FIGURE SUMMARY
Title

In vivo assessment of the permeability of the blood--brain barrier and blood-retinal barrier to fluorescent indoline derivatives in zebrafish

Authors
Watanabe, K., Nishimura, Y., Nomoto, T., Umemoto, N., Zhang, Z., Zhang, B., Kuroyanagi, J., Shimada, Y., Shintou, T., Okano, M., Miyazaki, T., Imamura, T., and Tanaka, T.
Source
Full text @ BMC Neurosci.

In vivo assessment of the permeability of the BBB to fluorescent compounds. Zebrafish larvae (casper line) at 7–8 dpf were immersed in egg water containing 1 μM of an ID or fluorescein for 1 h. In vivo fluorescence imaging of the zebrafish brain was performed using fluorescence microscopes. A: Schematic diagram showing the region observed using the fluorescence microscopes. B-H: In vivo fluorescence imaging of zebrafish larvae stained with ID possessing a rhodanine ring with an acetic acid group (ZMB996, ZMC213, and ZMJ018, B, C and D, respectively), with fluorescein (E), and with ID possessing a rhodanine ring with a propanoic acid group (ZMC808, ZMB740, and ZMB034, F, G and H, respectively). The OT was clearly visualized in zebrafish stained with IDs possessing a rhodanine moiety with a propanoic acid group. Scale bar: 100 μm. OT, optic tectum; CBV, cerebral blood vessel.

In vivo assessment of the permeability of the BRB to fluorescent IDs. Zebrafish larvae (albino line) at 7–8 dpf were immersed in egg water containing 1 μM of ID. In vivo fluorescence imaging of the zebrafish retina was performed using a CLSM. Schematic diagram (A and E) showing the region of the eye containing the HBV (B-D, I-K) and multiple layers of retina (F-H, L-N) observed using the CLSM. B-D and F-H: In vivo fluorescence imaging of zebrafish larvae stained with IDs possessing a rhodanine ring with an acetic acid group (ZMB996, ZMC213, and ZMJ018, B and F, C and G, D and H, respectively). The HBV in zebrafish stained with ZMJ018 were clearly visualized. I-N: In vivo fluorescence imaging of zebrafish larvae stained with IDs possessing a rhodanine ring with an propanoic acid group (ZMC808, ZMB740, and ZMB034, I and L, J and M, K and N, respectively). Both the HBV and multiple layers of retina were clearly visualized in zebrafish stained with ZMB034. Bar: 50 μm. HBV, hyaloid blood vessel; IPL, inner plexiform layer; OPL, outer plexiform layer; PCL, photoreceptor cell layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

In vivo assessment of the permeability of the BBB permeability to ZMB034 and ZMJ018 administered by intracardiac injection. ZMB034 or ZMJ018 was injected into the cardiac chamber of zebrafish larvae (MK001 line) at 7–8 dpf. In vivo imaging of zebrafish brain was performed using a CLSM. A-D: In vivo fluorescence imaging of zebrafish larvae injected with ZMB034 (A and C) or ZMJ018 (B and D) at 10 min (A and B) and 30 min (C and D) post-injection. While the CBV were clearly visualized in zebrafish injected with ZMB034 or ZMJ018, the OT was visualized only in zebrafish treated with ZMB034. E: Quantitative analysis of FI of the OT relative to the FI of CBV. The relative FI at 10 and 30 min after intracardiac injection was significantly higher in zebrafish injected with ZMB034 than in those injected with ZMJ018 (n = 4, *P < 0.05). Scale bar: 50 μm. FI, fluorescence intensity.

Effect of MK571 on the permeability of the BBB and BRB to ZMJ018. Zebrafish larvae (albino line) at 7-8 dpf were immersed in egg water containing 1 μM of ZMJ018 with and without 30 μM of MK571 for 4 h. A-C: Schematic diagram showing the regions observed using a CLSM. D-I: In vivo fluorescence imaging of the OT (D and G), HBV (E and H) and multiple layers of the retina (F and I) in zebrafish stained with ZMJ018 only (D-F) or ZMJ018 in the presence of MK571 (G-I). The OT and multiple layers of the retina were clearly visible in zebrafish stained with ZMJ018 in the presence of MK571. J-L: Quantitative analysis of the FI in the OT (J) and HBV (K), and the ratio of the FI (L). Both the FI in the OT and the relative FI (OT/HBV) were significantly higher in zebrafish stained with ZMJ018 in the presence of MK571 (n = 4, *P < 0.05). Scale bar: 50 μm. OT, optic tectum; CBV, cerebral blood vessel; HBV, hyaloid blood vessel; FI, fluorescence intensity.

Acknowledgments
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