Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues

Background

Collagen is the principal load-bearing polymer in all connective tissues ranging from skin to bone. Collagen networks strongly stiffen when a mechanical force is applied, thus preventing excessive deformation of the tissue. There are 16 types of collagen, of which the type I, II, and III nearly comprise the 80% of the collagen in the body that are packed together to form long thin fibrils. Collagen type IV forms a two-dimensional reticulum; while several other collagen types are associated with fibril-type collagen, linking them to each other or to other matrix components. These collagens along with the other components of the extracellular matrix (ECM) undergo constant remodeling to provide required biochemical properties like tensile strength and elasticity. This unique attribute of collagen is one of the influencing factors of the stability of reproductive tissues and its dysregulation can lead to adverse events such as abnormal placentation, rupture of membranes (Hampson et al., 1997; Marpaung, 2016) and pathological conditions of the reproductive tract, such as endometriosis (Shimizu and Hokano, 1990) etc.

In tissues, the basement membrane is rich in collagen, in addition it is found in the stroma and lining the connective tissues. Physical, mechanical or chemical damage of a tissue or organ would lead to disruption of collagen deposition, and organization. Hence, assessing the patterns of collagen distribution would provide us an idea about the tensile strength of the tissue/organs. Any alterations from normal patterns of collagen distribution would imply tissue damage. In this study, chorio-decidual tissue has been used as a model basement membrane. Changes in the biochemical properties of this feto-maternal membrane during pregnancy and various pathological conditions lead to its preterm rupture (Sebire, 2001; Fujimoto et al., 2002; Wang et al., 2004; Vega Sánchez et al., 2004; Surve et al., 2016).

Sirius red is a histology stain used to mark total collagen as well as differentiate between varying collagen types for evaluation of collagen distribution in tissues. The sulphonic acid group of Sirius red reacts with basic amino groups of lysine and hydroxylysine and guanidine group of arginine (present in the collagen molecule (Junqueira et al., 1979)). Thereby, being an anionic dye, it attaches to all the varying types of collagen isoforms. In bright field, collagen appears as bundles of pink to red fibers which get disturbed in pathological conditions. The same larger collagen fibers under polarized light appear bright yellow to orange and the thinner ones, including reticular fibers, look green. This birefringence or double refraction, whereby incident light is split by polarization into two different paths, is highly specific for collagen. The amount of polarized light absorbed by the Sirius red dye stringently depends on the orientation of the collagen bundles enabling to differentiate different collagen types (Junqueira et al., 1979; Lattouf et al., 2014). This method is very simple, quick, economic and reliable in comparison with other commonly used staining methods for collagen.