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2018, Critical Care
https://doi.org/10.1186/S13054-018-1973-5…
215 pages
1 file
Introduction: Conventional assay technique to quantify vascular permeability in animal studies requires sacrifice animals; this becomes a barrier to evaluate of temporal changes or responses to therapeutic approaches in a single individual. In vivo fluorescence imaging potentially quantifies vascular permeability without sacrifice animals. However, the use of this noninvasive approach for the assessment of vascular permeability in remote organ injury caused by systemic inflammatory disease such as sepsis has not been reported. Methods: Cecal ligation and puncture (CLP)-induced septic mouse model was compared to sham and hydrocortisone pretreated (CLP + HC) mouse models. The lung was assumed as an injured remote organ and the footpad was assumed as a noninvasive observational site. The mixture of Evans blue (EB) and fluorescent dye of Genhance 750 were injected into mice, and the extraction of EB in harvested lung was assessed as a conventional indicator of vascular permeability. Fluorescent intensities in the harvested lung or footpad were assessed and their correlation was analyzed to investigate this novel, noninvasive approach to estimation of lung vascular permeability. Results: EB extraction in the harvested lung in the CLP group was significantly higher than in the other groups (CLP vs. sham, P=0.0012; CLP vs. CLP + HC, P=0.011). Fluorescent intensity in the footpad and harvested lung in the CLP group was also significantly higher than in the other groups (footpad, CLP vs. sham, P<0.0001; CLP vs. CLP + HC, P=0.0004; lung, CLP vs. sham, P<0.0001; CLP vs. CLP + HC, P<0.0001). The fluorescent intensity of the footpad was strongly correlated with that of the lung (r=0.95). Conclusions: The fluorescence imaging technique may be useful for assessment of vascular permeability based on EB quantification. The footpad fluorescent intensity was strongly correlated with that of the lung, and may be a suitable indicator in noninvasive estimation of lung vascular permeability.
American Journal of Physiology-Lung Cellular and Molecular Physiology, 2021
To develop a dynamic in vivo near-infrared (NIR) fluorescence imaging assay to quantify sequential changes in lung vascular permeability-surface area product (PS) in rodents. Dynamic NIR imaging methods for determining lung vascular permeability-surface area product were developed and tested on non-irradiated and 13 Gy irradiated rats with/without treatment with lisinopril, a radiation mitigator. A physiologically-based pharmacokinetic (PBPK) model of indocyanine green (ICG) pulmonary disposition was applied to in vivo imaging data and PS was estimated. In vivo results were validated by five accepted assays: ex vivo perfused lung imaging, endothelial filtration coefficient (K f) measurement, pulmonary vascular resistance measurement, Evan’s blue dye uptake, and histopathology. A PBPK model-derived measure of lung vascular permeability-surface area product increased from 2.60 ± 0.40 [CL: 2.42–2.78] mL/min in the non-irradiated group to 6.94 ± 8.25 [CL: 3.56–10.31] mL/min in 13 Gy gro...
Journal of visualized experiments : JoVE, 2013
This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this ...
Clinical Science, 2008
Capillary leak accompanying systemic inflammatory response conditions is a significant clinical problem. In the present study, we describe and verify a method for studying capillary leak that is based on the injection of proteins that differ significantly in size and have spectrally distinguishable fluorophores. Control (n=11) and post-CLP (caecal ligation and puncture; n=14) Sprague–Dawley rats were injected with tracer amounts of albumin and PEG–Alb [albumin covalently linked to methoxy-poly(ethylene glycol)] labelled with fluorescein and Texas Red. Blood samples were withdrawn between 5 min and 144 h, and the fluorescence of the labelled proteins was determined. The relative retention of the PEG–Alb and albumin was assessed via measurement of the TER (transcapillary escape rate; in %/h) and the t50% estimate, defined as the time when the actual concentration reached 50% of its baseline. The concentration–time trends for both albumin and PEG–Alb tracers exhibited two-compartmental...
Thrombosis and Haemostasis, 2012
Vascular hyperpermeability contributes to morbidity in inflammation. Current methodologies for in vivo assessment of permeability based on extravasation of Evans Blue (EB)-bound albumin are cumbersome and often lack sensitivity. We developed a novel infrared fluorescence (IRF) methodology for measurement of EB-albumin extravasation to quantify vascular permeability in murine models. Vascular permeability induced by endotoxaemia was examined for all solid organs, brain, skin and peritoneum by IRF and the traditional absorbance-based measurement of EB in tissue extracts. Organ IRF increased linearly with increasing concentrations of intravenous EB (2.5-25 mg/kg). Tissue IRF was more sensitive for EB accumulation compared to the absorbance-based method. Accordingly, differences in vascular permeability and organ EB accumulation between lipopolysaccharide-treated and saline-treated mice were often significant when analysed by IRF-based detection but not by absorbance-based detection. EB was detected in all 353 organs analysed with IRF but only in 67% (239/353) of organs analysed by absorbance-based methodology, demonstrating improved sensitivity of EB detection in organs with IRF. In contrast, EB in plasma after EB administration was readily measured by both methods with high correlation between the two methods (n=116, r2=0.86). Quantitation of organ-specific EB-IRF differences due to endotoxin was optimal when IRF was compared between mice matched for weight, gender, and age, and with appropriate corrections for organ weight and EB plasma concentrations. Notably, EB-IRF methodology leaves organs intact for subsequent histopathology. In summary, EB-IRF is a novel, highly sensitive, rapid, and convenient method for the relative quantification of EB in intact organs of treatment versus control mice.
Journal of Biochemical and Biophysical Methods, 2007
Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemiareperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescencebased assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar®-Transwell® clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF 165 ) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 μm carboxylate-modified FluoSpheres® microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF 165 at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2 ± 2.4%, 16.0 ± 3.7%, 41.5 ± 4.9%, and 58.6 ± 10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactiveand impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated.
Journal of General Physiology, 1996
A B STRA CT A surface fluorescence method was developed to measure transalveolar transport of water, protons, and solutes in intact perfused lungs. Lungs from c57 mice were removed and perfused via the pulmonary artery (~2 mi/min). The airspace was filled via the trachea with physiological saline containing a membrane-impermeant fluorescent indicator (FITC-dextran or aminonapthalene trisulfonic acid, ANTS). Because fluorescence is detected only near the lung surface due to light absorption by lung tissue, the surface fluorescence signal is directly proportional to indicator concentration. Confocal microscopy confirmed that the fluorescence signal arises from fluorophores in alveolijust beneath the pleural surface. Osmotic water permeability (t f) was measured from the time course of intraalveolar FITC-dextran fluorescence in response to changes in perfusate osmolality. Transalveolar Pf was 0.017 _+ 0.001 cm/s at 23~ independent of the solute used to induce osmosis (sucrose, NaC1, urea), independent of osmotic gradient size and direction, weakly temperature dependent (Arrhenius activation energy 5.3 kcal/mol) and inhibited by HgC12. Pf was not affected by cAMP activation but was decreased by 43% in lung exposed to hyperoxia for 5 d. Diffusional water permeability (Pd) and Pf were measured in the same lung from intraalveolar ANTS fluorescence, which increased by 1.8-fold upon addition of 50% I)20 tO the perfusate. Pd was 1.3 • 10 -~' cm/s at 23~ Transalveolar proton transport was measured from FITC-dextran fluorescence upon switching perfusate pH between 7.4 and 5.6; alveolar pH half-equilibrated in 1.9 and 1.0 rain without and with HCO3-, respectively. These results indicate high transalveolar water permeability in mouse lung, implicating the involvement of molecular water channels, and establish a quantitative surface fluorescence method to measure water and solute permeabilities in intact lung. Key words: alveolus * osmosis * fluorescent indicator 9 aquaporin 9 water channel
Journal of Visualized Experiments, 2013
This method is based on the intravenous injection of Evans Blue in mice. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular, permeability endothelial cells partially lose the close contact and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues.
Journal of Controlled Release, 2002
The overall pulmonary disposition of various fluorescent probes was viewed by confocal imaging following intratracheal delivery in the rat in vivo. The green fluorescent dyes, coumarin-6, a 350 Da lipophilic molecule; calcein, a 623 Da hydrophilic molecule; or FITC-albumin, a 65 000 Da hydrophilic molecule; were insufflated as a dry powder or instilled as a solution in the lungs of rat in vivo. Immediately, 2 or 24 h following delivery, the lungs were colored with sulforhodamine and fixed by vascular perfusion. The lungs were then removed, grossly sliced and examined by confocal laser scanning fluorescence microscopy. Coumarin-6 diffused within minutes across the trachea, airways and alveolar tissue but was also retained for hours in type II alveolar epithelial cells. The diffusion of calcein across the tissue was fast as well, with no particular affinity for specific cells. FITC-albumin slowly permeated the tissue. It remained in the airspaces for hours and was intensively captured by alveolar macrophages. Compared to the powder, the solution bypassed dissolution and therefore shortened the lag time for diffusion and cellular capture. The technique allowed to obtain an overview of the fate of fluorescent probes locally in each region of the lungs and highlighted the strong dependence of the localization behavior on physico-chemical properties of molecules as well as a capture by particular cells of the pulmonary tissue.
Laboratory Investigation, 2010
We present a new lung imaging technique based on endoscopic confocal fluorescence microscopy (ECFM), which is a new method that is able to provide cellular and structural assessment of living tissue using a small confocal probe in direct contact with the visceral pleura. To observe distal airspace structure and cellular condition in normal and injured lungs (hyperoxic and bleomycin challenged), we used fluorescent-specific marker contrast and ECFM. Alveolar space ECFM with spectral analyses were performed at 488-nm excitation using FITC-labeled markers or naturally fluorescent dyes. The normal lung was compared with the sick lung, where our in vivo imaging experiments correlated well with results obtained with corresponding ex vivo conventional assays. Four main elements pertaining to the acute lung injury/ acute respiratory distress syndrome (ALI/ARDS) pathophysiology and established early key events were specifically studied: alveolar epithelial membrane phenotype, lung cell apoptosis, neutrophil recruitment, and edema. ECFM allowed visualization of (i) fine-tuned ultrastructural lectin (RCA-1) and sialoglycoprotein (RTI40) epithelial cell membrane expression, (ii) YO-PRO-1-related DNA linking of lung cell apoptosis, (iii) PKH2 green fluorescent cell linker-labeled neutrophil tracking in lung microcirculatory network and airspaces, (iv) FITC-dextran plasma contrast and extravasation with edema formation. ECFM provides reliable results to corresponding ex vivo fluorescent methods. ECFM, using the minimally invasive Five-1s optical instrument and specific fluorescent markers, is able to provide real-time potentially useful imaging of live unfixed normal and injured lung tissue with promising developments for improving bedside diagnostic and decision-making therapeutic strategy in patients with ALI.
Journal of Surgical Research, 1979
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