Quantitative analysis of microglia morphological changes in the hypothalamus of chronically stressed rats

Based on the successful establishment of a rat model of chronic restraint stress, we used multiple algorithms to quantify the morphological changes of rat hypothalamic microglia from various perspectives, providing a pathomorphological basis for the subsequent study of molecular mechanisms of hypothalamic stress injury, such as neuroinflammation. To verify the successful establishment of the chronic stress model, an enzyme-linked immunosorbent assay was performed to detect serum glucocorticoid levels. Microglia labeled with Iba1 in frozen sections of rat hypothalamus were scanned and photographed at multiple levels using confocal microscopy. Subsequently, images were processed for external contouring and skeletonization, and morphological indices of microglia were calculated and analyzed using fractal, skeleton, and Sholl analysis. In addition, the co-expression of CD68 (a marker that can reflect phagocytic activity) and Iba1 was observed by immunofluorescence technique. Compared with the control group, microglia in the chronic stress group displayed reduced fractal dimension and lacunarity, increased density and circularity, enlarged soma areas, and shortened and reduced branches. Sholl analysis confirmed the reduced complexity of microglia following chronic stress. Meanwhile, microglia CD68 increased significantly, indicating that the microglia in the chronic stress group have greater phagocytosis activity. In summary, chronic restraint stress promoted the conversion of microglia in the rat hypothalamus to a less complex form, manifested as larger soma, shorter and fewer branches, more uniform and dense texture, and increased circularity; indeed, the shape of these microglia resembled that of amoeba and they displayed strong phagocytosis activity.