ARTICLE |
Correspondence to: Shigeru Takahashi, Dept. of Oral Health Sciences, Hokkaido University Graduate School of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo 060-8586, Japan. E-mail: tshigeru@den.hokudai.ac.jp
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Summary |
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This study was designed to determine whether apoptosis and proliferation of myoepithelial cells occur in atrophic rat submandibular glands. The excretory duct of the right submandibular gland was doubly ligated with metal clips. The atrophic right submandibular glands removed after 128 days of duct ligation were investigated using immunohistochemical double staining for actin as a marker for myoepithelial cells and proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells, double staining for actin immunohistochemistry, nick end-labeling (TUNEL) as a marker for apoptotic cells, and transmission electron microscopy (TEM). A few PCNA- and no TUNEL-positive myoepithelial cells were found in the control submandibular glands taken from animals with no operation. In the experimental glands, PCNA-positive myoepithelial cells were common 2 and 3 days after duct ligation and then decreased in number. TUNEL-positive myoepithelial cells appeared at 2 days and were observed most frequently at 5 days. Apoptotic myoepithelial cells were also identified by TEM. These observations suggest that both apoptosis and proliferation of myoepithelial cells occur, especially in the early phase of atrophy, in the rat submandibular gland. (J Histochem Cytochem 49:15571563, 2001)
Key Words: apoptosis, proliferation, myoepithelial cell, submandibular gland, atrophy
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Introduction |
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Myoepithelial cells are observed in several exocrine glands, and processes from these cells surround acini and ducts. Although the structure and function of myoepithelial cells are similar to those of smooth muscle cells, they also display the characteristics of epithelium in that they are situated between secretory or duct cells and basal lamina (
Myoepithelial cells have been speculated to play an important role in the histogenesis of some salivary gland tumors, such as pleomorphic adenoma, myoepithelioma, adenoid cystic carcinoma, and certain other tumors (
Therefore, it is both important and interesting from the clinical aspect to determine whether or not proliferation and apoptosis of myoepithelial cells occur in atrophic submandibular gland. The purpose of the present study was to clarify this problem. To achieve this, rat submandibular glands with atrophy induced by doubly ligated ducts were examined, using immunohistochemical double staining for actin and proliferating cell nuclear antigen (PCNA), double staining for actin immunohistochemistry, and nick end-labeling (TUNEL) and TEM.
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Materials and Methods |
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Experimental Procedures
Seven-week-old male Wistar rats were used in this study. In the experimental rats, the right submandibular gland and its excretory duct were dissected under general anesthesia with ether. Then the excretory duct was doubly ligated with metal clips near the hilum of the gland (
All animal experimentation followed the Guide for the Care and Use of Laboratory Animals, Hokkaido University Graduate School of Dental Medicine.
Immunohistochemical Double Staining for Actin and PCNA
The sections were exposed to an anti-PCNA mouse monoclonal antibody (MAb) (PC-10; DAKO JAPAN, Kyoto, Japan), a marker for proliferating cells (-smooth muscle actin (1A4; DAKO JAPAN), a marker for myoepithelial cells. Actin localization was developed with a VIP Substrate Kit (Vector Laboratories; Burlingame, CA) and the sections were counterstained with methyl green.
Normal mouse serum was substituted for both primary antibodies as a negative control.
Single immunohistochemistry for actin was also performed, omitting the part with PCNA from the above immunohistochemical double staining.
Double Staining for Actin Immunohistochemistry and TUNEL
The apoptotic cells were identified by the modified TUNEL method (
Negative control sections were incubated with distilled water and normal mouse serum in the absence of TdT for TUNEL and anti-actin antibody for immunohistochemistry, respectively.
In both types of double staining, the actin-positive reaction was realized as purple cytoplasm and the PCNA- and TUNEL-positive reaction as brown nuclei.
Quantification
The labeling indices of PCNA and TUNEL in myoepithelial cells were calculated. In doubly stained sections, approximately 200 myoepithelial cells were randomly counted at a magnification of x400 (BH-2; OLYMPUS, Tokyo, Japan) and the percentage of double-positive cells was calculated. The mean of the percentages from four stained sections was used as a representative value for that animal. The means and standard error of the mean (SEM) were calculated for four experimental rats at each time point and four control rats, and then one-way analysis of variance (ANOVA), followed by the Fisher's protected least significant difference (PLSD) post-hoc test, were performed with StatView 4.5 statistical software (Abacus Concepts; Berkeley, CA).
Transmission Electron Microscopy
The fixed tissue was immersed in 1% osmium tetroxide, stained en bloc with 4% uranyl acetate, and embedded in Epon 812. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope (H-7000 electron microscope; HITACHI, Tokyo, Japan).
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Results |
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Histologically, ducts were dilated in the edematous glandular tissue and mitotic figures of duct cells were identified at 2 days after duct ligation. At 3 and 4 days, apoptosis of acinar cells was frequently observed and acinar cell numbers decreased. After 7 days most acinar cells had disappeared and the connective tissue with lymphoid cell infiltration increased around the residual ducts (
PCNA and TUNEL Observations
Single immunohistochemical staining for actin showed the distribution of myoepithelial cells in the submandibular glands. In the controls, myoepithelial cells surrounded acini and intercalated ducts. However, they were not observed at the periphery of striated ducts (Fig 1A). In the experimental glands, the atrophic acini were still surrounded by myoepithelial cells at 3 and 4 days (Fig 1B). With the disappearance of acini, many dilated ducts were surrounded by myoepithelial cells (Fig 1C).
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A few PCNA-positive myoepithelial cells were identified in the control glands (Fig 1D). In the duct ligated gland, PCNA-positive myoepithelial cells were often observed, especially at 2 and 3 days (Fig 1E). No TUNEL-positive myoepithelial cells were identified in the control glands (Fig 1F). However, in the experimental glands TUNEL-positive myoepithelial cells were observed at 2 days and later they were identified in all experimental specimens (Fig 1G). Negative control sections for both kinds of double staining showed no reaction.
The labeling indices of PCNA and TUNEL for myoepithelial cells are shown in Table 1 and Fig 2. The PCNA labeling index increased significantly between 1 and 2 days (p<0.0001) after duct ligation, reached a peak of 7.55% at 3 days, and significantly declined between 3 and 4 days (p<0.0005). The TUNEL- positive myoepithelial cells first appeared at 2 days. The TUNEL labeling index increased significantly between 4 and 5 days (p<0.0001) and was highest, 3.33%, at 5 days, after which it declined significantly between 5 and 7 days (p<0.0005). The PCNA and TUNEL indices in the control glands were 1.18% and 0%, respectively.
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TEM Observations
In the control glands, myoepithelial cells were located between the basal lamina and acinar or intercalated duct cells. In the cytoplasm, microfilaments, sometimes associated with dense bodies, were observed. There were caveolae along the basal plasma membrane (Fig 3A).
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In the experimental glands, ultrastructural changes of myoepithelial cells were remarkable from 3 days after duct ligation. Myoepithelial cells often showed bizarre structures, with cell processes protruding into interstitial spaces. The basement membrane of such myoepithelial cells tended to be thickened and arranged in many irregular folds (Fig 3B). Apoptosis of myoepithelial cells was rarely identified. In apoptotic myoepithelial cells, nuclear chromatin was aggregated into a dense mass and the electron density of the cytoplasm was increased (Fig 3C). Some myoepithelial cells had phagocytosed apoptotic bodies (Fig 3D).
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Discussion |
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In atrophy of the parotid gland (
It has been shown that precursors of or immature myoepithelial cells are able to proliferate in cultured and transplanted human mammary glands (
The maximum of the PCNA index of myoepithelial cells in the present study (7.55%) is much lower than that in atrophic parotid gland (23.1%) (
Using TEM and double staining for actin immunohistochemistry and TUNEL, this study demonstrates that apoptosis of myoepithelial cells occurs in atrophic rat submandibular glands. This is the first report to identify apoptotic myoepithelial cells in the salivary gland, although apoptosis of myoepithelial cells was reported in involution of lactating breasts in mice (
It has been understood that apoptotic bodies are phagocytosed by intraepithelial macrophages, duct cells, and acinar cells in atrophic salivary glands (
Recently, P2Y2 receptor, one of the receptors for extracellular nucleotides (P2 receptors), was reported to be an important component of the response to injury in submandibular gland from the fact that P2Y2 receptor activity and mRNA levels were increased in duct ligated submandibular gland (
In conclusion, the present study demonstrates that both apoptosis and proliferation of myoepithelial cells occur in atrophy of rat submandibular glands.
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (no. 13671961).
We gratefully acknowledge the assistance of Dr Neal I. Walker (Brisbane, Australia) with the manuscript.
Received for publication March 27, 2001; accepted June 19, 2001.
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