PYTHON-BASED SOFTWARE DEVELOPMENT FOR AUTOMATED HISTOMORPHOMETRY OF LIVER, KIDNEY, AND GASTROCNEMIUS MUSCLE IN PREGNANT AND LACTATING WISTAR RATS TREATED WITH BOTULINUM TOXIN TYPE A

Botulinum toxin type A (BT-A) is widely used due to its neuromuscular effects; however, its indirect impacts on visceral tissues and the influence of maternal physiological status still require further investigation. As a methodological innovation, this study describes the development and application of an automated software/pipeline in Python 3.11 for histomorphometric slide analysis, enabling standardization, traceability, and reproducible large-scale extraction of quantitative metrics. Digital microphotographs (2.5× and 10×) of liver, kidney, and gastrocnemius muscle from pregnant and lactating Wistar rats were evaluated and distributed into eight subgroups: controls (pregnant and lactating) and BT-A-treated groups at doses of 4U, 8U, and 16U. The computational system performed automatic file indexing, image preprocessing (resizing and normalization), segmentation using Otsu thresholding, identification of the largest connected component, and calculation of intensity parameters (mean and pixel standard deviation) and morphometric features (area, eccentricity, and circularity). Statistical analysis was conducted according to data normality (Shapiro–Wilk test), applying either Student’s t-test or Mann–Whitney U test (α=0.05). The results revealed an organ-specific pattern, with more pronounced alterations in the gastrocnemius muscle, the injection site, characterized by increased heterogeneity and reduced circularity, consistent with local tissue remodeling. In the kidney, greater morphological susceptibility was observed in lactating animals at higher doses, whereas the liver showed only subtle changes. In conclusion, the developed software increases the robustness and scalability of digital histomorphometry, strengthening comparative analyses and contributing to more accurate tissue safety assessments in experimental models.