Vol 82, No 3 (2023)
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Effects of liquid diet intake on nerve growth in salivary glands of growing rats

S. Takahashi1, Y. Nakamichi1, T. Yamamoto1
Pubmed: 36000590
Folia Morphol 2023;82(3):551-557.

Abstract

Background: The growth of parotid glands is inhibited by liquid diet intake
during growing period, while that of submandibular glands is not affected. This
study examined how liquid diet intake affects nerve growth in the parotid and
submandibular glands of growing rats, because nerves are closely involved in the
maintenance of salivary gland structure.
Materials and methods: Male Wistar rats were weaned at 21 days of age. Then,
rats were fed a pellet diet and a liquid diet in the control group and experimental
group, respectively. At 0, 2, 4, or 8 weeks, they were euthanised by isoflurane
overdose, and parotid and submandibular glands were removed. The frozen
sections were made and immuno-stained with anti-protein gene product 9.5
(PGP 9.5) antibody (general nerve marker), anti-tyrosine hydroxylase (TH) antibody
(sympathetic nerve marker), or anti-neuronal nitric oxide synthase (nNOS) antibody
(parasympathetic nerve marker).
Results: In control parotid glands, scattered punctate or short linear patterns of
PGP 9.5-positive sites were observed at week 0. After 2 weeks, PGP 9.5-positive
sites, some of which were arranged in long linear patterns, increased in number.
There were some TH-positive sites at week 0. After 2 weeks, there were increasing
numbers of TH-positive sites, often in long linear patterns. At week 0, there were
very few nNOS-positive sites, and nNOS immunoreactivity increased over time. After
week 4, they demonstrated linear patterns. In the experimental parotid glands,
there were fewer PGP 9.5- and nNOS-positive sites than in control parotid glands
at each time point, although TH immunoreactivity was similar between two groups
at each time point. In control submandibular glands, few punctate exhibited
PGP 9.5-positive site were observed at week 0. At week 4, PGP 9.5 immunoreaction
increased and showed linear patterns. TH-positive sites demonstrated punctate
or short linear patterns at week 0, and thereafter TH immunoreactivity increased
and were arranged in long linear patterns. There were few nNOS-positive sites
at week 0, and they gradually increased after week 4. The immunoreactivities of
all antibodies in the experimental submandibular glands were similar to those in
the control at each time point.
Conclusions: Parasympathetic nerve growth in rat parotid glands was inhibited by
liquid diet intake during the growth period, while liquid diet intake did not affect
parasympathetic nerve growth nor sympathetic nerve growth in rat submandibular
glands.

ORIGINAL ARTICLE

Folia Morphol.

Vol. 82, No. 3, pp. 551–557

DOI: 10.5603/FM.a2022.0074

Copyright © 2023 Via Medica

ISSN 0015–5659

eISSN 1644–3284

journals.viamedica.pl

Effects of liquid diet intake on nerve growth in salivary glands of growing rats

S. TakahashiY. NakamichiT. Yamamoto
Department of Oral Functional Anatomy, Hokkaido University Faculty of Dental Medicine, Sapporo, Japan

[Received: 4 July 2022; Accepted: 7 August 2022; Early publication date: 17 August 2022]

Background: The growth of parotid glands is inhibited by liquid diet intake during growing period, while that of submandibular glands is not affected. This study examined how liquid diet intake affects nerve growth in the parotid and submandibular glands of growing rats, because nerves are closely involved in the maintenance of salivary gland structure.
Materials and methods: Male Wistar rats were weaned at 21 days of age. Then, rats were fed a pellet diet and a liquid diet in the control group and experimental group, respectively. At 0, 2, 4, or 8 weeks, they were euthanised by isoflurane overdose, and parotid and submandibular glands were removed. The frozen sections were made and immuno-stained with anti-protein gene product 9.5 (PGP 9.5) antibody (general nerve marker), anti-tyrosine hydroxylase (TH) antibody (sympathetic nerve marker), or anti-neuronal nitric oxide synthase (nNOS) antibody (parasympathetic nerve marker).
Results: In control parotid glands, scattered punctate or short linear patterns of PGP 9.5-positive sites were observed at week 0. After 2 weeks, PGP 9.5-positive sites, some of which were arranged in long linear patterns, increased in number. There were some TH-positive sites at week 0. After 2 weeks, there were increasing numbers of TH-positive sites, often in long linear patterns. At week 0, there were very few nNOS-positive sites, and nNOS immunoreactivity increased over time. After week 4, they demonstrated linear patterns. In the experimental parotid glands, there were fewer PGP 9.5- and nNOS-positive sites than in control parotid glands at each time point, although TH immunoreactivity was similar between two groups at each time point. In control submandibular glands, few punctate exhibited PGP 9.5-positive site were observed at week 0. At week 4, PGP 9.5 immunoreaction increased and showed linear patterns. TH-positive sites demonstrated punctate or short linear patterns at week 0, and thereafter TH immunoreactivity increased and were arranged in long linear patterns. There were few nNOS-positive sites at week 0, and they gradually increased after week 4. The immunoreactivities of all antibodies in the experimental submandibular glands were similar to those in the control at each time point.
Conclusions: Parasympathetic nerve growth in rat parotid glands was inhibited by liquid diet intake during the growth period, while liquid diet intake did not affect parasympathetic nerve growth nor sympathetic nerve growth in rat submandibular glands. (Folia Morphol 2023; 82, 3: 551–557)
Key words: growth, liquid diet, nerve, salivary gland

Address for correspondence: Dr. S. Takahashi, Department of Oral Functional Anatomy, Hokkaido University Faculty of Dental Medicine, Kita 13, Nishi 7, Kita-ku, Sapporo, 060-8586, Japan, tel: +81 11 706 4218, fax: +81 11 706 4928, e-mail: tshigeru@den.hokudai.ac.jp

This article is available in open access under Creative Common Attribution-Non-Commercial-No Derivatives 4.0 International (CC BY-NC-ND 4.0) license, allowing to download articles and share them with others as long as they credit the authors and the publisher, but without permission to change them in any way or use them commercially.

INTRODUCTION

Salivary glands are exocrine glands that secrete saliva, which helps to maintain the oral environment. Salivary gland function is controlled through innervation by autonomic, sympathetic, and parasympathetic nerves [19]. Generally, sympathetic nerve stimulation evokes protein-rich secretion, while parasympathetic nerve stimulation evokes large volumes of saliva [20]; however, there are some variations in these effects among salivary glands and species [21]. Because innervation is important for salivary gland function, the effects of nerve damage on salivary glands have been experimentally investigated. Salivary glands with excretory duct ligation including parasympathetic nerves weighed less and exhibited more parenchymal atrophy, compared with salivary glands that were subjected to excretory duct ligation excluding parasympathetic nerves [8, 9]. Furthermore, the weight of parasympathectomised salivary glands decreased [1, 2, 12, 22], and acinar cells became atrophic in parasympathectomised salivary glands [1, 22]. Although sympathectomy had an atrophic effect on salivary glands, it was much weaker than the atrophic effect of parasympathectomy [23, 24, 35]. Thus, autonomic nerves presumably play important roles in maintaining normal salivary gland structure; parasympathetic nerves may have a greater effect, compared with sympathetic nerves [6].

There is clinical interest in how the daily intake of soft foods, which is a characteristic of modern eating habits, affects the oral maxillofacial region. Therefore, many experimental studies have been conducted in which experimental animals were fed a liquid diet; the salivary glands of liquid diet-fed animals have also been examined. In these studies, biochemical analysis showed decreases in salivary amylase [7, 11, 14, 15, 27] and salivary protein [11] levels, as well as a decreased salivary flow rate [4, 10, 25]; these findings indicated reduced parotid gland function. Histological examinations revealed atrophic changes in parotid glands, such as reduction of gland weight [3, 4, 7, 11, 14–17, 25–27, 29], shrinkage of acinar cells [23, 25–27, 29], reduction of proliferative activity [29], and apoptotic death of acinar cells [5, 29]. However, many studies demonstrated that atrophic changes were minimal or absent in the submandibular glands of liquid diet-fed animals [3, 14, 16, 17, 26, 30].

There is a need to clarify the effects of successive intake of soft food during growth periods on salivary gland growth because children tend to prefer soft food, rather than hard food [37]. In a previous study, increases in parotid gland weight were smaller in rats that were continuously fed a liquid diet immediately after weaning, compared with rats that were fed a pellet diet [28]. In the parotid glands of rats that were continuously fed a liquid diet, acinar cells did not grow larger and proliferative activity was reduced during the growth period, demonstrating that parotid gland growth was strongly inhibited by liquid diet intake during the growth period [28]. In contrast, submandibular gland growth was not inhibited by liquid diet intake during the growth period [31]. These results indicated that the parotid and submandibular glands differed in their response to continuous intake of a liquid diet during the growth period. Because autonomic nerves are closely involved in the maintenance of salivary gland structure, as described above, it was speculated that salivary glands, particularly parotid glands, would be negatively affected in animals that were fed a liquid diet during the growth period.

The purpose of this study was to determine how liquid diet intake influenced nerve growth in salivary glands during the growth period. For this purpose, rats were fed a liquid diet beginning immediately after weaning; their parotid and submandibular glands were examined by immunohistochemical analysis with anti-protein gene product 9.5 (PGP 9.5) antibody (a marker of all nerves), anti-tyrosine hydroxylase (TH) antibody (a sympathetic nerve marker), and anti-neuronal nitric oxide synthase (nNOS) antibody (a parasympathetic nerve marker).

MATERIALS AND METHODS

Ethics and animal welfare

Animal experiments in this study were carried out in accordance with the Hokkaido University Guide for the Care and Use of Laboratory Animals; the experimental protocol was approved by the Hokkaido University Laboratory Animal Committee (approval no. 14-0108). During the experiment, animals were housed in a temperature-controlled room (approximately 22°C) with a 12-h light/dark cycle and free access to drinking water; they were weighed and visually observed to confirm health status. Statistical comparisons in body weight between the control and experimental groups at each time point were made using Mann-Whitney U test (Ystat2008, Igakutosho, Tokyo, Japan). P < 0.05 was considered significant.

Experimental procedures

Twenty-eight 12-day-old male Wistar rats and their mothers were obtained from CLEA Japan Inc. (Tokyo, Japan); the male rats were weaned at 21 days of age and divided into two groups. Each control rat was fed a pellet diet (25 g/day; Labo MR Standard, Nosan Corp., Yokohama, Japan) for 0, 2, 4, or 8 weeks (n = 4 at each time point); each experimental rat was fed a liquid diet (prepared daily by mixing 25 g of the pellet diet in powder form with 50 mL of water) for 2, 4, or 8 weeks (n = 4 at each time point). At the end of the experimental period, all animals were subjected to food deprivation for 12 h at night to synchronise the salivary gland status. They were then euthanised by isoflurane overdose; the parotid and submandibular glands were immediately removed. Extracted glands were embedded in Tissue Tek OCT compound (Miles Scientific, Naperville, IL, USA), frozen in liquid nitrogen, and stored at –80°C until preparation.

Immunohistochemical analysis

Fresh frozen sections were prepared using a cryostat and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 5 min. After the sections had been washed with phosphate-buffered saline (PBS), they were incubated with the following primary antibodies overnight at 4°C: anti-PGP 9.5 rabbit monoclonal antibody (1:200; EPR4118, Abcam, Cambridge, UK) to detect all nerves, anti-TH rabbit polyclonal antibody (1:1000; AB152, Merck Millipore, Darmstadt, Germany) to detect sympathetic nerves, and anti-nNOS rabbit polyclonal antibody (1:100; Af480, Frontier Institute, Ishikari, Japan) to detect parasympathetic nerves. The sections were then incubated with biotinylated anti-rabbit goat polyclonal antibody (Histofine, Nichirei Bioscience, Tokyo, Japan) for 60 min at room temperature; subsequently, they were incubated with peroxidase-conjugated streptavidin (Histofine, Nichirei Bioscience) for 30 min at room temperature. Peroxidase activity was visualised using 3, 3’-diaminobenzidine; sections were lightly counterstained with Mayer’s haematoxylin. After each incubation step above, sections were thoroughly washed with PBS.

Negative control sections were incubated in PBS without primary antibodies, then subjected to all other treatments as above.

RESULTS

Body weights of animals

Animals in both groups appeared healthy during the experimental period, and their body weights increased as normal. There was no significant difference in body weights between the control and experimental groups at every time point (Fig. 1).

Figure 1. Box plot of body weights of the control (Cont) and experimental rats (Exp). There was no significant difference between two groups at each time point. Explanation of box plot: top of box, 75th percentile; bottom of box, 25th percentile; horizontal bar within box, median; upper whisker, maximum value; lower whisker, minimum value; W week.
Parotid glands

At week 0, thick nerve bundles in interlobular connective tissue exhibited PGP 9.5-positive sites. In glandular lobules, scattered punctate or short linear patterns of PGP 9.5-positive sites were observed (Fig. 2A). After 2 weeks of pellet diet intake, there were increasing numbers of PGP 9.5-positive sites around acini and ducts; some of these sites were arranged in long linear patterns (Fig. 2B). However, in the parotid glands of liquid diet-fed rats, there remained few PGP 9.5-positive sites at week 8 (Fig. 2C).

Furthermore, there were some TH-positive sites around acini in glandular lobules, as well as in thick nerve bundles in interlobular connective tissue, at week 0 (Fig. 2D). After 2 weeks of pellet diet intake, there were increasing numbers of TH-positive sites at the peripheries of acini and ducts, often in long linear patterns (Fig. 2E). TH immunoreactivity was similar between experimental and control groups at each time point (Fig. 2F).

At week 0, there were very few nNOS-positive sites in glandular lobules, most of which were punctate, although thick nerve bundles in interlobular connective tissues demonstrated extensive nNOS immunoreactivity (Fig. 2G). In control glands, nNOS immunoreactivity gradually increased. nNOS-positive sites were generally punctate at week 2, while they demonstrated linear patterns after week 4 (Fig. 2H). In experimental glands, nNOS immunoreactivity gradually increased. However, there were fewer nNOS-positive sites in experimental glands than in control glands at each time point; these sites mainly exhibited a punctate appearance (Fig. 2I).

Figure 2. Immunohistochemical analyses of parotid glands to determine distributions of protein gene product 9.5 (PGP 9.5; AC), tyrosine hydroxylase (TH; DF), and neuronal nitric oxide synthase (nNOS; GI). Normal parotid glands at week 0 (A, D, G); control parotid glands at week 8 (B, H) and week 4 (E); experimental parotid glands at week 8 (C, I) and week 4 (F). Scale bars = 30 µm. PGP 9.5 (A) and nNOS (G) immunoreactivities are minimal at week 0. Although PGP 9.5 and nNOS immunoreactivities both gradually increased in control glands (B, H), they remained minimal in experimental glands (C, I). TH immunoreactivity gradually increased in both control (E) and experimental glands (F), compared with parotid glands at week 0 (D).
Submandibular glands

At week 0, thick bundles of nerve fibres in interlobular connective tissue exhibited PGP 9.5-positive sites; few punctate exhibited PGP 9.5-positive sites were distributed around acini and ducts in glandular lobules (Fig. 3A). In control submandibular glands, PGP 9.5 immunoreactivity gradually increased around parenchymal tissue beginning at week 4; some PGP 9.5-positive sites demonstrated linear patterns (Fig. 3B). Immunoreactivity was similar between experimental and control groups at each time point (Fig. 3C).

Also at week 0, TH-positive sites were observed at the peripheries of acini and ducts, with a punctate or short linear appearance; they were also observed in thick nerve bundles in interlobular connective tissue (Fig. 3D). Thereafter, TH immunoreactivity gradually increased and TH-positive sites were present in long linear patterns (Fig. 3E). TH immunoreactivity was similar between experimental and control groups at each time point (Fig. 3F).

Finally, some thick nerve bundles in interlobular connective tissue exhibited nNOS immunoreactivity at week 0, while few nNOS-positive sites were present in glandular lobules (Fig. 3G). At week 2, nNOS immunoreactivity remained limited overall, although scattered punctate nNOS-positive sites were observed around acini and ducts in control glands. After week 4, nNOS immunoreactivity in glandular lobules gradually increased; some nNOS-positive sites demonstrated linear patterns (Fig. 3H). nNOS immunoreactivity was similar between experimental and control groups at each time point (Fig. 3I).

Figure 3. Immunohistochemical analyses of submandibular glands to determine distributions of protein gene product 9.5 (PGP 9.5; AC), tyrosine hydroxylase (TH; DF), and neuronal nitric oxide synthase (nNOS; GI). Normal submandibular glands at week 0 (A, D, G); control submandibular glands at week 4 (B, H) and week 2 (E); experimental submandibular glands at week 4 (C, I) and week 2 (F). Scale bars = 30 µm. At week 0, all immunoreactivities were minimal (A, D, G). Immunoreactivities were similar between experimental glands (B, E, H) and control glands (C, F, I).

Analysis of negative control sections for all primary antibodies revealed no reactions.

DISCUSSION

Protein gene product 9.5 is a ubiquitin carboxyl -terminal hydrolase [36]. It was originally identified as a new brain-specific protein; it has since been used as a marker of all nerves [32]. TH is an enzyme involved in amino acid and neurotransmitter metabolism; it is the rate-limiting enzyme in the synthesis of catecholamines (e.g. dopamine and noradrenaline) from tyrosine [18]. Therefore, TH can be used as a marker of sympathetic nerves [34]. Nitric oxide is formed from its precursor, L-arginine, by a family of nitric oxide synthases. nNOS is one of three nitric oxide synthase isoforms [33] and has been used as a parasympathetic nerve marker in various tissues [13]. For these reasons, PGP 9.5, TH, and nNOS for immunohistochemical analyses of nerve distribution were used in this study. While comparisons can be performed between control and experimental glands that have been stained with the same antibody, it is difficult to compare glands that have been stained with different antibodies because the degree of immunoreactivity depends on each antibody’s sensitivity.

In this study, PGP 9.5 and nNOS immunoreactivities were lower in experimental parotid glands than in control parotid glands, suggesting that liquid diet intake during the growth period inhibits parasympathetic nerve growth in parotid glands. A previous study [28] showed that liquid diet intake in the growth period suppressed the proliferative activity of the parotid gland; it also suppressed increases in parotid gland weight and acinar cell size. Therefore, present results are consistent with the previous finding that liquid diet intake adversely affects parotid gland growth.

In contrast, nerve growth in submandibular glands was not affected by liquid diet intake during the growth period, as demonstrated by the absence of a difference between control and experimental submandibular glands in all immunohistochemical staining assays in this study. This is consistent with a previous report [31] that investigated gland weight, acinar cell size, and acinar cell proliferation in the submandibular glands of liquid diet-fed growing rats, confirming that submandibular gland growth is not affected by liquid diet intake. Thus, parotid glands are much more sensitive to liquid diet intake, compared with submandibular glands. This discrepancy might be related to differences in parasympathetic nerve system innervation of the two glands [30, 31]. The parasympathetic fibres of the parotid glands arise from the inferior salivary nucleus and accompany the glossopharyngeal nerve, while the parasympathetic fibres of the submandibular glands arise from the superior salivary nucleus and accompany the facial nerve [38]. The expression of choline acetyltransferase, which is necessary for acetylcholine synthesis, is downregulated in the parotid glands of liquid diet-fed rats [3], suggesting suppression of the parasympathetic nerve system that is needed to maintain parotid gland structure. Thus, the parotid glands may exhibit degeneration. In contrast, the parasympathetic nerve system of the submandibular glands is unaffected by liquid diet intake. Our finding of a difference in parasympathetic nerve growth between parotid and submandibular glands might support the above theory that the different reactions to liquid diet intake are related to differences in parasympathetic nerve innervation between parotid and submandibular glands.

The previous study showed that liquid diet intake inhibited parotid gland growth by suppressing acinar cell enlargement and impeding acinar cell proliferation [28]; the present study showed that liquid diet intake inhibited parasympathetic nerve growth in parotid glands. The next question to address is whether the inhibition of parotid growth affects parasympathetic nerve growth in parotid glands, or whether the inhibition of parasympathetic nerve growth in parotid glands affects parotid growth. Although the present findings do not enable confirmation of either scenario, the inhibition of parasympathetic nerve growth might have a causative effect because parasympathetic nerves contribute to the maintenance of salivary gland morphology. Further studies are needed to investigate this sequence of events.

CONCLUSIONS

In conclusion, parasympathetic nerve growth in rat parotid glands was inhibited by liquid diet intake during the growth period, while liquid diet intake did not affect parasympathetic nerve growth nor sympathetic nerve growth in rat submandibular glands. Prudence is necessary to apply the conclusions obtained from rats to humans. However, they are considered to be a piece of evidence showing that the dietary habit requiring adequate mastication is important for healthy growth of the oral maxillofacial region. The conclusions of this study are significant in clinical dentistry to encourage patients to have the good dietary habit.

Acknowledgements

We thank Ryan Chastain-Gross, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Conflict of interest: None declared

REFERENCES

  1. Carpenter GH, Proctor GB, Garrett JR. Preganglionic parasympathectomy decreases salivary SIgA secretion rates from the rat submandibular gland. J Neuroimmunol. 2005; 160(1-2): 4–11, doi: 10.1016/j.jneuroim.2004.10.020, indexed in Pubmed: 15710452.
  2. Chaparro O, Yu WH, Shaw PA. Isoproterenol-induced expression of the cystatin S gene in submandibular glands of parasympathectomized rats. Brain Res Mol Brain Res. 1998; 61(1-2): 136–146, doi: 10.1016/s0169-328x(98)00223-x, indexed in Pubmed: 9795188.
  3. Eksrom J. Choline acethyltransferase and secretory responses of the rat’s salivary glands after liquid diet. Q J Exp Physiol. 1973; 58(2): 171–179, indexed in Pubmed: 448796.
  4. Ekström J, Templeton D. Difference in sensitivity of parotid glands brought about by disuse and overuse. Acta Physiol Scand. 1977; 101(3): 329–335, doi: 10.1111/j.1748-1716.1977.tb06014.x, indexed in Pubmed: 596206.
  5. ElGhamrawy TA. The effect of liquid diet on the parotid gland and the protective role of L-carnitine: immunohistochemical and ultrastructural study. Folia Morphol. 2015; 74(1): 42–49, doi: 10.5603/FM.2015.0007, indexed in Pubmed: 25792394.
  6. Garrett JR. Innervation of salivary glands: Neurohistological and functional aspects. In: Sreebny LM. The salivary system. CRC Press Inc., Boca Raton 1987: 69–93.
  7. Hall HD, Schneyer CA. Salivary gland atrophy in rat induced by liquid diet. Proc Soc Exp Biol Med. 1964; 117: 789–793, doi: 10.3181/00379727-117-29699, indexed in Pubmed: 14244956.
  8. Harrison JD, Fouad HM, Garrett JR. Variation in the response to ductal obstruction of feline submandibular and sublingual salivary glands and the importance of the innervation. J Oral Pathol Med. 2001; 30(1): 29–34, doi: 10.1034/j.1600-0714.2001.300105.x, indexed in Pubmed: 11140897.
  9. Harrison JD, Garrett JR. Histological effects of ductal ligation of salivary glands of the cat. J Pathol. 1976; 118(4): 245–254, doi: 10.1002/path.1711180407, indexed in Pubmed: 1271136.
  10. Ito K, Morikawa M, Inenaga K. The effect of food consistency and dehydration on reflex parotid and submandibular salivary secretion in conscious rats. Arch Oral Biol. 2001; 46(4): 353–363, doi: 10.1016/s0003-9969(00)00124-2, indexed in Pubmed: 11269869.
  11. Johnson DA. Effect of a liquid diet on the protein composition of rat parotid saliva. J Nutr. 1982; 112(1): 175–181, doi: 10.1093/jn/112.1.175, indexed in Pubmed: 6172574.
  12. Katsukawa H, Ninomiya Y, Funakoshi M. Effects of parasympathectomy on androgen responses of the rat submandibular gland. Arch Oral Biol. 1990; 35(4): 273–275, doi: 10.1016/0003-9969(90)90042-9, indexed in Pubmed: 2198865.
  13. Kiyokawa H, Katori Y, Cho KH, et al. Reconsideration of the autonomic cranial ganglia: an immunohistochemical study of mid-term human fetuses. Anat Rec (Hoboken). 2012; 295(1): 141–149, doi: 10.1002/ar.21516, indexed in Pubmed: 22095632.
  14. Kurahashi M. The effect of dietary consistency and water content on the parotid glands of submandibular and sublingual duct-ligated rats. Arch Oral Biol. 2002; 47(5): 369–374, doi: 10.1016/s0003-9969(02)00015-8, indexed in Pubmed: 12015217.
  15. Kurahashi M, Inomata K. Effects of dietary consistency and water content on parotid amylase secretion and gastric starch digestion in rats. Arch Oral Biol. 1999; 44(12): 1013–1019, doi: 10.1016/s0003-9969(99)00099-0, indexed in Pubmed: 10669079.
  16. Månsson B, Ekman R, Håkanson R, et al. Neuropeptides and disuse of the rat parotid gland. Exp Physiol. 1990; 75(4): 597–599, doi: 10.1113/expphysiol.1990.sp003435, indexed in Pubmed: 1699560.
  17. Månsson B, Nilsson BO, Ekström J. Effects of repeated infusions of substance P and vasoactive intestinal peptide on the weights of salivary glands subjected to atrophying influences in rats. Br J Pharmacol. 1990; 101(4): 853–858, doi: 10.1111/j.1476-5381.1990.tb14170.x, indexed in Pubmed: 1707705.
  18. Moss PA, Davies KE, Boni C, et al. Linkage of tyrosine hydroxylase to four other markers on the short arm of chromosome 11. Nucleic Acids Res. 1986; 14(24): 9927–9932, doi: 10.1093/nar/14.24.9927, indexed in Pubmed: 2880337.
  19. Pinkstaff CA. The cytology of salivary glands. Int Rev Cytol. 1980; 63: 141–261, doi: 10.1016/s0074-7696(08)61759-3, indexed in Pubmed: 395130.
  20. Proctor GB. The physiology of salivary secretion. Periodontol 2000. 2016; 70(1): 11–25, doi: 10.1111/prd.12116, indexed in Pubmed: 26662479.
  21. Proctor GB, Carpenter GH. Regulation of salivary gland function by autonomic nerves. Auton Neurosci. 2007; 133(1): 3–18, doi: 10.1016/j.autneu.2006.10.006, indexed in Pubmed: 17157080.
  22. Schneyer CA, Hall HD. Amylase and electrolyte changes after postganglionic parasympathectomy of parotid gland. Am J Physiol. 1964; 207: 308–312, doi: 10.1152/ajplegacy.1964.207.2.308, indexed in Pubmed: 14205339.
  23. Schneyer CA, Hall HD. Parasympathetic regulation of mitosis induced in rat parotid by dietary change. Am J Physiol. 1975; 229(6): 1614–1617, doi: 10.1152/ajplegacy.1975.229.6.1614, indexed in Pubmed: 1239958.
  24. Schneyer CA, Hall HD. Neurally mediated increase in mitosis and DNA of rat parotid with increase in bulk of diet. Am J Physiol. 1976; 230(4): 911–915, doi: 10.1152/ajplegacy.1976.230.4.911, indexed in Pubmed: 1267023.
  25. Scott J, Berry MR, Gunn DL, et al. The effects of a liquid diet on initial and sustained, stimulated parotid salivary secretion and on parotid structure in the rat. Arch Oral Biol. 1990; 35(7): 509–514, doi: 10.1016/0003-9969(90)90080-t, indexed in Pubmed: 2222254.
  26. Scott J, Gunn DL. A comparative quantitative histological investigation of atrophic changes in the major salivary glands of liquid-fed rats. Arch Oral Biol. 1991; 36(11): 855–857, doi: 10.1016/0003-9969(91)90035-s, indexed in Pubmed: 1763982.
  27. Sreebny LM, Johnson DA. Effect of food consistency and decreased food intake on rat parotid and pancreas. Am J Physiol. 1968; 215(2): 455–460, doi: 10.1152/ajplegacy.1968.215.2.455, indexed in Pubmed: 5665178.
  28. Takahashi S, Uekita H, Kato T, et al. Growth of rat parotid glands is inhibited by liquid diet feeding. Tissue Cell. 2015; 47(3): 336–341, doi: 10.1016/j.tice.2015.04.003, indexed in Pubmed: 25956847.
  29. Takahashi S, Uekita H, Kato T, et al. Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet. J Mol Histol. 2012; 43(6): 761–766, doi: 10.1007/s10735-012-9442-y, indexed in Pubmed: 22918835.
  30. Takahashi S, Uekita H, Kato T, et al. Immunohistochemical and ultrastructural investigation of acinar cells in submandibular and sublingual glands of rats fed a liquid diet. Tissue Cell. 2014; 46(2): 136–143, doi: 10.1016/j.tice.2014.01.001, indexed in Pubmed: 24553131.
  31. Takahashi S, Uekita H, Taniwaki H, et al. Acinar cell response to liquid diet during rats’ growth period differs in submandibular and sublingual glands from that in parotid glands. Tissue Cell. 2017; 49(2 Pt B): 275–284, doi: 10.1016/j.tice.2017.01.011, indexed in Pubmed: 28222888.
  32. Thompson RJ, Doran JF, Jackson P, et al. PGP 9.5: a new marker for vertebrate neurons and neuroendocrine cells. Brain Res. 1983; 278(1-2): 224–228, doi: 10.1016/0006-8993(83)90241-x, indexed in Pubmed: 6640310.
  33. Tsutsui M, Shimokawa H, Otsuji Y, et al. Pathophysiological relevance of NO signaling in the cardiovascular system: novel insight from mice lacking all NO synthases. Pharmacol Ther. 2010; 128(3): 499–508, doi: 10.1016/j.pharmthera.2010.08.010, indexed in Pubmed: 20826180.
  34. Warburton AL, Santer RM. Decrease in synapsin I staining in the hypogastric ganglion of aged rats. Neurosci Lett. 1995; 194(3): 157–160, doi: 10.1016/0304-3940(95)11769-s, indexed in Pubmed: 7478227.
  35. Wells H, Peronace AA. Functional hypertrophy and atrophy of the salivary glands of rats. Am J Physiol. 1967; 212(2): 247–251, doi: 10.1152/ajplegacy.1967.212.2.247, indexed in Pubmed: 6018002.
  36. Wilkinson KD, Lee KM, Deshpande S, et al. The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science. 1989; 246(4930): 670–673, doi: 10.1126/science.2530630, indexed in Pubmed: 2530630.
  37. Yamanaka R, Akther R, Furuta M, et al. Relation of dietary preference to bite force and occlusal contact area in Japanese children. J Oral Rehabil. 2009; 36(8): 584–591, doi: 10.1111/j.1365-2842.2009.01971.x, indexed in Pubmed: 19548957.
  38. Young JA, Lennep EWV. Gross anatomy. In The morphology of salivary glands. Academic Press Inc., London 1987: 8–21.