Regenerative Capacity of Atrophic Submandibular Gland by Duct Ligation in Mice

Abstract

Function degeneracy of the salivary gland leads to reduced protection against infection and reduced physiological function of the oral cavity, considerably diminishing the patient's quality of life. However, no method of treatment has yet been established to recover the secretory capacity of the salivary gland reduced by the degeneration or disappearance of the gland tissue. Ongoing salivary gland regeneration experiments using cell transplantation aim to introduce clinical regenerative treatment for the salivary gland. Because it is essential to understand the self-regenerative capacity that remains in the recipient's salivary gland, it is essential to search for optimal conditions for transplantation and establish a salivary gland atrophy model. In this context, we performed duct ligation of the submandibular gland in groups of mice for 7-, 14-, and 21-day ligation periods, compared immunohistochemical staining using acinar cell and stem cell markers, detected apoptosis using TUNEL staining, and analyzed gene expression using RT-PCR to search for the expression of factors related to regenerative capacity that remain in the atrophic salivary gland. All 3 periods of ligation led to remarkable atrophy/disappearance of acinar cells and the dominant presence of duct-like structures. These structures expressed stem cell markers most intensively after 14 days of ligation, and double immunostaining revealed the expressions of PSCA and c-Kit that coincided with them. AQP5 expression was detected in acinar cells and the apical membrane of the intercalated ducts in the normal submandibular gland and was also detected after 21 days of ligation in the remnant acinar cells and intercalated ducts. Conversely, the number of TUNELpositive apoptotic cells in the atrophic salivary gland was highest after 7 days of ligation. These results showed that the duct-like structures that exist after ligation expressed stem cell markers and AQP5, indicating the presence of a mechanism related to the regeneration of salivary gland tissue.

Expression of c-kit and Cytokeratin 5 in the Submandibular Gland after Release of Long-Term Ligation of the Main Excretory Duct in Mice

“…Histologic findings showed submandibular gland atrophy, disappearance of the acinar portion and an increase in duct-like structures and fibrous connective tissue after 2 months of ligation. These findings were similar to those of short-term ligation studies [1, 2, 31, 32, 35]. Gene expression analysis showed low levels of the acinar cell marker AQP5 [11, 13, 16, 25] and the functional marker α-amylase [11, 36], which is consistent with previous results [17].…”

“…It is well recognized that the atrophy provoked by obstruction of the salivary ducts induces changes in the structure [ 1 , 2 , 31 , 32 , 35 ], the residual function and the regenerative capacity of the glandular tissue [ 4 , 9 , 19 , 28 ]. Almost all the evidence is based on short-term experiments conducted over approximately 1 week, and there have been no longer-term observational studies.…”

Effects of Human Dental Pulp Stem Cell-Derived Conditioned Medium on Atrophied Submandibular Gland after the Release from Ligation of the Main Excretory Duct in Mice

“…In this study, the main duct of the right submandibular gland was ligated for 3 weeks. As presented in the 3W lig group, at the time of the release of ligation, sufficient atrophy of the salivary gland tissues was confirmed by HE and PAS staining, immunostaining for various proteins, and real-time PCR, which was consistent with the report by Akadomari et al 5) . On the other hand, studies on observation after the release of ligation have reported to be of various durations after the release, ranging from 1 week to as long as 2 months 7,8,29) .…”

“…Experiments using ligation of salivary glands have been reported to be of various lengths of duration, ranging from 1 week to as long as 3 months [5][6][7][8] . In this study, the main duct of the right submandibular gland was ligated for 3 weeks.…”

“…There have been numerous studies on the salivary gland tissue after ligation and ligation release as a model of duct obstruction by salivary gland stones [5][6][7][8] . The atrophied salivary glands after ligation release express stem cell and progenitor cell markers and exhibit the remaining self-renewal capacity [9][10][11][12][13][14] .…”