611 research outputs found
Letter to the editor: "Quantifying albumin permeability with multiphoton microscopy: why the difference?"
Intravital multiphoton microscopy as a tool for studying renal physiology and pathophysiology
The kidney is a complex and dynamic organ with over 40 cell types, and tremendous structural and functional diversity. Intravital multi-photon microscopy, development of fluorescent probes and innovative software, have rapidly advanced the study of intracellular and intercellular processes within the kidney. Researchers can quantify the distribution, behavior, and dynamic interactions of up to four labeled chemical probes and proteins simultaneously and repeatedly in four dimensions (time), with subcellular resolution in near real time. Thus, multi-photon microscopy has greatly extended our ability to investigate cell biology intravitally, at cellular and subcellular resolutions. Therefore, the purpose of the chapter is to demonstrate how the use in intravital multi-photon microscopy has advanced the understanding of both the physiology and pathophysiology of the kidney
Finding the bottom and using it: Offsets and sensitivity in the detection of low intensity values in vivo with 2-photon microscopy
Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required
Comparison of Vlasov-Uehling-Uhlenbeck model with 4 π Heavy Ion Data
Streamer chamber data for collisions of Ar + KCl and Ar + BaI2 at 1.2 GeV/nucleon are compared with microscopic model predictions based on the Vlasov-Uehling-Uhlenbeck equation, for various density-dependent nuclear equations of state. Multiplicity distributions and inclusive rapidity and transverse momentum spectra are in good agreement. Rapidity spectra show evidence of being useful in determining whether the model uses the correct cross sections for binary collisions in the nuclear medium, and whether momentum-dependent interactions are correctly incorporated. Sideward flow results do not favor the same nuclear stiffness parameter at all multiplicities
Microvascular inflammatory responses to ceramide and cigarette smoke in the intact rat assessed with intravital two-photon microscopy
poster abstractsmoke, characterized by alterations of the alveolar barrier function. We investigated this hypothesis by utilizing a novel application of intravital two-photon excitation microscopy (TPM) of the lung in a living, breathing animal.
Methods: We first developed a technique of TPM to permit imaging of the lung maintained within the thoracic cavity of an intact rat. To accomplish this, we optimized the lung-microscope interface with an imaging window uniquely designed to minimize cardiac and respiratory motion during TPM acquisitions. To investigate alveolar barrier disruption in real time, we utilized intravenous (i.v.) fluorescent probes to examine changes in alveolar leukocyte trafficking and microvascular barrier function in response to i.v. ceramide (C16:0 PEG, 10 mg/kg), and to cigarette smoke extract (CSE) delivered i.v. (1ml/kg) or intratracheally via a nebulizer (2mL/kg).
Results: We performed intravital TPM monitoring of the lung microcirculation of a living rat with maintained physiological cardio-pulmonary parameters for up to 3h. Time-lapse and 3-D reconstruction images revealed heterogeneous extravasation of FITC-labeled serum albumin from the alveolar microcirculation into the alveolar airspaces in response to ceramide, in a dose-dependent manner. Further, we noted that in response to both ceramide and to CSE, leukocytes accumulated in the lung parenchyma and demonstrated reduced mobility through the microcirculation, suggesting increased adhesion to the endothelium. Intratracheal administration of CSE caused increased extravasation of leukocytes into alveolar spaces within 10 minutes.
Conclusions: We developed approaches that permit the application of intravital TPM to lung with no motion artifacts from the breathing and cardiac cycles. This approach permits visualization of the lung subpleural parenchyma with a high resolution. Both gross and subtle inflammatory changes that reflect alveolar epithelial and/or endothelial barrier dysfunction can be assessed with this methodology
Sepsis-Induced Glomerular Endothelial Dysfunction Mediates Reductions in GFR and Increases in Protein Filtration
poster abstractBackground: Sepsis is now the leading cause of acute kidney injury (AKI) known to decrease Glomerular filtration rate (GFR) and increase proteinuria. There also exists a discrepancy between renal perfusion and GFR. Methods: To evaluate the potential role of the glomerulus in the overall pathogenesis of these abnormalities, we studied surface glomeruli in 8-10 week old Munich Wistar Frmter rats using intravital 2-photon microscopy in a cecal ligation and puncture (CLP) model of sepsis to ask targeted questions and compare the metric of measured GFR to serum creatinine changes at 24 hours post CLP. Results: Male rats undergoing CLP showed an increase in serum creatinine from 0.23 +/- 0.06 mg/dl to 0.80 +/-0.17 (P0.01) and a decrease in real time GFR from 0.69 +/- 0.06 ml/min/100gm body wt to 0.34 +/-0.15 (P0.01). Hemodynamic monitoring revealed normal and hyperdynamic cardiac status within the CLP group. Quantitative analysis of 15 glomeruli in three CLP septic rats revealed a reduction in red blood cell flow rates within capillary loops from 1,771 +/- 467 to 576 +/- 327 um/sec (P0.01); an increase in WBC adherence to glomerular capillary endothelial cells from 0.42 +/-0.33 to 7.25 +/- 5.82 WBC's/standardized glomerular volume (P0.05) in CLP rats; and an increase in the glomerular sieving coefficient (GSC) of a 150kD dextran from 0.007 +/- 0.003 to 0.097 +/- 0.046 (P0.05). Rouleaux formations were seen only in septic rats. Conclusions: These data indicate glomerular endothelial-WBC interactions during sepsis, in part, explain the reduction in GFR and increased filtration of large molecular weight proteins. The results from real time GFR accurately detected the drop in renal function for this model of sepsis
Chronic endothelin-1 infusion elevates glomerular sieving coefficient and proximal tubular albumin reuptake in the rat
AbstractAimWe have previously found that chronic endothelin-1 (ET-1) infusion in Sprague–Dawley rats increases glomerular permeability to albumin (Palb) as assessed in vitro independent of blood pressure with no observed albuminuria. In this study, we hypothesized that ET-1 increases glomerular albumin filtration with accompanied increase in albumin uptake via the proximal tubule, which masks the expected increase in urinary albumin excretion.Main methodsNonfasting Munich-Wistar Fromter rats were surgically prepared for in vivo imaging (n=6). Rats were placed on the microscope stage with the exposed kidney placed in a cover slip-bottomed dish bathed in warm isotonic saline. Rats were then injected i.v. with rat serum albumin conjugated to Texas Red that was observed to enter capillary loops of superficial glomeruli, move into Bowman's space, bind to the proximal tubular cell brush border and reabsorbed across the apical membrane. Glomerular sieving coefficient (GSC) was calculated as the ratio of conjugated albumin within the glomerular capillary versus that in Bowman's space. Rats were again studied after 2weeks of chronic ET-1 (2pmol/kg/min; i.v. osmotic minipump).Key findingsGlomerular sieving coefficient was significantly increased in rats following chronic ET-1 infusion (0.025±0.005 vs. 0.017±0.003, p<0.05). Mean fluorescence intensity for conjugated albumin within proximal tubules was increased by ET-1 infusion: 118.40±6.34 vs. 74.27±4.45 pixel intensity (p<0.01).SignificanceThese data provide in vivo evidence that ET-1 directly increases glomerular permeability to albumin and that albuminuria is prevented by increased PT albumin uptake in the rat
Collective Flow from the Intranuclear Cascade Model
The phenomenon of collective flow in relativistic heavy ion collisions is
studied using the hadronic cascade model ARC. Direct comparison is made to data
gathered at the Bevalac, for Au+Au at GeV/c. In contrast to the
standard lore about the cascade model, collective flow is well described
quantitatively without the need for explicit mean field terms to simulate the
nuclear equation of state. Pion collective flow is in the opposite direction to
nucleon flow as is that of anti-nucleons and other produced particles. Pion and
nucleon flow are predicted at AGS energies also, where, in light of the higher
baryon densities achieved, we speculate that equation of state effects may be
observable.Comment: 9 pages, 2 figures include
Isospin dependence of collective flow in heavy-ion collisions at intermediate energies
Within the framework of an isospin-dependent Boltzmann-Uehling-Uhlenbeck
(BUU) model using initial proton and neutron densities calculated from the
nonlinear relativistic mean-field (RMF) theory, we compare the strength of
transverse collective flow in reactions and
, which have the same mass number but different neutron/proton
ratios. The neutron-rich system () is found to show
significantly stronger negative deflection and consequently has a higher
balance energy, especially in peripheral collisions. NOTE ADDED IN PROOF: The
new phenomenon predicted in this work has just been confirmed by an experiment
done by G.D. Westfall et al. using the NSCL/MSU radioactive beam facility and a
spartan soccer. A paper by R. Pak et al. is submitted to PRL to report the
experimental result.Comment: Latex file, 9 pages, 4 figures availabe upon request; Phys. Rev.
Lett. (June 3, 1996) in pres
Inhibition of αvβ5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury
Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvβ5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvβ5 in a rat model of renal IRI. Pretreatment with this anti-αvβ5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvβ5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvβ5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvβ5 antibody treatment. Immunostaining for αvβ5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvβ5 in modulating vascular leak. Additional studies showed αvβ5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvβ5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvβ5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvβ5 inhibition as a promising therapeutic strategy for AKI
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