Isoflurane Prevents Acute Lung Injury Through ADP-Mediated Platelet Inhibition

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS signaling pathway. Conclusions: Inhalation of hydrogencan induce HO-1 and reduce hyperoxic lung injury. Our results suggest that hydrogen modulates Nrf2 and induces HO-1 and other antioxidant proteins. Administering hydrogen by providing gas for the patient to inhale is novel and may be feasible in clinical practice.

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hicle, and C75 treated groups are summarized in the table below. Conclusions: Stimulation of CPT1 activity by C75 administration enhanced ATP regeneration, prevented renal deterioration, lowered organ injury indexes, and inhibited the production of pro-inflammatory cytokines induced by renal I/R. Therefore, enhancing energy metabolism by C75 may provide a novel possible modality to treat patients with severe renal I/R injury.

TABLE 1 TABLE 2

Effects of Hydrogen on Hyperoxic Lung Injury.

Normoxia (N2) Normoxia (H2) Hyperoxia (N2) Hyperoxia (H2)

pO2 Level (mmHg)

Wet/ Dry Ratio

Pleural Effusion (ml)

415.8 6 36.0 442.8 6 28.7 200.2 6 27.8 318.9 6 25.6*

5.4 6 0.1 5.2 6 0.1 6.2 6 0.1 5.6 6 0.1*

0.0 6 0.0 0.0 6 0.0 4.1 6 0.6 3.5 6 0.5*

BAL Protein (mg/ml)

TNF-a mRNA (% GAPDH)

0.5 6 0.1 5.7 6 0.7 0.5 6 0.1 14.0 6 4.0 2.8 6 0.4 201.6 6 14.9 1.5 6 0.1* 82.6 6 11.0y

N¼5-10; Data are presented as mean 6SE ; y p < 0.01, * p < 0.05 when compared with Hyperoxia (N2). pO2 : oxygen partial pressure, BAL : bronchoalveolar lavage, TNF : tumor necrosis factor

Creatinine (mg/dL) BUN (mg/dL) AST (UI/L) LDH (U/L) Serum TNF-a (pg/ml) Renal TNF-a (mRNA) MPOa (U/g tissue) Renal Water Content (%)

Sham

I/R-Vehicle

I/R-C75

0.4 6 0.1 9.1 6 1.1 15.9 6 1.1 29.1 6 4.0 3.1 6 0.8 1.0 6 0.1 2.3 6 1.1 71.0 6 1.2

3.8 6 0.7* 102.3 6 10.4* 106.1 6 13.9* 381.9 6 47.5* 30.3 6 11.4* 2.7 6 0.1* 68.9 6 27.2* 79.0 6 2.1*

1.6 6 0.4# 50.6 6 7.5*# 51.2 6 8.5*# 33.0 6 1.9# 1.3 6 0.5# 1.5 6 0.2# 10.6 6 5.8# 72.03 6 2.3#

Mean6SEM (n¼5), one-way ANOVA: *p<.05 vs. Sham; #p<.05 vs. Vehicle. aMyeloperoxidase activity

29.4. Isoflurane Prevents Acute Lung Injury Through ADPMediated Platelet Inhibition. J. N. Harr,1 E. E. Moore,1,2 M. V. Wohlauer,1 M. Fragoso,1 F. Gamboni,1 J. Stringham,1 C. C. Silliman,1,3 A. Banerjee1; 1University of Colorado Denver, Aurora, CO; 2Denver Health Medical Center, Denver, CO; 3Bonfils Blood Center, Denver, CO 29.3. Stimulation of Carnitine Palmitoyltransferase 1 Improves Renal Function and Attenuates Tissue Damage After Ischemia/Reperfusion. J. P. Idrovo,1 W. L. Yang,1,2 J. Nicastro,1 G. F. Coppa,1 P. Wang1,2; 1Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY; 2The Feinstein Institute for Medical Research, Manhasset, NY Introduction: Renal injury as a result of ischemia/reperfusion (I/R) is a severe clinical problem with a high mortality rate and its treatment still remains on supportive therapy. During I/R, cellular homeostasis is disrupted due to energy depletion. Stimulating energy production in insulted organs may facilitate their recovery from I/R-induced injury. Fatty acid oxidation or b-oxidation is the major metabolic pathway for generating ATP in the kidneys. Carnitine palmitoyltransferase 1 (CPT1) is a key enzyme to control a rate-limiting step of this pathway. C75 is a synthetic cell-permeable a-methyleneg-butyrolactone compound that can stimulate CPT1 activity. Thus, we hypothesized that administration of C75 increases energy production and alleviates renal injury after I/R. Methods: Male adult rats were subjected to renal I/R by bilateral renal pedicle clamping with microvascular clips for 60 min, followed by i.v. administration of 8% DMSO (vehicle) or C75 (3 mg/kg BW). Blood and renal tissues were collected 24 h after reperfusion for measuring the levels of different markers of renal function, tissue damage and inflammation. Results: CPT1 activity and ATP levels decreased by 58% and 76%, respectively, in the vehicle group in comparison to the sham group, while those in the C75 treated group were restored to the levels comparable to the sham group (p < 0.05). C75 treatment significantly improved the microscopic structure of the kidneys in comparison to the vehicle group, judged by histological examination. Furthermore, C75 treatment also improved renal functions and reduced pro-inflammatory cytokine levels. the measurements among the sham, ve-

Introduction: Growing evidence suggests platelets play an essential role in post-traumatic acute lung injury (ALI) and multiple organ failure. Halogenated ethers have recently been shown to interfere with the formation of platelet-granulocyte aggregates, further supporting the theory of the coupling of coagulation and innate immunity in ALI. Isoflurane decreases ALI in sepsis models, but the mechanism is not known. the potential benefit of isoflurane has not been investigated in trauma/hemorrhagic shock (T/HS) models. Therefore, we hypothesized that isoflurane anesthesia attenuates T/HS-mediated ALI through platelet inhibition. Methods: Sprauge-Dawley rats (n¼32) were anesthetized by either 50 mg/kg pentobarbital or 0.5% inhaled isoflurane, and were subjected to control, trauma (laparotomy) and sham shock (T/SS), T/HS (laparotomy and hemorrhage-induced shock: MAP of 30 mmHg x 45 min), or were pre-treated with a P2Y12 (ADP) receptor antagonist and anesthetized with pentobarbital prior to T/HS (T/HS Pento ADP Inh). Animals were resuscitated with a combination of normal saline and returned shed blood. BALF protein was measured, and pulmonary immunofluorescence was performed to detect microthrombi. PlateletMappingÔ was used to determine specific thrombin-independent inhibition of the ADP and AA pathways of platelet activation. Results: Animals undergoing T/HS, and anesthetized with pentobarbital, developed significant lung leak compared to control and sham animals (*,# p < 0.001) (Figure 1). T/SS animals had a modest increase in ALI (p < 0.001), but isoflurane use abrogated both T/SS and T/HS provoked lung leak (p < 0.001) (Figure 1). Isoflurane prevented pulmonary microthrombi formation following T/HS compared to the pentobarbital group (0.00 vs. 1227.23 microns2) (p < 0.001). PlateletMappingÔ revealed specific platelet ADP-pathway inhibition with isoflurane (p < 0.001) without affecting the AA pathway (Table 1). Pentobarbital-anesthetized animals pre-treated with a P2Y12 (ADP) receptor antagonist decreased ALI to sham levels (Figure 1), and confirmed platelet ADP inhibition decreased lung

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

injury without isoflurane. Conclusions: Isoflurane attenuates ALI through an anti-platelet mechanism, in part, through inhibition of the platelet ADP pathway. This platelet inhibition uncouples coagulation from innate immunity, which protects against ALI, and highlights the potential applications of this protective therapy in various ischemia/reperfusion clinical scenarios.

TABLE 1 PlateletMappingÔ

Pentobarbital Isoflurane

% ADP Inh

% AA Inh

15.10 6 8.73 58.00 6 4.89 *

9.14 6 5.60 12.00 6 4.50

sulted in increased CaMKII activation. CaMKII inhibition with AC3I treatment resulted in protection from liver damage compared to vehicle treated control animals as measured by circulating ALT levels. Inhibition of CaMKII also resulted in decreased circulating levels of HMGB1 compared to controls. Using immunofluorescent staining, we found that hepatocytes of AC3I treated mice also demonstrated decreased HMGB1 nucleo-cytoplasmic translocation compared to control mice (Figure). to determine if the mechanism of CaMKII activation in liver I/R was mediated by oxidative stress, p47KO or WT mice treated with the antioxidant NAC were used. Both p47KO and WT mice treated with NAC were found to have decreased CaMKII activation compared to controls. in vitro, CaMKII activation was observed in hepatocyte and non-parenchymal cell (NPC) co-cultures exposed to hypoxia or H2O2. NPC cultures treated with AC3I and exposed to hypoxia had decreased inflammatory cytokine release compared to control cells. Transfer of cell culture media from oxidative-stressed NPCs to hepatocytes resulted in HMGB1 translocation and release, and this effect was abrogated by CaMKII inhibition in NPCs. Conclusions: CaMKII is activated during hepatic I/R and promotes organ damage through release of proinflammatory cytokines by NPCs and release of HMGB1 from hepatocytes. in addition, the activation of CaMKII occurs in response to NADPH oxidase-mediated oxidative stress. Inhibition of CaMKII leads to liver protection and therefore represents a potential therapeutic option against I/R injury.

29.5. Calcium/Calmodulin-Dependent Protein Kinase II Is Activated By Reactive Oxygen Species During Liver Ischemia/Reperfusion Injury and Promotes Organ Damage Through Release of High Mobility Group Box Protein 1 from Hepatocytes. J. R. Klune, J. Evankovich, R. Zhang, L. Zhang, J. Cardinal, M. R. Rosengart, A. Tsung; Department of Surgery, University of Pittsburgh, Pittsburgh, PA

29.6. The Lipid Mediator Lysophosphatidic Acid (LPA) Governs Microvascular Fluid Leak During Ischemia Reperfusion Injury. A. Garcia, A. Strumwasser, E. J. Miraflor, L. Y. Yeung, J. Sadjadi, G. P. Victorino; UCSF-East Bay Department of Surgery, Oakland, CA

Introduction: Hepatic ischemia/reperfusion (I/R) occurs in multiple clinical settings and involves activation of innate immunity, cytokine production, and release of endogenous danger signals such as HMGB1. the mechanisms that account for inflammation and local organ damage are only partially understood but include deranged calcium signaling and activation of Ca+2/Calmodulin Dependent Kinases (CaMK). the purpose of this study was to investigate the contribution of the CaMK II isoform to this process during liver I/R. Methods: WT, NADPH oxidase deficient (p47KO), or WT mice treated with the CaMKII inhibitor, AC3I, were subjected to partial warm ischemia of the left and median lobes of the liver. Liver damage was measured by serum ALT. Serum HMGB1 and tissue CaMKII activation determined by Western blot. HMGB1 localization was analyzed by immunofluorescent staining. in vitro, primary cell cultures were treated with AC3I or antioxidant and exposed to hypoxia (1% O2) or hydrogen peroxide (H2O2). Results: in vivo, hepatic I/R re-

Introduction: Lysophosphatidic Acid (LPA) is a lipid mediator that disturbs endothelial barrier function and can precipitate endothelial cell loss. LPA is synthesized through the conversion of lysophosphatidylcholine to LPA catalyzed by the enzyme autotaxin (ATX). LPA initiates intracellular cascades by binding G-protein receptors on endothelial cells which leads to deterioration of endothelial barrier function. Our hypothesis was that LPA governs microvascular fluid leak and plays a critical role during ischemiareperfusion injury (IRI). Our specific aims were: 1) to determine the effect of LPA on microvascular fluid leak, 2) to evaluate the impact of inhibiting LPA synthesis on endothelial cell monolayer permeability during anoxia/reoxygenation and on microvascular fluid leak during IRI, and 3) to evaluate the impact of LPA receptor blockade on microvascular fluid leak during IRI. Methods: in vivo mesenteric venular microvascular fluid leak (Lp) was examined using an intra-vital micro-occlusion technique in rats. First, Lp was