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Comparative histochemistry of posterior lingual salivary glands of mouse
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Comparative histochemistry of posterior lingual salivary glands of mouse Asterios Triantafyllou ∗ , David Fletcher Oral & Maxillofacial Pathology, School of Dentistry, University of Liverpool, Liverpool L3 5PS, United Kingdom
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Article history: Received 5 August 2016 Accepted 15 November 2016 Available online xxx Keywords: Enzymes Histochemistry Mouse Mucosubstances Nerves Salivary glands
a b s t r a c t Normal posterior deep and superficial salivary glands of tongue were examined in male mice by means of light microscopical histochemistry and neurohistology. Both glands showed acini and simple ducts. Demilunes were present in the superficial gland. Disulphides and neutral mucosubstances occurred in acini and demilunes. Tryptophan staining was seen in acini of the deep gland and demilunes, whereas acid mucosubstances were exclusively localised in the superficial gland. Dehydrogenase activities were widespread. Strong esterase activity occurred throughout the parenchyma of the deep gland and in demilunes; it was variably inhibited by E600, apart from acinar apical regions in the deep gland. Lipase was confined to acini of the deep gland and demilunes. Acid phosphatase staining was similarly localised; it was also seen in periluminal ductal rims of the deep gland, in which ouabain-sensitive Na,K-ATPase was localised basolaterally. Staining for alkaline phosphatase decorated occasional myoepithelial-like arrangements and interstitial capillaries. Acetylcholinesterase was associated with nerve fibres embracing glandular parenchyma. Adrenergic fibres were not seen. The results suggest that the acini of the posterior deep lingual gland secrete neutral glycoproteins, whereas the ducts transport ions and absorb luminal material. The posterior superficial lingual gland mainly secretes acid glycoproteins. Both glands produce lingual lipase, receive cholinergic-type innervation and have inconspicuous myoepithelium. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction The deep and superficial, minor salivary glands in the posterior part of the tongue, which were identified by von Ebner and Weber respectively, are of significance in digestion, taste and protection of taste buds (Nagato et al., 1997). They have been variously investigated in small carnivores and bats (Poddar and Jacob, 1980; Tandler et al., 1997; Triantafyllou et al., 1999); and intensely in man and rat (see: Hand et al., 1999; Redman, 2012). Less attention has been paid to the posterior lingual glands of mouse (Mus musculus), which are useful for studying glandular sexual dimorphism (Hanker et al., 1980) and harbouring amastigotes in experimental American trypanosomiasis (Lopes et al., 1991a, 1991b). Burstone (1953) applied early histochemical techniques to localise mucosubstances and nucleic acids in those glands; and the use of autoradiography, various sequences of basic dyes and vic-glycol methods in conjunction with pre-digestion and blocking of reactive moieties, and lectins enabled a more refined characterisation of carbohydrate residues therein (Spicer and Duvenci, 1964; Stoward et al., 1980; Schulte and
∗ Corresponding author. E-mail address: [email protected] (A. Triantafyllou).
Spicer, 1983). A more detailed histochemical profiling is, however, desirable. This prompted the present investigation wherein posterior lingual glands of male mouse were examined with the use of histological, protein, mucosubstance and enzyme histochemical, and neurohistological techniques, which have been found valuable while exploring minor salivary glands in other species (Harrison, 1974; Triantafyllou et al., 2001).
2. Materials and methods 2.1. Animals, glands and preservation Five mature male CD1 mice that had been fasted overnight and killed by neck dislocation (schedule 1) as part of an in vitro investigation (Smith et al., 2000), became available. Surplus tissues were donated for additional investigations to ensure best use of the material. Ethical committee approval was not required. The tongues of the mice were rapidly removed and cut into transverse, frontal slices. Posteriorly situated slices including the vallate papilla were further bisected. Halves were immediately quenched in isopentane cooled by solid carbon dioxide and then
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stored at −70 ◦ C. Other halves were immersion fixed in a formaldehyde solution and then processed for paraffin blocks.
2.2. Histology Sections of fixed tissue were cut at a thickness of 5 mm and were stained with haematoxylin and eosin.
2.3. Protein and amino acid histochemistry Sections of fixed tissue were cut at a thickness of 5 mm and were stained with the p-dimethylbenzaldehyde nitrite reaction for tryptophan (Adams, 1957) and the coupled maleimide reaction for thiol groups (staining blue) and disulphide groups (staining red) (Sippel, 1978).
2.4. Mucosubstance histochemistry Sections of fixed tissue were cut at a thickness of 5 mm and were stained with alcian blue at pH 2.5 followed by periodic acid-Schiff to demonstrate neutral mucosubstances staining red (periodate-reactive) and acid mucosubstances staining in varying shades of purple and royal blue (periodate-reactive and variably alcianophilic) (Mowry, 1956; Spicer et al., 1967). The high-iron diamine technique followed by alcian blue at pH 2.5 was used to demonstrate sulphated mucosubstances staining brown/black (high-iron diamine-reactive) and non-sulphated carboxylated mucosubstances staining sky blue (alcianophilic) (Spicer, 1965).
2.5. Enzyme histochemistry Sections of quenched pieces were cut at a thickness of about 10–12 mm on a cryostat and were incubated with substrates for the following oxidative enzymes: peroxidase with and without KCN to inhibit peroxidatic responses of respiratory enzymes (Graham and Karnovsky, 1966; Garret and Kidd, 1976), cytochrome oxidase (Kugler et al., 1988), succinate dehydrogenase (Nachlas et al., 1957), lactate dehydrogenase (Bancoft and Hand, 1987), and NADH and NAD(P)H dehydrogenases (Scarpelli et al., 1958). Cryostat sections were also incubated with substrates for the following hydrolytic enzymes: thiamine pyrophosphatase (Novikoff and Goldfischer, 1961), alkaline phosphatase (Stutte, 1967), Na,KATPase with and without ouabain to inhibit activity (Mayahara and Ogawa, 1988), acid phosphatase (Barka and Anderson, 1962), -glucoronidase (Hayashi et al., 1964), non-specific esterase with and without E600 inhibitor (Davis, 1959; Bancoft and Hand, 1987), and lipase with and without sodium taurocholate activator (Triantafyllou et al., 2002).
Fig. 1. Paraffin sections stained with haematoxylin and eosin. (a) Deep (von Ebner) gland; collections of acini (arrowhead) surround ducts (arrow) and are between skeletal muscle fibres (M). (b) Superficial (Weber) gland; tubulo-acini (asterisk), demilunes (arrowhead) and dilated duct (D) draining from two tubules. Objective magnification ×20.
3. Results 3.1. Histology The parenchyma of both glands was arranged in nonencapsulated lobular clusters interspersed between bundles of skeletal muscle (Fig. 1). The deep (von Ebner) gland showed acini and few intralobular ducts. The acini showed inconspicuous lumina and were composed of usually pyramidal cells with variably distinct borders, a prominent apical region replete with strongly eosinophilic granules and a basally aligned rounded vesicular nucleus with dispersed chromatin and inconspicuous nucleoli. The ducts were lined by simple small cuboidal cells with scanty, agranular, eosinophilic cytoplasm and central nuclei with dispersed chromatin (Fig. 1a). The superficial (Weber) gland showed acini and tubulo-acini opening into occasionally dilated, intralobular ducts lined by flattened epithelium. The acini and tubulo-acini were of a mixed appearance showing palely stained central cells with basal flattened nuclei, which were often capped by small, more densely stained demilunes (Fig. 1b). 3.2. Protein and amino acid histochemistry Variably intense, disulphide staining was localised in the apical region of acini of the deep gland, and in central cells and demilunes of the superficial gland; the remainder of the parenchyma showed moderate diffuse thiol staining (Fig. 2a and b). Moderate tryptophan staining was confined to acini and periluminal ductal rims of the deep gland, and demilunes of the superficial gland (Fig. 3a and b). 3.3. Mucosubstance histochemistry
2.6. Neurohistology Sections of quenched pieces were cut at a thickness of about 10–12 mm on a cryostat and were incubated for cholinesterases with and without eserine to inhibit activity (Karnovsky and Roots, 1964). In addition, cryostat sections of 30 mm thickness were treated by the glyoxylic acid method for catecholamines and were examined by fluorescence microscopy (de la Torre and Surgeon, 1976).
Neutral mucosubstances were present in both glands. Acid mucosubstances were localised in the superficial gland only (Figs. 4 and 5). The demilunar cells showed considerable variation in the staining with the high-iron diamine–alcian blue technique (Fig. 5). A few cells had a strong affinity for alcian blue alone, whereas others had a varying affinity for high-iron diamine. In some cells the affinities coexisted. The central cells were constantly stained by periodic acid–Schiff, and showed variable alcianophilia and moderate affin-
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Fig. 5. Paraffin section stained with the high-iron diamine-alcian blue procedure. Acini of the superficial gland are variously stained. Objective magnification ×20. Fig. 2. Paraffin sections stained with the coupled maleimide procedure. Moderate, weak and strong disulphide staining is seen in the acini (asterisks) of the deep gland (a) and central cells (arrow) and demilunes (arrowhead) of the superficial gland (b), respectively. Thiol groups are in ductal cells (D). Objective magnification ×40.
Fig. 6. Cryostat sections incubated in NADH dehydrogenase medium for 30 min at 37 ◦ C. (a) Reaction product is concentrated in ducts (D) of the deep gland where embraces unstained nuclei; lesser amounts of reaction product are in acini (asterisk); skeletal muscle fibres (M) are strongly stained. Objective magnification ×40. (b) Reaction product is concentrated in ductal lining (D) and demilunes (arrowhead) of the superficial gland; central cells (asterisk) appear weakly stained. Objective magnification ×20. Fig. 3. Paraffin sections stained with the p-dimethylbenzaldehyde-nitrite procedure. Staining is seen in acini of the deep gland (a) and demilunes (arrowheads) of the superficial gland (b). A duct with stained periluminal rim is arrowed. Central cells (asterisks) are unstained. Objective magnification ×40.
ity for high iron diamine (Figs. 4b and 5). Periluminal rims of ducts were strongly stained by periodic acid–Schiff (Fig. 4a). 3.4. Enzyme histochemistry
Fig. 4. Paraffin sections stained with the alcian blue-periodic acid-Schiff procedure. (a) Deep gland; (b) Superficial gland. Periodate reactive mucosubstances are in acinar cells of both glands and in periluminal rims (arrow) of ducts. Alcianophilic mucosubstances are confined to the superficial gland. Objective magnification ×40.
The parenchyma of both glands showed widespread, though variable, dehydrogenase and cytochrome oxidase activity (Fig. 6), and little or no peroxidase staining. The plasmalemma of acinar and ductal cells showed thiamine pyrophosphatase activity; cytoplasmic Golgi-like staining was inconspicuous (Fig. 7). Strong to intense diffuse esterase activity occurred in the parenchyma and ductal lumina of the deep gland, and demilunes (Fig. 8a and c); E600 inhibited the activity in acinar basal regions and ductal lining of the deep gland, and decreased staining of demilunes (Fig. 8b and d). Strong granular lipase activity was confined to acini of the deep gland and demilunes (Fig. 9), and markedly decreased when taurocholate was omitted from the incubation medium. Acid phosphatase activity was strong in acini of the deep gland and demilunes (Fig. 10); and moderate in periluminal ductal rims of the deep gland (Fig. 10a). Demilunes weakly stained for glucoronidase. Weak to moderate, ouabain-sensitive Na,K-ATPase activity was localised in the basolateral plasmalemma of ductal cells in the deep gland and demilunar cells (Fig. 11).
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Fig. 7. Cryostat section incubated in thiamine pyrophosphatase medium for 60 min at 37 ◦ C. Most of the reaction product is localised in borders between parenchymal cells, and in blood vessels (arrowheads) of the superficial gland. Objective magnification ×20. An acinus (A) is magnified in the inset; weak Golgi-like supranuclear staining is arrowed.
Fig. 9. Cryostat sections incubated in lipase medium for 60 min at 37 ◦ C. Reaction product is concentrated in acini (asterisks) of the deep gland (a) and demilunes (arrowhead) of the superficial gland (b). Central cells (arrow) are unstained. Objective magnification ×40.
Parenchymal alkaline phosphatase activity was confined to occasional periacinar and periductal myoepithelial-like arrangements, appearing stronger in the latter (Fig. 12). Interstitial thin-walled blood vessels strongly stained for thiamine pyrophosphatase and alkaline phosphatase (Figs. 7 and 12). 3.5. Neurohistology Acetylcholinesterase activity was associated with a network of nerve fibres embracing glandular parenchyma (Fig. 13). There were no differences in the density of nerve fibres between the glands. Fluorescent, adrenergic fibres were not seen. 4. Discussion To avoid effects of sexual dimorphism (Pinkstaff, 1998), only glands from male mice were examined in the present investigation. Features related to acini/tubulo-acini and demilunes, ducts, myoepithelial arrangements, vessels and innervation are examined in turn. The finding of acini and acini or tubulo-acini with demilunes in the deep and superficial glands, respectively corresponds to previous observations in other mammals (Hand et al., 1999; Redman,
Fig. 10. Cryostat sections incubated in acid phosphatase medium for 30 min at 37 ◦ C. Reaction product is localised in acini (asterisks) and around ductal lumina (open arrow) of the deep gland (a) (objective magnification ×40); and in demilunes (arrowhead) of the superficial gland (b) (objective magnification ×20). (D), duct.
Fig. 8. Cryostat sections incubated in non-specific esterase medium for 10 min at room temperature. (a) Reaction product is localised in acini (asterisk) and ducts (D) of the deep gland. (b) Incubation with E600 abolishes staining of ductal lining (arrow) and acinar basal regions (open arrow); stained material in ductal lumina (L) is seen. Objective magnification ×20. (c) Reaction product is concentrated in demilunes (arrowheads) of the superficial gland. (d) Incubation with E600 slightly reduces the demilunar staining (open arrow). Objective magnification ×40.
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Fig. 11. Cryostat sections incubated in Na,K-ATPase medium for 30 min at room temperature. Reaction product is localised to borders between ductal cells of deep gland (a) and demilunar cells of superficial gland (b) (arrowheads). Objective magnification ×20.
Fig. 12. Cryostat sections incubated in alkaline phosphatase medium for 30 min at room temperature. Reaction product is in periductal (arrow) and periacinar (open arrows) myoepithelial-like arrangements and blood vessels (arrowheads) of the deep gland. Objective magnification × 20.
Fig. 13. Cryostat sections incubated in cholinesterase medium for 30 min at 37 ◦ C. Reaction product is associated with nerve fibres (arrowheads) embracing parenchymal structures of the deep (a) and superficial (b) glands. Objective magnification ×20.
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2012). Rapid freezing and freeze-substitution technologies would be useful in assessing the possibility that demilunes are artefacts attributable to immersion fixation (Yamashina et al., 1999). The variable staining for disulphides in the acini and tubuloacini probably reflects bonds influencing conformation of proteins (James and Tas, 1984). The localisation of disulphides in acinar apical regions of the deep gland corresponds with findings in von Ebner’s gland of ferret (Triantafyllou et al., 2001). The high levels of disulphides in demilunes of the superficial gland contrast with their absence in demilunes of the submandibular gland of ferret where thiols are present (Triantafyllou et al., 1999). Differences in rates of synthesis, post-translational modification and turnover of proteins, being faster in submandibular demilunes and slower in central acinar cells of ferret, possibly account for their thiol and disulphide content, respectively (Triantafyllou et al., 1999). These rates may be similar in the superficial lingual gland of mouse, thereby allowing variable formation of disulphide bonds in demilunes and central cells. The present investigation localised tryptophan in acini of the deep gland and demilunes. Tryptophan histochemical staining is a feature of parotid acini in echidna (Fig. 28 in Young and Van Lennep, 1978) and conveniently demonstrates proteases selectively localised in apical regions of submandibular, granular and striated ducts of rodents and carnivores, respectively (Shori et al., 1997; Triantafyllou et al., 1999). Proteins with high tryptophan content in lingual glands of mouse remain to be biochemically identified. The varying localisation of neutral, and acid sulphated and non-sulphated carboxylated mucosubstances in the acini and tubulo-acini, corresponds to previous observations (Burstone, 1953; Spicer and Duvenci, 1964). Lectins are also variously binding therein (Stoward et al., 1980; Schulte and Spicer, 1983). The staining patterns appear reinforcing the demilunar and central acinar structural organisation of the superficial gland. Immersion fixation may have, however, effected increased hydration and swelling of acinar cells with high levels of sulphated mucosubstances, which would thus displace neighbours with lower levels or containing non-sulphated mucosubstances, towards distal parts of the glandular parenchyma where appear as demilunes (Yamashina et al., 1999). Proteins in apical regions of acini and tubulo-acini probably unite with mucosubstances to form secretory glycoproteins packed in granules. Events would depend on rough endoplasmic reticulum (RER) and Golgi complex (Hand, 1990). Electron microscopical histochemical investigations in salivary glands of rabbit and rat indicate that E600-sensitive non-specific esterase and thiamine pyrophosphatase staining is localised to cisternae of RER and Golgi saccules, respectively (Hand, 1971; Kyriacou and Garrett, 1985). That E600 inhibited esterase activity in acinar basal regions of the deep gland and decreased staining of demilunes therefore suggests the presence of RER therein, and corresponds to their pyroninophilia (Burstone, 1953). The present investigation indicates that cytoplasmic Golgi-like thiamine pyrophosphatase staining is inconspicuous in the glandular parenchyma. Hydration of sulphated mucosubstances could effect attenuation and/or dispersal of Golgi saccules and thus affect detection of their light microscopical, histochemical staining. Alternatively, the inconspicuous Golgi-like staining may reflect slow secretory cycles (Triantafyllou et al., 2001) and/or circadian rhythms (Field et al., 1999). These would enable post-translational modifications, including variable formation of disulphide bonds and incorporation of sulphate (Hand, 1987), and cytoplasmic accumulation of secretory glycoproteins; and account for the absence of thiols in demilunes and central cells of the superficial gland. A single type of acinar cell showing demilunar and central phenotypes may be considered (Triantafyllou et al., 2004). Post-translational
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modifications and steric relationships removing and/or masking amino acids may account for the selective localisation of tryptophan in demilunes. Masking events in central cells of the superficial gland are supported by their binding of the peanut lectin following digestion with sialidase (Stoward et al., 1980). Post-translational modifications of glycoproteins and crinophagy of accumulated secretory granules may involve lysosomal activities (Triantafyllou et al., 2001), which account for the acid phosphatase staining in acini of the deep gland and demilunes. Hydration of sulphated mucosubstances and dispersal of organelles may influence the apparent lack of staining in central cells of the superficial gland. Except for lysosomes, electron microscopical histochemistry indicates that acid phosphatase is localised in cytosol, GERL (Golgi associated endoplasmic reticulum) and secretory granules in parotid and submandibular acini of cat (Garret and Kidd, 1976). This technology would be useful in exploring similar localisations in lingual glands of mouse. As in other species (Triantafyllou et al., 2001), the acid phosphatase activities could be supported by the finding of variably E600-resistent non-specific esterase staining therein. Lysosomal localisation of such staining has been observed by electron microscopical histochemistry in submandibular glands of rabbits (Kyriacou and Garrett, 1985). Garret and Kidd (1976) suggested that lysosomes may inject substances into secretory granules, which is supported by the finding of E600-resistent non-specific esterase staining in ductal lumina. The selective localisation of -glucoronidase in demilunes would also accord with lysosomal activities (Triantafyllou et al., 2001). High levels of lingual lipase destined for secretion, have been biochemically demonstrated in mouse (DeNigris et al., 1988). The present investigation histochemically localised the enzyme in acini of the deep gland and demilunes. Although an overlap with E600resistent non-specific esterase activity cannot be excluded, the localisation corresponds with immunohistochemical and histochemical investigations in other species (Roberts and Jaffe, 1986; Hand et al., 1999; Triantafyllou et al., 2002). The finding of simple ducts in the glands also corresponds with the basic structural architecture of posterior lingual glands in mammals (Hand et al., 1999; Redman, 2012). Contraction of the lingual musculature effecting intermittent obstruction (Triantafyllou et al., 2001), would increase pressure in lumina and account for the finding of dilated ducts in the superficial gland. The absence of genuine ducts in the superficial gland of rat is noted (Nagato et al., 1997). The staining for neutral mucosubstances and tryptophan in ductal periluminal rims may reflect secretion or absorption followed by lysosomal processing of luminal secretions, both innate abilities of salivary ductal segments (Hand 1987, 1990). Secretion would accord with the finding of E600-sensitive non-specific esterase staining, indicative of RER cisternae (Kyriacou and Garrett, 1985), in the ductal cells; and the electron microscopical demonstration of ductal secretory granules in von Ebner’s gland of other species (Hand, 1970). Absorption would accord with the finding of periluminal ductal staining for acid phosphatase; and electron microscopical and histochemical demonstration of ductal phagosomes and lysosomal enzymatic staining in von Ebner’s glands of rat and ferret, respectively (Hand, 1970; Triantafyllou et al., 2001). Ductal cells of the deep gland and demilunes are probably transporting ions, which is reflected in their ouabain-sensitive staining for Na,K-ATPase. The finding accords with a basolateral plasmalemmal localisation of the enzyme (Garrett et al., 1992; Sims-Sampson et al., 1984; Triantafyllou et al., 1999), though contrasts with the absence of similar staining from von Ebner’s gland of ferret (Triantafyllou et al., 2001). Acini and ductal segments of other salivary glands in mouse also show Na,K-ATPase activity (SimsSampson et al., 1984).
In salivary glands, thiamine pyrophosphatase activity is associated with the Golgi complex (Hand, 1971; Auger and Harrison, 1982) and plasmalemmal staining in acini and ducts, as here, appears unusual. Electron microscopical histochemistry has, however, localised the enzyme on the plasmalemma of sinusoidal and canalicular surfaces in hepatocytes of mouse (Ogawa et al., 1982). A role in transport may be considered. The finding of widespread parenchymal localisation of dehydrogenases and cytochrome oxidase is variously attributable to cytosol and mitochondria, as in other species (Harrison, 1974; Bancoft and Hand, 1987). The copious and/or hydrated secretory material of central cells may explain their weaker staining. It is unlikely that the parenchymal staining for NADPH dehydrogenase reflects sites of nitric oxide synthase activity (Lohinai et al., 1995; Alm et al., 1997). Although electron microscopical confirmation is desirable, the present investigation suggests that alkaline phosphatase activity in relation to myoepithelial cells is inconspicuous in the posterior lingual glands of mouse where appears weaker in acini than ducts. Myoepithelial cells embracing acini have been found in lingual glands of rat, man and bats with electron microscopy (Hand, 1970; Tandler et al., 1994; Nagato et al., 1997; Tandler et al., 1997), and ferret with enzyme histochemistry (Triantafyllou et al., 2001). Absence of myoepithelial cells embracing acini is established in the parotid of rat (Garrett and Parsons, 1973) and myoepithelial cells are lacking in lingual glands of chameleon (Rabinowitz and Tandler, 1991). Possibly contraction of myoepithelial cells around ducts enables the rat parotid to resist distortion effected by mastication (Garrett and Parsons, 1973). The lingual musculature may exert a similar effect, thereby influencing an apparently stronger, periductal alkaline phosphatase activity in mouse. The variations illustrate the structural diversity in similar salivary glands between different species. The findings related to vessels and innervation are similar to those described in von Ebner’s gland of ferret (Triantafyllou et al., 2001) and readers are referred to this article for a relevant discussion. Applying technologies of classic histochemistry, the present investigation enabled particular localisations of protein-bound amino acid residues and enzymatic catalytic activities in posterior lingual glands of mouse. Except for yielding an insight into glandular secretory cycles and allowing comparative evaluation and appreciation of biodiversity, the localisations can be used for assessing glandular changes after experimental manipulation, and phylogeny. They should be interpreted in conjunction with further electron microscopical, investigations, and it would be of interest to immunohistochemically and biochemically explore whether the posterior lingual glands of mouse produce specific proteins like histatins and lipocalcins, similarly to their counterparts in other species (Piludu et al., 2006; Redman, 2012). Acknowledgements We are grateful to Mr A. R. Harmer and Dr P. M. Smith for effecting access to the mice. References Adams, C.W., 1957. A p-dimethylaminobenzaldehyde-nitrite method for the histochemical demonstration of tryptophane and related compounds. J. Clin. Pathol. 10, 56–62. Alm, P., Ekström, J., Larsson, B., Tobin, G., Andersson, K.E., 1997. Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation. Histochem. J. 29, 669–676. Auger, D.W., Harrison, J.D., 1982. Ultrastructural phosphatase cytochemistry of the intercalary ducts of the parotid and submandibular salivary glands of man. Arch. Oral Biol. 27, 79–81.
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Comparative histochemistry of posterior lingual salivary glands of mouse Asterios Triantafyllou ∗ , David Fletcher Oral & Maxillofacial Pathology, School of Dentistry, University of Liverpool, Liverpool L3 5PS, United Kingdom
a r t i c l e
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Article history: Received 5 August 2016 Accepted 15 November 2016 Available online xxx Keywords: Enzymes Histochemistry Mouse Mucosubstances Nerves Salivary glands
a b s t r a c t Normal posterior deep and superficial salivary glands of tongue were examined in male mice by means of light microscopical histochemistry and neurohistology. Both glands showed acini and simple ducts. Demilunes were present in the superficial gland. Disulphides and neutral mucosubstances occurred in acini and demilunes. Tryptophan staining was seen in acini of the deep gland and demilunes, whereas acid mucosubstances were exclusively localised in the superficial gland. Dehydrogenase activities were widespread. Strong esterase activity occurred throughout the parenchyma of the deep gland and in demilunes; it was variably inhibited by E600, apart from acinar apical regions in the deep gland. Lipase was confined to acini of the deep gland and demilunes. Acid phosphatase staining was similarly localised; it was also seen in periluminal ductal rims of the deep gland, in which ouabain-sensitive Na,K-ATPase was localised basolaterally. Staining for alkaline phosphatase decorated occasional myoepithelial-like arrangements and interstitial capillaries. Acetylcholinesterase was associated with nerve fibres embracing glandular parenchyma. Adrenergic fibres were not seen. The results suggest that the acini of the posterior deep lingual gland secrete neutral glycoproteins, whereas the ducts transport ions and absorb luminal material. The posterior superficial lingual gland mainly secretes acid glycoproteins. Both glands produce lingual lipase, receive cholinergic-type innervation and have inconspicuous myoepithelium. © 2016 Elsevier GmbH. All rights reserved.
1. Introduction The deep and superficial, minor salivary glands in the posterior part of the tongue, which were identified by von Ebner and Weber respectively, are of significance in digestion, taste and protection of taste buds (Nagato et al., 1997). They have been variously investigated in small carnivores and bats (Poddar and Jacob, 1980; Tandler et al., 1997; Triantafyllou et al., 1999); and intensely in man and rat (see: Hand et al., 1999; Redman, 2012). Less attention has been paid to the posterior lingual glands of mouse (Mus musculus), which are useful for studying glandular sexual dimorphism (Hanker et al., 1980) and harbouring amastigotes in experimental American trypanosomiasis (Lopes et al., 1991a, 1991b). Burstone (1953) applied early histochemical techniques to localise mucosubstances and nucleic acids in those glands; and the use of autoradiography, various sequences of basic dyes and vic-glycol methods in conjunction with pre-digestion and blocking of reactive moieties, and lectins enabled a more refined characterisation of carbohydrate residues therein (Spicer and Duvenci, 1964; Stoward et al., 1980; Schulte and
∗ Corresponding author. E-mail address: [email protected] (A. Triantafyllou).
Spicer, 1983). A more detailed histochemical profiling is, however, desirable. This prompted the present investigation wherein posterior lingual glands of male mouse were examined with the use of histological, protein, mucosubstance and enzyme histochemical, and neurohistological techniques, which have been found valuable while exploring minor salivary glands in other species (Harrison, 1974; Triantafyllou et al., 2001).
2. Materials and methods 2.1. Animals, glands and preservation Five mature male CD1 mice that had been fasted overnight and killed by neck dislocation (schedule 1) as part of an in vitro investigation (Smith et al., 2000), became available. Surplus tissues were donated for additional investigations to ensure best use of the material. Ethical committee approval was not required. The tongues of the mice were rapidly removed and cut into transverse, frontal slices. Posteriorly situated slices including the vallate papilla were further bisected. Halves were immediately quenched in isopentane cooled by solid carbon dioxide and then
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stored at −70 ◦ C. Other halves were immersion fixed in a formaldehyde solution and then processed for paraffin blocks.
2.2. Histology Sections of fixed tissue were cut at a thickness of 5 mm and were stained with haematoxylin and eosin.
2.3. Protein and amino acid histochemistry Sections of fixed tissue were cut at a thickness of 5 mm and were stained with the p-dimethylbenzaldehyde nitrite reaction for tryptophan (Adams, 1957) and the coupled maleimide reaction for thiol groups (staining blue) and disulphide groups (staining red) (Sippel, 1978).
2.4. Mucosubstance histochemistry Sections of fixed tissue were cut at a thickness of 5 mm and were stained with alcian blue at pH 2.5 followed by periodic acid-Schiff to demonstrate neutral mucosubstances staining red (periodate-reactive) and acid mucosubstances staining in varying shades of purple and royal blue (periodate-reactive and variably alcianophilic) (Mowry, 1956; Spicer et al., 1967). The high-iron diamine technique followed by alcian blue at pH 2.5 was used to demonstrate sulphated mucosubstances staining brown/black (high-iron diamine-reactive) and non-sulphated carboxylated mucosubstances staining sky blue (alcianophilic) (Spicer, 1965).
2.5. Enzyme histochemistry Sections of quenched pieces were cut at a thickness of about 10–12 mm on a cryostat and were incubated with substrates for the following oxidative enzymes: peroxidase with and without KCN to inhibit peroxidatic responses of respiratory enzymes (Graham and Karnovsky, 1966; Garret and Kidd, 1976), cytochrome oxidase (Kugler et al., 1988), succinate dehydrogenase (Nachlas et al., 1957), lactate dehydrogenase (Bancoft and Hand, 1987), and NADH and NAD(P)H dehydrogenases (Scarpelli et al., 1958). Cryostat sections were also incubated with substrates for the following hydrolytic enzymes: thiamine pyrophosphatase (Novikoff and Goldfischer, 1961), alkaline phosphatase (Stutte, 1967), Na,KATPase with and without ouabain to inhibit activity (Mayahara and Ogawa, 1988), acid phosphatase (Barka and Anderson, 1962), -glucoronidase (Hayashi et al., 1964), non-specific esterase with and without E600 inhibitor (Davis, 1959; Bancoft and Hand, 1987), and lipase with and without sodium taurocholate activator (Triantafyllou et al., 2002).
Fig. 1. Paraffin sections stained with haematoxylin and eosin. (a) Deep (von Ebner) gland; collections of acini (arrowhead) surround ducts (arrow) and are between skeletal muscle fibres (M). (b) Superficial (Weber) gland; tubulo-acini (asterisk), demilunes (arrowhead) and dilated duct (D) draining from two tubules. Objective magnification ×20.
3. Results 3.1. Histology The parenchyma of both glands was arranged in nonencapsulated lobular clusters interspersed between bundles of skeletal muscle (Fig. 1). The deep (von Ebner) gland showed acini and few intralobular ducts. The acini showed inconspicuous lumina and were composed of usually pyramidal cells with variably distinct borders, a prominent apical region replete with strongly eosinophilic granules and a basally aligned rounded vesicular nucleus with dispersed chromatin and inconspicuous nucleoli. The ducts were lined by simple small cuboidal cells with scanty, agranular, eosinophilic cytoplasm and central nuclei with dispersed chromatin (Fig. 1a). The superficial (Weber) gland showed acini and tubulo-acini opening into occasionally dilated, intralobular ducts lined by flattened epithelium. The acini and tubulo-acini were of a mixed appearance showing palely stained central cells with basal flattened nuclei, which were often capped by small, more densely stained demilunes (Fig. 1b). 3.2. Protein and amino acid histochemistry Variably intense, disulphide staining was localised in the apical region of acini of the deep gland, and in central cells and demilunes of the superficial gland; the remainder of the parenchyma showed moderate diffuse thiol staining (Fig. 2a and b). Moderate tryptophan staining was confined to acini and periluminal ductal rims of the deep gland, and demilunes of the superficial gland (Fig. 3a and b). 3.3. Mucosubstance histochemistry
2.6. Neurohistology Sections of quenched pieces were cut at a thickness of about 10–12 mm on a cryostat and were incubated for cholinesterases with and without eserine to inhibit activity (Karnovsky and Roots, 1964). In addition, cryostat sections of 30 mm thickness were treated by the glyoxylic acid method for catecholamines and were examined by fluorescence microscopy (de la Torre and Surgeon, 1976).
Neutral mucosubstances were present in both glands. Acid mucosubstances were localised in the superficial gland only (Figs. 4 and 5). The demilunar cells showed considerable variation in the staining with the high-iron diamine–alcian blue technique (Fig. 5). A few cells had a strong affinity for alcian blue alone, whereas others had a varying affinity for high-iron diamine. In some cells the affinities coexisted. The central cells were constantly stained by periodic acid–Schiff, and showed variable alcianophilia and moderate affin-
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Fig. 5. Paraffin section stained with the high-iron diamine-alcian blue procedure. Acini of the superficial gland are variously stained. Objective magnification ×20. Fig. 2. Paraffin sections stained with the coupled maleimide procedure. Moderate, weak and strong disulphide staining is seen in the acini (asterisks) of the deep gland (a) and central cells (arrow) and demilunes (arrowhead) of the superficial gland (b), respectively. Thiol groups are in ductal cells (D). Objective magnification ×40.
Fig. 6. Cryostat sections incubated in NADH dehydrogenase medium for 30 min at 37 ◦ C. (a) Reaction product is concentrated in ducts (D) of the deep gland where embraces unstained nuclei; lesser amounts of reaction product are in acini (asterisk); skeletal muscle fibres (M) are strongly stained. Objective magnification ×40. (b) Reaction product is concentrated in ductal lining (D) and demilunes (arrowhead) of the superficial gland; central cells (asterisk) appear weakly stained. Objective magnification ×20. Fig. 3. Paraffin sections stained with the p-dimethylbenzaldehyde-nitrite procedure. Staining is seen in acini of the deep gland (a) and demilunes (arrowheads) of the superficial gland (b). A duct with stained periluminal rim is arrowed. Central cells (asterisks) are unstained. Objective magnification ×40.
ity for high iron diamine (Figs. 4b and 5). Periluminal rims of ducts were strongly stained by periodic acid–Schiff (Fig. 4a). 3.4. Enzyme histochemistry
Fig. 4. Paraffin sections stained with the alcian blue-periodic acid-Schiff procedure. (a) Deep gland; (b) Superficial gland. Periodate reactive mucosubstances are in acinar cells of both glands and in periluminal rims (arrow) of ducts. Alcianophilic mucosubstances are confined to the superficial gland. Objective magnification ×40.
The parenchyma of both glands showed widespread, though variable, dehydrogenase and cytochrome oxidase activity (Fig. 6), and little or no peroxidase staining. The plasmalemma of acinar and ductal cells showed thiamine pyrophosphatase activity; cytoplasmic Golgi-like staining was inconspicuous (Fig. 7). Strong to intense diffuse esterase activity occurred in the parenchyma and ductal lumina of the deep gland, and demilunes (Fig. 8a and c); E600 inhibited the activity in acinar basal regions and ductal lining of the deep gland, and decreased staining of demilunes (Fig. 8b and d). Strong granular lipase activity was confined to acini of the deep gland and demilunes (Fig. 9), and markedly decreased when taurocholate was omitted from the incubation medium. Acid phosphatase activity was strong in acini of the deep gland and demilunes (Fig. 10); and moderate in periluminal ductal rims of the deep gland (Fig. 10a). Demilunes weakly stained for glucoronidase. Weak to moderate, ouabain-sensitive Na,K-ATPase activity was localised in the basolateral plasmalemma of ductal cells in the deep gland and demilunar cells (Fig. 11).
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Fig. 7. Cryostat section incubated in thiamine pyrophosphatase medium for 60 min at 37 ◦ C. Most of the reaction product is localised in borders between parenchymal cells, and in blood vessels (arrowheads) of the superficial gland. Objective magnification ×20. An acinus (A) is magnified in the inset; weak Golgi-like supranuclear staining is arrowed.
Fig. 9. Cryostat sections incubated in lipase medium for 60 min at 37 ◦ C. Reaction product is concentrated in acini (asterisks) of the deep gland (a) and demilunes (arrowhead) of the superficial gland (b). Central cells (arrow) are unstained. Objective magnification ×40.
Parenchymal alkaline phosphatase activity was confined to occasional periacinar and periductal myoepithelial-like arrangements, appearing stronger in the latter (Fig. 12). Interstitial thin-walled blood vessels strongly stained for thiamine pyrophosphatase and alkaline phosphatase (Figs. 7 and 12). 3.5. Neurohistology Acetylcholinesterase activity was associated with a network of nerve fibres embracing glandular parenchyma (Fig. 13). There were no differences in the density of nerve fibres between the glands. Fluorescent, adrenergic fibres were not seen. 4. Discussion To avoid effects of sexual dimorphism (Pinkstaff, 1998), only glands from male mice were examined in the present investigation. Features related to acini/tubulo-acini and demilunes, ducts, myoepithelial arrangements, vessels and innervation are examined in turn. The finding of acini and acini or tubulo-acini with demilunes in the deep and superficial glands, respectively corresponds to previous observations in other mammals (Hand et al., 1999; Redman,
Fig. 10. Cryostat sections incubated in acid phosphatase medium for 30 min at 37 ◦ C. Reaction product is localised in acini (asterisks) and around ductal lumina (open arrow) of the deep gland (a) (objective magnification ×40); and in demilunes (arrowhead) of the superficial gland (b) (objective magnification ×20). (D), duct.
Fig. 8. Cryostat sections incubated in non-specific esterase medium for 10 min at room temperature. (a) Reaction product is localised in acini (asterisk) and ducts (D) of the deep gland. (b) Incubation with E600 abolishes staining of ductal lining (arrow) and acinar basal regions (open arrow); stained material in ductal lumina (L) is seen. Objective magnification ×20. (c) Reaction product is concentrated in demilunes (arrowheads) of the superficial gland. (d) Incubation with E600 slightly reduces the demilunar staining (open arrow). Objective magnification ×40.
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Fig. 11. Cryostat sections incubated in Na,K-ATPase medium for 30 min at room temperature. Reaction product is localised to borders between ductal cells of deep gland (a) and demilunar cells of superficial gland (b) (arrowheads). Objective magnification ×20.
Fig. 12. Cryostat sections incubated in alkaline phosphatase medium for 30 min at room temperature. Reaction product is in periductal (arrow) and periacinar (open arrows) myoepithelial-like arrangements and blood vessels (arrowheads) of the deep gland. Objective magnification × 20.
Fig. 13. Cryostat sections incubated in cholinesterase medium for 30 min at 37 ◦ C. Reaction product is associated with nerve fibres (arrowheads) embracing parenchymal structures of the deep (a) and superficial (b) glands. Objective magnification ×20.
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2012). Rapid freezing and freeze-substitution technologies would be useful in assessing the possibility that demilunes are artefacts attributable to immersion fixation (Yamashina et al., 1999). The variable staining for disulphides in the acini and tubuloacini probably reflects bonds influencing conformation of proteins (James and Tas, 1984). The localisation of disulphides in acinar apical regions of the deep gland corresponds with findings in von Ebner’s gland of ferret (Triantafyllou et al., 2001). The high levels of disulphides in demilunes of the superficial gland contrast with their absence in demilunes of the submandibular gland of ferret where thiols are present (Triantafyllou et al., 1999). Differences in rates of synthesis, post-translational modification and turnover of proteins, being faster in submandibular demilunes and slower in central acinar cells of ferret, possibly account for their thiol and disulphide content, respectively (Triantafyllou et al., 1999). These rates may be similar in the superficial lingual gland of mouse, thereby allowing variable formation of disulphide bonds in demilunes and central cells. The present investigation localised tryptophan in acini of the deep gland and demilunes. Tryptophan histochemical staining is a feature of parotid acini in echidna (Fig. 28 in Young and Van Lennep, 1978) and conveniently demonstrates proteases selectively localised in apical regions of submandibular, granular and striated ducts of rodents and carnivores, respectively (Shori et al., 1997; Triantafyllou et al., 1999). Proteins with high tryptophan content in lingual glands of mouse remain to be biochemically identified. The varying localisation of neutral, and acid sulphated and non-sulphated carboxylated mucosubstances in the acini and tubulo-acini, corresponds to previous observations (Burstone, 1953; Spicer and Duvenci, 1964). Lectins are also variously binding therein (Stoward et al., 1980; Schulte and Spicer, 1983). The staining patterns appear reinforcing the demilunar and central acinar structural organisation of the superficial gland. Immersion fixation may have, however, effected increased hydration and swelling of acinar cells with high levels of sulphated mucosubstances, which would thus displace neighbours with lower levels or containing non-sulphated mucosubstances, towards distal parts of the glandular parenchyma where appear as demilunes (Yamashina et al., 1999). Proteins in apical regions of acini and tubulo-acini probably unite with mucosubstances to form secretory glycoproteins packed in granules. Events would depend on rough endoplasmic reticulum (RER) and Golgi complex (Hand, 1990). Electron microscopical histochemical investigations in salivary glands of rabbit and rat indicate that E600-sensitive non-specific esterase and thiamine pyrophosphatase staining is localised to cisternae of RER and Golgi saccules, respectively (Hand, 1971; Kyriacou and Garrett, 1985). That E600 inhibited esterase activity in acinar basal regions of the deep gland and decreased staining of demilunes therefore suggests the presence of RER therein, and corresponds to their pyroninophilia (Burstone, 1953). The present investigation indicates that cytoplasmic Golgi-like thiamine pyrophosphatase staining is inconspicuous in the glandular parenchyma. Hydration of sulphated mucosubstances could effect attenuation and/or dispersal of Golgi saccules and thus affect detection of their light microscopical, histochemical staining. Alternatively, the inconspicuous Golgi-like staining may reflect slow secretory cycles (Triantafyllou et al., 2001) and/or circadian rhythms (Field et al., 1999). These would enable post-translational modifications, including variable formation of disulphide bonds and incorporation of sulphate (Hand, 1987), and cytoplasmic accumulation of secretory glycoproteins; and account for the absence of thiols in demilunes and central cells of the superficial gland. A single type of acinar cell showing demilunar and central phenotypes may be considered (Triantafyllou et al., 2004). Post-translational
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modifications and steric relationships removing and/or masking amino acids may account for the selective localisation of tryptophan in demilunes. Masking events in central cells of the superficial gland are supported by their binding of the peanut lectin following digestion with sialidase (Stoward et al., 1980). Post-translational modifications of glycoproteins and crinophagy of accumulated secretory granules may involve lysosomal activities (Triantafyllou et al., 2001), which account for the acid phosphatase staining in acini of the deep gland and demilunes. Hydration of sulphated mucosubstances and dispersal of organelles may influence the apparent lack of staining in central cells of the superficial gland. Except for lysosomes, electron microscopical histochemistry indicates that acid phosphatase is localised in cytosol, GERL (Golgi associated endoplasmic reticulum) and secretory granules in parotid and submandibular acini of cat (Garret and Kidd, 1976). This technology would be useful in exploring similar localisations in lingual glands of mouse. As in other species (Triantafyllou et al., 2001), the acid phosphatase activities could be supported by the finding of variably E600-resistent non-specific esterase staining therein. Lysosomal localisation of such staining has been observed by electron microscopical histochemistry in submandibular glands of rabbits (Kyriacou and Garrett, 1985). Garret and Kidd (1976) suggested that lysosomes may inject substances into secretory granules, which is supported by the finding of E600-resistent non-specific esterase staining in ductal lumina. The selective localisation of -glucoronidase in demilunes would also accord with lysosomal activities (Triantafyllou et al., 2001). High levels of lingual lipase destined for secretion, have been biochemically demonstrated in mouse (DeNigris et al., 1988). The present investigation histochemically localised the enzyme in acini of the deep gland and demilunes. Although an overlap with E600resistent non-specific esterase activity cannot be excluded, the localisation corresponds with immunohistochemical and histochemical investigations in other species (Roberts and Jaffe, 1986; Hand et al., 1999; Triantafyllou et al., 2002). The finding of simple ducts in the glands also corresponds with the basic structural architecture of posterior lingual glands in mammals (Hand et al., 1999; Redman, 2012). Contraction of the lingual musculature effecting intermittent obstruction (Triantafyllou et al., 2001), would increase pressure in lumina and account for the finding of dilated ducts in the superficial gland. The absence of genuine ducts in the superficial gland of rat is noted (Nagato et al., 1997). The staining for neutral mucosubstances and tryptophan in ductal periluminal rims may reflect secretion or absorption followed by lysosomal processing of luminal secretions, both innate abilities of salivary ductal segments (Hand 1987, 1990). Secretion would accord with the finding of E600-sensitive non-specific esterase staining, indicative of RER cisternae (Kyriacou and Garrett, 1985), in the ductal cells; and the electron microscopical demonstration of ductal secretory granules in von Ebner’s gland of other species (Hand, 1970). Absorption would accord with the finding of periluminal ductal staining for acid phosphatase; and electron microscopical and histochemical demonstration of ductal phagosomes and lysosomal enzymatic staining in von Ebner’s glands of rat and ferret, respectively (Hand, 1970; Triantafyllou et al., 2001). Ductal cells of the deep gland and demilunes are probably transporting ions, which is reflected in their ouabain-sensitive staining for Na,K-ATPase. The finding accords with a basolateral plasmalemmal localisation of the enzyme (Garrett et al., 1992; Sims-Sampson et al., 1984; Triantafyllou et al., 1999), though contrasts with the absence of similar staining from von Ebner’s gland of ferret (Triantafyllou et al., 2001). Acini and ductal segments of other salivary glands in mouse also show Na,K-ATPase activity (SimsSampson et al., 1984).
In salivary glands, thiamine pyrophosphatase activity is associated with the Golgi complex (Hand, 1971; Auger and Harrison, 1982) and plasmalemmal staining in acini and ducts, as here, appears unusual. Electron microscopical histochemistry has, however, localised the enzyme on the plasmalemma of sinusoidal and canalicular surfaces in hepatocytes of mouse (Ogawa et al., 1982). A role in transport may be considered. The finding of widespread parenchymal localisation of dehydrogenases and cytochrome oxidase is variously attributable to cytosol and mitochondria, as in other species (Harrison, 1974; Bancoft and Hand, 1987). The copious and/or hydrated secretory material of central cells may explain their weaker staining. It is unlikely that the parenchymal staining for NADPH dehydrogenase reflects sites of nitric oxide synthase activity (Lohinai et al., 1995; Alm et al., 1997). Although electron microscopical confirmation is desirable, the present investigation suggests that alkaline phosphatase activity in relation to myoepithelial cells is inconspicuous in the posterior lingual glands of mouse where appears weaker in acini than ducts. Myoepithelial cells embracing acini have been found in lingual glands of rat, man and bats with electron microscopy (Hand, 1970; Tandler et al., 1994; Nagato et al., 1997; Tandler et al., 1997), and ferret with enzyme histochemistry (Triantafyllou et al., 2001). Absence of myoepithelial cells embracing acini is established in the parotid of rat (Garrett and Parsons, 1973) and myoepithelial cells are lacking in lingual glands of chameleon (Rabinowitz and Tandler, 1991). Possibly contraction of myoepithelial cells around ducts enables the rat parotid to resist distortion effected by mastication (Garrett and Parsons, 1973). The lingual musculature may exert a similar effect, thereby influencing an apparently stronger, periductal alkaline phosphatase activity in mouse. The variations illustrate the structural diversity in similar salivary glands between different species. The findings related to vessels and innervation are similar to those described in von Ebner’s gland of ferret (Triantafyllou et al., 2001) and readers are referred to this article for a relevant discussion. Applying technologies of classic histochemistry, the present investigation enabled particular localisations of protein-bound amino acid residues and enzymatic catalytic activities in posterior lingual glands of mouse. Except for yielding an insight into glandular secretory cycles and allowing comparative evaluation and appreciation of biodiversity, the localisations can be used for assessing glandular changes after experimental manipulation, and phylogeny. They should be interpreted in conjunction with further electron microscopical, investigations, and it would be of interest to immunohistochemically and biochemically explore whether the posterior lingual glands of mouse produce specific proteins like histatins and lipocalcins, similarly to their counterparts in other species (Piludu et al., 2006; Redman, 2012). Acknowledgements We are grateful to Mr A. R. Harmer and Dr P. M. Smith for effecting access to the mice. References Adams, C.W., 1957. A p-dimethylaminobenzaldehyde-nitrite method for the histochemical demonstration of tryptophane and related compounds. J. Clin. Pathol. 10, 56–62. Alm, P., Ekström, J., Larsson, B., Tobin, G., Andersson, K.E., 1997. Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation. Histochem. J. 29, 669–676. Auger, D.W., Harrison, J.D., 1982. Ultrastructural phosphatase cytochemistry of the intercalary ducts of the parotid and submandibular salivary glands of man. Arch. Oral Biol. 27, 79–81.
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Please cite this article in press as: Triantafyllou, A., Fletcher, D., Comparative histochemistry of posterior lingual salivary glands of mouse. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.11.007