Structure-stiffness relation of live mouse brain tissue determined by depth-controlled indentation mapping

The mechanical properties of brain tissue play a pivotal role in neurodevelopment and neurological disorders. Yet, at present, there is no consensus on how the different structural parts of the tissue contribute to its stiffness variations. Here, we have gathered depth-controlled indentation viscoelasticity maps of the hippocampus of isolated horizontal live mouse brain sections. Our results confirm the highly viscoelestic nature of the material and clearly show that the mechanical properties correlate with the different morphological layers of the samples investigated. Interestingly, the relative cell nuclei area seems to negatively correlate with the stiffness observed

Experimental and data analysis workflow for soft matter nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (μm). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young's Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with µm spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells. In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer's guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data

Biomechanics of cells and tissues: What can we learn when we combine mechanical stimuli with microscopy?

Understanding the mechanical properties of biological tissues can shed light on how those tissues work and why, at times, they lose their functionality. Furthermore, a full characterization of a tissue’s viscoelastic behavior may provide relevant hints for tissue reparation and tissue engineering. To measure these properties in in-vitro or ex-vivo experiments, researchers often make use of indentation instruments, which looks at how a material deforms under the effect of a calibrated mechanical load. In the first part of my talk, I will show how this technique can be used to determine the mechanical properties of brain slices, and I will comment on which kind of information those measurements can provide. I will show, for instance, that different regions of the brain have remarkably different viscoelastic properties, which seem to be correlated with the cell density measured, in a parallel experiment, via fluorescent microscopy. As this example highlights, indentation measurements alone are often not sufficient to understand why certain tissues have certain mechanical properties. Under a (not transparent) surface, biological materials are often inhomogeneous and anisotropic. Because the indentation stress propagates several microns deep into the sample, without a proper imaging tool coupled to the indentation instrument, it is impossible to extract useful information on the mechanics of the material the sample is made of. As a point in case, I will show our latest measurements of the mechanical properties of chick embryos, where, combining indentation with optical coherence tomography (OCT), we could precisely map the stiffness of the spine from head to tail – a measurement that may provide interesting cues in the analysis of somites formation and growth. I will also show how the combination of indentation and OCT might find its way in scar and burn classification, introducing a new instrument for skin characterization that our group has just recently completed. Finally, I will show some preliminary results on the use of multiphoton imaging for tissue mechanics characterization. In this last part of the talk, I will show that it is indeed possible to look at the displacement and deformation of cells in a thin slice of tissue while the tissue is compressed by a calibrated mechanical stroke. This approach may pave the way for a much more thorough analysis of the origin of certain mechanical properties of tissues, where the contribution of the individual cells to the viscoelastic features of the materials can be finally disentangle from that of the extracellular matrix. This project was supported by LASERLABEUROPE under the EC’s Seventh Framework Program (Grant agreement No. 284464), by the European Union’s Seventh Framework Programme (FP/20072013)/ERC grant agreement no. 615170, by the Dutch Technology Foundation (STW) under the OMNE program (13183 and under the iMIT program (P11–13). Declaration of interest: Davide Iannuzzi is founder, shareholder, and advisor of Optics11

Micro-indentation and optical coherence tomography for the mechanical characterization of embryos: Experimental setup and measurements on chicken embryos

The investigation of the mechanical properties of embryos is expected to provide valuable information on the phenomenology of morphogenesis. It is thus believed that, by mapping the viscoelastic features of an embryo at different stages of growth, it may be possible to shed light on the role of mechanics in embryonic development. To contribute to this field, we present a new instrument that can determine spatiotemporal distributions of mechanical properties of embryos over a wide area and with unprecedented accuracy. The method relies on combining ferrule-top micro-indentation, which provides local measurements of viscoelasticity, with Optical Coherence Tomography, which can reveal changes in tissue morphology and help the user identify the indentation point. To prove the working principle, we have collected viscoelasticity maps of fixed and live HH11-HH12 chicken embryos. Our study shows that the instrument can reveal correlations between tissue morphology and mechanical behavior. Statement of Significance: Local mechanical properties of soft biological tissue play a crucial role in several biological processes, including cell differentiation, cell migration, and body formation; therefore, measuring tissue properties at high resolution is of great interest in biology and tissue engineering. To provide an efficient method for the biomechanical characterization of soft biological tissues, we introduce a new tool in which the combination of non-invasive Optical Coherence Tomography imaging and depth-controlled indentation measurements allows one to map the viscoelastic properties of biological tissue and investigate correlations between local mechanical features and tissue morphology with unprecedented resolution

Viscoelastic mapping of mouse brain tissue:Relation to structure and age

There is growing evidence that mechanical factors affect brain functioning. However, brain components responsible for regulating the physiological mechanical environment are not completely understood. To determine the relationship between structure and stiffness of brain tissue, we performed high-resolution viscoelastic mapping by dynamic indentation of the hippocampus and the cerebellum of juvenile mice brains, and quantified relative area covered by neurons (NeuN-staining), axons (neurofilament NN18-staining), astrocytes (GFAP-staining), myelin (MBP-staining) and nuclei (Hoechst-staining) of juvenile and adult mouse brain slices. Results show that brain subregions have distinct viscoelastic parameters. In gray matter (GM) regions, the storage modulus correlates negatively with the relative area of nuclei and neurons, and positively with astrocytes. The storage modulus also correlates negatively with the relative area of myelin and axons (high cell density regions are excluded). Furthermore, adult brain regions are ∼ 20%-150% stiffer than the comparable juvenile regions which coincide with increase in astrocyte GFAP-staining. Several linear regression models are examined to predict the mechanical properties of the brain tissue based on (immuno)histochemical stainings

Regional variations in stiffness in live mouse brain tissue determined by depth-controlled indentation mapping

The mechanical properties of brain tissue play a pivotal role in neurodevelopment and neurological disorders. Yet, at present, there is no consensus on how the different structural parts of the tissue contribute to its stiffness variations. Here, we have gathered depth-controlled indentation viscoelasticity maps of the hippocampus of acute horizontal live mouse brain slices. Our results confirm the highly viscoelestic nature of brain tissue. We further show that the mechanical properties are non-uniform and at least related to differences in morphological composition. Interestingly, areas with higher nuclear density appear to be softer than areas with lower nuclear density

Regional variations in stiffness in live mouse brain tissue determined by depth-controlled indentation mapping

The mechanical properties of brain tissue play a pivotal role in neurodevelopment and neurological disorders. Yet, at present, there is no consensus on how the different structural parts of the tissue contribute to its stiffness variations. Here, we have gathered depth-controlled indentation viscoelasticity maps of the hippocampus of acute horizontal live mouse brain slices. Our results confirm the highly viscoelestic nature of brain tissue. We further show that the mechanical properties are non-uniform and at least related to differences in morphological composition. Interestingly, areas with higher nuclear density appear to be softer than areas with lower nuclear density

Dynamic indentation reveals differential viscoelastic properties of white matter versus gray matter-derived astrocytes upon treatment with lipopolysaccharide

Astrocytes in white matter (WM) and gray matter (GM) brain regions have been reported to have different morphology and function. Previous single cell biomechanical studies have not differentiated between WM- and GM-derived samples. In this study, we explored the local viscoelastic properties of isolated astrocytes and show that astrocytes from rat brain WM-enriched areas are ~1.8 times softer than astrocytes from GM-enriched areas. Upon treatment with pro-inflammatory lipopolysaccharide, GM-derived astrocytes become significantly softer in the nuclear and the cytoplasmic regions, where the F-actin network appears rearranged, whereas WM-derived astrocytes preserve their initial mechanical features and show no alteration in the F-actin cytoskeletal network. We hypothesize that the flexibility in biomechanical properties of GM-derived astrocytes may contribute to promote regeneration of the brain under neuroinflammatory conditions