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Optimization of Evans Blue Quantitation in Limited Rat Tissue Samples

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Optimization of Evans Blue Quantitation in Limited Rat Tissue Samples

Hwai-Lee Wang et al. Sci Rep.

Abstract

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Figures

Figure 1
Figure 1. Evans blue dye (EBD) fluorescence (620 nm excitation/680 nm emission) detection.
Standard curve showing EBD fluorescence as a function of dye concentration in clear (a–c) and black (c) 96-well plates. (a) Full EBD standard curve based on a 1:3 ethanol dilution. Each 30 µl replicate of EBD (in 50% TCA/0.9% saline) was supplemented with 90 µl of 95% ethanol to ensure consistent optic path length. Concentrations of the 30 µl replicates are indicated on the x-axis. (b) EBD standard curves for the 120 µl undiluted dye (in 50% TCA/0.9% saline) and 30 µl dye (in 50% TCA/0.9% saline) diluted to 120 µl with one solvent: 95% ethanol, 50% TCA, 0.9% saline, or water. Concentrations of the 120 µl undiluted dye and the 30 µl dye (prior to dilution) are indicated on the x-axis. (c) The maximum and minimum detectable EBD concentrations in the clear and black 96-well plates were determined by Tukey's multiple comparisons tests. A significant difference (***P<0.001) indicates a detectable concentration difference, and no significance (ns; P>0.05) indicates that the concentration difference was not detectable.
Figure 2
Figure 2. False-positive fluorescence readings (620 nm excitation/680 nm emission) from biological solutes.
(a–c) Auto-fluorescence of biological solutes from the brain (a), blood (b), and plasma (c) samples (diluted to 500 µl in saline), with or without protein-precipitation by 1:1–1:3 50% trichloroacetic acid (TCA). [TCA final] following the addition of 1:1, 1:2, and 1:3 volume ratios of 50% TCA were 25, 33, and 37.5%, respectively.
Figure 3
Figure 3. Evans blue dye (EBD) optical density (absorbance of 620 nm) detection.
Standard curve showing EBD absorbance as a function of dye concentration. (a) Full EBD standard curve based on a 1:3 ethanol dilution. Each 30 µl replicate of EBD (in 50% TCA/0.9% saline) was supplemented with 90 µl of 95% ethanol to ensure consistent optic path length. Concentrations of the 30 µl replicates are indicated on the x-axis. (b) EBD standard curves for the 120 µl undiluted dye (in 50% TCA/0.9% saline) and 30 µl dye (in 50% TCA/0.9% saline) diluted to 120 µl with one solvent: 95% ethanol, 50% TCA, 0.9% saline, or water. Concentrations of the 120 µl undiluted dye and the 30 µl dye (prior to dilution) are indicated on the x-axis. (c) The maximum and minimum detectable EBD concentrations in the clear 96-well plate were determined by Tukey's multiple comparisons tests. A significant difference (***P<0.001 and **P<0.01) indicates a detectable concentration difference, and no significance (ns; P>0.05) indicates that the concentration difference was not detectable.
Figure 4
Figure 4. False-positive optical density readings (absorbance of 620 nm) from biological solutes.
(a–c) Absorbance of biological solutes from the brain (a), blood (b), and plasma (c) samples (diluted to 500 µl in saline), with or without protein-precipitation by 1:1–1:3 of 50% trichloroacetic acid (TCA). [TCA final] following the addition of 1:1, 1:2, and 1:3 volume ratios of 50% TCA were 25, 33, and 37.5%, respectively.
Figure 5
Figure 5. Extraction of Evans blue dye (EBD) from the protein-EBD complex in vitro.
(a) Illustration showing the experimental protocol for (b–e). EBD formed a complex with plasma proteins. Thereafter, the dye was extracted from the protein with 50% trichloroacetic acid (TCA), and its concentration was detected by spectroscopy in a 96-well plate. (b–e) The percentage yields of EBD that were extracted from the protein-EBD complex by different volume-ratios of 50% TCA (in 0.9% saline), which resulted in different [TCA final], were compared. The extracted EBD was measured by spectroscopy and normalized to the total EBD added to the plasma samples to generate the protein-EBD complex solutions. The total EBD concentrations were as follows: 0.5 µg/ml (b), 5 µg/ml (c), 50 µg/ml (d), and 500 µg/ml (e).
Figure 6
Figure 6. Extraction of Evans blue dye (EBD) from the protein-EBD complex and peripheral/central organs in vivo.
(a) Illustration showing the experimental protocol for (b–d). Each rat was injected with EBD; EBD was extracted from the central/peripheral organs 2 h later, and its concentration was measured by spectroscopy in a 96-well plate. (b–d) EBD was extracted by different volume-ratios of 50% TCA (in 0.9% saline) from the brain parenchyma (b), liver parenchyma (c), and plasma (d) in rats injected with the dye 2 h prior to sample collection.

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References

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