Use of Multiphoton Intravital Imaging and a Multiscale Computational Model of Thrombus Development to Study the Role of FVII in Thrombogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 391-391
Author(s):  
Malgorzata Kamocka ◽  
Zhiliang Xu ◽  
Nan Chen ◽  
Mark Alber ◽  
Elliot D. Rosen
Keyword(s):  
High Resolution ◽  
Computational Model ◽  
Imaging System ◽  
Structural Information ◽  
Cellular Interactions ◽  
Wild Type ◽  
Cellular Potts Model ◽  
Modeling Framework ◽  
Deficient Mice

Abstract Using 2-photon intravital microscopy we have generated high resolution, near real-time 3-dimensional images of a developing thrombus. Following Titanium - Sapphire laser-induced injuries in mouse mesenteric vessels, the developing thrombus was monitored by collecting stacks of confocal images through the developing thrombus. Data were collected in 3 channels for fluorescently labeled platelets, fibrinogen and 70,000 MW dextran. By including fluorescently labeled dextran in the blood we were able to monitor flow (plasma) and not labeled cells (leukocytes and erythrocytes) forming black silhouettes in the plasma. Since each 3-D reconstruction involves a series of scans, we were able to generate approximately 2–3 reconstructions per minute. Thus, the system sacrifices temporal resolution for high resolution structural information revealing the changing, heterogeneous sub-domain structure of the developing thrombus. The imaging system has been used to study the consequences of FVII-deficiency. Following injury, of the luminal surface of the vessel, a thin layer of platelets and fibrin accumulated at the injury site. Unlike injuries in wild-type mice where the thrombus continues to grow, the injuries in FVII deficient mice failed to grow as a result of frequent embolization from the developing structure. Interestingly, in a model of ferric chloride induced injury of the carotid artery, thrombi in FVII deficient mice form large structures capable of reducing flow although, unlike wild type mice, the FVII deficient animals fail to form stable occlusive clots. In parallel with the modified experimental vascular injury model, we have begun development of a computational model of thrombus development. The modeling framework consists of a stochastic and discrete Cellular Potts Model (CPM) to describe platelet and cellular interactions and continuous submodels to describe hydrodynamic and biochemical reactions. Our multiscale model includes the vessel wall, platelets (in resting and activated states), blood cells, coagulation reactions, fibrin formation, and hydrodynamic parameters as components. By comparing the in vivo experimental results with those of simulations varying the concentration of FVII we are able to refine and validate the computational model of thrombogenesis.

MR-GUIDED PULSE OXIMETRY IMAGING OF BREAST IN VIVO

2011 ◽  
Vol 04 (02) ◽  
pp. 199-208
Author(s):  
ZHIQIU LI ◽  
SHUDONG JIANG ◽  
VENKATARAMANAN KRISHNASWAMY ◽  
SCOTT C. DAVIS ◽  
SUBHADRA SRINIVASAN ◽  
...  
Keyword(s):  
Breast Tissue ◽  
Near Infrared ◽  
Imaging System ◽  
Structural Information ◽  
Human Subjects ◽  
Nir Spectroscopy ◽  
Tomography System ◽  
Finger Pulse ◽  
Lock In

A near-infrared (NIR) tomography system with spectrally-encoded sources in two wavelength bands was built to quantify the temporal oxyhemoglobin and deoxyhemoglobin contrast in breast tissue at a 20 Hz bandwidth. The system was integrated into a 3 T magnetic resonance (MR) imaging system through a customized breast coil interface for simultaneous optical and MRI acquisition. In this configuration, the MR images provide breast tissue structural information for NIR spectroscopy of adipose and fibro-glandular tissue in breast. Spectral characterization performance of the NIR system was verified through dynamic phantom experiments. Normal human subjects were imaged with finger pulse oximeter (PO) plethysmogram synchronized to the NIR system to provide a frequency-locked reference. Both the raw data from the NIR system and the recovered absorption coefficients of the breast at two wavelengths showed the same frequency of about 1.3 Hz as the PO output. The frequency lock-in approach provided a practical platform for MR-localized recovery of small pulsatile variations of oxyhemoglobin and deoxyhemoglobin in the breast, which are related to the heartbeat and vascular resistance of the tissue.

scholarly journals Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Adria Carbo ◽  
Danyvid Olivares-Villagómez ◽  
Raquel Hontecillas ◽  
Josep Bassaganya-Riera ◽  
Rupesh Chaturvedi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
T Cell ◽  
Wild Type ◽  
Content Type ◽  
Interleukin 21 ◽  
H Pylori ◽  
Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

Urokinase-dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1703-1711 ◽  
Author(s):  
Frederic Lluı́s ◽  
Josep Roma ◽  
Mònica Suelves ◽  
Maribel Parra ◽  
Gloria Aniorte ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Plasminogen Activator ◽  
Muscle Regeneration ◽  
Plasminogen Activators ◽  
Skeletal Muscle Regeneration ◽  
Plasminogen Activation ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Deficient Mice

Plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are extracellular proteases involved in various tissue remodeling processes. A requirement for uPA activity in skeletal myogenesis was recently demonstrated in vitro. The role of plasminogen activators in skeletal muscle regeneration in vivo in wild-type, uPA-deficient, and tPA-deficient mice is investigated here. Wild-type and tPA−/− mice completely repaired experimentally damaged skeletal muscle. In contrast, uPA−/− mice had a severe regeneration defect, with decreased recruitment of blood-derived monocytes to the site of injury and with persistent myotube degeneration. In addition, uPA-deficient mice accumulated fibrin in the degenerating muscle fibers; however, the defibrinogenation of uPA-deficient mice resulted in a correction of the muscle regeneration defect. A similar severe regeneration deficit with persistent fibrin deposition was also reproducible in plasminogen-deficient mice after injury, suggesting that fibrinolysis by uPA-mediated plasminogen activation plays a fundamental role in skeletal muscle regeneration. In conclusion, the uPA-plasmin system is identified as a critical component of the mammalian skeletal muscle regeneration process, possibly because it prevents intramuscular fibrin accumulation and contributes to the adequate inflammatory response after injury. These studies demonstrate the requirement of an extracellular proteolytic cascade during muscle regeneration in vivo.

scholarly journals In vivo multi-parametric manganese-enhanced MRI for detecting senile plaques in rodent models of Alzheimer’s disease

2021 ◽  
Author(s):  
Eugene Kim ◽  
Davide Di Censo ◽  
Mattia Baraldo ◽  
Camilla Simmons ◽  
Ilaria Rosa ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
High Resolution ◽  
Senile Plaques ◽  
Rodent Models ◽  
Wild Type ◽  
Enhanced Mri ◽  
Manganese Uptake ◽  
5Xfad Mice ◽  
Manganese Enhanced Mri

AbstractSenile plaques are a hallmark of Alzheimer’s disease (AD) that develop in its earliest stages. Thus, non-invasive detection of these plaques would be invaluable for diagnosis and the development and monitoring of treatments, but this remains a challenge due to their small size. Here, we investigated the utility of manganese-enhanced MRI (MEMRI) for visualizing plaques in transgenic rodent models of AD across two species: 5xFAD mice and TgF344-AD rats.Fourteen mice (eight transgenic, six wild-type) and eight rats (four transgenic, four wild-type) were given subcutaneous injections of MnCl2 and imaged in vivo using a 9.4T Bruker scanner. Susceptibility-weighted images, transverse relaxation rate (R2*) maps, and quantitative susceptibility maps were derived from high-resolution 3D multi-gradient-echo (MGE) data to directly visualize plaques. Longitudinal relaxation rate (R1) maps were derived from MP2RAGE data to measure regional manganese uptake. After scanning, the brains were processed for histology and stained for beta-amyloid (4G8 antibody), iron (Perl’s), and calcium/manganese (Alizarin Red).MnCl2 improved signal-to-noise ratio (1.55±0.39-fold increase in MGE images) as expected, although this was not necessary for detection of plaques in the high-resolution images. Plaques were visible in susceptibility-weighted images, R2* maps, and quantitative susceptibility maps, with increased R2* and more positive magnetic susceptibility compared to surrounding tissue.In the 5xFAD mice, most MR-visible plaques were in the hippocampus, though histology confirmed plaques in the cortex and thalamus as well. In the TgF344-AD rats, many more plaques were MR-visible throughout the hippocampus and cortex. Beta-amyloid and iron staining indicate that, in both models, MR visibility was driven by plaque size and iron load.Voxel-wise comparison of R1 maps revealed increased manganese uptake in brain regions of high plaque burden in transgenic animals compared to their wild-type littermates. Interestingly, in contrast to plaque visibility in the high-resolution images, the increased manganese uptake was limited to the rhinencephalon in the TgF344-AD rats (family-wise error (FWE)-corrected p < 0.05) while it was most significantly increased in the cortex of the 5xFAD mice (FWE-corrected p < 0.3). Alizarin Red staining suggests that manganese bound to plaques in 5xFAD mice but not in TgF344-AD rats.Multi-parametric MEMRI is a simple, viable method for detecting senile plaques in rodent models of AD. Manganese-induced signal enhancement can enable higher-resolution imaging, which is key to visualizing these small amyloid deposits. We also present in vivo evidence of manganese as a potential targeted contrast agent for imaging plaques in the 5xFAD model of AD.HighlightsThis is the first study to use manganese-enhanced MRI (MEMRI) for direct visualization of senile plaques in rodent models of Alzheimer’s disease, in vivo.Manganese enhancement is not necessary to detect plaques but improves image contrast and signal-to-noise ratio.Manganese binds to plaques in 5xFAD mice but not in TgF344-AD rats, demonstrating potential as a targeted contrast agent for imaging plaques in certain models of AD.

scholarly journals An in vivo inflammatory loop potentiates KRAS blockade

2019 ◽  
Author(s):  
Kristina A.M. Arendt ◽  
Giannoula Ntaliarda ◽  
Vasileios Armenis ◽  
Danai Kati ◽  
Christin Henning ◽  
...  
Keyword(s):  
Interleukin 1Β ◽  
Wild Type ◽  
Poor Survival ◽  
Murine Bone Marrow ◽  
Chemokine Ligand ◽  
Isoprenylcysteine Carboxylmethyltransferase ◽  
Chemokine Ligand 2 ◽  
Deficient Mice

ABSTRACTKRAS inhibitors perform inferior to other targeted drugs. To investigate a possible reason for this, we treated cancer cells with KRAS inhibitors deltarasin (targeting phosphodiesterase-δ), cysmethynil (targeting isoprenylcysteine carboxylmethyltransferase), and AA12 (targeting KRASG12C), and silenced/overexpressed mutant KRAS using custom vectors. We show that KRAS-mutant tumor cells exclusively respond to KRAS blockade in vivo, because the oncogene co-opts host myeloid cells via a C-C-motif chemokine ligand 2/interleukin-1β signaling loop for sustained tumorigenicity. Indeed, KRAS-mutant tumors did not respond to deltarasin in Ccr2 and Il1b gene-deficient mice, but were deltarasin-sensitive in wild-type and Ccr2-deficient mice adoptively transplanted with wild-type murine bone marrow. A KRAS-dependent pro-inflammatory transcriptome was prominent in human cancers with high KRAS mutation prevalence and predicted poor survival. Hence the findings support that in vitro systems are suboptimal for anti-KRAS drug screens, and suggest that interleukin-1β blockade might be specific for KRAS-mutant cancers.

Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis

2001 ◽  
Vol 280 (2) ◽  
pp. G291-G297 ◽  
Author(s):  
Cameron W. Lush ◽  
Gediminas Cepinskas ◽  
William J. Sibbald ◽  
Peter R. Kvietys
Keyword(s):  
Leukocyte Adhesion ◽  
Cecal Ligation ◽  
Leukocyte Rolling ◽  
Wild Type ◽  
Endothelial Adhesion Molecule ◽  
Acute Peritonitis ◽  
Leukocyte Accumulation ◽  
Deficient Mice

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)−/− mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS−/− and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS−/− mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS−/− mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS−/− mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS−/− mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.

scholarly journals A Role for Mac-1 (CDIIb/CD18) in Immune Complex–stimulated Neutrophil Function In Vivo: Mac-1 Deficiency Abrogates Sustained Fcγ Receptor–dependent Neutrophil Adhesion and Complement-dependent Proteinuria in Acute Glomerulonephritis

1997 ◽  
Vol 186 (11) ◽  
pp. 1853-1863 ◽  
Author(s):  
Tao Tang ◽  
Alexander Rosenkranz ◽  
Karel J.M. Assmann ◽  
Michael J. Goodman ◽  
Jose-Carlos Gutierrez-Ramos ◽  
...  
Keyword(s):  
Immune Complex ◽  
Mutant Mice ◽  
Polymorphonuclear Neutrophil ◽  
Complement C3 ◽  
Acute Glomerulonephritis ◽  
Wild Type ◽  
Filamentous Actin ◽  
Deficient Mice

Mac-1 (αmβ2), a leukocyte adhesion receptor, has been shown in vitro to functionally interact with Fcγ receptors to facilitate immune complex (IC)–stimulated polymorphonuclear neutrophil (PMN) functions. To investigate the relevance of Mac-1–FcγR interactions in IC-mediated injury in vivo, we induced a model of Fc-dependent anti–glomerular basement membrane (GBM) nephritis in wild-type and Mac-1–deficient mice by the intravenous injection of anti-GBM antibody. The initial glomerular PMN accumulation was equivalent in Mac-1 null and wild-type mice, but thereafter increased in wild-type and decreased in mutant mice. The absence of Mac-1 interactions with obvious ligands, intercellular adhesion molecule 1 (ICAM-1), and C3 complement, is not responsible for the decrease in neutrophil accumulation in Mac-1– deficient mice since glomerular PMN accumulation in mice deficient in these ligands was comparable to those in wild-type mice. In vitro studies showed that spreading of Mac-1–null PMNs to IC-coated dishes was equivalent to that of wild-type PMNs at 5–12 min but was markedly reduced thereafter, and was associated with an inability of mutant neutrophils to redistribute filamentous actin. This suggests that in vivo, Mac-1 is not required for the initiation of Fc-mediated PMN recruitment but that Mac-1–FcγR interactions are required for filamentous actin reorganization leading to sustained PMN adhesion, and this represents the first demonstration of the relevance of Mac-1–FcγR interactions in vivo. PMN-dependent proteinuria, maximal in wild-type mice at 8 h, was absent in Mac-1 mutant mice at all time points. Complement C3–deficient mice also had significantly decreased proteinuria compared to wild-type mice. Since Mac-1 on PMNs is the principal ligand for ic3b, an absence of Mac-1 interaction with C3 probably contributed to the abrogation of proteinuria in Mac-1–null mice.

Differential response to myocardial reperfusion injury in eNOS-deficient mice

2002 ◽  
Vol 282 (6) ◽  
pp. H2422-H2426 ◽  
Author(s):  
Brent R. Sharp ◽  
Steven P. Jones ◽  
David M. Rimmer ◽  
David J. Lefer
Keyword(s):  
Ischemia Reperfusion ◽  
Myocardial Necrosis ◽  
In Vivo Model ◽  
Pcr Analysis ◽  
Inos Mrna ◽  
Mrna Levels ◽  
Wild Type ◽  
Significant Difference ◽  
Deficient Mice

Two strains of endothelial nitric oxide synthase (eNOS)-deficient (−/−) mice have been developed that respond differently to myocardial ischemia-reperfusion (MI/R). We evaluated both strains of eNOS−/− mice in an in vivo model of MI/R. Harvard (Har) eNOS−/− mice ( n = 12) experienced an 84% increase in myocardial necrosis compared with wild-type controls ( P < 0.05). University of North Carolina (UNC) eNOS−/−( n = 10) exhibited a 52% reduction in myocardial injury versus wild-type controls ( P < 0.05). PCR analysis of myocardial inducible NO synthase (iNOS) mRNA levels revealed a significant ( P < 0.05) increase in the UNC eNOS−/− mice compared with wild-type mice, and there was no significant difference between the Har eNOS−/− and wild-type mice. UNC eNOS−/− mice treated with an iNOS inhibitor (1400W) exacerbated the extent of myocardial necrosis. When treated with 1400W, Har eNOS−/− did not exhibit a significant increase in myocardial necrosis. These data demonstrate that two distinct strains of eNOS−/− mice display opposite responses to MI/R. Although the protection seen in the UNC eNOS−/− mouse may result from compensatory increases in iNOS, other genes may be involved.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo

2019 ◽  
Vol 12 (574) ◽  
pp. eaao7232 ◽  
Author(s):  
Katsuhiko Itoh ◽  
Gen Kondoh ◽  
Hitoshi Miyachi ◽  
Manabu Sugai ◽  
Yoshiyuki Kaneko ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Sperm Maturation ◽  
Sperm Cells ◽  
Chromatin Binding ◽  
Wild Type ◽  
Underlying Mechanisms ◽  
Deficient Mice ◽  
Protamine 2

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l−/−; Prm2S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l−/− mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

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