11.1 Background and development

The respiratory system consists of the nasal cavity, larynx, trachea, major airways and lung parenchyma. The respiratory system is studied routinely in phenotyping studies and in more detail to test inhaled compounds for toxicity and for the evaluation of mouse models of human disease. For these reasons it is important to understand the anatomy and histology of the mouse respiratory tract. Nasal and pulmonary epithelia are able to metabolize xenobiotics and frequently demonstrate lesions from these compounds (Renne et al. 2009). Identification of the metabolic functions of the respiratory epithelia has resulted in great interest in the structures of the respiratory system.

11.2 Embryology

In the mouse, three stages of development are recognized in the embryonic lung. The glandular period lasts until about day 10 and the lung at this stage consists of poorly defined connective tissue and proliferating columnar epithelium. During the cannalicular phase, which occurs from day 10 until a couple of days before birth, the bronchial tree and vascular systems start to develop. Alveoli start to form in the last couple of days before birth (alveolar period), but do not become fully differentiated until a few days postnatally. Postnatally (Figure 11.1) the alveolar spaces become defined by increased numbers of septae and the thickness of the septal walls decreases.

Figure 11.1 Section of lung from a mouse on postnatal Day 2. Note the relative thickness of the alveolar septae due to incomplete septation.

Considerable differences in lung volume and morphometric parameters exist between inbred mice. Adult C3H/HeJ mice (C3) have a 50% larger lung volume and 30% greater mean linear intercept than C57BL/6J (B6) mice (Soutiere and Mitzner 2006]). These differences are thought to occur because of lung air volume differences and different rates of alveolar septation (Soutiere and Mitzner 2006).

11.3 Anatomy and histology of the respiratory system

11.4 Upper respiratory tract

The cutting of standardised upper respiratory tract sections and the recognition of the normal histologic anatomy of cell populations within those sections is very important as it enables more accurate interpretation of the effects on cell types that will vary with location of the level of the section. The variations in the distribution of different epithelia within the nasal turbinates require consistent sectioning of the nasal turbinates and larynx. (Kittel., et al. 2004; Renne et al. 1992) (Figure 11.2a and b).

Figure 11.2 (a) Illustration of a longitudinal section of the nasal cavity and the four levels of sections, which may be taken to aid complete examination of the nasal cavity. (b) The distribution of epithelial types is illustrated progressing from squamous epithelium (orange) at the entrance through a narrow band of transitional epithelium (pink), to respiratory epithelium (lilac) and extensive areas of olfactory epithelium (blue) caudally.

At necropsy, the lower jaw, calvarium and brain are removed and the nasal cavities should be perfused through the nasopharynx with formaldehyde. The entire head is then placed in fixative. Decalcification is performed in formic acid or Christensen’s fixative and then tissues are removed from the decalcifying solution, processed and microtomed by standard procedures.

For diagnostic purposes, for instance to check for rhinitis, a single section through the nasal cavity may be made at the level of the upper incisor teeth.

If a complete evaluation of the nasal cavity is required then three or four standard sections of the mouse nasal turbinates may be cut with reference to the upper palate (Young 1981) and teeth (Renne et al. 2007) (Figure 11.2a). The first section (level 1) is cut at the level of the upper incisor teeth (Figure 11.3). The roots of the teeth are shown laterally. The paired vomeronasal organs (Jacobson’s organ) and nasolacrimal ducts are present in level 1. A high volume of air passes through the central region of the nasal cavity so it is vulnerable to inhaled compounds and pathogens and is a common site for lesions (Renne et al. 2007).

Figure 11.3 The first nasal cavity section (level 1) is cut at the level of the upper incisor teeth and includes the vomeronasal organ (not shown in this image). The most rostral location of level 1 is lined by stratified squamous epithelium in the ventral meatus. In level 1, transitional epithelium lines the lateral walls and lateral turbinate surfaces and olfactory epithelium can line the dorsal meatus. Respiratory epithelium lines the septum, dorsal meatus and medial aspects of the turbinates.

The second section is taken at the level of the incisive papilla (Figure 11.4). At this level, bilateral communication with the oral cavity via the incisive ducts can be observed (Reznik 1990). The incisor teeth are still present in this section and the nasolacrimal duct may still be observed. The nasolacrimal duct has moved lateral to the incisor tooth. The olfactory epithelium in this level is especially susceptible to injury (Renne et al. 2007).

Figure 11.4 The second nasal cavity section is taken at the level of the incisive papilla. The incisor teeth are still present in this section and the nasolachrymal duct may still be observed. The nasolachcrymal duct has moved lateral to the incisor tooth. The dorsal meatus is lined by olfactory epithelium. Squamous epithelium lines the nasopalatine duct ventrally. The respiratory epithelium lines the nasal septum, the turbinates and lateral walls and the transitional epithelium has disappeared at this level.

The third section is taken at the level of the second palatal ridge (Figure 11.5). This section includes the maxillary sinuses and Steno’s glands. The glands surrounding the maxillary sinuses are known as Steno’s glands. They are similar to serous salivary glands. The most frequent site of injury at level 3 in mice is the olfactory epithelium lining the dorsal medial meatus (Renne et al. 2007).

Figure 11.5 The third nasal cavity section is taken at the level of the second palatal ridge. This section includes the maxillary sinuses and Steno’s glands. The majority of the ethmoid turbinates are lined by olfactory ∗ epithelium. The maxillary sinuses are lined by respiratory epithelium.

The fourth section is taken at the level of the first upper molar tooth (Figure 11.6). Here the nasopharyngeal duct is visible with the nasal-associated lymphoid tissue (NALT) present on either side of the nasopharyngeal duct. Bowman’s glands produce mucus, are highly metabolic and are found in the lamina propria below the olfactory epithelium. Large and prominent nerve bundles in the lamina propria can be observed as well as the olfactory bulbs of the brain.

Figure 11.6 The fourth nasal cavity section is taken at the level of the first upper molar tooth. Here the nasopharyngeal duct is visible and is lined by respiratory epithelium. NALT is visible on ventrally either side of the nasopharyngeal duct. The epithelium in this region covering the ethmoid turbinates is almost entirely olfactory.

11.4.2 The nasal cavity

The nasal turbinates, larynx, trachea and tracheal bifurcation and associated lymphoid tissue are often only examined histologically in inhalation toxicity studies. However consideration should be given to examining the nasal cavity in primary phenotyping screens, in cases of rhinitis secondary to ciliary defects or in accidental inhalation of bedding material or dusty food stuff. Inhalation of foreign material is (Figure 11.7) common and may be a cause of otherwise unexplained death in mice. Mice are obligate nose breathers and so obstruction to airflow due to rhinitis (or other space occupying lesion) can result in swallowing of air leading to a distended stomach and intestines which eventually compress the diaphragm causing death by asphyxiation.

Figure 11.7 Accidental inhalation of bedding is common and causes rhinitis and this may be a cause of otherwise unexplained death in mice.

The positions of the ethmoid turbinate, hard palate, incisor tooth, maxilloturbinate, naris and nasoturbinate nasal vestibule in the mouse are illustrated in Figure 11.2.

The mouse nasal cavity is divided into two main air passages by the nasal septum (Harkema et al. 2011). Each nasal passage extends from the nares to the nasopharynx caudally (Harkema et al. 2011). The nasal cavities contain a dorsal meatus, which becomes the dorsal ethmoid recess, a middle meatus that becomes the maxillary sinus and a ventral meatus, which terminates at the nasopharyngeal duct. The vestibule is located immediately after the nares before the main chambers of the nose (Harkema et al. 2011). Four epithelial types are found in the nasal cavity / nasal turbinates (Monticello et al. 1990). These include squamous epithelium, transitional epithelium, respiratory epithelium and olfactory epithelium. Stratified squamous epithelium lines the vestibule (Figure 11.8). It has a basal layer covered with several layers of squamous epithelial cells that become successively flatter towards the mucosal surface. The nasal vestibule requires squamous epithelium to protect the underlying tissues from injurious, inhaled substances. Squamous epithelium lines the ventral meatus, although at this level most of the turbinates are lined by respiratory epithelium.

Figure 11.8 Stratified squamous epithelium lines the vestibule.

Inhaled air moves from the vestibule to the nasal chambers, which are made up of nasal turbinates. The turbinates are bony structures that project into the lumen from the lateral walls and increase the surface area of the nose (Harkema et al. 2011). The squamous epithelium of the vestibule gives rise to transitional epithelium (Figure 11.9) before becoming respiratory epithelium, which lines the rostral turbinates. Thus, transitional epithelium lines the tips and lateral aspects of parts of the naso and maxilloturbinates and the lateral wall of the anterior nasal cavity. Transitional epithelium is one to two cells thick and is primarily made up of nonciliated cuboidal and short columnar cells resting on basal cells, covered with microvilli. Transitional epithelium contains few goblet cells (Harkema et al. 2011). The cells of the transitional epithelium contain abundant smooth endoplasmic reticulum (SER) in the cytoplasm and are an important store for xenobiotic-metabolizing enzymes such as cytochrome p450. The lamina propria below the squamous and transitional epithelia contains blood vessels, connective tissue, nerves and serous and mucous glands. Prominent ‘swell bodies’ (venous plexuses) are found in the maxilloturbinates and the lateral wall of the proximal nasal passage in mice (Harkema et al. 2011). These structures function to rehydrate the mucosa (Gartner and Hiatt 1997).

Figure 11.9 The squamous epithelium of the vestibule gives rise to transitional epithelium.

Respiratory epithelium (Figure 11.10) covers most of the naso and maxilloturbinates medially, the nasal septum and ventral ethmoid turbinates. Respiratory epithelium is composed largely of pseudostratified, ciliated and nonciliated cuboidal to columnar cells, goblet cells and basal epithelial cells. Solitary chemoreceptor and brush cells are also observed in respiratory epithelium (Harkema 2011). There is a gradual increase in goblet cells as the respiratory epithelium moves from the front of the nasal turbinates to the more posterior turbinates. The lamina propria below the respiratory epithelium contains serous and mucous glands. Steno’s gland is situated in the lateral walls of the maxillary sinus (Figure 11.11).

Figure 11.10 Respiratory epithelium covers most of the naso and maxilloturbinates medially.

Figure 11.11 Steno’s gland is situated in the lateral walls of the maxillary sinus.

Olfactory epithelium lines the ethmoid turbinates and some of the anterior dorsal meatus. Olfactory epithelium (Figure 11.12) is a pseudostratified columnar epithelium. It is composed of tall sustentacular cells, olfactory neurons and basal cells. The sustentacular cells are columnar, secretory cells and support the olfactory neurons. In addition to supporting the olfactory neurons, the sustentacular cells also possess microvilli, which act together with the microvilli of the olfactory neurons. Large amounts of cytochrome p450 are found in the cytoplasm of the sustentacular cells (Harkema et al. 2011). The olfactory neurons are lined up between the sustentacular cells and their nuclei form a prominent layer, which is five to six nuclei thick. The olfactory sensory neurons are bipolar neuronal cells. The dendrites of the olfactory neurons extend above the epithelial surface and end in an olfactory knob which in turn, extends into the cilia onto the surface. The cilia and microvilli of the sensory neuron are involved in the function of smell. The axon of the olfactory sensory neuron extends through the basement membrane and joins other axons to form the nonmyelinated nerves bundles. The nonmyelinated nerve bundles penetrate the bony cribriform plate and extend into the olfactory bulb of the brain. The olfactory epithelium overlays a lamina propria made up of nerve bundles, blood vessels and tubulo-alveolar Bowman’s glands. The Bowman’s glands produce enzymes for the metabolism of xenobiotic compounds and secrete mucus that coats the surface epithelium (Renne et al. 2007). The paired vomeronasal glands (organ of Jacobson) (Figure 11.13) are situated in the base of the nasal septum. The vomeronasal organs are not present in humans. In animals, they are thought to play a role in recognition of pheromones and food flavour perception (Reznik 1990; Renne et al. 2009). The vomeronasal organs are lined by respiratory epithelium dorsal-medially and olfactory epithelium ventral-laterally. Neutrophils are found commonly in normal vomeronasal glands (Reznik 1990).

Figure 11.12 Olfactory epithelium is a pseudostratified columnar epithelium composed of tall sustentacular cells, olfactory neurons, and basal cells.

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