This study was designed to evaluate the distribution of mast cells in the lung tissues of rats exposed to biomass  smoke. Fifty six female Wistar albino adult rats were used. They were divided into two experimental groups  (control and biomass smoke-treated), each containing 28 animals. Control rats were not exposed to the  biomass smoke at any time during the experiment. Rats in the treatment group were exposed daily (one hour)  to biomass smoke for 3, 6 or 9 months. Lung tissues samples were obtained under deep anesthesia from the  randomly selected 7 animals in both groups. Lung tissues were fixed in Mota’s fixative (BLA) for 24 h and  embedded in paraffin. Sections of 6 μm thickness were cut and stained with 0.5% toluidine blue in 0.5 N  hydrochloric acid at pH 0.5 for 30 min. The numbers of mast cell in lung tissues of the animals exposed to  the biomass for 6 or 9 months were significantly (P&lt;0.05) higher than controls. This study showed that long  term exposure to biomass smoke was associated with the increased number of mast cells in the lung.

Acar, S

Coskun, O

Kanter, M

Karaca, T

Yörük, M

Özbay, B

English

Journals from University of Tartu

IntroductionBiomass smoke is produced by burning biomassmade of animal manure/dung. In some areas,biomass is used, under primitive and inefficientconditions, as the major source of domestic energy.Exposure to biomass smoke, during cooking, bak-ing and heating, in the developing countries wassuggested to be a potential risk factor for obstruc-tive airway disease (OAD) and bronchopulmonarydisease (Dennis and Maldonado, 1996). Severalreports suggest that exposure to biomass smokeincreases the prevalence of chronic bronchitis andproduces lung damage (Perez-Padilla et al. 1996.,Özbay et al., 2001). Biomass smoke includes NO2(oxides of nitrogen), NH3 (ammonia), hydrogencyanide, aldehydes, ketones, acrolein, etc. and par-ticles (Pandey et al., 1989). The role of mast cells (MCs) of the lung tissue hasnot been studied previously in those exposed tobiomass smoke. The MCs in the respiratory tractare now considered to play a pivotal role not only inallergic reaction but also in a number of inflamma-tory disorders by releasing a variety of biologicallyactive substances, such as histamine, leukotrienes,platelet-activating factors and prostaglandins(Kaliner, 1985; Marone, 1985).MCs play a fundamental role in the initiation ofallergic reactions and are believed to play an impor-tant role in the pathogenesis of bronchial asthma.Their localization at vascular, mucosal, and connec-tive tissue sites, coupled with their elevated num-bers at sites of chronic inflammation, has led to thehypothesis that mast cells are also central to theoccurrence of numerous other inflammatoryresponses (Katz et al., 1991) The secretory productsof mast cells have important regulatory effects onthe immune system (Vannier et al.,1991) and onhomeostasis in general (Lewis and Austen 1981).Mast cells from bronchoalveolar lavage, however,are more sensitive to immunological stimulationand do not require sensitization for maximalresponse (Hunt et al. 1991).            Scand. J. Lab. Anim. Sci. No. 2. 2004. Vol. 31.Distribution of mast cells in lung tissues of rats exposed tobiomass smokeby M. Kanter*1, M.Yörük2, B. Özbay3, T.Karaca2, S. Acar3& O. Coskun11Dept. of Histology and Embryology, Faculty of Medicine, University of Zonguldak Karaelmas, Zonguldak, 2Dept of Histology and Embryology, Faculty of Veterinary Medicine, University of Yuzuncu Yil, Van, 3Dept of Respiratory Diseases, Faculty of Medicine, University of Yuzuncu Yil, Van, Turkey. SummaryThis study was designed to evaluate the distribution of mast cells in the lung tissues of rats exposed to biomasssmoke. Fifty six female Wistar albino adult rats were used. They were divided into two experimental groups(control and biomass smoke-treated), each containing 28 animals. Control rats were not exposed to thebiomass smoke at any time  during the experiment. Rats in the treatment group were exposed daily (one hour)to biomass smoke for 3, 6 or 9 months. Lung tissues samples were obtained under deep anesthesia from therandomly selected 7 animals in both groups. Lung tissues were fixed in Mota’s fixative (BLA) for 24 h andembedded in paraffin. Sections of 6 µm thickness were cut and stained with 0.5% toluidine blue in 0.5 Nhydrochloric acid at pH 0.5 for 30 min.  The numbers of mast cell in lung tissues of the animals exposed tothe biomass for 6 or 9 months were significantly (P<0.05) higher than controls. This study showed that longterm exposure to biomass smoke was associated with the increased number of mast cells in the lung.67*Correspondence: Dr. M. Kanter, Dept. of Histology andEmbryology, Faculty of Medicine, University of ZonguldakKaraelmas, Zonguldak, Turkey, e-mail:mehmetkan-ter65@hotmail.com ,Scand.  J. Lab. Anim. Sci.  No. 2.  2004. Vol.  3168.In previous studies, it has been shown that there isa relation between biomass smoke exposure andchronic obstructive lung disease (Dennis and Mal-donado, 1996; Perez-Padilla et al., 1996; Özbay etal., 2001). It is also well known that the mast cellis one of the important cells and  plays a funda-mental role in the pathogenesis of asthma. There-fore, our aim was to investigate the distribution ofMCs in lung tissues of rats exposed to biomasssmoke.Materials and MethodsAnimalsFifty six adult inbred female Wistar albino rats (n=7x 8) weighing about 300 g were obtained from theLaboratory of Animal Science, Medical School,Yuzuncu Yil University, Van, Turkey. The animalswere given standard rat pellets (Van Food Factory,Van, Turkey) and tap water ad libitum. The rats werehoused in individual cages (360 x 200 x 190 mm),each containing 3 animals, 1 month before the startof the experiments. They were divided into twogroups (control and biomass smoke-treated), eachcontaining 28 animals. Rats in the treatment groupwere exposed to the biomass smoke daily (onehour) for 3, 6 or 9 months. Animals in the treatedgroup were exposed to biomass smoke in inhalationchambers equipped with a trap, and designed tosustain dynamic and adjustable airflow. The ani-mals were housed in an individual cage within theinhalation chamber for 1 h/day (from 09.00 a.m. to10.00 a.m. h). Neither food nor water was given tothe animals during the exposure. The control groupwas housed in identical chambers ventilated withfresh air. There were no deaths in any of the groups.All animals were housed in stainless cages understandard laboratory conditions (light period 07.00a.m. to 8.00 p.m. h, 21±2°C, relative humidity55%), and received humane care according to thecriteria outlined in the “Guide for the Care and Useof Laboratory Animals” prepared by the NationalAcademy of Sciences and published by the Nation-al Institutes of Health. Histological procedureAt the end of the experiment, the rats were anes-thetized with an intraperitoneal injection of chloralhydrate (6 ml of 7% chloral hydrate/kg body wt)and sacrificed by decapitation. Lung tissue sampleswere taken from the randomly chosen seven rats ineach group for histological examination at thebeginning, and at 3, 6 or 9 months of the experi-ments. Lung tissues were fixed in Mota’s fixative(BLA) for 24 h, and embedded in paraffin. Sectionsof 6 µm thickness were cut and stained with 0.5%toluidine blue (Gurr, England) in 0.5 N hydrochlo-ric acid at pH 0.5 for 30 min (Enerback, 1986).  Microscopic ExaminationMicroscopic examination was carried out at a mag-nification of 400 and the counts of mast cells weredetermined per square millimeter by using a stan-dardized ocular grid. Intact or partially degranulat-ed mast cells were counted. The density and distri-bution of mast cells were examined in the sectionsstained with toluidine blue. Tissue sections wereexamined under light microscopy (x400) and thenumber of mast cells counted in random high-pow-er fields using a Nikon Optiphot 2 light microscopeincorporating a square graticule in the eyepiece(eyepiece x10, objective x40, a total side length of0.225mm). MC density was assessed by countingthe number of cells in 200 high power fields in lungtissue preparations of each group. The MC densityin each site was calculated and recorded as MCnumbers /mm2. The tissue compartments used torecord the MC distribution in the lung were the sub-pleural and interalveolar connective tissues, peri-bronchiolar areas, perivascular regions, alveolarsepta and alveolar space.StatisticsStudent’s t-test was used for comparing groups. ResultsAt the end of the experiment, no morphological dif-ferences were found in the mast cells of control (Fig.1) and experimental group in the lung  parenchyma.Scand.  J. Lab. Anim. Sci.  No. 2.  2004. Vol.  3169.However, these cells were  extremely large in thesubpleural area. The MCs varied in shape fromovoid to elongate and were predominantly located inthe intralung bronchial lamina propria, subpleural,and alveolar wall and septa. The majority of MCscontained many strongly stained granules, whichfrequently obscured the nucleus (Fig. 2). Some ofthe MCs in the lung tissues of the biomass experi-mental group were degranulated. The number of MCs in the lung tissue of  bothgroups is shown in Table 1. Although  numbers ofMCs were counted in the intralung bronchial lami-na propria, subpleura, and alveolar wall and septaregion of lung tissues, because the distribution ofMCs in 3 regions were linear in both groups, theirmeans are shown in the table as the numbers ofMCs in the lung tissues. The numbers of mast cellin lung tissues of the animals exposed to thebiomass smoke for 6 or  9 months were significant-ly (P<0.05) higher than controls. DiscussionMany pathological characteristics of lung disease,such as asthma,  can be attributed to the actions ofmast cell mediators (Kaliner 1985; Holgate & Kay,1991). Therefore, lung diseases increase the num-ber of MCs. MCs in asthmatic lung or such condi-tions secrete a greater amount of histamine and other mediators than in healthy individuals, thuscausing constriction of airways. Mast cells also playa role in the etiology of many inflammatory diseases especially of the lung and intestine. Ercanet al. (1997) suggested that  under stressful condi-tions the number of mast cells in the mucosa of theurinary bladder are increased. In addition to theirrole in hypersensitivity responses, mast cells can beinvolved in the generation of the immune responsein the respiratory tract, in the central arm of theimmune response where histamine is a potent mo-dulator (Rocklin et al, 1978), and also at the effec-tor stage of cytotoxicity (McDermott et al, 1982).Goto et al.(1984) suggested that after intratrachealadministration of bleomycin, lung histamine levelsincreased as much as 14-fold by day 50.  Pulmonarymast cells changes were present early in the fibrot-ic process, and by day 14 the mast cell density in the parenchyma was 10 times higher than normal.Such an increase of the mast cells was  found to becoherent with that in chronic bronchitis (Pesci etal., 1994), fibrosis (Goto et al., 1984), asthma(Casala et al., 1987) or when exposed to radiation(Bjermer et al., 1993). The current experiment indicated (Table 1) thatbiomass-smoke exposure for 6 or 9 months Table 1.  Number of mast cells in the lungs rat*.               Groups (n=28)    Exposure time of  biomass smoke (Month) Control Experimental Groups0 25.58±4.3 a 25.07±4.3a3 24.81± 2.4a 27.05±2.4a6 26.10 ±1.8a 31.74±3.1b9 25.32±3.2a 37.92±5.6c* All samples stained with toluidine blue, pH 0.5.Values are mean ± Standard deviation (SE) (Experimental groups n = 7, control n = 7) a,b,c Mast cells density in the second and third biomass smoke-exposed rats was significantly greater thanthat in control specimens (p<0.05).Fig.1. Toluidine blue staining, lung tissues of rats control group, mast cells (arrows), x360.Fig. 2. Toluidine blue staining, lung tissues of rats 6 months biomass-exposed group, mast cells (arrows), x360Scand.  J. Lab. Anim. Sci.  No. 2.  2004. Vol.  3170.Scand.  J. Lab. Anim. Sci.  No. 2.  2004. Vol.  3171.increased the number of MCs in the lung of rats.Our study is the first one to show a linear increasein the lung tissue mast cells with the elapse of time.In a quantitative and qualitative study by Fox et al.,normal human lungs had an average concentrationof 350 mast cells/ mm2 of alveolar wall whereasabnormal lungs showed an average of 523/mm2(Fox et al. 1981). Warton et al. (1986) identifiedthat the numerical density of MCs per square mil-limeter of alveolar wall in tissue section was299±258 mast cells / mm2 in normal and 366±260in asthmatic lung. Pesci et al. (1994) found,15.51±13.39 and 2.9±3.65, the numbers of mastcell per square millimeter of epithelium in patientswith chronic bronchitis and normal control sub-jects, respectively.It is concluded that long-term biomass smoke expo-sure increases the number of MCs in the lung tis-sue, which might lead to lung diseases such aschronic bronchitis, fibrosis and  asthma. 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Distribution of mast cells in lung tissues of rats exposed to biomass smoke

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Distribution of mast cells in lung tissues of rats exposed to biomass smoke

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