Cell death and cell proliferation during atrophy of the rat parotid gland induced by duct obstruction

Rat parotid gland atrophy after unilateral duct ligation was studied by light and electron microscopy. Death of secretory acinar cells, which took the form of apoptosis, resulted in their complete disappearance within 5 days. The remnants of the dying cells were mostly phagocytosed and degraded by macrophages within the glandular epithelium; a few were taken up by adjoining epithelial cells. The acinar cell deletion was accompanied by increased mitosis of striated and intercalated duct epithelial cells. However, over many weeks, there was enhanced apoptosis of duct cells, which eventually led to marked shortening of intercalated ducts. Apoptosis of capillary endothelial cells was observed and may account for the reduction in the capillary bed known to accompany gland atrophy. The end-stage lesion comprised small numbers of ducts in a condensed stroma. Compensatory hyperplasia, involving proliferation of duct and acinar cells, was demonstrated in the contralateral glands.     

Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction

Acinar cell regeneration in the rat parotid gland after atrophy induced by a one week period of duct obstruction was examined using histology, immunohistochemistry and transmission electron microscopy (TEM). For immunohistochemistry, antibodies to 5-bromo-2'-deoxyuridine (BrdU), injected one hour before tissue collection, and cytokeratin were employed. When clips were removed from the duct, only ductal epithelial cells remained; all acinar cells had been deleted. Some duct cells were BrdU positive. After three days, newly-formed acini comprising immature acinar cells had appeared; many of the cells were BrdU positive and mitotic figures were readily identified. Thereafter progressive acinar cell maturation and proliferation occurred, parotid gland weight returning to control levels by 7 days. Peak BrdU labelling indices for duct and acinar cells were on days 0 and 4, respectively. By TEM, cytoplasmic organelles in epithelial cells of transitional duct-acinar structures seen at 2 days were poorly developed. Immature acinar cells seen on day 3 contained zymogen granules and had increased endoplasmic reticulum and mitochondria. By day 5, maturing acinar cells had abundant endoplasmic reticulum and zymogen granules, resembling acinar cells in control glands. These observations indicated origin of acinar cell precursors from duct cells during regeneration of the acinar cell-free atrophic gland. Subsequent expansion of the acinar cell population was dependent on maturation and proliferation of these newly-formed cells.     

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.     

Histogenesis of acinic cell carcinoma of the major and minor salivary glands. An ultrastructural study

This study describes the light microscopic, histochemical and electron-microscopic findings of 10 acinic cell carcinomas from the major and minor salivary glands. Ultrastructurally, four cell types were identified: secretory acinar cells, intercalated duct-like cells, pluripotential reserve/stem cells and myoepithelial cells. This cellular composition suggests that the tumours are derived from neoplastic proliferation, cytodifferentiation and functional maturation of pluripotential reserve/stem cells which normally reside at the acinar-intercalated duct junctions and/or in the intercalated ducts proper of adult salivary glands. This study further supports the concept that different salivary gland tumours recapitulate various developmental stages in the normal embryogenesis of the salivary glands.     

The development of the granular convoluted duct in the rat submandibular gland

The development of the granular convoluted duct in the submandibular gland of male rats, 4 to 12 weeks of age, was investigated. During this period, the average weight of the gland increased from 213 to 526 mg, the total DNA and RNA contents doubled, and the protein content tripled. Radioautographs were prepared from Epon embedded sections of the gland of the rats given ³H-thymidine and stained with toluidine blue. The glands of 4-week-old rats consisted mainly of acinar cells (45%), intercalated ductal cells (20%) and striated ductal cells (16%). A few granular convoluted ductal cells were seen in the striated duct close to the intercalated duct. The frequency (and absolute number) of granular convoluted ductal cells increased linearly from 1% (3 × 10⁶) at four weeks to 26% (68 × 10⁶) at eight weeks, while the calculated number of striated ductal cells remained stationary. The absolute number of acinar cells and intercalated ductal cells nearly doubled between four to eight weeks of age. The proliferative activity of all cell types declined with age but between six and ten weeks of age the rate of proliferation of ductal cells was relatively higher than the rate of proliferation of the acinar cells. Morphologically the size and number of granules in the granular convoluted ductal cells increased with age. Based on the above data it is concluded that the granular convoluted ductal cells developed from that segment of the striated ductal cells which is in close proximity with the intercalated ductal cells. The heterogeneity of the granules in the granular convoluted ductal cells observed from six weeks of age might denote the functional diversity of the cells.     

The development of the granular convoluted duct in the rat submandibular gland.

The development of the granular convoluted duct in the submandibular gland of male rats, 4 to 12 weeks of age, was investigated. During this period, the average weight of the gland increased from 213 to 526 mg, the total DNA and RNA contents doubled, and the protein content tripled. Radioautographs were prepared from Epon embedded sections of the gland of the rats given 3-H-thymidine and stained with toluidine blue. The glands of 4-week-old rats consisted mainly of acinar cells (45%), intercalated ductal cells (20%) and striated ductal cells (16%). A few granular convoluted ductal cells were seen in the striated duct close to the intercalated duct. The frequency (and absolute number) of granular convoluted ductal cells increased linearly from 1% (3 X 10-6) at four weeks to 26% (68 X 10-6) at eight weeks, while the calculated number of striated ductal cells remained stationary. The absolute number of acinar cells and intercalated ductal cells nearly doubled between four to eight weeks of age. The proliferative activity of all cell types declined with age but between six and ten weeks of age the rate of proliferation of ductal cells was relatively higher than the rate of proliferation of the acinar cells. Morphologically the size and number of granules in the granular convoluted ductal cells increased with age. Based on the above data it is concluded that the granular convoluted ductal cells developed from that segment of the striated ductal cells which is in close proximity with the intercalated ductal cells. The heterogeneity of the granules in the granular convoluted ductal cells observed from six weeks of age might denote the functional diversity of the cells.     

Ultrastructural study of acinar and intercalated duct organization of submandibular and parotid salivary gland

According to some current hypotheses, the morphology and organization of the intercalated duct/acinar interface of salivary gland have implications for the induction of tumors in this organ. However, this region has received limited detailed investigation. To study the organization of the terminal ductal segments of salivary gland, conventional transmission electron microscopy of human parotid and submandibular glands and canine submandibular gland was combined with 3-dimensional observations of polymer casts of the canine submandibular ductal system; the latter were prepared by retrograde injection of acrylic resin via the main excretory duct with subsequent digestion of the gland tissue. The division of intercalated ducts, into first- and second-order branches, and acinar arrangement is more complex than previously suggested. The entire surface of each elongated second-order intercalated duct is covered with acini projecting in all directions. In the human gland, some acini abut directly on the intercalated duct surface, whereas others are connected by a short stalk of intercalated duct cells; in comparison with canine submandibular gland, the latter may be a modification producing a third-order of the intercalated duct unit. All of these features combine to produce a highly efficient secretory apparatus with a large proportion of acinar cells to each intercalated duct.     

Dynamics of parenchymal cell division, differentiation, and apoptosis in the young adult female mouse submandibular gland

The submandibular salivary gland of the young adult female mouse has two secretory cell types, acinar and granular duct, which are separated by intercalated ducts. Based on the occurrence of autologous cell division in these cells, they have been traditionally classified as expanding populations. However, differentiation from stem or progenitor cells in the intercalated ducts, usually associated with renewing populations, has also been detected. The question of renewing or expanding populations is resolved by quantitating and integrating the rates of autologous cell division, differentiation, and apoptosis for each cell type. The integrated data shows that both acinar and granular duct cell populations exhibit a substantial positive growth index, whereas the growth index for the intercalated duct cells is moderately negative. On balance, it suggests that the submandibular gland of the young adult female mouse is still growing. Comparison of young female mice with older females suggests that, although overall parenchymal growth slows with age, there is no longer a net loss of intercalated duct cells. Comparison with young adult male submandibular glands indicates that gender differences exist in the rates and mechanisms used for maintaining the different cell populations. The acinar and granular duct cell populations in young adult female mouse submandibular glands are expanding at the expense of the intercalated duct cell population, which appears to be contracting.     

An electron microscopic study of acetylcholinesterase-activity and vasoactive intestinal peptide- and neuro-peptide Y-immunoreactivity of the intraepithelial nerve fibers in the nasal gland of the guinea pig

In combination with transmission electron microscopy (TEM), histochemistry for acetylcholinesterase (AChE) and immunohistochemistry for vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were carried out on the intraepithelial nerve fibers of the guinea-pig nasal gland. AChE-positive nerve profiles and VIP-immunoreactive nerve profiles were detected in abundance within the epithelium of the glandular acini and within the epithelium of intralobular excretory ducts including the intercalated and striated ducts. Intraepithelial NPY-immunoreactive nerve profiles were also considerably large in number in the nasal gland, but less frequent than the other two types of nerve profiles; furthermore, the NPY-immunoreactive nerve profiles appeared absent within the epithelium of the striated duct. All the intraepithelial nerve varicosities were in close spatial contact with the epithelial cells of the acinus and the duct and also with the myoepithelial cells, which were commonly seen in the acinus and the intercalated duct. Throughout the present study, however, no membranous specializations could be found between the nerve varicosities and the epithelial cells or the myoepithelial cells. The present results suggest an intense and delicate regulation through the collaboration among ACh, VIP and NPY of the secretory activity of the guinea-pig nasal gland, including the emission of acinar secretions into the duct through contraction of the myoepithelium and modification of the secretion contents by the duct epithelium.     

Changing myoepithelial cell distribution during regeneration of rat parotid glands

The distribution of the myoepithelial cells during regeneration of the rat parotid gland after atrophy induced by one week of parotid duct ligation was investigated by immunohistochemistry for actin and transmission electron microscopy (TEM). Immunohistochemically, residual ducts were surrounded by actin-positive cells when clips were removed from the duct. Three days later, most of the newly formed acini originating from the residual ducts were also embraced by actin-positive cells. After 10 days, actin-positivity tended to be seen as dots around acini that decreased in number day by day. On day 21 actin-positive cells mainly surrounded intercalated ducts with only a few positive reactions identified at the acinar periphery. Electron microscopically, residual ducts and newly formed acini were peripherally embraced by myoepithelial cells before day 5. After day 7, shift of myoepithelial cells from the periphery of acini to the duct-acinar junctional region was identified. Then few myoepithelial cells were identified at the periphery of acini. These observations indicate that myoepithelial cells migrate from the acinar periphery to the duct-acinar junctional region during rat parotid regeneration, and that such behaviour is closely related to that seen during rat parotid development.     

Transient occurrence of 27 kDa heat‐shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland

It has been suggested that the 27 kDa heat‐shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27‐immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3‐week‐old rats for 7 days, the number of Hsp27‐positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3–4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants. Anat Rec 264:358–366, 2001. © 2001 Wiley‐Liss, Inc.     

Localization of Hsp27 in the Rat Submandibular Gland Following the Application of Various Surgical Treatments

Salivary glands repair and regenerate following various types of injuries and surgical procedures. However, the tissue responses induced in the contralateral glands have yet to be elucidated in detail. Hsp27, a member of the heat-shock protein (Hsp) family, is strongly expressed in physiological environments, particularly during development. Hsp27 was previously shown to play a role in the regulation of acinar cell proliferation and differentiation in the rat submandibular gland.The present study performed the following surgical treatments on the right submandibular glands of adult rats: 1) duct ligation followed by unligation after one week; 2) partial sialoadenectomy; and 3) total sialoadenectomy. Immunohistochemistry for Hsp27 and Ki67 was performed in the experimental and normal contralateral glands, and localization was histologically and morphometrically analyzed.The results obtained revealed the localization of Hsp27 to the intercalated duct in the submandibular glands of non-treated rats. The expression of Hsp27 was strongly induced in both the uninjured contralateral control glands as well as treated glands of experimental rats regardless of the surgical procedure performed. The number of Hsp27-immunopositive cells increased rapidly following surgery, and subsequently returned to the same level as that in non-treated rats after 4 weeks. However, no marked changes were observed in the number of Ki67-immunopositive proliferating cells. Therefore, the change in the number of Hsp27-immunopositive cells may have contributed to compensatory hypertrophy. The results of the present study indicate that the expression of Hsp27 in the intercalated duct in the submandibular gland may play a role in the differentiation of acinar cells.     

Histopathological study of von Ebner's lingual salivary glands in Trypanosoma cruzi-infected mice

In the present study we describe the histopathological alterations induced by RC strain of Trypanosoma cruzi in the mouse von Ebner's lingual salivary glands during the acute period of infection: Amastigotes were found in von Ebner's gland acini cells, excretory duct cells, intralobular connective tissue, inside the acini lumen and muscle fibres. Desorganized parenchyma with impairment at the acinar and duct level, and intense lymphoplasmocytic infiltrate were seen.     

腮腺主导管结扎诱导萎缩后的组织转归

背景：人体大唾液腺常因受到头颈部肿瘤放射治疗、舍格伦综合征及涎腺炎等因素的影响发生腺体萎缩,目前对长期萎缩性腮腺内组织形态变化的观察仍较少。 目的：观察腮腺主导管结扎诱导腮腺萎缩后的组织转归。 方法：通过结扎SD大鼠右侧腮腺主导管诱导腺体萎缩,采用苏木精-伊红染色观察正常腮腺及导管结扎后0(对照),1,3,7,14,30,60 d萎缩性腮腺组织内腺泡、导管细胞的面积;免疫组织化学染色定量分析肌上皮细胞在腮腺萎缩不同时间点的数量分布变化。 结果与结论：结扎腮腺主导管后腺泡细胞出现快速凋亡,至14 d时已基本消失。随着腺体萎缩,间质逐渐纤维化并伴随炎性细胞浸润,组织内形成大量导管样结构,导管面积逐渐增加,到14 d时达到顶峰,随后逐渐减少,导管样结构呈典型的双套层结构,结扎各时间点腺泡、导管面积与对照组比较差异均有显著性意义(P < 0.05)。结扎后7 d内肌上皮细胞数量快速增加,随后肌上皮细胞数量增长缓慢,维持在一定的范围。表明腮腺主导管结扎诱导腺体萎缩早期腺泡细胞快速消失,出现大量导管样结构,肌上皮反应性增殖,随着腺体的萎缩由导管样结构及肌上皮细胞组成“双套层”结构可能抑制腺体的进一步萎缩。     

The organization of the salivary gland microcirculation

1. The microvasculature of the rabbit submandibular salivary gland has been investigated employing in vivo microscopy, blood flow measurements, latex casts, microsphere injections and examination of fixed sections of the gland.2. Two principal microcirculations were distinguished in the living gland, one supplying the acini and the other the intralobular ducts. Parasympathetic nerve stimulation (2, 5 or 10 sec(-1)) elicited different responses in each of the two microcirculations. Flow in the capillaries around the acini slowed initially before increasing. In contrast, flow in the intralobular duct capillaries increased soon after beginning stimulation.3. In some experiments both whole gland flow and microvascular flow were monitored simultaneously. Whole gland flow increased at the same time as flow in the acinar capillaries was decreasing and as flow in the intralobular duct capillaries increased. Flow in acinar capillaries increased about 5 sec after glandular flow started to increase.4. These observations could be explained if either the vascular beds of the acini and the intralobular ducts were arranged in parallel or if arteriovenous anastomoses were to shunt the acinar circulation. No such anastomoses were found in latex casts made of the gland vasculature, and microspheres injected into the artery supplying the gland were not found in the venous effluent.5. The intraglandular distribution of microspheres was measured in histological sections of the injected glands to give an estimate of the distribution of blood flow between the duct and acinar microcirculations. At rest and during maintained stimulation about 55% of the blood flow passed through the intralobular duct microcirculations, whilst during this initial 15 sec of stimulation this proportion was increased to over 70%. This finding is consistent with a parallel arrangement of the two microcirculations.6. The conclusions drawn from these observations are that the duct and acinar microcirculations are arranged in parallel, that there are differences in the way the vasodilatation is mediated in these circulations, and that arterio-venous anastomoses play no significant role in this gland.     

Transient occurrence of 27 kDa heat-shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants.     

A granular cell at the acinar-intercalated duct junction of the rat submandibular gland

A granular cell was present at the acinar-intercalated duct junction in submandibular gland of adult rats of several different strains. It occurred more frequently in females thanin males. It was a small pyramidalshaped cell, usually forming the most proximal part of the intralobular duct system. The cell contained a relatively large, basally located nucleus. Numerous heterogeneous granules, usually exhibiting both electron-dense and electron-lucent regions, were present in the apical cytoplasm. Exocytosis of the granules at the apical cell surface was occasionally seen. Dilated cisternae of endoplasmic reticulum were frequently observed between the apical granules and in the basal and lateral cytoplasm. The function of the granular cell is unknown. Their structure and location suggest that they may (1) constitute a mature secretory cell population, or (2) represent progenitor cells for acinar and/or intercalated duct cells.     

Keratinocyte growth factor prevents radiation damage to salivary glands by expansion of the stem/progenitor pool

Irradiation of salivary glands during radiotherapy treatment of patients with head and neck cancer evokes persistent hyposalivation. This results from depletion of stem cells, which renders the gland incapable of replenishing saliva to produce acinar cells. The aim of this study was to investigate whether it is possible to expand the salivary gland stem/progenitor cell population, thereby preventing acinar cell depletion and subsequent gland dysfunction after irradiation. To induce cell proliferation, keratinocyte growth factor (DeltaN23-KGF, palifermin) was administered to C57BL/6 mice for 4 days before and/or after local irradiation of salivary glands. Salivary gland vitality was quantified by in vivo saliva flow rates, morphological measurements, and a newly developed in vitro salisphere progenitor/stem cell assay. Irradiation of salivary glands led to a pronounced reduction in the stem cells of the tissues, resulting in severe hyposalivation and a reduced number of acinar cells. DeltaN23-KGF treatment for 4 days before irradiation indeed induced salivary gland stem/progenitor cell proliferation, increasing the stem and progenitor cell pool. This did not change the relative radiation sensitivity of the stem/progenitor cells, but, as a consequence, an absolute higher number of stem/progenitor cells and acinar cells survived after radiation. Postirradiation treatment with DeltaN23-KGF also improved gland function, and this effect was much more pronounced in DeltaN23-KGF pretreated animals. Post-treatment with DeltaN23-KGF seemed to act through accelerated expansion of the pool of progenitor/stem cells that survived the irradiation treatment. Overall, our data indicate that DeltaN23-KGF is a promising drug to enhance the number of salivary gland progenitor/stem cells and consequently prevent radiation-induced hyposalivation. Disclosure of potential conflicts of interest is found at the end of this article.     

Changes in the distribution and fine structure of the intralobular blood vessels of the submandibular gland in the postnatally developing mouse

Previous studies have shown that the blood vessels supplying the endocrine organs and the mucosa of the intestinal canals change in terms of not only their distribution but also their structure with the development and growth of each organ. We examined changes in the distribution and structure of intralobular blood vessels, including capillaries, throughout the postnatal development of the submandibular gland, an exocrine organ. The mouse submandibular gland from days 0 (birth) to 49 was investigated chronologically and ultrastructurally. The capillaries changed from continuous to fenestrated on day 10, coincident with an increase in the number of acini to more than the number of terminal tubules. The number of sections of intralobular blood vessels per unit area gradually decreased with increasing acinar size and was lowest on day 21 when pups were weaned; the same number was maintained from then on. In contrast with the reduction in the number of intralobular blood vessels, the number of capillary pores appeared to increase gradually. Acinar size increased further till day 28. Capillary pore number also increased further, till day 35, apparently in relation to the increasing acinar size. These findings suggest that the changes in distribution and structure of the intralobular blood vessels in the submandibular gland of the postnatally developing mouse are closely related to the development of the parenchymal cells in preparation for weaning and sexual maturity.