NIR-II window tracking of hyperglycemia induced intracerebral hemorrhage in cerebral cavernous malformation deficient mice

2020 ◽  
Vol 8 (18) ◽  
pp. 5133-5144 ◽  
Author(s):  
Abdul K. Parchur ◽  
Zhi Fang ◽  
Jaidip M. Jagtap ◽  
Gayatri Sharma ◽  
Christopher Hansen ◽  
...  
Keyword(s):  
Intracerebral Hemorrhage ◽  
Cavernous Malformation ◽  
Ex Vivo ◽  
Cerebral Cavernous Malformation ◽  
Micro Ct ◽  
Kinetic Imaging ◽  
Mice Brain ◽  
Deficient Mice

Left panel: Pseudocolor map of 3 principle components from NIR-II kinetic imaging, Right panel (top to bottom): In vivo Ag2S QD NIR-II fluorescence, ex vivo iodine micro-CT, FITC dextran perfusion, and H&E staining in control vs CCM1+/− mice brain.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Platelet ADP receptors contribute to the initiation of intravascular coagulation

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 594-600 ◽  
Author(s):  
Catherine Leon ◽  
Meike Alex ◽  
Antje Klocke ◽  
Eberhard Morgenstern ◽  
Christine Moosbauer ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Intravascular Coagulation ◽  
Adp Receptors ◽  
Adp Receptor ◽  
Deficient Mice ◽  
Storage Pools

Abstract While the adenosine 5′-diphosphate (ADP) pathway is known to enhance thrombus formation by recruiting platelets and leukocytes to the primary layer of collagen-adhering platelets, its role for the initiation of coagulation has not been revealed. Ex vivo inhibition of the P2Y12 ADP receptor by clopidogrel administration diminished the rapid exposure of tissue factor (TF), the major initiator of coagulation, in conjugates of platelets with leukocytes established by the contact of whole blood with fibrillar collagen. Under in vitro conditions, the P2Y12 and P2Y1 ADP receptors were both found to be implicated in the exposure of TF in collagen-activated whole blood. Immunoelectron-microscopy revealed that collagen elicited the release of TF from its storage pools within the platelets. Functional activation of the intravascular TF was reduced by inhibition of the ADP receptors, partially due to the disruption of the platelet-neutrophil adhesions. Injection of collagen into the venous system of mice increased the number of thrombin-antithrombin complexes, indicative for the formation of thrombin in vivo. In P2Y1-deficient mice, the ability of collagen to enhance the generation of thrombin was impaired. In conclusion, the platelet ADP pathway supports the initiation of intravascular coagulation, which is likely to contribute to the concomitant formation of fibrin at the site of the growing thrombus.

Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 1911-1918 ◽  
Author(s):  
Matt W. Goschnick ◽  
Lai-Man Lau ◽  
Janet L. Wee ◽  
Yong S. Liu ◽  
P. Mark Hogarth ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Fluorescein Isothiocyanate ◽  
Clot Retraction ◽  
In Vivo Evaluation ◽  
Integrin Αiibβ3 ◽  
Normal Platelet ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Deficient Mice

AbstractWe investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin αIIbβ3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin αIIbβ3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal “insideout” integrin αIIbβ3 signaling (fluorescein isothiocyanate [FITC]–fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin αIIbβ3 “outside-in” signaling events in platelets and is necessary for stability of arterial thrombi in vivo.

scholarly journals Targeted Deletion of the Lipopolysaccharide (LPS)-binding Protein Gene Leads to Profound Suppression of LPS Responses Ex Vivo, whereas In Vivo Responses Remain Intact

1997 ◽  
Vol 186 (12) ◽  
pp. 2051-2056 ◽  
Author(s):  
Mark M. Wurfel ◽  
Brian G. Monks ◽  
Robin R. Ingalls ◽  
Russell L. Dedrick ◽  
Russell Delude ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Ex Vivo ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Cellular Responses ◽  
Tnf Α ◽  
Deficient Mice ◽  
Lps Binding

Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-α. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-α levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.

scholarly journals Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice

2018 ◽  
Vol 148 (9) ◽  
pp. 1415-1420 ◽  
Author(s):  
Xiaoying Wang ◽  
Yang Yuan ◽  
Inka C Didelija ◽  
Mahmoud A Mohammad ◽  
Juan C Marini
Keyword(s):  
Ex Vivo ◽  
Transcript Abundance ◽  
Mutant Mice ◽  
Ex Vivo Model ◽  
Endogenous Production ◽  
Gene Coding ◽  
Cycle Disorders ◽  
Deficient Mice

Abstract Background The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. Objective The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. Methods Six-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Results Liver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg−1 · h−1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA−1 · d−1; P < 0.001). Conclusions Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

scholarly journals Multimodality Approach to Characterizing the Distinctive Hallmarks of Lung Fibrosis: A Mouse Model with Bleomycin and Indocyanine Green

2021 ◽  
Author(s):  
Andrea Grandi ◽  
Erica Ferrini ◽  
Roberta Ciccimarra ◽  
Martina Mambrini ◽  
Laura Mecozzi ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Time Course ◽  
Ex Vivo ◽  
Imaging Techniques ◽  
Lavage Fluid ◽  
Pharmacological Therapy ◽  
Micro Ct ◽  
Oropharyngeal Aspiration ◽  
Multimodality Approach

Abstract Background.Idiopathic Pulmonary Fibrosis is a progressive disease with short life expectancy and no disease-modifying pharmacological therapy. The continuous refinement of animal models and the integration of in-vivo imaging techniques is fundamental for the selection of new antifibrotic drugs.Indocyanine Green (ICG), a fluorescent dye, was administered by oropharyngeal aspiration (OA) to mice with Bleomycin (BLM) to map the lung exposure.Methods.Female mice C57bl/6 were treated via OA with BLM+ICG or ICG. Animals were imaged at 7, 14 and 21 days either with the fluorescent system or Micro-CT. At each time point subsets of mice were sampled for ex-vivo assessment. Histological assessment of fibrosis by Ashcroft score, airspace enlargements and mean linear intercept (MLI) were evaluated at 7, 14 and 21 days. Leukocytes and cytokines were measured in bronchoalveolar lavage fluid. Results.Fluorescence imaging revealed a persistent lung signal in both groups until 21 days. In BLM+ICG group, Micro-CT detected a marked increase in hypo- and non-aerated tissues throughout the study. At later time points hyper-inflated tissue was detected. Histology revealed high Ashcroft score throughout the time-course with a prominent increase in airspace size and MLI at day 21. ICG mice had healthy lungs.Conclusions.We showed that ICG can be used as a tracer to map the distribution of BLM in lungs. However, BLM+ICG produced unexpected severe lung changes different from pure BLM model, such as emphysema-like features which progressively worsened. The multimodalities approach warranted characterization of the distinctive features of this new pulmonary fibrosis model and provided fundamentals for in-vivo translation.

scholarly journals Enhanced Susceptibility of ADAP-Deficient Mice to Listeria monocytogenes Infection Is Associated With an Altered Phagocyte Phenotype and Function

2021 ◽  
Vol 12 ◽  
Author(s):  
Martha A. L. Böning ◽  
Gerald P. Parzmair ◽  
Andreas Jeron ◽  
Henning P. Düsedau ◽  
Olivia Kershaw ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Ex Vivo ◽  
Transcriptional Profiling ◽  
Fatal Outcome ◽  
Flow Cytometric Analysis ◽  
Adaptor Protein ◽  
Phagocytic Capacity ◽  
Inflammatory Monocytes ◽  
Deficient Mice

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection.

scholarly journals Caspase-3/NLRP3 signaling in the mesenchymal stromal niche regulates myeloid-biased hematopoiesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Zhang ◽  
Yaozhen Chen ◽  
Dandan Yin ◽  
Fan Feng ◽  
Qunxing An ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Knockout Mice ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Gene Knockout ◽  
Hematopoietic Stem ◽  
Nlrp3 Gene ◽  
Gene Knockout Mice ◽  
Deficient Mice

Abstract Background The fate of hematopoietic stem cells (HSCs) is determined by a complex regulatory network that includes both intrinsic and extrinsic signals. In the past decades, many intrinsic key molecules of HSCs have been shown to control hematopoiesis homeostasis. Non-hematopoietic niche cells also contribute to the self-renewal, quiescence, and differentiation of HSCs. Mesenchymal stromal cells (MSCs) have been identified as important components of the niche. However, the regulatory role of MSCs in hematopoiesis has not been fully understood. Methods Caspase-3 and NLRP3 gene knockout mice were generated respectively, and hematopoietic development was evaluated in the peripheral circulation and bone marrow by flow cytometry, colony formation assay, and bone marrow transplantation. Bone-associated MSCs (BA-MSCs) were then isolated from gene knockout mice, and the effect of Caspase-3/NLRP3 deficient BA-MSCs on hematopoiesis regulation was explored in vivo and ex vivo. Results We report that Caspase-3 deficient mice exhibit increased myelopoiesis and an aberrant HSC pool. Ablation of Caspase-3 in BA-MSCs regulates myeloid lineage expansion by altering the expression of hematopoietic retention cytokines, including SCF and CXCL12. Interestingly, NLRP3 gene knockout mice share phenotypic similarities with Caspase-3 deficient mice. Additionally, we found that NLRP3 may play a role in myeloid development by affecting the cell cycle and apoptosis of hematopoietic progenitors. Conclusions Our data demonstrate that the Caspase-3/NLRP3 signaling functions as an important regulator in physiological hematopoiesis, which provides new insights regarding niche signals that influence hematopoiesis regulation in the bone marrow.

Rapid, Exponential Extra-Splenic Platelet Consumption in WASP Deficient Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2174-2174
Author(s):  
Ted S. Strom ◽  
Jim Y. Wan ◽  
Haiming Du ◽  
Carl W. Jackson
Keyword(s):  
Platelet Count ◽  
Consumption Rate ◽  
Ex Vivo ◽  
Fold Increase ◽  
Extramedullary Hematopoiesis ◽  
Plateau Phase ◽  
Platelet Production ◽  
Normal Platelet ◽  
Deficient Mice

Abstract The thrombocytopenia seen in patients with the Wiskott-Aldrich Syndrome (WAS) is thought to be due primarily to rapid platelet consumption, and is markedly improved by splenectomy. While a murine model of WAS shows only mild thrombocytopenia, we have previously demonstrated rapid platelet turnover in this model; that splenectomy improves the platelet count in WASP-(C57Bl/6J) mice; and that the latter mice do not achieve the same platelet count found in splenectomized WT mice. Here we show that CMFDA-labeled WASP- platelets are consumed rapidly and exponentially in WT recipients, with an estimated lifespan of 18 hours (vs. 87 hours for WT platelets). WASP- platelets are consumed only slightly more slowly in splenectomized WT recipients (lifespan 28 hours, vs. 102 hr for WT platelets). On the C3H background, WASP- mice have normal platelet counts but show a similar rapid, exponential platelet consumption rate (lifespan 25 hours in either C3H or C57Bl/6J recipients, vs 85 hours for WT platelets). In vivo platelet biotinylation studies demonstrate less effective labeling of WASP- (C57Bl/6J) platelets than of WT platelets. After a plateau phase, in vivo labeled WASP- platelets show the same kind of rapid platelet turnover seen with ex vivo labeling (figure 1). Our results imply a three-fold increase in platelet production rate in WASP- mice on either background, consistent with the markedly increased splenic extramedullary hematopoiesis seen in WASP- mice. WASP- mice also show an increased number of bone marrow megkaryocytes, the ploidy distribution of which is normal. We conclude that WASP- mice demonstrate a significantly increased rate of extra-splenic platelet consumption that is largely (on the C57Bl/6 background) or completely (on the C3H background) compensated by increased platelet production. The ability of WASP- mice to compensate for their rapid platelet consumption, and the normal ploidy of their megakaryocytes, suggests that platelet production is not impaired by WASP deficiency in this model. In vivo biotinylation of WASP-and WT platelets In vivo biotinylation of WASP-and WT platelets

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