Infrared fluorescence for vascular barrier breach in vivo – A novel method for quantitation of albumin efflux

Abstract

Summary 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 endotoxemia 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 i.v. 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 analyzed by IRF-based detection but not by absorbance-based detection. EB was detected in all 353 organs analyzed with IRF but only in 67% (239/353) of organs analyzed 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.

Organ-Specific Protection Against Lipopolysaccharide-Induced Vascular Leak Is Dependent on the Endothelial Protein C Receptor

“…Recently we found that in C57Bl/6J (control) mice areas of LPS-induced EB dye extravasation in the liver corresponded to focal areas of liver necrosis. 27 Histological analysis of livers of EPCR low mice injected with LPS (medium dose) showed similar findings of focal areas of liver necrosis corresponding to areas of EB dye extravasation (Supplemental Figure 3). Moreover, at baseline (saline injection) livers of EPCR low and control mice appeared similar (data not shown).…”

“…The role of EPCR for vascular leakage in vivo was determined using our recently developed IRF technology for quantification of EB accumulation in organs. 27 Pair wise analysis of EPCR low and control mice that were matched for gender, age and weight allowed for the specific detection of EPCR-dependent differences in vascular barrier function in an LPS-induced mouse model of endotoxemia. Proof of plasma leak as the predominant cause of EB tissue accumulation was provided by demonstration of profound intravascular volume loss at the same extent of weight loss in EPCR low mice compared to wild-type mice, thus excluding dehydration for the volume contraction, or blood pooling for accumulation of EB dye in central organs.…”

“…27 Application of this methodology to study the contribution of EPCR to vascular leakage, tissue edema and organ injury provide unique insights into the role of EPCR in the pathophysiological induction of vascular leakage during endotoxemia. Low EPCR expression resulted in profound plasma tissue extravasation, lung injury, severe renal hemorrhage and albuminuria.…”

Mitochondrial DNA Damage Initiates Acute Lung Injury and Multi-Organ System Failure Evoked in Rats by Intra-Tracheal Pseudomonas Aeruginosa

“…The mixture was then centrifuged at 4000 × g for 30 minutes at room temperature. A 200 μL aliquot of supernatant was collected into a Costar™ clear polystyrene 96-well plate (Thermo Fisher Scientific Inc., MA, USA) and dual-wavelength spectrophotometry using a Spectramax® M5 microplate reader (Molecular Devices, LLC, CA, USA) at 620 and 740 nm used to estimate dye content; correction for heme content used the formula Abs corr = Abs 620 − (1.426 * Abs 740 ) + 0.03) as previously described (17). …”

Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation

“…32 Sixty minutes after IV infusion of Evans blue-albumin (25 mg/kg of body weight), a blood sample was drawn for measurement of plasma dye concentration, animals were perfused with 30 mL of warmed saline, and organs were dissected. Evans blue content in blood and organs was determined by imaging on an Odyssey infrared imager (LI-COR Biosciences, Lincoln NE) with Odyssey Application software, version 3.0.…”