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Histology of respiratory system Aj. Poonyawee Jirarattanawan Respiration is the mechanism of gas exchange, occurring within the lungs.It is necessary for every organism as it provides the source of oxygen and helps ineliminating the waste products, carbon dioxide. Respiratory tract is composed of 2 main portions; 1. Conduction portion which is the cavity that inspired air pass through (airway). No gas exchange occurs within this portion. 2. Respiratory portion which is the site of gas exchangeFunctions of respiratory system • Air conduction • Air filtration • Gas exchange Conducting portion • Warm • Moisten • Filter inspired air Respiratory portion gas exchangeFigure 1: Structures of respiratory system (Ross & Pawlina, 2011) Passageway of the inspired air Nasal cavity nasopharynxlarynx trachea 1Figure 2: Left lateral view of the airway, midsagittal section (Ross & Pawlina, 2011) Histology of Respiratory System

Nose and nasal cavity A CThe structures of nose and nasal cavity are skin tissue and Bepithelium covering the bony and cartilaginous frameworks; cartilage attip of nose & nasal septum, and bones nasal & maxilla.Nose (external): skin tissue (keratinized stratified squamous epithelium) ➢ Nostrils (anterior nares)Nasal cavity (A);1. Vestibule (B): anterior part of the nasal cavity, continuingfrom the external skin and posteriorly continue to the Olfactory region respiratory region. Respiratory region2. Respiratory region (C): nasal mucosa3. Olfactory region (D) contains smell receptors. vestibuleNasal cavity ➢ Nasal septum (NS): medial wall ofA the cavity ➢ Paranasal sinuses (PS): air cavities that lie lateral to the nasal cavity. • Communicate with nasal cavity • Lined with respiratory epithelium • Named for bone that they are found ➢ Conchae or turbinate: three shelf- like folds at the lateral wall of the cavity, formed by turbinate bone (TB) that project into the cavity. ItFigure 3: Nasal cavity, kitten coronal slice (Young et at,2014) increases surface area to contact the air.VestibuleB ➢ Epithelium: keratinized (continuing from skin) and non-keratinized Figure 4: Nasal vestibule (Dongmei et al, 2011) (continuing to respiratory region) stratified squamous epithelium • Vibrisisae (vibrissal follicles): short hair that entraps large particulate matter in the inspired air before it is carried into the nasal cavity • Sebaceous gland: The secretion of the gland helps in entrapping the matter in the air. ➢ Framework: hyaline cartilage Histology of Respiratory System 2

Respiratory region➢ Mucosa warms, moistens, and filters the inspired air. ✓ Epithelium: pseudostratified ciliated columnar epithelium with goblet cells, also called respiratory epithelium • Ciliated cells: tall columnar cells with cilia • Goblet cells: secrete mucus for entrapping the small particles • Basal cells (conical shape): stem cells of epithelium • Brush cells: : columnar cells with microvilli at the apical surface that play a role in transduction the general sensation by synaptic contact with nerve fiber of trigeminal nerve (CN V) • Small granular cells (Kulchitsky cells): enteroendocrine cells that contain secrestory granulesThe epithelium lies on basement membrane which cannot be seen under LM. ✓ Lamina propria is rich of vascular network (blood vessels) which help in warming the inspired air by blood flowing through part of loop close to the surface.➢ Submucosa: loose connective C tissue contains seromucous glands.** Submucosa cannot beabsolutely separated fromlamina propria➢ Deep layer of the tissue attaches to periosteum of the bone and perichondrium of the cartilage. Figure 5: Nasal mucosa, x42 (Dongmei et al, 2011) C Olfactory region Respiratory region vestibule A C BFigure 6: Nasal mucosa, high power (Young et at, 2014) 3 Histology of Respiratory System

Olfactory region Olfactory region (D) This region is located in part of Respiratorydome of nasal cavity and lined with regionolfactory mucosa. vestibule➢ Mucosa; ✓ Olfactory epithelium; Pseudostratified ciliated columnar epithelium • No goblet cell • Olfactory receptor cells (bipolar neurons) receive the smell and give axons to the CNS. • Supporting cells (sustentacular cell): The columnar cells with numerous microvilli that provide metabolic and physical support for the olfactory cells (act as neuroglia). • Basal cells (conical shape) is the stem cells for olfactory receptor cells • Duct of olfactory (Bowman’s) gland: cuboidal cells • Brush cells✓ Lamina propria: directly continuous Figure 7: Diagram of olfactory epithelium (Dongmei et al, 2011) to periosteum of underlying bony framework = **No submucosa • Blood vessels • Lymphatic vessels • Myelinated nerves • Unmyelinated olfactory nerves • **Olfactory (Bowman’s) glands: branched tubuloacinar serous glandsThe secretion of the glands into thesurface of olfactory epithelium serves asa trap and solvent for odoriferoussubstances (smell).Figure 8: (a) Diagram of the olfactory epithelium 4 (b) Olfactory mucosa (Ross, 2011) Histology of Respiratory System

Nasopharynx and oropharynx Nasopharynx (E) E ➢ Mucosa; ✓ Epithelium: respiratory epithelium F ✓ Lamina propria contains • Seromucous glands • **Waldeyer ring of lymphoid tissue which bulges outward into the nasopharynx, called nasopharyngeal tonsil or adenoid ➢ Submucosa contains numerous of seromucous glands. Figure 9: Nasopharynx (Young et at, 2014)Oropharynx (F)➢ Mucosa; ✓ Nonkeratinized stratified squamous epithelium ✓ Lamina propria contains lymphatic tissue, called palatine tonsil➢ Submucosa contains numerous of seromucous glands.Histology of Respiratory System 5

Larynx The larynx mainly consists oflaryngeal cartilages (hyaline & elasticcartilage) as skeletal framework andlaryngeal (intrinsic & extrinsic)muscles, covered with the membrane.Epiglottis (one of the laryngeal a bcartilage): Elastic cartilage coveredwith mucous membrane on both sides➢ Mucosa; ✓ Anterior surface and upper half ofposterior surface is covered with stratified squamous epithelium c d✓ Lower half of posterior surface is covered with respiratory epithelium Figure 10: (a) Anterior view , (b) lateral view and✓ Lamina propria: lymphatic tissue (c) posterior view with the (d) coronal section of larynx& seromucous gland (Netter, 2014)➢ Submucosa contains seromucous gland Anterior surface Posterior surface Stratified Taste buds in squamous epitheliumnonkeratinized epithelium Lingual mucosa Elastic cartilage Seromucous gland of epiglottis in lamina propriaPerichondrium of Laryngealepiglottis cartilage mucosa Pseudostratified 6 ciliated columnar epithelium Figure 11: Epiglottis, longitudinal section (Eroschenko, 2013) Histology of Respiratory System

It is divided into 3 compartments;1. Upper compartment: superior to vestibular fold (false vocal cord), continuing fromlower half of posterior surface of epiglottis. It is lined with respiratory epithelium2. Middle compartment: from vestibular fold to vocal foldFramework: thyroid cartilage➢ Vestibular fold: • Epithelium: respiratory epithelium • Lamina propria & submucosa: numerous seromucous glands, blood vessels, adipose cells, and lymphatic nodules cartilage:➢ Ventricle: • Epithelium: respiratory epithelium • Lamina propria blend with perichondrium of laryngeal numerous seromucous glands and blood vessels➢ Vocal fold (true vocal cord): • Epithelium: stratified squamous epithelium • Vocalis ligament and muscle3. Lower compartment: inferior to the vocal fold • Epithelium: respiratory epithelium • Lamina propria: devoid (a few) seromucous glands & lymphatic nodule• Framework: cricoid cartilage and skeletal muscles of larynx Part of epiglottisFigure 12: Larynx, coronal section (Eroschenko, 2013) 7 Histology of Respiratory System

Figure 13: (a) Larynx (b) Mucosa of ventricular fold (c) Mucosa of vocal cord (Ross, 2011) 8 Histology of Respiratory System

Overview of lower airwayFigure 14: Diagram of components in each part of respiratory tract (Ross, 2011)Histology of Respiratory System 9

Trachea Trachea inferiorly extends from larynx to middle of thorax where it dividesinto 2 main (primary) bronchi. To keep the lumen of the trachea permanently open, theC-shaped cartilaginous rings act as the framework of the trachea to prevent collapse.The wall of trachea consists of 4 definable layers;1. Mucosa: pseudostratified ciliated columnar epithelium and an elastic, rich-fibers lamina propria2. Submucosa: composed of lightly denser connective tissue than lamina propria.3. Cartilaginous layer: anteriorly composed of C-shaped hyaline cartilages (which can partially be replaced by bone)4. Adventitia: composed of connective tissue that attaches to adjacent structuresFigure 15: Trachea, bronchi and cross section of trachea (Netter, 2014) 10 Histology of Respiratory System

Figure 16: (a) Trachea and esophagus, x3.25 (b) trachea, x100 (Ross, 2011) Figure 17: Epithelium of trachea, x284 & x403 (Dongmei, 2011)➢ Mucosa; ✓ Epithelium: respiratory epithelium • Goblet cells • Ciliated columnar cells: move the entrapped particles out from the airway toward the mouth (serve as mucociliary escalator) • Basal cells: stem cells of epithelium that lie closely on basement membrane • Neuroendocrine cells (DNES): secrete serotonin, and peptide hormones that act as local mediators. • Small granule cells: the function is not well understood ✓ Lamina propria: typical loose connective tissue with numerous lymphocytes, mast cells, plasma cells, eosinophils, and fibroblasts. • Lymphatic tissue (diffuse & nodular form)Histology of Respiratory System 11

➢ Submucosa: loose connective tissue, containing lymphatic tissue, lymphatic & blood vessels, and seromucous glands➢ Cartilaginous layer; ✓C-shaped hyaline cartilages on the anterior side, of which the arrangement provides flexibility to the trachea and maintain the lumen. ✓Trachealis muscle is the smooth muscle located between the open end of cartilaginous ring on the posterior side. The muscle attaches directly at the perichondrium of the cartilage. The contraction and expansion of trachealis muscle adjust the diameter of tracheal lumen.➢ Adventitia, the outermost layer, lies peripheral to the cartilaginous ring and trachealis muscle. It is the connective tissue that binds trachea to adjacent structures in the neck and mediastinum. It contains large blood vessels and lymphatic vessels.G = goblet cell GlEp = epitheliumLp = lamina propriaBM = basement membraneSM = submucosaGl = seromucous glandFigure 18: Tracheal mucosa and submucosa, x400 (Ross, 2011) C = cartilage 12 T = trachealis muscle LM = longitudinal smooth muscleFigure 19: Trachea, stained with H&E and Alcian blue (Young, 2011) Histology of Respiratory System

Figure 20: Schema of bronchus, bronchiole, and alveoli (Netter, 2014)Histology of Respiratory System 13

Bronchus Trachea divides into left and right main (primary) bronchi. Then on enteringthe lung hilum, each main bronchus divides into lobar (secondary) bronchi. Thereare 2 lobes in the left lung and 3 lobes in the right. Each lobar bronchus divides intosegmental (tertiary) bronchi. carina Figure 21: Bronchial tree (Netter, 2014) 14➢ Mucosa: similar to trachea, but less tall columnar epithelium, fewer goblet cells, more elastic fiber in lamina propria. Lamina propria decrease the thickness as the diameter of bronchi decrease.➢ Submucosa: separated from lamina propria by complete circular layers of smooth muscle which become more prominent in more distal bronchi. It contains fewer seromucous glands.***Between mucosa andsubmucosa of bronchi,there is the muscularislayer, smooth musclelayer. The layer ofsmooth muscle increasesas the cartilage decrease.But in smaller bronchi,the smooth muscle ismore attenuated andloosely organized. It mayappears discontinuous. Figure 22: Secondary bronchi, x37 (Dongmei, 2011) Histology of Respiratory System

➢ Cartilaginous layer: flattened interconnected plates➢ Adventitia: moderately dense connective tissue, continuous with adjacent structures, such as pulmonary artery and lung parenchyma. Respiratory Smooth muscle epitheliumLamina propria Smooth muscleSerous gland SubmucosaBlood vesselCartilage plate Figure 23: Primary bronchi (Young, 2014)Figure 24: Tertiary bronchi (Dongmei, 2011) Figure 25: Scanning electron micrograph of lumen surface of bronchus, x1200 (Ross, 2011) Histology of Respiratory System 15

Bronchiole➢ Mucosa: pseudostratified ciliated columnar epithelium gradually transforms into a simple ciliated columnar and simple cuboidal epithelium as the bronchioles become more narrow. ✓ Goblet cells is still present in large bronchiole, but absent in terminal bronchiole. ✓ The connective tissue underlies the epithelium.➢ Thick layer of smooth muscle underlies the connective tissue.➢ Submucosa: become the thin layer with no glands➢ Cartilaginous layer: cartilage plates are absent.➢ Adventitia: lung tissueFigure 26: (a) Small tertiary bronchus (b) Bronchiole 16 (Dongmei, 2011) Histology of Respiratory System

Terminal bronchiole➢ Epithelium: simple ciliated cuboidal epithelium ✓ No goblet cells ✓ Clara cells: nonciliated rounded or dome-shape apical surface projection They secrete surfactant.➢ Small amount of connective tissue underlies epithelium.➢ Circumferential layer of smooth muscle underlies the connective tissue.Figure 27: Terminal bronchiole, x284 & x1181 (Dongmei, 2011) TB = terminal bronchiole 17 RB = respiratory bronchiole AD = alveolar duct AS = alveolar sacFigure 28: respiratory portion of bronchial tree (Ross, 2011) Histology of Respiratory System

Figure 29: Schema of intrapulmonary blood circulation (Netter, 2014)Histology of Respiratory System 18

Respiratory bronchiole Respiratory bronchiole is the first part of respiratory portion and containsa small number of single alveoli, opening to the respiratory bronchiole. Eachrespiratory bronchiole divides into several alveolar ducts which have numerousalveoli opening along their length. The end of each alveolar duct is alveolar sac.➢ Epithelium: simple ciliated cuboidal epithelium ✓ Clara cells: • Secrete surfactant • Act as stem cells • Contain enzymes➢ Incomplete layer of smooth muscle surrounded by fibroelastic connective tissueAlveolar ductThe wall of alveolar duct consists of;• Epithelium: simple squamous epithelium (type I pneumocytes) & simple cuboidal epithelium (at the knobs or alveolar rings)• Underlying the epithelium, there is a small aggregation of smooth muscle, collagen and elastic fibers which form alveolar ring surrounding the alveolar ducts and the opening of alveolar sacs and alveoli.Figure 30: Bronchial tree (Young, 2011)T = terminal bronchiole AD = alveolar duct AR = alveolar ringR = respiratory bronchiole AS = alveolar sacHistology of Respiratory System 19

Alveolus Figure 31: Diagram of respiratory portion (Dongmei, 2011)Figure 32: Diagram of blood-air barrier (Dongmei, 2011) The end of alveolar Figure 33: Blood-air barrier (Young, 2014) duct is the alveolar sac which contains several alveoli. The alveolus is the terminal unit of respiratory system. The wall of alveolus is composed of; 1. Type I pneumocytes: primary cells which form the alveolar wall and contact with the capillary walls The site of gas exchange is the blood-air barrier, composed of type I pneumocyte (P1), endothelium of capillary (E) and fused basement membrane (BM) of these cells. 2. Type II pneumocytes: septal cells which are located in the connective tissue septum. They produce the surfactant. Histology of Respiratory System 20

The red arrows point at black-stained elastic 3. Dust cells, macrophage of fibers within the interalveolar septum alveolus can be found within the interalveolar septum, near Ma = dust cells the type II pneumocytes. They serve as phagocytic cells, Figure 34: Pulmonary elastic tissue, eliminating the unfriendly stained with Elastic van Giemsa (Young, 2014) particles from the inspired air. 4. Fibroblasts, elastic and collagen fibers Between adjacent alveoli, the interalveolar septa contain small opening, called alveolar pore. The pore provides movement of air between the alveoli. It helps in adjusting the air pressure to protect alveolar collapse.Figure 35: alveoli with interalveolar septa (Dongmei, 2011)Figure 36: Cells in alveolus (Ross, 2011) 21 Histology of Respiratory System

A = alveolusC = capillarySC = septal cell (type II pneumocyte)SM = smooth muscleabFigure 37: (a) Alveolar knob composed of smooth muscle and simple cuboidal epithelium (b) Interalveolar septa composed of capillary and pneumocytes (Ross, 2011)Figure 38: Pneumocytes (Dongmei, 2011)Histology of Respiratory System 22

References1. Ross, M.H. & Pawlina, W. (2011). Histology: A text and atlas: with correlated cell and molecular biology 6th edition. China: Lippincott Williams & Wilkins, a Wolters Kluwer business.2. Young, B., O’Dowd, G. & Woodford, P. (2011). Wheater’s Functional Histology: A text and colour atlas 6th edition. Pennsylvania: Churchill Livingstone, an imprint of Elservier Ltd.3. Eroschenko, V.P. (2013). diFiore’s atlas of Histology with functional correlations. China: Lippincott Williams & Wilkins, a Wolters Kluwer business.4. Gartner, L.P. & Hiatt, J.L. (2014). Color atlas and text of Histology 6th edition. China: Lippincott Williams & Wilkins, a Wolters Kluwer business.5. Cui, D., Naftel, J.P., Daley, W.P., Lynch, J.C., Haines, D.E., Yang, G. & Fratkin, J.D. (2011). Atlas of Histology: with functional and clinical correlation. China: Lippincott Williams & Wilkins, a Wolters Kluwer business.6. Netter, F.H. (2014). Atlas of Human Anatomy 6th edition. Pennsylvania: Saunders, an imprint of Elservier Inc.Histology of Respiratory System 23