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Keywords:

  • developmental change;
  • distribution of intralobular blood vessel;
  • fine structure of intralobular blood vessel;
  • submandibular gland;
  • mouse

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

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. © 2005 Wiley-Liss, Inc.

It has been reported that the distribution and fine structure of the blood vessels supplying tissues or organs change with the development and growth of the tissue or organ. In particular, the capillaries in almost all endocrine organs (Eurenius, 1977; Bertossi and Poncali, 1981; Bertossi and Ribatt, 1983; Galabov and Schiebler, 1983), the small intestinal mucosa (Milici and Bankston, 1981), and the renal glomerulus (Larsson and Maunsbach, 1980) change from the continuous to the fenestrated type. The distribution and fine structure of the blood vessels of the salivary glands have also been reported in various animals (Takada, 1969, 1970; Sato and Miyoshi, 1990; Sato et al., 1993; Akimoto, 1994), including mice (Ban, 1959). However, there has been no detailed report of the architecture, distribution, and structure of blood vessels in the developing salivary glands and, in particular, of the fine structure of the capillaries. Our study was designed to examine the distribution and structure of intralobular blood vessels in the submandibular gland of the postnatally developing mouse, paying special attention to changes in capillary structure with development. In addition, we also investigated the relationship between the development and growth of parenchymal cells and the distribution and structure of the intralobular blood vessels.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Animals

All animal experiments followed the Guidelines for the Care and Use of Laboratory Animals of the Nippon Dental University School of Dentistry Research Center for Odontology Section of Biological Science. Male ICR-strain mice aged 1, 3, 5, 7, 10, 14, 21, 28, 35, or 49 days were used. The day of birth was counted as day 0. All the mice were maintained at an ambient temperature of 23°C with a 12-hr light/dark cycle and were allowed free access to food and water. Pups were separated from their dams at 21 days.

Tissue Preparation for Light Microscopy

Mice from each age group were killed under deep anesthesia with an intraperitoneal injection of pentobarbital sodium at between 14:00 and 15:00 hr. The submandibular glands were removed and cut into small pieces, fixed in 10% (v/v) neutral buffered formalin, then dehydrated in a graded series of ethanol solutions and embedded in paraffin. The paraffin-embedded samples were cut into 5 μm sections, and the sections were stained with hematoxylin and eosin (H&E).

Tissue Preparation for Transmission Electron Microscopy

Under deep anesthesia with an intraperitoneal injection of pentobarbital, mice from each age group were killed by vascular perfusion with a fixative composed of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M phosphate-buffered saline (PBS; pH 7.4). The submandibular glands were removed and cut into small pieces. The pieces were postfixed with 1% osmium tetraoxide in PBS, dehydrated with a graded ethanol series, and embedded in Epon. Before ultrathin sectioning, semithin sections (1 μm thick) were made and stained with toluidine blue to detect parts suitable for ultrastructural observation. Ultrathin sections were stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (JEM-2000 EX II; Jeol, Japan). To make it easy to observe the capillaries, those crossing sections were used. In addition, for mice of all age groups, fenestrated capillaries were observed in those submandibular gland serial sections in which the pores appeared most numerous.

Measurement of Size and Number of Terminal Tubules and Acini

Changes in the size and number of terminal tubules and acini were assessed in H&E-stained sections of the submandibular gland in mice aged 1–49 days. Three glands were used from mice of the same age, and then three sections were sampled from one gland at random. Subsequently, three areas were selected from one section arbitrarily and were photographed under a light microscope at 400×. Each photograph was processed on a computer with the Scicon Image program, and the number and cross-sectional area of terminal tubules and acini per unit area (200 × 150 μm) were calculated. Then the average value for every age group was computed. The numbers of terminal tubules and acini were added together in those samples in which both were intermingled. Measurement of the size was performed in the terminal tubules and acini used for measurement of the number. The cross-sectional area of terminal tubules and acini of each age group was calculated and then the average value of the area was computed respectively. In the samples having terminal tubules and acini intermingled, the average value of area was measured without discriminating these cells. The average value of the cross-sectional area was presented as the size of the terminal tubule and acinus.

Measurement of Number of Intralobular Blood Vessels

Changes in the number of intralobular blood vessels, including blood capillaries, were assessed in the same samples as those used to measure the size and number of terminal tubules and acini. The number of animals, sections, and areas used and the measuring method were the same as those used to measure the number of terminal tubules and acini. Sections of the intralobular blood vessels were identified by finding erythrocytes stained with eosin. Capillaries were not distinguished from other intralobular blood vessels because of the difficulty in differentiating capillaries from small blood vessels such as arterioles and venules under light microscopy.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Light Microscopy

On day 1, cell masses with cytoplasm showing positive staining for H&E were scattered in the lobules (Fig. 1a). The cell masses were distinguishable from the terminal tubules and ducts because of the presence of a wide lumen. Among the cell masses, cross- or obliquely sectioned intralobular blood vessels containing a few eosinophilic erythrocytes were observed, surrounded by loose connective tissue. On day 3, a small number of cell masses stained almost palely, like the acini of mature glands, although most of the cell masses showed almost the same stainability as on day 1 (Fig. 1b) and the number of blood vessel sections seemed to have decreased a little in number compared with day 1. By day 7, the number of cell masses showing pale staining had increased (Fig. 1c). Therefore, it seemed that in many of the developing terminal portions the terminal tubules were changing to acini. In addition, reduction in the number of blood vessels and the quantity of connective tissue had progressed further since day 3. On day 10, the number of acini seemed greater than on day 7 (Fig. 1d). By contrast, terminal tubules were barely to be seen. The blood vessels were distributed over the acini and the ducts, which were approaching each other because of further reduction in the area of connective tissue. On day 21, the acini seemed to have become larger than on day 10 (Fig. 1e), whereas cut sections of blood vessels appeared to have decreased further in number. On days 28, 35, and 49, the acini appeared to have increased further in size, but the number of blood vessel sections seemed to have changed little compared with day 21 (Fig. 1f).

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Figure 1. Light micrograph of the lobules of the submandibular gland in the postnatally developing mouse. H&E staining. Scale bars = 20 μm. a: Day 1. There are loosely distributed terminal tubules and developing ducts and intralobular blood vessels with erythrocytes surrounding them. b: Day 3. The intralobular blood vessels have decreased slightly in number compared with those on day 1, although the parenchymal cells show little change in structure and distribution. c: Day 7. Acini, composed of cells with stainability similar to that of mature acinar cells, have increased in number. The number of intralobular blood vessels appears to have decreased further compared with day 3. d: Day 10. The majority of the acini show almost the same stainability as mature acini, although they are still small. The number of vessels appears to have decreased slightly further. e: Day 21. The acini appear to have increased in size and have also approached each other. The number of intralobular blood vessels looks to have decreased further compared with day 10. f: Day 49. Acini slightly bigger than on day 21 can be seen. The number of intralobular blood vessels looks almost the same as on day 21.

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Electron Microscopy

Because light microscopy showed that the number of intralobular blood vessel sections changed with the development of the terminal tubules and acini, we investigated the structure of the blood vessels, and in particular the capillaries, in more detail by transmission electron microscopy. On day 1, terminal tubules were composed of cells with polymorphilic granules and blood capillaries surrounding them. The capillaries were continuous and composed of endothelial cells with thick cytoplasm (Fig. 2). On day 7, the capillaries were still continuous but now had cytoplasm that was a little thinner (Fig. 3). In addition, many of the endothelial cells had pinocytotic vesicles. On day 10, electron microscopy showed many more acini, in addition to the developing acini composed of proacinar cells. With the appearance of many acini, electron microscopy also demonstrated fenestrated capillaries, although pores were still scarce (Fig. 4). On day 14, the acini appeared to have increased in number and the fenestrated capillaries had more pores (Fig. 5). On day 21, the acini had increased in size and the fenestrated capillaries appeared to have more pores (Fig. 6). On day 28, the number of pores in the capillaries seemed to have increased further, although light microscopy showed almost no change in the number of blood vessels per unit area after day 21 (not shown). On day 35, the pore number seemed to have almost peaked, because the number of pores looked almost the same on days 35 and 49 (Fig. 7).

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Figure 2. Electron micrographs of capillaries in a lobule of the submandibular gland of a postnatally developing mouse (Figs. 2–6). Day 1. Only continuous capillaries can be seen. T, terminal tubule cell. Scale bar = 2 μm.

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Figure 3. Day 7. This continuous blood capillary shows many pinocytotic vesicles (arrows) in the cytoplasm. Scale bar = 1 μm.

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Figure 4. Day 10. Fenestrated capillaries have appeared, showing a few pores. Scale bar = 1 μm. Inset is a high-magnification view of the pores (arrows). Scale bar = 500 nm.

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Figure 5. Day 14. This fenestrated blood capillary appears to have more pores than on day 10. Scale bar = 2 μm. Inset is a high-magnification view of the pores (arrows). Scale bar = 500 nm.

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Figure 6. Day 21. This fenestrated blood capillary appears to have more pores than on day 14. A, acinar cell. Scale bar = 2 μm. Inset is a high-magnification view of the pores (arrows). Scale bar = 500 nm.

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Figure 7. Day 49. Capillary pores appear to have increased more in number than on day 21. Scale bar = 2 μm. Inset is a high-magnification view of the pores (arrows). Scale bar = 500 nm.

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Measurement of Number of Terminal Tubules, Acini, and Small Blood Vessels

Light microscopy gave the impression that the number of terminal tubules, acini, and intralobular blood vessels changed with the development of the submandibular glands. Accordingly, the number of their sectioned profiles per unit area was measured in H&E-stained sections sampled from glands of each age (Fig. 8). The average number of terminal tubules was 25.00 ± 0.01 on day 1, when only terminal tubules were observed. The number of developing terminal portions then increased gradually day by day, with an increase in the number of acini in contrast with the decrease in number of terminal tubules, and by day 14 it had reached 34.33 ± 3.79, by which time the acini were virtually the only terminal portions seen. Thereafter, the average number of acini decreased to 22.00 ± 2.00 by day 21 and then remained almost the same right up to day 49. The average number of blood vessel sections was 30.07 ± 1.53 on day 1, decreasing gradually day by day with the increase in numbers of acini, until it reached 16.67 ± 1.15 on day 14. By day 21 the average number of blood vessels had decreased further to 8.67 ± 2.08 with the reduction in number of acini, and thereafter it remained almost the same until day 49.

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Figure 8. Changes in the number of terminal tubules, acini, and intralobular blood vessels in the mouse submandibular gland lobule with postnatal development. IBV, intralobular blood vessel; TT, terminal tubule cell; AC, acinar cell.

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Measurement of Size of Terminal Tubules and Acini

As light microscopy strongly suggested that the size of the acini changed with development of the glands following a change in the number of blood vessels, the area of terminal tubules and acini was measured in H&E-stained sections sampled from glands of each age (Fig. 9). The average area of terminal tubules on day 1, when only terminal tubules were observed, was 33.52 ± 2.50 μm2. After that, the average area increased gradually with the appearance and increase in numbers of acini until day 14. Subsequently, on day 21, by which time the number of acini and blood vessels per unit area had decreased rapidly, the average area had risen considerably to 53.30 ± 3.15 μm2; it peaked at 68.49 ± 5.01 μm2 on day 28. A similar high value was obtained on day 49.

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Figure 9. Changes in the area of terminal tubules and acini in the mouse submandibular gland lobule with postnatal development.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Changes in Number of Terminal Tubules, Acini, and Intralobular Blood Vessels With Postnatal Development

For several days after birth, the mouse submandibular gland showed terminal tubules distributed loosely in the lobules. It has been reported that submandibular glands in the early postnatal mouse (Watanabe et al., 1997) and rat (Dvorak, 1969; Kim et al., 1970; Yamashina and Barka, 1972) have terminal tubules that change to acini with further development. Around the terminal tubules, many cross- or obliquely sectioned small blood vessels, including capillaries, were observed. However, with the appearance and increase in number of acini, the number of blood vessels seemed to decrease. Measurement of the number of blood vessel sections per unit area also showed that the number of blood vessels decreased gradually until day 14, coincident with the increase in the number of acini. Moreover, the number of blood vessels per unit area abruptly decreased on day 21, as if it were aligned with a rapid reduction in the number of acini per unit area at this time. Measurement of the area of acini clarified the fact that the acini rapidly increased in size on day 21, and that an increase in acinar size continued until day 28. Therefore, it seemed that the decrease in number of acini per unit area was caused by an increase in acinar size. It has been reported that the capillaries distributed in the lobules of the rat salivary gland form an extensive capillary network, in which each capillary is loop-shaped and communicates extensively with the others; usually three or four acini are surrounded by one loop (Suddick and Dowd, 1969; Nagato et al., 1980; Ohtani et al., 1983). Therefore, it can be considered that the numerous cross- or oblique sections of blood vessels seen on light microscopy were in fact part of a three-dimensional network of looped capillaries. Moreover, it seems that the decrease in number of blood vessel sections per unit area is related to the increase in number of acini within each capillary loop in addition to the increase in acinar size. In the mouse (Takahashi, 1951; Rugh, 1994; Hetherington et al., 2000) and the rat (Redman and Sreebny, 1971; Grand et al., 1975; Redman and Sweney, 1976), the switchover from milk to solid food begins around day 14 and is completed by about day 30, and usually pups are weaned on day 21. Accordingly, in the present study, the pups were also separated from the dams on day 21. The marked increase in size of the acini, which started on day 21, appears to be related to morphological and functional maturation of the submandibular gland acinar cells, as it has been reported that solid food increases sensitivity to mastication more than liquid food, thus promoting growth of the acinar cells (Redman and Sreebny, 1976; Johnson et al., 1977). In addition, it has been demonstrated that in relation to the eating cycle of the animals, the acinar cell size increases progressively between 05:00 and 17:00 hr, together with an apparent increase in the number of the secretory granules (Sreebny and Johnson, 1969; Redman and Sweney, 1976). In the present study, the glands were removed at between 14:00 and 15:00 hr. Therefore, it seems that there was an accumulation of acini that had considerably increased in size. After day 21, the number of blood vessels per unit area remained almost the same for the rest of the observation period, as the number of acini per unit area had become fixed. This suggests that the acini and the blood vessels in the lobule enter a state of harmony in number and distribution.

Changes in Structure of Capillaries With Postnatal Development

Light microscopy showed that the number of intralobular blood vessels per unit area decreased with growth of the terminal tubules and acini. This was especially remarkable on day 21, when the increase in size of the acini became conspicuous. Therefore, we investigated the structure of the blood vessels and, in particular, the capillaries. Electron microscopy showed continuous capillaries on days 1 and 7. The continuous blood capillaries on day 1 were composed of endothelial cells with thick cytoplasm, but by day 7 the endothelial cells had become a little thinner and had pinocytotic vesicles.

It has been reported that the capillaries of the intestinal mucosa are continuous in the early stage of development and gradually come to have pinocytotic vesicles (Milici and Bankston, 1981). It seemed that the capillaries in the submandibular gland parenchyma follow a developmental process similar to that of the capillaries of the intestinal mucosa. On day 10, fenestrated capillaries appeared, although pores were scarce. The appearance of many acinar cells in addition to proacinar cells has been reported in the submandibular gland of the rat (Yamashina and Barka, 1973; Denny et al., 1988) and mouse (Srinivasan and Chang, 1979) from day 10 onward. The transformation from continuous to fenestrated capillaries at this time may be useful in raising the permeability of the submandibular gland parenchyma to substances and in promoting differentiation and development of the parenchymal cells. The number of pores appeared to increase further between days 14 and 21. On day 21, light microscopy showed that the acini had increased in size, whereas the number of blood vessels per unit area had decreased in number. The increase in the number of pores may have been fully compensated for by the decrease in number of blood vessels per unit area. It has been reported that, in the mouse submandibular gland from days 21–25, the terminal portions are almost all composed of acinar cells, and granular convoluted ducts appear (Srinivasan and Chang, 1979). As described above, in the present study, the mice were weaned on day 21. Therefore, it seemed that the capillaries developed further by increasing the number of pores so that growth and function of the parenchymal cells might be promoted. The number of capillary pores seemed to have almost peaked on day 35. Therefore, we consider that the capillaries in the mouse submandibular gland parenchyma have matured morphologically and functionally by day 35 every day, by which time the animal has advanced further toward sex maturity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Mrs. Sumie Sato for help with histology preparation.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED