Differential immunohistochemical expression of type I collagen and matrix metalloproteinase 2 among major salivary glands of young and geriatric mice

Abstract Objective This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice. Material and Methods Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson’s Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2. Results We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001). Conclusions This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.


Introduction
Three pairs of major salivary glands produce saliva (the parotid, submandibular, and sublingual glands) as well as minor salivary glands located throughout the oral cavity 1  Although the sublingual gland is a mixed gland, the mucous cells are predominant, and therefore it is rich in mucin secretion 7 .
Matrix metalloproteinases (MMPs) play an important role in physiological conditions such as tissue remodeling, morphogenesis, wound healing and normal developmental processes, as well as pathological processes such as tumor cell invasion, angiogenesis and metastasis 8 . Some publications have shown that MMP-2 (Gelatinase A), which is found at a molecular weight of 72 kDa in its pro-form and 66 kDa in the active form, degrades gelatin and type IV collagen as well as type I, II and III collagen 9 .
Collagens are the principal proteins of the extracellular matrix, which constitute 30% of the dry body weight. They provide a great balance of flexibility and strength to the tissue in which they are found.
Type I collagen is the member of the collagen family, which is the most abundant and widely distributed in fibril form 10 .
There are many publications examining the changes in the structure and function of organs during aging. In publications about human and animal salivary glands, only a single gland is usually emphasized when mentioning the changes of salivary glands with aging.
However, specifying differences between the major and minor glands as well as evaluating the major salivary glands individually among themselves increases the accuracy of structural and functional studies to be performed on salivary glands. Since these glands, the functions of their secretions and the quantities are different from each other, rates and contents of their secretions also vary according to the gland. This indicates that they must be assessed separately rather than together as in previous studies. This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 nd type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) with aging in mice.

Immunohistochemical examination
The 4-5-mm-thick sections taken from paraffin blocks were transferred onto poly-lysinized lamellae. We performed imunohistochemical assay using the H-score analysis 11 . The intensity of MMP-2 and type 1 collagen immunoreactivity was evaluated semi-quantitatively by using the categories of stain intensity during follow-up: 0 (No staining), 1+ (Weak but detectable staining), 2+ (Moderate staining), and 3+ (Intensive staining). Firstly, an H-score value was obtained for each tissue by calculating the sum of the percentages of cells according to the categories of stain intensity. Then, this value was multiplied by the weighted intensity of staining using the H-score formula =ΣPi (i 1+) ("i" represents the intensity scores and "Pi" represents the relative percentage of cells).
Each slide was evaluated in 5 randomly selected areas in light microscope (40X magnification). The percentage of cells in each intensity in these areas was identified at different times by two researchers who were unaware of the species and source of the tissues.
The average scores of both researchers were used.

Statistical analysis
The Shapiro-Wilk test was used for testing normality. The data were normally distributed. P values <0.05 were considered statistically significant. The independent two-sample t-test was used to compare groups.

Histological findings
Hematoxylin-eosin staining Moreover, blood vessels decreased and shrank due to aging. Increased fat tissue in the parotid glands of the old mice was observed. In the parotid gland, we also observed that the parenchymal tissue was divided into many lobules by the stromal connective tissue. Blood vessels and large interlobular ducts were seen in the stromal connective tissue. Interlobular channels were lined with pseudostratified epithelium.

Masson's trichrome staining
To assess type I collagen, Masson's trichrome staining was used 12 . Fibrotic areas are rich in collagens and, therefore, they appear in blue upon Masson trichrome staining (asterisks). We observed that the total collagen density was lower in young mice than in older mice. The collagen fibers were looser and disorganized in older mice than in young mice.
Moreover, acinar atrophy and dilated interlobular ducts were seen in old mice (Figure 2).

Immunohistochemical findings
Immunohistochemical staining for type I collagen  Aging is one of the main physiological factors for saliva reduction. Among its many function, salivary secretion can protect the integrity of oral tissues 13 . It is very effective in protecting teeth against decay, tasting, swallowing, talking, and adapting to prosthetics 14 . We observed in this study that MMP2 and type 1 collagen expression in the major salivary glands significantly increased in older mice (eighteen-month-old) than in young mice (two-month-old). According to this information, in many studies examining salivary glands       in dysfunctions in oral tissue integrity, dental decay, swallowing, talking, and adapting to prosthetics. In this context, serious pain complications and functional disturbances may occur with losses of physiological activity due to age-related degeneration in the major salivary glands.

Funding
No funding was received