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Anatomia, Histologia, Embryologia

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Histological Characteristics of the Tracheobronchial Tree of the Least Shrew (Cryptotis Parva) F. Arodaki1, W. Khamas2, N. Darmani3 and M. Al-Tikriti3* Addresses of authors: 1 Mountain Vista Medical Center, 1301 S. Crismon Ave, Mesa, AZ 85209, USA; 2 College of Veterinary Medicine, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766-1854, USA; 3 Department of Anatomy and Basic Medical Sciences, College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766-1854, USA

*Correspondence: Tel.: +9094695305; fax: +9094695698; e-mail: [email protected] With 9 figures Received September 2016; accepted for publication March 2017 doi: 10.1111/ahe.12272

Summary The least shrew (Cryptotis parva) is a small vomit-competent insectivorous species which has recently been introduced as an emesis animal model in the laboratory. In this study, the respiratory system of the least shrew was examined and compared with the well-established larger species routinely used in the laboratory. Five least shrews (4–5 g body weight, 45–60 days old) were used. Standard histological procedures were followed for light microscopic examination. The lining epithelium of the trachea was found to be pseudostratified ciliated columnar (PSCC). Three types of cells were easily identified, basal and ciliated as well as few goblet cells interspersed among the ciliated cells and they were not clearly recognizable. A few tracheal seromucous glands were located at the free end of the C-shaped cartilaginous rings. The cartilaginous rings are replaced by smooth muscle cells before the bronchi enter into the lung. The lining epithelium of tracheobronchial tree gradually changes into simple cuboidal epithelium that lacks goblet cells. However, the division of the tracheobronchial tree is similar to other mammalian species. On the other hand, the principal bronchus lacks cartilaginous plaques as it becomes intrapulmonary bronchus. The wall of the bronchi is supported by thick layers of spirally arranged smooth muscles. Two types of cells were readily recognizable: basal and ciliated cells, with rarely observed goblet cells. In addition, the PSCC epithelium changes into simple cuboidal much earlier in the bronchial division relative to other species.

Introduction The least shrew has been used as an experimental model for laboratory investigation because of its small size, short reproductive cycle and other physiological similarities to primates, including its ability to vomit in response to emetogens (Darmani and Ray, 2009). In fact, the least shrew has been shown to be an excellent animal model for toxicological (Mock et al., 2005) and reproductive (Bedford et al., 1997) studies as well as mechanistic emesis studies involving emetics and antiemetic agents (Darmani, 1998; Ray and Darmani, 2007). These studies have led to the need for further examination of the anatomic,

histological and physiological characteristic features in this species (Mock et al., 2001). Previous studies on emesis involving the brainstem and gastrointestinal systems have been carried out in larger vomit-competent species such as dogs, cats and ferrets by numerous investigators (Hawthorn et al., 1988). These studies provide a good foundation to explore the comparative histomorphology of the least shrew in relation to the larger emesis models including humans. Such studies are essential especially when applying basic conclusions derived from the use of shrews to the clinical setting. As emesis involves multiple systems and emetogens used in emesis studies such as cisplatin can also affect other

© 2017 The Authors. Anatomia, Histologia, Embryologia published by Blackwell Verlag GmbH Anat. Histol. Embryol. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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systems, including the respiratory system. Recently, it has been demonstrated that the intratracheal route of administration can reduce cisplatin-evoked toxicity and to increase its concentration and retention within lung tumours (Xie et al., 2010). Therefore, it becomes essential to study and understand the normal histology of the least shrew respiratory system and compare it to those of other species.

Materials and Methods Animals Five least shrews were used in this study. Adult least shrews were bred in the Animal Facility of Western University of Health Sciences. Previous studies had demonstrated no gender differences, so both males and females were used. Shrews were housed in groups of 5–10 on a 14-h:10-h light/dark cycle, fed with food and water ad libitum as described previously (Darmani et al., 1999). All the shrews used were 45–60 days old and weighed between 4 and 5 g. This study was carried out in strict accordance with the recommendations in the guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Department of Health and Human Services Publication, revised, 1985). The protocol was approved by the Western University of Health Sciences IACUC. The feeding and maintenance of shrews are fully described elsewhere (Darmani, 1998; Darmani et al., 1999). The animals were sacrificed following deep anaesthesia via inhalation of isoflurane. The entire lung with the trachea was removed and placed in 8% neutral-buffered formalin solution for 72 h. Tissue samples were taken from the trachea, from principal bronchus and from each lobe of the lung. The specimens were cut into small pieces and immersed in the same fixative for 48 h before further processing. Fixed tissues were washed three times in phosphate buffer (pH 7.4, 0.1 M). Subsequently, the samples were dehydrated in a graded series of ethanol, which were then embedded in paraffin block. Four- to five-micrometrethick sections were collected and mounted on glass slides. Tissue sections were mounted and stained with haematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) stains (Bancroft and Gamble, 2005).

These lobes are separated by deep inter-lobar fissures (Figs. 1 and 2) and are ventilated each separately by lobar bronchi (Fig. 3). The left lung consists of only one large caudal lobe that is fused almost completely with the small cranial lobe leaving no line of demarcation that grossly separates the two lobes (Fig. 1). The trachea of the least shrew is lined by pseudostratified ciliated columnar (PSCC) epithelium (Fig. 4). The cells of the bronchus are of low columnar in contrast to those described in the mouse (Hyde et al., 2009). Two types of cells were readily recognized as basal and ciliated cells. The goblet cells are not globular in shape as in other species and thus cannot be easily distinguished from the ciliated columnar cells without special stain. PAS-stained sections showed goblet cells interspersed among the ciliated cells (Fig. 5). They were oblong in shape and contained few minute granules in their apical portion. In the rhesus monkey (Macaca mulatta), the PSCC epithelium continues into the lining of the tracheobronchial tree to the level of the bronchiole where it still retains its three cellular components (basal, ciliated and goblet cells) (Wilson et al., 1997). In contrast, in the human lung, the epithelium changes because the cilia persist further down than goblet cells to ensure that mucous does not accumulate in the distal regions of the lungs (Wilson et al., 1997). Likewise, similar findings were described in nonprimate mammals such as the opossum (Didelphis virginiana) (Krause and Leeson, 1973) and pigs (Winkler and Cheville, 1984). However, in the least shrew, the PSCC lining epithelium of the tracheobronchial tree ended at the level of the lobar bronchus and continued from there to the terminal bronchioles as low columnar to simple cuboidal type of epithelium. In addition, the cellular elements of the epithelium are drastically reduced in the number of ciliated and goblet cells towards the bronchioles.

Results and Discussion The least shrew shares some characteristic features with the larger primates; however, because of its small size, it is a better animal model to be used as a laboratory animal especially when it comes to emesis. The right lung consists of cranial, middle, caudal and accessory lobes.

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Fig. 1. Gross morphology of the lung lobation of the least shrew.

© 2017 The Authors. Anatomia, Histologia, Embryologia published by Blackwell Verlag GmbH Anat. Histol. Embryol.

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Fig. 2. Gross anatomy and histological montage showing anterior (AnL), middle (ML) and caudal (CdL) lobes of the right lung.

Fig. 3. PAS-stained histological montage of the left lung showing the division of tracheobronchial tree of the least shrew. Bv – blood vessel; IPB – intrapulmonary bronchus. 1, 2, 3 are segmental bronchi. Bar 200 lm.

The submucosa of the trachea contains a thin vascular connective tissue layer that lies immediately underneath the basement membrane (Fig. 4). A few tracheal seromucous glands were mainly concentrated at the free end of the C-shaped cartilaginous rings. Their ducts are lined by simple cuboidal epithelium (Fig. 4). The cartilaginous rings continues down to the principal bronchus (Fig. 6). In contrast, in humans and primates, the cartilage ends at the level of the bronchioles (Maina, 1987; Tyler et al., 1988).


Fig. 4. Histological section of the tracheal wall showing pseudostratified ciliated columnar epithelium (PSCCE), submucosal glands (G) with duct (D) and cartilaginous ring (C). Ciliated cell (CC), basal cell (BC). (H&E stain), bar 20 lm.

Fig. 5. Histological section of the tracheal wall stained with (PAS) showing pseudostratified ciliated columnar epithelium (PSCCE) with goblet cells (GC). (PAS stain), bar 10 lm.

The lobar bronchi branch monopodially as they enter the lung, where the branches are few and of small diameters (Fig. 3). Each lobar bronchus was recognized by its thick wall lined with pseudostratified columnar epithelium with few cilia and no goblet cells (Fig. 7). The wall of the intrapulmonary bronchus is surrounded by several layers of smooth muscles that replace the cartilaginous plates (Fig 6). The intrapulmonary bronchus further divides into few segmental bronchi before it ends into a few terminal bronchioles (Fig. 3 and 8). The terminal bronchioles eventually give rise to respiratory bronchiole before continuing to become the alveolar duct (Fig. 9). Many laboratory mammals lack or have


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small respiratory bronchioles while carnivores possess extensive respiratory bronchioles (Tyler et al., 1988). The least shrew may not have respiratory bronchioles or have a short one, which can be overlooked due to sectioning orientation in a similar fashion to most mammalian species (Tyler and Julian, 1992; in humans; Plopper and Hyde, 1992; Hyde et al., 2009 in the mouse). The lining epithelium of the bronchioles gradually changes from PSC into simple cuboidal epithelium with dome-like apical surface (Fig. 9). Although the division of the tracheobronchial tree in the least shrew is similar in principle to other mammalian species (Hyde et al., 2009), a few remarkable variations have

been found. Among these variations, the lobar bronchus lacks cartilaginous plaques even before it becomes intrapulmonary. Therefore, instead of the cartilaginous plaques, the wall of the bronchi is supported by thick layers of smooth muscles. These changes within the respiratory airways of the least shrew facilitate breathing through control of the distribution of the inspired air by the action of the smooth muscle due to the lack of the hyaline cartilages much earlier in the bronchial tree than in other animal species. This will also contribute to the least shrew small weight. The tracheobronchial tree division is much less significant compared to other animal species (Hyde et al., 2009). The respiratory bronchioles may or may not be present and

Fig. 6. Histological section of the lung showing principal (PB) and lobar bronchus (LB), cartilaginous plate (C), smooth muscle (SM). (PAS stain), bar 200 lm.

Fig. 8. PAS-stained histological section of the lung, showing segmental bronchus (SB) divided into two terminal bronchioles (TB); alveolar duct (AD). (H&E) stain, bar 100 lm.

Fig. 7. High magnification of the lobar bronchus lined by pseudostratified columnar epithelium resting on PAS-positive basement membrane (BM). PAS stain), bar 10 lm.

Fig. 9. A PAS-stained histological section of the lung, showing terminal bronchiole (TB) opens into respiratory bronchiole (RB) that continues into the alveolar duct (AD). (PAS stain), bar 50 lm.

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therefore is similar to the mouse (Hyde et al., 2009). Furthermore, the PSCC epithelium changes into simple cuboidal much earlier in the bronchial division relative to other species. Two types of cells were readily recognized: basal and ciliated cells; however, the third type, goblet cells, was observed when stained with PAS. Ciliated and goblet cells are reduced in number gradually distal to the level of secondary bronchus. In conclusion, the least shrew has unique histological features which should be further investigated at the ultrastructural level for functional aspects of the observed cellular and structural changes. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgements This research was supported by start-up fund from the College of Osteopathic Medicine of the Pacific, Western University of Health Sciences. The authors thank Ms. Seetha Chebolu for her technical assistance. References Bancroft, J. D., and M. Gamble, 2005: Theory and Practice of Histological Techniques. Philadelphia, PA: Elsevier Limited Churchill Livingstone. Bedford, J. M., O. B. Mock, and D. M. Phillips, 1997: The unusual site of sperm storage and behavior of the cumulus oophorus in the oviduct of the least shrew, Cryptotis parva. Biol. Reprod. 56, 1255–1267. Darmani, N. A., 1998: Serotonin 5 HT3 receptor antagonists prevent cisplatin-induced emesis in cryptotis parva: a new experimental model of emesis. J. Neural Trans. 105, 1143– 1154. Darmani, N. A., W. Zhao, and B. Ahmad, 1999: The role of D2 and D3 Dopamine receptors in the mediation of emesis in Cryptotis parva (the Least Shew). J. Neural. Transm. 106, 1045–1061. Darmani, N. A., and A. P. Ray, 2009: Evidence for a re-evaluation of the neurochemical and anatomical bases of chemotherapy-induced vomiting. Chem. Rev. 109, 3158– 3199. Hawthorn, J., K. J. Ostler, and P. L. Andrew, 1988: The role of the abdominal visceral innervation and 5-hydroxytryptamine M-receptors in vomiting induced by the cytotoxic drugs cyclophosphamide and cisplatin in the ferret. Q. J. Exp. Physiol. 73, 7–21.


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