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Protective Effects of Montelukast Against Stress-Induced Degeneration of the Urinary Bladder/Stres ile Uyarilmis Mesane Hasarina Karsi Montelukastin Koruyucu Etkileri.


Interstitial cystitis (IC) is one of the inflammatory bladder diseases that occurs mostly in women (90%) with urinary frequency, urgency, burning and suprapubic pain (1). Although the pathophysiological basis of IC is not been fully understood, experimental and epidemiological data show that urothelial damage, increase and degranulation of mast cells and activation of afferent fibers have a role in this disease. It was indicated that stress conditions aggravates IC symptoms (2). Continuous exposure to stressful stimuli has been directly related to the onset, progression or outcome of many pathological processes in several organs such as stomach and urinary bladder. should be corrrected as "In a previous study it was observed that cold-restraint stress induced urothelial damage, activated and increased mast cells and activation of afferent fibers in rats (3). When activated, mast cells release many inflammatory mediators as histamine, proteases, prostaglandins, leukotrienes and cytokines (3). It was reported that the mast cells have a critical role in inflammatory and allergic reactions including stress-induced inflammatory diseases (4). Water avoidance stress (WAS), one of the used stress models, mimics psychological stimuli so it may be a model of life stress as its duration is 5 days (5). In recent studies it is shown that WAS cause both histopathological (6) and functional abnormalities as micturition frequency, interval and volume of rat urinary bladder (7).

Leukotrienes are lipid mediators involved in allergic and inflammatory diseases (8). There are two classes of leukotrienes, leukotriene B4 (LTB4) and the cysteinyl leukotrienes C4 (LTC4), D4 (LTD4) and E4 (LTE4) (9). The cysteinyl leukotrienes are potent pro-inflammatory mediators, and are produced by inflammatory cells, such as mast cells, eosinophils, basophils and macrophages (10). Leukotrienes are potent spasmogens that promote mucous secretion and have primarily been studied with regard to their action on reactive preparations from guineapig and human airways, and guinea-pig ileum (11). Leukotriene receptor antagonists and synthesis inhibitors have been shown to be effective on several inflammatory models in rats, such as experimentally induced mucosal damage of stomach (12), colitis (13), burn induced multiorgan damage (14) and renal ischemia/reperfusion injury (15). Montelukast is cysteinyl leukotriene receptor 1 antagonist, selectively inhibited the LTD4 mediated calcium response and used to treat asthma and other diseases (16, 17). Oral treatment daily has inhibited bronchoconstriction and decreased blood eosinophils. (18). Recent studies were shown that montelukast decreased urinary frequency and pain in eosinophilic cystitis (19) and IC (17).

It was aimed to evaluate the protective effects of montelukast on WAS induced urinary baldder damage in rats. Morphologic alterations, mast cell activity, uroplakin distribution, GAG layer and tight junctions were evaluated by histological techniques, oxidative stress markers; malondialdehyde (MDA) and glutathione (GSH) were analysed by biochemical methods.



Wistar Albino adult female rats (180-200 gr) were used in this study. They were housed individually in standard laboratory conditions and fed with standard pellet laboratory chow and water ad libitum. All experimental protocols were approved by the Marmara University, Animal Care and Use Committee (33.2017.mar9).

Water avoidance stress procedure

Rats were handled daily by the same investigator for 2 weeks before the study and then subjected to WAS according to the Santos et all. (5). The procedure works by placing the rat on a glass platform (8 X 6 [cm.sup.2]) in the middle of a plastic container with 90 cm diameter and 50 cm height, filled with warm water (25 [degrees] C) to 1 cm below the height of the platform. Rats avoided the aversive stimulus (water) by remaining on the platform for 2 h (5).

Experimental Groups

In the study four groups were present as control, montelukast (ML), WAS (WAS) and WAS exposed ML-treated (WAS+ML) groups. Eight rats were used in each group. In control and ML groups rats were placed on the platform in a waterless container for 2h daily for 5 days afterwards, each day received an i.p injection of physiological saline or 10 mg/kg ML. In WAS and WAS+ ML groups rats were exposed to WAS for 2h daily for 5 days. Right after WAS exposure, rats received either physiological saline or 10 mg/kg ML injected i.p. for 5 days. The animals were fed with a standard pellet lab chow and water ad libitum during the experimental procedures. Rats were euthanized by decapitation 2 h after the last stress exposure and/or treatments, the urinary bladders were removed from the animals and processed for microscopic and biochemical investigations.

Light Microscopic Preparation

The urinary bladder samples were fixed in 10% buffered formalin for 48 hours and processed routine paraffin embedding. Approximately 5mm thick paraffin sections were stained with hematoxylin and eosin (H&E) for morphological observation.

Triptase and uroplakin Immunohistochemistry

For triptase immunohistochemistry, paraffine sections were rehydrated, endogenous peroxidase activity was quenched with 3% [H.sub.2][O.sub.2] in methanol 10 min and the sections were rinsed in phosphate buffered saline (PBS). Non-specific labeling was blocked by incubation in a blocking solution (Super Block, Scy Tek UltraTek HRP, Anti-Polivalent, UHP 125, USA) for 25 min and then the sections were incubated with a mouse anti-mast cell tryptase (diluted 1:100, Novo Castra, NCL-MCTRYP, UK), overnight at 4 [degrees]C. In the negative controls, the sections were incubated with PBS without mouse antimast cell tryptase overnight at 4 [degrees]C. An anti-polyvalent biotinylated antibody (Scy Tek Ultra Tek HRP, Anti--Polivalent, UHP 125, and USA) was applied as a secondary antibody (supplied ready-touse) for 30 min. The sections were then incubated with horseradish peroxidase (HRP)--labeled avidin (supplied ready-to-use, Scy Tek UltraTek HRP, Anti-Polivalent, UHP 125, USA) for 25 min, and then labeling revealed by incubation with a freshly prepared solution of 3-amino-9-ethylcarbazole (AEC, 20 ml AEC chromogen with each 1 ml of AEC substrate, Scy Tek ACJ 125 AEC Chromogen/Substrate Bulk Pack, USA) for 5-10 min. Sections were coverslipped with glycerol gelatine (20).

For the uroplakin immunohistochemistry, tissue sections were deparaffinized and rehydrated and endogenous peroxidase activity was quenched with 1% [H.sub.2][O.sub.2] in methanol for 5 minutes and the sections were rinsed in PBS two times. The sections were treated with trypsin solution for epitope retrieval for 10 minutes at 37[degrees]C, incubated with normal goat serum (1:50) in PBS, and then incubated with mouse monoclonal antibodies UPIII (the AU1 antibody, undiluted, catalog# RDI-PRO651108, Fitzgerald, USA) overnight at 4[degrees]C. After then the other procedures were similar as triptase immunohistochemistry mentioned above (21).

Light microscopic sections were evaluated and photographed with a digital camera (Olympus C-5060; Olympus, Tokyo, Japan) attached to a photomicroscope (BX51; Olympus, Tokyo, Japan).

Electron Microscopic Preparation

Transmission electron microscopy (TEM) was used to demonstrate the glycosaminoglycan (GAG) layer and leakage of the thight junction. The urinary bladder samples were fixed in 4% phosphate buffered glutaraldehyde (0.13 M and pH 7.4), stained en bloc with ruthenium red (RR) and postfixed with OsO4 (ratio used 1 part of stock RR solution: 4 parts of 1% OsO4) for 1 hour. Samples were stained en bloc within a range of 1/5 stock RR solution and 2% uranyl acetate (22), then dehydrated in a ascending alcohol series and embedded in epon 812. Sixty nanometer thick sections were stained with uranyl acetate and lead citrate. These sections were investigated and photographed with digital camera (Olympus Morada Soft Imaging System) attached to a transmission electron microscope (Jeol 1200 EXII,Tokyo, Japan).

Scanning electron microscopy (SEM) was used to demonstrate the alterations of luminal surface of urinary bladder. The samples were fixed in 4% phosphate buffered glutaraldehyde (0.13M, pH: 7.4) for 4 h, postfixed with 1% Os[O.sub.4] for 1h, and processed for routine scanning electron micrsocopy. Then, the specimens were examined under Jeol JSM SEM (Tokyo, Japan).

Mast Cell Counts

The paraffin sections stained with triptase immunohistochemistry were used for mast cell counting. Similar ten areas in each sample were selected and tryptase immunoreactive (ir) mast cells were counted at X400 magnification in the mucosa.

Measurement of Malondialdehyde (MDA) and Glutathione (GSH) Levels

Tissue samples were homogenized in ice cold 150 mM KCl (w/v, 10 %) for determination of MDA and GSH levels. The MDA levels were used as index of the products of lipid peroxidation and used as a biomarker of oxidative damage in urinary bladder. Results are expressed as nmol MDA/g tissue (23). GSH was determined by the spectrophotometric method using the Ellman's reagent (24). Results are expressed as mmol GSH/g tissue.


Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software, San Diego; CA; USA). All data were expressed as means [+ or -] SEM. Groups of MDA and GSH data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Mast cells evaluation was done by the Mann-Whitney non-parametric U-test. Values of p<0.05 were regarded as significant.


Morphological Results

The control group had a regular layout of mucosa with urothelium (Fig.1A1) and a few numbers of granulated mast cells (Fig. 1A2). Uroplakin immunoractivity (ir) was observed throughout the urothelium as a brown color (Figure 1A3). Regular luminal mucosal topography at SEM level (Fig. 1A4), GAG layer and tight junctions (Fig. 1A5) were obsereved at TEM level.

Biochemical results

WAS induction caused an increase in MDA levels (P<0.001), compared to control group showed a significant decrease in MDA levels compared to WAS group (P<0.001). The GSH level (P<0.001) diminished in the WAS group and increased in WAS +ML group (P<0.01, Fig. 3).


In our study we showed that induction of WAS caused urothelial infiltration and urothelial damage, increased number of both granulated and activated mast cells increased malondialdehyde and decreased endogenous glutathione levels. The leukotriene antagonist montelukast ameliorated both oxidative damage and morphology in urinary bladder.

Stress conditions worsen the symptoms of IC in patients (25) and experimental studies aim to explain the mechanism of the stress conditions on urinary bladder. One of the experimental studies that could explain the life stress with duration of 5 days is WAS including psychological stimuli (5). Stress conditions induce the increase and activity of mast cells, moreover mast cell activity increases in many diseases as irritable bowel disease, psoriasis and IC (4). The mast cell activation has been indicated in many different stress models as well in WAS. Acute immobilization stress induces mast cell activation in urinary bladder (26) and dura mater (27). Moreover, cold-restraint stress causes an increase and activation of mast cells in urinary bladder mucosa (3). In this study, WAS exposure caused an increase in the number and activation of mast cells in urinary bladder. These results suggest that WAS-induced activation of mast cells occurs by activating an inflammatory reaction and stimulating mast cells, to release inflammatory mediators such as histamine and leukotriens to the extracellular matrix, and this can be prevented by montelukast treatment via its anti-inflammatory and antioxidant effects.

Surface glycosaminoglycan (GAG) layer maintains the integrity of the urothelium, tight junctions and urothelial permeability (28-30). Generally, degeneration in the mucous layer of the urothelium has an initiating role in IC (29). In IC patients dilatations in the cells of the superficial and intermediate layers were demonstrated (31). A decreased mucous layer, degeneration of the cell junctions and increased number of microvilli (32) were observed in the SEM observations of IC patients. Our light and electron microscopic observations of WAS group showed a reduction of the GAG layer, decrease of uroplakin-ir density, leaky tight juntions, desquamation and ulceration at the apical surface. Because of the reduction of GAG layer, dilatation of tight junctions, reduction of density of microridges may be related to inflammation.

MDA is a good indicator of the rate of lipid peroxidation and it is related to inflammation in tissue (33). GSH is one of the endogenous antioxidant against a number of noxious stimuli including free oxygen radicals and several reports indicate that reduction of cellular GSH is accompanied by lipid peroxidation. It was shown that montelukast suppresses the release of oxidative stress markers and enhances enzymatic antioxidant activities in urinary bladder in spinal cord injury induced secondary degeneration (34) and kidney in cisplatin induced renal disfunction (35). In this study, the MDA level was significantly increased and GSH level was significantly decreased by WAS exposure. However, montelukast treatment causes significant decrease of MDA level indicating a reduction in lipid peroxidation and cellular damage, and prevention of GSH reduction, might be related by its free radical scavenging activity.

It was shown that cysteinyl leukotrienes induce the activation of mast cells. Cysteinyl leukotrienes can provoke interleukine 5 and tumor necrotic factor-a production in mast cells, and this effect is blocked by cysteinyl leukotrienes inhibition (36). One of cysteinyl leukotriene antagonist montelukast down regulates the human monocyte chemotaxis induced by monocyte chemotactic protein-1 which is chemotactic for eosinophils and mast cells realize the release of histamine and leukotriene (37). Cysteinyl leukotrienes mediate histamine hypersensitivity by increase of histamine H1 receptors (38). Cysteinyl leukotrienes initiate histamine responses by recruiting additional histamine receptors in immunologically related cells (38). Moreover, montelukast was effective for micturation symptoms and urinary bladder pain in IC patients (39). In the present study it was observed that, montelukast treatment reduces the number and activity of mast cells in WAS induced bladder damage. In accordance to the inhibition of mast cell activation regular urothelium was observed in both light and electron microscopy levels. These results might be related by the anti-inflammatory effects of montelukast.


It is demonstrated that montelukast, a cysteinyl leukotriene receptor 1 antagonist, alleviates WAS-induced bladder damage by inhibiting mast cell activation and preserve the urothelial mucous layer through its antioxidant effects. Thus, montelukast is a highly promising agent for the protection of urinary bladder from pathologic conditions in which oxidant-antioxidant balance is altered. Moreover, montelukast might be also used as therapeutic agent for IC symptoms which are worsening by stressful conditions.

Acknowledgement: This study was supported by L'Oreal Turkey, the Supporting Grant of the Young Women Scientists (2005).


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Esra Cikler Dulger (1), Yasemin Ersoy Canillioglu (2), Sule Cetinel (3), Goksel Sener (4), Feriha Ercan (3)

(1) Istanbul Bilim University, School of Medicine, Department of Histology&Embryology, Istanbul

(2) Bahcesehir University, School of Medicine, Department of Histology&Embryology, Istanbul

(3) Marmara University, School of Medicine, Department of Histology&Embryology, Istanbul

(4) Marmara University, School of Pharmacy, Department of Pharmacology, Istanbul

ORCID IDw of the authors: F.E. 0000-0003-2339-5669, Y.E.C. 0000-0002-3839-6931

Cite this article as: Dulger E.C, Ersoy Canillioglu Y., Cetinel S., Sener G., Ercan F. Protective Effects of Montelukast Against Stress-Induced Degeneration of the Urinary Bladder. Clin Exp Health Sci 2018; DOI: 10.5152/clinexphealthsci.2017.691

Correspondence Author/Sorumlu Yazar: Feriha Ercan E-mail/E-posta:
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Title Annotation:Original Article / Ozgun Arastirma
Author:Dulger, Esra Cikler; Canillioglu, Yasemin Ersoy; Cetinel, Sule; Sener, Goksel; Ercan, Feriha
Publication:Clinical and Experimental Health Sciences
Article Type:Report
Date:Sep 1, 2018
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