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Cooling Treatment Transiently Increases the Permeability of Brain Capillary Endothelial Cells Through Translocation of Claudin-5

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Abstract

The blood–brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5 °C for 1 h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15 °C) and recovered to normal levels after rewarming to 37 °C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.

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Acknowledgments

We thank Prof Takeshi Yamakawa for his critical comments and Miss Ayami Osaki for her excellent technical assistance. This work was supported by a Grant-in-Aid for Specially Promoted Research (No. 20001008 to T.Y. and M.S.), a Grant-in-Aid for Exploratory Research (No. 22659261 to M.S.), and a Grant-in-Aid for Scientific Research (C) (No. 21590215 to Y.O.) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), and by the Yamaguchi University Research Project on STRESS.

Conflict of interest

The authors report no conflict of interest concerning the materials and methods used in this study or the findings specified in this paper.

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Authors and Affiliations

  1. Department of Neurosurgery, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan

    Akinori Inamura, Yasuhiro Adachi, Takao Inoue, Yeting He, Satoshi Shirao, Sadahiro Nomura, Masami Fujii & Michiyasu Suzuki

  2. Department of Organ Anatomy, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan

    Yasuhiro Adachi, Nobuko Tokuda, Takashi Nawata & Yuji Owada

  3. Department of Pathology, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan

    Eiji Ikeda

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  1. Akinori Inamura
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Correspondence to Yuji Owada.

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11064_2013_1066_MOESM1_ESM.pdf

Restored claudin-5 expression on membrane protein fraction after rewarming. Western blot analysis of membrane protein fraction from bEnd3 cells revealed that a claudin-5 expression was decreased after the cells were cooled at 15 °C for 1 h (1 h) and restored to its original level 1 h after rewarming to 37 °C (+1 h). The expression level of membranous claudin-5 remained for the next 24 h (+24 h). Na–K-ATPase was used as the internal control for membranous protein fraction. Three independent experiments were performed and representative data were shown (PDF 34 kb)

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Inamura, A., Adachi, Y., Inoue, T. et al. Cooling Treatment Transiently Increases the Permeability of Brain Capillary Endothelial Cells Through Translocation of Claudin-5. Neurochem Res 38, 1641–1647 (2013). https://doi.org/10.1007/s11064-013-1066-4

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