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Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ
Published on: June 5, 2018
Quantitative analysis of microglia morphological changes in the hypothalamus of chronically stressed rats
Ke Chen1, Xin Qi1, Lin-Lin Zhu1
1Hebei Key Laboratory of Forensic Medicine, Collaborative Innovation Center of Forensic Medical Molecular Identification, College of Forensic Medicine, Hebei Medical University, No.361 Zhongshan Dong Road, 050017 Shijiazhuang, China.
Abstract:
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.
Insights
Chronic stress reshapes hypothalamic microglia in rats, altering their morphology and increasing phagocytic activity. This provides a pathomorphological basis for understanding stress-induced neuroinflammation.
Area of Science:
- Neuroscience
- Stress Research
- Cell Biology
Background:
- Chronic stress is a significant risk factor for various health issues.
- Hypothalamic dysfunction is implicated in stress-related disorders.
- Microglia, the brain's immune cells, play a crucial role in neuroinflammation and neuronal health.
Purpose of the Study:
- To investigate the morphological changes of hypothalamic microglia in a rat model of chronic restraint stress.
- To establish a pathomorphological basis for studying molecular mechanisms of hypothalamic stress injury, including neuroinflammation.
- To quantify microglial morphological alterations using advanced imaging and analysis algorithms.
Main Methods:
- Establishment of a rat model of chronic restraint stress.
- Quantification of serum glucocorticoid levels to verify the stress model.
- Confocal microscopy and immunofluorescence staining of Iba1-labeled microglia in rat hypothalamus.
- Image processing for contouring and skeletonization.
- Morphological analysis using fractal, skeleton, and Sholl analysis.
- Assessment of CD68 co-expression to evaluate phagocytic activity.
Main Results:
- Microglia in the chronic stress group exhibited reduced fractal dimension and lacunarity.
- Increased density, circularity, and enlarged soma areas were observed in stressed microglia.
- Branches of microglia became shorter and fewer, indicating reduced complexity (confirmed by Sholl analysis).
- Significant increase in CD68 expression indicated heightened phagocytic activity in stressed microglia.
Conclusions:
- Chronic restraint stress induces significant morphological changes in hypothalamic microglia, characterized by reduced complexity and increased phagocytosis.
- These alterations suggest a shift towards an activated, amoeboid-like microglial phenotype.
- The findings provide crucial pathomorphological insights into stress-induced hypothalamic neuroinflammation.
