a causal relationship between ketamine toxicity and the development of cystitis. In addition, there are currently no studies describing the effects of the urinary metabolites of ketamine on bladder function. The aim of this study was to fully explore the pathogenesis of ketamine cystitis in vivo, in vitro and in clinic.
Methods and materials
Sv-HUC-1cells was cultivated in vitro to the logarithmic growth phase. Then, Sv-HUC-1cells was exposed to100nM、1μM、10μM、100μM dose of ketamine respectively. MTT assay was used to measure the proliferative activity of the urothelial cells with the effect of ketamine. Amplex Red assay was used to measure the generation of free radicals produced by the urothelial cells when exposed to ketamine.
Twenty-four2-month-old male Sprague-Dawley (SD) rats weighing180-200g had free access to standard laboratory rodent chow and water. Groups of six rats were randomly assigned to control, normal saline (NS), low-dose (5mg.kg-1) and high-dose (50mg.kg-1) ketamine groups. The two experimental groups received a single intraperitoneal (i.p.) injection of ketamine hydrochloride dissolved in500μL saline at09:00h each day. The NS group received an i.p. injection of vehicle the same time:rats in the control group were untreated. All rats were weighed weekly to adjust the quantity of ketamine administrated.
Urinary frequency was determined by housing the rats individually in modified metabolic cages that had a fine stainless steel mesh, which retained feces but allowed urine to pass, through its base. Paper, impregnated with saturated copper sulfate solution (CuSO45H2O) and dehydrated at200℃for1h, was placed under the base of the cage. The urine falling onto this paper, rehydrated the anhydrous CuSO4, turning it blue. The number of urinations was determined by counting the number of blue spots observed. Baseline urinary frequency was measured3days before ketamine administration, and then at consecutive3day periods after4,8and16weeks of treatment.
Urine samples were collected from the metabolic cage, but with the CuSO4paper removed. The short-term effect of ketamine was assessed by analyzing urine samples for APF and NO. A6h baseline urine sample was collected immediately before the first ketamine injection and subsequent urine samples were collected at6h intervals for30h. The longer term effects of ketamine were monitored using Gp-51and potassium measurements at baseline and after4,8,16weeks of treatment. Urine was collected for24h, immediately before daily ketamine administration and thereafter24-h urinary volume was recorded daily. Aliquots of urine were stored at-80℃and were thawed once before analysis. The levels of APF and Gp51in urine were measured using an ELISA assay. Levels of NO were estimated via the Griess reaction. Urinary potassium concentration was determined using an ion selective electrode technique in an automated chemistry analyzer.
Rats were killed by spine dislocation, and the bladders were excised. Some of the bladder specimens were stained with hematoxylin and eosin (H&E), and examined under a light microscope. Immunohistochemical studies were performed using the DAKO ChemMateTM EnVisionTM Detection Kit. The following primary antibodies were used:anti-iNOS, anti-occludin antibody and anti-ZO-1. The integrated optical density of cytoplasm and cell membranes containing brown-yellow granules was detected by using Image-Pro Plus v6.0software. Western blot analysis was used to examinate the expression of iNOS, Occludin and ZO-1in the bladder. Protein signals were quantified by scanning densitometry using a FluorChem Q system. The results of western blotting were quantified by Quantity One4.4.0software.
43ketamine-associated cystitis patients (male29cases, female14cases) were analyzed.32patients without indwelling urinary catheter were categorized as group A, while the other11patients with indwelling urinary catheter were in group B. The therapy regimes consisted of anti-inflammatory, antioxidant, relieving spasm and pain, improving the microcirculation and repairing the bladder epithelium barrier.30healthy adults were selected as the controls. Urinary potassium(K), sodium (Na) and creatinine (Cr) were determined in24h urine samples from all groups before and after treatments.24h urinary Cr was used as the internal standard.24h urinary K and Na in the individual patients were calculated as concentrations relative to the Cr concentrations. The pelvic pain and urgency/frequency patient symptom (PUF) were used for evaluation before and after the treatments. The differences of urinary K were compared with in each group and between groups before and after treatments. In addition, relationship of urinary K and PUF were assessed by statistics.
In part two, statistical analysis was performed using SPSS13.0software. One-way ANOVA, with LSD test were used to test for differences between groups. In part three, statistical analysis was performed using Prism version5.0. Repeated measures ANOVA, with Dunnett's correction, was used to test for differences from the control values. In part four, statistical analysis was performed using SPSS13.0software. The data were analyzed by using t test-way ANOVA and Pearson product-moment correlation analysis.
Results
Sv-HUC-1cells was cultivated to the logarithmic growth phase. After treated with100mM、1μM、10μM、100μM of ketamine respectively for24h and48h, MTT assay was used to measure the proliferative activity of the Sv-HUC-1cells between ketamine treated groups and control group. Although the results had statistical difference, no obvious inhibitory effects of ketamine on the proliferation of cells were observed. It means that ketamine was not cytoxic to Sv-HUC-1cells. Amplex Red assay was used to measure the generation of free radicals produced by the urothelial cells when exposed to ketamine. The results showed that, in60mins duration, with cells and no cells plates had similar increased fluorescent rates with time. There were no significant difference between with cells and no cells groups, which indicated that ketamine had no effect on the generation of free radicals in Sv-HUC-1cells.
Rats presented with cataleptic immobility within1min of administration of50mg.kg-1ketamine i.p. This was followed by ataxia (head and body swaying) after about10minutes, and falling over and staying still for approximately1h until recovery. Rats in the LK group became mildly excited and active within15min of ketamine injection. Three of six rats in the HK group developed hematuria after16weeks of ketamine treatment. At Week12, high dose ketamine significantly increased micturition frequency compared with that in the NS group (24-hour frequency20.4±0.9vs16.4±0.6, P<0.01), and both low and high-dose ketamine groups showed a significant increase in frequency at Week16(24-hour frequency21.0±0.7vs16.4±0.8, P<0.05;28.6±1.4vs16.4±0.8, P<0.001; n=6). Weight gain at Week16was significantly slower in the HK group than in the NS group (15.6±4.4g vs53.1±4.0g,P<0.05;n=6).
Urinary nitric oxide levels were measured in triplicate samples immediately before and6h after ketamine administration. The fractional change in NO in relative to un-injected controls was1.19±0.05in the NS group, LK1.73±0.09in the LK group and2.77±0.06in the HK group. There was no significant difference in NO levels between control and NS groups. Rat urinary APF was measured at6h intervals for42h. The mean APF concentration at the initial collection point before injection was22.3±0.13pg.mL-1, with no differences being seen between groups. Neither the control nor the NS injected animals deviated from this over the42h collection period, whereas, both low and high dose ketamine increased APF concentrations, with the high dose producing a more rapid response. After12h a similar magnitude of response was seen with both doses of ketamine and the response lasted until the end of the collection period.
Urinary potassium:creatinine ratios significantly decreased with time in both the HK and LK groups (HK:at0weeks46±0.8vs.8weeks36±0.6and16weeks31.5±1.4P<0.05, P<0.001; NK:at0weeks:44.7±0.7vs16weeks36.8±0.8, P<0.05,n=6). There were no changes in the ratios in either the control or NS groups. Urinary GP-51levels were significantly reduced from baseline levels after8and16weeks of treatment with low and high dose ketamine.(HK:initial38.98±0.35pg/ml vs.8weeks32.83±0.33pg/ml and16weeks15.74±1.22pg/ml P<0.05, P<0.001; LK:initial37.35±0.49pg/ml vs.8weeks33.68±0.32pg/ml and16weeks34.40±0.30pg/ml P<0.01, P<0.05, n=6). There were no significant changes over time in GP-51levels in the control or NS groups.
Rats receiving high dose ketamine had a thicker and more compact bladder epithelial layer compared to control rats. Inflammatory cell infiltration and collagen deposition (fibrosis) was present in the bladder submucosa in the HK group. The normal loose connective tissue within the submucosal layer was diminished in the HK group. In both the LK and HK groups there was increased staining for iNOS in the bladder epithelial layer as shown by quantified immunohistochemistry and Western blot densitometry. NS rats also presented increased iNOS expression compared with control rats. The tight junction protein ZO-1was chiefly confined to the bladder epithelial layer, but the staining density and the relative protein expression of ZO-1was decreased in the LK and HK groups compared to the NS group. Expression of occludin mainly occurred in the endothelial cells of the bladder submucosa. The staining density and the relative protein expression of occludin were significantly increased in both LK and HK rats compared to NS rats.
Conclusion
Ketamine, in the dose between100nM to100μM, had not cytoxic effect on Sv-HUC-1cells. Moreover, it can not induce oxidative stress reaction on the cells. Therefore, we thought it was its metabolites rather than ketamine itself affect the urinary system.
Due the results of the rat model of ketamine cystitis, we hypothesized that metabolites of ketamine in urine had a direct toxic effect on bladder epithelial cells, generating nitric oxide and APF. The cumulative toxicity and sustained release of APF impaired the integrity of the bladder epithelial barrier resulting in decreased expression of glycoprotein GP-51and ZO-1within the bladder epithelial layer. The resulting increase in permeability enabled urine constituents, such as urea, and potassium to penetrate the bladder interstitium and muscle layers, resulting in mononuclear cell infiltration, fibrosis, angiogenesis and hypervascularity. Potassium diffusion depolarized nerves and muscles causing symptoms, such as frequency, hematuria and pain. With further development our rat model of ketamine induced cystitic symptoms may help to uncover the etiology and evaluate new methods for treating IC.
In clinic, urinary potassium measurement had a role in evaluating the disease status and efficacy of treatments of patients who suffered from ketamine-associated cystitis.
引文
1. Domino, E. F.:Taming the ketamine tiger.1965. Anesthesiology,113:678,2010
2. Harrison, N. L., Simmonds, M. A.:Quantitative studies on some antagonists of N-methyl D-aspartate in slices of rat cerebral cortex. Br J Pharmacol,84:381,1985
3. Moore, K. A., Sklerov, J., Levine, B. et al.:Urine concentrations of ketamine and norketamine following illegal consumption. J Anal Toxicol,25:583,2001
4. Quibell, R., Prommer, E. E., Mihalyo, M. et al.:Ketamine*. J Pain Symptom Manage,41: 640,2011
5. Siegel, R. K.:Phencyclidine and ketamine intoxication:a study of four populations of recreational users. NIDA Res Monogr:119,1978
6. Chu, P. S., Kwok, S. C., Lam, K. M. et al.:'Street ketamine'-associated bladder dysfunction:a report of ten cases. Hong Kong Med J,13:311,2007
7. Shahani, R., Streutker, C., Dickson, B. et al.:Ketamine-associated ulcerative cystitis:a new clinical entity. Urology,69:810,2007
8. Yang, J., Bian, S. Z.:[A reviewing for abusing of ketamine]. Fa Yi Xue Za Zhi,23:312, 2007
9. Schifano, F., Corkery, J., Oyefeso, A. et al.:Trapped in the "K-hole":overview of deaths associated with ketamine misuse in the UK (1993-2006). J Clin Psychopharmacol,28: 114,2008
10. Homma, Y., Ueda, T., Tomoe, H. et al.:Clinical guidelines for interstitial cystitis and hypersensitive bladder syndrome. Int J Urol,16:597,2009
11. Wood, D., Cottrell, A., Baker, S. C. et al.:Recreational ketamine:from pleasure to pain. BJU Int,2011
12. Abrams, P., Artibani, W., Cardozo, L. et al.:Reviewing the ICS 2002 terminology report: the ongoing debate. Neurourol Urodyn,28:287,2009
13. Gillenwater, J. Y., Wein, A. J.:Summary of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases Workshop on Interstitial Cystitis, National Institutes of Health, Bethesda, Maryland, August 28-29,1987. J Urol,140:203,1988
14. Parsons, C. L., Boychuk, D., Jones, S. et al.:Bladder surface glycosaminoglycans:an epithelial permeability barrier. J Urol,143:139,1990
15. Parsons, C. L., Hurst, R. E.:Decreased urinary uronic acid levels in individuals with interstitial cystitis. J Urol,143:690,1990
16. Parsons, C. L.:The role of the urinary epithelium in the pathogenesis of interstitial cystitis/prostatitis/urethritis. Urology,69:9,2007
17. Parsons, C. L.:The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int,107:370,2011
18. Logadottir, Y. R., Ehren, I., Fall, M. et al.:Intravesical nitric oxide production discriminates between classic and nonulcer interstitial cystitis. J Urol,171:1148,2004
19. Birder, L. A., Wolf-Johnston, A., Buffington, C. A. et al.:Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis. J Urol,173:625,2005
20. Hanno, P. M., Parsons, C. L., Shrom, S. H. et al.:The protective effect of heparin in experimental bladder infection. J Surg Res,25:324,1978
21. Byrne, D. S., Sedor, J. F., Estojak, J. et al.:The urinary glycoprotein GP51 as a clinical marker for interstitial cystitis. J Urol,161:1786,1999
22. Acharya, P., Beckel, J., Ruiz, W. G. et al.:Distribution of the tight junction proteins ZO-1, occludin, and claudin-4,-8, and-12 in bladder epithelium. Am J Physiol Renal Physiol, 287:F305,2004
23. Khandelwal, P., Abraham, S. N., Apodaca, G.:Cell biology and physiology of the uroepithelium. Am J Physiol Renal Physiol,297:F1477,2009
24. Liu, H. T., Shie, J. H., Chen, S. H. et al.:Differences in mast cell infiltration, E-cadherin, and zonula occludens-1 expression between patients with overactive bladder and interstitial cystitis/bladder pain syndrome. Urology,80:225 e13,2012
25. Lavelle, J. P., Meyers, S. A., Ruiz, W. G. et al.:Urothelial pathophysiological changes in feline interstitial cystitis:a human model. Am J Physiol Renal Physiol,278:F540,2000
26. Keay, S., Zhang, C. O., Trifillis, A. L. et al.:Decreased 3H-thymidine incorporation by human bladder epithelial cells following exposure to urine from interstitial cystitis patients. J Urol,156:2073,1996
27. Erickson, D. R.:Urine markers of interstitial cystitis. Urology,57:15,2001
28. Keay, S. K., Szekely, Z., Conrads, T. P. et al.:An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide. Proc Natl Acad Sci U S A,101:11803,2004
29. Keay, S., Zhang, C. O., Shoenfelt, J. L. et al.:Decreased in vitro proliferation of bladder epithelial cells from patients with interstitial cystitis. Urology,61:1278,2003
30. Zhang, C. O., Wang, J. Y, Koch, K. R. et al.:Regulation of tight junction proteins and bladder epithelial paracellular permeability by an antiproliferative factor from patients with interstitial cystitis. J Urol,174:2382,2005
31. Kim, J., Keay, S. K., Dimitrakov, J. D. et al.:p53 mediates interstitial cystitis antiproliferative factor (APF)-induced growth inhibition of human urothelial cells. FEBS Lett,581:3795,2007
32. Keay, S., Kleinberg, M., Zhang, C. O. et al.:Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production. J Urol,164:2112,2000
33. Keay, S. K., Zhang, C. O., Shoenfelt, J. et al.:Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis. Urology,57:9,2001
34. Keay, S., Leitzell, S., Ochrzcin, A. et al.:A mouse model for interstitial cystitis/painful bladder syndrome based on APF inhibition of bladder epithelial repair:a pilot study. BMC Urol,12:17,2012
35. Keay, S., Seillier-Moiseiwitsch, F., Zhang, C. O. et al.:Changes in human bladder epithelial cell gene expression associated with interstitial cystitis or antiproliferative factor treatment. Physiol Genomics,14:107,2003
36. Shahjee, H. M., Koch, K. R., Guo, L. et al.:Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells. J Exp Clin Cancer Res,29:160,2010
37. Keay, S., Kaczmarek, P., Zhang, C. O. et al.:Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor. Chem Biol Drug Des,77:421,2011
38. Smet, P. J., Jonavicius, J., Marshall, V. R. et al.:Distribution of nitric oxide synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry. Neuroscience, 71:337,1996
39. Xu, D. Z., Lu, Q., Deitch, E. A.:Nitric oxide directly impairs intestinal barrier function. Shock,17:139,2002
40. Chertin, B., Rolle, U., Solari, V. et al.:The role of nitric oxide in bladder urothelial injury after bladder outlet obstruction. BJU Int,94:392,2004
41. Muijsers, R. B., Folkerts, G., Henricks, P. A. et al.:Peroxynitrite:a two-faced metabolite of nitric oxide. Life Sci,60:1833,1997
42. Forstermann, U.:Nitric oxide and oxidative stress in vascular disease. Pflugers Arch,459: 923,2010
43. Darley-Usmar, V., Wiseman, H., Halliwell, B.:Nitric oxide and oxygen radicals:a question of balance. FEBS Lett,369:131,1995
44. Ho, M. H., Bhatia, N. N., Khorram, O.:Physiologic role of nitric oxide and nitric oxide synthase in female lower urinary tract. Curr Opin Obstet Gynecol,16:423,2004
45. Stothers, L., Laher, I., Christ, G. T.:A review of the L-arginine-nitric oxide-guanylate cyclase pathway as a mediator of lower urinary tract physiology and symptoms. Can J Urol,10:1971,2003
46. Romih, R., Korosec, P., Sedmak, B. et al.:Mitochondrial localization of nitric oxide synthase in partially differentiated urothelial cells of urinary bladder lesions. Appl Immunohistochem Mol Morphol,16:239,2008
47. Rovner, E., Propert, K. J., Brensinger, C. et al.:Treatments used in women with interstitial cystitis:the interstitial cystitis data base (ICDB) study experience. The Interstitial Cystitis Data Base Study Group. Urology,56:940,2000
48. Hanno, P. M., Buehler, J., Wein, A. J.:Use of amitriptyline in the treatment of interstitial cystitis. J Urol,141:846,1989
49. van Ophoven, A., Hertle, L.:Long-term results of amitriptyline treatment for interstitial cystitis. J Urol,174:1837,2005
50. Sant, G. R., Theoharides, T. C., Letourneau, R. et al.:Interstitial cystitis and bladder mastocytosis in a woman with chronic urticaria. Scand J Urol Nephrol,31:497,1997
51. Theoharides, T. C., Sant, G. R.:Hydroxyzine therapy for interstitial cystitis. Urology,49: 108,1997
52. Nickel, J. C., Barkin, J., Forrest, J. et al.:Randomized, double-blind, dose-ranging study of pentosan polysulfate sodium for interstitial cystitis. Urology,65:654,2005
53. Hampson, S. J., Woodhouse, C. R.:Sodium pentosanpolysulphate in the management of haemorrhagic cystitis:experience with 14 patients. Eur Urol,25:40,1994
54. Toren, P. J., Norman, R. W.:Cyclophosphamide induced hemorrhagic cystitis successfully treated with pentosanpolysulphate. JUrol,173:103,2005
55. Portenoy, R. K., Foley, K. M.:Chronic use of opioid analgesics in non-malignant pain: report of 38 cases. Pain,25:171,1986
56. Sant, G. R.:Intravesical 50% dimethyl sulfoxide (Rimso-50) in treatment of interstitial cystitis. Urology,29:17,1987
57. Khanna, O. P., Loose, J. H.:Interstitial cystitis treated with intravesical doxorubicin. Urology,36:139,1990
58. Rajkumar, G. N., Conn, I. G.:Botulinum toxin:a new dimension in the treatment of lower urinary tract dysfunction. Urology,64:2,2004
59. Vemulakonda, V. M., Somogyi, G. T., Kiss, S. et al.:Inhibitory effect of intravesically applied botulinum toxin A in chronic bladder inflammation. J Urol,173:621,2005
60. Omura, Y:Basic electrical parameters for safe and effective electro-therapeutics [electro-acupuncture, TES, TENMS (or TEMS), TENS and electro-magnetic field stimulation with or without drug field] for pain, neuromuscular skeletal problems, and circulatory disturbances. Acupunct Electrother Res,12:201,1987
1. Kowalski, J., Spychala, A.:[Mean transit time (MTT)-a new test of forced expiration]. Pneumonol Pol,51:1,1983
2. Massa, E. M., Farias, R. N.:Effect of auto-oxidized phospholipids on oxidative enzyme assays based on tetrazolium salt reduction. Biochim Biophys Acta,746:209,1983
3. Rosa, C. G, Tsou, K. C.:The use of tetranitro-blue tetrazolium for the cytochemical localization of succinic dehydrogenase. Cytochemical and cytological studies of sarcoma 37 ascites tumor cells. J Cell Biol,16:445,1963
4. Mohanty, J. G, Jaffe, J. S., Schulman, E. S. et al.:A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods,202:133,1997
5. Arroyo, A., Modriansky, M., Serinkan, F. B. et al.:NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance. J Biol Chem,277:49965,2002
6. Thor, H., Smith, M. T., Hartzell, P. et al.:The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells. J Biol Chem,257:12419,1982
7. Warren, M. C., Bump, E. A., Medeiros, D. et al.:Oxidative stress-induced apoptosis of endothelial cells. Free Radic Biol Med,29:537,2000
8. Reznikoff, C. A., Johnson, M. D., Norback, D. H. et al.:Growth and characterization of normal human urothelium in vitro. In Vitro,19:326,1983
9. Schmidt, W. W., Messing, E. M., Reznikoff, C. A.:Cultures of normal human urothelial cells from ureters of perfused cadaver transplant kidneys. J Urol,132:1262,1984
10. Reznikoff, C. A., Loretz, L. J., Johnson, M. D. et al.:Quantitative assessments of the cytotoxicity of bladder carcinogens towards cultured normal human uroepithelial cells. Carcinogenesis,7:1625,1986
11. Petzoldt, J. L., Leigh, I. M., Duffy, P. G. et al.:Culture and characterisation of human urothelium in vivo and in vitro. Urol Res,22:67,1994
12. Bishai, M. B., Kagawa, S., Narayan, K. S. et al.:Growth of a malignant human urothelial cell line (J82) in a serum-free medium (HMRI-1) developed previously for normal human urothelium. J Urol,134:1287,1985
13. Cilento, B. G., Freeman, M. R., Schneck, F. X. et al.:Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro. J Urol,152:665,1994
14. Christian, B. J., Loretz, L. J., Oberley, T. D. et al.:Characterization of human uroepithelial cells immortalized in vitro by simian virus 40. Cancer Res,47:6066,1987
15. Defendi, V, Naimski, P., Steinberg, M. L.:Human cells transformed by SV40 revisited: the epithelial cells. J Cell Physiol Suppl,2:131,1982
16. Stiles, C. D., Desmond, W., Jr., Sato, G. et al.:Failure of human cells transformed by simian virus 40 to form tumors in athymic nude mice. Proc Natl Acad Sci U S A,72: 4971,1975
17. Balda, M. S., Whitney, J. A., Flores, C. et al.:Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol,134:1031,1996
18. Cross, W. R., Eardley, I., Leese, H. J. et al.:A biomimetic tissue from cultured normal human urothelial cells:analysis of physiological function. Am J Physiol Renal Physiol, 289:F459,2005
19. Madara, J. L.:Regulation of the movement of solutes across tight junctions. Annu Rev Physiol,60:143,1998
20. Gaillard, P. J., Voorwinden, L. H., Nielsen, J. L. et al.:Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur J Pharm Sci,12:215,2001
21. Hu, P., Meyers, S., Liang, F. X. et al.:Role of membrane proteins in permeability barrier function:uroplakin ablation elevates urothelial permeability. Am J Physiol Renal Physiol, 283:F1200,2002
22. Tschugguel, W., Zhegu, Z., Gajdzik, L. et al.:High precision measurement of electrical resistance across endothelial cell monolayers. Pflugers Arch,430:145,1995
1. Persson, J.:Wherefore ketamine? Curr Opin Anaesthesiol,23:455,2010
2. Chu, P. S., Kwok, S. C., Lam, K. M. et al.:'Street ketamine'-associated bladder dysfunction:a report of ten cases. Hong Kong Med J,13:311,2007
3. Chu, P. S., Ma, W. K., Wong, S. C. et al.:The destruction of the lower urinary tract by ketamine abuse:a new syndrome? BJU Int,102:1616,2008
4. Yeung, L. Y., Rudd, J. A., Lam, W. P. et al.:Mice are prone to kidney pathology after prolonged ketamine addiction. Toxicol Lett,191:275,2009
5. Tan, S., Chan, W. M., Wai, M. S. et al.:Ketamine effects on the urogenital system-changes in the urinary bladder and sperm motility. Microsc Res Tech,2011
6. Wood, D., Cottrell, A., Baker, S. C. et al.:Recreational ketamine:from pleasure to pain. BJU Int,2011
7. Ratliff, T. L., Klutke, C. G., McDougall, E. M.:The etiology of interstitial cystitis. Urol Clin North Am,21:21,1994
8. Parsons, C. L., Boychuk, D., Jones, S. et al.:Bladder surface glycosaminoglycans:an epithelial permeability barrier. J Urol,143:139,1990
9. Matter, K., Balda, M. S.:Signalling to and from tight junctions. Nat Rev Mol Cell Biol,4: 225,2003
10. Slobodov, G., Feloney, M., Gran, C. et al.:Abnormal expression of molecular markers for bladder impermeability and differentiation in the urothelium of patients with interstitial cystitis. J Urol,171:1554,2004
11. Keay, S. K., Zhang, C. O., Shoenfelt, J. et al.:Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis. Urology,57:9,2001
12. Erickson, D. R.:Urine markers of interstitial cystitis. Urology,57:15,2001
13. Sant, G. R., Kempuraj, D., Marchand, J. E. et al.:The mast cell in interstitial cystitis:role in pathophysiology and pathogenesis. Urology,69:34,2007
14. Parsons, C. L.:The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int,107:370,2011
15. Archer, S.:Measurement of nitric oxide in biological models. FASEB J,7:349,1993
16. Nomiya, A., Nishimatsu, H., Homma, Y.:Interstitial cystitis symptoms associated with ketamine abuse:The first Japanese case. Int J Urol,18:735,2011
17. Xu, D. Z., Lu, Q., Deitch, E. A.:Nitric oxide directly impairs intestinal barrier function. Shock,17:139,2002
18. Lundberg, J. O., Ehren, I., Jansson, O. et al.:Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis. Urology,48:700,1996
19. Birder, L. A., Wolf-Johnston, A., Buffington, C. A. et al.:Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis. J Urol,173:625,2005
20. Keay, S., Zhang, C. O., Trifillis, A. L. et al.:Decreased 3H-thymidine incorporation by human bladder epithelial cells following exposure to urine from interstitial cystitis patients. J Urol,156:2073,1996
21. Keay, S., Kaczmarek, P., Zhang, C. O. et al.:Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor. Chem Biol Drug Des,77:421,2011
22. Keay, S., Leitzell, S., Ochrzcin, A. et al.:A mouse model for interstitial cystitis/painful bladder syndrome based on APF inhibition of bladder epithelial repair:a pilot study. BMC Urol,12:17,2012
23. Zacharias, D. A., Mullen, M., Planey, S. L.:Antiproliferative factor-induced changes in phosphorylation and palmitoylation of cytoskeleton-associated protein-4 regulate its nuclear translocation and DNA binding. Int J Cell Biol,2012:150918,2012
24. Byrne, D. S., Sedor, J. F., Estojak, J. et al.:The urinary glycoprotein GP51 as a clinical marker for interstitial cystitis. J Urol,161:1786,1999
25. Parsons, C. L., Bautista, S. L., Stein, P. C. et al.:Cyto-injury factors in urine:a possible mechanism for the development of interstitial cystitis. J Urol,164:1381,2000
26. Parsons, C. L., Greene, R. A., Chung, M. et al.:Abnormal urinary potassium metabolism in patients with interstitial cystitis. J Urol,173:1182,2005
27. Parsons, C. L., Stein, P. C., Bidair, M. et al.:Abnormal sensitivity to intravesical potassium in interstitial cystitis and radiation cystitis. Neurourol Urodyn,13:515,1994
28. Parsons, C. L., Hurst, R. E.:Decreased urinary uronic acid levels in individuals with interstitial cystitis. J Urol,143:690,1990
29. Moaddel, R., S. L. Venkata, et al.:A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome. Talanta 82(5): 1892-1904,2010
30. Ng, S. H., H. K. Lee, et al.:Dilated common bile ducts in ketamine abusers. Hong Kong Med J 15(2):157; 2009
31. Wong, S. W., K. F. Lee, et al.:Dilated common bile ducts mimicking choledochal cysts in ketamine abusers." Hong Kong Med J 15(1):53-56.2009
1. Parsons CL, Boychuk D, Jones S, et al. Bladder surface glycosaminoglycans:an epithelial permeability barrier. J Urol,1990.143(1):139-142.
2. Parsons CL., Greene AR, Chung M, et al. Abnormal urinary potassium metabolism in patients with interstitial cystitis. J Urol,2005.173(4):1182-1185.
3. Parsons CL. The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int,2011.107(3):370-375.
4. Parsons CL, Rosenberg MT, Sassani P, et al. Quantifying symptoms in men with interstitial cystitis/prostatitis, and its correlation with potassium-sensitivity testing. BJU Int,2005; 95(1):86-90.
5. Keller, M. L., McCarthy, D.0. and Neider, R. S. Measurement of symptoms of interstitial cystitis:a pilot study. Urol. Clin. North Am,1994; 21(1):67-71
6. Goin JE, Olaleye D, Peters KM, et al. Psychometric analysis of the University of Wisconsin Interstitial Cystitis Scale:implications for use in randomized clinical trials. J Urol 1998; 159(3):1085-90
7. Parsons CL, Dell J, Stanford EJ, et al. Increased prevalence of intersitial cystitis: Previously unrecognized urologic and gynecologic cases indentified using a new symptom questionnaire and intravesical potassium sensitivity. Urology,2002; (60) 573-578.
8. Mulholland SG, Hanno P, Parsons CL, et al. Pentosan polysulfate sodium for therapy of interstitial cystitis:A double-blind placebo-controlled clinical study. Urology,1990; 35(6) 552-8.
9. Parsons CL, Benson G, Childs SJ, et al. A quantitatively controlled method to study prospectively interstitial cystitis and demonstrate the efficacy of pentosanpolysulfate:J Urol,1993;150(3):845-8
10. Hanno PM, Fritz R, Wein AJ,et al. Heparin as antibacterial agent in rabbit bladder: Urology,1978; 12(4):411-5
11. Parsons CL. Successful downregulation of bladder sensory nerves with combination of heparin and alkalinized lidocaine in patients with interstitial cystitis:Urology,2005; 65(1):45-8.
12. 魏辉,黄英,张晓忠等.碱化利多卡因膀胱灌注治疗氯胺酮相关性膀胱炎[J].中华泌尿外科杂志.2010；31(9)：621-623
13. 王亮,陈昭颉,王庆堂等.肝素膀胱灌注治疗间质性膀胱炎[J].中华泌尿外科杂志,2004,25(9)：625-626.
2. Harrison, N. L., Simmonds, M. A.:Quantitative studies on some antagonists of N-methyl D-aspartate in slices of rat cerebral cortex. Br J Pharmacol,84:381,1985
3. Moore, K. A., Sklerov, J., Levine, B. et al.:Urine concentrations of ketamine and norketamine following illegal consumption. J Anal Toxicol,25:583,2001
4. Quibell, R., Prommer, E. E., Mihalyo, M. et al.:Ketamine*. J Pain Symptom Manage,41: 640,2011
5. Siegel, R. K.:Phencyclidine and ketamine intoxication:a study of four populations of recreational users. NIDA Res Monogr:119,1978
6. Chu, P. S., Kwok, S. C., Lam, K. M. et al.:'Street ketamine'-associated bladder dysfunction:a report of ten cases. Hong Kong Med J,13:311,2007
7. Shahani, R., Streutker, C., Dickson, B. et al.:Ketamine-associated ulcerative cystitis:a new clinical entity. Urology,69:810,2007
8. Yang, J., Bian, S. Z.:[A reviewing for abusing of ketamine]. Fa Yi Xue Za Zhi,23:312, 2007
9. Schifano, F., Corkery, J., Oyefeso, A. et al.:Trapped in the "K-hole":overview of deaths associated with ketamine misuse in the UK (1993-2006). J Clin Psychopharmacol,28: 114,2008
10. Homma, Y., Ueda, T., Tomoe, H. et al.:Clinical guidelines for interstitial cystitis and hypersensitive bladder syndrome. Int J Urol,16:597,2009
11. Wood, D., Cottrell, A., Baker, S. C. et al.:Recreational ketamine:from pleasure to pain. BJU Int,2011
12. Abrams, P., Artibani, W., Cardozo, L. et al.:Reviewing the ICS 2002 terminology report: the ongoing debate. Neurourol Urodyn,28:287,2009
13. Gillenwater, J. Y., Wein, A. J.:Summary of the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases Workshop on Interstitial Cystitis, National Institutes of Health, Bethesda, Maryland, August 28-29,1987. J Urol,140:203,1988
14. Parsons, C. L., Boychuk, D., Jones, S. et al.:Bladder surface glycosaminoglycans:an epithelial permeability barrier. J Urol,143:139,1990
15. Parsons, C. L., Hurst, R. E.:Decreased urinary uronic acid levels in individuals with interstitial cystitis. J Urol,143:690,1990
16. Parsons, C. L.:The role of the urinary epithelium in the pathogenesis of interstitial cystitis/prostatitis/urethritis. Urology,69:9,2007
17. Parsons, C. L.:The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int,107:370,2011
18. Logadottir, Y. R., Ehren, I., Fall, M. et al.:Intravesical nitric oxide production discriminates between classic and nonulcer interstitial cystitis. J Urol,171:1148,2004
19. Birder, L. A., Wolf-Johnston, A., Buffington, C. A. et al.:Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis. J Urol,173:625,2005
20. Hanno, P. M., Parsons, C. L., Shrom, S. H. et al.:The protective effect of heparin in experimental bladder infection. J Surg Res,25:324,1978
21. Byrne, D. S., Sedor, J. F., Estojak, J. et al.:The urinary glycoprotein GP51 as a clinical marker for interstitial cystitis. J Urol,161:1786,1999
22. Acharya, P., Beckel, J., Ruiz, W. G. et al.:Distribution of the tight junction proteins ZO-1, occludin, and claudin-4,-8, and-12 in bladder epithelium. Am J Physiol Renal Physiol, 287:F305,2004
23. Khandelwal, P., Abraham, S. N., Apodaca, G.:Cell biology and physiology of the uroepithelium. Am J Physiol Renal Physiol,297:F1477,2009
24. Liu, H. T., Shie, J. H., Chen, S. H. et al.:Differences in mast cell infiltration, E-cadherin, and zonula occludens-1 expression between patients with overactive bladder and interstitial cystitis/bladder pain syndrome. Urology,80:225 e13,2012
25. Lavelle, J. P., Meyers, S. A., Ruiz, W. G. et al.:Urothelial pathophysiological changes in feline interstitial cystitis:a human model. Am J Physiol Renal Physiol,278:F540,2000
26. Keay, S., Zhang, C. O., Trifillis, A. L. et al.:Decreased 3H-thymidine incorporation by human bladder epithelial cells following exposure to urine from interstitial cystitis patients. J Urol,156:2073,1996
27. Erickson, D. R.:Urine markers of interstitial cystitis. Urology,57:15,2001
28. Keay, S. K., Szekely, Z., Conrads, T. P. et al.:An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide. Proc Natl Acad Sci U S A,101:11803,2004
29. Keay, S., Zhang, C. O., Shoenfelt, J. L. et al.:Decreased in vitro proliferation of bladder epithelial cells from patients with interstitial cystitis. Urology,61:1278,2003
30. Zhang, C. O., Wang, J. Y, Koch, K. R. et al.:Regulation of tight junction proteins and bladder epithelial paracellular permeability by an antiproliferative factor from patients with interstitial cystitis. J Urol,174:2382,2005
31. Kim, J., Keay, S. K., Dimitrakov, J. D. et al.:p53 mediates interstitial cystitis antiproliferative factor (APF)-induced growth inhibition of human urothelial cells. FEBS Lett,581:3795,2007
32. Keay, S., Kleinberg, M., Zhang, C. O. et al.:Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production. J Urol,164:2112,2000
33. Keay, S. K., Zhang, C. O., Shoenfelt, J. et al.:Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis. Urology,57:9,2001
34. Keay, S., Leitzell, S., Ochrzcin, A. et al.:A mouse model for interstitial cystitis/painful bladder syndrome based on APF inhibition of bladder epithelial repair:a pilot study. BMC Urol,12:17,2012
35. Keay, S., Seillier-Moiseiwitsch, F., Zhang, C. O. et al.:Changes in human bladder epithelial cell gene expression associated with interstitial cystitis or antiproliferative factor treatment. Physiol Genomics,14:107,2003
36. Shahjee, H. M., Koch, K. R., Guo, L. et al.:Antiproliferative factor decreases Akt phosphorylation and alters gene expression via CKAP4 in T24 bladder carcinoma cells. J Exp Clin Cancer Res,29:160,2010
37. Keay, S., Kaczmarek, P., Zhang, C. O. et al.:Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor. Chem Biol Drug Des,77:421,2011
38. Smet, P. J., Jonavicius, J., Marshall, V. R. et al.:Distribution of nitric oxide synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry. Neuroscience, 71:337,1996
39. Xu, D. Z., Lu, Q., Deitch, E. A.:Nitric oxide directly impairs intestinal barrier function. Shock,17:139,2002
40. Chertin, B., Rolle, U., Solari, V. et al.:The role of nitric oxide in bladder urothelial injury after bladder outlet obstruction. BJU Int,94:392,2004
41. Muijsers, R. B., Folkerts, G., Henricks, P. A. et al.:Peroxynitrite:a two-faced metabolite of nitric oxide. Life Sci,60:1833,1997
42. Forstermann, U.:Nitric oxide and oxidative stress in vascular disease. Pflugers Arch,459: 923,2010
43. Darley-Usmar, V., Wiseman, H., Halliwell, B.:Nitric oxide and oxygen radicals:a question of balance. FEBS Lett,369:131,1995
44. Ho, M. H., Bhatia, N. N., Khorram, O.:Physiologic role of nitric oxide and nitric oxide synthase in female lower urinary tract. Curr Opin Obstet Gynecol,16:423,2004
45. Stothers, L., Laher, I., Christ, G. T.:A review of the L-arginine-nitric oxide-guanylate cyclase pathway as a mediator of lower urinary tract physiology and symptoms. Can J Urol,10:1971,2003
46. Romih, R., Korosec, P., Sedmak, B. et al.:Mitochondrial localization of nitric oxide synthase in partially differentiated urothelial cells of urinary bladder lesions. Appl Immunohistochem Mol Morphol,16:239,2008
47. Rovner, E., Propert, K. J., Brensinger, C. et al.:Treatments used in women with interstitial cystitis:the interstitial cystitis data base (ICDB) study experience. The Interstitial Cystitis Data Base Study Group. Urology,56:940,2000
48. Hanno, P. M., Buehler, J., Wein, A. J.:Use of amitriptyline in the treatment of interstitial cystitis. J Urol,141:846,1989
49. van Ophoven, A., Hertle, L.:Long-term results of amitriptyline treatment for interstitial cystitis. J Urol,174:1837,2005
50. Sant, G. R., Theoharides, T. C., Letourneau, R. et al.:Interstitial cystitis and bladder mastocytosis in a woman with chronic urticaria. Scand J Urol Nephrol,31:497,1997
51. Theoharides, T. C., Sant, G. R.:Hydroxyzine therapy for interstitial cystitis. Urology,49: 108,1997
52. Nickel, J. C., Barkin, J., Forrest, J. et al.:Randomized, double-blind, dose-ranging study of pentosan polysulfate sodium for interstitial cystitis. Urology,65:654,2005
53. Hampson, S. J., Woodhouse, C. R.:Sodium pentosanpolysulphate in the management of haemorrhagic cystitis:experience with 14 patients. Eur Urol,25:40,1994
54. Toren, P. J., Norman, R. W.:Cyclophosphamide induced hemorrhagic cystitis successfully treated with pentosanpolysulphate. JUrol,173:103,2005
55. Portenoy, R. K., Foley, K. M.:Chronic use of opioid analgesics in non-malignant pain: report of 38 cases. Pain,25:171,1986
56. Sant, G. R.:Intravesical 50% dimethyl sulfoxide (Rimso-50) in treatment of interstitial cystitis. Urology,29:17,1987
57. Khanna, O. P., Loose, J. H.:Interstitial cystitis treated with intravesical doxorubicin. Urology,36:139,1990
58. Rajkumar, G. N., Conn, I. G.:Botulinum toxin:a new dimension in the treatment of lower urinary tract dysfunction. Urology,64:2,2004
59. Vemulakonda, V. M., Somogyi, G. T., Kiss, S. et al.:Inhibitory effect of intravesically applied botulinum toxin A in chronic bladder inflammation. J Urol,173:621,2005
60. Omura, Y:Basic electrical parameters for safe and effective electro-therapeutics [electro-acupuncture, TES, TENMS (or TEMS), TENS and electro-magnetic field stimulation with or without drug field] for pain, neuromuscular skeletal problems, and circulatory disturbances. Acupunct Electrother Res,12:201,1987
1. Kowalski, J., Spychala, A.:[Mean transit time (MTT)-a new test of forced expiration]. Pneumonol Pol,51:1,1983
2. Massa, E. M., Farias, R. N.:Effect of auto-oxidized phospholipids on oxidative enzyme assays based on tetrazolium salt reduction. Biochim Biophys Acta,746:209,1983
3. Rosa, C. G, Tsou, K. C.:The use of tetranitro-blue tetrazolium for the cytochemical localization of succinic dehydrogenase. Cytochemical and cytological studies of sarcoma 37 ascites tumor cells. J Cell Biol,16:445,1963
4. Mohanty, J. G, Jaffe, J. S., Schulman, E. S. et al.:A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods,202:133,1997
5. Arroyo, A., Modriansky, M., Serinkan, F. B. et al.:NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance. J Biol Chem,277:49965,2002
6. Thor, H., Smith, M. T., Hartzell, P. et al.:The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells. J Biol Chem,257:12419,1982
7. Warren, M. C., Bump, E. A., Medeiros, D. et al.:Oxidative stress-induced apoptosis of endothelial cells. Free Radic Biol Med,29:537,2000
8. Reznikoff, C. A., Johnson, M. D., Norback, D. H. et al.:Growth and characterization of normal human urothelium in vitro. In Vitro,19:326,1983
9. Schmidt, W. W., Messing, E. M., Reznikoff, C. A.:Cultures of normal human urothelial cells from ureters of perfused cadaver transplant kidneys. J Urol,132:1262,1984
10. Reznikoff, C. A., Loretz, L. J., Johnson, M. D. et al.:Quantitative assessments of the cytotoxicity of bladder carcinogens towards cultured normal human uroepithelial cells. Carcinogenesis,7:1625,1986
11. Petzoldt, J. L., Leigh, I. M., Duffy, P. G. et al.:Culture and characterisation of human urothelium in vivo and in vitro. Urol Res,22:67,1994
12. Bishai, M. B., Kagawa, S., Narayan, K. S. et al.:Growth of a malignant human urothelial cell line (J82) in a serum-free medium (HMRI-1) developed previously for normal human urothelium. J Urol,134:1287,1985
13. Cilento, B. G., Freeman, M. R., Schneck, F. X. et al.:Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro. J Urol,152:665,1994
14. Christian, B. J., Loretz, L. J., Oberley, T. D. et al.:Characterization of human uroepithelial cells immortalized in vitro by simian virus 40. Cancer Res,47:6066,1987
15. Defendi, V, Naimski, P., Steinberg, M. L.:Human cells transformed by SV40 revisited: the epithelial cells. J Cell Physiol Suppl,2:131,1982
16. Stiles, C. D., Desmond, W., Jr., Sato, G. et al.:Failure of human cells transformed by simian virus 40 to form tumors in athymic nude mice. Proc Natl Acad Sci U S A,72: 4971,1975
17. Balda, M. S., Whitney, J. A., Flores, C. et al.:Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol,134:1031,1996
18. Cross, W. R., Eardley, I., Leese, H. J. et al.:A biomimetic tissue from cultured normal human urothelial cells:analysis of physiological function. Am J Physiol Renal Physiol, 289:F459,2005
19. Madara, J. L.:Regulation of the movement of solutes across tight junctions. Annu Rev Physiol,60:143,1998
20. Gaillard, P. J., Voorwinden, L. H., Nielsen, J. L. et al.:Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur J Pharm Sci,12:215,2001
21. Hu, P., Meyers, S., Liang, F. X. et al.:Role of membrane proteins in permeability barrier function:uroplakin ablation elevates urothelial permeability. Am J Physiol Renal Physiol, 283:F1200,2002
22. Tschugguel, W., Zhegu, Z., Gajdzik, L. et al.:High precision measurement of electrical resistance across endothelial cell monolayers. Pflugers Arch,430:145,1995
1. Persson, J.:Wherefore ketamine? Curr Opin Anaesthesiol,23:455,2010
2. Chu, P. S., Kwok, S. C., Lam, K. M. et al.:'Street ketamine'-associated bladder dysfunction:a report of ten cases. Hong Kong Med J,13:311,2007
3. Chu, P. S., Ma, W. K., Wong, S. C. et al.:The destruction of the lower urinary tract by ketamine abuse:a new syndrome? BJU Int,102:1616,2008
4. Yeung, L. Y., Rudd, J. A., Lam, W. P. et al.:Mice are prone to kidney pathology after prolonged ketamine addiction. Toxicol Lett,191:275,2009
5. Tan, S., Chan, W. M., Wai, M. S. et al.:Ketamine effects on the urogenital system-changes in the urinary bladder and sperm motility. Microsc Res Tech,2011
6. Wood, D., Cottrell, A., Baker, S. C. et al.:Recreational ketamine:from pleasure to pain. BJU Int,2011
7. Ratliff, T. L., Klutke, C. G., McDougall, E. M.:The etiology of interstitial cystitis. Urol Clin North Am,21:21,1994
8. Parsons, C. L., Boychuk, D., Jones, S. et al.:Bladder surface glycosaminoglycans:an epithelial permeability barrier. J Urol,143:139,1990
9. Matter, K., Balda, M. S.:Signalling to and from tight junctions. Nat Rev Mol Cell Biol,4: 225,2003
10. Slobodov, G., Feloney, M., Gran, C. et al.:Abnormal expression of molecular markers for bladder impermeability and differentiation in the urothelium of patients with interstitial cystitis. J Urol,171:1554,2004
11. Keay, S. K., Zhang, C. O., Shoenfelt, J. et al.:Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis. Urology,57:9,2001
12. Erickson, D. R.:Urine markers of interstitial cystitis. Urology,57:15,2001
13. Sant, G. R., Kempuraj, D., Marchand, J. E. et al.:The mast cell in interstitial cystitis:role in pathophysiology and pathogenesis. Urology,69:34,2007
14. Parsons, C. L.:The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int,107:370,2011
15. Archer, S.:Measurement of nitric oxide in biological models. FASEB J,7:349,1993
16. Nomiya, A., Nishimatsu, H., Homma, Y.:Interstitial cystitis symptoms associated with ketamine abuse:The first Japanese case. Int J Urol,18:735,2011
17. Xu, D. Z., Lu, Q., Deitch, E. A.:Nitric oxide directly impairs intestinal barrier function. Shock,17:139,2002
18. Lundberg, J. O., Ehren, I., Jansson, O. et al.:Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis. Urology,48:700,1996
19. Birder, L. A., Wolf-Johnston, A., Buffington, C. A. et al.:Altered inducible nitric oxide synthase expression and nitric oxide production in the bladder of cats with feline interstitial cystitis. J Urol,173:625,2005
20. Keay, S., Zhang, C. O., Trifillis, A. L. et al.:Decreased 3H-thymidine incorporation by human bladder epithelial cells following exposure to urine from interstitial cystitis patients. J Urol,156:2073,1996
21. Keay, S., Kaczmarek, P., Zhang, C. O. et al.:Normalization of proliferation and tight junction formation in bladder epithelial cells from patients with interstitial cystitis/painful bladder syndrome by d-proline and d-pipecolic acid derivatives of antiproliferative factor. Chem Biol Drug Des,77:421,2011
22. Keay, S., Leitzell, S., Ochrzcin, A. et al.:A mouse model for interstitial cystitis/painful bladder syndrome based on APF inhibition of bladder epithelial repair:a pilot study. BMC Urol,12:17,2012
23. Zacharias, D. A., Mullen, M., Planey, S. L.:Antiproliferative factor-induced changes in phosphorylation and palmitoylation of cytoskeleton-associated protein-4 regulate its nuclear translocation and DNA binding. Int J Cell Biol,2012:150918,2012
24. Byrne, D. S., Sedor, J. F., Estojak, J. et al.:The urinary glycoprotein GP51 as a clinical marker for interstitial cystitis. J Urol,161:1786,1999
25. Parsons, C. L., Bautista, S. L., Stein, P. C. et al.:Cyto-injury factors in urine:a possible mechanism for the development of interstitial cystitis. J Urol,164:1381,2000
26. Parsons, C. L., Greene, R. A., Chung, M. et al.:Abnormal urinary potassium metabolism in patients with interstitial cystitis. J Urol,173:1182,2005
27. Parsons, C. L., Stein, P. C., Bidair, M. et al.:Abnormal sensitivity to intravesical potassium in interstitial cystitis and radiation cystitis. Neurourol Urodyn,13:515,1994
28. Parsons, C. L., Hurst, R. E.:Decreased urinary uronic acid levels in individuals with interstitial cystitis. J Urol,143:690,1990
29. Moaddel, R., S. L. Venkata, et al.:A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome. Talanta 82(5): 1892-1904,2010
30. Ng, S. H., H. K. Lee, et al.:Dilated common bile ducts in ketamine abusers. Hong Kong Med J 15(2):157; 2009
31. Wong, S. W., K. F. Lee, et al.:Dilated common bile ducts mimicking choledochal cysts in ketamine abusers." Hong Kong Med J 15(1):53-56.2009
1. Parsons CL, Boychuk D, Jones S, et al. Bladder surface glycosaminoglycans:an epithelial permeability barrier. J Urol,1990.143(1):139-142.
2. Parsons CL., Greene AR, Chung M, et al. Abnormal urinary potassium metabolism in patients with interstitial cystitis. J Urol,2005.173(4):1182-1185.
3. Parsons CL. The role of a leaky epithelium and potassium in the generation of bladder symptoms in interstitial cystitis/overactive bladder, urethral syndrome, prostatitis and gynaecological chronic pelvic pain. BJU Int,2011.107(3):370-375.
4. Parsons CL, Rosenberg MT, Sassani P, et al. Quantifying symptoms in men with interstitial cystitis/prostatitis, and its correlation with potassium-sensitivity testing. BJU Int,2005; 95(1):86-90.
5. Keller, M. L., McCarthy, D.0. and Neider, R. S. Measurement of symptoms of interstitial cystitis:a pilot study. Urol. Clin. North Am,1994; 21(1):67-71
6. Goin JE, Olaleye D, Peters KM, et al. Psychometric analysis of the University of Wisconsin Interstitial Cystitis Scale:implications for use in randomized clinical trials. J Urol 1998; 159(3):1085-90
7. Parsons CL, Dell J, Stanford EJ, et al. Increased prevalence of intersitial cystitis: Previously unrecognized urologic and gynecologic cases indentified using a new symptom questionnaire and intravesical potassium sensitivity. Urology,2002; (60) 573-578.
8. Mulholland SG, Hanno P, Parsons CL, et al. Pentosan polysulfate sodium for therapy of interstitial cystitis:A double-blind placebo-controlled clinical study. Urology,1990; 35(6) 552-8.
9. Parsons CL, Benson G, Childs SJ, et al. A quantitatively controlled method to study prospectively interstitial cystitis and demonstrate the efficacy of pentosanpolysulfate:J Urol,1993;150(3):845-8
10. Hanno PM, Fritz R, Wein AJ,et al. Heparin as antibacterial agent in rabbit bladder: Urology,1978; 12(4):411-5
11. Parsons CL. Successful downregulation of bladder sensory nerves with combination of heparin and alkalinized lidocaine in patients with interstitial cystitis:Urology,2005; 65(1):45-8.
12. 魏辉,黄英,张晓忠等.碱化利多卡因膀胱灌注治疗氯胺酮相关性膀胱炎[J].中华泌尿外科杂志.2010；31(9)：621-623
13. 王亮,陈昭颉,王庆堂等.肝素膀胱灌注治疗间质性膀胱炎[J].中华泌尿外科杂志,2004,25(9)：625-626.