Histomorphology of the lower respiratory tract in the Indian crested porcupine (Hystrix indica)
Abstract
The Indian crested porcupine (Hystrix indica) (ICP) is widely distributed in Asia; however, compared with other rodents, little is known about the structures of its respiratory system. The aim of this study was to evaluate the histomorphology of the lower respiratory portion of the ICP to provide a basis for the identification of the normal structure of this organ. The larynx, trachea and lungs of four carcasses of adult Indian crested porcupines (two males and two females) were dissected and fixed in 10% neutral-buffered formalin. The gross anatomy and histology of all specimens were evaluated. A macroscopic evaluation showed unique structures in the ICP respiratory system, including the presence of a chamber-like structure at the origin of the bronchi and a difference in epiglottis shape between males and females. Histologically, the stratified squamous epithelium covered the epiglottis and arytenoid cartilage, and the pseudostratified ciliated columnar epithelium covered the internal part of the thyroid and cricoid cartilages. Histomorphological studies showed a few goblet cells in the tracheal epithelium. In the bronchi and larger bronchioles, pseudostratified ciliated columnar epithelia were observed. Bronchi were surrounded by segments of cartilage. Distal bronchioles had a simple cuboidal/columnar epithelium with club (Clara) cells, lacked cartilaginous tissue in their walls and had a complete smooth muscle layer. These results revealed histomorphological differences between the ICP and other rodents.
1 INTRODUCTION
The Indian crested porcupine (Hystrix indica) (ICP) is a large hystricomorph rodent (order Rodentia) belonging to the family Hystricidae. It is widely distributed from central to southwestern Asia and is the largest rodent in Iran (Arzamani et al., 2018; Albaba, 2016; Khan et al., 2000; Rajabloo et al., 2015). It has various defence mechanisms and nocturnal behaviour and spend the days in natural caves, excavated burrows or crevices (Rajabloo et al., 2015). It is globally listed as a species of Least Concern by the IUCN Red List of Threatened Species (Amori et al., 2018); however, in some countries, including Turkey, Jordan, India, Pakistan and Iran, it is threatened by poaching for its meat and to minimise crop damage (Khan et al., 2000; Al Dhaheri et al., 2018).
Morphological studies of the structure of the respiratory system are essential to understand vertebrate behaviour and physiology. Accordingly, characterisation of the morphological and histological features of the respiratory system is a major research goal (Fonseca et al., 2017; Mario et al., 2018; Oliveira et al., 2012). Since rodents have the potential to spread zoonotic respiratory diseases, studies of the respiratory system have focused on rats, guinea pigs, mice and others (Enria & Pinheiro, 2000; Calisher et al., 2003; Hill & Brown, 2011; Blagojevic et al., 2018; Ilgun et al., 2014; Treuting & Dintzis, 2012; Yarto-Jaramillo, 2011). However, to our knowledge, the respiratory system of the ICP, belonging to the ‘Old World’ porcupines, has not been evaluated.
Therefore, the aim of this study was to evaluate the histomorphology of the lower respiratory portion of ICP to provide the necessary information for the identification of the normal structure of this organ.
2 MATERIALS AND METHODS
2.1 Cadaver preparation
In cooperation with the Department of Environment, four carcasses of adult ICPs (two males and two females; average weight, 16 kg) that recently died in an accident or were abandoned after hunting were referred to the anatomy laboratory of the Faculty of Veterinary Medicine. After examining the corpses to ensure no disease, the larynx, trachea and lungs were dissected and fixed in 10% neutral-buffered formalin. Images of specimens were obtained using a digital camera (Canon EOS 4000D), and the morphological characteristics of the larynx, trachea and lungs were evaluated. To study small structures, a stereoscopic microscope (ZeissStemi SV6) was used. The measurements were obtained using a digital ruler caliper (sensitivity: 0.01 mm; Digimatic Caliper).
2.2 Tissue preparation and staining
For light microscopy, tissue samples were collected from the laryngeal cartilages, three areas of the trachea (cranial third, middle third, and caudal third), and the cranial and caudal lobes of both lungs of each animal. These specimens were then dehydrated in increasing concentrations of ethanol solution, rinsed with xylene and embedded in paraffin. Serial sections of the blocks were cut at 5 μm and stained routinely with haematoxylin and eosin (H&E), periodic acid–Schiff (PAS) and Masson's trichrome (Luna, 1986). Histological sections were observed using a light microscope (Olympus SX-21) equipped with a digital camera (TrueChrome II).
2.3 Immunohistochemistry
Immunohistochemistry (immunofluorescence) was applied for the identification of CC10 (a club cell marker). After deparaffinisation, the lung tissue sections (5 μm) were washed four times with PBS (5 min each). The histological sections were placed in 2 N hydrochloric acid for antigen retrieval for 30 min, and borate buffer solution was then used for neutralisation. After washing once with PBS, tissue sections were incubated in 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 30 min at room temperature. After washing once with PBS, tissue sections were incubated with 10% normal goat serum in PBS (blocking buffer) for 30 min at room temperature. Mouse anti-CC10 primary antibody (1:100 dilution; sc-365992) was applied in a humidified chamber at 4°C overnight. Lung tissue sections were washed four times with PBS (5 min each) and were then incubated with the goat anti-Mouse IgG (FITC) secondary antibody (1:150 dilution; ab6785, Abcam) for 90 min at 37°C. After rinsing with PBS, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Merck KGaA, Darmstadt, Germany). Immunolabelled club cells were assessed using a fluorescence microscope (Olympus).
3 RESULTS
3.1 Morphological findings
3.1.1 Larynx
The larynx of the ICP included cricoid, thyroid, arytenoid and epiglottis cartilages. The cricoid cartilage had a wide quadrilateral lamina (blade), a median crest on the caudal half and a very shallow notch on the caudal border (Figure 1a). The T-shaped arytenoid cartilage consisted of a corniculate process in the dorsal part, a long and sharp vocal process ventrally, and a muscular process on the lateral surface (Figure 1b). The thyroid cartilage body and laminae were approximately the same diameter. The notches of the rostral and caudal borders of the body were shallow, and the laryngeal prominence was not seen on the ventral surface. At the junction of the first and second fifths of the thyroid laminae of the lateral surface, an oblique line extended from the rostral border to the caudal border. In the thyroid cartilage, the thyroid fissure became the foramen (Figure 1c). The epiglottis cartilage was a short quadrilateral plate in males and leaf-shaped in females, with a prominent stalk in both genders (Figure 1d).
3.1.2 Trachea
The total length of the ICP trachea was 18.5 ± 0.76 cm. The trachea was composed of 36–40 incomplete rings. The tracheal rings were diverse in shape and diameter and did not show a single pattern. However, the dorsal half was consistently wider than the ventral half and overlapped on adjacent rings and the opposite end, although the direction of overlap along the trachea differed, from right to left or left to right. The free ends of the tracheal rings were irregular in shape (Figure 2b). In addition, a few anastomoses between adjacent rings were observed. The trachealis muscle was attached to the internal surface of the tracheal rings. The diameter of the trachea and size of the dorsal gap varied among parts. The diameter of the first ring was 18.26 mm but then varied from 13.11 to 21.79 mm. Two dilations were observed throughout the trachea. The first dilation was located in the second quarter, and the second dilation was located at the level of the origin of the principal bronchi (Figure 2a), where the free borders of tracheal rings were more distant, and a chamber-like structure formed, with tracheal rings as the floor and loose connective tissue and trachealis muscle as the roof (Figure 2b,c). From each side of this chamber, two bronchi originated and entered the cranial and caudal lobes of the right and left lungs (Figure 2a). The bronchi were short and consisted of 7–8 incomplete rings.
3.1.3 Lungs
The lungs of the ICP were located between the 2nd and 6th intercostal spaces. The left and right lungs were subdivided into cranial and caudal lobes by deep interlobar fissures creating pars cranialis and caudalis of the respective lobes (Figure 2b). Ventral edges of the lungs were divided by short incisures, creating a serrated appearance (Figure 2b). The cranial and caudal lobes of each lung were almost equal in size.
3.2 Histological findings
3.2.1 Larynx
The larynx of the ICP involved two epithelium types, a stratified squamous epithelium covering the epiglottis and arytenoid cartilages (Figures 3 and 4), and pseudostratified ciliated columnar epithelium covering the internal part of the thyroid and cricoid cartilages. The epiglottis consisted of elastic cartilage with thin elastic fibres in the extracellular matrix (Figure 3). This cartilage was surrounded by the perichondrium. Under the keratinised squamous stratified epithelium, loose connective tissue with seromucinous glands was observed (Figures 3 and 4). The mucus units were more prevalent than serous units, and the serous cells were located on peripheral mucus units and formed serous demilunes. Thyroid, arytenoid and cricoid cartilages were hyaline and epiglottis-like. Under the epithelium, many seromucinous glands with loose connective tissue were found with many secretory units (Figures 3 and 4).
3.2.2 Trachea
The inner surface of the ICP trachea was lined by a pseudostratified columnar epithelium (Figure 3). The propria submucosa was loose connective tissue with seromucinous glands, where the mucus units were considerably higher in number. Histologically, the propria submucosa was similar to that of other species and consisted of elastic fibres. The C-shaped rings of the tracheal cartilage were hyaline and surrounded by a perichondrium. The outer aspect of the trachea was surrounded by the adventitia (Figures 3 and 4). A few goblet cells were observed by PAS staining of the tracheal epithelium. Under the tracheal epithelium, a highly vascularised tissue with loose connective tissue was also observed (Figure 5). The chamber-like structure in the distal end of trachea was lined by a pseudostratified columnar epithelium. The propria submucosa was smooth muscles and loose connective tissue with fat cells. Furthermore, the chamber-like structure showed a floor of hyaline cartilage (Figure 3c).
3.2.3 Bronchi
At the origin of bronchi, in the internal membrane of the trachea, the epithelium was pseudostratified ciliated columnar with ciliated and non-ciliated cells. Goblet cells were not observed by PAS staining of the bronchial epithelium. A loose connective tissue layer was detected under the epithelium. The smooth muscle fibres were oblique, circular, and longitudinal and did not follow a regular arrangement. The outer tunic layer was a serous membrane (Figures 3 and 5).
The lung sections consisted of intrapulmonary conducting airways, gas exchange areas, blood vessels and connective tissue. The epithelia of bronchi and larger bronchioles were pseudostratified ciliated columnar. Bronchi were surrounded by segments of cartilage. Between the epithelium and cartilages of the bronchi, a few smooth muscle fibres were observed (Figure 5). Bronchioles had a simple ciliated columnar epithelium, with no cartilaginous tissue in their walls, and a complete smooth muscle layer with collagen fibres (Figures 5 and 6). Terminal bronchioles had a simple cuboidal epithelium with club cells (Figures 5 and 7). CC10-positive cells (i.e. club cells) were detected in the distal airways, including the terminal and respiratory bronchioles (Figure 7).
Respiratory bronchioles were long and abundant in the sections. They had a similar histological structure with terminal bronchioles but with open alveoli in their walls. These structures led to alveolar ducts and alveolar sacs (Figures 6 and 7). The alveoli had two cell types: (1) alveolar squamous cells or type I pneumocytes; (2) alveolar cuboidal cells or type II pneumocytes. Macrophages were seen in the lumen of alveoli. Between alveoli, a rich bed of capillaries was observed (Figure 5).
4 DISCUSSION
In the present study, considering the importance of respiratory system anatomy, the detailed histomorphological features of the lower portion of the respiratory tract of the ICP were evaluated.
The larynx of the ICP included cricoid, thyroid and arytenoid cartilages with the corniculate process and epiglottis. The morphology was similar to those for some other rodents, such as Oligoryzomys nigripes, Mus musculus and Rattus norvegicus (Mario et al., 2018; Borgard et al., 2019; Oliveira et al., 2012). In the thyroid cartilage of the ICP, there was a thyroid foramen instead of a thyroid fissure found in some other rodents, including the laboratory rat (Rattus norvegicus), kangaroo rat (Dipodomys ordii), grasshopper mouse (Onychomys. arenicola, Onychomys. torridus and Onychomys. leucogaster) and laboratory mouse (Mus musculus) (Riede et al., 2017). The rostral horn of the thyroid cartilage was short and narrow, and the caudal horn was short and broad. The rostral horn is broad in the genera Mus, Rattus, and Onychomys, and is smaller and pointed in Dipodomys (Riede et al., 2017). The shape of the epiglottis of the ICP differed between males and females, with quadrilateral and foliate shapes, respectively. Shape differences in the epiglottis between sexes have not been reported to date; however, Ozkadif et al. (2016) reported a significantly larger epiglottis in male New Zealand rabbits than in females. The cartilage was without a cuneiform process in the ICP, unlike in some other members of the rodent family, such as the grasshopper mouse and laboratory rat (Riede et al., 2017). The arytenoid cartilage of the ICP included muscular, vocal and corniculate processes, which have been observed in other rodents, such as agouti (Dasyprocta sp.), kangaroo rat and grasshopper mouse (Silva et al., 2014; Riede et al., 2017). The cricoid cartilage of the ICP was very similar to those in agoutis, laboratory mice and kangaroo rats (Silva et al., 2014; Riede et al., 2017).
The trachea of the porcupine was composed of 36–40 incomplete rings with variation in shape and diameter along the length. The number of rings was higher than in other rodents such Oligoryzomys nigripes, with 11 rings (Mario et al., 2018); Cricetomys gambianus, with 21–33 rings; and mole rates, with 30.5 ± 4.5 rings (Ibe et al., 2011; Ilgun et al., 2014).
Widening of the trachea at the level of the origin of the principal bronchi has not been observed in other rodents or porcupines, to the best of our knowledge, and appears to be a unique structure specific to ICP. Its function remains to be elucidated. It might serve as a reservoir structure to improve ventilation as the animal burrows. In the African giant pouched rat, the morphology of the respiratory system is attributed to the design of its burrow, so that the presence of several openings and the small number of inhabitants in each cavity provide suitable ventilation and lead to a poorly developed respiratory system in the animal (Ibe et al., 2011).
Each right and left lung of the ICP included cranial and caudal lobes subdivided into cranial and caudal parts. The crested porcupine (Hystrix cristata) (Ozdemir et al., 2006) has a right lung with a bilobed cranial lobe and middle, caudal and accessory lobes, with a bilobed middle lobe and a caudal lobe in the left lung. In H. cristata, the caudal lobe is the largest, which is inconsistent with our results. The mole rat, Wistar rat and African giant-pouched rat have five lung lobes, one in the left lung and four in the right lung (Suckow et al., 1975; Ibe et al., 2011; Ilgun et al., 2014), different from the structure of the ICP.
The epiglottises of the mouse and rat are elastic cartilage lined with a squamous stratified epithelium (Treuting et al., 2018), consistent with the epiglottis of the ICP. The epiglottises of Oligoryzomys nigripes, nine-banded armadillo and brown-nosed coati are elastic cartilage with a squamous stratified epithelium with connective tissue and seromucinous glands (Oliveira et al., 2012; Fonseca et al., 2017; Mario et al., 2018). The presence of seromucinous glands under the squamous stratified epithelium provides moisture to the mucosa of this part of the larynx. In the mouse, rat and house shrew, taste buds are distributed in the epithelium lining of the epiglottis (Shrestha et al., 1995; Treuting et al., 2018). We did not detect taste buds on the laryngeal surface of the epiglottis epithelium in the ICP. Furthermore, taste buds are not present on the surface of the epiglottis in the nine-banded armadillo (Fonseca et al., 2017). These studies suggested obvious species differences, probably linked to feeding habits. It has been suggested that laryngeal taste buds are numerous in ruminants and are rare in insectivorous species, while an intermediate number seems to be found in omnivorous animals (Sbarbati et al., 2004).
The arytenoid epithelia in coatis (Oliveira et al., 2012) and the ICP are covered with a squamous stratified epithelium, compared with a pseudostratified columnar epithelium in the nine-banded armadillo (Fonseca et al., 2017). In the mouse and rat, an intermediate epithelium (composed of non-ciliated, cuboidal-to-flattened cells) is present on the surface of the arytenoid cartilage (Treuting et al., 2018).
We detected seromucinous glands in the lamina propria (subepithelial glands) throughout the larynx, as observed in rats (Treuting et al., 2018), coatis (Oliveira et al., 2012) and agoutis (Silva et al., 2014), different from the nine-banded armadillo, which lacks glands (Fonseca et al., 2017). Furthermore, subepithelial mucous or seromucinous glands are distributed only within portions of the larynx in mice (Treuting et al., 2018).
The thyroid, cricoid and arytenoid cartilages in the rat and mouse, nine-banded armadillo (Fonseca et al., 2017), and ICP are hyaline and have a perichondrium and connective tissue with a pseudostratified ciliated columnar epithelium. The ICP did not have an osteoid matrix, as observed in the nine-banded armadillo (Fonseca et al., 2017).
The trachea was covered with a pseudostratified ciliated columnar epithelium with hyaline cartilage in Oligoryzomys nigripes (Mario et al., 2018) and ICP. ICP trachea and bronchi epithelium composed of non-ciliated cells (basal, columnar and cuboidal) and scattered ciliated cells resting on a basement membrane. The epithelia of the trachea in the mouse and rat are mostly composed of non-ciliated cells. In rodent airways, non-ciliated cells are the principal sources of secretions (Treuting et al., 2018). Furthermore, it has been reported that the primary progenitor cell of rat airway epithelium is the total non-ciliated columnar cell population (columnar secretory cells), which can divide and form new secretory and ciliated cells (Evans et al., 1986). The tracheal rings of Siphonops annulatus (kuehne & Junqueira, 2000) are similarly hyaline but have unique fattened or cubic cells. The number of goblet cells in the tracheal epithelium of the ICP was low, similar to other rodents, such as the mouse and rat (Treuting et al., 2018). In the ICP, in the loose connective tissue under the epithelium of the trachea, vascular tissue was observed, similar to observations in the rat and mouse (Treuting et al., 2018).
The bronchi of the ground squirrel (Blagojevic et al., 2018), rat (Koptyev et al., 2014) and mouse (Chaturvedi & Lee, 2005; Thiesse et al., 2010) have cartilage segments around the duct, consistent with our findings for the ICP. In the ground squirrel (Blagojevic et al., 2018), the epithelia of the larger bronchi and bronchioles are pseudostratified ciliated columnar; in the ICP, the epithelium of the terminal bronchioles was simple columnar or cuboidal with club cells.
Respiratory bronchioles were long and abundant in the ICP but are not well-developed in other rodents (e.g. mouse and rat) (Treuting et al., 2018).
Our immunohistochemical findings revealed that club cells are predominant in the terminal and respiratory bronchioles, in line with the terminal and respiratory bronchioles of mice (Ryerse et al., 2001; Treuting & Dintzis, 2012).
We did not detect bronchus-associated lymphoid tissue (BALT) in the ICP. However, Blagojevic et al. (2018) reported this tissue type in the ground squirrel interstitial stroma of bronchioles.
The alveolar epithelium of the ICP had two types of cells, including many squamous cells (type I pneumocytes) and few cubic cells (type II pneumocytes). Type II pneumocytes are columnar in the ground squirrel (Blagojevic et al., 2018). In the ICP, we detected macrophage cells in the interstitial stroma between the alveoli and alveoli cavity, unlike in Siphonops annulatus (kuehne & Junqueira, 2000). Pulmonary macrophage populations divide into alveolar macrophages, strategically positioned in the airways, and interstitial macrophages, located within the lung parenchymal tissue (Byrne et al., 2015). In the mouse and rat, macrophage cells have been observed in the alveoli (Treuting et al., 2018). In fossorial rodents, the mole rat Tachyoryctes splendens, alveolar macrophages have been frequently observed on the alveolar surface; but these macrophages have been rarely observed on the alveolar surface in the lung of naked mole rat Heterocephalus glaber (Maina et al., 1992). These cells are the first line of defence against pathogens, and their presence in the wall of the alveoli impairs the detection of cuboidal alveolar epithelial cells (Treuting et al., 2018). It has been suggested that presence of these macrophages may be more related to the defence mechanisms of the pulmonary tissue in the burrow environment which contains a high concentration of dust and possibly disease causing agents (Maina et al., 1992).
5 CONCLUSIONS
Our results showed that the lower respiratory portion of the ICP (H. indica) is essentially similar to those of other rodents, with the exception of several histomorphological properties, like the different shape of epiglottis between sexes, unique anatomy of the trachea with its widening at the level of origin of main bronchi, absence of taste buds on laryngeal surface of the epiglottis epithelium, and long and abundant respiratory bronchioles. Accordingly, further anatomical and histological comparisons of the respiratory system in the ICP involving animals representing a larger number of gender and age groups may be useful, in addition to analyses of other parts of the body, such as the digestive system. Our histomorphological analyses emphasise the importance of additional embryological, physiological and behavioural studies of the ICP, given the multifunctional and complex characteristics of the respiratory system.
ACKNOWLEDGMENTS
The authors would like to thank the Department of Environment, Mazandaran province, Iran for collecting the specimens. This research was supported by a research grant from the Amol University of Special Modern Technologies, Amol, Iran.
CONFLICT OF INTEREST
None.
ETHICAL APPROVAL
This was a study carried out on animals that were considered cadavers, and therefore, no ethical approval was needed.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
