Exploring the mechanical microenvironment of the brain by dynamic indentation

The physiological processes of cells and tissues are regulated not only by biochemical and electrical signaling but also by mechanics. The mechanical microenvironment is involved in the progression of diseases and is one of the key elements in fabricating physiologically relevant 3D tissues. The complexity of biological tissue structure leads to complex mechanical behavior. However, our ability to characterize the mechanical properties of biomaterials is limited due to technical challenges. Mechanosensation of brain cells and the mechanical microenvironment of brain tissue are relevant for healthy functioning, neurodevelopment, neurodegenerative diseases, and regeneration. There are many challenges associated with the mechanical testing of brain tissue such as restricted access to the brain, lack of available material, difficult sample preparation procedures, and preservation of its viability. Furthermore, mechanical testing can be performed at various scales, from single cells to the whole organ and with various mechanical testing modalities. As seen from previous studies, variation of the experimental parameters contribute to the observed variability in mechanical data. Finally, understanding which structural components of tissues and cells give rise to certain mechanical behavior remains an unmet objective. To address above mentioned issues, three objectives were set for this thesis: Objective 1: Develop indentation setup and protocols to measure mechanical properties of the brain slices in a reproducible manner. Novel indentation protocols using ferrule-top indentation device are established in this thesis. Chapter 2 introduces contact mechanics theory. As there are many different models available, general guidelines are given for selecting indentation profiles for soft tissue measurements. Chapter 3 shows experimental observations when indenting on the brain slices such as the influence of the indentation-depth, indentation-speed, oscillatory-ramp, dynamic mechanical analysis, tissue mounting, degradation, swelling, and conditioning. Objective 2: Understand the relationship between the structure of the brain and its mechanical properties. With the established novel measurement protocols, viscoelastic maps of the hippocampus of the mouse brain are reported in Chapter 4. For the first time in the literature, clear differences between subregions are observed, which agrees with anatomical region boundaries. Surprisingly, high cell-density regions are softer than low-cell density regions. Chapter 5 shows viscoelastic maps of the hippocampus and cerebellum of the juvenile mouse, where mechanical contrast overlaps with anatomical regions. Comparison between juvenile and adult shows that adult hippocampus is stiffer than juvenile. Correlations are found between the amount of different brain components such as nuclei, myelin, astrocytes, and viscoelastic parameters, and a linear regression model is suggested. Finally, Chapter 6 shows that the hippocampus of the Alzheimer's disease mouse model is stiffer than healthy controls. In summary, progress has been made in understanding the mechanical brain microenvironment in terms of viscoelasticity and structural composition by introducing novel dynamic indentation protocols. Objective 3: Adapt indentation setup and methodology from tissue characterization to single cells with the aim to study astrocytes and microglia in an inflammatory environment. Indentation protocols are adapted to single cells. Chapter 7 compares astrocytes derived from gray matter (GM) and white matter (WM) regions where the latter are found to be softer. As a response to treatment with pro-inflammatory lipopolysaccharide (LPS), GM astrocytes become softer, where the F-actin network appears rearranged, whereas WM astrocytes preserve their initial features. Chapter 8 compares microglia derived from GM and WM regions where the latter are more viscoelastic. When treated with LPS, the increase in viscoelasticity in GM microglia is accompanied by an increase in Tnf-alpha mRNA and reorganization of F-actin which is absent in WM microglia which decreases viscoelasticity. Together, these both studies show that glial cells have region-dependent phenotypes which can be observed not only in their biochemical responses but also in biomechanical.