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Jan R. Thiele Kurt Goerendt G. Bjoern Stark Steffen U. Eisenhardt 《Journal of visualized experiments : JoVE》2012,(66)
Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological
conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as
following transplant and cardiovascular surgery.1 Reperfusion of previously
ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits
excessive inflammation of the affected tissue. Adjacent to the production of reactive
oxygen species, activation of the complement system and increased microvascular
permeability, the activation of leukocytes is one of the principle actors in the
pathological cascade of inflammatory tissue damage during reperfusion.2, 3
Leukocyte activation is a multistep process consisting of rolling, firm adhesion and
transmigration and is mediated by a complex interaction between adhesion molecules in
response to chemoattractants such as complement factors, chemokines, or
platelet-activating factor.4While leukocyte rolling in postcapillary venules is predominantly mediated by the
interaction of selectins5 with their counter ligands, firm adhesion of
leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion
molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7Gold standard for the in vivo observation of leukocyte-endothelial
interaction is the technique of intravital microscopy, first described in
1968.8Though various models of IRI (ischemia-reperfusion injury) have been described for
various organs, 9-12 only few are suitable for direct visualization of
leukocyte recruitment in the microvascular bed on a high level of image
quality.8We here promote the digital intravital epifluorescence microscopy of the postcapillary
venule in the cremasteric microcirculation of the rat 13 as a convenient method
to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in
striated muscle tissue and provide a detailed manual for accomplishing the technique. We
further illustrate common pitfalls and provide useful tips which should enable the reader
to truly appreciate, and safely perform the method.In a step by step protocol we depict how to get started with respiration controlled
anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer
periods of time. We then describe the cremasteric preparation as a thin flat sheet for
outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that
has been well established in our laboratories.Download video file.(88M, mov) 相似文献
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Ferruh Artunc Christian Mueller Tobias Breidthardt Raphael Twerenbold Andreas Peter Claus Thamer Peter Weyrich Hans-Ulrich Haering Bjoern Friedrich 《PloS one》2012,7(10)
Background
In hemodialysis patients, elevated plasma troponin concentrations are a common finding that has even increased with the advent of newly developed sensitive assays. However, the interpretation and relevance of this is still under debate.Methods
In this cross-sectional study, we analyzed plasma concentrations of sensitive troponin I (TnI) and troponin T (TnT) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality.Results
In all of the enrolled patients, plasma TnI or TnT was detectable at a median concentration of 14 pg/ml (interquartile range: 7–29) using the Siemens TnI ultra assay and 49 pg/ml (31–74) using the Roche Elecsys high sensitive TnT assay. Markedly more patients exceeded the 99th percentile for TnT than for TnI (95% vs. 14%, p<0.0001). In a multivariate linear regression model, TnT was independently associated with age, gender, systolic dysfunction, time on dialysis, residual diuresis and systolic blood pressure, whereas TnI was independently associated with age, systolic dysfunction, pulse pressure, time on dialysis and duration of a HD session. During a follow-up period of nearly two years, TnT concentration above 38 pg/mL was associated with a 5-fold risk of death, whereas elevation of TnI had a gradual association to mortality.Conclusion
In hemodialysis patients, elevations of plasma troponin concentrations are explained by cardiac function and dialysis-related parameters, which contribute to cardiac strain. Both are highly predictive of increased risk of death. 相似文献34.
Arens R Wang P Sidney J Loewendorf A Sette A Schoenberger SP Peters B Benedict CA 《Journal of immunology (Baltimore, Md. : 1950)》2008,180(10):6472-6476
CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-A(b)-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-gamma, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-gamma production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen. 相似文献
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For experiments at the torque meter, flies are kept on standard fly medium at 25°C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly''s intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly''s control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser. 相似文献
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Lopez D Niesen M Bedi M McLean MP 《Prostaglandins, leukotrienes, and essential fatty acids》2008,78(2):131-135
Sterol carrier protein X (SCPx) is a peroxisomal protein with both lipid transfer and thiolase activity. Treating with the fatty acid, lauric acid, induced SCPx mRNA levels in rat liver and in rat hepatoma H4IIE cells but enhanced protein levels of SCPx and the thiolase produced as a post-translational modification of SCPx were only seen in H4IIE cells. Further investigation revealed that the presence of insulin can mask lauric acid effects on the SCPx gene especially at the protein level. These data are in agreement with the findings that diabetes, a medical condition characterized by high levels of fatty acids in an insulin deficient environment, enhances the hepatic expression of SCPx. 相似文献
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Stepwise assembly of functional C‐terminal REST/NRSF transcriptional repressor complexes as a drug target
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