The Expression And Clinical Significance Of Histamine Receptors In Ketamine-associated Urinary System Dysfunction

Research background:Lower urinary tract symptoms(LUTS) caused by long term ketamine addiction, which was reported by Hong Kong scholars, began to spring up since2007. Usually, the onset symptoms is LUTS in the exposed populations. At present, domestic scholars used to call it "Ketamine-associated Urinary System Dysfunction (KAUD)". The clinical manifestation of KAUD is very similar to interstisal cystitis(IC). Clinical guidelines for interstitial cystitis and hypersensitive bladder syndrome(2009) claimed that the pathogenic mechanisms of them may be similarly. Mold confirmed in the bladder tissues of IC patients, mast cell were activated and histamine and its metabolite levels were significantly higher compare with normal bladder tissues. Boucher and his coleagues confirmed that in IC mice models, mast cells were activated, histamine and other chemical substances secretion. What’s more sodium hyaluronate have inhibitory effect on mast cell activation.In order to explore the pathogenesis of KAUD, confirmed that the mast cells and histamine receptor is involved in the pathological changes of the disease process, this study made the following approach:1. From November2011to September2012, in the people’s liberation army181hospital uropoiesis surgical department10KAUD patients were selected. Special staining methods were used to observe the mast cell, histamine and histamine receptors expression in bladder tissue. In order explore the roles they play in KAUD pathological changes.2. The animal model was established, at the aim at the relation of histamine receptor expression and upper urinary damage, to provide new ideas for the treatment of the disease and new therapeutic targets.3. To explore the treatment strategies of the disease.Objective1. To investigate the expression and clinical significance of four histamine receptors:H1R, H2R, H3R and H4R in urinary bladder of patients of Ketamine associated Urinary System Dysfunction.2. To investigate the expression and clinical significance of H1repceptor in kidney and bladder tissue of mice after prolonged ketamine intraperitoneal injection.3. To investigate the treatment strategy for Ketamine-associated Urinary System Dysfunction.Methods1. Immunohistochemical S-P method was used to detect the expression of histamine receptors:H1R, H2R, H3R and H4R in the bladder tissues from the experimental group (bladder tissues from10patients with ketamine-induced cystitis) and control group (we chose10patients’, whose bladder was full cut because of bladder tumor, bladder tissue away from the bladder tumor). The average optical density (OD value) of four kinds of different histamine receptors in the two groups were separately calculated by Imagepro-plus6.0. At the same time, mast cells were marked by toluidine blue special dyeing and counted. PSS13.0was used in the statistic analysis. The data followed normal distribution was expressed with x±s. Two-sample t-test was used for statistical analysis. The data which is not subject to normal distribution was expressed with M (P25, P75) and compared with rank sum test. The P value smaller than0.05was thought to be statistical significance.2. We did the study from May2012to December2012. Sixty male2-month-old SD rats, weighted200±10g, were randomly divided into Group A and Group B. In the Group A, Ketamine,100mg/kg, was given as intraperitoneal injection every other day, while in Group B100mg/kg normal saline was given. The dosage was adjusted every week according the weight of rats. After2,4and6months,10rats from each group were randomly chosen. First, record the Micturition number during2hours. Second, collect urine samples over a24h period and the content of Na+and K+were determined. Third, blood samples, kidneys and urine bladders were harvested. Creatinine were determined in the blood samples. After fixed with4%neutral formalin overnight, the tissue samples were finally embedded in paraffin blocks with a sagittal orientation and serial sections of1.8μm were obtained. HE staining were conducted on all the tissues, Immunohistochemical S-P method was used to detect the expression of H1receptor in the bladder tissues from Group A and Group B. The average optical density in both groups were separately calculated by Imagepro-plus6.0. One-way ANOVA was used for analysis urine volume of24h, micturition number during2hour, the loss of sodium from urine for24h and the loss of potassium from urine in the two groups. PSS13.0was used in the statistic analysis. Factorial analysis was used for the average optical density statistic analysis. The P value smaller than0.05was thought to be statistical significance.3. A retrospective analysis of35patients with ketamine-induced cystitis,31male,4female, was undertaken in our department during November2011and March2013.15patients in group A, whose cystometric capacity (MCC) greater than200ml below narcotism, received treatment I:prednisone20mg, bid, po; solifenacin succinate5mg, qd, po; amitriptyline50mg, qd, cimetidine0.2g,bid, po. There are20patients with a MCC smaller than200ml in group B received treatment Ⅱ:being given bladder hydrodistention therapy, before treatment I. There are only two patients in group C, whose MCC still less than200ml after hydrodistention therapy, recieved treatment III, augmentation cystoplasty. PUF and Micturition time interval were compared before and after treatment. PSS13.0was used in the statistic analysis. The statistic method was adopted the matched t-test. The P value smaller than0.05was thought to be statistical significance.Results:1.(1)Results of HE staining:The urinary bladder of the experimental group displayed pathology when compared with control. The small blood vessels under mucosal dilated. The epithelium in the experimental group was much thiner than those of control. Infiltration of mononuclear cells was observed in the submucosa of the bladder wall.(2) Toluidine blue staining and mast cell count:In the experimental group, the infiltration of mast cells in the urinary bladder submucosal and bladder smooth muscle, along a line with vascular distribution. The mast cell which was count for each100x microscopic field in experimental group were (488.5±255.1), comparing with the control group (88.0-46.2). The difference between the two groups in mast cell count was statistically significant (P<0.05).(3)Comparing with control group, the expression of H1R, H2R, and H4R in test group were significantly raised (P<0.05). The expression of H3R in bladder tissue had no significance (P>0.05). Mast cells were increased in experimental group (P<0.05).2.10SD rats were executed after six months ketamine use. Nine in ten successed establish rabbit models.(1)HE staining of kidney tissue:there were lots of red blood cells in renal tubule and collecting duct. After four months ketamine addiction, rats of Group A renal interstitial vascular expansion and endothelial cell proliferation was observed. The renal tubules were broaden and wall became thin. The ones with six months ketamine addiction, Infiltration of mononuclear cells was observed in the renal distal tubules, collecting tubules and tubulointerstitial lesions.(2)HE staining of bladder tissue:2months after the treatment, bladder mucosa edema in rats. Four months after the treatment, lymphocytes, monocytes were found in submucosal, muscle and perivascular. For months after the rats bladder transitional epithelial cells thinned or disappeared, and the cell boundaries is not clear or collapse.The expression of H1receptor in the kidney followed three characteristics:(1) the degree of the expression of H1in renal proximal convoluted tubules was reduced than that of distal convoluted tubules.(2) The expression level of interstitial small vascular endothelial cell is higher than other part.(3) expression level. The results indicated that there were more H1receptors in cortical nephron than medullary nephron.Comparing with group B, urine volume of24h and Micturition number during2hours were significantly increased in group A (P<0.05). The loss of sodium from urine for24h increased significantly, while the loss of potassium from urine reduced significantly (P<0.05). But the serum creatinine level had no difference among the three groups (P<0.05). The expression of H1receptor in kidneys and urine bladder in group A was significantly raised compared with group B (P<0.05). In the group A, the expression of H1receptor in kidneys harvested after2months,4months and6months drug use are significantly differenced (P<0.05) and goes up with the drug use time prolonged. While in the bladders,4months and6months H1receptor expression were significantly raised than2months’(P<0.05).3. In group A, the PUF declined by an average of14.93±1.67, and Micturition time interval prolonged by an average of84.60±25.79min. Both of the difference were significance (P<0.05).5individuals in group B received treatment I had no effect after the treatment (P>0.05). The other15individuals received treatment II. The PUF declined by an average of15.40±3.09, and Micturition time interval prolonged by an average of67.07±23.94min. Both of the difference were significance(P<0.05). In group C, the quality of life of them was improved significantly after augmentation cystoplasty.Conclusions:1. Mast cells, H1R, H2R, and H4R are closely related to the ketamine-induced cystitis. Cortisol may be an effective medical for ketamine-induced cystitis.2. Inhibition of mast cell activation may be a new therapy idea for KAUD.3. H1R, H2R, and H4R may be new diagnostic indicators and new treatment targets of ketamine-induced cystitis.4. Prolong ketamine addiction exerts toxicity not only on the bladder but also on the kidney. The increased expression of H1receptor in rats kidney and bladder tissues of group A indicates that H1receptor may be related to ketamine-induced urinary system dysfunction. H1receptor may be a new treatment target for ketamine-induced dysfunction. The loss of sodium and potassium from urine for24h may be a sensitive index for the assessment of degree of kidney damage in the early stages of ketamine-induced dysfunction.5. The treatment we chose for the ketamine-induced cystitis should be based on the MCC. If MCC>200ml, conservative medication is the best choice. If MCC<200ml, hydrodistention therapy is needed before conservative medication. For the individual whose MCC still less than200ml after hydrodistention therapy, augmentation cystoplasty may be recommended.