Alteration of mechanical stress in the mouse brain with age and hemorrhagic stroke

Introduction: Residual mechanical stresses, also known as solid stresses, arise during tissue growth or remodeling, primarily observed during organogenesis and tumor growth. Although these stresses typically decrease in healthy adult organs, they persist at elevated levels in mature brains, with their progression and impacts remaining unclear. The brain undergoes significant morphological changes throughout life and in diseases like Alzheimer's and stroke, potentially affecting solid stress. However, mapping these stresses is challenging. Unanswered questions include how residual solid stress changes in these diseases and whether it contributes to disease progression. Our limited understanding of solid stress-associated mechanobiology in the brain is partly due to mapping challenges. Unlike stiffness, measuring solid stress requires either mechanical intervention or embedding microgels. Our previously established method (Nat. Biomedical Eng. 2016) involved slicing brain sections to measure stress-induced deformations, providing insights into stress distribution across ages and conditions. We used this method to map solid stresses in mice brains across three age groups to better understand their spatiotemporal distribution and alterations with aging and neuropathologies like stroke. We observed a rapid increase in solid stress from early to mature stages, persisting in aged brains. Lowering ionic strength increased solid stresses, indicating the role of charged extracellular matrix constituents, while hemorrhagic stroke reduced solid stress levels. These findings shed light on solid stress alterations in healthy and diseased brains, contributing to our understanding of their biological and immunological implications. This study aids in unraveling the role of solid stress as a potential biomarker in brain aging and pathology.

Materials and

Methods: Based on the first principle that tissues containing residual solid stress deform upon release of physical confinement, we harvested mice brains across three age groups: 5–7 days, 8–12 weeks, and 22 months. Briefly, the harvested brain was embedded in agarose and then sliced into 250 µm thick sections that were left in phosphate-buffered saline (PBS) and separated from the surrounding agarose to allow the residual solid stresses to relax, and then fixed in formalin prior to imaging the structural changes using 3-D fluorescent microscopy. The extent of change was assessed by (i) normalized deformation, (ii) changes in surface area, and (iii) local curvature of the tissue slice. Each parameter served as an index of the solid stress in the tissue providing a 3-D map for spatial heterogeneity of these stresses in the brain. We validated our methodology to rule out effects of fixation, buoyancy, and have both positive- and negative-controls for the heart and kidney that have large and no known solid stresses.

Results, Conclusions, and Discussions: Utilizing our previously developed slicing method (Fig. 1a) we successfully mapped the spatial heterogeneity of solid stress in mouse brains from very young to very old mice. We validated our measurements were not affected due to fixation or buoyancy, and that our method provided expected results in kidney (little to no solid stress) and heart (high degree of solid stress) tissue slices taken from mice from each age group (Fig. 1b). We found a substantial increase in residual solid stress within the murine brain from infancy to adulthood, persisting at heightened levels throughout aging (Fig. 1c). This trend may be associated with alterations in brain components, particularly the heterogeneities of gray and white matter. Additionally, our investigation revealed age-dependent variations in the viscoelastic properties of the murine brain, likely stemming from the distinct viscoelasticity of brain constituents and their age-related changes. We produced the initial map of solid stress through serial coronal sections up to 5 mm into the brain, noting higher solid stresses in posterior sections of mature mouse brains compared to anterior sections (Fig. 1d), possibly due to heterogeneous ECM distribution and region-specific mechanical properties. Furthermore, we found that solutions with lower ionic strength amplify solid stress levels in the mouse brain, although further investigation is needed to understand the effects of charged ECM components and changes in cell volume due to osmosis. Lastly, our study demonstrated a reduction in solid stress in the case of hemorrhagic stroke in mature murine brains (Fig. 1e), potentially linked to alterations in ECM composition and morphology. These insights enhance our understanding of solid stress as a potential biomarker for aging and various brain pathologies.

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