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. 2018 Apr 27;15(7):696-702.
doi: 10.7150/ijms.24257. eCollection 2018.

Low-Dose Evans Blue Dye for Near-Infrared Fluorescence Imaging in Photothrombotic Stroke Model

Hye-Won Ryu  1 , Wonbong Lim  2 , Danbi Jo  3 , Subin Kim  3 , Jong Tae Park  1 , Jung-Joon Min  4 , Hoon Hyun  3   5 , Hyung-Seok Kim  1   5
Affiliations

Low-Dose Evans Blue Dye for Near-Infrared Fluorescence Imaging in Photothrombotic Stroke Model

Hye-Won Ryu et al. Int J Med Sci. .

Abstract

Background: Evans blue dye (EBD) is the most common indicator to analyze the extent of blood-brain barrier (BBB) breakdown in several neurological disease models. However, the high-dose of EBD (51.9 mg/kg) is usually required for visualization of blue color by the human eye that brings potential safety issues. Methods: To solve this problem, low-dose of EBD was applied for the near-infrared (NIR) fluorescence-assisted quantitation of BBB breakdown in photothrombotic stoke model. Animals were allocated to seven dose groups ranging from 1.35 nmol (5.19 μg/kg) to 13.5 μmol (51.9 mg/kg) EBD. Results: EBD was undetectable in the non-ischemic brain tissue, and the fluorescence signals in the infarcted hemisphere seemed proportional to the injected dose in the dose range. Although the maximum fluorescence signals in brain tissue were obtained with the injections of 1.35 nmol ~ 13.5 μmol EBD, the background signals in the neighboring brain tissues were significantly increased as well. Since the high concentration of EBD is necessary for color-based identification of the infarcted lesion in brain tissues, even 10-fold diluted could not be distinguished visually by naked eye. Conclusions: NIR fluorescence-assisted method could potentially provide new opportunities to study BBB leakage just using small amount of EBD in different pathological conditions and to test the efficacy of various therapeutic strategies to protect the BBB.

Keywords: Evans blue dye; blood-brain barrier; near-infrared fluorescence; photothrombotic stroke model; signal-to-background ratio.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
(A) Chemical structure and optical properties of EBD . (B) In vivo NIR imaging of brain tissue (left) and biodistribution (right) using the conventional concentration of 4% EBD in rats. 13.5 μmol of EBD was injected intraperitoneally into 250 g SD-rats 6 h prior to imaging and resection. Abbreviations used are: Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen. Scale bars = 1 cm. Images are representative of N = 6 independent experiments. All NIR fluorescence images have identical exposure and normalizations.
Figure 2
Figure 2
In vivo dose-dependent imaging of infarcted brain tissue using by diluted EBD in rats. Each concentration of EBD was injected intraperitoneally into 250 g SD-rats in the range of 1.35 nmol to 1.35 μmol 6 h prior to imaging. Scale bars = 1 cm. Images are representative of N = 6 independent experiments. NIR fluorescence images for each condition have identical exposure times and normalizations.
Figure 3
Figure 3
Dose-response plotting of SBR (mean ± S.D.) for infarcted brain tissue. SBR was calculated by the fluorescence intensity of infarcted brain tissue versus the signal intensity of neighboring brain tissue obtained at 6 h post-injection.
Figure 4
Figure 4
In vivo dose-dependent biodistribution of the EBD in rats. Each concentration of EBD was injected intraperitoneally into 250 g SD-rats in the range of 1.35 nmol to 1.35 μmol 6 h prior to imaging and resection. Abbreviations used are: Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa, pancreas; Sp, spleen. Scale bars = 1 cm. Images are representative of N = 6 independent experiments. All NIR fluorescence images have identical exposure and normalizations.
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