Keiji Ohata, MD, Toyofumi F

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Protective Effect of Inhaled Rho-Kinase Inhibitor on Lung Ischemia-Reperfusion Injury Keiji Ohata, MD, Toyofumi F. Chen-Yoshikawa, MD, PhD, Toshi Menju, MD, PhD, Ei Miyamoto, MD, Satona Tanaka, MD, Mamoru Takahashi, MD, Hideki Motoyama, MD, PhD, Kyoko Hijiya, MD, Akihiro Aoyama, MD, PhD, Hiroshi Date, MD, PhD The Annals of Thoracic Surgery Volume 103, Issue 2, Pages 476-483 (February 2017) DOI: 10.1016/j.athoracsur.2016.07.067 Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 A diagram of (A) the isolated rat lung perfusion model and (B) the experimental protocol. Cannulas were inserted into the pulmonary artery and left atrium and were connected to the perfusion circuit driven by 2 roller pumps. The lung was placed in an artificial thorax and ventilated. (RKI = Rho-kinase inhibitor group; WI = warm ischemia group.) The Annals of Thoracic Surgery 2017 103, 476-483DOI: (10.1016/j.athoracsur.2016.07.067) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Diagram shows the molecular mechanism of Rho-kinase in cell contraction. Rho-kinase inactivates myosin phosphatase by phosphorylation and increases the phosphorylation ratio of myosin light chain (MLC), which results in cell contraction. The Annals of Thoracic Surgery 2017 103, 476-483DOI: (10.1016/j.athoracsur.2016.07.067) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Physiologic data after reperfusion is shown for (a) pulmonary vascular resistance, (b) dynamic compliance, (c) partial pressure of arterial oxygen, and (d) wet/dry lung weight ratio in the sham, Rho-kinase inhibitor (RKI), and warm ischemia (WI) groups. The error bars show the standard error of the mean. ***p < 0.001 for differences between the two groups indicated. (BL = baseline.) The Annals of Thoracic Surgery 2017 103, 476-483DOI: (10.1016/j.athoracsur.2016.07.067) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 (A) Macroscopic and (B) histologic (hematoxylin and eosin staining, original magnification ×200) findings in rat lungs after reperfusion. Macroscopic findings showed the lungs of the warm ischemia (WI) group were reddish and edematous. This was not present in the sham or Rho-kinase inhibitor (RKI) groups. Microscopically, mild edema in the perivascular space was observed in the sham and RKI groups (arrow). In contrast, severe perivascular edema with sporadic hemorrhage was observed in the WI group (arrowhead). The Annals of Thoracic Surgery 2017 103, 476-483DOI: (10.1016/j.athoracsur.2016.07.067) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Representative protein bands for phosphorylated (p)-myosin phosphatase, total myosin phosphatase, and β-actin as the protein loading control and the mean values of p-myosin phosphatase expression, total myosin phosphatase expression, and the phosphorylation ratio of myosin phosphatase, which reflects Rho-kinase activity. Rho-kinase activity and p-myosin phosphatase expression were greater in the warm ischemia (WI) group than in the sham and Rho-kinase inhibitor (RKI) groups. The error bars show the standard error of the mean. **p < 0.01, and ***p < 0.001 for differences between two groups. The Annals of Thoracic Surgery 2017 103, 476-483DOI: (10.1016/j.athoracsur.2016.07.067) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Representative protein bands for phosphorylated (p) endothelial nitric oxide synthase (eNOS), total eNOS, and β-actin and the mean values of p-eNOS expression, total eNOS expression, and the phosphorylation ratio of eNOS. Expression of p-eNOS in the warm ischemia (WI) group was significantly lower than in the sham and Rho-kinase inhibitor (RKI) groups. The error bars show the standard error of the mean. *p < 0.05, **p < 0.01 between groups. The Annals of Thoracic Surgery 2017 103, 476-483DOI: (10.1016/j.athoracsur.2016.07.067) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions