Morphology Of Bronchial Tree System Of Yak Lung At Different Age

Objectives:With the increasing of altitude, the oxygen content in the atmosphere will gradually reduce, mammals lived in sea level will get high-altitude diseases when they exposed to the high altitude. Yak (Bos grunniens), lived in 3000-5000 m altitude, gets stable features in physiological, biochemical and morphological functions in adapting to the chronic hypoxia of high plateau, no high-altitude diseases are occured. This adaptability is determined by hereditary factors, that is, the yak's lung has the adaptive structure to the chronic hypoxia conditions. In this study, we use several histochemical methods and transmission electron microscope technique to study the structures of bronchial tree in different age yak; then, use immunohistochemistry technique to study the morphology and distribution of pulmonary neuroendocrine cells in the lung, in order to provide basic information for probing the adaptability of yak in the chronic hypoxia environment.Methods:Samples were got from the apical and caudal lobes of left lung in fetus (pregnancy 7-8 months; immunohistochemistry), newborn (1-3 days), juvenile (5-6 months) and adult yaks (3-5 years). After conventional sections preparation, (1) using hematoxylin and eosin staining, Periodic Acid Schiff's reaction, Aliclin blue staining, Verhoeffe's Van Gession and Grimileus'stainings to study the general morphology, the ciliated, goblet and Clara cells of epithelium, the submucosal glands, elastic fiber, smooth muscle and mast cells around the airways in yak's lung; (2) using Image-Pro Express to measure and count the diameter of various generation of bronchial airways, the thickness of bronchial epithelium, the densities of goblet and Clara cells of airway epithelium in 100μm of basal lamina lining each airway, the number of pulmonary neuroendocrine cells and neuroepithelial bodies in per square centimeter of tissue sections, thickness of muscularis, cartilage and adventitia around airways; (3) using transmission electron microscope to observe the ultrastructure of bronchial, bronchiolar, terminal bronchiolar and respiratory bronchiolar epithelium; and (4) using immunohistochemistry to examine the distribution of pulmonary neuroendocrine cells and neuroepithelial bodies in different age yak's lung.Results:1. The airways branching in the yak were by unequal dichotomy, that is, the smaller branch formed a larger angle with the parent tube, and occasionally trichotomy was found. The diameters of bronchial airways were increased with the age growing, and decreased with repeated bifurcation stemming from the parent tube. The height of bronchial epithelium were also increased with the age growing, and declined progressively with airways branching. The thickest epithelium was present at axial bronchi airways (newborn:29.77±9.96μm; juvenile:40.27±10.39μm; adult: 41.18±9.48μm), and then gradually declined with the diameter diminished to the thinnest in respiratory bronchiolar epithelium (newborn:8.37±2.08μm; juvenile: 8.17±1.98μm; adult:8.92±3.09μm). The study also showed that the variation of diameters in airways was always greater than that of height of epithelium, and the variation in older group was greater than that of younger group.2. This study quantitatively defined the distribution and abundance of goblet, Clara and pulmonary neuroendocrine cells and neuroepithelial body in the conducting airway epithelium in different age yak.Goblet cell mainly distributed in trachea, various generations of bronchi and bronchioles. The highest density of goblet cell was noted in tracheal and axial bronchial epithelium, then reduced in bronchi and the lowest was in bronchioles. There was no goblet cell emerged in distal bronchioles. The shifts between each group gradually reduced with age growing.Clara cell, firstly appeared in bronchioles, principally observed in epithelium of terminal and respiratory bronchioles, especially in latter (newborn:15.3±5.0/100μm; juvenile:14.7±3.8/100μm; adult:13.9±2.6/100μm). The densities of Clara cell among groups were inclined to decline with age.Pulmonary neuroendocrine cells were randomly present in whole lung, especially in the epithelium of airways and pulmonary mesenchyme. Neuroepithelial bodies were typically found at points of branching of airways. The overall number of pulmonary neuroendocrine cell and neuroepithelial body were found by sharp decrease at juvenile. With neurone specific enolase immunostaining, the density of neuroepithelial body reached the peak in newborn (29.1±5.1/cm2) and declined significantly in adults (5.5±1.9/cm2). The density of solitary pulmonary neuroendocrine cell was highest in fetus (285.8±17.6/cm2) and lowest in adults (111.7±39.1/cm2). At all ages, about 55-60% of the total neuroendocrine cells were localized within bronchi and bronchioles. Besides, with chromogranin A immunostaining, the density of neuroepithelial body reached the peak in fetus (16.8±2.9/cm2) and decreased significantly in adults (3.4±0.93/cm2). The density of solitary pulmonary neuroendocrine cell was also highest in fetus (229.2±24.8/cm2) and lowest in adults (71.0±20.4/cm2). Additional, approximately 60% of the total neuroendocrine cells were localized within bronchi and bronchioles at all groups.3. Most ciliated cells in bronchial epithelium had electron-dense cytoplasm and an elongated nucleus. Many small oval mitochondria and well-developed smooth endoplasmic reticulum frequently observed in cytoplasm, together with occasional strands of rough endoplasmic reticulum, together with ribosomes and vesicles. The other ciliated cells had slightly electron-lucent cytoplasm than that of the former type, and numerous mitochondria. Some RER and a few distended SER were observed, but no vesicles were found. However, both of the cells contained rich glycogen granules in younger yak than that of the older. Occasional Golgi apparatus and lysosomes also observed.4. Goblet cells had characteristic features:dentate nuclear in dense cytoplasm, with stacks of rough endoplasmic reticulum and numerous dense membrane-bounded mucous droplets which were round or oval, often with an electron-lucent core which was different from that of other animals. The droplets were not confluent. A few oblong mitochondria, numerous glycogen and occasional Golgi complex were present. Some, less-frequent, goblet cells were noted in juvenile yak lung containing few mitochondria, numerous specific vacuoles, amounts of distended rough endoplasmic reticulum and smooth endoplasmic reticulum, and few numbers membrane-bound dense granules. The structure of mucous cells in submucosal gland was similar to that of goblet cells in epithelium of airways.5. Clara cells were a major cell type in distal bronchioles. They had dome shape and usually protruded into the airway lumen. Large amounts of rough endoplasmic reticulum and smooth endoplasmic reticulum, abundant glycogen granules and many secretory droplets occured within the cytoplasm, otherwise several typical "clefts" were also found in cytoplasm of yak.6. The yak airways had extensive submucosal glands throughout the whole airways except in distal bronchioles. The number of submucosal glands decreased with decreasing airway caliber. Two distinct forms of serous cells in glands were found: one type was filled with many round dense secretory granules, plentiful RER and few other organelles; the other type contained some oval mitochondrial and distended RER, the granules resembled the former. No serous cells were observed throughout the whole airways epithelium in three age group yaks.7. Histochemically, a majority of goblet and mucous cells, presented in the surface epithelium of bronchi and glands, secreted neutral and acid mucosubstances, only a little sulfated mucosubstances were present. Few mucosubstances were observed in the surface of bronchiolar epithelium. No mucous secretions were observed from the terminal to respiratory bronchiolar level.Conclusions:1. The airways branching in the yak were mainly by unequal dichotomy and occasionally trichotomy. The diameters of bronchial airways were increased with the age growing, and decreased progressively with airways branching. The height of epithelium were also increased with the age growing, and decreased with repeated bifurcation stemming from the parent tube. The variation of diameters in airways was always greater than that of height of epithelium, and the variation in older group was greater than that of younger group.2. This study was the first to quantitatively define the distribution and abundance of goblet, Clara and pulmonary neuroendocrine cells in the conducting airway epithelial cells in different age yak. The density of goblet cell was gradually reduced in bronchial epithelium with the declining of airway diameter. The density of Clara cell was gradually increased in distal bronchiolar epithelium with the declining of airway diameter. The quantitative studies of pulmonary neuroendocrine cells in different age yak revealed significantly age-related changes in term of their distribution.3. The morphology and distribution of various cells (including ciliated, goblet, Clara, pulmonary neuroendocrine cells, and so on) were important for maintaining the normal functions of the respiratory system, which included acting as a barrier to various insults; facilitating mucociliary clearance; secreting substances for airway surface protection, revealed the adaptability of yaks' lung to the natural environment.4. Goblet and mucous cells in the surface epithelium and submucosal glands of bronchi secreted neutral, acid and a little sulfated mucosubstances. It contributes to protecting the mucous membrane of respiratory system to adapt the high-altitude and droughty conditions.