Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

Abstract

Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741.

Zebrafish as a systems toxicology model for developmental neurotoxicity testing

“…; Nishimura et al. ). Tight junctions between endothelial cells and efflux transporters located at the apical side of endothelial cells mainly contribute to the distribution of chemicals to the brain through the BBB (Loscher and Potschka ; Ek et al.…”

“…; Nishimura et al. ) and efflux transporters are functional by 8 dpf (Watanabe et al. ; Fleming et al.…”

“…Therefore, an animal model possessing a BBB that is similar to those found in mammals is advantageous for DNT and may be relevant for developmental neurotoxicity in humans. It has been reported that zebrafish have a BBB that is similar to those in mammals (Jeong et al 2008;Xie et al 2010;Watanabe et al 2012;Fleming et al 2013;Nishimura et al 2013). Tight junctions between endothelial cells and efflux transporters located at the apical side of endothelial cells mainly contribute to the distribution of chemicals to the brain through the BBB (Loscher and Potschka 2005;Ek et al 2012).…”

Complement and blood–brain barrier integrity

“…In addition, a quick and easy method of assessing the BBB integrity in vitro is by determining the electrical resistance across it. Further, with advancing technology, different modalities of MRI along with contrast agents including the most recent indoline compound ID, ZMB741 (Nishimura et al, 2013) are very valuable in studying alterations of the BBB. More recently, fluorescently labeled tracers are being explored as possible future models to accelerate research advances in this area.…”

Long‐Wavelength Fluorescent Reporters for Monitoring Protein Kinase Activity

“…Fortunately, a wide array of fluorophores are available that are visualized in the red and near IR [2] and several of these have been covalently attached to antibodies for diagnostic purposes. [3–4] However, with the prominent exception of proteases, [5] there are few long wavelength probes for enzymes, which is especially problematic with respect to intracellular erythrocyte biochemistry. [6] Unlike other mammalian cells, the high hemoglobin content of erythrocytes optically obscures subcellular monitoring at wavelengths less than 600 nm.…”