Mechanosensitivity of the Nervous System (Mechanosensitivity in Cells and Tissues)

Mechanosensing

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Forewords by Nektarios Tavernarakis and Pontus Persson

Currently, investigations of the effects of mechanical stress on nervous system are focused on several issues. The majority of studies investigate the effects of mechanical stimulation on mechanosensitive channels, as its primary target and interactive agent, and aim on description of downstream intracellular signaling pathways together with addressing general issues of biomechanics of the nervous system. Knowledge of biomechanics, and mechanisms, which underlie it on organism, organ, tissue and cellular level, is necessary for understanding of the normal functioning of living organisms and allows to predict changes, which arise due to alterations of their environment, and possibly will allow to develop new methods of artificial intervention.

The book brings up the problem closer to the experts in related medical and biological sciences as well as practicing doctors besides just presenting the latest achievements in the field. Mechanosensitivity of the Nervous System. Andre Kamkin , Irina Kiseleva. Ion Channels with Mechanosensitivity in the Nervous System.

Neuronal Mechanosensitivity in the Gastrointestinal Tract. After subsidence of an acute injury response, implanted foreign bodies become chronically surrounded by activated microglial cells [6], which release proinflammatory and immunoregulatory substances [7], followed by a layer of reactive astrocytes [8] with an increased production of intermediate filaments particularly glial fibrillary acidic protein, GFAP [4,5]. The resulting glial scar may have a toxic effect on local neurons and acts as a physical barrier around the implant [6], thus insulating it from the remaining neurons and, in case of an electrode, detrimentally increasing its impedance [9].

The universal occurrence of the FBR is not well correlated with the chemical nature of the implant [10], which is generally selected to be inert. This poses the question about the trigger of an FBR. Importantly, CNS cells not only respond to chemical but also to mechanical signals i. For example, most neuronal and glial cell types adapt their morphology and cytoskeletal composition to the stiffness of their surrounding in vitro [11—17]. Published by Elsevier Ltd. All rights reserved. CNS tissue belongs to the softest tissues in nature [17], and neural implants are usually orders of magnitude stiffer than the physiological cell environment.

Hence, it seemed possible that local cells respond to a mismatch of mechanical compliance between nervous tissue and the implant. To pursue this idea, we exposed microglia and astrocytes, the main contributors to FBRs in the CNS, to materials of different stiffness but same chemical properties and tested their morphological and inflammatory responses to these mechanical signals in vitro and in vivo.

Compliant culture substrates were fabricated from polyacrylamide as described previously [13] and functionalized with poly-D-lysine solution PDL; Sigma see Supporting information for details and Fig. S5 for control measurements of rheo-logical properties and PDL coating.

Mechanosensation

All animal experiments were conducted in accordance with the United Kingdom Animals Scientific Procedure Act of and institutional guidelines. Cell cultures were prepared from neonatal Sprague-Dawley rat cerebral cortices as previously described [18]. All in vitro experiments were done after one day in culture. Phase contrast images of cells cells per gel in fields of view, 3 cultures per cell type were taken.

According to changes in morphology, four morphological categories were defined for microglia and five for astrocytes and arbitrary scores of 1 for the round cell shape to 4 or 5 for the most spread cell shape were assigned to each category [13] Fig. For IL-1P, the blocking step was performed with normal donkey serum Stratech.

Primary antibodies or cocktails of primary antibodies; for details see Supporting information were prepared in proper dilutions in PBS-TX and added to the cells. Cells were incubated for 2 h at room temperature, or alternatively overnight. After 1 h incubation at room temperature, cells were washed and mounted with FluorSave mounting reagent.

For Figs. In Fig.

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The hybridized probe arrays were stained with streptavidin phycoerythrin conjugate and scanned using an Affymetrix GeneChip 7G scanner. Each independent experiment included three arrays from three biological replicates.

Statistical analysis was performed using a one-sample Student's t-test, which was applied to the mean of each normalized value against the baseline value of 1. Genes regulated differently by more than 1. The significance of the association between the data set and the pathway was measured by two means: 1 A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed and 2 Fisher's exact test was used to calculate a P value to determine the probability that the association between the genes in the data set and the pathway is due to chance.

Glial cell morphology depends on substrate stiffness. Both primary microglial cells A-C and astrocytes D-F change their morphology in response to the stiffness of the substrate. Moreover, astrocytes occasionally showed star-like morphologies, resembling their in vivo shape arrow in D.

Importance of mechanosensitivity for the peripheral nervous system

An activated phenotype was frequently observed on stiff gels arrowheads in B. Scale bars: 50 mm. Scale bars: 30 mm.

Inflammatory responses of microglial cells to stiff substrates. Scale bar: 50 mm. G Western blots showing intracellular protein concentrations after 1 day in culture.

Forewords by Nektarios Tavernarakis and Pontus Persson

A TaqMan real-time PCR method was applied to confirm changes in expression profiles of a selection of genes in microglia i. Real-time PCR was run using TaqMan primers and reagents Applied Biosystems and arbitrary units of gene quantities were extracted from Ct values using the standard curves. Astrocytes were lysed using. Inflammatory response of astrocytes to stiff substrates. Scale bar: mm.

Cells were pelleted and lysed with 30 ml of ice-cold lysis buffer Cell Signaling supplemented with additional protease inhibitors Complete Lysis-M kit, Roche for 30—60 min. Intermittent vortexing and sonication was performed every 15 min. In microglial samples, avidin-biotin amplification Vectastain, Vector labs was used. Western blots were quantified using ImageJ software. Briefly, images were inverted, boxes of equal size were drawn around the bands of interest, and mean white values MWV measured; background was measured accordingly and subtracted from the band values.

The method was confirmed by Gaussian fitting analysis using Origin. Briefly, plot profiles of inverted blots were plotted and fitted to a Gaussian. Background was subtracted from the base of the curve and the integrated value of the curve was determined. PAA premixes of 30 kPa and Pa gels were polymerized on top of each other in a 0.

After opening the skull, a composite foreign body was inserted into brain tissue using fine tweezers Fig. The insertion point was located 1.

The brain was covered with the excised bony slab and the skin was sutured. Animals received analgesic injections for the following 2—3 days. Images were taken using a confocal laser scanning microscope and stitched together using Corel Draw X3 software. The interface between the brain tissue and different gel types was determined using bright field images Fig.

Fluorescence images were analyzed using Adobe Photoshop CS5 software.

To capture local responses to foreign body stiffness, the edges of the lesion around the stiff and soft gels were separately found using the magic lasso tool, the border of the mask was extended to 50 pixels, and average gray values of these selections determined. This way, protein levels around the stiff and soft parts of the implant in the same animal were normalized for the contact area of implant and tissue Fig. In vitro data was collected in all cases from at least three different cultures. Origin software Version 8; OriginLab was used to analyze the statistical significance of the data.

Kolmogorov—Smirnov tests confirmed a normal distribution of values before any parametric test was employed. In order to examine the putative effect of contact compliance on glial cells, we cultured primary rat microglia and astrocytes on polyacrylamide substrates of different compliance but identical poly-D-lysine surface coating [13] Fig. We studied morphological, gene and protein expression changes as a function of substrate stiffness after one day in culture.

Microglial cells growing on Pa surfaces mostly showed spherical morphologies, with some short processes and. A quantitative analysis [13] showed significant morphological differences Fig. Consistent with previous studies [12,13], astrocytes on compliant gels either had a rounded shape or a star-like morphology with fine processes, quite unlike their usual in vitro appearance on rigid substrates, but resembling their in vivo morphology Fig.

Quantitative shape analysis confirmed that also differences in astrocyte morphologies were significant Fig. Hence, both microglia and astrocytes displayed morphological characteristics of an activated phenotype on stiffer substrates. We then investigated whether glial responses to mechanical stiffness might also involve the production of molecules that could trigger an inflammatory reaction. We first analyzed the transcrip-tional profile of microglial cells and astrocytes as a function of substrate compliance using DNA microarrays.

In microglial cells 73 genes were significantly upregulated on stiff substrates compared to compliant ones Table S1 and genes downregulated Table S2. In total, 15 pathways related to different inflammatory and pathogenic functions were upregu-lated on stiff substrates Table S5 , and 8 were attenuated Table S6. In order to confirm these mRNA changes, we selected some key inflammation-related molecules, for which antibodies exist, for examination at the protein level by immunocy-tochemistry and Western blot.

Introduction

Astrocytes growing on stiff substrates contained significantly higher levels of pro-caspase In vivo FBR is triggered by the stiffness of an implant. B Schematic coronal section and top view of a rat brain indicating the location of the cFB dotted lines. The dashed gray line indicates the region visible in C , which shows brain tissue at the end of an experiment. Scale bar: 1 mm.

D Immunohistochemistry of brain tissue in the vicinity of the compliant and E stiff part of a cFB asterisks implanted for three weeks; blue: Hoechst stained nuclei, red: CD11b OX 42 showing activated microglial cells, green: GFAP showing activated astrocytes. Inflammatory and astrogliotic reactions are considerably increased around the stiff material cf.

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