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Postnatal growth of the rat palatine gland
Tissue & Cell, 2001 33 (6) 614±620 ß 2001 Harcourt Publishers Ltd DOI: 10.1054/tice.2001.0216, available online at http://www.idealibrary.com
Tissue&Cell
Postnatal growth of the rat palatine gland S. Nakamura, S. Takahashi, M. Wakita, M. Morita Abstract. To elucidate how the palatine glands grow postnatally, the palatine glands of rats from 0 to 8 weeks of age were investigated histologically and immunohistochemically. Under light microscope, three dimensions of the right part of the palatine glands were measured and the total number of excretory ducts of the glands was counted from the parasagittal serial sections. Immunohistochemistry with anti-5-bromo-20 -deoxyuridine (BrdU) monoclonal antibody was also employed to detect the cellular proliferative activity. At birth (0 weeks), the palatine glands consisted of ducts and immature acini. The ducts in the glands were connected with excretory ducts. After 2 weeks, there was no duct in the glands. Most acinar cells became mature as mucous cells and took the form of tubulo-acini connected directly with excretory ducts. In the posterior region of the glands, serous acinar cells forming demilunes were occasionally seen. All three dimensions of the palatine glands became longer, and the number of excretory ducts tended to increase. Immunohistochemistry showed acinar and duct cells were highly proliferative in early stage of postnatal life and their proliferative activity decreased thereafter. This study demonstrated that immature rat palatine glands of newborn rats grow three-dimensionally during maturation, and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from the excretory ducts formed postnatally. ß 2001 Harcourt Publishers Ltd Keywords: postnatal growth, palatine gland, cell proliferation, excretory duct
Introduction Development of major salivary glands such as parotid, submandibular, and sublingual glands has been extensively studied and many data have been accumulated. Scant attention has been given to the development of the minor salivary glands, however. There are some reports concerning von Ebner's glands (Baumgartner, 1917; Hamosh & Hand, 1978; Kock et al., 1992; Adi et al., 1995), buccal glands (Zimmerman & Zeidenstein, 1951; Redman, 1972), and labial glands (Zimmerman & Department of Oral Health Sciences, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan Received 1 May 2001 Accepted 3 September 2001 Correspondence to: Dr Shiro Nakamura, Department of Oral Health Sciences, Hokkaido University Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo, 060-8586, Japan. Tel. & Fax: 81 11 706 4225; E-mail: [email protected]
614
Zeidenstein, 1951; Goodman & Stern, 1972; Adi et al., 1994, 1995). The palatine gland has many short excretory ducts passing from the gland to oral cavity and forms an almost compact glandular body situated in the submucous layer of the hard and soft palates (DuBrul, 1988). Clinically, the palate is the most common site for benign or malignant neoplastic changes of minor salivary gland origin (Hjertman & Eneroth, 1970; Chung et al., 1978; Weisberger et al., 1979). Because of this it is of interest to investigate the morphogenesis of the palatine glands. Nielsen and Westergaard (1971) described that the development of the human palatine glands begins at the 11th week of fetal life and that, after branching and canalization of epithelial cord, mucous terminal portions ®rst appear at the 15th week. Srivastava and Vyas (1979) reported that the palatine gland of rats developed from tubular invaginations of the oral epithelium lining the soft palate and that it grows by pouching, resulting in
PALATINE GLAND POSTNATAL GROWTH
the development of glands with large irregular pouches. However, there are few studies of the development of the palatine glands apart from those mentioned above, and little information has been accumulated as for other minor salivary glands. The present study aimed to determine how the palatine glands grow postnatally. To achieve this, rat palatine glands in postnatal life were examined, using histological analysis and immunohistochemistry with anti-5-bromo20 -deoxyuridine (BrdU) antibody to observe the proliferative activity.
Materials and methods Histology Male Wistar rats were used in this study. The animals were killed in groups of three or four at ages of 0, 1, 2, 3, 4, 6, or 8 weeks postnatally by an overdose of pentobarbital sodium. The BrdU, which is incorporated into DNA by cells traversing the S-phase of the cell cycle, was administrated at a dose of 50mg/kg body weight by intraperitoneal injection 1 h prior to the sacri®ce. The whole head was ®xed in 4% buffered paraformaldehyde (pH 7.4) for 24 h and decalci®ed in a mixture of 50% formic acid and 20% tri sodium citrate dehydrate for 4±9 days. After decalci®cation, the head was cut into exact halves at the medial plane and the right half was embedded in paraf®n. Parasagittal 5 m-m thick serial sections were made and stained with haematoxylin-eosin (HE) or Alcian blueKernechtrot. All animal experimentation followed the Guidelines for the Care and Use of Laboratory Animals, Hokkaido University Graduate School of Dental Medicine. Histological measurement Histological measurement with parasagittal serial sections were performed. In the right half of the palatine gland of each animal, the maximum of length and height were measured using eyepiece micrometer discs (Fig. 1a), and the maximum of width was calculated by multiplying thickness of section (5 mm) by the total number of sections from the medial plane to the lateral margin of the palatine gland (Fig. 1b). The total number of excretory ducts of the right half of the palatine gland in each animal was counted out by observing all parasagittal serial sections. In all items, the mean and standard deviation (SD) were calculated for three or four animals at each time point. Immunohistochemistry Five sections from each animal were randomly selected for the immunohistochemistry, and were pretreated with 0.1% trypsin for 20 min at 378C and later with 3N HCl for 10 min at room temperature. After pretreatment, the sections were successively reacted with anti-BrdU mouse monoclonal antibody (DAKO), anti-mouse rabbit
615
biotinylated IgG (DAKO), and streptavidin-biotin peroxidase complex. The sections were rinsed thoroughly with phosphate-buffered saline after each incubation. Peroxidase activity was visualized using diaminobenzidine tetrahydrochloride. Sections were lightly counterstained with haematoxylin. Normal mouse serum was substituted for the primary antibody in the negative control sections. After immunohistochemical staining, a total of 2500 acinar cells and 750 excretory or other types of duct cells were counted for each animal, and the BrdU labeling indices (percentage of cells labeled) were calculated at each time point.
Results Histological observations The palatine glands of all rats were situated from the posterior end of the hard palate to the soft palate. At birth (0 weeks), the palatine glands consisted of ducts and immature acini surrounded by connective tissue (Fig. 2a). The ducts in the glands were composed of cuboidal or squamous epithelial cells and showed no character of ductal components seen in mature salivary glands such as intercalated, striated, or interlobular ducts. These ducts were connected with excretory ducts opening into oral cavity. Each immature acinar cell had a round nucleus situated basally and the cytoplasm around the nucleus was eosinophilic. The luminal cytoplasm of the immature acinar cells was clear and stained positively with Alcian blue. Mitotic ®gures were often seen in ducts and immature acinar cells (Fig. 2b). At 1 week, the number of ducts in the glands was smaller, while acini numbers increased and maturation of the acini had progressed. The acini mainly consisted of mucous cells with rich clear cytoplasm pressing the spindle nuclei against the basal area (Fig. 2c). The shape of the acini was mainly round, and there were also some tubuloacinar structures. The glandular tissue was occasionally organized into a lobule like structure separated by connective tissue. At more than 2 weeks, most acinar cells were mature mucous cells and took the form of tubuloacini. Now there was no duct in the glands, and the palatine glands were composed of only tubulo-acini. The tubulo-acini were joined in the palatine gland and then, at the glandular periphery, they were connected with excretory ducts which opened into the oral cavity (Fig. 2d). In the posterior region of the palatine glands, serous acinar cells were very rarely observed. These cells had capped mucous acini and formed demilunes (Fig. 2e). At all experimental periods, there were no epithelial cords extending from the palatal epithelium toward the palatine glands. Figure 3 shows the changes in the dimensions of the palatine glands. The length, width, and height of the right part of the palatine gland constantly increased from 0 to 8
616
NAKAMURA ET AL.
1a A
NC
H
P
PG L OC A
1b
PG
W
P
Fig. 1 Schematic figures of histological measurement. Throughout PG, palatine gland; A, anterior; P, posterior. a: parasagittal view of the palate. Length (L) and height (H) of the right half of the palatine gland. NC, nasal cavity; OC, oral cavity. b: Surface view of the palate. Width (W) of the right half of the palatine gland.
weeks of age. At 8 weeks the approximate length was four times, the width was twice, and the height was 10 times that at 0 weeks. The changes in the number of the excretory ducts of the palatine glands, not including other types of ducts, are presented in Figure 4 There were many excretory ducts at 0 weeks (average 23.0). This number increased sharply up to 3 weeks of age and thereafter the increase was moderate. Immunohistochemical observations At 0 weeks, a number of BrdU positive cells were seen in immature acini (Fig. 5a), ducts in the palatine glands (Fig. 5b) and excretory ducts. Fibroblast-like cells in the
stroma were also often BrdU positive. At 1 week, many maturing acinar and duct cells were still BrdU positive (Fig. 5c). However, after that, these BrdU positive cells gradually decreased (Fig. 5d). The BrdU labeling indices of acinar and duct cells are demonstrated in Figure 6. At 0 and 1 week, the labeling index for duct cells included the ducts in the palatine glands and the excretory ducts, while after 2 weeks it included only excretory ducts owing to disappearance of the ducts in the glands. At 1 week the index for acinar cells reached a peak of 10.1%, after which it declined to 0.2% at 8 weeks. The index for duct cells was highest at 10.1% at 0 weeks and thereafter it decreased to 2.5% at 8 weeks.
PALATINE GLAND POSTNATAL GROWTH
2a
2d
2b
2e
617
2c
Fig. 2 Histology of the rat palatine gland during postnatal development (HE). a: 0 weeks. The palatine gland consists of ducts (arrows) and immature acini
(arrowheads). x160. b: 0 weeks. A mitotic figure of immature acinar cell (arrowhead). x480. c: 1 week. Acini increase in number and their maturation has progressed. x320. d: 8 weeks. The palatine gland is composed of only mucous tubulo-acini. x80. e: A serous acinar cell forming a demilune (arrow). x400.
Discussion In the early stage of postnatal development of rat palatine glands, immature acinar cells, as transitional structures, were often observed in the present study. This suggests that in this period differentiation from duct to acinar cells occur actively in the palatine glands of rats.
During postnatal development of the palatine glands, the length, width, and height of the palatine glands increased constantly. Both acinar and duct cells were highly proliferative in the early stage of postnatal growth and maintained the proliferative activity throughout all stages of postnatal life. These ®ndings indicate that proliferation of the parenchymal cells
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10
8
6 mm
length width
4
height
2
0 0
1
2
3
4 5 Time (weeks)
6
7
8
9
Fig. 3 Postnatal growth of length (solid line), width (broken line), and height (dotted line) of the right part of the palatine glands. Error bars show SD.
The number of excretory ducts
60
50
40
30
20
10
0
0
1
2
3
4 5 Time (weeks)
6
7
8
9
Fig. 4 Postnatal changes in the number of excretory ducts of the palatine glands. The results are expressed as means + SD.
contribute to the growth of the palatine glands. The proliferative activity of acinar and duct cells has also been identi®ed during the development of parotid (Redman & Sreebny, 1970; Klein, 1982; Redman, 1995), submandibular (Chang, 1974; Alvares & Sesso, 1975; Klein, 1982), and sublingual glands (Taga & Sesso, 1998), and in the regeneration of parotid (BurfordMason et al., 1993; Cummins et al., 1994; Takahashi et al., 1998) and submandibular glands (Takahashi & Wakita, 1993). This allows the conclusion that parenchymal cell proliferation is very important for the rapid increase in the size of salivary glands. It is known that there are many excretory ducts of the palatine glands. However, it is not known how the number of the excretory ducts changes during development. This study demonstrated that the excretory
ducts increase in number during the postnatal period. The process of developmental events of salivary glands is initiated by the proliferation of oral epithelial cells and their downgrowth into the underlying mesenchyme, resulting in lumen formation of the epithelial rudiment and differentiation of the duct cells to acinar cells (Redman, 1987). As the excretory ducts originate from the oral epithelium, the increase in excretory duct numbers implies that the excretory ducts arise from the epithelium of the palate during postnatal life and suggests that glandular tissue is newly formed from these excretory ducts. Therefore, the increase in the excretory duct numbers is possibly another factor in the growth of the palatine glands. However, no epithelial cords extending from the oral epithelium toward the palatine glands were observed in this study. It may be dif®cult to identify
PALATINE GLAND POSTNATAL GROWTH
5a
5c
5b
5d
619
Fig. 5 Immunohistochemistry for BrdU. a: 0 weeks. Immature acinar cells (arrows) are BrdU-positive. x600. b: 0 weeks. There are BrdU-positive duct cells
in the palatine gland (arrowheads). x600. c: 1 week. Many maturing acinar cells are BrdU-positive. x400. d: 6 weeks. A few BrdU positive cells are seen in acinus (arrowhead) and excretory duct (arrows). x200.
12
Labeling index of BrdU (%)
10
Acinar cell
8
Duct cell 6
4
2
0
0
1
2
3
4 5 Time (weeks)
6
7
8
9
Fig. 6 BrdU labeling indices of acinar cells (dotted line) and duct cells (solid line) of the palatine glands. The results are expressed as means + SD.
620
NAKAMURA ET AL.
extending epithelial cords, because the distance between the oral epithelium and the palatine glands is very short and the extending epithelial cords reach the palatine glands in a short time. According to some oral histology textbooks (Hand, 1986; Dale, 1998), human palatine glands are classi®ed as pure mucous. Recently, Riva et al. (1999) observed seromucous acinar cells and de®ned the human palatine glands as mixed glands. There are two suggestions for rat palatine glands, that they have no (Odajima, 1975) or some serous acinar cells (Leeson & Leeson, 1968). In this study, serous acinar cells forming demilunes were identi®ed by the thorough observation of the serial sections of the rat palatine glands. Serous acinar cells were rare and located in the posterior portion of the palatine glands and it may be dif®cult to detect these cells by electron microscope (Odajima, 1975). In conclusion, this study demonstrated that immature palatine glands of newborn rats grow threedimensionally as they mature and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from excretory ducts formed postnatally. ACKNOWLEDGEMENTS We thank Emeritus Professor H. Tani, Hokkaido University Graduate School of Dental Medicine, for his stimulating suggestions regarding this study. REFERENCES Adi, M.M., Chisholm, D.M. and Waterhouse, J.P. 1994. Stereological and immunohistochemical study of development of human fetal labial salivary glands and their S-100 protein reactivity. J. Oral Pathol. Med., 23, 36±40. Adi, M.M., Chisholm, D.M. and Waterhouse, J.P. 1995. Histochemical study of lectin binding in the human fetal minor salivary glands. J. Oral Pathol. Med., 24, 130±135. Alvares, E.P. and Sesso, A. 1975. Cell proliferation, differentiation and transformation in the rat submandibular gland during early postnatal growth. A quantitative and morphological study. Arch. Histol. Jap., 38, 177±208. Baumgartner, E.A. 1917. The development of the serous glands (von Ebner's) of the vallate papillae in man. Am. J. Anat., 22, 365±383. Burford-Mason, A.P., Cummins, M.M., Brown, D.H., Mackay, A.J. and Dardick, I. 1993. Immunohistochemical analysis of the proliferative capacity of duct and acinar cells during ligationinduced atrophy and subsequent regeneration of rat parotid gland. J. Oral Pathol. Med., 22, 440±446. Chang, W.W.L. 1974. Cell population changes during acinus formation in the postnatal rat submandibular gland. Anat. Rec., 178, 187±202.
Chung, C.K., Rhaman, S.M. and Constable, W.C. 1978. Malignant salivary gland tumors of the palate. Arch. Otolaryngol., 104, 501±504. Cummins, M., Dardick, I., Brown, D. and Burford-Mason, A. 1994. Obstructive sialadentis: a rat model. J. Otolaryngol., 23, 50±56. Dale, A.C. 1998. Salivary glands. In: Ten Cate, A.R. (ed). Oral Histology, Development, structure, and function, 5th edn. C.V. Mosby, St Louis, pp 315±344. DuBrul, E.L. 1988. The oral viscera. In: DuBrul, E.L. (ed) Sicher and DuBrul's Oral Anatomy, 8th edn. Ishiyaku EouroAmerica, St Louis, pp 172±176. Goodman, A.S. and Stern, I.B. 1972. Morphologic development of the human fetal labial salivary glands. J. Dent. Res., 51, 990±999. Hamosh, M. and Hand, A.R. 1978. Development of secretory activity in serous cells of the rat tongue. Dev. Biol., 65, 100±113. Hand, A.R. 1986. Salivary glands. In: Bhaskar, S.N. (ed) Orban's Oral Histology and Embryology, 10th edn. C.V. Mosby, St Louis, pp 328±360. Hjertman, L. and Eneroth, C.M. 1970. Tumours of the palate. Acta Otolaryngol. Suppl., 263, 179±182. Klein, R.M. 1982. Acinar cell proliferation in the parotid and submandibular salivary glands of the neonatal rat. Cell Tissue Kinet., 15, 187±195. Kock, K., BlaÈker, M. and Schmale, H. 1992. Postnatal development of von Ebner's gland: accumulation of a protein of the lipocalin superfamily in taste papillae of rat tongue. Cell Tissue Res., 267, 313±320. Leeson, C.R. and Leeson, T.S. 1968. Fine structure and possible secretory mechanism of rat palatine glands. J. Dent. Res., 47, 653±662. Nielsen, G. and Westergaard, E. 1971. The development of the palatine glands in human foetuses with a crown-rump length of 32±145 mm. Acta Odontol. Scand., 29, 231±250. Odajima, M. 1975. An ultrastructural study of the palatine glands of the rat. Jpn. J. Oral Biol., 17, 268±294. Redman, R.S. and Sreebny, L.M. 1970. Proliferative behavior of differentiating cells in the developing rat parotid gland. J. Cell Biol., 46, 81±87. Redman, R.S. 1972. The anterior buccal gland of the rat: a mucous salivary gland which develops as a branch of Stensen's duct. Anat. Rec., 172, 167±178. Redman, R.S. 1987. Development of the salivary gland. In: Sreebny, L.M. (ed) The Salivary System. Boca Raton, FL, CRC Press, pp 2±17. Redman, R.S. 1995. Proliferative activity by cell type in the developing rat parotid gland. Anat. Rec., 241, 529±540. Riva, A., Loffredo, F., Puxeddu, R. and Riva, F.T. 1999. A scanning and transmission electron microscope study of the human minor salivary glands. Arch. Oral Biol., 44, Suppl. 1, S27±S31. Srivastava, H.C. and Vyas, D.C. 1979. Postnatal development of rat soft palate. J. Anat., 128, 97±105. Taga, R. and Sesso, A. 1998. Postnatal development of the rat sublingual glands. A morphometric and radioautographic study. Arch. Histol. Cytol., 61, 417±426. Takahashi, S. and Wakita, M. 1993. Regeneration of the intralobular duct and acinus in rat submandibular glands after YAG laser irradiation. Arch. Histol. Cytol., 56, 199±206. Takahashi, S., Schoch, E. and Walker, N.I. 1998. Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction. Int. J. Exp. Path., 79, 293±301. Weisberger, E., Luna, M.A. and Guillamondegui, O.M. 1979. Salivary gland cancers of the palate. Am. J. Surg., 138, 584±587. Zimmerman, A.A. and Zeidenstein, S. 1951. The origin and distribution of the labial and buccal glands in the human fetus. J. Dent. Res., 30, 587±598.
Tissue&Cell
Postnatal growth of the rat palatine gland S. Nakamura, S. Takahashi, M. Wakita, M. Morita Abstract. To elucidate how the palatine glands grow postnatally, the palatine glands of rats from 0 to 8 weeks of age were investigated histologically and immunohistochemically. Under light microscope, three dimensions of the right part of the palatine glands were measured and the total number of excretory ducts of the glands was counted from the parasagittal serial sections. Immunohistochemistry with anti-5-bromo-20 -deoxyuridine (BrdU) monoclonal antibody was also employed to detect the cellular proliferative activity. At birth (0 weeks), the palatine glands consisted of ducts and immature acini. The ducts in the glands were connected with excretory ducts. After 2 weeks, there was no duct in the glands. Most acinar cells became mature as mucous cells and took the form of tubulo-acini connected directly with excretory ducts. In the posterior region of the glands, serous acinar cells forming demilunes were occasionally seen. All three dimensions of the palatine glands became longer, and the number of excretory ducts tended to increase. Immunohistochemistry showed acinar and duct cells were highly proliferative in early stage of postnatal life and their proliferative activity decreased thereafter. This study demonstrated that immature rat palatine glands of newborn rats grow three-dimensionally during maturation, and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from the excretory ducts formed postnatally. ß 2001 Harcourt Publishers Ltd Keywords: postnatal growth, palatine gland, cell proliferation, excretory duct
Introduction Development of major salivary glands such as parotid, submandibular, and sublingual glands has been extensively studied and many data have been accumulated. Scant attention has been given to the development of the minor salivary glands, however. There are some reports concerning von Ebner's glands (Baumgartner, 1917; Hamosh & Hand, 1978; Kock et al., 1992; Adi et al., 1995), buccal glands (Zimmerman & Zeidenstein, 1951; Redman, 1972), and labial glands (Zimmerman & Department of Oral Health Sciences, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan Received 1 May 2001 Accepted 3 September 2001 Correspondence to: Dr Shiro Nakamura, Department of Oral Health Sciences, Hokkaido University Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo, 060-8586, Japan. Tel. & Fax: 81 11 706 4225; E-mail: [email protected]
614
Zeidenstein, 1951; Goodman & Stern, 1972; Adi et al., 1994, 1995). The palatine gland has many short excretory ducts passing from the gland to oral cavity and forms an almost compact glandular body situated in the submucous layer of the hard and soft palates (DuBrul, 1988). Clinically, the palate is the most common site for benign or malignant neoplastic changes of minor salivary gland origin (Hjertman & Eneroth, 1970; Chung et al., 1978; Weisberger et al., 1979). Because of this it is of interest to investigate the morphogenesis of the palatine glands. Nielsen and Westergaard (1971) described that the development of the human palatine glands begins at the 11th week of fetal life and that, after branching and canalization of epithelial cord, mucous terminal portions ®rst appear at the 15th week. Srivastava and Vyas (1979) reported that the palatine gland of rats developed from tubular invaginations of the oral epithelium lining the soft palate and that it grows by pouching, resulting in
PALATINE GLAND POSTNATAL GROWTH
the development of glands with large irregular pouches. However, there are few studies of the development of the palatine glands apart from those mentioned above, and little information has been accumulated as for other minor salivary glands. The present study aimed to determine how the palatine glands grow postnatally. To achieve this, rat palatine glands in postnatal life were examined, using histological analysis and immunohistochemistry with anti-5-bromo20 -deoxyuridine (BrdU) antibody to observe the proliferative activity.
Materials and methods Histology Male Wistar rats were used in this study. The animals were killed in groups of three or four at ages of 0, 1, 2, 3, 4, 6, or 8 weeks postnatally by an overdose of pentobarbital sodium. The BrdU, which is incorporated into DNA by cells traversing the S-phase of the cell cycle, was administrated at a dose of 50mg/kg body weight by intraperitoneal injection 1 h prior to the sacri®ce. The whole head was ®xed in 4% buffered paraformaldehyde (pH 7.4) for 24 h and decalci®ed in a mixture of 50% formic acid and 20% tri sodium citrate dehydrate for 4±9 days. After decalci®cation, the head was cut into exact halves at the medial plane and the right half was embedded in paraf®n. Parasagittal 5 m-m thick serial sections were made and stained with haematoxylin-eosin (HE) or Alcian blueKernechtrot. All animal experimentation followed the Guidelines for the Care and Use of Laboratory Animals, Hokkaido University Graduate School of Dental Medicine. Histological measurement Histological measurement with parasagittal serial sections were performed. In the right half of the palatine gland of each animal, the maximum of length and height were measured using eyepiece micrometer discs (Fig. 1a), and the maximum of width was calculated by multiplying thickness of section (5 mm) by the total number of sections from the medial plane to the lateral margin of the palatine gland (Fig. 1b). The total number of excretory ducts of the right half of the palatine gland in each animal was counted out by observing all parasagittal serial sections. In all items, the mean and standard deviation (SD) were calculated for three or four animals at each time point. Immunohistochemistry Five sections from each animal were randomly selected for the immunohistochemistry, and were pretreated with 0.1% trypsin for 20 min at 378C and later with 3N HCl for 10 min at room temperature. After pretreatment, the sections were successively reacted with anti-BrdU mouse monoclonal antibody (DAKO), anti-mouse rabbit
615
biotinylated IgG (DAKO), and streptavidin-biotin peroxidase complex. The sections were rinsed thoroughly with phosphate-buffered saline after each incubation. Peroxidase activity was visualized using diaminobenzidine tetrahydrochloride. Sections were lightly counterstained with haematoxylin. Normal mouse serum was substituted for the primary antibody in the negative control sections. After immunohistochemical staining, a total of 2500 acinar cells and 750 excretory or other types of duct cells were counted for each animal, and the BrdU labeling indices (percentage of cells labeled) were calculated at each time point.
Results Histological observations The palatine glands of all rats were situated from the posterior end of the hard palate to the soft palate. At birth (0 weeks), the palatine glands consisted of ducts and immature acini surrounded by connective tissue (Fig. 2a). The ducts in the glands were composed of cuboidal or squamous epithelial cells and showed no character of ductal components seen in mature salivary glands such as intercalated, striated, or interlobular ducts. These ducts were connected with excretory ducts opening into oral cavity. Each immature acinar cell had a round nucleus situated basally and the cytoplasm around the nucleus was eosinophilic. The luminal cytoplasm of the immature acinar cells was clear and stained positively with Alcian blue. Mitotic ®gures were often seen in ducts and immature acinar cells (Fig. 2b). At 1 week, the number of ducts in the glands was smaller, while acini numbers increased and maturation of the acini had progressed. The acini mainly consisted of mucous cells with rich clear cytoplasm pressing the spindle nuclei against the basal area (Fig. 2c). The shape of the acini was mainly round, and there were also some tubuloacinar structures. The glandular tissue was occasionally organized into a lobule like structure separated by connective tissue. At more than 2 weeks, most acinar cells were mature mucous cells and took the form of tubuloacini. Now there was no duct in the glands, and the palatine glands were composed of only tubulo-acini. The tubulo-acini were joined in the palatine gland and then, at the glandular periphery, they were connected with excretory ducts which opened into the oral cavity (Fig. 2d). In the posterior region of the palatine glands, serous acinar cells were very rarely observed. These cells had capped mucous acini and formed demilunes (Fig. 2e). At all experimental periods, there were no epithelial cords extending from the palatal epithelium toward the palatine glands. Figure 3 shows the changes in the dimensions of the palatine glands. The length, width, and height of the right part of the palatine gland constantly increased from 0 to 8
616
NAKAMURA ET AL.
1a A
NC
H
P
PG L OC A
1b
PG
W
P
Fig. 1 Schematic figures of histological measurement. Throughout PG, palatine gland; A, anterior; P, posterior. a: parasagittal view of the palate. Length (L) and height (H) of the right half of the palatine gland. NC, nasal cavity; OC, oral cavity. b: Surface view of the palate. Width (W) of the right half of the palatine gland.
weeks of age. At 8 weeks the approximate length was four times, the width was twice, and the height was 10 times that at 0 weeks. The changes in the number of the excretory ducts of the palatine glands, not including other types of ducts, are presented in Figure 4 There were many excretory ducts at 0 weeks (average 23.0). This number increased sharply up to 3 weeks of age and thereafter the increase was moderate. Immunohistochemical observations At 0 weeks, a number of BrdU positive cells were seen in immature acini (Fig. 5a), ducts in the palatine glands (Fig. 5b) and excretory ducts. Fibroblast-like cells in the
stroma were also often BrdU positive. At 1 week, many maturing acinar and duct cells were still BrdU positive (Fig. 5c). However, after that, these BrdU positive cells gradually decreased (Fig. 5d). The BrdU labeling indices of acinar and duct cells are demonstrated in Figure 6. At 0 and 1 week, the labeling index for duct cells included the ducts in the palatine glands and the excretory ducts, while after 2 weeks it included only excretory ducts owing to disappearance of the ducts in the glands. At 1 week the index for acinar cells reached a peak of 10.1%, after which it declined to 0.2% at 8 weeks. The index for duct cells was highest at 10.1% at 0 weeks and thereafter it decreased to 2.5% at 8 weeks.
PALATINE GLAND POSTNATAL GROWTH
2a
2d
2b
2e
617
2c
Fig. 2 Histology of the rat palatine gland during postnatal development (HE). a: 0 weeks. The palatine gland consists of ducts (arrows) and immature acini
(arrowheads). x160. b: 0 weeks. A mitotic figure of immature acinar cell (arrowhead). x480. c: 1 week. Acini increase in number and their maturation has progressed. x320. d: 8 weeks. The palatine gland is composed of only mucous tubulo-acini. x80. e: A serous acinar cell forming a demilune (arrow). x400.
Discussion In the early stage of postnatal development of rat palatine glands, immature acinar cells, as transitional structures, were often observed in the present study. This suggests that in this period differentiation from duct to acinar cells occur actively in the palatine glands of rats.
During postnatal development of the palatine glands, the length, width, and height of the palatine glands increased constantly. Both acinar and duct cells were highly proliferative in the early stage of postnatal growth and maintained the proliferative activity throughout all stages of postnatal life. These ®ndings indicate that proliferation of the parenchymal cells
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10
8
6 mm
length width
4
height
2
0 0
1
2
3
4 5 Time (weeks)
6
7
8
9
Fig. 3 Postnatal growth of length (solid line), width (broken line), and height (dotted line) of the right part of the palatine glands. Error bars show SD.
The number of excretory ducts
60
50
40
30
20
10
0
0
1
2
3
4 5 Time (weeks)
6
7
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9
Fig. 4 Postnatal changes in the number of excretory ducts of the palatine glands. The results are expressed as means + SD.
contribute to the growth of the palatine glands. The proliferative activity of acinar and duct cells has also been identi®ed during the development of parotid (Redman & Sreebny, 1970; Klein, 1982; Redman, 1995), submandibular (Chang, 1974; Alvares & Sesso, 1975; Klein, 1982), and sublingual glands (Taga & Sesso, 1998), and in the regeneration of parotid (BurfordMason et al., 1993; Cummins et al., 1994; Takahashi et al., 1998) and submandibular glands (Takahashi & Wakita, 1993). This allows the conclusion that parenchymal cell proliferation is very important for the rapid increase in the size of salivary glands. It is known that there are many excretory ducts of the palatine glands. However, it is not known how the number of the excretory ducts changes during development. This study demonstrated that the excretory
ducts increase in number during the postnatal period. The process of developmental events of salivary glands is initiated by the proliferation of oral epithelial cells and their downgrowth into the underlying mesenchyme, resulting in lumen formation of the epithelial rudiment and differentiation of the duct cells to acinar cells (Redman, 1987). As the excretory ducts originate from the oral epithelium, the increase in excretory duct numbers implies that the excretory ducts arise from the epithelium of the palate during postnatal life and suggests that glandular tissue is newly formed from these excretory ducts. Therefore, the increase in the excretory duct numbers is possibly another factor in the growth of the palatine glands. However, no epithelial cords extending from the oral epithelium toward the palatine glands were observed in this study. It may be dif®cult to identify
PALATINE GLAND POSTNATAL GROWTH
5a
5c
5b
5d
619
Fig. 5 Immunohistochemistry for BrdU. a: 0 weeks. Immature acinar cells (arrows) are BrdU-positive. x600. b: 0 weeks. There are BrdU-positive duct cells
in the palatine gland (arrowheads). x600. c: 1 week. Many maturing acinar cells are BrdU-positive. x400. d: 6 weeks. A few BrdU positive cells are seen in acinus (arrowhead) and excretory duct (arrows). x200.
12
Labeling index of BrdU (%)
10
Acinar cell
8
Duct cell 6
4
2
0
0
1
2
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4 5 Time (weeks)
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9
Fig. 6 BrdU labeling indices of acinar cells (dotted line) and duct cells (solid line) of the palatine glands. The results are expressed as means + SD.
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extending epithelial cords, because the distance between the oral epithelium and the palatine glands is very short and the extending epithelial cords reach the palatine glands in a short time. According to some oral histology textbooks (Hand, 1986; Dale, 1998), human palatine glands are classi®ed as pure mucous. Recently, Riva et al. (1999) observed seromucous acinar cells and de®ned the human palatine glands as mixed glands. There are two suggestions for rat palatine glands, that they have no (Odajima, 1975) or some serous acinar cells (Leeson & Leeson, 1968). In this study, serous acinar cells forming demilunes were identi®ed by the thorough observation of the serial sections of the rat palatine glands. Serous acinar cells were rare and located in the posterior portion of the palatine glands and it may be dif®cult to detect these cells by electron microscope (Odajima, 1975). In conclusion, this study demonstrated that immature palatine glands of newborn rats grow threedimensionally as they mature and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from excretory ducts formed postnatally. ACKNOWLEDGEMENTS We thank Emeritus Professor H. Tani, Hokkaido University Graduate School of Dental Medicine, for his stimulating suggestions regarding this study. REFERENCES Adi, M.M., Chisholm, D.M. and Waterhouse, J.P. 1994. Stereological and immunohistochemical study of development of human fetal labial salivary glands and their S-100 protein reactivity. J. Oral Pathol. Med., 23, 36±40. Adi, M.M., Chisholm, D.M. and Waterhouse, J.P. 1995. Histochemical study of lectin binding in the human fetal minor salivary glands. J. Oral Pathol. Med., 24, 130±135. Alvares, E.P. and Sesso, A. 1975. Cell proliferation, differentiation and transformation in the rat submandibular gland during early postnatal growth. A quantitative and morphological study. Arch. Histol. Jap., 38, 177±208. Baumgartner, E.A. 1917. The development of the serous glands (von Ebner's) of the vallate papillae in man. Am. J. Anat., 22, 365±383. Burford-Mason, A.P., Cummins, M.M., Brown, D.H., Mackay, A.J. and Dardick, I. 1993. Immunohistochemical analysis of the proliferative capacity of duct and acinar cells during ligationinduced atrophy and subsequent regeneration of rat parotid gland. J. Oral Pathol. Med., 22, 440±446. Chang, W.W.L. 1974. Cell population changes during acinus formation in the postnatal rat submandibular gland. Anat. Rec., 178, 187±202.
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