Alteration of muscarinic and purinergic receptors in urinary bladder of rats with cyclophosphamide-induced interstitial cystitis

We characterized muscarnic and purinergic receptors and urodynamic parameters in the bladder of cyclophosphamide (CYP)-treated rats to clarify the mechanisms involved in the pathophysiology of interstitial cystitis (IC). In the cystometry of CYP-treated rats compared with control rats, the micturition interval and micturition volume were significantly (55% and 77%, respectively) decreased and the frequency of micturition and basal pressure were significantly (3 and 2.3 times, respectively) increased. These changes in urodynamic parameters may characterize the detrusor overactivity occurring in CYP-treated rats. The maximal number of binding sites (B(max)) for specific binding of [N-methyl-(3)H]scopolamine methyl chloride ([(3)H]NMS) and alphabeta-methylene ATP [2,8-(3)H]tetrasodium salt ([(3)H]alphabeta-MeATP) was significantly (43% and 31%, respectively) decreased in the bladder of CYP-treated rats compared with control rats. On the other hand, the apparent dissociation constant (K(d)) for neither radioligand was significantly altered by the CYP treatment. K(i) value for the inhibition of bladder [(3)H]NMS binding by antimuscarinic agents (oxybutynin, tolterodine, darifenacin, and AF-DX 116) did not differ significantly between control and CYP-treated rats. The inhibition constant (K(i)) for the inhibition of bladder [(3)H]alphabeta-MeATP binding by purinergic antagonists (A-317491, PPADS) was significantly higher in CYP-treated rats than control rats. In conclusion, CYP treatment has been shown to cause down-regulation of pharmacologically relevant (muscarinic and purinergic) receptors in the bladder of rats. Thus, the present study offers further pharmacological evidence that both muscarinic and purinergic mechanisms contribute significantly to the urinary dysfunction due to IC.     

Interstitial cystitis/bladder pain syndrome: An update

Interstitial cystitis, or painful bladder syndrome, is a condition characterized by bladder pain, urinary frequency, urgency, and nocturia. The cause of the condition remains obscure and it remains a diagnosis of exclusion. Current theories of pathogenesis include a chronic or subclinical infection, autoimmunity, neurogenic inflammation or bladder urothelial defects.     

Abnormal urothelial HLA-DR expression in interstitial cystitis.

Interstitial cystitis is a chronic inflammatory disorder of the urinary bladder that predominantly afflicts middle-age women. The end stage of the disease is ulceration of the urothelium, the so-called Hunner's ulcer. The aetiology of interstitial cystitis remains obscure. We have studied bladder biopsies from 22 cases of interstitial cystitis and control groups consisting of six cases of bacterial cystitis and eight healthy women. Indirect immunofluorescence was performed on the biopsies using murine MoAbs to human HLA class I molecules, and class II molecules, HLA-DP, HLA-DQ and HLA-DR. In interstitial cystitis, bacterial cystitis and normal controls most cells expressed HLA class I products. In six cases of interstitial cystitis and one case of bacterial cystitis there was evidence of HLA class I hyperexpression. In normal bladder and bacterial cystitis HLA class II expression was restricted to submucosal dendritic cells, Langerhans cells macrophages, vascular endothelial cells and activated lymphocytes. All but two cases of interstitial cystitis showed surface expression of HLA-DR (but not HLA-DP or DQ). In all cases of interstitial cystitis there was an increase in the numbers of macrophages, activated lymphocytes and vascular endothelial cells expressing HLA class II molecules within the submucosa. These findings provide further evidence for the importance of inappropriate HLA molecule expression in a disease suspected of having an autoimmune pathogenesis and where cellular autoimmune mechanisms play a decisive role in the destruction of the target cells--the bladder urothelium.     

Possible application of Raman microspectroscopy to verify the interstitial cystitis diagnosis after potassium sensitivity test: phenylalanine or tryptophan as a biomarker

There is lack of a worldwide standard technique for clinical diagnosis of interstitial cystitis (IC). Raman spectroscopy with higher specificity and sensitivity has been extensively used to act as a non-destructive analytical technique without special sample preparation. In this preliminary study, possible use of Raman microspectroscopy as an IC diagnostic tool was attempted. Twenty-two participants were screened by clinical features, history, urodynamic evaluations and potassium sensitivity test (PST). The freeze-dried water samples voided from all the participants after PST were directly determined by using a confocal Raman microspectroscopy to search the biomarker. Participants with or without IC symptom were separated into control and clinical groups, according to the above screening. The participants in the clinical group were further divided into mild and severe subgroups by PST. The symptom of urinary pain and urgency was significant difference between the mild and severe subgroups (p<0.05). A significant increase in urinary frequency but a marked reduction in bladder capacity, maximum cystometric capacity and maximum voiding flow rate were obtained for clinical group of IC participants, as compared with the result of control group (p<0.05). By using Raman microspectroscopic determination, the band near 1003 or 1005 cm(-1) assigned to phenylalanine was respectively detected from the freeze-dried water sample of control group or mild subgroup, but the band at 1010 cm(-1) due to tryptophan was found in the freeze-dried water sample of severe subgroup. The result of this preliminary study first suggests a possible application of Raman microspectroscopy to strongly certify the results of PST for IC diagnosis. Phenylalanine or tryptophan might be acted as a biomarker to assist the diagnosis of IC after PST. Particularly, the appearance of tryptophan might be used to discriminate the severity of IC symptom.     

Pharmacological validation of a model of cystitis pain in the mouse

Interstitial cystitis (IC) is a chronic pelvic-perineal pain syndrome of unknown etiology that mainly targets the lower urinary tract. Pain is the most prominent feature of IC and current therapies provide limited relief. Novel treatment options for IC could be identified if more predictive animal models were available. A rat model based on administration of cyclophosphamide (CP) mimics the symptoms of IC and has been well characterized. However, experiments in mice have not consistently reported both the spontaneous and evoked pain behaviors. The current series of studies demonstrate that CP (200-400mg, i.p.) increased both spontaneous and evoked pain behaviors in mice. Additionally, clinically relevant compounds: morphine (1-10mg/kg), ketorolac (1-5.6mg/kg) and duloxetine (3-30mg/kg) all significantly reversed pain behaviors. In contrast, gabapentin (56mg/kg) had no effect. Thus, CP-induced cystitis in mice may be used to evaluate novel therapeutics for the treatment of pain due to interstitial cystitis.     

Nerve growth factor enhances cholinergic innervation and contractile response to electric field stimulation in a murine in vitro model of chronic asthma

Background Asthma is a chronic inflammatory disease characterized by airway hyper‐responsiveness. Alterations in the neurogenic control are believed to contribute to the pathogenesis. Yet, the long‐term interaction between nerves and inflammatory mediators, such as the neurotrophin nerve growth factor (NGF), are not fully understood much due to the absence of appropriate experimental assays. Objective To develop an ex vivo mouse organ culture assay and to investigate the effects of NGF on nerve‐mediated airway contractions. Method Mouse tracheal segments were cultured in periods of up to 16 days. Their contractile responses to electric field stimulation (EFS) were investigated. In addition, the effect of 4 days of NGF treatment was analysed using EFS and immunohistochemistry. Results EFS (0.2–25.6 Hz) induced reproducible and frequency‐dependent cholinergic contractions of both fresh and cultured tracheal segments. The main part of the EFS response was blocked by tetrodotoxin or atropine. After 4 days in culture, regional differences appeared, with stronger EFS responses in distal than in proximal segments. More nerve fibres were seen in distal segments than in proximal segments. Treatment with NGF during 4 days of culture increased the innervation of the proximal segments, at the same time as the cholinergic contractile responses to EFS were enhanced dose‐dependently. Conclusion Culture of tracheal segments appears to be a suitable assay for the examination of long‐term effects induced by inflammatory mediators on neurally mediated airway contractions. NGF treatment enhanced the cholinergic, nerve‐dependent contractions and increased the amount of nerve fibres seen in the murine tracheal segments, suggesting a role for NGF in the development of airway hyper‐responsiveness.     

不同年龄牦牛睾丸蛋白基因产物9.5和神经肽Y的分布比较

目的 探讨蛋白基因产物9.5(PGP9.5)和神经肽Y(NP-Y)在不同年龄牦牛睾丸的分布特征。方法 采取睾丸摘除术收集3~4月龄睾丸9对,3、4岁牦牛睾丸10对,10~12岁牦牛睾丸7对,常规石蜡包埋、切片,免疫组织化学SP法观察PGP 9.5和NP-Y在不同年龄牦牛睾丸的分布特点。结果 PGP 9.5和NP-Y显著分布于幼龄及青年牦牛睾丸Leydig细胞及间质血管周围或血管壁内,两者在青年牦牛睾丸初级精母细胞为阴性,其余生精细胞为阳性;且阳性信号在睾丸体最强,其次为头部,尾部最弱;各年龄段Sertoli 细胞中NP-Y为阳性表达,PGP 9.5均为阴性;老龄牦牛睾丸血管PGP 9.5阳性信号明显,而NP-Y基本无表达。结论 青年牦牛睾丸组织PGP 9.5及NP-Y的显著分布对睾丸生殖功能调节发挥着重要的调控作用;提示NP-Y在老龄牦牛睾丸血管的分布显著降低。     

豚鼠膀胱中二聚体Cajal间质细胞的分布特点及意义

目的　观察二聚体Cajal间质细胞(interstitial cells of Cajal,ICC)在豚鼠膀胱不同组织及部位的分布特点,并探讨其意义。 方法　电子显微镜下观察豚鼠膀胱壁组织切片黏膜层,黏膜下层,肌层内二聚体ICC分布情况;对膀胱组织切片进行免疫荧光染色,用c-Kit抗体标记ICC,激光共聚焦显微镜下观察二聚体ICC在膀胱顶部、体部、颈部的分布特点。 结果　在电子显微镜下见二聚体ICC主要分布于黏膜下层,而肌层主要以单体ICC为主。免疫荧光染色发现每高倍镜视野下膀胱顶部二聚体ICC平均数量为（3.47±0.53）个,体部和颈部为（1.57±0.45）个和(0.49±0.19)个。膀胱顶部二聚体ICC数量明显高于体部和颈部（P<0.01）。 结论　二聚体ICC主要分布于豚鼠膀胱顶部的黏膜下层,可能是感受黏膜张力刺激,引发顶部膀胱自发兴奋的起搏细胞。     

Immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in the ovary of the sexually immature ostrich (Struthio camelus)

The innervation of the ovary has been studied in various species of birds and mammals. Despite the fact that the innervation of any organ is an essential factor in controlling its growth and function, no information is available on the distribution of nerve fibers in the ovary of the sexually immature ostrich. Thus, the present study was undertaken to investigate the distribution of nerve fibers in the ovary of the sexually immature ostrich, using antibodies against neurofilament protein type M of 160 kD (NP), protein gene product 9.5 (PGP 9.5) and neuron specific enolase (NSE). A total of 26 sexually immature female ostriches, aged between 12 and 14 months were used in the present study. Immunostaining was performed using a LSAB plus kit (Dakocytomation, Denmark). Antibodies against NP and PGP 9.5 were used at dilutions of 1:25 and 1:50, respectively. A ready-to-use solution containing antibodies against NSE was also used. Strong immunostaining for NP, PGP 9.5 and NSE was observed in nerve bundles, which coursed through the ovarian stalk and extended into the medulla and cortex. In addition, NSE immunoreactive nerve cell bodies were observed in the cortex and medulla. NP, PGP 9.5 and NSE immunoreactive nerve fibers were present in the thecal layer of the follicular wall. The current study has highlighted the distribution of NP, PGP 9.5 and NSE-immunoreactive nerve fibers in the ovary of the sexually immature ostrich. The findings of the present study suggest that the distribution of nerve fibers in the immature ostrich is similar to that of the domestic fowl.     

膀胱过度活动症及间质性膀胱炎膀胱灌注治疗的研究进展

膀胱过度活动症(overactive bladder,OAB)与间质性膀胱炎/膀胱疼痛综合征(interstitial cystitis/painful bladder syndrome,IC/PBS)是妇科泌尿临床上缺乏特异性诊断指标的两种常见膀胱疾病,均有尿频、尿急、夜尿增多症状,给患者的日常生活、身心健康带去严重困扰,然而其病因均不明确.近年来,随着人们对生活质量要求的提高,OAB及IC/PBS越来越得到重视.     

蛋白基因产物9.5与宫颈癌临床病理表现的关系及其作用机制的实验研究

目的:研究蛋白基因产物9.5(PGP9.5)在宫颈癌病理标本中的表达与宫颈癌临床病理特征及预后的关系,并初步探讨利用PGP9.5-siRNA靶向治疗宫颈癌的潜在价值。方法:回顾性分析2008年1月~2015年6月在天津市第五中心医院妇科及天津市中心妇产科医院接受手术治疗并经病理确诊的180例宫颈癌患者临床资料。所有患者宫颈癌病理标本制作病理切片并进行SP法免疫组化染色。以PGP9.5染色后评分为依据将患者分为PGP9.5高表达组和PGP9.5低表达组。观察2组患者年龄、HPV感染、病理分级、肿瘤直径、淋巴结转移、浸润深度、累及脉管和临床分期等临床病理学参数的关系。采用Kaplan-Meier法和log-rank检验进行生存分析。采用PGP9.5-siRNA、NC-siRNA、PGP9.5真核表达质粒和对照空质粒按照脂质体载体试剂说明书转染Si Ha细胞,设立si-PGP9.5组、NC组、PGP9.5组和vector组。同时设立Si Ha细胞空白对照(control)组。分别采用Western blot实验、平板克隆形成实验和Transwell实验分析5组细胞PGP9.5蛋白的表达水平、克隆形成能力和侵袭能力。结果:2组患者病理分级、肿瘤直径、淋巴结转移、浸润深度、累及脉管和临床分期等临床病理参数与PGP9.5表达水平有关。PGP9.5高表达组患者3、5年总体生存率明显低于PGP9.5低表达组患者。与control组相比,si-PGP9.5组SiHa细胞中PGP9.5蛋白的表达量明显降低,克隆形成数量明显减少,过膜细胞数量明显减少;PGP9.5组Si Ha细胞中PGP9.5蛋白的表达量明显升高,克隆形成数量明显增多,过膜细胞数量明显增加。结论:PGP9.5蛋白高表达预示宫颈癌患者预后不良,可能成为预测宫颈癌患者预后的良好指标,对其表达进行抑制可能实现对宫颈癌的有效基因治疗。     

Follicular cystitis in urine cytology: A clinical and cytopathologic study

Despite being an important differential diagnosis of bladder tumor on cystoscopy, follicular cystitis (FC) is rarely diagnosed on cytologic material. We performed a retrospective study on cases of FC diagnosed on bladder biopsy and/or urine cytology in our institution. A total of 35 cases of FC were identified with a female predominance (F:M = 2:1). Hematuria was the most common clinical presentation. Cystoscopic findings included mass lesions, yellow plaques, and surface erythema. History of urinary tract infection was reported in 48% of the patients, and majority of those patients had positive concurrent urine culture, most commonly with beta‐hemolytic streptococcus, Group B. A total of 17 out of 35 patients had urine cytology specimens. When the presence of follicular dendritic cells in clusters of variously sized lymphocytes is used as the cytological diagnostic criterion, 6 out of 17 cases were diagnosed as FC and 5 out of 6 were confirmed by concurrent biopsy. This retrospective study not only analyzed the clinical characteristics of FC but also elucidated the cytological diagnostic criteria of FC and confirmed its specificity.     

Colloid osmotic pressure of the subcapsular interstitial fluid of rat kidneys during hydropenia and volume expansion

Summary To determine the colloid osmotic pressure of subcapsular interstitial fluid in rat kidneys two different methods were used. Collection of subcapsular fluid with glass pipettes or implanted microcatheters and subsequent protein analysis resulted in a protein concentration of 1.8g%±0.6 and 2.0g%±0.8, respectively. Lymph protein concentration was not significantly different from that of subcapsular fluid samples. During extracellular volume expansion both subcapsular and lymph protein concentration fell to 0.42g%±0.23 and 0.7g%±0.5. Application of anin vivo oncometric method resulted in an effective oncotic pressure about twice that estimated from protein determinations. Using average values for intratubular and intracapillary oncotic and hydrostatic pressures a tubulo-interstitial net driving force of 20 mm Hg and an interstitial-capillary net driving force of 13 mm Hg is estimated in hydropenic animals. During volume expansion net transtubular pressure gradient is reduced to about 60–70% of control while the transcapillary gradient is virtually unchanged.     

Immunocytochemical expression of protein gene product (PGP) 9.5 in the cat bronchopulmonary neuroendocrine cells and nerves

Variously fixed, wax-embedded lung and gastrointestinal serial tissue sections from newborn to adult cats were stained with hematoxylin-eosin (H&E), Grimelius' silver, and immunohistochemical techniques using antisera to protein gene product (PGP) 9.5, a neuron-specific protein under strong evolutionary constraints. PGP 9.5 is revealed as a pan-neuroendocrine marker useful for tracing the pulmonary diffuse neuroendocrine system (PDNES) and studying the relationships between neuronal and neuroendocrine elements at various stages of life. Its occurrence is also compared in the pulmonary and the gastrointestinal tract. In spite of a close resemblance to already described neuroepithelial bodies (NEB) of other mammals, cat NEB feature typical constitutional and distributional difference, illustrating interspecies differences. The number of PGP 9.5 immunopositive pulmonary neuroendocrine cells declines gradually after 3 weeks and throughout adult life. Immunoreactivity in neuronal elements is lost after 1 week of age. In gastrointestinal tissues, only neuronal lelements immunostain, suggesting functional variations or a separate embryological origin for enteroendocrine cells. © 1993 Wiley-Liss, Inc.     

Cytologic manifestations of cystitis follicularis in urine specimens

Four cases of cystitis follicularis diagnosed by urine cytology are presented, and are the first reported in the cytologic literature. Cystitis follicularis (follicular cystitis) is characterized by formation of lymphoid follicles in the lamina propria of the trigonal region of the bladder, and is considered to be the result of repeated bouts of urinary tract infection, usually bacterial, with other pathologic processes contributing to the development and prolongation of the infection. Cytologically it differs from chronic cystitis with prominent lymphocytosis by the presence of cellular elements from the germinal centers of lymphoid follicles, reminiscent of the cytologic findings in follicular cervicitis, with possible additional epithelial cytologic atypias from the overlying urothelium, which frequently undergoes reactive changes (hyperplastic, metaplastic, and ulcerative). The practical aspect of recognition of this entity in cytologic specimens is avoiding diagnostic errors of possible malignancy (lymphoid or other), and also of other forms of inflammatory disease, such as granulomatous type, with a different clinical significance.     

Connective tissue stroma in bladder papillary transitional cell carcinoma, carcinoma in situ and benign cystitis

The stromal characteristics in papillary and non-papillary tumours of the urinary bladder were investigated in an attempt to improve the accuracy of histopathological diagnosis. It appeared to be possible to differentiate true papillary tumours from pseudopapillary structures lined by carcinoma in situ. Stromal differences were not found in cases of carcinoma in situ accompanied by denuding cystitis and cystitis due to other aetiological factors. It is concluded that histopathological examination of the stroma of bladder tumours improves diagnostic accuracy.     

Immunocytochemical investigation of neurovascular relationships in human tooth pulp

This study sought to explore the anatomical relationships between peptidergic nerves and blood vessels within human primary and permanent teeth. Extracted primary and permanent molars (n = 120) were split longitudinally, placed in Zamboni's fixative and the coronal pulps were processed for indirect immunofluorescence. Ten-micrometre-thick serial frozen pulp sections were triple-labelled using combinations of the following antisera: (1) protein gene-product 9.5 (PGP 9.5), a general neuronal marker; (2) one of the neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) or neuropeptide Y (NPY); and (iii) the lectin Ulex europeus, a label for vascular endothelium. The mid-coronal pulp region was examined, using fluorescence microscopy, to determine the proportion of blood vessels showing a positive innervation (recorded when PGP 9.5-labelled nerves appeared to intersect the vessel wall). In addition, the percentage of these vascular-related nerves expressing each of the above neuropeptides was recorded. Overall, 20% of pulpal blood vessels appeared to have a positive innervation. In the main these were thick-walled arterioles. Capillaries, venules and lymphatics were mostly devoid of an associated innervation. Ninety-two per cent of vascular-related nerves expressed CGRP, 87% expressed SP, 15% expressed VIP and 80% expressed NPY. There were no significant differences in overall innervation or peptide-related innervation between primary and permanent teeth (P < 0.05, ANOVA), indicating that pulpal blood flow is likely to be subject to similar neurological control mechanisms in both dentitions.     

Immunohistochemical localisation of regulatory neuropeptides in human circumvallate papillae

The occurrence and distribution of neuropeptide-containing nerve fibres in the human circumvallate papillae were examined by the peroxidase–antiperoxidase immunolocalisation method using surgical specimens that had not been subjected to radiotherapy, and the abundance of neuropeptide-containing fibres was expressed as the percentage of total nerve fibres demonstrated by protein gene product (PGP) 9.5 immunoreactivity for a quantitative representation of these peptidergic fibres. Substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (IR) nerve fibres were densely distributed in the connective tissue core of the circumvallate papillae, and some SP and CGRP-IR fibres were associated with the taste buds. A moderate number of vasoactive intestinal polypeptide (VIP)-IR fibres and a few galanin (GAL)-IR fibres were also seen in the connective tissue core and subepithelial layer. There were, however, no VIP-IR or GAL-IR fibres associated with the taste buds. Neuropeptide Y (NPY)-IR fibres were few and were associated with the blood vessels. Within the epithelium of the circumvallate papillae, no peptidergic fibres were found, although a number of PGP 9.5-IR fibres were detected. The abundance of SP, CGRP, VIP, and GAL-IR fibres expressed as the percentage of total PGP 9.5 IR fibres was 25.35±3.45%, 22.18±3.26%, 10.23±1.18%, and 4.12±1.05%, respectively. The percentage of NPY-IR fibres was below 3%. In a deeper layer of the papillae, a few VIP, GAL, and NPY-IR ganglion cells were found, and VIP immunoreactivity was detected in a few cells of the taste buds. There was no somatostatin, leucine enkephalin, or methionine enkephalin immunoreactivity in the circumvallate papillae. These results suggest that the dense SP and CGRP-IR fibres within the connective tissue core of the human circumvallate papillae may be involved in the deep sensation of the tongue.