Normal Mouse Bladder Research Articles

Murine β-defensin-2 may regulate the effect of bacillus Calmette-Guérin (BCG) in normal mouse bladder

PurposeWe investigated whether bacillus Calmette-Guérin (BCG)–induced secretion of murine β-defensin-2 (mBD2) and determined whether mBD2 regulated BCG effects in the normal mouse bladder. Materials and methodsA total of 140 C57BL/6 female mice were divided into 28 groups, and the experiment was performed over 3 steps. In the first step (20 groups), mice bladders were stimulated with different doses of BCG (multiplicity of infection [MOI] 0, 1, 10, 30, and 100) and histological analysis was conducted in bladder specimens isolated at different times (0, 4, 8, and 24h after instillation) to determine optimal dose and time point of BCG internalization and urine mBD2 and cytokine concentration. In the second step (4 groups), BCG internalization and urine cytokine levels were measured after pretreatment of different recombinant mBD2 (rmBD2) (0, 1, 2.5, and 5ng/ml) at optimal dose and time point. In the third step (4 groups), BCG internalization and urine cytokine levels were compared between pretreatment conditions (control, rmBD2, anti-mBD2 Ab, and rmBD2+anti-mBD2 Ab). Urine was collected for estimating mBD2 levels and a multiplex analysis for 9 cytokines. Real-time polymerase chain reaction assay was used for estimating the relative BCG cell number in mice bladder tissue. ResultsBladder edema was induced by BCG (MOI 30 and 100), which progressed to an inflammatory infiltrate composed primarily of neutrophils and increased mBD2 secretion at 4 hours after instillation. Relative BCG cell number and urinary cytokine levels (interferon-γ and interleukins [IL]-2, -4, -6, and -10) response pattern was characterized by a peak at 4 hours after instillation followed by rapid decline. The levels of interferon-γ, and IL-1β, -2, -4, -6, and -10 and relative BCG cell numbers decreased in a dose-dependent manner according to pretreatment with rmBD2 protein, and the responses were potentiated in the anti-mBD2 pretreatment group at 4 hours after BCG (MOI 30) instillation. ConclusionThe present results suggest that the mouse urothelium produces mBD2 in response to intravesicular BCG as a defense mechanism against BCG, and blocking mBD2 by an anti-mBD2 antibody increased the effectiveness of BCG.

Exogenous glycosaminoglycans coat damaged bladder surfaces in experimentally damaged mouse bladder

BackgroundInterstital cystitis is often treated with exogenous glycosaminoglycans such as heparin, chondroitin sulphate (Uracyst), hyaluronate (Cystistat) or the semi-synthetic pentosan polysulphate (Elmiron). The mechanism of action is presumed to be due to a coating of the bladder surface to replace the normally present chondroitin sulphate and heparan sulphate lost as a result of the disease. This study used fluorescent labelled chondroitin sulphate to track the distribution of glycosaminoglycans administered intravesically to mouse bladder that had been damaged on the surface.MethodsThe surfaces of mouse bladders were damaged by 3 mechanisms – trypsin, 10 mM HCl, and protamine sulphate. Texas Red-labeled chondroitin sulphate was instilled into the bladders of animals with damaged bladders and controls instilled only with saline. Bladders were harvested, frozen, and sectioned for examination by fluorescence.ResultsThe normal mouse bladder bound a very thin layer of the labelled chondroitin sulphate on the luminal surface. Trypsin- and HCl-damaged bladders bound the labelled chondroitin sulphate extensively on the surface with little penetration into the bladder muscle. Protamine produced less overt damage, and much less labelling was seen, presumably due to loss of the label as it complexed with the protamine intercalated into the bladder surface.ConclusionGlycosaminoglycan administered intravesically does bind to damaged bladder. Given that the changes seen following bladder damage resemble those seen naturally in interstitial cystitis, the mechanisms proposed for the action of these agents is consistent with a coating of damaged bladder.

Altered Gene Expression Profile in Mouse Bladder Cancers Induced by Hydroxybutyl(butyl)nitrosamine

A variety of genetic alterations and gene expression changes are involved in the pathogenesis of bladder tumor. To explore these changes, oligonucleotide array analysis was performed on RNA obtained from carcinogen-induced mouse bladder tumors and normal mouse bladder epithelia using Affymetrix (Santa Clara, CA) MGU74Av2 GeneChips. Analysis yielded 1164 known genes that were changed in the tumors. Certain of the upregulated genes included EGFR-Ras signaling genes, transcription factors, cell cycle-related genes, and intracellular signaling cascade genes. However, downregulated genes include mitogen-activated protein kinases, cell cycle checkpoint genes, Rab subfamily genes, Rho subfamily genes, and SH2 and SH3 domains-related genes. These genes are involved in a broad range of different pathways including control of cell proliferation, differentiation, cell cycle, signal transduction, and apoptosis. Using the pathway visualization tool GenMAPP, we found that several genes, including TbR-l, STAT1, Smad1, Smad2, Jun, NFκB, and so on, in the TGF-β signaling pathway and p115 RhoGEF, RhoGDl3, MEKK4A/MEKK4B, P13KA, and JNK in the G13 signaling pathway were differentially expressed in the tumors. In summary, we have determined the expression profiles of genes differentially expressed during mouse bladder tumorigenesis. Our results suggest that activation of the EGFR-Ras pathway, uncontrolled cell cycle, aberrant transcription factors, and G13 and TGF-β pathways are involved, and the cross-talk between these pathways seems to play important roles in mouse bladder tumorigenesis.

Radiation tolerance of normal mouse bladders after intravesical chemotherapy

The aim of this study was to compare functional damage in normal mouse bladder after various initial intravesical therapies and to investigate tolerance to subsequent irradiation. Six consecutive weekly intravesical instillations of Mitomycin C (MMC) or doxorubicin (DOX) were used as the initial therapy. Irradiation with single doses of 10–25 Gy (X-rays) was given at 4 or 12 weeks after intravesical treatment: Functional bladder damage was assessed from changes in the micturition frequency, expressed as frequency index (FI, number of urination events/ml urine in a 24-h test period) and from cystometry measurements of bladder volume at 52–56 weeks. Irradiation alone caused a temporary acute response (increased FI) within the first 4 weeks and a progressive late response starting from 15 to 37 weeks, depending on the radiation dose. A reduced bladder capacity was also evident at 52–56 weeks after 25 Gy. Intravesical MMC or DOX caused a 3-fold increase in FI during intravesical therapy with recovery to control levels within 2–3 weeks after cessation of treatment. Irradiation 4 weeks after MMC, or 4 or 12 weeks after DOX resulted in acute responses very similar to irradiation alone. There was no difference in time of onset or extent of late bladder damage when irradiation was given after DOX, compared with irradiation alone as assessed from repeated measurements of FI or cystometry at 52–56 weeks. In contrast, irradiation 12 weeks after MMC led to a decrease in acute radiation response compared with X-rays alone. In addition, the onset of late increases in FI was delayed (almost doubled) after pretreatment with MMC and there was no decrease in bladder volume at 52–56 weeks after irradiation compared with untreated controls. These data suggest that bladder tolerance to irradiation was not compromised by either of these drugs and may have even been improved by MMC.

Late changes in the normal mouse bladder after irradiation alone or in combination with cis‐DDP or cyclophosphamide, assessed by stereological analysis

Stereological analysis has been performed to investigate the late changes in the normal mouse bladder one year after radiation alone (10-25 Gy) or radiation (10-15 Gy) combined with cyclophosphamide (CTX), 100 mg/kg, or cis-diamminedichloroplatinum (II) (cis-DDP), 6 mg/kg. A significant increase in total volume of urothelium was found in the group treated with CTX combined with radiation compared with the control. The total volume of connective tissue in the muscular layer in the groups which received X-rays only showed a significant increase compared to the control. There also seemed to be an increase in the amount of connective tissue in muscular tissue, although this was not statistically significant, in the groups treated with CTX and irradiation compared with CTX alone. This indicates the importance of radiation in the development of late damage. Results from bladders treated with cis-DDP alone or combined with radiation were statistically inconclusive due to the small number of animals in each group. The group treated with CTX and radiation (15 Gy) showed a significant decrease in the total volume of lamina propria, but no difference was found in the other treatment groups when comparing connective tissue, the muscular layer or the amount of vessels and capillaries with their corresponding control groups. Stereological analysis seems a feasible method to demonstrate late bladder tissue changes encountered after irradiation alone or combined with chemotherapy.

A Pharmacological in Vitro Study of the Mouse Urinary Bladder at the Time of Acute Change in Bladder Reservoir Function after Irradiation

Mouse urinary bladder strips were investigated as to whether the acute change in bladder reservoir function seen after irradiation might be due to major changes in basic nerve and smooth muscle functions. The release mechanism of acetylcholine, cholinergic and non-cholinergic nerve activation explored by indomethacin and potassium channel activation were investigated. It was concluded that the normal mouse bladder is partly cholinergically and partly non-cholinergically innervated. The role of acetylcholine is of the same importance as in other rodents. However, it was not possible to distinguish any difference between normal and irradiated mouse bladders in respect to nerve and smooth muscle function.

Early and late changes in the normal mouse bladder reservoir function due to irradiation and cis-DDP.

Early and late changes in the reservoir function of the mouse bladder were investigated after radiation alone or a combination of radiation and cisplatinum (cis-DDP). Bladder function was investigated by repeated cystometries. Treatments consisted of either single fraction radiation (5-10-15-25-30 Gy) or 20 Gy in combination with cis-DDP (6 mg kg-1; i.p.) administered at various time intervals from 14 days before until 14 days after radiation. At two selected time intervals (15 min and 4 h before) radiation was given at different dose levels (5-10-15-20 Gy). Within 30 days after irradiation a dose-dependent early response was noticed both in the radiation alone group and the group where cis-DDP was administered 15 min before radiation. The dose-response curve showed a slight but non-significant shift to the left in the combined treatment group (dose effect factor (DEF) = 1.18). Investigation of the early change in bladder reservoir function in the animals treated with 20 Gy alone or a combination of 20 Gy plus cis-DDP at various intervals in relation to irradiation demonstrated a significant increase in response when cis-DDP was administered 24 h and 15 min before and 4 h, 72 h and 336 h after 20 Gy (P less than 0.05). The reversible nature of the early damage was demonstrated. Late response was irreversible and significantly increased in most groups were cis-DDP was administered from 168 h before until 72 h after compared to radiation alone. Comparing groups treated with radiation alone with groups where cis-DDP was administered 15 min and 4 h before radiation revealed DEF values up to 1.45 (P less than 0.05), reflecting the significantly larger response in combined treatment groups. Survival was significantly decreased in all combined treatment groups compared to groups treated with radiation only and likewise survival was decreased in the group treated by cis-DDP alone compared to control (no treatment at all).

The ability of normal mouse cells to reduce the malignant potential of transformed mouse bladder epithelial cells depends on their somatic origin.

Somatic cell hybrids have been made between a transformed mouse bladder carcinoma cell line and normal mouse bladder epithelium and mesenchyme. In the epithelial tumour/mesenchyme hybrids the malignant phenotype was expressed dominantly whereas in the carcinoma/normal epithelium hybrids the malignant potential was greatly reduced. In both cases the dominant in vitro and in vivo phenotype was that of the normal parental cells. All hybrid tumours were first palpable after 4-7 days, demonstrating that the tumours had not arisen as a result of in vivo selection of a sub-population of tumorigenic cells. Chromosome analysis showed that the carcinoma/normal epithelium hybrids were all in the hypertetraploid range but the large variation in the karyotypic profile of each hybrid made it impossible to implicate any specific chromosomes in the control of expression of the malignant phenotype. During normal development in bladder epithelium, terminal differentiation is associated with tetraploid formation by cell fusion. The reduction in malignancy of the carcinoma/normal epithelium hybrids may perhaps be due to the expression of genes associated with normal terminal differentiation after cell fusion and tetraploid formation. This is also supported by the more differentiated phenotype of the hybrid tumours. Of the 10 mesenchyme/epithelium hybrids analysed cytogenetically , four were in the hypertetraploid range from which little meaningful data could be obtained about specific chromosome losses. Chromosome analysis of the cells from the near-tetraploid hybrids showed only minor differences from what might have been expected from the input of the two parents; these differences appeared to be due to random chromosome loss. The maximum number of chromosomes lost from any of the hybrids was five, although one, two or three was more usual. The only consistent chromosome loss was of a single copy of chromosome 4, which in two of the hybrids represented the only chromosome change. The possibility that this loss might facilitate re-expression of the malignant phenotype is discussed.

E. coli adherence to bladder epithelial cells of mice.

Adherence of E. coli to bladder cells was studied by mixing E. coli with cells scraped from the surface of the normal mouse bladder. E. coli adherence to bladder epithelium did not correlate with renal infection, the ability of E. coli to resist phagocytosis, the growth of the strains, the presence of K-antigen or dulcitol fermentation. There was also no correlation with proportion of deaths, motility, or rough mutation. Pili were observed in three of the 22 strains of E. coli and their presence was not associated with increased virulence. In this model of renal infection neither adherence of E. coli to bladder epithelial cells nor the presence of pili were significant virulence factors.

Alkaline phosphatase as a marker of transformation in adult mouse bladder epithelium after [formula omitted] exposure to 7, 12-dimethylbenz(a)anthracene

Loss of alkaline phosphatase from bladder epithelium has been suggested as a specific preneoplastic change induced by chemical carcinogens in rats (Stiller and Rauscher 1971; Kunze and Schauer 1971; Kunze et al. 1975), and mice (Highman et al. 1975). Using an in vitro bladder epithelial cell transformation system established by treating primary cultures of mouse bladder epithelium from young adult and old donors with 7,12-dimethylbenz-(a) anthracene (DMBA) (Summerhayes and Franks in press) and electron microscope cytochemistry, the localisation of alkaline phosphatase was compared in normal mouse bladder, in epithelial cell cultures before and after transformation with DMBA and in tumours derived from implants of these cells in syngeneic mice.

Development and characterization of cell lines of normal mouse bladder epithelial cells and 2-acetylaminofluorene-induced urothelial carcinoma cells grown in monolayer tissue culture

Development and characterization of cell lines of normal mouse bladder epithelial cells and 2-acetylaminofluorene-induced urothelial carcinoma cells grown in monolayer tissue culture

Development and characterization of cell lines of normal mouse bladder epithelial cells and 2-acetylaminofluorene-induced urothelial carcinoma cells grown in monolayer tissue culture.

Normal urinary bladder epithelial cells and cells derived from 2-acetylaminofluorene-induced urothelial carcinomas from male Balb/c mice were grown in monolayer culture and were characterized. Cell lines of normal bladder epithelium were mononucleated, sheet-forming cells, with a modal chromosome number of 40. Bladder epithelial carcinoma cells induced by 2-acetylaminofluorene were polynucleate, relatively fast growing, grew in soft agar, demonstrated a higher cloning efficiency than normal cells and formed tumors when inoculated into syngeneic hosts. Differences in morphology were recorded by photomicrography using phase optics and scanning electron microscopy.