International Journal of Medical Sciences

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23 February 2019

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Int J Med Sci 2018; 15(7):696-702. doi:10.7150/ijms.24257

Research Paper

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

Hye-Won Ryu1,*, Wonbong Lim4,*, Danbi Jo2, Subin Kim2, Jong Tae Park1, Jung-Joon Min3, Hoon Hyun2,5,✉, Hyung-Seok Kim1,5,✉

1. Department of Forensic Medicine,
2. Department of Biomedical Sciences and
3. Department of Nuclear Medicine, Chonnam National University Medical School, Gwangju 61469, South Korea
4. Department of Premedical Program, School of Medicine, Chosun University, Gwangju 61452, South Korea
5. Center for Creative Biomedical Scientists, Chonnam National University Medical School, Gwangju 61469, South Korea
*These authors contributed equally to this work.


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, photothrombotic stroke model, blood-brain barrier, near-infrared fluorescence, signal-to-background ratio

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license ( See for full terms and conditions.
How to cite this article:
Ryu HW, Lim W, Jo D, Kim S, Park JT, Min JJ, Hyun H, Kim HS. Low-Dose Evans Blue Dye for Near-Infrared Fluorescence Imaging in Photothrombotic Stroke Model. Int J Med Sci 2018; 15(7):696-702. doi:10.7150/ijms.24257. Available from