Myelination dictates axonal viscoelasticity.

During development, dramatic changes in myelination, growth of neural networks and changes in grey-to-white matter ratio build up the astonishingly plastic brain of a child. The progressive increase in myelination insulates the nervous system, which, in turn, modifies the mechanical microenvironment of the brain spatiotemporally. A growing body of evidence demonstrates the role of mechanical forces in growth, differentiation, maturation and electrical properties of neurons. However, due to limitations in imaging resolution, the exact relationship between myelination, axonal organization and the mechanical properties of nerves at the cellular level is still unknown. Here, we propose a novel approach to study the direct relationship between axonal viscoelasticity with changing fibre anisotropy and myelination during development. With the use of atomic force microscopy (AFM) with in situ fluorescent imaging of the primary neuron-oligodendrocyte co-cultures, we found that as axons are progressively myelinated in vitro, their stiffness increases. Direct quantification of myelin along axons using immunofluorescence also demonstrated a positive correlation between increased myelination over time and increased axonal stiffness (p = .001). Notably, AFM measurements along a single axon showed that the Young's modulus measured across myelinated regions were significantly higher than those of adjacent unmyelinated segments at all time points (p < .0001). Force-relaxation analysis also demonstrated that myelin sheath dominates the regulation of viscoelasticity of axons temporally. Collectively, our findings indicate a direct link between myelination, axonal orientation and viscoelasticity, providing important insights about the mechanical environment in the paediatric brain, with direct implications for our understanding of developmental brain disorders and paediatric brain injury.

Viscoelasticity of children and adolescent brains through MR elastography.

Magnetic Resonance Elastography (MRE) is an elasticity imaging technique that allows a safe, fast, and non-invasive evaluation of the mechanical properties of biological tissues in vivo. Since mechanical properties reflect a tissue's composition and arrangement, MRE is a powerful tool for the investigation of the microstructural changes that take place in the brain during childhood and adolescence. The goal of this study was to evaluate the viscoelastic properties of the brain in a population of healthy children and adolescents in order to identify potential age and sex dependencies. We hypothesize that because of myelination, age dependent changes in the mechanical properties of the brain will occur during childhood and adolescence. Our sample consisted of 26 healthy individuals (13 M, 13 F) with age that ranged from 7-17 years (mean: 11.9 years). We performed multifrequency MRE at 40, 60, and 80 Hz actuation frequencies to acquire the complex-valued shear modulus G = G' + iG″ with the fundamental MRE parameters being the storage modulus (G'), the loss modulus (G″), and the magnitude of complex-valued shear modulus (|G|). We fitted a springpot model to these frequency-dependent MRE parameters in order to obtain the parameter α, which is related to tissue's microstructure, and the elasticity parameter k, which was converted to a shear modulus parameter (µ) through viscosity (η). We observed no statistically significant variation in the parameter µ, but a significant increase of the microstructural parameter α of the white matter with increasing age (p < 0.05). Therefore, our MRE results suggest that subtle microstructural changes such as neural tissue's enhanced alignment and geometrical reorganization during childhood and adolescence could result in significant biomechanical changes. In line with previously reported MRE data for adults, we also report significantly higher shear modulus (µ) for female brains when compared to males (p < 0.05). The data presented here can serve as a clinical baseline in the analysis of the pediatric and adolescent brain's viscoelasticity over this age span, as well as extending our understanding of the biomechanics of brain development.

Amplified Flow Imaging (aFlow): A Novel MRI-Based Tool to Unravel the Coupled Dynamics Between the Human Brain and Cerebrovasculature.

With each heartbeat, periodic variations in arterial blood pressure are transmitted along the vasculature, resulting in localized deformations of the arterial wall and its surrounding tissue. Quantification of such motions may help understand various cerebrovascular conditions, yet it has proven technically challenging thus far. We introduce a new image processing algorithm called amplified Flow (aFlow) which allows to study the coupled brain-blood flow motion by combining the amplification of cine and 4D flow MRI. By incorporating a modal analysis technique known as dynamic mode decomposition into the algorithm, aFlow is able to capture the characteristics of transient events present in the brain and arterial wall deformation. Validating aFlow, we tested it on phantom simulations mimicking arterial walls motion and observed that aFlow displays almost twice higher SNR than its predecessor amplified MRI (aMRI). We then applied aFlow to 4D flow and cine MRI datasets of 5 healthy subjects, finding high correlations between blood flow velocity and tissue deformation in selected brain regions, with correlation values r = 0.61 , 0.59, 0.52 for the pons, frontal and occipital lobe ( ). Finally, we explored the potential diagnostic applicability of aFlow by studying intracranial aneurysm dynamics, which seems to be indicative of rupture risk. In two patients, aFlow successfully visualized the imperceptible aneurysm wall motion, additionally quantifying the increase in the high frequency wall displacement after a one-year follow-up period (20%, 76%). These preliminary data suggest that aFlow may provide a novel imaging biomarker for the assessment of aneurysms evolution, with important potential diagnostic implications.

Micromechanical heterogeneity of the rat pia-arachnoid complex.

To better understand the onset of damage occurring in the brain upon traumatic events, it is essential to analyze how external mechanical loads propagate through the skull and meninges and down to the brain cortex. However, despite their crucial role as structural dampers protecting the brain, the mechanical properties and dynamic behavior of the meningeal layers are still poorly understood. Here, we characterized the local mechanical heterogeneity of rat pia-arachnoid complex (PAC) at the microscale via atomic force microscopy (AFM) indentation experiments to understand how microstructural variations at the tissue level can differentially affect load propagation. By coupling AFM mechanical testing with fresh tissue immunofluorescent staining, we could directly observe the effect of specific anatomical features on the local mechanical properties of tissue. We observed a two-fold stiffening of vascularized tissue when compared to non-vascularized PAC (with instantaneous Young's modulus distribution means of 1.32  ±â€¯ 0.03 kPa and 2.79  ±â€¯ 0.08 kPa, respectively), and statistically significant differences between regions of low- and high-vimentin density, reflecting trabecular density (with means of 0.67  ±â€¯ 0.05 kPa and 1.29  ±â€¯ 0.06 kPa, respectively). No significant differences were observed between cortical and cerebellar PAC. Additionally, by performing force relaxation experiments at the AFM, we identified the characteristic time constant τ1 of PAC tissue to be in the range of 2.7  ±â€¯ 1.2 s to 3.1  ±â€¯ 0.9 s for the different PAC regions analyzed. Taken together, the results presented point at the complex biomechanical nature of the meningeal tissue, and underscore the need to account for its heterogeneity when modeling its behavior into finite element simulations or other computational methods enabling the prediction of load propagation during injury events. STATEMENT OF SIGNIFICANCE: The meningeal layers are pivotal in shielding the brain during injury events, yet the mechanical properties of this complex biological interface are still under investigation. Here, we present the first anatomically-informed micromechanical characterization of mammalian pia-arachnoid complex (PAC). We developed a protocol for the isolation and fresh immunostaining of rat PAC and subjected the tissue to AFM indentation and relaxation experiments, while visualizing the local anatomy via fluorescence microscopy. We found statistically significant variations in regional PAC stiffness according to the degree of vascularization and trabecular cell density, besides identifying the tissue's characteristic relaxation constant. In essence, this study captures the relationship between anatomy and mechanical heterogeneity in a key component of the brain-skull interface for the first time.

Isolation and Immunofluorescent Staining of Fresh Rat Pia-Arachnoid Complex Tissue for Micromechanical Characterization.

In this article, we describe a protocol for the isolation and staining of fresh tissue of the inner rat meningeal layers, or pia-arachnoid complex (PAC). The PAC is believed to act as a mechanical damper offering a fundamental layer of protection against brain injury; however, its overall mechanical properties are still rather unexplored. In order to perform micromechanical measurements on the PAC, the tissue must be extracted and characterized while maintaining its native mechanical properties (i.e., avoiding any chemical or physical modification that could alter it). In light of this need, we developed a protocol for the immunofluorescent staining of fresh PAC tissue that does not require any fixation or permeabilization step. This approach will allow researchers to investigate important properties of the anatomy of ex vivo PAC tissue while at the same time offering a platform for the mechanical analysis of this complex material. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of fresh rat pia-arachnoid complex tissue Basic Protocol 2: Fresh immunofluorescent staining of rat pia-arachnoid complex tissue Alternate Protocol: Adhesion of pia-arachnoid complex tissue to glass slides for micromechanical characterization.

Nanoscale Topography and Poroelastic Properties of Model Tissue Breast Gland Basement Membranes.

Basement membranes (BMs) are thin layers of condensed extracellular matrix proteins serving as permeability filters, cellular anchoring sites, and barriers against cancer cell invasion. It is believed that their biomechanical properties play a crucial role in determining cellular behavior and response, especially in mechanically active tissues like breast glands. Despite this, so far, relatively little attention has been dedicated to their analysis because of the difficulty of isolating and handling such thin layers of material. Here, we isolated BMs derived from MCF10A spheroids-three-dimensional breast gland model systems mimicking in vitro the most relevant phenotypic characteristics of human breast lobules-and characterized them by atomic force microscopy, enhanced resolution confocal microscopy, and scanning electron microscopy. By performing atomic force microscopy height-clamp experiments, we obtained force-relaxation curves that offered the first biomechanical data on isolated breast gland BMs to our knowledge. Based on enhanced resolution confocal microscopy and scanning electron microscopy imaging data, we modeled the system as a polymer network immersed in liquid and described it as a poroelastic material. Finite-element simulations matching the experimental force-relaxation curves allowed for the first quantification, to our knowledge, of the bulk and shear moduli of the membrane as well as its water permeability. These results represent a first step toward a deeper understanding of the mechanism of tensional homeostasis regulating mammary gland activity as well as its disruption during processes of membrane breaching and metastatic invasion.

Effects of Plectin Depletion on Keratin Network Dynamics and Organization.

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin ß4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and ß1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

Keratins as the main component for the mechanical integrity of keratinocytes.

Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.