Viscoelastic properties of white and gray matter-derived microglia differentiate upon treatment with lipopolysaccharide but not upon treatment with myelin

Background The biomechanical properties of the brain have increasingly been shown to relate to brain pathology in neurological diseases, including multiple sclerosis (MS). Inflammation and demyelination in MS induce significant changes in brain stiffness which can be linked to the relative abundance of glial cells in lesions. We hypothesize that the biomechanical, in addition to biochemical, properties of white (WM) and gray matter (GM)-derived microglia may contribute to the differential microglial phenotypes as seen in MS WM and GM lesions. Methods Primary glial cultures from WM or GM of rat adult brains were treated with either lipopolysaccharide (LPS), myelin, or myelin+LPS for 24 h or left untreated as a control. After treatment, microglial cells were indented using dynamic indentation to determine the storage and loss moduli reflecting cell elasticity and cell viscosity, respectively, and subsequently fixed for immunocytochemical analysis. In parallel, gene expression of inflammatory-related genes were measured using semi-quantitative RT-PCR. Finally, phagocytosis of myelin was determined as well as F-actin visualized to study the cytoskeletal changes. Results WM-derived microglia were significantly more elastic and more viscous than microglia derived from GM. This heterogeneity in microglia biomechanical properties was also apparent when treated with LPS when WM-derived microglia decreased cell elasticity and viscosity, and GM-derived microglia increased elasticity and viscosity. The increase in elasticity and viscosity observed in GM-derived microglia was accompanied by an increase in Tnfα mRNA and reorganization of F-actin which was absent in WM-derived microglia. In contrast, when treated with myelin, both WM- and GM-derived microglia phagocytose myelin decrease their elasticity and viscosity. Conclusions In demyelinating conditions, when myelin debris is phagocytized, as in MS lesions, it is likely that the observed differences in WM- versus GM-derived microglia biomechanics are mainly due to a difference in response to inflammation, rather than to the event of demyelination itself. Thus, the differential biomechanical properties of WM and GM microglia may add to their differential biochemical properties which depend on inflammation present in WM and GM lesions of MS patients.

Modeling of Deformable Cell Separation in a Microchannel with Sequenced Pillars

Embedded pillar microstructures are an efficient approach for controlling and sculpting shear flow in a microchannel but have not yet demonstrated to be effective for deformability-based cell separation and sorting. Although simple pillar configurations (lattice, line sequence) worked well for size-based separation of rigid particles, they had a low separation efficiency for circulating cells. The objective of this study was to optimize sequenced microstructures for separation of deformable cells. This was achieved by numerical analysis of pairwise cell migration in a microchannel with multiple pillars, which size, longitudinal spacing, and lateral location as well as the cell elasticity and size varied. This study revealed two basic pillar configurations optimized for deformability-based separation: 1) “duplet” that consists of two closely spaced pillars positioned far below the centerline and above the centerline halfway to the wall; and 2) “triplet” composed of three widely-spaced pillars located below, above and at the centerline, respectively. The duplet configuration is well suited for deformable cell separation in short channels, while the triplet or a combination of duplets and triplets provides even better separation in long channels. These optimized pillar microstructures can dramatically improve microfluidic methods for sorting and isolation of blood and rare circulating tumor cells.

Single-Cell Elasticity Measurement with an Optically Actuated Microrobot

A cell elasticity measurement method is introduced that uses polymer microtools actuated by holographic optical tweezers. The microtools were prepared with two-photon polymerization. Their shape enables the approach of the cells in any lateral direction. In the presented case, endothelial cells grown on vertical polymer walls were probed by the tools in a lateral direction. The use of specially shaped microtools prevents the target cells from photodamage that may arise during optical trapping. The position of the tools was recorded simply with video microscopy and analyzed with image processing methods. We critically compare the resulting Young’s modulus values to those in the literature obtained by other methods. The application of optical tweezers extends the force range available for cell indentations measurements down to the fN regime. Our approach demonstrates a feasible alternative to the usual vertical indentation experiments.

Rac1 Promotes Cell Motility by Controlling Cell Mechanics in Human Glioblastoma

The failure of existing therapies in treating human glioblastoma (GBM) mostly is due to the ability of GBM to infiltrate into healthy regions of the brain; however, the relationship between cell motility and cell mechanics is not well understood. Here, we used atomic force microscopy (AFM), live-cell imaging, and biochemical tools to study the connection between motility and mechanics in human GBM cells. It was found thatRac1 inactivation by genomic silencing and inhibition with EHT 1864 reduced cell motility, inhibited cell ruffles, and disrupted the dynamics of cytoskeleton organization and cell adhesion. These changes were correlated with abnormal localization of myosin IIa and a rapid suppression of the phosphorylation of Erk1/2. At the same time, AFM measurements of the GBM cells revealed a significant increase in cell elasticity and viscosity following Rac1 inhibition. These results indicate that mechanical properties profoundly affect cell motility and may play an important role in the infiltration of GBM. It is conceivable that the mechanical characters might be used as markers for further surgical and therapeutical interventions.

Epithelial Cell-like Elasticity Modulates E-cadherin Adhesion Organization

E-cadherin adhesions are essential for cell-to-cell cohesion and mechanical coupling between epithelial cells and reside in a micro-environment that comprises the adjoining epithelial cells. While E-cadherin has been shown to be a mechanosensor, it is unknown if E-cadherin adhesions can differentially sense stiffness within the range of that of epithelial cells. A survey of literature shows that epithelial cells’ Young’s moduli of elasticity lie predominantly in the sub kPa to few kPa range, with cancer cells often being softer than non-cancerous ones. Here, we devised oriented E-cadherin-coated soft silicone substrates with sub kPa or few kPa elasticity, but with similar viscous moduli, and found that E-cadherin adhesions differentially organize depending on the magnitude of epithelial cell-like elasticity. Linearly shaped E-cadherin adhesions associated with radially oriented actin, but not irregularly shaped E-cadherin adhesions associated with circumferential actin foci, were much more numerous on 2.4 kPa E-cadherin substrates compared to 0.3 kPa E-cadherin substrates. However, the total amount of E-cadherin in both types of adhesions taken together was similar on the 0.3 kPa and 2.4 kPa E-cadherin substrates, across many cells. Furthermore, while the average extent of nuclear recruitment of the mechanoresponsive transcription factor YAP on the 0.3 kPa E-cadherin substrate was undiminished relative to that on the 2.4 kPa substrate, a fraction of cells on the softer substrate displayed relatively high levels of YAP nuclear recruitment. Our results show that E-cadherin adhesions can be regulated by epithelial cell-like elasticity and have significant implications for disease states like carcinomas characterized by altered epithelial cell elasticity.

Pen-strep influence macrophage mechanical property and mechano-response to microenvironment

Penicillin-streptomycin (Pen-strep) is a common antibiotic used to prevent bacterial infection in cell culture and clinical treatment. Current research found pen-strep increased macrophage modulus but limited influence on cell adhesion. Phalloidin statin image indicates pen-strep mediate cell morphology on different extracellular matrix coated surface. The roundness analyzes further illustrated pen-strep promote cell spread on PDMS rubber, type I collagen, laminin, poly amino acid, poly-RGD peptides. Finally, YAP-1 and TAZ upregulation and β1 integrin downregulation may be the causes of cell elasticity and mechano-response to extracellular matrix (ECM) change.