Histopathological changes in parotid gland following submandibular gland failure: an experimental animal study

Introduction Submandibular glands are exposed to many effects due to diseases and therapeutic interventions. A study evaluating the effect of submandibular gland dysfunction on the parotid gland has not been presented in the literature. Objective The aim of this study was to evaluate the histopathological changes in the parotid gland following submandibular gland failure. Methods Three groups of seven randomly selected female New Zealand rabbits weighing 2500–3000 g were studied. Unilateral and bilateral submandibular glands were removed in Groups 1 and 2, respectively. No procedure was performed in Group III, the control group. The parotid glands were removed 30 days later. Histological parameters were evaluated and graded between 0 (none) and 3 (severe). Differences between groups were compared using the Mann–Whitney U test. Results Mean mucus accumulation in acinar cells was 2.57 ± 0.53 and 1.71 ± 0.75 in Groups 1 and 2, respectively (p < 0.05). This value was 0.57 ± 0.53 in Group 3, which was significantly lower than in Groups 1 and 2 (p < 0.05). Mean dilatation of the intercalated ducts’ lumen was 1.28 ± 0.48 and 1.57 ± 0.53 in Groups 1 and 2, respectively (p > 0.05). This value was 0.28 ± 0.48 in Group 3, which was significantly lower than in Groups 1 and 2 (p < 0.05). Mean mucus accumulation in the intercalated ducts’ lumen was 2.00 ± 0.81 and 1.00 ± 0.57 in Groups 2 and 3, respectively (p < 0.05). Conclusion The findings of this study indicate that only 1 month after submandibular gland failure, the parotid glands exhibit significant changes.


Conclusion:
The findings of this study indicate that only 1 month after submandibular gland failure, the parotid glands exhibit significant changes. © 2018 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

Introduction
Three pairs of major salivary glands are positioned around the oral cavity: the sublingual glands under the tongue, the submandibular glands under the floor of the mouth, and the parotid glands in the posterior aspect of the mouth at the retromandibular fossae. 1 The major salivary glands are exposed to a number of factors which leads to loss of function. Some of these factors such as botulinum toxin injection, gland excision, and gland transfer are intended for therapeutic purposes. 2 ---4 Salivary gland stones, radiotherapy applied to the head and neck region, and salivary gland trauma cause significant dysfunctions. 5 ---7 In the lack of a study evaluating the effect of any major salivary gland (or glands) dysfunction on the other salivary glands, our aim was to evaluate the histopathological changes of the parotid gland in a rabbit model of impaired submandibular gland function.

Study design and animals
This study was performed on 21 female New Zealand rabbits (2500---3000 g) in the Center for Experimental Research of the University of Firat, after obtaining approval from the Ethics Board of the School of Medicine of the University of Firat (Number of Document: 142563/14). The animals were randomly divided into three groups, with seven in each group: • Group I: Unilateral submandibular glands were resected, and bilateral parotid glands were removed 30 days later for histopathological examination. • Group II: Bilateral submandibular glands were resected, and bilateral parotid glands were removed 30 days later for histopathological examination.
• Group III (control group): No surgical intervention was performed on the submandibular glands, and bilateral parotid glands were removed for histopathological examination.

Surgical procedure
The same surgical procedure was performed in all Group I and II rabbits. The animals were anesthetized using 10 mg/kg xylazine hydrochloride (Rompun ® ; Bayer AG, Germany) and 50 mg/kg ketamine hydrochloride (Ketalar ® ; Eczacibasi Ilac, Turkey). A 3 cm long horizontal incision was created 1 cm under the corpus of the mandible. The submandibular glands were exposed after elevating skin and subcutaneous flaps (Fig. 1). Unilateral and bilateral submandibular glands were excised from the animals in Groups I and II, respectively. Prophylactic 20---40 mg/kg cephazolin sodium (Sefazol Flk ® ; Mustafa Nevzat, Turkey) was administered 1 h before and 1 h after surgery. All animals were followed for 30 days after the surgical procedure.
The rabbits were anesthetized again using 10 mg/kg xylazine hydrochloride and 50 mg/kg ketamine hydrochloride on the 30th postoperative day. An incision approximately 3 cm in length was made in the region of the parotid gland. The parotid gland was exposed by elevating skin and subcutaneous flaps and then excised. The same surgical procedure was performed on the contralateral parotid gland.

Specimen preparation
The parotid glands were fixed in 10% glutaraldehyde for 4 ---6 h. Fixated specimens were gradually dehydrated in ethanol after being maintained in 1% osmium tetroxide for 0.5 hour and then placed in Epon. Ultratome III glass knives (Shandan Finesse, United Kingdom) were used to obtain 1.5 m thick sections. The sections were stained in hematoxylin and eosin and examined at 40×, 100×, 200×, and 1000× magnification using a light microscope (Olympus, BX51, Japan).

Specimen evaluation
Mucus accumulation in acinar cells, dilatation of intercalated ducts' lumen, mucus accumulation in the lumen of intercalated ducts, cytoplasmic granule accumulation, and myoepithelial cell count were assessed using Eyepieces graticule (an ocular micrometer with 100 equal squares measuring 1 × 1 mm each) mounted on an Olympus light microscope.
From each rabbit, four sections were collected. Within each section, four distinct areas were examined at four magnifications (40×, 100×, 200×, and 1000×) and graded as follows: none = 0, mild = 1, intermediate = 2, and severe = 3. The mean score was calculated using all four examined areas of all four sections from each rabbit.

Statistical analysis
A database was created using the histopathological data, which was statistically analyzed using SPSS 11.5 package program (SPSS Inc, ABD). Values of p less than 0.05 were accepted as statistically significant. The variables had a nonnormal distribution since each group was composed of seven rabbits and the total number of rabbits was 21; thus, nonparametric tests were used in the statistical analyses. The Mann---Whitney U test was used to compare groups.

Results
Mean mucus accumulation in acinar cells was 2.57 ± 0.53 in Group 1 and 1.71 ± 0.75 in Group 2 (p < 0.05). This value was 0.57 ± 0.53 in Group 3, which was significantly lower than in Groups 1 and 2 (p < 0.05). Mean dilatation of the intercalated ducts' lumen was 1.28 ± 0.48 in Group 1 and 1.57 ± 0.53 in Group 2 (p > 0.05). This value was 0.28 ± 0.48 in Group 3, which was significantly lower than in Groups 1 and 2 (p < 0.05). Mean mucus accumulation in the intercalated ducts' lumen was 2.00 ± 0.81 in Group 2 and 1.00 ± 0.57 in Group 3 (p < 0.05). This value was 1.42 ± 0.53 in Group 1, which was not significantly different from the intercalated ducts mucus accumulation in either of the other two groups. There were no significant differences between groups in terms of cytoplasmic granule accumulation or number of myoepithelial cells (Table 1).

Discussion
Secretions are produced in salivary glands by structures composed of mucous and serous cells called acini. Each acinus opens into an intercalated duct, which in turn drains into a striated duct. Myoepithelial cells are found in the basal laminae of the epithelium near the acinus and in the basal lamina of the canal epithelium. Acini and intercalated ducts are surrounded by myoepithelial cells. 8,9 The parotid gland is composed of serous acinar cells containing intense intracytoplasmic translucent granules that produce a pure serous secretion. 10 Alterations in the histological structure of the parotid gland are seen in tumors, inflammatory conditions, systemic diseases, and following radiation treatment; thus,   the secretory function of the gland deteriorates. 11 In the present study, loss of submandibular gland function was shown to cause a proportional dilatation of the intercalated ducts' lumen ( Fig. 2). Similar to our study, intercalated ducts' lumen dilatation is also seen in sialolithiasis and excessive alcohol consumption. 12,13 Squamous metaplasia in the epithelium, intermediate to severe chronic inflammation, and varying degrees of acinar destruction were reported in biopsy material obtained from a gland with sialolithiasis. 12 These extra findings in sialolithiasis are not presented in our own work which is probably due to the fact that the time of study limited as little as a month. In contrast to our study, intracytoplasmic granule accumulation is an important finding of excessive alcohol consumption. 13 The main ductal ectasia of salivary gland is one of the histopathological findings of Sjögren's syndrome and infectious diseases. 14,15 Also, interstitial fibrosis and metaplastic changes are the mainstays of radiotherapy-related gland damage. 16 While these findings are not observed in our study, mucus accumulation is another important finding of the histopathological examination. Mucus accumulation in acinar cells was greater in animals with unilateral and bilateral submandibular gland excision compared to the control group, and there was also a statistically significant difference between the unilateral and bilateral groups (Fig. 3). Mucus accumulation in the lumen of intercalated ducts was also greater in animals with bilateral submandibular gland excision compared to the control group.

Conclusion
Unilateral or bilateral excision of the submandibular gland results in histopathological changes in the parotid gland after a short period of time. When the histopathological changes in other diseases affecting the salivary gland are considered, the dilatation of the intercalated ducts' lumen in our study was consistent with findings in salivary gland diseases related to infectious and overuse of alcohol. The cause of histopathological changes in the parotid glands after a short time (1 month) might be explained by the parotid glands being overworked, as they must compensate for the large workload previously shared by the submandibular gland.
The present study can be considered a good model for evaluating the short-term effects of decreased submandibular gland function on the parotid gland. Nevertheless, additional studies are required to evaluate long-term effects such as metaplasia and etc.