Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation

Summary This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.     

Apoptosis and proliferation of myoepithelial cells in atrophic rat submandibular glands.

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 1-28 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.     

The roles of apoptosis and mitosis in atrophy of the rat sublingual gland

The roles of apoptosis and mitosis of acinar and duct cells in the atrophy of the sublingual gland of rat induced by double duct ligation was investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), and transmission electron microscopy (TEM). Many PCNA-positive duct cells were observed 3 days after duct ligation, and the numbers decreased thereafter. At 3 and 5 days, several TUNEL-positive acinar cells were observed and typical apoptotic acinar cells were identified by TEM. Necrotic acinar cells were also observed ultrastructurally. After 7 days, there were few acini but many ducts, as well as many structures representing transition from acinus to duct. These observations demonstrate that acinar cell loss by apoptosis and duct cell proliferation by mitosis occur in atrophic sublingual glands as well as in other atrophic salivary glands. In addition, it appears that the transition from acinar to duct cell and the necrosis of acinar cells play important roles in the atrophy of the sublingual gland.     

Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.     

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Immunofluorescence-microscopic demonstration of myosin and actin in salivary glands and exocrine pancreas of the rat

Summary Actin and myosin were localized in various salivary glands (parotid, submandibular, sublingual, lingual and Harderian gland) and the exocrine pancreas of rats by indirect immunofluorescence microscopy using specific rabbit antibodies against chicken gizzard myosin and actin. A bright immunofluorescent staining with both antibodies was observed at three main sites: (1) In myoepithelial cells of all salivary glands, (2) in secretory gland cells underneath the cell membrane bordering the acinar lumen (except Harderian and mucous lingual gland), and (3) in epithelial cells of the various secretory ducts (of all glands) in similar distribution as in acinar cells. The present immunohistochemical findings in acinar cells could lend further support to a concept suggesting that myosin and actin are involved in the process of transport and exocytosis of secretory granules.Supported by grants form Deutsche Forschungsgemeinschaft (Dr. 91/1, Ste. 105/19 and U. 34/4). We thank Mrs. Ursula König, Mrs. Christine Mahlmeister and Miss Renate Steffens for excellent technical assistance.     

Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet

Parotid glands of experimental animals fed a liquid diet are reported to show atrophy (Hall and Schneyer 1964; Wilborn and Schneyer 1970; Hand and Ho 1981; Scott et al. 1990; Scott and Gunn 1991). To clarify whether apoptosis and proliferation of acinar cells participate in atrophy of rat parotid glands induced by liquid diet, rats were fed a liquid diet and compared to pellet-fed controls. Parotid glands were removed at 3, 7, 14 or 21?days, weighed, and examined using transmission electron microscopy (TEM), and studied immunohistochemically for cleaved-caspase-3 (Casp-3), a marker of apoptotic cells, and 5-bromo-2′-deoxyuridine (BrdU), a marker for proliferating cells. Body weights of experimental rats fed liquid diets were not significantly different from controls fed pellet diets; however weights of experimental parotid glands were smaller than those of controls. In the experimental parotid glands, structures like apoptotic bodies were histologically observed in acini at each time point; more Casp-3-positive acinar cells were identified in experimental parotid glands than in the controls on days 3, 7, and 14. Experimental glands showed fewer BrdU-positive acinar cells at each time point. TEM confirmed typical apoptotic acinar cells in the atrophic glands. These findings suggest that increased acinar cell apoptosis and reduced acinar cell proliferation occur in atrophic parotid glands of rats fed a liquid diet.     

Immunohistochemical and ultrastructural investigation of acinar cells in submandibular and sublingual glands of rats fed a liquid diet

In atrophic parotid glands induced by liquid diet, acinar cell apoptosis is increased while proliferative activity is reduced. This study aimed to clarify how liquid diet affects submandibular and sublingual glands, including acinar cell apoptosis and proliferation. Seven-week-old male Wistar rats were fed either a liquid (experimental group) or pellet diet (control group) from 3 to 21 days, respectively. Submandibular and sublingual glands were weighed and examined histologically, ultrastructurally, and immunohistochemically using antibodies to cleaved caspase-3 (Casp-3) and 5-bromo-2′-deoxyuridine (BrdU). Weights of submandibular and sublingual gland from the experimental group were not significantly different from controls at any time point. Histological and ultrastructural characteristics of experimental acinar cells in both glands were normal. Acinar cells in control and experimental submandibular glands were positively stained with periodic acid Schiff (PAS) and weakly stained by alcian blue (AB). In control and experimental sublingual glands, mucous acinar cells were PAS-positive and strongly AB-positive. Although Casp-3- and BrdU-positive acinar cells were identified in both glands in the experimental group, their labeling indices were not significantly different from controls. In conclusion, liquid diet in rats does not induce atrophic alterations to acinar cells, including apoptosis and proliferative activity in submandibular and sublingual glands.     

p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas

The mechanisms linking acinar cell apoptosis and ductal epithelial proliferation remain unknown. To determine the relationship between these events, pancreatic duct ligation (PDL) was performed on p53(+/+) and p53(-/-) mice. In mice bearing a wild-type p53 allele, PDL resulted in upregulation of p53 protein in both acinar cells and proliferating duct-like epithelium. In contrast, upregulation of Bcl-2 occurred only in duct-like epithelium. Both p21(WAF1/CIP1) and Bax were also upregulated in duct-ligated lobes. After PDL in p53(+/+) mice, acinar cells underwent widespread apoptosis, while duct-like epithelium underwent proliferative expansion. In the absence of p53, upregulation of p53 target genes and acinar cell apoptosis did not occur. The absence of acinar cell apoptosis in p53(-/-) mice also eliminated the proliferative response to duct ligation. These data demonstrate that PDL-induced acinar cell apoptosis is a p53-dependent event and suggest a direct link between acinar cell apoptosis and proliferation of duct-like epithelium in duct-ligated pancreas.     

P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues.

p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.     

Immunohistological study of small Rho GTPases and β-catenin during regeneration of the rat submandibular gland

Our study immunohistochemically evaluated the localization patterns of small Rho GTPases and β-catenin during regeneration of the rat submandibular gland. After 7?days of obstruction, regenerating glands were collected at days 0, 3, 7, 11 and 14 after duct release to study regeneration. RhoA was detected strongly and RhoC was detected weakly in the cytoplasm of newly formed acinar cells from day 3 to 7, and both RhoA and RhoC at the basal site and cytoplasm were detected moderately from day 11 to 14. RhoB was detected strongly and moderately in the cytoplasm of newly formed and matured acinar cells, respectively, and detected strongly in duct-like structures (DLSs) and intercalated ducts (ICDs). Rac1 was detected at the cell–cell and subcellular region, but β-catenin was not observed in newly formed acinar cells. Rac1 immunolabeling gradually reduced, and the β-catenin staining pattern became stronger. p-Rac1, a phosphorylated form of Rac1, was observed in the cytoplasm of newly formed acinar cells. At apical and subcellular region of DLSs and ICDs, Rac1 and β-catenin were detected. These findings suggest that RhoA and RhoC might be involved in the actin cytoskeleton at the basolateral site of regenerating acinar cells, and RhoB might play a unique role in regenerating acinar cells and in DLSs and ICDs. Rac1 and β-catenin at the cell–cell region might play important roles in cell–cell adhesion and the differentiation of regenerating acinar cells, as well as actin reconstruction at apical and subcellular regions of DLSs and ICDs.     

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice.

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.     

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70–100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the compostion of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell–cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

Exogenous thyroid hormone affects myoepithelium and proliferation in the developing rat parotid gland

Abstract

In the mature rat parotid gland, myoepithelial cells (MEC) invest intercalated ducts, but not acini. During postnatal development, however, these cells differentiate around both intercalated ducts and acini, then translocate to only intercalated ducts during weaning. Previously, we found that thyroxine (T₄) accelerates translocation of cells with small secretory granules from acini into intercalated ducts and the number of apoptotic cells increased tremendously with high doses. We present here additional analysis of the effects of T₄ on developing rat parotid gland, namely, the distribution of MEC and the proliferation of parenchymal cells. Beginning at age four days, pups were given daily subcutaneous injections of low, medium, and high doses of T₄ or vehicle or no injection. At ages 4, 7, 10, and 15 days, glands were excised and processed for light microscopy. Sections were double-immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and actin, and counterstained with hematoxylin. Proliferative activity was assessed via PCNA histochemistry and MEC were identified using actin histochemistry. MEC in the T₄ groups invested mostly acini at 15 days in vehicle/normal glands and mostly intercalated ducts after 10 days in the T₄ groups. The proliferative activity of acinar cells and MEC in vehicle/normal glands declined progressively with age and T₄ increased the rate of this decline in the MEC in a dose-dependent manner. We conclude that T₄ accelerates the translocation of MEC from acini to intercalated ducts and that an important mechanism is the more rapid decline in the proliferative activity of MEC than in acinar cells in the T₄ groups. Some of the decline in the proliferative activity of all cells in the high and medium dose T₄ groups after seven days may have been due to dose-related thyroxine toxicity.     

The common bile duct ligation in rat: a relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing α-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.     

Growth of rat pancreatic acinar cells quantitated with a monoclonal antibody against the proliferating cell nuclear antigen

The monoclonal antibody PC10 raised against the proliferating cell nuclear antigen (PCNA) was used to study acinar cell replication in the pancreas of rats under different functional conditions. In Western blots, the antibody recognized a single band of 37 kDa in pancreatic homogenates indicating its specificity in this particular species and organ. Three conditions of growth were chosen for immunohistochemical analysis: pancreatic preand postnatal development, pancreatic regeneration after injury, and cholecystokinin-stimulated acinar cell proliferation. The time course of acinar cell replication under each condition was the same as that obtained after tritiated thymidine incorporation with subsequent autoradiography, indicating that the percentage of PCNA-positive cells reflects the pool of cycling cells in the models investigated. However, the absolute number of PCNA-positive cells was two to ten times higher than comparable labeling indices from ³H-thymidine autoradiography. This finding might reflect the half life of PCNA, which exceeds the duration of the S-phase. Thus, PCNA-positive cells not only represent S-phase cells, but also cells that have recently completed the cell cycle.     

Time course of differentiation of different cell types in 3D-reconstructed eccrine sweat glands

Epidermal basal cells invaginate into the dermis to form sweat ducts, which then grow downwards further to form secretory coils during the ontogenesis of eccrine sweat glands, but the time course of differentiation of different cell types in 3D-reconstructed eccrine sweat glands remain unclear. In this study, secretory cell-specific marker K7, clear secretory cell-specific marker CA II, dark secretory cell-specific marker GCDFP-15, myoepithelial cell-specific marker α-SMA, inner duct cell-specific marker S100P and outer duct cell-specific marker S100A2 were detected by immunofluorescence staining. The results showed that S100P and S100A2 were first detected at 2 weeks post implantation, K7 and α-SMA at 3 weeks, and GCDFP-15 and CA II at 4 weeks. The differentiation of ducts preceded secretory coils in 3D-reconstructed eccrine sweat glands. After 8 weeks post implantation, the distribution of these markers in 3D-reconstructed eccrine sweat glands was similar to that in native ones, and the percentage of the 3D-reconstructed glands expressing these markers maintained steady. We conclude that although the 3D-reconstructed and native eccrine sweat glands originated from different cells, the differentiation of different cell types in 3D-reconstructed eccrine sweat glands parallels the sequence observed during embryonic development.