Mast cells of the bovine trachea: staining characteristics, dispersion techniques and response to secretagogues.

Sections of the lower trachea of cattle, fixed in either Carnoy''s or formalin, were stained with toluidine blue, alcian blue, or alcian blue and safranin O to study the mast cell population. After toluidine blue staining, about twice as many cells in tissue fixed in Carnoy''s contained dark blue granules compared with tissue fixed in formalin. In addition, for the first time in cattle, a population of cells containing red granules was identified after staining with alcian blue and safranin O. Most of these red granules were formalin sensitive. An enzymatic dispersal technique for mast cells is described that yielded 9.4+/-0.4% mast cells (percentage of nucleated cells) with a viability of 92.3+/-0.6%. Spontaneous histamine release was 3.3+/-0.8%. Dispersed mast cells were challenged with various immunological and nonimmunological secretagogues. The calcium ionophores, A23187, ionomyocin, and BrX537A, were effective in releasing up to 94% of histamine in mast cells in a dose-response relationship. Pasteurella haemolytica culture supernate caused about 10% histamine release at a dose of 0.5 mg/mL after correction for spontaneous release. The average histamine content of the mast cells was 6.6+/-1.0 pg/cell. Cytospins of dispersed cells fixed in Carnoy''s and stained with alcian blue and safranin O contained mast cells with blue and red granules, and a few cells with a mixture of both granule types. Based on the effects of type of fixation, staining characteristics and histamine content, a mix of subtypes of mast cells is present in the bovine trachea. However, functionally they respond to secretagogues differently than rodent mast cells. Without an immunological secretagogue, studies to determine compounds that will be effective in blocking mast cell degranulation will be limited.     

日粮中缺硒对鸡空肠肥大细胞组织化学和超微结构的影响

为探讨硒缺乏对鸡空肠中肥大细胞(MC)的形态学和超微结构的影响,将10只鸡随机分成低硒试验组(饲喂低硒日粮0.032 mg/kg)和正常对照组,在75日龄时断头处死立即取出空肠,进行甲苯胺蓝、长时间甲苯胺蓝、阿利新蓝-藏花红染色、阿利新蓝-高碘雪夫氏反应和醛品红-阿利新蓝5种染色并进行透射电镜观察。结果显示,采用不同染色方法所获得的MC数量、大小、形态、颜色及颗粒的黏多糖成分均不相同。电镜下,MC细胞胞浆内充盈大量的特征性颗粒,颗粒内容物呈均质状,电子密度高低不等,核圆形或椭圆形,胞质中可见高尔基复合体和较多的线粒体;试验组可见MC数量显著增加并与变性细胞紧密相邻,异染色质增多,核偏位或固缩,胞质稀疏,同时可见MC内颗粒内容物释放后的空隙或空泡。因此,组化染色提示肥大细胞存在不同亚型;试验组MC数量明显增加,这提示MC是重要的免疫细胞之一,在机体的免疫调节中发挥着重要的作用。     

日粮中缺硒对鸡空肠肥大细胞组织化学和超微结构的影响

为探讨硒缺乏对鸡空肠中肥大细胞(MC)的形态学和超微结构的影响,将10只鸡随机分成低硒试验组(饲喂低硒日粮0.032 mg/kg)和正常对照组,在75日龄时断头处死立即取出空肠,进行甲苯胺蓝、长时间甲苯胺蓝、阿利新蓝-藏花红染色、阿利新蓝-高碘雪夫氏反应和醛品红-阿利新蓝5种染色并进行透射电镜观察.结果显示,采用不同染色方法所获得的MC数量、大小、形态、颜色及颗粒的黏多糖成分均不相同.电镜下,MC细胞胞浆内充盈大量的特征性颗粒,颗粒内容物呈均质状,电子密度高低不等,核圆形或椭圆形,胞质中可见高尔基复合体和较多的线粒体;试验组可见MC数量显著增加并与变性细胞紧密相邻,异染色质增多,核偏位或固缩,胞质稀疏,同时可见MC内颗粒内容物释放后的空隙或空泡.因此,组化染色提示肥大细胞存在不同亚型;试验组MC数量明显增加,这提示MC是重要的免疫细胞之一,在机体的免疫调节中发挥着重要的作用.     

Distribution, density and histochemical profiles of the lung mast cells during the post-hatching period of Japanese quails (Coturnix coturnix japonica)

The distribution, density and histochemical characteristics of mast cells in the lungs of the Japanese quail were investigated during the post-hatching period. In the period starting from the first to the 60th day post-hatching, based on proteoglycan content, three types of mast cells, which were alcian blue-positive, safranin O-positive and alcian blue/safranin O-positive, were found to exist in the lungs. The application of staining with berberine sulphate demonstrated that, similar to the distribution of safranin O-positive cells, the heparin-containing cells were located in the periphery of large blood vessels. The percentages of mast cells in different localization sites of the lungs were found to vary with age in the post-hatching period with toluidine blue staining. The lack of any statistically significant increase/decrease in the number of mast cells per unit area of the right and left lung lobes is partially in favour of the proposal that the mast cell number increases with the growth of the lung volume in the post-hatching period.     

奶牛乳腺肥大细胞免疫形态学的增龄性变化

应用改良甲苯胺蓝染色法研究不同年龄奶牛乳腺肥大细胞分布特点的增龄性变化，并用Alcian bluc-safranin染色法对肥大细胞进行细胞化学分型研究。结果：青年奶牛乳腺中肥大细胞数量较少，中年奶牛乳腺中肥大细胞的数量增多，有趣的是，老年奶牛乳腺肥大的数量更多，AB-S染色显示，各年龄奶牛乳腺中只有黏膜型肥大细胞。结果表明：奶牛乳腺肥大细胞数量具有随年龄增长而增加的特点，但肥大细胞的类型没有发生改变。     

Morphological and immunocytochemical investigations on mast cells in porcine ureter

Morphological, morphometric, histochemical and immunocytochemical investigations on mast cells, located in the wall of ureter of 8 months aged pigs were performed. Mast cells were found in all three layers of ureteral wall, but their distribution was irregular and the number unequal. It was established that alcian blue (AB)-positive mast cells were significantly more than toluidine blue (TB)-positive mast cells. A statistically significant smaller number of both AB and TB-stained mast cells were observed in the tunica mucosa. The largest number of mast cells was found in the tunica muscularis. In the adventitia, mast cells were higher in number in the main connective tissue than in the connective tissue near the blood vessels. Mast cells stained with TB showed variably expressed gamma-metachromasia, which was best visible in those situated in the lamina propria of the mucosa. The prevailing parts of mast cells, however, were AB-positive after AB-safranin staining. This was mostly found in mast cells of the tunica muscularis and in mast cells of perivascular location in the tunica adventitia. Immunocytochemically, mast cells were found to be positive for histamine and vasoactive intestinal polypeptide in the muscle coat, and to histamine in the adventitia, as well. On the basis of obtained results it was presumed that the mast cells in porcine ureter most probably took part not only in keeping of local homeostasis, but played also an important role of mobility of smooth muscle cells in the middle layer of ureter on one hand, and, on the other, in the adventitial blood vessels.     

Canine Transmissible Venereal Tumour: Asessment of Mast Cell Numbers as Indicators of the Growth Phase

Mast cells are immune cells that are involved mainly in type 1 hypersensitivity reactions, and they have been implicated in tumour angiogenesis. In this study we assessed the presence of mast cell numbers and microvessel density during the progression and regression stages of natural spontaneous canine transmissible venereal tumours (CTVT). Mast cells were demonstrated by histochemical staining with toluidine blue, alcian blue and safranin O. Microvessel counts were demonstrated by immunohistochemical labelling with an antibody against the endothelial cell marker factor VIII. Mitotic cells, apoptotic cells and tumour infiltrating lymphocytes were counted from haematoxylin–eosin-stained sections. Tumour fibrosis was evaluated on Masson's trichome-stained sections. The results showed that progressing tumours had significantly higher mast cell counts and microvessel counts at the invasive edges of the tumours than did regressing tumours. In both the progressing and regressing tumours, microvessel counts were significantly positively correlated with mast cell counts. Regressing tumours had significantly higher mast cell counts of the whole tumour than progressing tumours. The results also showed that progressing tumours had significantly higher mitotic rate than regressing tumours, and fibrosis and apoptosis were significantly higher in regressing tumours than progressing tumours. There were no significant differences between the biochemical and haematological values of dogs with progressing and regressing tumours. These results suggests that mast cells play a role in CTVT progression probably by promoting vascularization at the invasion front during the progression phase, and that mast cell count could be used as one of the histological factors to indicate growth stage of CTVT.     

Comparison of four staining methods for detection of mast cells in equine bronchoalveolar lavage fluid

Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules.     

Multicentric mast cell tumors in a horse

Abstract: A 6‐year‐old female Rocky Mountain horse was presented for evaluation of draining tracts and distal limb subcutaneous edema on the left front and left hind limbs that had been present for 2 weeks. Direct smears of fluid collected by fine‐needle aspiration of subcutaneous fluid from both limbs were highly cellular with a predominance of eosinophils accompanied by numerous, moderately atypical, variably granulated mast cells. The cytologic diagnosis was mast cell tumor (MCT) with prominent eosinophilic infiltration with a differential diagnosis of eosinophilic granuloma. Histologic evaluation of surgical biopsies of lesions from both limbs was performed on sections stained with H&E, toluidine blue, and Luna stains. The histologic diagnosis was MCT, and staining with toluidine blue and Luna stains confirmed the presence of mast cells and eosinophils, respectively. In addition, the mast cells strongly expressed CD117. This is the first reported case of cutaneous mast cell neoplasia in a horse in which primary presenting complaints were draining tracts and distal limb subcutaneous edema involving multiple limbs. This case illustrates the utility of staining for CD117 expression in combination with traditional stains, such as toluidine blue and Luna, in differentiating MCTs from other eosinophilic lesions in horses.     

Mast cell numbers in normal and glaucomatous canine eyes

Numbers of mast cells in the cornea, sclera, choroid, ciliary body, iris, and retina of sections of globes from 35 clinically normal dogs and 34 dogs with secondary glaucoma was determined. Fixed globes were trimmed along a vertical midsagittal plane and embedded in paraffin. Tissue sections, approximately 6 microns thick, were stained with toluidine blue for identification of mast cells. In normal globes, most of the mast cells were observed in the anterior portion of the uvea, and fewer mast cells were seen in the choroid and sclera. Mast cells were not observed in the retina and were seldom observed in the cornea of dogs with or without glaucoma. In sections of glaucomatous globes, mast cells were distributed evenly in the uvea and sclera, and fewer mast cells were present than in normal globes, regardless of the cause of glaucoma.     

小鼠胃、小肠和精囊黏膜肥大细胞的组织学观察

取健康小鼠胃、肠管和精囊放入生理盐水中冲洗干净,放入10%中性甲醛固定液中固定、透明、浸蜡、包埋,对胃、肠管和精囊进行连续切片,并用苏木精-伊红和甲苯胺蓝染色。然后,用光学显微镜观察小鼠胃、肠管和精囊的肥大细胞并计数,再用生物统计学方法分析、比较了胃、肠管和精囊肥大细胞的分布规律。结果发现,胃内的肥大细胞最多,小肠较少,而精囊中几乎看不到。胃和小肠内肥大细胞主要分布在血管和腺管周围。     

Connective tissue-type mast cell leukemia in a dog

An unusual case diagnosed as connective tissue-type mast cell leukemia with marked mastocyte infiltration into visceral organs in a seven-year-old female Curly-Coated retriever is presented. Acute circulatory collapse, emesis, diarrhea, abdominal enlargement, icterus, cyanosis, dyspnea, pulmonary edema, hepatomegary, ascites, and right ventricular enlargement were observed. Hematologic and biochemical examinations revealed mast cell leukemia, mature neutrophilia, monocytosis, thrombocytopenia, hemolytic hyperbilirubinemia, hyperhistaminemia, renal and hepatic injuries. Mast cells were distributed systemically, but predominantly in the diaphragm and liver with a large mass among the serosa of ileum, cecum and colon. Mast cells were stained intensely by both safranin and berberine sulfate.     

Mast cells in the salivary glands and tongue of the ferret: demonstration and some histochemical observations

Previous investigations failed to demonstrate mast cells in the alimentary tract and extraparietal glands of the ferret. It was decided therefore to test this and assess factors that may be of influence. Major salivary glands and tongues of mature ferrets, which had been fixed in formalin-calcium, were examined by means of light microscopical histochemistry. Staining of paraffin sections with techniques depending on basic dyes or esterolytic activity was carried out for conventional times with and without previous oxidation, hot acid hydrolysis, and trypsin and beta-glucuronidase digestion. Aldehyde fuchsin and high iron diamine consistently revealed the presence of few mast cells in interstitial stroma of salivary glands and lingual musculature, and in the lamina propria of lingual mucosa. Alcian blue at 0.5 M MgCl2 and safranin produced less consistent results, and even fewer metachromatic mast cells were detected. No staining of mast cells was obtained with the technique for naphthol AS-D chloroacetate esterase. Pretreatment did not increase the numbers and/or staining reactions of mast cells. The results refute the previous misconception and suggest that ferret is a species with a low incidence of mast cells largely expressing a connective-tissue phenotype.     

二乙基己烯雌酚对成年仓鼠附睾肥大细胞数量分布的影响

成年雄性仓鼠经皮下连续注射二乙基己烯雌酚(DES)7 d后,用改良甲苯胺蓝染色法(MTB)和阿尔新蓝番红染色法(AB S)研究DES对附睾肥大细胞的形态大小、类型和数量分布的影响。结果表明:仓鼠附睾肥大细胞为结缔组织型肥大细胞,多分布于附睾头与附睾尾的被膜和间质中。试验组的肥大细胞常见于附睾管近旁,多呈脱颗粒状。虽然试验组与对照组的肥大细胞大小相近,但试验组的肥大细胞数量增多,尤其是附睾尾间质中肥大细胞增加明显(P<0.01)。     

Distribution and Heterogeneity of Mast Cells in Female Reproductive Tract and Ovary on Different Days of the Oestrus Cycle in Angora Goats

The physiological distribution of mast cells (MCs) in the reproductive tract and ovary of 12 Angora goats was determined using light microscopic histochemical techniques. Uterus (corpus uteri and cornu uteri), uterine cervix, uterine tubes (isthmus and ampulla) and ovary samples were obtained by laparatomy from groups of animals during metoestrus, dioestrus and proestrus (days 5, 10 and 16 of the oestrous cycle). Tissues were fixed in Mota's fixative (basic lead acetate) for 48 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 1% aqueous solution at pH 1.0 for 5 min and alcian blue-Safranin at pH 1.0 for 30 min. MCs were generally associated with blood vessels in all reproductive organs. In the uterus, they were concentrated mainly in the close of the uterine gland and deep stroma in the endometrium. Higher MC numbers were observed by toluidine blue staining in the uterus, uterine cervix and uterine tubes on days 10 (corpus uterine: 4.7 ± 3.8 and cornu uterine: 4.9 ± 3.5) and 16 (corpus uterine: 5.9 ± 4.5 and cornu uterine: 5.4 ± 2.4) of the oestrous cycle compared with day 5 (p < 0.05). Mast cells were not observed in the follicles, the corpus luteum and the underside of the surface epithelium of the ovarian cortex, but were observed in the interstitial cortical stroma and the ovarian medulla. In the ovary, MC numbers were significantly higher on day 16 of the oestrous cycle (cortex: 3.4 ± 2.4 and medulla: 5.7 ± 4.5, p < 0.05). Safranin-positive connective tissue MCs were not observed in the uterine tube on any occasion. These results indicate oestrous cycle-related changes in the number and location of MCs in goat reproductive organs.     

Mast cell distribution,epidermal thickness and hair follicle density in normal canine skin: possible explanations for the predilection sites of atopic dermatitis?

Mast cell counts, epidermal thickness and hair follicle density were quantified in toluidine blue stained sections of normal skin from 20 different body regions in 10 dogs and compared to the predilection sites of canine atopic dermatitis. Mast cell distribution varied significantly from site to site (P < 0.0001) and counts in the superficial dermis were significantly higher than the deeper dermis (P < 0.05). Mast cell counts were highest in the medial and lateral pinna (mean 10.4–11.3 per high power field, HPF) and in the ventral interdigital skin of the hind and fore feet (mean 9.2–9.5 per HPF). Counts in these regions were at least 150% higher than all the other sites (means ranging between 2.9 and 6.0 per HPF). Variations in mast cell counts, epidermal thickness or hair follicle density did not adequately explain the predilection sites of canine atopic dermatitis. However, the results provide some evidence that cutaneous mast cell distribution may be involved in the frequent occurrence of ear and foot pruritus in this disease.     

Morphological Investigations of the Glandulae Profundae Plicae Semilunares Conjuctivae in the Domestic Swine (Sus scrofa domesticus) and the Wild Hog (Sus scrofa ferus)

Samples of glandulae profundae plicae semilunares (Harderian glands) from five domestic swine and five wild hogs were used for this research. The gland samples were fixed in Bouin solution and mounted in paraffin. The paraffin slices were stained with haematoxylin and eosin, according to the periodic-acid-Schiff (PAS) method, with alcian blue (pH 2.5), with toluidine blue (pH 4.0), and applying a combination of staining with alcian blue (pH 2.5) and the PAS method. In domestic swine and wild hogs, these glands are tubular-alveolar with wide glandular lumina. A great deal of acid mucopolysaccharides and PAS-positive substances were noted within swines’ glandular cells, while, in wild hogs, PAS-positive substances were not frequent, acid mucopolysaccharides being noted in only a few glandular-acini cells. The appearance of the metachromatic phenomenon was not noted either in domestic swine or in wild hogs. In domestic swine, the level of acid mucopolysaccharides is probably due either to the housing method or to the influence of the alkaline substances that may appear on an eye's mucous conjunctive membrane during intensive breeding, such that the Harderian glands protect the mucous membrane by extracting acid mucopolysaccharides.     

Use of histochemical techniques in the characterisation of osteochondroses affecting pigs

Growth cartilages with dyschondroplastic foci (osteochondrosis) or areas of chondrolysis were selected from the ribs and bones of the appendicular skeleton of 132 commercial pigs euthanased between one and 169 days old or at a liveweight of 100 kg. Histochemical staining techniques that identified proteoglycans, collagen and deposits of calcium enhanced the visualisation of the lesions, were valuable for recognising the distribution of lesions, and helped to elucidate the development of the lesions. On the basis of the histochemical and morphological differences, it was considered that the lesions associated with growth plates and the lesions associated with articular-epiphyseal cartilage complexes should be considered as different entities. Lesions were identified in the articular-epiphyseal cartilage complexes of pigs at 15 days old, earlier than has been reported previously. Toluidine blue and safranin O were more useful than alcian blue and, in terms of staining intensity, toluidine blue gave more consistent results than safranin O.     

大鼠乳腺局部免疫的调节机制--泌乳期与静止期乳腺肥大细胞分布的比较

应用改良甲苯胺蓝染色法(MTB)观察了大鼠泌乳期和静止期乳腺肥大细胞的分布、形态、数量变化规律．并用阿尔新蓝一番红鉴别染色法(AB-S)进行了细胞化学分型研究。结果：AB-S染色显示，大鼠乳腺只存在黏膜型肥大细胞；无论是泌乳期还是静止期。乳腺肥大细胞大多分布于腺泡间和小叶问结缔组织中。细胞的形态各异，但细胞数量在静止期和泌乳期有显著差异。泌乳期的乳腺肥大细胞数量明显减少(P＜0．01)。乳腺肥大细胞的动态变化，可能与乳腺泌乳期腺泡上皮的生长和静止期腺泡问结缔组织细胞增生等结构变化有关。     

Age-related changes in the number of mast cells in the avian lymphoid organs

The distribution of mast cells (MCs) was studied in the lymphoid organs (thymus, bursa of Fabricius and spleen) of 0-, 7-, 21-, 30- and 120-day-old chickens, using light microscopic histochemical techniques. Tissues samples were obtained under deep anaesthesia from animals in five groups. Tissues were fixed in Mota's fixative (basic lead acetate) for 24 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 0.5% aqueous solution at pH 1.0 for 5 min and Alcian blue/Safranine at pH 1.42 for 30 min. MCs were found in the organs, mostly associated with sinuses and blood vessels. A large increase in MCs was observed in both thymus and spleen of 21-day-old chickens compared with 0-, 7-, 30- and 120-day-old chickens. However, in the bursa of Fabricius, numbers of MCs were significantly higher in the 7-day-old group compared with other age groups. Safranine-positive MCs were not observed in all organs and age groups. These results showed age-related changes in the number of MCs in avian lymphoid tissues.