Quantitative Histomorphometric Analysis of Collagen Bundles in Masson's Trichrome Stained Rat ( Rattus norvegicus ) Skin: A Methodological Study

ABSTRACT Background and Aims While Masson's Trichrome is widely used for collagen visualization, its qualitative nature limits reproducibility and statistical comparison. A methodological gap persists in the quantitative assessment of collagen in Rattus norvegicus skin, an important model for dermatological research. This study's objective was to develop a novel, quantitative protocol to precisely measure collagen features, enhancing the study of tissue remodeling and fibrosis. Methods Formalin‐fixed, paraffin‐embedded skin tissue from Wistar rats was stained with Masson's Trichrome. Digital images were analyzed using ImageJ. The protocol involved creating custom stain vectors for precise color deconvolution, separating the image into blue–green (collagen) and red–pink (non‐collagenous) channels. Quantitative features, including area, intensity, skewness, and kurtosis, were extracted from the thresholded regions of each stain. Results Analysis of 168 images showed that the blue–green (collagen) stained area was significantly larger than the red–pink area ( p < 0.001). The median collagen area was 1.273 mm² compared to 0.313 mm² for the red–pink area. Collagen stain intensity was also significantly higher ( p < 0.001). Distribution shape analysis confirmed distinct signatures: the blue–green stain was highly symmetrical (skewness about 0.07), while the red–pink stain was platykurtic and positively skewed, reflecting its binding to more heterogeneous components. Conclusion This work provides a validated framework for the objective and reproducible quantification of collagen in rat skin. By bridging traditional histology with computational analysis, this protocol offers a robust tool for future studies on collagen dynamics in skin pathologies and wound healing, with potential applications in evaluating therapeutic strategies.