Changes in P-Selectin Expression on Cardiac Microvessels in Blood-Perfused Rat Hearts Subjected to Ischemia-Reperfusion Andrew O. Chukwuemeka, FRCS,

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Changes in P-Selectin Expression on Cardiac Microvessels in Blood-Perfused Rat Hearts Subjected to Ischemia-Reperfusion Andrew O. Chukwuemeka, FRCS, K. Alun Brown, PhD, Graham E. Venn, MS, David J. Chambers, PhD The Annals of Thoracic Surgery Volume 79, Issue 1, Pages 204-211 (January 2005) DOI: 10.1016/j.athoracsur.2004.06.105 Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 The recirculating Langendorff blood perfusion circuit. A plastic reservoir was inserted into a temperature-controlled heart chamber; tubing from the chamber passed through a peristaltic pump and into a home-made oxygenator consisting of coils of thin-walled, gas-permeable silicon tubing. The oxygenator was encased in a temperature-controlled glass chamber through which 95% O2:5% CO2 was passed countercurrent to the flow of the perfusate. It was connected to a temperature-controlled aortic cannula which returned the perfusate to the reservoir (for further details, see Methods). During ischemia, a three-way tap before the aortic cannula diverted the perfusate directly into the reservoir and maintains continuous circulation of the perfusate. The heart was sealed within a separate chamber and the temperature maintained at 37°C. The Annals of Thoracic Surgery 2005 79, 204-211DOI: (10.1016/j.athoracsur.2004.06.105) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Expression of P-selectin on cardiac microvessels. Sections of myocardial tissue were treated with a primary antibody against P-selectin and the staining developed by the avidin-antiavidin peroxidase complex. Panels A and B show blood vessels in normal heart tissue do not express P-selectin; panels C and D demonstrate the marked expression of P-selectin (indicated by arrows) on the luminal walls of blood vessels in hearts subjected to 15 minutes of reperfusion after ischemia. (Panels A and C: magnification ×100; panels B and D: magnification ×400.) The Annals of Thoracic Surgery 2005 79, 204-211DOI: (10.1016/j.athoracsur.2004.06.105) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Upregulation of P-selectin on blood vessel walls during perfusion, ischemia, and reperfusion. After 30 minutes of control perfusion with modified Krebs-Henseleit buffer solution with Gelofusine (MKH/G) plus blood at a flow rate of 4.5 mL/min, isolated rat hearts were subjected to 60 minutes of ischemia (shaded area) and 60 minutes of reperfusion. Hearts were sampled throughout the experimental duration and processed for immunohistochemical staining. Results are expressed as the percentage of vessels expressing P-selectin. Values are mean ± SEM of at least 18 measurements per time point (3 hearts, each cut into 3 segments and at least 2 sections per segment). *p less than 0.05 compared with 30-minute value. The Annals of Thoracic Surgery 2005 79, 204-211DOI: (10.1016/j.athoracsur.2004.06.105) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Upregulation of P-selectin on blood vessel walls during continuous perfusion. Isolated rat hearts were perfused with either modified Krebs-Henseleit buffer solution with Gelofusine (MKH/G) plus blood (solid line) or MKH/G plus blood depleted of leukocytes and platelets (dashed line) at a flow rate of 4.5 mL/min for 120 minutes. Hearts were sampled throughout the experimental duration and processed for immunohistochemical staining. Results are expressed as the percentage of vessels expressing P-selectin. Values are mean ± SEM of at least 18 measurements per time point (3 hearts, each cut into 3 segments and at least 2 sections per segment). *p less than 0.05 compared with 0-minute value. The Annals of Thoracic Surgery 2005 79, 204-211DOI: (10.1016/j.athoracsur.2004.06.105) Copyright © 2005 The Society of Thoracic Surgeons Terms and Conditions