Inflammatory Activation after Experimental Cardiac Tamponade

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1 Received: Jnury 17, 2013 Accepted fter revision: My 14, 2013 Published online: July 12, X/13/ $38.00/0 Originl Pper Inflmmtory Activtion fter Experimentl Crdic A. Vss b G. Süveges c D. Érces M. Nógrády G. Vrg I. Földesi d M. Futkuchi e M. Imi f N. Okd f H. Okd f M. Boros J. Kszki Institute of Surgicl Reserch, b Second Deprtment of Medicine, nd Deprtments of c Trumtology nd d Lbortory Medicine, University of Szeged, Szeged, Hungry; Deprtments of e Moleculr Toxicology nd f Immunology, Ngoy City University Grdute School of Medicl Sciences, Ngoy, Jpn Key Words Crdic tmponde Big-endothelin High-mobility group box protein-1 Histmine Troponin T Intestinl microcircultion Abstrct Bckground/Purpose: Crdic tmponde is medicl emergency sitution ssocited with high rte of life-thretening complictions, even fter immedite interventions. Our im ws to chrcterize the cute inflmmtory consequences of this event in cliniclly relevnt lrge niml model. Methods: Crdic tmponde ws induced for 60 min in nesthetized, ventilted nd thorcotomized minipigs by intrpericrdil fluid dministrtion, the men rteril pressure (MAP) being mintined in the intervl of mm Hg (n = 8). A further group (n = 7) served s shm-operted control. The globl mcrohemodynmics, including the rightnd left-hert end-distolic volumes (RHEDV nd LHEDV), the pulmonry vsculr resistnce index (PVRI) nd the superior mesenteric rtery (SMA) flow, were monitored for 240 min, nd the intestinl microcircultory chnges (pco 2 gp) were evluted by indirect tonometry. Blood smples were tken for the determintion of crdic troponin T nd vsoctive inflmmtory meditors, including histmine, nitrite/nitrte, big-endothelin, superoxide nd highmobility group box protein-1 levels in ssocition with intestinl leukocyte nd complement ctivtion. Results: The crdic tmponde induced significnt decreses in MAP, crdic output, LHEDV nd SMA flow, while the PVRI nd the pco 2 gp incresed significntly. After the removl of fluid from the pericrdil sc, the MAP nd the LHEDV were decresed, while A.V. nd G.S. contributed eqully to this work. Mihály Boros, MD, PhD, DSc Institute of Surgicl Reserch, University of Szeged Pécsi u. 6 HU 6722 Szeged (Hungry) E-Mil med.u-szeged.hu

2 2 the PVRI nd the pco 2 gp remined elevted when compred with those in the shm-operted group. In the posttmponde period, the brupt relese of inflmmtory meditors ws ccompnied by significnt splnchnic leukocyte ccumultion nd complement ctivtion. Conclusions: The mcrocircultory nd splnchnic microcircultory disturbnces were ccompnied by significnt proinflmmtory rection; endothelin nd the complement system my be significnt components of the inflmmtory cscde tht is ctivted in this porcine model of pericrdil tmponde. Introduction Crdiogenic shock my occur in ptients with crdic or extrcrdic filling disorders such s crdic tmponde [1, 2]. The cute rise in pericrdil pressure compresses the tri nd the right ventricle nd hinders the distolic filling of the hert [3], thereby leding to decresed crdic output (CO). Vsoconstriction is generl compenstory rection in the forms of shock tht involve low CO, occurring s n pproprite response tht serves to restore the declining rteril flow nd pressure towrds former vlues. The vsoconstriction is nonuniform, nd the redistributed blood flow cn mintin the blood supply for the hert nd brin t reltively norml level in the short run. This process is medited through ctivtion of the sympthetic nervous system nd vrious humorl meditors [4], leding to systemic vsoconstriction, tchycrdi nd fluid retention [2]. However, the price for the potentilly beneficil effects of selective vsoconstriction is severe hypoxi in the underperfused orgns [5 7]. Moreover, the incresed fterlod my subsequently worsen the peripherl hypoperfusion nd lso increses the oxygen demnd of the myocrdium. Dringe of the pericrdil sc through pericrdiocentesis or surgicl pericrdiotomy is the first choice for therpy [8], but removl of the pericrdil fluid cn led to reperfusion phenomenon with further peripherl rections. As consequence of hypoxi nd reoxygention, rective oxygen species re generted nd polymorphonucler neutrophil (PMN) leukocytes re ctivted. The formtion of these rdicls my led to disintegrtion of the cell membrnes, structurl dmge nd decresed cellulr functions [9, 10]. Highmobility group box protein-1 (HMGB1), relesed pssively by necrotic nd dmged cells, ws recently identified s n importnt signl for leukocyte recruitment [11]. Further fctors identified in the bckground of PMN leukocyte ccumultion re n incresed level of endothelin-1 (ET-1) formtion nd decresed level of nitric oxide (NO) formtion, which coexist in ischemi-reperfusion syndromes [12]. ET-1, one of the most powerful endogenous vsoctive meditors, my contribute to the impirment of the microcircultion through its vsoconstrictor nd prodhesive effects [13]. Moreover, it results in histmine relese from resident mst cells [14], nd influences the ctivtion of the complement cscde [15]. With regrd to this bckground, we hypothesized tht cute filure of the myocrdil pump function is ccompnied by significnt inflmmtory ctivtion, which cn ply importnt roles in further clinicl complictions. Our primry im ws to investigte the immedite effects of crdic tmponde on the systemic nd peripherl circultions in cliniclly relevnt, lrge niml model. We lso imed to chrcterize the mjor components of the proinflmmtory profile of the posttmponde phse, in ssocition with the chnges in overll hemodynmics. We ssumed tht better understnding of the elements of this mechnism my led to new prospects for interventions designed to dmpen or reverse the secondry detrimentl consequences of cute hert filure.

3 3 Mterils nd Methods The experiments were crried out in strict dherence to the NIH guidelines for the use of experimentl nimls nd the study ws pproved by the Ethicl Committee for the Protection of Animls in Scientific Reserch t the University of Szeged (V./148/2013). Animls nd Instrumenttion Inbred Vietnmese minipigs of both sexes (n = 15, weighing 24 ± 3 kg) were fsted for 12 h preopertively, but received wter d libitum. Anesthesi ws induced with ketmine (20 mg kg 1 ) nd xylzine (2 mg kg 1 ) i.m. nd mintined with continuous infusion of propofol (50 μl min 1 kg 1 i.v.; 6 mg kg 1 h 1 ) supplemented with ketmine (5 mg kg 1 ) xylzine (0.5 mg kg 1 ) mixture i.m. before thorcotomy nd fter 90 min of the observtion period. After endotrchel intubtion, the nimls were mechniclly ventilted with the tidl volume set t 9 ± 2 ml kg 1, nd the respirtory rte ws djusted to mintin the end-tidl pressure of CO 2 nd rteril pco 2 (PCO 2 ) in the rnge mm Hg. Positive end-expirtory pressure ws not pplied during the crdic tmponde. The nimls were plced in supine position on heting pd for mintennce of the body temperture between 36 nd 37 C, nd received n infusion of Ringer s lctte t rte of 10 ml kg 1 h 1 during the experiments. The right jugulr vein ws cnnulted (7 F; Edwrds Lifesciences LLC, Irvine, Clif., USA) for the mesurement of centrl venous pressure (CVP) nd for fluid dministrtion. The right femorl rtery nd vein were dissected nd thermodilution ctheter (PULSION Medicl Systems SE, Munich, Germny) ws plced in the femorl rtery for the mesurement of men rteril pressure (MAP) nd CO by trnspulmonry thermodilution method (PiCCO; PULSION Medicl Systems SE). A pulmonry rtery ctheter (PV2057 VoLEF Ctheter; PULSION Medicl Systems SE) ws inserted vi the right femorl vein into the pulmonry rtery by trcing the pressure signls. After midline bdominl incision, the root of the superior mesenteric rtery (SMA) ws dissected free. An ultrsonic flow probe (Trnsonic Systems Inc., Ithc, N.Y., USA) ws plced round the exposed SMA to mesure the mesenteric blood flow. In ll protocols, the nimls were monitored continuously nd period of 30 min ws llowed for recovery from surgery. The nimls were rndomly divided into 2 experimentl groups. Group 1 (n = 7) served s shmoperted control, with the sme time-frme nd smpling s in group 2 (n = 8) with the induction of crdic tmponde. Left lterl thorcotomy ws performed nd cnnul ws fixed into the pericrdil cvity in both groups. A pericrdil tmponde ws induced for 60 min by the intrpericrdil dministrtion of colloid solution (60 90 ml hydroxyethyl strch 6%; Fresenius Kbi Deutschlnd GmbH, Homburg, Germny), while the MAP ws kept in the intervl mm Hg. After this period, the fluid ws removed from the pericrdil sc nd the nimls were monitored for 180 min posttmponde. Peripherl blood smples were tken t bseline, fter 75 min, fter 150 min nd t the end of the observtion period (240 min) to detect the levels of vsoctive nd inflmmtory meditors (big-et, histmine, nitrite/nitrte, HMGB1 nd whole-blood superoxide production). Smll intestinl tissue biopsies were tken t the end of the experiments for myeloperoxidse (MPO) ctivity mesurements nd immunohistochemicl nlysis of the complement C3 deposit. Hemodynmic Mesurements CVP nd blood flow signls were monitored continuously nd registered with computerized dtcquisition system (SPELL Hemosys; Experimetri Ltd., Budpest, Hungry). For further hemodynmic monitoring, we used combintion of PiCCO Plus V nd VoLEF V 1.0 (PULSION Medicl Systems SE) monitors [16]. The MAP, CO, hert rte (HR) nd extrvsculr lung wter index (EVLWI) were mesured with the PICCO Plus monitoring system, while the PiCCO VoLEF monitor system ws pplied to mesure the pulmonry rteril pressure (PAP), nd clculte the right- nd left-hert end-distolic volumes (RHEDV nd LHEDV), systemic vsculr resistnce index (SVRI) nd pulmonry vsculr resistnce index (PVRI). All hemodynmic prmeters were indexed for body surfce re or body weight. A detiled description of the trnspulmonry thermodilution nd volumetric nlysis is provided elsewhere [17]. Evlution of Intestinl Microcircultion: pco 2 Gp Mesurements A difference between locl tissue nd PCO 2 levels is sensitive prmeter with which to evlute the effectiveness of therpy imed t countercting microcircultory dysfunction in the gstrointestinl trct [18]. A silstic blloon-free tonometric probe (Tonosoft Medicl Technicl nd R&D Ltd., Hungry) ws intro-

4 4 duced into the intestinl lumen through smll enterotomy to monitor intrmucosl pco 2 levels by cpnometry [19]. For clcultion of the pco 2 gp vlues, the simultneously tken PCO 2 levels were subtrcted from the tonometric pco 2 levels. Arteril nd venous blood smples were tken t the bseline nd once n hour, nd blood-gs prmeters were mesured with blood-gs nlyzer (Cobs b121; Roche, Austri). Biochemicl Mesurements MPO Activity The ctivity of MPO, mrker of PMN leukocyte ctivtion, ws determined in ilel biopsy smples by the method of Kuebler et l. [20]. Briefly, the smple ws homogenized with Tris-HCl buffer (0.1 M, ph 7.4) contining 0.1 m M phenylmethylsulfonyl fluoride to block tissue proteses, nd then centrifuged t 4 C for 20 min t 2,000 g. The MPO ctivities of the smples were mesured t 450 nm (UV-1601 spectrophotometer; Shimdzu, Jpn) nd the dt were referred to the protein content. Whole-Blood Superoxide Production For the whole-blood superoxide production mesurements, the chemiluminometric method of Zimmermnn et l. [21] ws used. During the mesurements, 10 μl of whole blood ws dded to 1 ml of Hnk s solution nd the mixture ws kept t 37 C until ssy. The chemiluminometric response ws mesured with Lumt LB9507 luminometer (Berthold, Vienn, Austri) over 30 min fter the ddition of 100 μl of lucigenin. Plsm Nitrite/Nitrte Level Mesurements The levels of plsm nitrite/nitrte (NO x ), stble end-products of NO, were mesured by mens of the Griess rection. The ssy depends on the enzymtic reduction of nitrte to nitrite, which is then converted into colored zo compound detected spectrophotometriclly t 540 nm [22]. HMBG1, Big-ET nd Histmine Mesurements in Plsm Blood smples of 4 ml were drwn from the jugulr vein into chilled polypropylene tubes contining EDTA (1 mg ml 1 ) nd then centrifuged t 1,200 g for 10 min t 4 C. The plsm smples were next collected nd stored t 70 C until ssy. The plsm concentrtion of HMGB1 ws mesured with commercilly vilble HMGB1 ELISA kit (Shino-Test Corportion, Kngw, Jpn). Plsm levels of big-et, 38-mino-cid precursor protein of ET-1, were mesured with commercilly vilble kit (Biomedic Hungri Kft., Budpest, Hungry). Plsm histmine concentrtions were determined by commercilly vilble enzyme-linked immunossy (Quntikine ultrsensitive EIA kit for histmine; Biomedic Hungri Kft.). Plsm Troponin T Level Mesurements Crdic troponin T levels were mesured from plsm smples by highly sensitive electrochemiluminescent immunossy (ECLIA; Elecsys 2010, Roche Dignostics GmbH, Mnnheim, Germny). The nlyticl sensitivity ws 5 ng/l, the intr- nd interssy vritions t the mesured concentrtion rnge were 3.2 nd 6.2 CV%, respectively. Immunohistochemistry The presence of complement C3 deposit in the smll intestinl mucos ws detected by immunohistochemistry (IHC) [23] on formlin-fixed, prffin-embedded smll intestine sections, using rbbit polyclonl nticomplement frgment C3c primry ntibody (Bioss Inc., Woburn, Mss., USA). The sections were deprffinized for 5 min, which ws followed by ntigen retrievl with citrte buffer for 20 min. The ctivity of endogenous peroxidses ws blocked with 5% H 2 O 2 for 10 min. The nonspecific interctions were inhibited during the next 30 min of incubtion. The primry ntibody ws diluted 1: 500 in ntibody diluent for IHC (BD Phrmingen, Sn Diego, Clif., USA). After wshing of the sections, the secondry ntibody ws diluted 1: 500 nd the smples were incubted for 8 min. Sections were counterstined with 3,3 -diminobenzidine nd hemtoxylin. The entire IHC investigtion ws crried out with n utomtic Leic Bond-mx IHC mchine (Leic Microsystems, Tokyo, Jpn). For quntittive nlysis, immunostined sections were exmined under light microscope. The coded sections were nlyzed by n independent specilist in histopthology (M.F.). The numbers of cpillries positive for complement frgment C3c were ssessed t mgnifiction of 400.

5 Crdic tmponde Shm-operted MAP (mm Hg) * CVP (mm Hg) b Fig. 1. Chnges in MAP ( ) nd CVP ( b ) in the shm-operted nd crdic-tmponde groups. The grey boxes indicte the durtion of the crdic tmponde. The plots demonstrte the medin vlues nd the 25th (lower whisker) nd 75th (upper whisker) percentiles. * p < 0.05 within groups vs. bseline vlues, + p < 0.05 between groups vs. shm-operted group vlues. Sttisticl Anlysis Dt nlysis ws performed with sttisticl softwre pckge (SigmStt for Windows, Jndel Scientific, Erkrth, Germny). The distribution of our experimentl dt ws nlyzed by the Kolmogorov-Smirnov normlity test. Filure of the normlity test indicted nonprmetric distribution of the dt. The Friedmn repeted-mesures nlysis of vrince on rnks ws pplied within groups. Time-dependent differences from the bseline for ech group were ssessed by the Dunn method, nd differences between groups were nlyzed with the Mnn-Whitney test. In the figures, medin vlues nd 25th nd 75th percentiles re given; p vlues <0.05 re considered sttisticlly significnt. Results He mo d y nmic s In the shm-operted group, there were no significnt hemodynmic chnges compred to the bseline vlues, nd the meditor levels did not chnge significntly during the observtion period. The MAP ws mintined in the intervl mm Hg during the tmponde for 60 min ( fig. 1 ) by the infusion of colloid fluid into the pericrdil sc, nd this resulted in significnt decline of pproximtely 60% in CO in the group undergoing crdic tmponde. The SVRI nd HR were incresed significntly (by 32 nd 66%, respectively; tble 1 ). After relief of the tmponde, the MAP ws significntly lower in the crdic tmponde group compred with the control group, while the CO nd HR returned to the bseline, despite the reduced MAP. The decline in the venous return during the tmponde ws evidenced by the incresed CVP ( fig. 1 b). This process ws ccompnied by decreses in RHEDV nd LHEDV ( fig. 2, b). After relief of the tmponde, the CVP nd RHEDV were normlized, but the LHEDV did not rech the bseline vlue in the tmponde group; it remined significntly lower compred with the shm-operted group ( fig. 2, b). These chnges demonstrte the long-lsting impirment of the left ventriculr function following the crdic tmponde. The crdic tmponde resulted in significnt, trnsient decrese in PAP nd 2-fold elevtion of the PVRI ( tble 2 ). In the posttmponde period, further long-lsting significnt

6 6 Tble 1. Effects of crdic tmponde on CO, HR nd SVRI 5 min 30 min 60 min 90 min 120 min 180 min 240 min CO, l min 1 m 2 Shm-operted 2.60 (2.39; 2.97) Crdic tmponde 2.82 (2.46; 3.01) 2.73 (2.25; 3.34) 1.36*, ** (0.95; 1.89) 2.74 (2.30; 3.14) 1.19*, ** (1.03; 1.30) 2.85 (2.60; 3.28) 2.44 (2.29; 3.01) 2.94 (2.65; 3.30) 2.48 (2.16; 3.03) 2.85 (2.51; 3.35) 2.40 (2.13; 2.74) 2.82 (2.62; 3.33) 2.40 (2.07; 2.79) HR, bet min 1 Shm-operted 124 (120; 137) Crdic tmponde 113 (103; 118) 128 (118; 134) 175*, ** (136; 203) 125 (119; 134) 188*, ** (173; 206) 126 (115; 137) 125 (122; 143) 127 (104; 135) 118 (113; 120) 120 (104; 128) 114 (105; 116) 122 (106; 126) 113 (98; 122) SVRI, mm Hg ml 1 min 1 1 kg Shm-operted 0.79 (0.73; 0.83) Crdic tmponde 0.78 (0.71; 0.92) 0.81 (0.75; 0.85) 0.91 (0.80; 0.94) 0.74 (0.69; 0.79) 1.03* (0.84; 1.06) 0.68 (0.66; 0.77) 0.83 (0.72; 0.85) 0.64 (0.58; 0.68) 0.83** (0.69; 0.93) 0.66 (0.57; 0.71) 0.82** (0.73; 0.91) 0.67 (0.61; 0.75) 0.79 (0.71; 0.91) Vlues re medins (25th percentile; 75th percentile). * p < 0.05 within group; ** p < 0.05 between groups vs. control group. Tble 2. Effects of crdic tmponde on PAP, PVRI nd EVLWI 5 min 30 min 60 min 90 min 120 min 180 min 240 min PAP, mm Hg Shm-operted 30.6 (27.0; 35.0) Crdic tmponde 30.4 (27.3; 34.2) 31.1 (27.3; 35.4) 24.3*, ** (21.1; 27.8) 28.9 (27.5; 35.6) 30.0 (19.2; 35.6) 31.2 (26.0; 36.0) 38.4 (30.0; 41.3) 30.3 (27.4; 34.8) 36.5 (34.4; 42.7) 30.3 (26.8; 32.5) 41.2*, ** (35.3; 43.2) 30.8 (28.3; 33.6) 37.9 (31.9; 42.8) PVRI, mm Hg ml 1 min 1 1 kg Shm-operted 0.37 (0.31; 0.39) Crdic tmponde 0.41 (0.26; 0.50) 0.35 (0.30; 0.43) 0.75*, ** (0.66; 0.98) 0.31 (0.29; 0.35) 0.88*, ** (0.81; 0.95) 0.29 (0.25; 0.35) 0.48** (0.42; 0.53) 0.29 (0.25; 0.32) 0.51** (0.44; 0.63) 0.32 (0.24; 0.36) 0.54** (0.49; 0.60) 0.31 (0.23; 0.38) 0.49** (0.47; 0.54) EVLWI, ml kg 1 Shm-operted 8.0 (7.8; 9.0) Crdic tmponde 8.0 (8.0; 9.0) 8.2 (8.0; 9.0) 8.0 (7.5; 8.8) 8.5 (8.0; 9.0) 8.0 (7.3; 8.9) 8.5 (8.0; 9.1) 8.5 (8.0; 10.8) 8.5 (8.0; 9.1) 9.0 (8.3; 10.8) 8.4 (8.0; 9.0) 9.5* (8.5; 10.0) 8.4 (8.0; 9.0) 10.0*, ** (9.0; 11.0) Vlues re medins (25th percentile; 75th percentile). * p < 0.05 within group; ** p < 0.05 between groups vs. control group.

7 LHEDV (ml m 2 ) * * * * RHEDV (ml m 2 ) Crdic tmponde Shm-operted b Fig. 2. Chnges in LHEDV ( ) nd RHEDV ( b ) in the shm-operted nd crdic-tmponde groups. The grey boxes indicte the durtion of the crdic tmponde. The plots demonstrte the medin vlues nd the 25th (lower whisker) nd 75th (upper whisker) percentiles. * p < 0.05 within groups vs. bseline vlues, + p < 0.05 between groups vs. shm-operted group vlues. increses in PVRI nd PAP occurred when compred with the shm-operted group, while the EVLWI ws significntly elevted t the end of the observtion period ( tble 2 ). The significnt decrese in SMA blood flow during the tmponde indicted deteriorted mesenteric circultion. After the removl of the pericrdic fluid, the SMA flow returned to the control vlues ( fig. 3 ). The pco 2 gp, the difference between the locl tissue nd the rteril pco 2, is relible index of locl tissue perfusion. The pco 2 gp incresed significntly during the tmponde, while relief of the tmponde resulted in significntly lower gp, though the vlues remined significntly higher thn tht for the shm-operted control group throughout the posttmponde period ( fig. 3 b; online suppl. tble 1, B io c he mic l Pr me t e r s Peripherl blood smples were tken t bseline, fter 75 min, fter 150 min nd t the end of the observtion period (240 min). In the crdic tmponde group, incresed superoxide rdicl production ws observed t the beginning of the posttmponde phse ( fig. 4 ). In prllel, the plsm histmine levels were incresed significntly t 75 nd 240 min of the observtion period: M = 16.2 n M (p25 = 15.5; p75 = 16.6) nd M = 10.3 n M (p25 = 9.1; p75 = 12.8), respectively, versus the bseline (M = 7.5 n M ; p25 = 6.4; p75 = 8.8) or versus the corresponding vlue for the shm-operted group (M = 7.4 n M ; p25 = 5.7; p75 = 9.4). The NO x concentrtion in the plsm llows n estimte of the chnges in NO production. The consequence of the crdic tmponde ws slight, but sttisticlly significnt increse in NO x level t the end of the posttmponde period when compred with the bseline level nd with tht for the shm-operted group ( fig. 4 b). Big-ET is stble precursor of ET-1 with longer hlf-life. The plsm big-et level incresed significntly, 4- to 5-fold, in response to the crdic tmponde ( fig. 5 ). HMGB1 is n effective signl for leukocyte ctivtion, which cuses n escltion of the inflmmtory process. The plsm level of HMGB1 ws elevted significntly fter the compression of the hert ( fig. 5 b).

8 8 SMA blood flow (ml min 1 ) * Intestinl pco2 gp (mm Hg) b Crdic tmponde Shm-operted Fig. 3. Chnges in SMA blood flow ( ) nd intestinl pco 2 gp ( b ) in the shm-operted nd crdic-tmponde groups. The grey boxes indicte the durtion of the crdic tmponde. The plots demonstrte the medin vlues nd the 25th (lower whisker) nd 75th (upper whisker) percentiles. * p < 0.05 within groups vs. bseline vlues, + p < 0.05 between groups vs. shm-operted group vlues. Whole-blood superoxide production (RLU) 80,000 60,000 40,000 20, Plsm NOx (μmol l 1 ) b Crdic tmponde Shm-operted Fig. 4. Chnges in whole-blood superoxide production ( ) nd plsm NO x level ( b ) in the shm-operted nd crdic-tmponde groups. The grey boxes indicte the durtion of the crdic tmponde. The plots demonstrte the medin vlues nd the 25th (lower whisker) nd 75th (upper whisker) percentiles. * p < 0.05 within groups vs. bseline vlues, + p < 0.05 between groups vs. shm-operted group vlues. The troponin T level in the plsm llows n estimte of the crdiomyocyte dmge. The men concentrtions of troponin T were significntly incresed fter the compression of the hert nd t the end of the posttmponde period when compred with the bseline nd with those for the shm-operted group ( fig. 6 ). The rte of PMN leukocyte ccumultion ws determined through mesurement of the MPO ctivity of the intestinl tissue smples tken t the end of the experiments. The level of

9 9 Plsm big-et level (fmol ml 1 ) * Plsm HMGB1 level (ng ml 1 ) b *+ Crdic tmponde Shm-operted Fig. 5. Chnges in plsm big-et concentrtion ( ) nd HMGB1 level ( b ) in the shm-operted nd crdictmponde groups. The grey boxes indicte the durtion of the crdic tmponde. The plots demonstrte the medin vlues nd the 25th (lower whisker) nd 75th (upper whisker) percentiles. * p < 0.05 within groups vs. bseline vlues, + p < 0.05 between groups vs. shm-operted group vlues. Fig. 6. Chnges in plsm troponin T level in the shm-operted nd crdic-tmponde groups. The grey box indictes the durtion of the crdic tmponde. The plots demonstrte the medin vlues nd the 25th (lower whisker) nd 75th (upper whisker) percentiles. * p < 0.05 within groups vs. bseline vlues, + p < 0.05 between groups vs. shm-operted group vlues. Plsm troponin T level (ng l 1 ) Crdic tmponde Shm-operted MPO ctivity ws significntly higher in the smll intestine tissue smples of the crdic tmponde group, indicting the incresed ccumultion of PMN leukocytes (crdic tmponde: M = 5.37 (p25 = 4.8; p75 = 6.22; U mg protein 1 ) versus the shm-operted group: M = 2.84 (p25 = 2.19; p75 = 3.22). Activtion of the complement cscde ws evluted by the presence of com - ple ment C3 deposit in the smll intestinl mucos with the IHC method ( fig. 7 ). In ech field of view of the slides, the number of cpillries showing C3 deposit positivity ws counted. The number of C3 deposits ws significntly higher in the tmponde group thn in the shm-operted group (M = 3; p25 = 1; p75 = 4.5 vs. M = 0; p25 = 0; p75 = 1 deposits/field of view).

10 10 b Color version vilble online c d Fig. 7. Chnges in complement C3 deposition demonstrted by IHC nlyses in the smll intestinl mucosl biopsies tken t the end of the experiments: HE stining in the shm-operted ( ) nd crdic-tmponde ( c ) groups; extensive C3 deposition with IHC stining in the crdic-tmponde group ( d ) compred to the shm-operted group ( b ). Discussion Pericrdil tmponde is ccompnied by high mortlity nd postopertive compliction rtes, even in the event of dequte tretment. In the cse of blunt chest trum, the mortlity might exceed 80% [24], while 22% mortlity rte ws found fter elective open-hert surgery with cliniclly proven cses of loclized crdic tmponde [25]. Our gol ws to chrcterize the hemodynmic effects of temporry mechnicl compression of the hert nd to outline the proinflmmtory elements which my ply role in the cscde of events induced by relief of the tmponde. Our mjor finding is tht the posttmponde period is chrcterized by decresed level of systemic perfusion nd by n impired pulmonry circultion, s evidenced by the MAP, LHEDV, PAP, PVRI nd EVLWI dt. The pco 2 gp chnges suggested tht, in prllel, significnt intestinl microcircultory dysfunction evolved in this porcine model. More importntly, these responses were ssocited with brupt increses in superoxide rdicl production, big-et, troponin T, HMGB1, histmine, intestinl MPO ctivity nd complement ctivtion during the posttmponde phse. The crdic filling disorder induced different vsoconstrictive compenstory rections in the systemic nd pulmonry circultions: the increse in PVRI ws much higher (112%) thn tht in SVRI (32%). Previous experimentl nd clinicl dt suggest tht the incresing pericrdil pressure cuses continuous decline in coronry blood flow due to n increse in coronry vsculr resistnce [26, 27]. Sklidis et l. [27] observed decresed hyperemic flow under incresed pericrdil pressure, which implies n ugmented susceptibility to myocrdil ischemi. The significntly elevted troponin T during the cute phse of the

11 11 tmponde lso demonstrtes the deteriortion of oxygention nd dmge of the crdic muscle cells. After the relief of the tmponde, the MAP ws decresed, while the CO ws kept compensted, nd there were no significnt differences compred to the control group. This my be explined by the normlized prelod, s evidenced by the normlized CVP nd RHEDV nd the moderte elevtion of the fterlod. However, the significnt decrese in LHEDV indictes left ventriculr dysfunction during the posttmponde phse. The persistent elevtions in PAP nd PVRI could contribute to this process, together with the lung edem s reveled by the elevted EVLWI. These conclusions re supported by clinicl observtions on erly crdic filure nd pulmonry edem fter removl of the pericrdil effusion [28, 29]. In crdiogenic shock, prompt tretment of hypotension is needed in order to void the genertion of inflmmtory meditors [30]. Indeed, the pericrdil tmponde triggered chrcteristic mcro- nd microcircultory chnges in the intestines. While the SMA flow, which reflects the blood supply of the smll intestine nd colon, ws diminished during the tmponde, prolonged impirment of the mucosl microcircultion ws detected. This is in ccordnce with previous ssumptions tht the mcro- nd microhemodynmics my chnge reltively independently, or my be dissocited in stress conditions [31]. Aginst the bckground of these hemodynmic ltertions, multi-fceted role of humorl meditors, including ET-1, is proposed. Hypoxi is considered to be one of the bsic stimuli for ET-1 synthesis. This peptide is produced predominntly by the endothelium, but in pthophysiologicl sttes, other cell types such s leukocytes, mcrophges, smooth muscle cells, crdiomyocytes nd mesngil cells cn lso serve s sources of its relese [12]. The incresed plsm level of ET-1 could be responsible for the decresed coronry perfusion [32] nd pulmonry hypertension. The ctivtion of vsoconstrictor ET receptors cn further ply decisive role in cute microcircultory disorders of the peripherl crdiovsculr system. It hs been shown tht selective ET-A receptor ntgonism increses the CO, decres - es the peripherl resistnce [14, 33] nd reduces intestinl microvsculr injury nd PMN leukocyte ccumultion during ischemi-reperfusion [34]. In ddition to its independent role s dominnt vsoconstrictor, the peptide my lso influence the functions of other cell types in the circultory system. ET-1 hs been reported to induce leukocyte rolling nd dherence through predominntly ET-A receptor-medited mechnism [13]. There is close reltionship between compromised mucosl blood flow nd the mgnitude of PMN leukocyte-endothelil cell interctions in the intestines [34]. On the other hnd, ET-1 lso cuses histmine relese from mst cells [14], which my led to enhnced vsculr permebility nd reltive blood loss into the dilted vessels. Histmine relese in the pulmonry circultion contributes to the increse in EVLWI [35], while in the splnchnic re histmine relese probbly plys role in the counterregultion of the excessive, prolonged vsoconstriction tht contributes to the lethl outcome [4]. Furthermore, ET-1 ctivtes NADPH oxidse, resulting in n incresed superoxide rdicl production [36], which cn simultneously reduce NO production, leding to the formtion of the highly cytotoxic peroxynitrite [37]. From this point of view, the excessive relese of ET-1 cn be the key plyer s concerns the spreding inflmmtory responses, when intensive complement ctivtion is lso ignited. The presence of complement C3 deposits ws verified in this tmponde model nd we found incresed plsm levels of HMGB1 too, the relese of which is dditionlly directly medited by the complement cscde. In this scenrio, HMGB1 relese is further dnger signl to responsive cells; it mplifies the production nd secretion of other proinflmmtory meditors nd finlly induces excessive inflmmtion [38, 39]. This study hs some limittions. Firstly, thorcotomy cuses severe surgicl trum. Diphrgmtic window through lprotomy could be possible lterntive to rech the peri-

12 12 crdil cvity [30]. Nevertheless, sfe ctheteriztion of the pericrdil sc nd quick guidnce of the dignostic instrumenttion into correct positions into the hert cvities re dvntges of the open-chest model. Secondly, the inflmmtory rection my be nonspecific becuse crdiogenic nd hypovolemic shock components re not mutully exclusive. Decreses in MAP, CO, splnchnic perfusion nd microcircultory dmge cn occur in nerly ll forms of circultory shock (e.g. hypovolemic, crdiogenic or distributive). Nevertheless, there re tmponde-specific consequences s well, such s elevtion of the CVP, decrese in LHEDV nd incresed plsm troponin T level, which could ll be direct signs nd consequences of tmponde-induced crdic ischemi. In conclusion, we hve demonstrted chrcteristic mcrohemodynmic chnges, together with pprent signs of splnchnic inflmmtory rection fter the relief of tmponde. The evidence further suggests tht ET-1 nd the complement system my be significnt components of the inflmmtory cscde tht is ctivted in this porcine model of pericrdil tmponde. Acknowledgements The uthors re grteful to Ms. Ágnes Fekete, Andre Bus, Csill Mester nd Nikolett Beretk for skillful ssistnce. This study ws supported by reserch grnts OTKA K104656, TÁMOP A-11/1/KONV nd TÉT JP 16/09. Disclosure Sttement The uthors declre no conflicts of interest. References 1 Bodson L, Bouferrche K, Vieillrd-Bron A: Crdic tmponde. Curr Opin Crit Cre 2011; 17: Toplin S, Ginsberg F, Prrillo JE: Crdiogenic shock. Crit Cre Med 2008; 36:S66 S74. 3 Tyson GS, Mier GW, Olsen CO, Dwis JW, Rnkin JS: Pericrdil influences on ventriculr filling in the conscious dog. Circ Res 1984; 54: Kszki J, Ngy S, Tárnoky K, Lczi F, Vecsernyés M, Boros M: Humorl chnges in shock induced by crdic tmponde. Circ Shock 1989; 29: Biley RW, Bulkley GB, Hmilton SR, Morris JB, Hglund UH, Meilhn JE: The fundmentl hemodynmic mechnism underlying gstric stress ulcertion in crdiogenic shock. Ann Surg 1987; 205: Bulkley GB, Kvietys PR, Perry MA, Grnger DN: Effects of crdic tmponde on colonic hemodynmics nd oxygen uptke. Am J Physiol 1983; 244:G604 G Buerke M, Lemm H, Dietz S, Werdn K: Pthophysiology, dignosis, nd tretment of infrction-relted crdiogenic shock. Herz 2011; 36: Seferovic PM, Ristic AD, Imzio M, Mksimović R, Simeunović D, Trinchero R, Pnkuweit S, Misch B: Mngement strtegies in pericrdil emergencies. Herz 2006; 31: Prsd K, Klr J, Chudhry AK, Debnth D: Polymorphonucler leukocyte ctivtion nd crdic function t orgn nd cellulr level. Am Hert J 1990; 119: Boros M, Kszki J, Ngy S: Oxygen-free rdicl induced histmine relese during intestinl ischemi-reperfusion. Eur Surg Res 1989; 21: Scffidi P, Misteli T, Binchi ME: Relese of chromtin protein HMGB1 by necrotic cells triggers inflmmtion. Nture 2002; 418: Boros M: Microcircultory dysfunction during intestinl ischemi-reperfusion. Act Physiol Hung 2003; 90: Boros M, Mssberg S, Brnyi L, Okd H, Messmer K: Endothelin-1 induces leukocyte dhesion in submucosl venules of the rt smll intestine. Gstroenterology 1998; 114: Kszki J, Czóbel M, Szly L, Ngy S, Boros M: Endothelin-1 induces orgn-specific histmine libertion nd neutrophil grnulocyte ccumultion in the rt. Inflmm Res 2008; 57:

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