scholarly journals A Shift in Tissue Stiffness During Hippocampal Maturation Correlates to the Pattern of Neurogenesis and Composition of the Extracellular Matrix

2021 ◽  
Vol 13 ◽  
Author(s):  
Youngjae Ryu ◽  
Misato Iwashita ◽  
Wonyoung Lee ◽  
Kenji Uchimura ◽  
Yoichi Kosodo
Keyword(s):  
Extracellular Matrix ◽  
Chondroitin Sulfate ◽  
Enzymatic Digestion ◽  
Granule Cell Layer ◽  
Neural Cells ◽  
Tissue Stiffness ◽  
Differentiation Markers ◽  
Differential Signal ◽  
Force Microscopy

Aging changes the mechanical properties of brain tissue, such as stiffness. It has been proposed that the maintenance and differentiation of neural stem cells (NSCs) are regulated in accordance with extracellular stiffness. Neurogenesis is observed in restricted niches, including the dentate gyrus (DG) of the hippocampus, throughout mammalian lifetimes. However, profiles of tissue stiffness in the DG in comparison with the activity of NSCs from the neonatal to the matured brain have rarely been addressed so far. Here, we first applied ultrasound-based shear-wave elasticity imaging (SWEI) in living animals to assess shear modulus as in vivo brain stiffness. To complement the assay, atomic force microscopy (AFM) was utilized to determine the Young’s modulus in the hippocampus as region-specific stiffness in the brain slice. The results revealed that stiffness in the granule cell layer (GCL) and the hilus, including the subgranular zone (SGZ), increased during hippocampal maturation. We then quantified NSCs and immature neural cells in the DG with differentiation markers, and verified an overall decrease of NSCs and proliferative/immature neural cells along stages, showing that a specific profile is dependent on the subregion. Subsequently, we evaluated the amount of chondroitin sulfate proteoglycans (CSPGs), the major extracellular matrix (ECM) components in the premature brain by CS-56 immunoreactivity. We observed differential signal levels of CSPGs by hippocampal subregions, which became weaker during maturation. To address the contribution of the ECM in determining tissue stiffness, we manipulated the function of CSPGs by enzymatic digestion or supplementation with chondroitin sulfate, which resulted in an increase or decrease of stiffness in the DG, respectively. Our results illustrate that stiffness in the hippocampus shifts due to the composition of ECM, which may affect postnatal neurogenesis by altering the mechanical environment of the NSC niche.

scholarly journals Enzymatic Digestion of Hyaluronan-Based Brain Extracellular Matrix in vivo Can Induce Seizures in Neonatal Mice

2019 ◽  
Vol 13 ◽  
Author(s):  
Alena Balashova ◽  
Vladimir Pershin ◽  
Olga Zaborskaya ◽  
Natalia Tkachenko ◽  
Andrey Mironov ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Enzymatic Digestion ◽  
Neonatal Mice ◽  
Brain Extracellular Matrix

scholarly journals Simultaneous in vivo time-lapse stiffness mapping and fluorescence imaging of developing tissue

2018 ◽  
Author(s):  
Amelia J. Thompson ◽  
Iva K. Pillai ◽  
Ivan B. Dimov ◽  
Christine E. Holt ◽  
Kristian Franze
Keyword(s):  
Fluorescence Imaging ◽  
Temporal Dynamics ◽  
Time Lapse ◽  
Tissue Mechanics ◽  
Tissue Stiffness ◽  
Time Resolved ◽  
Force Microscopy ◽  
Atomic Force ◽  
Spatio Temporal

AbstractTissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that in the developing Xenopus brain, a stiffness gradient evolves over time because of differential cell proliferation. Subsequently, axons turn to follow this gradient, underpinning the importance of time-resolved mechanics measurements.

scholarly journals Predicting local tissue mechanics using immunohistochemistry

2018 ◽  
Author(s):  
David E. Koser ◽  
Emad Moeendarbary ◽  
Stefanie Kuerten ◽  
Kristian Franze
Keyword(s):  
Nervous Tissue ◽  
Tissue Mechanics ◽  
Tissue Stiffness ◽  
Force Microscopy ◽  
Local Tissue ◽  
Atomic Force ◽  
Local Stiffness ◽  
Sophisticated Technology

AbstractLocal tissue stiffness provides an important signal to which cells respond in vivo. However, assessing tissue mechanics is currently challenging and requires sophisticated technology. We here developed a model quantitatively predicting nervous tissue stiffness heterogeneities at cellular resolution based on cell density, myelin and GFAP fluorescence intensities. These histological parameters were identified by a correlation analysis of atomic force microscopy-based elasticity maps of spinal cord sections and immunohistochemical stainings. Our model provides a simple tool to estimate local stiffness distributions in nervous tissue, and it can easily be expanded to other tissue types, thus paving the way for studies of the role of mechanical signals in development and pathology.

scholarly journals Microelastic mapping of the rat dentate gyrus

2016 ◽  
Vol 3 (4) ◽  
pp. 150702 ◽  
Author(s):  
Tomás Luque ◽  
Michael S. Kang ◽  
David V. Schaffer ◽  
Sanjay Kumar
Keyword(s):  
Stem Cells ◽  
Dentate Gyrus ◽  
Cell Layer ◽  
Granule Cell Layer ◽  
Lineage Commitment ◽  
Force Microscopy ◽  
Living Slices ◽  
Atomic Force ◽  
Threefold Increase

The lineage commitment of many cultured stem cells, including adult neural stem cells (NSCs), is strongly sensitive to the stiffness of the underlying extracellular matrix. However, it remains unclear how well the stiffness ranges explored in culture align with the microscale stiffness values stem cells actually encounter within their endogenous tissue niches. To address this question in the context of hippocampal NSCs, we used atomic force microscopy to spatially map the microscale elastic modulus ( E ) of specific anatomical substructures within living slices of rat dentate gyrus in which NSCs reside during lineage commitment in vivo . We measured depth-dependent apparent E -values at locations across the hilus (H), subgranular zone (SGZ) and granule cell layer (GCL) and found a two- to threefold increase in stiffness at 500 nm indentation from the H (49 ± 7 Pa) and SGZ (58 ± 8 Pa) to the GCL (115 ± 18 Pa), a fold change in stiffness we have previously found functionally relevant in culture. Additionally, E exhibits nonlinearity with depth, increasing significantly for indentations larger than 1 µm and most pronounced in the GCL. The methodological advances implemented for these measurements allow the quantification of the elastic properties of hippocampal NSC niche at unprecedented spatial resolution.

scholarly journals microRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis

2018 ◽  
Author(s):  
Albertomaria Moro ◽  
Tristan Discroll ◽  
William Armero ◽  
Liana C. Boraas ◽  
Dionna M. Kasper ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Tissue Stiffness ◽  
Matrix Synthesis ◽  
Tissue Properties ◽  
Multiple Systems ◽  
Regulatory Pathways ◽  
Genome Wide ◽  
Mechanical Homeostasis

SummaryThe mechanical properties of tissues, which are determined primarily by their extracellular matrix (ECM), are largely stable over time despite continual turnover of ECM constituents 1,2. These observations imply active homeostasis, where cells sense and adjust rates of matrix synthesis, assembly and degradation to keep matrix and tissue properties within the optimal range. However, the regulatory pathways that mediate this process are essentially unknown3. Genome-wide analyses of endothelial cells revealed abundant microRNA-mediated regulation of cytoskeletal, adhesive and extracellular matrix (CAM) mRNAs. High-throughput assays showed co-transcriptional regulation of microRNA and CAM genes on stiff substrates, which buffers CAM expression. Disruption of global or individual microRNA-dependent suppression of CAM genes induced hyper-adhesive, hyper-contractile phenotypes in multiple systems in vitro, and increased tissue stiffness in the zebrafish fin-fold during homeostasis and regeneration in vivo. Thus, a network of microRNAs and CAM mRNAs mediate tissue mechanical homeostasis.

scholarly journals Organ sculpting by patterned extracellular matrix stiffness

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Justin Crest ◽  
Alba Diz-Muñoz ◽  
Dong-Yuan Chen ◽  
Daniel A Fletcher ◽  
David Bilder
Keyword(s):  
Extracellular Matrix ◽  
Genetic Manipulation ◽  
Tissue Expansion ◽  
Differential Resistance ◽  
Mechanical Anisotropy ◽  
Force Microscopy ◽  
Egg Chambers ◽  
Extracellular Matrix Stiffness ◽  
Asymmetric Force

How organ-shaping mechanical imbalances are generated is a central question of morphogenesis, with existing paradigms focusing on asymmetric force generation within cells. We show here that organs can be sculpted instead by patterning anisotropic resistance within their extracellular matrix (ECM). Using direct biophysical measurements of elongating Drosophila egg chambers, we document robust mechanical anisotropy in the ECM-based basement membrane (BM) but not in the underlying epithelium. Atomic force microscopy (AFM) on wild-type BM in vivo reveals an anterior–posterior (A–P) symmetric stiffness gradient, which fails to develop in elongation-defective mutants. Genetic manipulation shows that the BM is instructive for tissue elongation and the determinant is relative rather than absolute stiffness, creating differential resistance to isotropic tissue expansion. The stiffness gradient requires morphogen-like signaling to regulate BM incorporation, as well as planar-polarized organization to homogenize it circumferentially. Our results demonstrate how fine mechanical patterning in the ECM can guide cells to shape an organ.

scholarly journals Hippo Pathway Deregulation Drives Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma

2018 ◽  
Author(s):  
Daniela Pankova ◽  
Yanyan Jiang ◽  
Iolanda Vendrell ◽  
Jon N. Buzzelli ◽  
Anderson Ryan ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Matrix ◽  
Lung Adenocarcinoma ◽  
Hippo Pathway ◽  
Metastatic Progression ◽  
Differentiation Markers ◽  
Cancer Stemness ◽  
Metastatic Dissemination

AbstractLung cancer remains the leading cause of cancer-related death due to poor treatment responses arising from tumor heterogeneity and epigenetic aberrations. Here we show that adverse prognosis associated with epigenetically silenced tumour suppressor RASSF1A is a consequence of increased extracellular matrix, tumour stiffness and metastatic dissemination in vivo and in vitro. We find that lung cancer cells with methylated RASSF1A display constitutive nuclear YAP1 and expression of prolyl4hydroxylase2 (P4HA2) into extracellular matrix that correlates with increases collagen deposition. Furthermore, we identify an epigenetic axis in tumour cells where elevated ECM impedes the intrinsic suppression of WNT signaling (via TPBG/5T4) triggering b-catenin-YAP1 activation and thus results in a cancer stem-like programming. As key drivers, we identified RASSF1A and P4HA2 mediating the ECM-dependent stemness and metastatic dissemination in vivo. Re-expression of RASSF1A or inhibition of P4HA2 activity reverse these effects and increase levels of lung differentiation markers (TTF-1, Mucin5B) in vivo and in vitro. Our study identifies an epigenetic program to cancer stemness and metastatic progression of lung adenocarcinoma and P4HA2 as potential target for uncoupling ECM signals towards cancer stemness.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Closure of Fetoscopic Access Sites with Amniotic Extracellular Matrix Scaffolds in an In Vivo Model

2006 ◽  
Vol 66 (S 01) ◽  
Author(s):  
N Ochsenbein-Kölble ◽  
J Jani ◽  
G Verbist ◽  
L Lewi ◽  
K Marquardt ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
In Vivo Model ◽  
Extracellular Matrix Scaffolds

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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