Investigation of Receptor-Mediated Cyanocobalamin (Vitamin B ₁₂ ) Transport across the Inner Blood-Retinal Barrier Using Fluorescence-Labeled Cyanocobalamin

The blood-to-retina supply of cyanocobalamin (vitamin B ₁₂ ) across the blood-retinal barrier (BRB) was investigated by synthesizing a fluorescence-labeled cyanocobalamin (Cy5-cyanocobalamin). In the in vivo analysis following internal jugular injection of Cy5-cyanocobalamin, confocal microscopy showed the distribution of Cy5-cyanocobalamin in the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the retinal pigment epithelium (RPE). In the in vitro analysis with TR-iBRB2 cells, an in vitro model cell line of the inner BRB, Cy5-cyanocobalamin uptake by TR-iBRB2 cells exhibited a time-dependent increase after preincubation with transcobalamin II (TCII) protein, during its residual uptake without preincubation with TCII protein. The Cy5-cyanocobalamin uptake by TR-iBRB2 cells was significantly reduced in the presence of unlabeled cyanocobalamin, chlorpromazine, and chloroquine and was also significantly reduced under Ca ²⁺ -free conditions. Confocal microscopy of the TR-iBRB2 cells showed fluorescence signals of Cy5-cyanocobalamin and GFP-TCII protein, and these signals merged with each other. The RT-PCR, Western blot, and immunohistochemistry clearly suggested the expression of TCII receptor (TCII-R) in the inner and outer BRB. These results suggested the involvement of receptor-mediated endocytosis in the blood-to-retina transport of cyanocobalamin at the inner BRB with implying its possible involvement at the outer BRB.