Chronic Changes in the Atrophied Submandibular Gland after Long-term Ligation of the Main Excretory Duct in Mice

Abstract

To examine the pathology of salivary glands that have undergone atrophy or hypofunction due to old age or disease, the duct ligation model is used. This model has been used in studies on the course of glandular parenchyma atrophy and the potential for repair and regeneration. However, the period of ligation was short in most of these studies, and none have examined long-term progress. Therefore, we investigated long-term ligation of the submandibular gland in mice and examined the chronic changes in atrophied salivary glands. The ligation periods were 1, 2 and 3 months, and atrophied salivary glands were resected after each period. Resected glands were examined by histology, immunohistochemistry, RT-PCR, and transmission electron microscopy. Histologically, disappearance of acinar cells and increases in duct-like structures were observed over time in atrophied salivary glands, resulting from long-term submandibular gland main excretory duct ligation. Acinar cell markers (-amylase, aquaporin 5) showed marked weakness in expression after ligation, but expression was still observed after 3 months of ligation. Stem cell markers (c-kit, Sca-1) showed greater expression at 1 month of ligation, compared with controls, but expression subsequently decreased with time. Expression of the precursor cell marker cytokeratin 5 was retained throughout long-term ligation. Atrophied salivary gland tissue resulting from long-term ligation showed increases in specific duct-like structures over time, and the expression of stem cell markers and progenitor cell markers in the area of these structures suggests that the repair capability remained intact.

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

“…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]. While obstruction causes histologic atrophy and changes the morphology of the acinar portion, low levels of marker proteins were nonetheless maintained inside the cells.…”

“…It is an epithelial basal cell protein with a molecular weight of 58 kDa. It is recognized as an exocrine gland stem cell marker in the mammary gland, and it has recently been used as a precursor-cell marker [3, 17]. While we found that c-kit expression was elevated after 2 months of ligation, it decreased significantly 1 week after clip removal before rising significantly over the next 2 months.…”

“…The same approach was used to remove the clip 2 months later. The duration of ligation was informed by a previous study that reported no significant difference in the expression of c-kit, Sca-1, or cytokeratin 5 (CK5), which were judged to participate in salivary gland regeneration, after 2 or 3 months of obstruction [ 17 ]. Submandibular gland tissue samples were collected after 2 months of ligation, and then 1 week, 1 month or 2 months after the release of ligation.…”

“…The structural changes and the functional improvements in atrophied salivary glands after release from long-term obstruction, for example by salivary calculi, is still not known. To date, the longest period of experimental obstruction of the main salivary gland duct has been 3 months, in a study that identified the expression of a stem cell marker in atrophied salivary glands [ 17 ]. The regenerative capacity of salivary glands after the release of long-term obstruction has not yet been examined.…”

Age-Dependent Differential Expression of Apoptotic Markers in Rat Oral Mucosa

“…The cornified layer diminished in thickness and the epithelial ridges became more regular and shorter than in younger rats (Lindhe et al,2008). Epithelial and connective tissue atrophy of the oral mucosa in older rats correlated with a decrease in vascularity and salivary gland secretion (Murayama et al, 2017).…”