Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3′,5′-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.
|Number of pages||9|
|Journal||Journal of Controlled Release|
|State||Published - Mar 2022|
Bibliographical noteFunding Information:
This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI [ JP19K07160 (P.I., Shin-ichi Akanuma)], by The Research Foundation for Pharmaceutical Sciences (P.I., Shin-ichi Akanuma), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (P.I., Shin-ichi Akanuma), and the JSPS KAKENHI [Grant Numbers JP20H03403 (P.I., Ken-ichi Hosoya)].
This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI [JP19K07160 (P.I., Shin-ichi Akanuma)], by The Research Foundation for Pharmaceutical Sciences (P.I., Shin-ichi Akanuma), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (P.I., Shin-ichi Akanuma), and the JSPS KAKENHI [Grant Numbers JP20H03403 (P.I., Ken-ichi Hosoya)].The authors thank Dr. Masahiko Watanabe (Hokkaido University) for kindly providing GLUT1 antibody and Mr. Ryuta Jomura (University of Toyama) for help with retinal capillary isolations and X.L. oocyte uptake studies.
© 2022 Elsevier B.V.
- Breast cancer resistance protein
- Inner blood-retinal barrier
- Multidrug resistance-associated protein
- Retinal capillaries
ASJC Scopus subject areas
- Pharmaceutical Science