scholarly journals Sexual Dimorphism in Extracellular Matrix Composition and Viscoelasticity of the Healthy and Inflamed Mouse Brain

2021 ◽  
Author(s):  
Clara Sophie Batzdorf ◽  
Anna Sophie Morr ◽  
Gergely Bertalan ◽  
Ingolf Sack ◽  
Rafaela Vieira Silva ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Sexual Dimorphism ◽  
Mouse Brain ◽  
Brain Regions ◽  
Matrix Composition ◽  
Specific Reference ◽  
Autoimmune Encephalomyelitis ◽  
Healthy Female ◽  
Membrane Components

Magnetic resonance elastography (MRE) has revealed sexual dimorphism in brain stiffness in healthy individuals and multiple sclerosis (MS) patients. In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), we showed previously that inflammation-induced brain softening was associated with alterations of the extracellular matrix (ECM). However, it remained unclear whether the brain ECM presents sex-specific properties that can be visualized by MRE. Therefore, we aimed here at quantifying sexual dimorphism in brain viscoelasticity in association with ECM changes in healthy and inflamed brains. Multifrequency MRE was applied to the midbrain of healthy and EAE mice of both sexes to quantitatively map regional stiffness. To define differences in brain ECM composition, gene expression of the key basement membrane components laminin (Lama4, Lama5), collagen (Col4a1, Col1a1) and fibronectin (Fn1) was investigated by RT-qPCR. We showed that the healthy male cortex expressed less Lama4, Lama5, Col4a1 but more Fn1 (all p < 0.05) than the healthy female cortex, which was associated with 9% softer properties (p = 0.044) in that region. At peak EAE, cortical softening was similar in both sexes compared to healthy tissue, with an 8% difference remaining between males and females during EAE (p < 0.001). Cortical Lama4, Lama5 and Col4a1 expression increased 2 to 3-fold in EAE in both sexes while Fn1 decreased only in males (all p < 0.05). No significant sex differences in stiffness were detected in other brain regions. In conclusion, sexual dimorphism in the ECM composition of cortical tissue in the mouse brain is reflected by in vivo stiffness measured with MRE and should be considered in future studies by sex-specific reference values.

scholarly journals Alternative Splicing of FN (Fibronectin) Regulates the Composition of the Arterial Wall Under Low Flow

2020 ◽  
Author(s):  
Patrick A. Murphy ◽  
Noor Jailkhani ◽  
Sarah-Anne Nicholas ◽  
Amanda M. Del Rosario ◽  
Jeremy L. Balsbaugh ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Alternative Splicing ◽  
Low Flow ◽  
Matrix Composition ◽  
Splice Isoforms ◽  
Vitro Assay ◽  
Disturbed Flow ◽  
The Impact

Objective: Exposure of the arterial endothelium to low and disturbed flow is a risk factor for the erosion and rupture of atherosclerotic plaques and aneurysms. Circulating and locally produced proteins are known to contribute to an altered composition of the extracellular matrix at the site of lesions, and to contribute to inflammatory processes within the lesions. We have previously shown that alternative splicing of FN (fibronectin) protects against flow-induced hemorrhage. However, the impact of alternative splicing of FN on extracellular matrix composition remains unknown. Approach and Results: Here, we perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. We also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. We found flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in our in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments. Conclusions: Our results reveal the extent of the dynamic alterations in the matrisome in the acute response to low and disturbed flow and show how changes in the splicing of FN, a common response in vascular inflammation and remodeling, can affect matrix composition.

scholarly journals Extracellular matrix modulates the biological effects of melatonin in mesenchymal stem cells

2014 ◽  
Vol 223 (2) ◽  
pp. 167-180 ◽  
Author(s):  
Fan He ◽  
Xiaozhen Liu ◽  
Ke Xiong ◽  
Sijin Chen ◽  
Long Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Mesenchymal Stem Cells ◽  
Biological Effects ◽  
Matrix Composition ◽  
Calcium Deposition ◽  
Specific Gene ◽  
Skeletal Tissue ◽  
Underlying Mechanisms

Both self-renewal and lineage-specific differentiation of mesenchymal stem cells (MSCs) are triggered by theirin vivomicroenvironment including the extracellular matrix (ECM) and secreted hormones. The ECM may modulate the physiological functions of hormones by providing binding sites and by regulating downstream signaling pathways. Thus, the purpose of this study was to evaluate the degree of adsorption of melatonin to a natural cell-deposited ECM and the effects of this interaction on the biological functions of melatonin in human bone marrow-derived MSCs (BM-MSCs). The fibrillar microstructure, matrix composition, and melatonin-binding affinity of decellularized ECM were characterized. The cell-deposited ECM improved melatonin-mediated cell proliferation by 31.4%, attenuated accumulation of intracellular reactive oxygen species accumulation, and increased superoxide dismutase (SOD) mRNA and protein expression. Interaction with ECM significantly enhanced the osteogenic effects of melatonin on BM-MSCs by increasing calcium deposition by 30.5%, up-regulating osteoblast-specific gene expression and down-regulating matrix metalloproteinase (MMP) expression. The underlying mechanisms of these changes in expression may involve intracellular antioxidant enzymes, because osteoblast-specific genes were down-regulated, whereas MMP expression was up-regulated, in the presence of SOD-specific inhibitors. Collectively, our findings indicate the importance of native ECM in modulating the osteoinductive and antioxidant effects of melatonin and provide a novel platform for studying the biological actions of growth factors or hormones in a physiologically relevant microenvironment. Moreover, a better understanding of the enhancement of MSC growth and osteogenic differentiation resulting from the combination of ECM and melatonin could improve the design of graft substitutes for skeletal tissue engineering.

scholarly journals Noninvasive, in vivo imaging of subcortical mouse brain regions with 17  μm optical coherence tomography

2015 ◽  
Vol 40 (21) ◽  
pp. 4911 ◽  
Author(s):  
Shau Poh Chong ◽  
Conrad W. Merkle ◽  
Dylan F. Cooke ◽  
Tingwei Zhang ◽  
Harsha Radhakrishnan ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Mouse Brain ◽  
In Vivo Imaging ◽  
Brain Regions ◽  
Optical Coherence

scholarly journals Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 470 ◽  
Author(s):  
Anish Deshpande ◽  
Remitha M. Vinayakamoorthy ◽  
Brijesh K. Garg ◽  
Jaya Prakash Thummapudi ◽  
Gauri Oza ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Splice Variants ◽  
Brain Regions ◽  
Nucleotide Polymorphisms ◽  
Western Blots ◽  
Alpha Bungarotoxin

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

scholarly journals Quantification of the Cannabinoid Type 1 Receptor Availability in the Mouse Brain

2020 ◽  
Vol 14 ◽  
Author(s):  
Isabelle Miederer ◽  
Viktoria Wiegand ◽  
Nicole Bausbacher ◽  
Petra Leukel ◽  
Stephan Maus ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Mouse Brain ◽  
Brain Regions ◽  
Cb1 Receptor ◽  
Receptor Ligand ◽  
Addictive Disorders ◽  
Cannabinoid Type 1 Receptor ◽  
Slow Kinetics

Introduction: The endocannabinoid system is involved in several diseases such as addictive disorders, schizophrenia, post-traumatic stress disorder, and eating disorders. As often mice are used as the preferred animal model in translational research, in particular when using genetically modified mice, this study aimed to provide a systematic analysis of in vivo cannabinoid type 1 (CB1) receptor ligand-binding capacity using positron emission tomography (PET) using the ligand [18F]MK-9470. We then compared the PET results with literature data from immunohistochemistry (IHC) to review the consistency between ex vivo protein expression and in vivo ligand binding.Methods: Six male C57BL/6J (6–9 weeks) mice were examined with the CB1 receptor ligand [18F]MK-9470 and small animal PET. Different brain regions were evaluated using the parameter %ID/ml. The PET results of the [18F]MK-9470 accumulation in the mouse brain were compared with immunohistochemical literature data.Results: The ligand [18F]MK-9470 was taken up into the mouse brain within 5 min after injection and exhibited slow kinetics. It accumulated highly in most parts of the brain. PET and IHC classifications were consistent for most parts of the telencephalon, while brain regions of the diencephalon, mesencephalon, and rhombencephalon were rated higher with PET than IHC.Conclusions: This preclinical [18F]MK-9470 study demonstrated the radioligand’s applicability for imaging the region-specific CB1 receptor availability in the healthy adult mouse brain and thus offers the potential to study CB1 receptor availability in pathological conditions.

scholarly journals Capturing relevant extracellular matrices for investigating cell migration

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1408 ◽  
Author(s):  
Patricia Keely ◽  
Amrinder Nain
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Three Dimensional ◽  
3D Culture ◽  
Extracellular Matrices ◽  
Matrix Composition ◽  
Disease States ◽  
Stromal Microenvironment ◽  
Different Tissues

Much progress in understanding cell migration has been determined by using classic two-dimensional (2D) tissue culture platforms. However, increasingly, it is appreciated that certain properties of cell migration in vivo are not represented by strictly 2D assays. There is much interest in creating relevant three-dimensional (3D) culture environments and engineered platforms to better represent features of the extracellular matrix and stromal microenvironment that are not captured in 2D platforms. Important to this goal is a solid understanding of the features of the extracellular matrix—composition, stiffness, topography, and alignment—in different tissues and disease states and the development of means to capture these features

scholarly journals Human cerebral organoids establish subcortical projections in the mouse brain after transplantation

2020 ◽  
Author(s):  
Xin Dong ◽  
Shi-Bo Xu ◽  
Xin Chen ◽  
Mengdan Tao ◽  
Xiao-Yan Tang ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Therapeutic Potential ◽  
Neurological Diseases ◽  
Brain Regions ◽  
Synaptic Connections ◽  
Glutamatergic Neurons ◽  
Human Brain Development ◽  
Basal Brain

Abstract Numerous studies have used human pluripotent stem cell-derived cerebral organoids to elucidate the mystery of human brain development and model neurological diseases in vitro, but the potential for grafted organoid-based therapy in vivo remains unknown. Here, we optimized a culturing protocol capable of efficiently generating small human cerebral organoids. After transplantation into the mouse medial prefrontal cortex, the grafted human cerebral organoids survived and extended projections over 4.5 mm in length to basal brain regions within 1 month. The transplanted cerebral organoids generated human glutamatergic neurons that acquired electrophysiological maturity in the mouse brain. Importantly, the grafted human cerebral organoids functionally integrated into pre-existing neural circuits by forming bidirectional synaptic connections with the mouse host neurons. Furthermore, compared to control mice, the mice transplanted with cerebral organoids showed an increase in freezing time in response to auditory conditioned stimuli, suggesting the potentiation of the startle fear response. Our study showed that subcortical projections can be established by microtransplantation and may provide crucial insights into the therapeutic potential of human cerebral organoids for neurological diseases.

scholarly journals Perlecan Knockdown Significantly Alters Extracellular Matrix Composition and Organization During Cartilage Development

2020 ◽  
Vol 19 (7) ◽  
pp. 1220-1235 ◽  
Author(s):  
Alexander R. Ocken ◽  
Madeline M. Ku ◽  
Tamara L. Kinzer-Ursem ◽  
Sarah Calve
Keyword(s):  
Extracellular Matrix ◽  
Matrix Composition ◽  
Pericellular Matrix ◽  
Post Translational Modifications ◽  
Healthy Cartilage ◽  
Liquid Chromatography Tandem Mass ◽  
Cartilage Biomechanics ◽  
Insight Into

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y−Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y−Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

scholarly journals In vivo metabolic labeling of sialoglycans in the mouse brain by using a liposome-assisted bioorthogonal reporter strategy

2016 ◽  
Vol 113 (19) ◽  
pp. 5173-5178 ◽  
Author(s):  
Ran Xie ◽  
Lu Dong ◽  
Yifei Du ◽  
Yuntao Zhu ◽  
Rui Hua ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Mouse Brain ◽  
Large Scale ◽  
Neuronal Cell ◽  
Brain Regions ◽  
Metabolic Labeling ◽  
Proteomic Profiling ◽  
Associated Proteins ◽  
Sialylated Glycoproteins

Mammalian brains are highly enriched with sialoglycans, which have been implicated in brain development and disease progression. However, in vivo labeling and visualization of sialoglycans in the mouse brain remain a challenge because of the blood−brain barrier. Here we introduce a liposome-assisted bioorthogonal reporter (LABOR) strategy for shuttling 9-azido sialic acid (9AzSia), a sialic acid reporter, into the brain to metabolically label sialoglycoconjugates, including sialylated glycoproteins and glycolipids. Subsequent bioorthogonal conjugation of the incorporated 9AzSia with fluorescent probes via click chemistry enabled fluorescence imaging of brain sialoglycans in living animals and in brain sections. Newly synthesized sialoglycans were found to widely distribute on neuronal cell surfaces, in particular at synaptic sites. Furthermore, large-scale proteomic profiling identified 140 brain sialylated glycoproteins, including a wealth of synapse-associated proteins. Finally, by performing a pulse−chase experiment, we showed that dynamic sialylation is spatially regulated, and that turnover of sialoglycans in the hippocampus is significantly slower than that in other brain regions. The LABOR strategy provides a means to directly visualize and monitor the sialoglycan biosynthesis in the mouse brain and will facilitate elucidating the functional role of brain sialylation.

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.

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