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Hypothermic Circulatory Arrest Increases Permeability of the Blood Brain Barrier in Watershed Areas Toru Okamura, MD, Nobuyuki Ishibashi, MD, T. Susheel Kumar, MD, David Zurakowski, PhD, Yusuke Iwata, MD, Hart G.W. Lidov, MD, Richard A. Jonas, MD The Annals of Thoracic Surgery Volume 90, Issue 6, Pages 2001-2008 (December 2010) DOI: 10.1016/j.athoracsur.2010.06.118 Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Whole brain photographs of sagittal view for (A) 25°C hypothermic circulatory arrest (HCA) group, (B) 15°C HCA group, and (C) control group. Blood brain barrier (BBB) disruption around watershed area at 25°C HCA is clearly apparent. The leakage of Evans blue dye (blue area on 25°C HCA) as a disruption tracer of BBB was not observed under other conditions: 15°C HCA, 25°C low flow, 37°C full flow, and control. The Annals of Thoracic Surgery 2010 90, 2001-2008DOI: (10.1016/j.athoracsur.2010.06.118) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Ratio of absorbance of Evans blue dye in cortical and cerebellar tissue versus plasma Evans blue dye level 3 hours after end of bypass. The cortex/plasma ratio was significantly higher for 25°C hypothermic circulatory arrest (HCA) than 37°C full flow (FF [p = 0.03]) and 37°C OFF ([p = 0.005]). Cerebellar tissue/plasma ratio was significantly higher for 25°C HCA than 37°C OFF (p = 0.005). (LF = low flow.) The Annals of Thoracic Surgery 2010 90, 2001-2008DOI: (10.1016/j.athoracsur.2010.06.118) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Immunohistochemistry in brain tissue. (A) Extravasation of Evans blue dye (red) was observed. The lectin (green) on endothelial cells was stained as a marker of vascularity. The nucleus (blue) was stained using 4',6'-diamidino-2-phenylindole hydrochloride (DAPI). (B) Leakage of blood brain barrier was not found. The Annals of Thoracic Surgery 2010 90, 2001-2008DOI: (10.1016/j.athoracsur.2010.06.118) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Blood brain barrier leakage for cortex, caudate nucleus, thalamus, hippocampus, and cerebellum using Evans blue dye at 3 hours after cardiopulmonary bypass. Pixel intensity in 25°C hypothermic circulatory arrest (HCA) group was significantly higher than control group for each brain area (*p < 0.05 versus control; one-way analysis of variance). (FF = full flow; LF = low flow; OFF = control.) The Annals of Thoracic Surgery 2010 90, 2001-2008DOI: (10.1016/j.athoracsur.2010.06.118) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 (A) Immunohistochemistry of cortex to demonstrate colocalization of Evans blue dye and albumin. (B) Evans blue dye shows red. (C) Albumin is green with fluorescein-conjugated polyclonal rabbit anti-albumin. (D) Nuclei are blue using 4',6-diamidino-2-phenylindole. The Annals of Thoracic Surgery 2010 90, 2001-2008DOI: (10.1016/j.athoracsur.2010.06.118) Copyright © 2010 The Society of Thoracic Surgeons Terms and Conditions