Induction of a specific (LM) protein in the submandibular gland of the rat by repeated amputation of the lower incisor teeth

Chronic administration of the beta-adrenergic agonist isoproterenol (IPR) leads to marked hyperplastic/hypertrophic enlargements of the parotid and submandibular glands in rats and mice with concomitant changes in the composition of both the glands and the saliva. Conspicuous among the alterations of the submandibular saliva is the appearance of a 13,000 Mr protein, termed LM (large mobile) protein. Repeated amputation of the lower incisor teeth also causes enlargements of the major salivary glands in rats. In this study, we have compared the enlargements of submandibular glands of rats produced by IPR administration or teeth amputation with respect to the relative levels of the LM protein in gland extracts and saliva. Administration of IPR-HCl (40 mg/kg) twice daily for 5 days or amputation of the lower incisor teeth 3 times a week for 3 weeks resulted in a 2.2-fold increase in the weight of the submandibular gland. Amputation for one week led to a 1.4-fold increase in gland weight. Double immunodiffusion in agar antibodies against the purified LM protein gave a single precipitin line with gland extracts and saliva of IPR-treated and teeth-amputated rats, indicating immunological identity of the reacting antigens. No precipitin lines were seen with gland extracts or saliva of untreated rats. Immunoblots of pooled saliva obtained from IPR-treated or teeth-amputated rats revealed a single protein band of the same electrophoretic mobility in SDS-polyacrylamide gels when stained using anti-LM antibodies. The relative concentrations of LM protein in gland extracts and saliva were measured by a solid-phase enzyme-linked immunoabsorption assay using antibodies against the purified LM protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenomenon of hypertrophy of the testes after amputation of the lower incisors in rats]

Repeated amputation of the lower incisors in rats carried out at the early postnatal on togenesis causes an enlargement of the salivary glands and hypertrophy of the testis with the phenomena of acceleration of their differentiation. Hypertrophy of the testes withthe mentioned action was much greater (76%) than in the case of unilateral castration at the same age. In adult animals repeated amputation of the lower incisors caused hypertrophy of the salivary glands, but failed to influence the hypertrophy of the testes.     

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.     

Clonal proliferation of multipotent stem/progenitor cells in the neonatal and adult salivary glands

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD=+/-7.02)h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2+/-4.18 vs. 4.5+/-1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands.     

Rescue of salivary gland function after stem cell transplantation in irradiated glands

Head and neck cancer is the fifth most common malignancy and accounts for 3% of all new cancer cases each year. Despite relatively high survival rates, the quality of life of these patients is severely compromised because of radiation-induced impairment of salivary gland function and consequential xerostomia (dry mouth syndrome). In this study, a clinically applicable method for the restoration of radiation-impaired salivary gland function using salivary gland stem cell transplantation was developed. Salivary gland cells were isolated from murine submandibular glands and cultured in vitro as salispheres, which contained cells expressing the stem cell markers Sca-1, c-Kit and Musashi-1. In vitro, the cells differentiated into salivary gland duct cells and mucin and amylase producing acinar cells. Stem cell enrichment was performed by flow cytrometric selection using c-Kit as a marker. In vitro, the cells differentiated into amylase producing acinar cells. In vivo, intra-glandular transplantation of a small number of c-Kit(+) cells resulted in long-term restoration of salivary gland morphology and function. Moreover, donor-derived stem cells could be isolated from primary recipients, cultured as secondary spheres and after re-transplantation ameliorate radiation damage. Our approach is the first proof for the potential use of stem cell transplantation to functionally rescue salivary gland deficiency.     

Influence of submandibular salivary glands on hormone responsiveness of mouse mammary glands

Surgical removal of the submandibular salivary glands (sialoadenectomy) of female Balb/c mice significantly (P less than 0.05) reduced mammary development as judged by development scores and mammae DNA levels. Reduction in mammae development score by sialoadenectomy was observed in both mice saline injected and mice treated with estradiol and progesterone. Autografts of submandibular salivary tissue or daily administration of EGF to sialoadenectomized mice partly alleviated the atrophy of the mammary gland induced by sialoadenectomy (P less than 0.05). The results of our studies are consistent with a model of mammary gland developmental regulation that includes the submandibular salivary gland as a mediator of mammogenesis via secretion of EGF.     

Microvascular transplantation of the rat submandibular gland

Xerostomia results from salivary gland irradiation during treatment of head and neck malignancies. In addition to having difficulty with speech and swallowing, these patients experience loss of taste, dental caries, and chronic fungal infections. The paired submandibular glands provide 70 percent of the normal salivary flow and are difficult to shield during radiation therapy. Another sicca condition, xerophthalmia, may result from facial nerve injury or other medical disorders and results in pain, corneal ulceration, and possible vision loss. Treatment options for xerostomia are limited, and management of xerophthalmia usually focuses on the eyelids, rather than the fundamental problem of inadequate secretory protection. In this study, a rat model for submandibular gland microvascular transplantation was developed to assess the feasibility of salivary tissue transfer. Sixteen rats underwent submandibular gland transplantation from the neck to the groin. Fourteen of these rats underwent microvascular anastomosis of the vascular pedicle. Ten glands were assessed for viability at 4 days after transplantation, and four glands were examined after 7, 10, 14, or 21 days. By gross and histologic examination, 93 percent of transplanted glands showed expected long-term viability after at least 4 postoperative days. Microvascular techniques were shown to be applicable to the transplantation of submandibular gland salivary tissue. This has not previously been shown in a rat model. It is possible that submandibular glands could be transplanted to the eye for treatment of xerophthalmia and out of the neck during irradiation of the head and neck, with subsequent replantation after treatment as a means of preventing permanent xerostomia.     

Irradiation-induced effects on the innervation of rat salivary glands: Changes in enkephalin- and bombesin-like immunoreactivity in ganglionic cells and intraglandular nerve fibers

When treating head and neck for cancer with the use of radiotherapy the salivary glands are usually within the treatment volume with ensuing dryness and discomfort. Since the autonomic nervous system is of pivotal importance for the salivary gland function and integrity, the irradiation-induced effects may involve an influence on the innervation of salivary glands. Therefore, the rat submandibular gland, including the submandibular ganglionic cells, has been subjected to immunohistochemical examination with respect to expression of neuropeptides following fractionated irradiation with high energy photons. A markedly enhanced expression of bombesin- and leu-enkephalin-(ENK)-like immunoreactivities (LI) in the ganglionic cells and a pronounced increase in the number of nerve fibers showing these immunoreactivities in the submandibular gland tissue following irradiation were observed 10 days after treatment. On the other hand, no changes in the patterns of VIP (vasoactive intestinal polypeptide)- and NPY (neuropeptide Y)-immunoreactivities occurred. Thus, the present study shows that alterations in the expression of certain neuropeptides take place in the submandibular gland and its associated ganglionic cells in response to irradiation of the head and neck region. These changes may add further explanation to the inherent radiosensitivity of salivary glands.     

Characteristics of sialidase in the rat salivary glands

Using 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as substrate, we measured sialidase activity in the salivary glands and other organs of the rat. The pH optima of salivary gland sialidase were between 4.0 and 4.5, which were similar to those of the enzyme in the brain, liver and kidney. Among the salivary glands, the submandibular one showed the highest sialidase activity followed by the parotid and the sublingual glands. However, sialidase activity in these glands was lower when compared with the activity in the brain, liver and kidney. From the subcellular distribution study, salivary gland sialidase was found to be mainly localized in the lysosomes. The pH optima of the lysosomal sialidase of the salivary glands were between 4.0 and 4.5; and Km values for 4MU-NeuAc approximately 0.09 mmol/l. In the submandibular and parotid glands, a soluble sialidase with a different pH optimum (5.5) and Km value (0.25 mmol/l) was also detected.     

The sympathetic and parasympathetic nature of neuropeptide Y-immunoreactive nerve fibres in the major salivary glands of the rat

Summary The distribution and origin of neuropeptide Y in the major salivary glands of the rat was studied by indirect immunofluorescence technique. Numerous nerve fibres immunoreactive for the peptide were seen in the parotid and sublingual glands. Most of the fibres were located around blood vessels and salivary acini. In the submandibular gland the number of immunoreactive nerve fibres around the acini was lower in comparison with that in the parotid and sublingual glands. Some immunoreactive nerve fibres were also found around or along intra- and interlobular ducts in all major salivary glands.A large number of the neuropeptide-containing neuronal cell bodies and nerve fibres were detected in the sympathetic superior cervical ganglion. Sympathetic postganglionic nerve trunks of this ganglion contained numerous immunoreactive nerve fibres as well. A subpopulation of the neuronal cell bodies in the submandibular ganglion were immunoreactive to neuropeptide Y.Both uni- and bilateral superior cervical ganglionectomies caused a significant decrease in the number of immunoreactive nerve fibres around the blood vessels in all the major salivary glands. However, these denervations did not affect the density of nerve fibres around the acini and ducts. On the contrary, unilateral parasympathetic denervation by sectioning the auriculotemporal nerve reduced the fibres around the secretory acini in the parotid gland remarkably, while only a minor reduction in the density of immunoreactive fibres associated with the blood vessels of the gland was detected. Unilateral electrocoagulation of the trigeminal nerve branches caused no detectable change in the density of immunoreactive nerve fibres in any of the major salivary glands.On the basis of the present findings it is concluded that neuropeptide Y-reactive nerve fibres present in all major salivary glands around the blood vessels seem to be mainly sympathetic, whereas those around the acini and ducts seems to be of parasympathetic origin.     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.     

Xylose-linked proteoglycan synthesis does not have a primary role in the control of secretory cell differentiation in salivary glands

Xylose-linked proteoglycans, particularly chondroitin sulfate proteoglycan, have been shown to play a significant role in the regulation of salivary gland morphogenesis. The purpose of this study was to determine if xylose-linked proteoglycans are involved in the regulation of differentiation of salivary gland secretory cells. Embryonic rat submandibular salivary gland rudiments were cultured for 120 hr in the presence or absence of 0.75 to 1.0 mM p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of xylose-linked proteoglycan assembly. beta-D-Xyloside has been shown to block submandibular gland morphogenesis (Thompson and Spooner, 1982). In the present study glandular morphogenesis was blocked in 93.3% of the rudiments cultured in the presence of beta-D-xyloside. However, secretory cell differentiation was observed in 71.4% of those rudiments in which morphogenesis had been inhibited. Biochemical evaluation confirmed that xylose-linked proteoglycan assembly had been inhibited by xyloside. These results indicate that while xylose-linked proteoglycans play a significant role in the control of salivary gland morphogenesis these molecules are not primary regulators for secretory cell differentiation within developing salivary glands.     

High levels of GM1-ganglioside beta-galactosidase in the salivary glands and GM1-like-ganglioside storage in parotids of deficient mice.

We have previously demonstrated high levels of GM1-ganglioside beta-galactosidase (beta-gal) in the salivary glands of Swiss-Webster mice (Nowroozi et al., J Craniofac Genet Dev Biol 18:51, 1998), and suggested that this activity reflects an important role for the lysosome in catabolism of salivary glycoconjugates. Here, we characterized and compared activities of lysosomal glycosidases among the salivary glands, spleen, and muscle of C57BL/6 mice, beta-gal hexosaminidase, and beta-glucuronidase activities are high in all three glands relative to muscle. Enzyme activities in the sublingual gland were substantially higher than in the submandibular and parotid glands. Spleen displays levels of activity that are comparable or higher (for beta-glucuronidase) than those in the salivary glands, whereas muscle displays substantially lower levels of these lysosomal glycosidases. In order to investigate the role of beta-gal in the salivary glands, we further characterized the salivary phenotype of knock-out mice deficient in this enzyme, mimicking human GM1-gangliosidosis. In contrast with the relative levels of beta-gal specific-activity among the salivary glands, only the parotid developed severe, generalized, degenerative histopathological changes in beta-gal-deficient knock-out mice. GM1-like-ganglioside, typically found at high levels only in the nerve tissue, where its exact function is still not clear, was demonstrated in storage vacuoles of the parotid glands of the deficient mice by binding of cholera toxin subunit B. Thus, beta-gal activity observed in the parotid gland most likely reflects its role in GM1-ganglioside catabolism, and this ganglioside, never previously reported in the salivary glands, may have a role in parotid exocrine secretory functions. beta-gal may also serve in secretory glycoprotein catabolism in other salivary glands, but this function may be non-essential for these glands.     

Use of Biometric Characteristics of Major Salivary Glands as a Baseline to Develop a Formula for the Quantitative Estimation of Posttraumatic Regeneration of Glandular Tissue

In experiments conducted on 903 rats, we studied the biometric characteristics of the major salivary glands (parotid, submandibular, and sublingual glands) during ontogenesis. We calculated the indices of nondirectional fluctuation asymmetry for the submandibular and sublingual glands and determined correlation coefficients and the coefficients of linear regression between salivary glands, rat body weight, and the weight of the femoral bone (the largest bone in the rat). The strongest correlation was found between the dry weight of the submandibular gland and the rat body weight. Mathematical analysis of the growth of the submandibular gland after sialotomy allowed us to derive a formula for the quantitative estimation of regeneration, taking into account the natural growth of the rat.     

Use of biometric characteristics of major salivary glands as a base line to develop a formula for the quantitative estimation of posttraumatic regeneration of glandular tissue

In experiments conducted on 903 rats, we have studied biometric characteristics of the major salivary glands (parotid, submandibular, and sublingual glands) during ontogenesis. We have calculated the indices of non-directional fluctuation asymmetry for the submandibular and sublingual glands and determined correlation coefficients and coefficients of linear regression between salivary glands, rat body weight, and the weight of the femoral bone (the largest bone in the rat). The strongest correlation was found between the dry weight of the submandibular gland and the rat body weight. Mathematical analysis of the growth of the submandibular gland after sialotomy allowed us to derive a formula for the quantitative estimation of regeneration, taking into account the natural growth of the rat.     

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats

Salivary gland neoplasms exhibit complex histopathology in a variety of tumor types and treatment options depend largely on the stage of the cancer. Induced pluripotent stem cells (iPS) have been investigated for treating induced salivary gland cancer and for restoring salivary gland function. We investigated iPS treatment for salivary gland cancer both in vitro and in vivo. For our study in vitro, we re-programmed human skin fibroblasts to form iPS cells using a plasmid containing Oct4, Sox2, L-MYC and LIN28. For our study in vivo, we used 30 white male albino rats divided into the following groups of 10: group 1 (control): rats were injected with phosphate-buffered saline (PBS), group 2 induced squamous cell carcinoma (SCC): rat submandibular glands were injected with squamous carcinoma cells (SCC), group 3 (induced SCC/iPS): SCC treated rats treated with 5 × 106 iPS cells. Submandibular glands from rats of all groups were examined histologically and real time PCR was performed for amylase, and COX I and COX II gene expression. We confirmed that submandibular gland specimens included tumor tissue before starting treatment with iPS. iPS treated cases exhibited regeneration of salivary glands, although minor degenerative and vascularization changes remained. The acinar cells regained their proper organization, but continued to exhibit abnormal activity including hyperchromatism. iPS cells may be useful for treating salivary gland carcinomas.     

Influence of streptozotocin-induced diabetes on hexokinase activity of rat salivary glands

The influence of diabetes on the enzyme hexokinase (HK) was examined in the salivary glands of rats. Diabetes was induced by an intraperitoneal injection of streptozotocin (60 mg/Kg body weight) in overnight fasted rats (180-200 g). The animals were killed 48 hours and 30 days after the induction of diabetes and the submandibular and parotid salivary glands extracted for use. Hyperglycemia was evaluated by determining the blood sugar. The area occupied by each intralobular component, acini, ducts, total parenchyma and stroma was measured, and no differences were observed compared with control. In the soluble fraction of the submandibular gland, no difference in the specific activity of HK was observed, between the diabetic and control animals, however, the activity per gland and per g of tissue showed lower values than control. The specific activity of the bound form was reduced in the diabetic gland. The results obtained for the parotid gland were different from the submandibular. The specific activity of both the soluble and bound forms were increased in the diabetic animals. The DEAE-cellulose column chromatography of the soluble and bound forms of the enzyme from both glands showed a first peak appearing during the washing of the column and two other peaks were eluted by the gradient. Thus, three isoenzymes in the submandibular and parotid salivary glands for the control and diabetic rats have been found.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.