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Carlos Otavio Corso, MD, PhD Dept. of Morphological Sciences

TOPICAL HYPOTHERMIA PLUS ISCHEMIC PRECONDITIONING: LIVER INJURIES, HEPATIC FUNCTION AND OXIDANT DEFENSES AT INITIAL REPERFUSION IN RATS. Carlos Otavio Corso, MD, PhD Dept. of Morphological Sciences Post-graduation Course of Surgical Sciences Universidade Federal do Rio Grande do Sul.

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Carlos Otavio Corso, MD, PhD Dept. of Morphological Sciences

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  1. TOPICAL HYPOTHERMIA PLUS ISCHEMIC PRECONDITIONING: LIVER INJURIES, HEPATIC FUNCTION AND OXIDANT DEFENSES AT INITIAL REPERFUSION IN RATS Carlos Otavio Corso, MD, PhD Dept. of Morphological Sciences Post-graduation Course of Surgical Sciences Universidade Federal do Rio Grande do Sul

  2. HEPATIC ISCHEMIA Oxygen reduction Anaerobic metabolism Mithocondrial dysfunction ATP reduction Na+/K+ pump dysfunction Cellular edema NO and ET-1 imbalance Major Liver Ressection Shock Hepatic Trauma Liver Transplantation

  3. INITIAL REPERFUSION 2 - 4 hs Vasoconstriction (ET-1 x ON) Microcirculation impairment Endothelial edema Platelet aggregation Reduced perfusion NO REFLOW Release RLO, TNF-α IL-1, PAF

  4. LATE REPERFUSION 6 - 24 hs

  5. REPERFUSION Wiesner RH. Conference in Annual International Liver Transplantation Society Congress, April 5-6, 2008. www.ilts.org

  6. STRATEGIES TO REDUCE I/R DAMAGE Antioxidants (N-acetilcistein, Alfa-tocoferol, Allopurinol) Anti TNF-α, Anti ICAM-1, Anti P-selectin Adenosine, L-arginine, Cerulein Genetic therapy (HO-1, IL-13, NFκB) Intermitent Occlusion Ischemic Preconditioning (IPC) Hypothermia

  7. HYPOTHERMIA Mechanisms of Action Metabolism reduction RLO release reduction Less neutrophilic infiltration Reduced inflammation Body temperature reduction or temperature reduction of an organ submitted to ischemia

  8. ISCHEMIC PRECONDITIONING Mechanisms of action ATP consum reduction Cellular metabolism reduction RLO release reduction Mitochondrial integrity preservation Reduce citokines release Leukocyte activity attenuation Apoptosis reduction From Pasupathy et al,2001 Short periods of ischemia and reperfusion protect the organ during long periods of ischemia Murray et al, 1986

  9. Molecular Bases of PCI Cutrín JC et al. Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning. Free Rad Biol Med, 2002, 33: 1200–8.

  10. AIM OF THE STUDY • Combination of Hypothermia and PCI

  11. Experimental Model Animals and Anesthesia Male Wistar Rats Weight: 200 - 250 g Ketamine 75mg/Kg /Xilazine10mg/Kg i.p. SurgicalProcedure Carotidarterycatheter – MAP Rectaltemperature Laparotomy – Liver exposure Bile duct cannulation Liver temperature Iced saline superfusion Right and median lobes ischemia (90min) Whole liver reperfusion (120min)

  12. Experimental protocol 1. 10min Ischemia and 10min Reperfusion before Ischemia PCI e PCI+H (Peralta et al, 2000) 2. Reduction of liver temperature (26º C) by iced saline superfusion H e PCI+H (Biberthaler et al, 2001, Khandoga et al, 2003)

  13. Experimental Protocol C (n=4) 210 min IN (n=7) 90 min 120 min PCI (n=7) 10 min 10 min 90 min 120 min H + PCI (n=7) 10 min 10 min 90 min 120 min H (n=7) 90 min 120 min Reperfusion Ischemia

  14. Topical Hypothermia

  15. Measurements Body temperature Mean Arterial Pressure (MAP) Bile flow Plasmatic AST ALT LDH Hepatic Markers TBARS Catalase (CAT) Superoxid Dismutase (SOD) Histhopatologic Score (Degeneração Hidrópica, Necrose, Infiltração neutrofílica)

  16. MAP One-way ANOVA, p> 0,05

  17. Body Temperature One-way ANOVA, p> 0,05

  18. Transaminases AST and ALT at the end of reperfusion. *p<0,05 vs. C. Data are Means ± SEM. One-way ANOVA, Tukey for multiple comparisons.

  19. LDH LDH at the end of reperfusion. *p<0,05 vs. C. Data are Means ± SEM. One-way ANOVA, Tukey for multiple comparisons.

  20. Bile Flow * p<0,05 vs. IN and PCI. Data are Means ± SEM. One-way ANOVA, Tukey for multiple comparisons.

  21. Bile Flow * p<0,05 vs. IN and PCI. Data are Means ± SEM. One-way ANOVA, Tukey for multiple comparisons.

  22. Bile Flow * p<0,05 vs. IN and PCI. Data are Means ± SEM. One-way ANOVA, Tukey for multiple comparisons.

  23. TBARS TBARS at the end of reperfusion*p<0,05 vs. H+PCI, #P<0,01 vs. H+PCI and H. Data are Means ± SEM. One-way ANOVA, Kruskall-Wallis, Dunn for multiple comparisons

  24. SOD SOD at the end of reperfusion. #p<0,01 vs. H, *P<0,05 vs. H. Data are Means ± SEM. One-way ANOVA, Student-Neuman-Keuls for multiple comparisons

  25. CATALASE Catalase at the end of reperfusion.*p<0,05 vs. PCI, #P<0,01 vs. PCI. Data are Means ± SEM. One-way ANOVA, Student-Neuman-Keuls for multiple comparisons

  26. HISTHOPATOLOGIC SCORE HE stained sections . p<0,05 vs. C. Data are Means ± SEM. One-way ANOVA, Tukey for multiple comparisons.

  27. CONCLUSIONS • Model stable and reliable • Synergism on membrane lipidic peroxidation with combined Hypothermia and PCI • Induction of 26ºC Hypothermia, associated or not to PCI, leads to attenuation of hepatocellular damage and early recovery of hepatic function • Mechanisms are very likely related to reduced oxidative stress and ATP preservation

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