Early markers of regeneration following ductal ligation in rat submandibular gland

Rat submandibular glands can recover their function and secretory protein content following ductal ligation-induced atrophy. Morphological studies have established that following ligation, deligation of the gland allows the regeneration of new salivary gland tissue. However, little is known about changes happening during early regeneration following intra-oral duct ligation, which does not damage the parasympathetic nerves. Glands that had been 2 weeks ligated or 2 weeks ligated + 3 days deligated were compared. Tissue was prepared for histological, immunohistochemical (SMG-B and Ki-67) and immunocytochemical analyses (smooth muscle actin, aquaporin 5). Haematoxylin and eosin staining of deligated glands showed that some acini regained their cytoplasmic volume; moreover, the loss of Alcian blue/periodic acid-Schiff’s staining from the lumen of ducts suggested successful deligation. The deligated gland was characterized by atypical acinar-ductal branched structures, which were less frequent in the ligated gland and rarely seen in normal unoperated tissue. Myoepithelial cells were also investigated since changes in their morphology reflected changes in the acini morphology not readily detected by conventional staining. Actin staining revealed the presence of some shrunken acini in the atrophic tissue, whereas they had regained their normal morphology in the deligated gland suggesting that the acini were recovering. Some acini during deligation regained aquaporin 5 expression, which had decreased during atrophy. SMG-B protein, located in the pro-acinar cell during gland development and usually found in the intercalated duct cells in the adult, was detected in the newly formed acini of the deligated gland. This study suggests that morphological markers of regeneration appear as early as 3 days following ligation removal. The authors thank the Wellcome Trust for funding.     

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.     

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.     

Mitogen-activated protein kinase up-regulation and activation during rat parotid gland atrophy and regeneration: role of epidermal growth factor and beta2-adrenergic receptors

Abstract The rat secretory ductal obstruction model has been widely used to assess salivary gland injury, growth, and differentiation. In this study, a novel ductal obstruction and release procedure was used to explore the signaling pathways leading to salivary gland regeneration. Rats underwent bilateral parotid ductal obstruction in which the duct was occluded against a plastic disk subcutaneously and released by external ligature removal. This ductal obstruction/release procedure was validated to produce glandular atrophy and regeneration with histological analysis and periodic acid-Schiff staining. Immunoblot analysis indicated that during ductal obstruction and the early post-release period (day 7), expression of immunoreactive proliferating cell nuclear antigen and vimentin was increased in the parotid glands compared with sham-operated animals. Immunohistochemical staining and immunoblots revealed up-regulation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated receptor kinase (ERK)1/2, and p38 during the atrophic and regeneration phases of ductal obstruction/release. Similarly, increases in activated, i.e., phosphorylated, ERK1/2 (pERK1/2) and p38 (phospho-p38) were demonstrable in both ductal and recovering acinar cells, with pERKs expressed predominantly in the nuclei and phospho-p38 distributed throughout the cells. Furthermore, levels of epidermal growth factor (EGF) receptor and β2-adrenergic receptor (β2-AR) were elevated in the ligated glands and at day 7 post-release; β1-AR levels did not change over the same time period. These results support the view that cell proliferation is involved in duct ligation-induced atrophy of the rat parotid gland and gland recovery upon ligature removal. Up-regulation of ERKs and p38, and the activation of these MAPKs by up-regulated EGF and β2-ARs, may be important signaling components underlying glandular atrophy and subsequent regeneration.     

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands

 Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands.With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay. Accepted: 19 December 1996     

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.     

Ultrastructural and immunoelectron microscopic studies on infiltrating mononuclear cells in lymphocytic submandibulitis in NOD mice

The ultrastructural relations of the infiltrating mononuclear cells to the parenchymal tissues were studied in the submandibular gland of the female non-obese diabetic (NOD) mouse. In addition, the phenotype of mononuclear cells infiltrating the submandibular gland has been determined by light and electron microscopy by using monoclonal antibodies against T-cell subsets (Thy1.2, Lyt1, Lyt2). Ultrastructurally, lymphoid cells were frequently observed around and in the acini and ducts. Some of the lymphoid cells observed in the acini and ducts were irregular in shape and sometimes sent spike-like projections into acinar and ductal cells. Immunohistochemical study demonstrated that Thy1.2+ cells were predominant among the infiltrating cells, and the majority of these infiltrating T-cells were composed of Lyt1+ cells with a small proportion of Lyt2+ cells. By immunoelectron microscopy, lymphocytes carrying Thy1.2, Lyt1 or Lyt2 antigen were identified, as is demonstrated by an electron-dense reaction product on the entire cell surface, and these immunopositive cells were frequently observed around and in the acini and ducts. Some of the Thy1.2+, Lyt1+ or Lyt2+ cells observed in the acini and ducts demonstrated a close contact with acinar and ductal cells and both Lyt1+ and Lyt2+ cells sent spike-like projections into them. Occasionally, a partial degeneration of acinar cell adjacent to the invading lymphocytes was observed. These observations suggest that T-lymphocytes are involved in the direct destruction of acinar and ductal cells in the NOD mouse submandibular glands.     

Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation

Summary The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0–14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.     

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.     

Salivary glands of the cat: A histochemical study

Synopsis The submandibular, sublingual and parotid glands of the cat have been studied. Mucosubstance histochemistry demonstrated acidic mucosubstances with varying properties in the acini. Thiamine pyrophosphatase and nucleoside diphosphatase reaction products were seen with a Golgi-like appearance in acinar cells. Granules of acid phosphatase, -glucuronidase and E600-resistant esterase reaction products, presumably representing lysosomal enzyme activities, were seen in acinar and ductal cells. Diffuse acid phosphatase and -glucuronidase reaction products were seen in central cells of the submandibular acini, and diffuse non-specific esterase reaction product was seen in acinar and ductal cells. Arylamidase reaction product was associated with some acinar cells. Reaction product from a peroxidase technique was seen in demilunar cells of the submandibular acini, in parts of the sublingual acini, in parotid acini, and in ductal cells. Cytochrome oxidase and succinate dehydrogenase reaction products were seen most strongly in striated ducts, whereas NADH- and NADPH-diaphorase reaction products were seen at a high level throughout the ducts.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.     

Localization of kallikrein in porcine pancreas and submandibular gland as revealed by the indirect immunofluorescence technique.

The glandular kininogenase kallikrein is known to occur in many mammalian organs and glands but direct histochemical localization has been achieved in only a few cases. We have now been able to localize porcine kallikrein in the acinar cells of the pancreas and in the striated and collecting duct cells of the submandibular gland. Incubation of frozen and fixed sections with one of the crossreacting antibodies, anti-pancreatic, anti-submandibular or anti-urinary kallikrein IgG resulted in the same immunofluorescence pattern. There was evidence of a specific fluorescence neither in the acinar cells, nor in the interstitial tissue or blood cells of the submandibular gland nor in the islets of Langerhans, the interlobular ducts or blood vessels of the pancreas. From all data now available about glandular kallikreins, it seems that the kallikreins in these organs are very similar.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

The effect of pilocarpine and food uptake on the rate of incorporation of [3H]-leucine in vivo was measured by means of quantitative radioautography in three exocrine cells of the rat: the acinar and the granular duct cells of the submandibular and the acinar cells of the parotid gland. The three cell types react differently. The submandibular acinar cells showed a decrease in incorporation rate after pilocarpine administration but not after feeding. The incorporation rate of the granular duct cells of the submandibular gland remains constant after both stimulations. The acinar cells of the parotid gland show an increase in incorporation rate of [3H]-leucine in response to both. The contrast between the submandibular and the parotid gland could also be demonstrated radiobiochemically, the results reflecting the incorporation rates of the acinar cells of both glands, giving no information on the contribution of other cell types. The decrease in incorporation rate of the submandibular gland acinar cells is accompained by a shift of polyribosomes towards monomers.     

Fine structure of the mandibular gland of the Djungarian hamster (Phodopus sungarus)

The mandibular gland of the Djungarian hamster was examined by light microscopy, and transmission and scanning electron microscopies. Its acinar cells reacted with periodic acid-Schiff (PAS) and were weakly stained with alcian blue (AB). There were intercellular canaliculi between the acinar cells. These cells therefore appeared to be seromucous. The acinar epithelium was composed of light cells containing various spherical secretory granules. The granular cells of the mandibular gland possessed many acidophilic granules exhibiting a positive reaction to PAS stain. They were frequently observed at the junction of the acini and intercalated ducts in all mandibular glands examined. All of these cells were light and contained secretory granules of varying size and density. The intercalated ducts consisted exclusively of light cells possessing a few round granules of high density in the apical region. The striated ducts were comprised of two portions--a secretory portion and a typical striated portion without secretory granules. The secretory portion consisted of light, dark and specifically light epithelial cells containing acidophilic granules, which exhibited a strongly positive PAS reaction. The epithelium of typically striated portions was composed of light and dark cells containing fine vacuoles in the apical region. The mandibular gland of the Djungarian hamster revealed no histological differences between sexes.     

Occurrence and Nuclear Localization of cAMP Response Element- Binding Protein in the Post-natal Development of the Rat Submandibular Gland

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5 promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the post-natal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1–2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the r352;-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar ce lls that are regulated by r352;-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

The intact parasympathetic nerve promotes submandibular gland regeneration through ductal cell proliferation

ObjectivesSalivary gland regeneration is closely related to the parasympathetic nerve; however, the mechanism behind this relationship is still unclear. The aim of this study was to evaluate the relationship between the parasympathetic nerve and morphological differences during salivary gland regeneration.Materials and MethodsWe used a duct ligation/deligation‐induced submandibular gland regeneration model of Sprague‐Dawley (SD) rats. The regenerated submandibular gland with or without chorda lingual (CL) innervation was detected by haematoxylin–eosin staining, real‐time PCR (RT‐PCR), immunohistochemistry and Western blotting. We counted the number of Ki67‐positive cells to reveal the proliferation process that occurs during gland regeneration. Finally, we examined the expression of the following markers: aquaporin 5, cytokeratin 7, neural cell adhesion molecule (NCAM) and polysialyltransferases.ResultsIntact parasympathetic innervation promoted submandibular gland regeneration. The process of gland regeneration was significantly repressed by cutting off the CL nerve. During gland regeneration, Ki67‐positive cells were mainly found in the ductal structures. Moreover, the expression of NCAM and polysialyltransferases‐1 (PST) expression in the innervation group was significantly increased during early regeneration and decreased in the late stages. In the denervated submandibular glands, the expression of NCAM decreased during regeneration.ConclusionsOur findings revealed that the regeneration of submandibular glands with intact parasympathetic innervation was associated with duct cell proliferation and the increased expression of PST and NCAM.