SEARCH

SEARCH BY CITATION

Keywords:

  • soft palate;
  • brachycephalic dog;
  • histology;
  • brachycephalic airway obstructive syndrome (BAOS)

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

In brachycephalic dogs, the skull bone shortening is not paralleled by a decreased development of soft tissues. Relatively longer soft palate is one of the main factors contributing to pharyngeal narrowing during normal respiratory activity of these dog breeds, which are frequent carriers of the brachycephalic airway obstructive syndrome (BAOS), which affects most part of them during their postnatal life. No histological studies assessing the morphology and the normal tissue composition of the soft palate in brachycephalic dogs are available, neither has ever been determined whether the elongated soft palate is a primary or secondary event. Aim of this study was to describe the morphology of the caudal soft palate in brachycephalic dogs with Grade I BAOS to identify potential features possibly favoring the pathogenesis of BAOS. Specimens from brachycephalic dogs (N = 11) that underwent preventive surgery were collected from surgery, processed for histology, and examined at six transversal levels. The brachycephalic soft palates showed peculiar features such as thickened superficial epithelium, extensive oedema of the connective tissue, and mucous gland hyperplasia. Several muscular alterations were evidenced in addition. The results of this investigation add to the general knowledge of the anatomy of soft palate in the canine species and establish baseline information on the morphological basis of the soft palate thickening in brachycephalic dogs. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

Size variation in the domestic dog is extreme and surpasses that of all other living and extinct species in the dog family, Canidae (Wayne, 1986). The dog's skull comes in many shapes and sizes than any other mammal (Evans, 1993), so that the canine skull is classified into long and narrow (dolichocephalic), short and wide (brachycephalic), or of medium proportions (mesaticephalic; Evans, 1993). Morphology and morphometry of the skull are much studied as they bring forth important consequences in the canine species, especially regarding the breathing activity in the different breeds (Craven et al., 2007).

In brachycephalic dogs, the short skull base structure involves the reduction of pharyngeal length (Stockard, 1941). Moreover, the skull bone shortening is not paralleled by a decreased development of soft tissues, leading to a relatively longer soft palate (Harvey, 1989). The increased length of soft palate is one of the main factors contributing to pharyngeal narrowing during normal respiratory activity in brachycephalic dogs, so that the resection of the caudal part of the soft palate (palatoplasty) represents the conventional surgical technique to prevent and/or resolve the main clinical problems related to this condition (Farquharson and Smith, 1942; Harvey, 1982; Wykes, 1991; Koch et al., 2003; Brdecka et al., 2007; Riecks et al., 2007). Recently, a novel surgical approach characterized by palatoplasty has introduced the reduction in both the length and thickness of soft palate in brachycephalic dogs (Dupré et al., 2005).

Actually, brachycephalic dogs are frequent, if not obligated, carriers of the brachycephalic airway obstructive syndrome (BAOS), which affects the most part of them during their postnatal life. This syndrome is characterized by various severe combinations of stenotic nares, long soft palate, everted laryngeal saccules, and laryngeal collapse. The main clinical signs associated with BAOS are stertor, snoring, stridor, exercise intolerance, tachypnea, inspiratory efforts, cyanosis, and in severe cases, loss of consciousness due to temporary inhability to breath in properly.

The thick, redundant, and hyperplastic appearance of soft palate in brachycephalic breeds has been macroscopically and clinically described (Aron and Crowe, 1985; Hendricks et al., 1987; Wykes, 1991). Although histology of the soft palate in the average dog's head has been generically described (Evans, 1993), and was analytically reported by us in the companion article dealing with the mesaticephalic dog breeds (Arrighi et al., 2011), no microanatomical studies on the brachycephalic dog soft palate seem to be available.

In the current study, we have investigated the microscopic anatomy of the distal soft palate in adult brachycephalic dogs with Grade I BAOS, to evaluate the thick, redundant, and hyperplastic soft palate, which has been typically observed in these breeds and can be involved in the BAOS development. The morphological characteristics of the dog soft palate in adult brachycephalic breeds add to the information given in the companion article (Arrighi et al., 2011) dealing with the caudal soft palate morphology in mesaticephalic dogs.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Palatoplasty was performed in 11 brachycephalic dogs at the Section of Surgery, Department of Clinical Veterinary Sciences of the University of Milan (Italy). The dog's age ranged from 8 months to 4 years and the breeds were Pug dogs (N = 5), English Bulldog (N = 3), and French Bulldogs (N = 3; mean age = 21 months, median = 17 months). Each dog underwent clinical examination, lateral pharyngeal radiographs, and pharyngoscopy. Dogs had no or mild clinical signs of BAOS, so they were clinically classified as Grade I according to Poncet et al. ( 2006).

All the specimens consisted of the distal part of the soft palate, resected along a curved line crossing transversally from the caudal border of the fossa tonsillaris to the same point on the opposite side (Fig. 1), as previously described (Hobson, 1995).

thumbnail image

Figure 1. Palatoplasty surgery. The resection line is indicated in a, starting from the caudal ridge of the tonsillar fossa.

Download figure to PowerPoint

After surgical resection, the soft palate specimens were distended, pinned out on a thin cardboard, fixed, processed, and stained as described by Arrighi et al., 2011.

One soft palate from an English Bulldog was subdivided into transverse pieces, snap-frozen in liquid nitrogen-cooled isopentane, and kept at −80°C. To assess the presence and distribution of adipose tissue, progressive cryostatic sections (4–6 μm thick) were obtained, put onto glass slides, dried at 37°C for 3min, then incubated for 30 min at room temperature with Sudan black B (saturated solution in 70% alcohol).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The macroscopical aspect of the soft palate collected from brachycephalic dogs diagnosed as Grade I BAOS was thick and irregularly shaped. During specimen collection it was difficult to orientate the samples properly, due to marked sliding between tissues. In particular, the mucosal layer tended to slip over the musculo-connective axis of the organ due to a particular imbibition of the connective tissues.

Several features characterized the brachycephalic soft palate microscopically, major of which being: hyperplasia of the superficial epithelia, extensive swell of interfibrillar matrix and edema of the lamina propria, mucous gland hyperplasia, and structurally degenerated muscle fibres. From the examination of the data concerning frequency and severity of these features no correlations can be evidenced with age and/or breed of the subjects.

The mucosal epithelial lining was thickened due to moderate to severe mucosal hyperplasia. Additionally, keratinocyte swelling with intracellular oedema were particularly evident multifocally at the oral side (Fig. 2). A mild mucosal hyperplasia was also evident on the nasopharyngeal side but with no obvious signs of cell swelling.

thumbnail image

Figure 2. Morphology of the oral mucosa. H and E. (a) A high number of cell layers builds up a thickened pluristratified squamous epithelium, accompanied by an underlying loose connective tissue. (b) Intracellular edema can be seen in the oral epithelium. Scale bars: a = 100 μm and b = 20 μm.

Download figure to PowerPoint

In brachycephalic dogs, collagen fibers of the lamina propria were irregularly disposed and distanced because of edema, nevertheless, a sub-epithelial dense connective tissue with thin fibres and an underlying thicker connective were evident (Fig. 2).

Edema also affected the glandular stroma and the perimisium between the bundles of muscular fibers, at the level the musculo-connective axis of the organ. In the more aboral sections, the edematous connective tissue was the only component of the soft palate, because axial tissue had disappeared orally.

The palatine aponeurosis was observed only in three brachycephalic dogs and only in the first third of the soft palate.

Glandular hyperplasia was present either at the nasopharyngeal (Fig. 3a) and oral (Fig. 3b) side. Palatine glands of the nasopharyngeal side were mostly composed by serous acini or prevalently serous mixed acini. Glands of the oral side were mostly composed by mucous acini. In the glandular lobules of both sides, mucous acini were composed of large mucous cells, containing abundant intracytoplasmic secretory material and with apical margin loss (Fig. 3a,b). These cells had a mixed Alcian and PAS positivity, with prevalent Alcian positivity indicating a production of acidic glycoconjugates. Within the acidic Alcian-positive glycoconjugates, only the sulphated component stained black-brownish with HID/AB reaction, both in mucous cells and ducts. Serous cells showed PAS positivity with AB/PAS staining. Glandular ducts were often moderately to severely dilated in the oral side (Fig. 3b), the secretory material was mostly Alcian-positive with a small amount of PAS-positive glycoconjugates. The gland lumen frequently contained exfoliated ductal epithelial cells. Ductal epithelium was often flattened and frequently pluristratified in specimens from brachycephalic dogs. In four brachycephalic dogs, a focal mucous spilling was found associated with areas of severe lumen dilation and flattening of mucosal gland epithelium but with no sign of inflammation was present.

thumbnail image

Figure 3. (a and b) Palatine glands. H and E. (a) Degeneration of mucous component can be seen in the hyperplasic glands at the nasal side. (b) Salivary gland hyperplasia, duct dilation, and mucus stasis mixed with exfoliated epithelial cells can be seen in the oral glands. (ch) Palatine musculature. H and E. (c) Area of muscular fiber disproportion can be seen, with groups of severe hypotrophy (arrows) associated with compensatory hypertrophy (arrowheads) and severe fibrosis. (d) Segmental hypercontraction (arrow) and floccular degeneration, in which fibres start to fragment (arrowhead). (e) Angular and twisted fibres can be seen, longitudinally sectioned. Darker stained and larger diameter fibres can also be seen in transverse section during hyaline degeneration. (f) Hyaline degeneration with fibre atrophy. (g) Increased number of activated satellite cells and internal nuclei can be seen, suggesting a regenerative process. (h) Pale cytoplasm and loss of striations can be seen in muscle hyaline degeneration, together with regenerative attempts (arrow). Scale bars: a = 200 μm; b = 100 μm; c = 200 μm; d = 50 μm; e = 100 μm; f = 100 μm; g and h = 50 μm.

Download figure to PowerPoint

Several histopathological features characterized the soft palate muscles of the brachycephalic dogs (Fig. 3c–h). Severe muscular fibre size variation was observed. Angular, atrophic fibers alternating with normal and hypertrophic ones (Fig. 3c) were frequent. Intensely eosinophylic and homogenous fibres (hyaline degeneration) and hyaline fragmented fibres (Zenker necrosis) were also common (Fig. 3d–h). Segmental hypercontraction was randomly observed (Fig. 3e). Multinucleated giant cell fibres were also observed, possibly representing attempts to regenerate. Activation of satellite cells with displacement of the nuclei from the periphery to the centre of the cells was also a significant finding (Fig. 3g). These aspects were often associated with severe endomysial and perimysial fibrosis. In brachycephalic dogs, the palatine muscles could be occasionally identified but the severe muscular disorganization together with the oedema made the recognition impossible for the other pairs of muscles.

No lobules of adipose tissue were evidenced in any section of the brachycephalic soft palate. This observation was confirmed by the Sudan black stained specimen.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Soft palate abnormalities have been pointed out as fundamental components of BAOS, however, the anatomical bases of soft palate thickening, which is considered as one of the factors implicated in nasopharyngeal obstruction, have not been investigated on a microanatomical basis in brachycephalic dogs (Aron and Crowe, 1985; Hendricks et al., 1987; Harvey, 1989; Hendricks, 1992; Dupré et al., 2005). According to our study, conspicuous microscopic features characterized most tissue components of brachycephalic soft palate that account for the enlarged width and thickness of the organ. The most important microscopic aspects were hyperplasia and intracellular edema of mucosal lining, diffuse oedema, and amplified myxoid matrix in the lamina propria. These changes surprisingly affected all dogs in brachycephalic group classified as Grade I BAOS patients. Similar microscopical findings characterize the soft palate in human patients with obstructive sleep apnea syndrome (OSAS) (Woodson et al., 1991; Hamans et al., 2000; Berger et al., 2002).

Structural changes were observed in the soft palate mucosa of human patients with snoring and obstructive sleep apnea (OSA) and interpreted as a consequence of the trauma of snoring (Paulsen et al., 2002). Inspiratory effort occur in brachycephalic dogs since the beginning of their postnatal life, even if no or mild clinical signs are present (Hendricks et al., 1987). Features like acanthosis and reduction of connective tissue papillae have been invoked as possible contributory factor to upper airway collapsibility in human patients with OSA (Paulsen et al., 2002). In the specimens of brachycephalic dog soft palate we observed epithelial hyperplasia together with keratinocyte swelling and intracellular oedema. Moreover, accumulation of intercellular fluid and myxoid matrix were noticed at the level of mucosal lamina propria, which might collaborate to the reduction of soft tissue cohesiveness. Significantly thicker covering epithelium and increased amount of intercellular space considered as expression of edema was described also in OSA patients and snorers (Hamans et al., 2000). Mechanical trauma can produce changes in epithelial surfaces under the influence of unphysiological mechanical stress, resulting in different modifications of the overlying epithelium or underlying connective tissue (Paulsen et al., 2002). These findings could explain both the enlarged thickness of the soft palate in brachycephalic dogs and the marked sliding between tissues we found during sample collection, that made orientation more difficult to obtain compared with mesaticephalic dogs.

An additional contributor to thickening of the soft palate was palatine gland hyperplasia together with luminal mucin accumulation. The function of palatine glands is to ensure constant humidity and lubrication of the palatine mucosa, to prevent friction damage during food intake (Kuehn and Moon, 2005). Salivary glands have been demonstrated to change their metabolic state according with tissue needs and diet content of fibers, becoming either hyperplastic or atrophic (Hall and Schneyer, 1977). Since palatine gland hyperplasia was present in all the brachycephalic dogs considered in this study, diet factors are less likely responsible for this feature. Vibrations and friction of soft palate during inspiratory efforts and snoring could represent valid causes for palatine gland hyperplasia to occur as defensive mechanism (Woodson et al., 1991).

An additional finding was the dilation of acini and ducts that were filled by mucin rich in acidic Alcian-positive glycoconjugates containing only sulphated components. The higher content in sulphated glycoconjugates is known to make the mucous material thicker. Although no signs of ductal obstruction were observed, epithelial thickening, acinar hyperplasia, and production of mucins having increased viscosity were likely responsible of reduced mucus flow and mucin stasis. In this study, four cases of mucin spilling in soft tissues were evidenced in brachycephalic dogs. This feature was only occasionally seen in mesaticephalic dogs (Arrighi et al., 2011). However, this finding was not interpreted as intravital mucocele since the spilling was not associated with inflammation or reactive tissue changes and was hypothesized to be a traumatic consequence of sample collection during surgery.

The anatomy of the palatine musculature of brachycephalic dogs enrolled in this study suffered from extensive degenerative lesions involving the majority of muscular fibers. All these findings were consistent with a chronic and persistent traumatic insult to the palatine muscles, and contrasted with the extremely rare and focal areas of muscular degeneration present in mesaticephalic dogs (Arrighi et al., 2011). Experimental observations confirm that an abnormal rise in muscular activity may damage muscular fibers in human (Dennett and Fray, 1988), particularly so in case of prolonged contractions in persistently activated muscles (McCully and Faulkner, 1985; Stauber et al., 1988; Faulkner et al., 1989; Lieber et al., 1991). In brachycephalic dogs with BAOS negative pressure is abnormally increased during inspiratory efforts, and pulls soft tissues towards the glottis. Therefore, in brachycephalic dogs with BAOS snoring and stertor, together with the increased negative pressure, could promote an abnormal muscular activity in activated soft palate muscles (Van der Touw et al., 1994). Even if we enrolled in this study dogs classified as Grade I BAOS, it is likely that inspiratory effort also characterize those subjects with no or mild clinical signs. As no differences in severity of lesions were found between younger versus older dogs, it is likely that negative pressure works in these breeds since the early phase of their life.

The major pitfall of this study is that only tissues obtained during palatoplasty were examined. However, there is evidence that also the cranial portion of the soft palate may be implicated in airway obstruction in brachycephalic dogs (Hendricks et al., 1987). An additional drawback could be that in this study we enrolled only brachycephalic dogs diagnosed Grade I BAOS. On the other hand, it is known that it is difficult to distinguish among healthy and diseased brachycephalic dogs. Nowadays, an increasing number of brachycephalic dogs that were considered “normal” once experience surgical treatment as preventive therapy to decrease the possibility for BAOS to occur or weaken the secondary changes, such as laryngeal collapse in initial forms of BAOS. In our study, we enrolled only brachycephalic dogs submitted for preventive surgery, in which clinical signs and secondary changes were mild or not present yet, thus considering brachycephalic dogs undergoing preventive surgery as representative of a “normal” group of brachycephalic dogs.

In conclusion, by description of microscopic lesions, this study validates the gross findings of a “long, thick, redundant and/or, hyperplastic” soft palate, which was clinically reported in brachycephalic dogs affected by BAOS (Hendricks et al., 1987; Wykes, 1991; Hendricks, 1992; Dupré et al., 2005). According to our findings, brachycephalic soft palate is characterized by variable microscopical features that contribute to the increased thickness since the earliest Grade of the respiratory syndrome, which affects most part of the individuals belonging to these breeds during their postnatal life. The progressive volume increase of the soft palate, and changes in consistency and elastic resistance, together with muscular injury might be involved in functional impairment during the respiratory cycle. However, it was not possible from this study to establish whether part of the increased thickness of soft palate is due to an inherited defect. Future studies on the microanatomy of soft palate in newborn brachycephalic dogs would be useful to better state this point.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Mr. Paolo Stortini (VSA, Università degli Studi di Milano) is gratefully acknowledged for his experienced and skilful technical support. Excellent technical assistance in image editing made by Mr. Marco M. Colombo (digital creative and webmaster, Università degli Studi di Milano) is specially acknowledged. Part of this work was presented at the XXVIII Congress of the European Association of Veterinary Anatomists (EAVA), Paris, France, July 28–31, 2010 (Proceedings in Anat. Histol. Embryol., 39/4, 263-264, 2010).

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  • Aron DN, Crowe DT. 1985. Upper airway obstruction. Vet Clin North Am Small Anim Pract 15: 902904.
  • Arrighi S, Pichetto M, Roccabianca P, Romussi S. The anatomy of the dog soft palate. I. Histological evaluation of the caudal soft palate in mesaticephalic breeds. Anat Rec 294: 12611266.
  • Berger G, Gilbey P, Hammel I, Ophir D. 2002. Histopathology of the uvula and the soft palate in patients with mild, moderate, and severe obstructive sleep apnea. Laryngoscope 112: 357363.
  • Brdecka D, Rawlings C, Howerth E, Cornell K, Stiffle K. 2007. A histopathological comparison of two techniques for soft palate resection in normal dogs. J Am Anim Hosp Assoc 43: 3944.
  • Craven BA, Neuberger T, Paterson EG, Webb AG, Josephson EM, Morrison EE, Settles GS. 2007. Reconstruction and morphometric analysis of the nasal airway of the dog (Canis familiaris) and implications regarding olfactory airflow. Anat Rec 290: 13251340.
  • Dennett X, Fray HJ. 1988. Overuse syndrome: a muscle biopsy study. Lancet 2: 903908.
  • Dupré G, Findji L, Poncet C. 2005. The folded flap palatoplasty: a new technique for treatment of elongated soft palate in dogs. In: Proceedings of the 14th Annual Scientific Meeting of European College of Veterinary Surgeons, Marcy L'Etoile, France, E 19, p 3.
  • Evans HE. 1993. Miller's anatomy of the dog. 3rd ed. Philadelphia: Saunders.
  • Farquharson J, Smith KW. 1942. Resection of the soft palate in the dog. J Am Vet Med Assoc 100: 427430.
  • Faulkner JA, Jones DA, Round JM. 1989. Injury to skeletal muscles of mice by forced lengthening during contractions. Q J Exp Physiol 74: 661670.
  • Hall HD, Schneyer CA. 1977. Functional mediation of compensatory enlargement of the parotid gland. Cell Tissue Res 184: 249254.
  • Hamans EPPM, Van Marck EA, De Backer WA, Creten W, Van de Heyning PH. 2000. Morphometric analysis of the uvula in patients with sleep-related breathing disorders. Eur Arch Otorhinolaryngol 257: 232236.
  • Harvey CE. 1982. Upper airway obstruction surgery. 2. Soft palate resection in Brachycephalic dogs. J Am Anim Hosp Assoc 18: 538544.
  • Harvey CE. 1989. Inherited and congenital airway conditions. J Small Anim Pract 30: 184187.
  • Hendricks JC. 1992. Brachycephalic airway syndrome. Vet Clin North Am Small Anim Pract 22: 11451153.
  • Hendricks JC, Kline LR, Kovalski RJ, O'Brien JA, Morrison AR, Pack AI. 1987. The English Bulldog: a natural model of sleep-disordered breathing. J Appl Physiol 63: 13441350.
  • Hobson HP. 1995. Brachycephalic syndrome. Seminars in Veterinary Medicine. Small Anim Surg 10: 109114.
  • Koch DA, Arnold S, Huber M, Mantovam PM. 2003. Brachycephalic syndrome in dogs. Compend Contin Educ Pract Vet 25: 4855.
  • Kuehn DP, Moon JB. 2005. Histologic study of intravelar structures in normal human adult specimens. Cleft Palate Craniofac J 42: 481489.
  • Lieber RL, Woodburn TM, Friden J. 1991. Muscle damage induced by eccentric contractions of 25% strain. J Appl Physiol 77: 24982507.
  • McCully KK, Faulkner JA. 1985. Injury to skeletal muscle fibers of mice following lengthening contractions. J Appl Physiol 59: 119126.
  • Paulsen FP, Steven P, Tsokos M, Jungmann K, Müller A, Verse T, Pirsig W. 2002. Upper airway epithelial structural changes in obstructive sleep-disordered breathing. Am J Respir Crit Care Med 166: 501509.
  • Poncet CM, Dupré GP, Freiche VG, Bouvy BM. 2006. Long-term results of upper respiratory syndrome surgery and gastrointestinal tract medical treatment in 51 brachycephalic dogs. J Small Anim Pract 47: 137142.
  • Riecks TW, Birchard SJ, Stephens JA. 2007. Surgical correction of brachycephalic syndrome in dogs: 62 cases (1991–2004). J Am Vet Med Assoc 230: 13241328.
  • Stauber WT, Fritz WH, Vogelbach DW, Dahlmann B. 1988. Characterization of muscles injured by forced lengthening. I. Cellular infiltrates. Med Sci Sports Exerc 20: 345353.
  • Stockard CR. 1941. The genetic and endocrinic basis for differences in form and behaviour: as elucidated by studies of contrasted pure-line dog breeds and their hybrids. The American Anatomical Memoirs, 19. Philadelphia: The Wistar Institute of Anatomy and Biology.
  • Van der Touw T, O'Neill N, Brancatisano A, Amis T, Whetley J, Engel LA. 1994. Respiratory-related activity of soft palate muscles: augumentation by negative upper airway pressure. J Appl Physiol 76: 424432.
  • Wayne RK. 1986. Cranial morphology of domestic and wild canids—the influence of development on morphological change. Evolution 40: 243261.
  • Woodson BT, Garancis JC, Toohill RJ. 1991. Histopathologic changes in snoring and obstructive sleep apnea syndrome. Laryngoscope 101: 13181322.
  • Wykes PM. 1991. Brachycephalic airway obstructive syndrome. Probl Vet Med 3: 188197.