Acta Anaesthesiol Scand 2014; 58: 1015–1024 Printed in Singapore. All rights reserved
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd ACTA ANAESTHESIOLOGICA SCANDINAVICA
doi: 10.1111/aas.12355
Pulmonary function after hemorrhagic shock and resuscitation in a porcine model T. K. Nielsen1,2, C. L. Hvas1, G. P. Dobson3, E. Tønnesen1 and A. Granfeldt1,4
1 Department of Anesthesiology and Intensive Care Medicine, Aarhus University Hospital, Aarhus, Departments of 2Anesthesiology, 4Internal Medicine, Regional Hospital of Randers, Randers, Denmark and 3Heart and Trauma Research Laboratory, Department of Physiology and Pharmacology, James Cook University, Townsville, QLD, Australia
Background: Hemorrhagic shock may trigger an inflammatory response and acute lung injury. The combination adenosine, lidocaine (AL) plus Mg2+ (ALM) has organ-protective and anti-inflammatory properties with potential benefits in resuscitation.The aims of this study were to investigate: (1) pulmonary function and inflammation after hemorrhagic shock; (2) the effects of ALM/AL on pulmonary function and inflammation. Methods: Pigs (38 kg) were randomized to: sham + saline (n = 5); sham + ALM/AL (n = 5); hemorrhage control (n = 11); and hemorrhage + ALM/AL (n = 9). Hemorrhage animals bled to a mean arterial pressure (MAP) of 35 mmHg for 90 min, received resuscitation with Ringer’s acetate and 20 ml of 7.5% NaCl with ALM to a minimum MAP of 50 mmHg, after 30 min shed blood and 0.9% NaCl with AL were infused. Hemorrhage controls did not receive ALM/AL. Primary endpoints were pulmonary wet/dry ratio, PaO2/FiO2 ratio (partial pressure of arterial oxygen to the fraction of inspired oxygen), cytokine and protein measurements in bronchoalveolar lavage fluid (BALF)
and lung tissue, neutrophil invasion and blood flow in lung tissue. Results: In the hemorrhage groups, wet/dry ratio increased significantly compared with the sham groups. PaO2/FiO2 ratio decreased during shock but normalized after resuscitation. BALF did not indicate significant pulmonary inflammation, oxidative stress or increased permeability. Intervention with ALM caused a temporary increase in pulmonary vascular resistance and reduced urea diffusion across the alveolar epithelia, but had no effect on wet/dry ratio. Conclusion: Hemorrhagic shock and resuscitation did not cause acute lung injury or pulmonary inflammation. The question whether ALM/AL has the potential to attenuate acute lung injury is unanswered.
H
cardioplegic agent to arrest the heart and protect the myocardium during cardiac surgery.12,13 The combination of adenosine and lidocaine (AL) reduces activation and priming of neutrophils.14 Furthermore, in a small-animal model of hemorrhagic shock and hypotensive resuscitation, a bolus administration of 7.5% NaCl with ALM improved survival, hemodynamic function and reversed acute coagulopathy.15,16 This study is based on material from an animal series, from which results on cardiac and renal functioning after hemorrhagic shock and resuscitation have previously been published.17 The aims of this study were to investigate: (1) pulmonary function and inflammation after hemorrhagic shock and resuscitation in a largeanimal model; and (2) the effects of ALM/AL on pulmonary function and inflammation.
emorrhage is a major cause of trauma deaths.1,2 Resuscitation with fluid and blood restores blood pressure but leaves organs susceptible to reperfusion injury. This may result in multiple organ failure (MOF), the leading cause of late postinjury mortality.3,4 Acute lung injury (ALI) is often the first stage of MOF.5 Previous small-animal studies have described the presence of pulmonary inflammation following hemorrhagic shock alone and subsequently investigated the effects of anti-inflammatory interventions.6–8 Few studies have been carried out in large-animal models, in which the relevant pathophysiology and hemodynamic variables can be described in detail,9 and they have revealed conflicting results.10,11 The combination of adenosine, lidocaine and magnesium (ALM) is currently in use as a
Accepted for publication 25 May 2014 © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
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Methods The study was approved by the National Committee on Animal Research Ethics and conducted in accordance with the ‘Principles of Laboratory Animal Care’.18
Animal preparation Thirty-eight female Danish Landrace (crossbred Yorkshire/Duroc) pigs, 35–42 kg, were fasted overnight, but allowed free access to water. Anesthesia was induced with 20 mg intravenous (i.v.) midazolam and 250 mg i.v. s-ketamine, and maintained with a continuous infusion of fentanyl 60 μg/ kg/h and midazolam 6 mg/kg/h. The animals were mechanically ventilated with a positive endexpiratory pressure of 5 cm H2O, FiO2 0.35 and tidal volume 10 ml/kg. The ventilation rate was adjusted to maintain PaCO2 between 5.0–6.0 kPa. Body temperature was maintained at 38.5°C. All animals received a bolus of Ringer’s acetate (20 ml/kg) before surgical procedures. Sheaths were inserted into the carotid artery, the external jugular vein and the femoral vessels. A pulmonary artery catheter (CCOmbo; Edwards Lifesciences, Irvine, CA, USA) was inserted to monitor cardiac output (CO), core temperature, mean pulmonary artery pressure (MPAP), central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP) (Vigilance Monitor; Edwards Lifesciences). Pulmonary vascular resistance (PVR) (dyn s/cm5) was calculated using the equation: 80 × (MPAP − PCWP) / (CO). A pigtail catheter (Medtronic, Minneapolis, MN, USA) was placed in the left ventricle via the femoral artery sheath for injection of neutron-activated microspheres. After surgery, all animals were given boluses of heparin 200 U/kg and 100 U/kg hourly to prevent blood clotting.
Arterial blood gases were analyzed every 0.5 h (ABL 725, Radiometer, Brønshøj, Denmark). All animals rested for 1 h before the experiment started.
Experimental protocol The animals were randomly assigned to four groups: sham + saline (n = 5), sham + ALM/AL (n = 5), hemorrhage control (n = 11), hemorrhage + ALM/AL (n = 9) (Fig. 1). Sham animals underwent anesthesia, surgery and instrumentation only. Hemorrhage was performed by drawing blood via the femoral artery catheter at a rate of 2.15 ml/ kg/min for 7 min, and then 1.15 ml/kg/min, until target MAP of 35 mmHg was reached.19 A MAP of 30–35 mmHg was maintained for 90 min by withdrawing or infusing shed blood. The shed blood was stored in a citrated glucose solution at 37°C. After hemorrhagic shock, pre-hospital resuscitation and transport was simulated by resuscitating the hemorrhage control and hemorrhage + ALM/AL animals with Ringer’s acetate at an infusion rate of 120 ml/min to reach a target MAP of 50 mmHg for 30 min. The hemorrhage control group recieved 20 ml of 7.5% NaCl, while the hemorrhage + ALM/AL group received an infusion of 20 ml 7.5% NaCl supplemented with ALM/AL (Fig. 1). The ALM contained adenosine (0.23 mg/kg), lidocaine (0.64 mg/kg) and MgCl2 (0.41 mg/kg). If MAP decreased below 50 mmHg, 30-ml boluses of Ringer’s acetate were infused. After 30 min of hypotensive resuscitation, 75% of the shed blood volume was re-infused at a rate of 60 ml/min, and the pigs were observed for 6 h after reperfusion. During infusion of shed blood, 10 ml of 0.9% NaCl containing a higher concentration of adenosine (0.82 mg/ kg) and lidocaine (1.66 mg/kg) was given as a bolus injection in the hemorrhage + ALM/AL group, while hemorrhage control pigs received 10 ml of
Sham + saline (n = 5) Hemorrhage control (n = 11) HS
NS
Sham + ALM/AL (n = 5) Hemorrhage + ALM/AL (n = 9) ALM –60 min Baseline
1016
0
45
90 Fluid
AL 0 3060 120 180 240 300360 min
Shock Fluid Permissive hypotension
Reinfusion of shed blood + observation
Fig. 1. Experimental protocol. HS (hypertonic saline, 7.5% NaCl, 20 ml); NS (normal saline, 10 ml); adenosine, lidocaine and magnesium [ALM; 20 ml of 7.5% NaCl + adenosine (0.23 mg/kg), lidocaine (0.64 mg/kg) and MgCl2 (0.41 mg/kg)]; adenosine and lidocaine [AL; 10 ml 0.9% NaCl + adenosine (0.82 mg/kg), lidocaine (1.66 mg/kg)].
Hemorrhagic shock and acute lung injury
0.9% NaCl. All four groups received a maintenance infusion of Ringer’s acetate at a rate of 10 ml/kg/h starting 1 h after return of shed blood.
Lung tissue blood flow Lung tissue blood flow was measured using 15-μm neutron-activated microspheres (BioPhysics Assay Laboratory, Inc, Worcester, MA, USA) as previously described.20,21 Measurements were at baseline, during hemorrhage, during fluid resuscitation with Ringer’s acetate and 1.5 h after reperfusion (resuscitation with blood). Lung tissue blood flow is presented as an average of the upper and lower lobes in ml/min/g of tissue.
PaO2/FiO2 ratio PaO2/FiO2 ratio was calculated based on PaO2 and FiO2.
Wet/dry lung tissue weight ratio Representative samples of the right upper and lower lung lobes were weighed and placed in an oven at 70°C until no further weight loss. Ratios are presented as a mean of the upper and lower lobes.
Bronchoalveolar lavage (BAL) Performed as a single-cycle procedure with a fiberoptic bronchoscope (Olympus Medical Systems Corp, Tokyo, Japan).22 By visual guidance, 60 ml of 37°C saline was instilled into the left lower lobe over 15 s and immediately withdrawn. The last 5 ml of the withdrawn BAL fluid (BALF) was spun at 440 g for 10 min at 4°C. The supernatant was frozen at −80°C. BALF was analyzed for total protein (biuret method), urea, lactate dehydrogenase (LDH) and microalbumin according to Siemens® Clinical Methods for ADVIA 1650 (Tarrytown, NY, USA), and for malondialdehyde (MDA) by a colorimetric assay (Northwest Life Science Specialties, Vancouver, WA, USA). Standards and samples were determined at 532 nm, corrected for both substrate (substrate blind) and unspecific background at 570 nm. Intra-assay precision was 10.7% (coefficient of variance, CV). BALF cytokine analysis (IL-1β, IL-6, IL-8, IL-10 and TNF-α) was performed with a multiplex assay (Procarta® Porcine Cytokine Assay Kit; Panomics, Fremont, CA, USA), according to the manufacturer’s instructions and as previously reported.23,24 Detection limits (pg/ml) were IL-1β (3.14), IL-6 (2.28), IL-8 (34.4), IL-10 (102.36) and TNF-α (2.75). Inter-assay variations averaged 10–19%, and intra-assay variations averaged 3–9%.
Lung tissue cytokines IL-6, IL-8, IL-10 and TNF-α from the right middle lobe were analyzed using an in-house time-resolved immunofluorometric assay (TRIFMA) based on porcine-specific matched pairs of anti-cytokine antibodies in combination with recombinant cytokine standards (R&D Systems, Minneapolis, MN, USA).25 Detection limits (pg/mg of protein) were IL-6 (19.2), IL-8 (18), IL-10 (20.4) and TNF-α (80.8).
Immunohistochemistry Quantification of neutrophil infiltration and apoptosis in pulmonary tissue was performed. Neutrophil infiltration was evaluated using a rabbit monoclonal myeloperoxidase (MPO) antibody (Clone sp 72, Cell Marque Corp, Rocklin, CA, USA). Amplification was carried out using ultraView Universal DAB amplication system (Ventana Medical Systems, Inc, Tucson, AZ, USA). Apoptosis was quantified using an antibody for activated caspase 3 as described previously.26 Sections from porcine spleen served as positive controls. Stained cells were quantified using light microscopy by an examiner blinded to group assignment. Cells were counted in random non-overlapping microscope fields using 400× magnification (500 × 500 μm). MPO and caspase 3 are presented as cells per 10 and 15 fields of vision, respectively.
Statistics It was predetermined to analyze the temporal data in three phases: (1) the entire study, (2) the fluid resuscitation phase and (3) the blood resuscitation phase (reperfusion). All data were included in the statistical analyses, while graphs and tables were simplified with less time points presented. For continuous variables, a repeated two-way ANOVA was used to analyze data for time-dependency and between-group differences. The assumptions of the models were verified by inspecting scatter plots of the residuals vs. fitted values and normal quantile plots of the residuals. The following variables needed logarithmic transformation: heart rate (HR), MAP, PVR, MPAP and tissue blood flow. All variables were presented on the original scale of measurement. Single endpoint values were compared using a two-way ANOVA. Data not normally distributed were square root transformed to ensure normality. Variables were presented as medians with 95% confidence intervals (CI). Measurements considered not to be normally distributed even after transformation were analyzed by Kruskal–Wallis one-way ANOVA, and presented as median and
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25% and 75% interquartile range (IQR). P-values less than 0.05 were considered statistically significant. The analyses were performed using Sigmaplot 11 (Systat Software, Inc, San Jose, CA, USA) and Stata 11.0 (StataCorp LP, College Station, TX, USA). This study represents an analysis of pulmonaryrelated data collected as part of another study focused on the cardiovascular and renal effects of ALM and AL on hemorrhagic shock.17 For completeness, descriptive data on hemodynamic variables were presented from that study. Data on PVR, pulmonary blood flow, PaO2/FiO2 ratio, wet/dry ratio, BAL, lung cytokines and lung immunohistochemistry have not been published before. Hence, the number of animals included in the present study was based on an a priori power analysis of the primary endpoint from the previous study: Absolute difference in fluid treatment during hypotensive resuscitation between controls and ALM group (Diff: 9 ml/kg; SD = 5.8; α = 0.05 and β = 0.1, n = 9). Power calculation was also performed with respect to the primary outcome of this study’s wet/dry ratio (Diff: 0.3 g; SD = 0.17; α = 0.05 and β = 0.1, n = 7).
Results Blood loss and mortality Blood loss was equal in the hemorrhage groups; mean [95%CI] (ml/kg), hemorrhage control group: 49 [43–56] vs. hemorrhage + ALM/AL group: 49 [44–56], amounting to a total blood loss of 74% of total blood volume.27 Eight pigs were excluded as they developed ventricular fibrillation during the shock phase prior to randomization. Two pigs in the hemorrhage control group developed irreversible shock during the fluid resuscitation phase and could not be resuscitated with Ringer’s acetate.
Hemodynamic variables Less crystalloid fluid was necessary to maintain the target MAP in the hemorrhage + ALM/AL group (median [95%CI] (ml/kg): 24.7 [19.4–31.5]) compared with the hemorrhage control group (41.5 [27.7–61.8]). After re-infusion of shed blood, both hemorrhage groups showed an equal decline in MAP over time (Table 1). During hemorrhage, MPAP decreased in both groups. Infusion of Ringer’s acetate and shed blood resulted in a substantial increase in MPAP without group differences. MPAP returned to baseline value after resuscitation. Sham animals were hemodynamically stable throughout the study (Table 1).
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PVR increased in the hemorrhage groups prior to randomization without group differences; median [95%CI] (dyn s/cm5), sham + saline: 164 [124–215], sham + ALM/AL: 186 [142–245], hemorrhage control: 524 [424–646], hemorrhage + ALM/AL: 461 [376–565], (P < 0.0001). During fluid resuscitation and permissive hypotension, PVR was significantly higher in the hemorrhage + ALM/AL group compared with the hemorrhage control group; hemorrhage + ALM/AL: 1291 [1053–1583] vs. hemorrhage control: 1052 [838–1321], (P = 0.04), (Fig. 2). The higher PVR was related to a higher MPAP and lower cardiac index in the hemorrhage + ALM/AL group. No difference in PVR existed between hemorrhage groups after resuscitation with blood.
PaO2/FiO2 ratio PaO2/FiO2 ratio decreased significantly during the 90 min shock phase in both hemorrhage groups compared with sham groups (P < 0.0001). There was no difference between hemorrhage groups. PaO2/ FiO2 returned to sham group levels after blood resuscitation. All four groups showed a progressive decrease in PaO2/FiO2 ratio during the reperfusion phase with no group differences (P = 0.28, Fig. 3).
Pulmonary blood flow Lung parenchymal tissue blood flow decreased by 90% in the hemorrhage control group and by 94% in the hemorrhage + ALM/AL group during hemorrhage (no significant group difference). In both hemorrhage groups, blood flow was decreased 1.5 h after reperfusion (blood resuscitation) compared with baseline levels (P < 0.05). Treatment with ALM and AL had no effect on pulmonary blood flow (Table 1).
Wet/dry lung tissue ratio In the two hemorrhage groups, wet/dry tissue ratio increased significantly compared with the sham groups (P = 0.011).There was no difference between the two hemorrhage groups (P = 0.311, Fig. 4).
Bronchoalveolar lavage In all four groups, 45–65% of infused volumes were recovered, with no difference between groups (P = 0.46, Table 2). Urea BAL concentration was significantly lower in the two ALM/AL-treated groups as opposed to the non-treated groups (P = 0.04, Table 2). No effect of ALM/AL was observed on plasma urea levels.
Median [CI]
Median [CI]
Median [CI]
Statistics
1.09 [0.52–2.28] 0.56 [0.29–1.11]
17 [14–20] 20 [17–23] 18 [16–21] 18 [16–21]
54 [46–63] 59 [50–68] 57 [51–64] 58 [52–65]
94 [86–104] 98 [89–107] 90 [84–96] 91 [85–98]
Baseline
0.11 [0.05–0.24] 0.03 [0.02–0.07]
17 [14–20] 18 [16–22] 15 [13–17] 14 [12–15]
53 [45–62] 57 [49–67] 138 [123–155] 138 [123–155]
91 [83–100] 97 [88–106] 34 [32–37] 34 [31–36]
45 min
Shock
17 [14–20] 19 [16–22] 18 [16–20] 19 [16–21]
55 [47–64] 57 [49–67] 176 [156–198] 178 [159–201]
89 [81–98] 92 [83–101] 31 [29–34] 33 [31–35]
90 min
0.11 [0.05–0.24] 0.08 [0.04–0.16]
18 [16–22] 21 [18–24] 33 [29–37] 35 [31–39]
55 [47–65] 60 [51–70] 150 [133–168] 155 [137–174]
87 [79–96] 94 [86–103] 52 [48–56] 55 [51–59]
Fluid
Resuscitation
18 [15–21] 19 [16–23] 24 [22–28] 23 [21–27]
55 [47–64] 60 [51–70] 161 [143–181] 169 [150–190]
85 [78–94] 90 [82–99] 51 [47–54] 54 [50–58]
Blood
0.30 [0.14–0.62] 0.21 [0.11–0.41]
17 [15–21] 18 [16–22] 32 [28–36] 30 [27–34]
53 [46–63] 59 [50–69] 109 [97–122] 105 [93–118]
83 [75–91] 90 [82–99] 78 [73–84] 81 [76–87]
0.5 h
17 [15–20] 19 [16–22] 24 [21–27] 23 [20–26]
54 [46–63] 60 [52–71] 90 [80–101] 89 [79–100]
82 [75–90] 89 [81–98] 70 [65–75] 75 [70–80]
1h
After reperfusion
17 [15–21] 18 [16–23] 20 [17–22] 18 [16–20]
54 [46–64] 56 [48–65] 73 [65–82] 78 [70–88]
77 [70–85] 82 [72–87] 67 [62–72] 68 [63–73]
3h
19 [16–22] 21 [17–25] 20 [17–22] 18 [16–21]
52 [45–61] 57 [49–67] 73 [65–82] 72 [64–81]
73 [66–80] 76 [86–83] 58 [54–62] 62 [58–67]
6h
AL, adenosine and lidocaine; ALM, adenosine, lidocaine and magnesium; CI, confidence interval; HR, heart rate; MAP, mean arterial pressure; MPAP, mean pulmonary artery pressure.
Blood flow (ml/min/g of tissue) Hemorrhage control Median [CI] Hemorrhage + ALM/AL
Hemorrhage + ALM/AL
Hemorrhage control
Sham + ALM/AL
MPAP (mmHg) Sham + saline
Hemorrhage + ALM/AL
Hemorrhage control
Sham + ALM/AL
HR (bpm) Sham + saline
Hemorrhage + ALM/AL
Hemorrhage control
Sham + ALM/AL
MAP (mmHg) Sham + saline
Hemodynamic variables.
Table 1
Hemorrhagic shock and acute lung injury
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Fig. 2. Pulmonary vascular resistance (PVR) shown as median and 95%CI of groups. Adenosine, lidocaine and magnesium (ALM) treatment during fluid resuscitation augmented the increase in PVR caused by hemorrhagic shock #P < 0.05 for hemorrhage control vs. sham + saline, ¤P < 0.05 for hemorrhage + ALM/AL vs. hemorrhage control (analysis of variance).
Fig. 4. Wet/dry ratio shown as mean of upper and lower lobe wet/dry ratio for each individual, together with group mean. Ratio was higher in hemorrhage groups compared with sham groups; ¤P < 0.05 (two-way analysis of variance). Mean [95% confidence interval], sham + saline: 5.95 [5.70–6.19], sham + ALM/AL: 5.81 [5.56–6.06] vs. hemorrhage control: 6.06 [5.94–6.19], hemorrhage + ALM/AL: 6.07 [5.95–6.19]; (P = 0.011). AL, adenosine and lidocaine; ALM, adenosine, lidocaine and magnesium.
Protein, albumin, LDH and MDA concentrations in BALF were not significantly different between groups (Table 2). The levels of IL-1β and IL-8 were not significantly different between groups. The cytokines IL-6, IL-10 and TNF-α in BALF were below the detection limits.
Tissue cytokines
Fig. 3. Partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2)/FiO2 ratio shown as median and 95%CI of groups. PaO2/FiO2 ratio decreased significantly during the 90 min shock phase in both hemorrhage groups compared with sham groups (P < 0.0001). All four groups decreased in PaO2/FiO2 ratio from baseline to end of observation. For both non-treated groups, this reached statistical significance (P < 0.05 for time group effect, paired t-test), whereas ALM/AL-treated groups tended only to decrease.
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Shock and resuscitation were not associated with increased lung tissue levels of cytokines in the sham groups. IL-8 was significantly lower in the hemorrhage control group; median [25–75%IQR] (pg/mg of protein), 61.64 [48.3–73.0] compared with sham + saline 119.51 [90.0–281.4]. There was no difference in IL-6, IL-10 and TNF-α.
Immunohistochemistry MPO-stained cell count tended to be lower in the ALM/AL-treated groups; mean [95%CI] (cells/10 fields of vision), hemorrhage + ALM/AL: 173 [90– 256], hemorrhage control: 212 [131–293], sham + ALM/AL: 103 [39–167], sham + saline: 176 [87–265]; (P = 0.13). Activated caspase 3 was not significantly different among groups; (P = 0.28).
*Statistical significant lower urea levels i ALM/AL treated groups. ANOVA, analysis of variance; AL, adenosine and lidocaine; ALM, adenosine, lidocaine and magnesium; CI, confidence interval.
0.41
0.45
0.04*
0.65
0.12
0.26
0.44
0.63
0.37
Median [CI]
0.32 [0.11–0.53] 15.50 [–1.07–32.07] 0.65 [0.26–1.04] 0.14 [0.01–0.28] 5.00 [–0.66–10.66]
0.28 [–0.03–0.59] 17.20 [4.90–29.50] 0.45* [0.32–0.58] 0.14 [0.01–0.27] 10.00 [4.59–15.41]
0.42 [0.21–0.63] 26.30 [11.13–41.47] 0.63 [0.35–0.91] 0.26 [0.12–0.41] 12.00 [5.03–18.97]
0.35 [0.21–0.49] 20.60 [8.71–32.49] 0.44* [0.26–0.63] 0.093 [–0.06–0.24] 13.00 [1.06–24.94]
0.35
Discussion
Protein (g/l) Albumin (mg/l) Urea (mM) MDA (μM) LDH (U/l)
ALM/AL (two-way ANOVA)
0.46 (one-way ANOVA) 28.2 [16.7–39.6]
Hemorrhage + ALM/AL Hemorrhage control
34.3 [27.6–41.1] 31.8 [16.9–46.7]
Sham + ALM/AL Sham + saline
Mean [CI] BAL fluid (ml)
Bronchoalveolar lavage (BAL) fluid analysis.
Table 2
38.7 [24.7–52.7]
P-value
Hemorrhage (two-way ANOVA)
Hemorrhagic shock and acute lung injury
In the present study, resuscitation after hemorrhage did not cause ALI or significant pulmonary inflammation. There was a small but significant increase in lung water (wet/dry weight ratio) that did not affect oxygenation. Treatment with ALM and AL during fluid resuscitation and return of shed blood, respectively, were associated with a significant increase in PVR. Furthermore, in both ALM/AL groups, urea diffusion across the alveolar epithelia decreased. However, because hemorrhagic shock and resuscitation caused only minor alterations in pulmonary function in the current study, the effect of ALM/AL on ALI remains unanswered. Few studies have investigated the impact of hemorrhagic shock without concomitant tissue injury in a large-animal model. Regarding pulmonary function and inflammation, results are conflicting.11,28–31 The inconsistent findings may relate to the different animal models of hemorrhage and resuscitation.32–34 Moreover, there is a lack of a consensus of which features or outcome variables define ALI in the experimental animal model.9 In the present study of hemorrhage and resuscitation, we evaluated ALI with an extensive palette of features characterizing ALI, according to an international consensus workshop9 encompassing histology, alveolar capillary barrier permeability, inflammatory response and physiological dysfunction evidence. Furthermore, we used a model of severe hemorrhage with a realistic resuscitation protocol with permissive hypotension. Permissive hypotensive resuscitation is gaining acceptance in the initial phase after severe hemorrhage.35,36 The decrease in the PaO2/FiO2 ratio during hemorrhage can be explained by a change in the distribution of blood within the lung and a mismatch between ventilation and perfusion.37 Roesner et al.10 found a marked decrease in PaO2/ FiO2 ratio 3–5 h after hemorrhagic shock in a pressure-controlled pig model, indicating ALI. This is in contrast to our study, despite an average blood loss of 74% in our study compared with 60% in the study by Roesner et al. The difference may relate to the more extensive surgery for instrumentation (laparotomy). Although not intended, the surgical trauma may have acted as a second hit in addition to reperfusion injury.38,39 Another explanation for the lack of an effect of hemorrhage on pulmonary function could be the protective effect of hypotensive resuscitation, reducing volume overloading and subsequent develop-
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ment of ALI. Treatment with ALM/AL reduced epithelial leakage (BALF urea concentration) regardless of hemorrhagic shock because the reduction was also present in the sham ALM/AL group. This indicates that in the present study the impact of anesthesia and mechanical ventilation exceeds the impact of hemorrhage and resuscitation. This observation is substantiated by the decrease in PaO2/FiO2 ratio seen in all four groups during the experiment. PVR increased significantly when pigs were subjected to hemorrhagic shock, as found by other investigators,11,40,41 and decreased to baseline levels after resuscitation. The increase in PVR was due to a lower CO and PCWP and a higher MPAP. The more pronounced incline in PVR in the hemorrhage + ALM/AL is in accordance with the known vasoconstricting effects of adenosine in the pulmonary circulation,42,43 but the increase in PVR could also be related to the lower volume of crystalloid fluid during resuscitation in the ALM/AL group. The augmented increase in PVR in the hemorrhage + ALM/AL group did not affect oxygenation (PaO2/FiO2 ratio) or wet/dry ratio and was only temporary.
Limitations This study used pressure-controlled bleeding without tissue injury, which is clearly different from the clinical setting. The lack of concomitant tissue trauma avoids the triggering and elaboration of local and systemic inflammatory responses that may remotely prime the pulmonary inflammatory state of the lungs or cause direct injury to the lungs. In addition, the 6 h observation period after reperfusion might be too short to for fulminant ALI to develop, although other studies have demonstrated the presence of ALI early after hemorrhage.10 Heparin has been reported to possess antiinflammatory properties;44,45 hence, we cannot rule out that heparin may have reduced the inflammatory response in the current model. In conclusion, the minimal degree pulmonary damage found in this study questions whether global ischemia–reperfusion injury induced by hemorrhage without concomitant trauma and resuscitation is sufficient to elicit ALI in a large-animal model. Whether treatment with ALM/AL can prevent pulmonary inflammation and subsequent ALI will require adding a second hit, e.g. tissue trauma. Conflicts of interests: Geoffrey P. Dobson is the inventor of the ALM technology in cardiac surgery and preservation including trauma and infection;
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PCT patents pending. The remaining authors declare no competing interests. Funding: This work was supported by the Tryg Foundation, Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis’ Foundation, Aase and Ejner Danielsens Foundation, Holger and Ruth Hesses Foundation, the Danish Society of Anesthesiology and Intensive Care Medicine Foundation, A. P. Moeller Foundation for the Advancement of Medical Science, The Danish Medical Association Research Fund / The Søren Segels & Johanne Wiibroe Segels Fund, Helga and Peter Kornings Foundation and Health Research Fund of Central Denmark Region, Viborg, Denmark.
References 1. Heckbert SR, Vedder NB, Hoffman W, Winn RK, Hudson LD, Jurkovich GJ, Copass MK, Harlan JM, Rice CL, Maier RV. Outcome after hemorrhagic shock in trauma patients. J Trauma 1998; 45: 545–9. 2. Kauvar DS, Lefering R, Wade CE. Impact of hemorrhage on trauma outcome: an overview of epidemiology, clinical presentations, and therapeutic considerations. J Trauma 2006; 60: S3–11. 3. Durham RM, Moran JJ, Mazuski JE, Shapiro MJ, Baue AE, Flint LM. Multiple organ failure in trauma patients. J Trauma 2003; 55: 608–16. 4. Tsukamoto T, Chanthaphavong RS, Pape HC. Current theories on the pathophysiology of multiple organ failure after trauma. Injury 2010; 41: 21–6. 5. Regel G, Grotz M, Weltner T, Sturm JA, Tscherne H. Pattern of organ failure following severe trauma. World J Surg 1996; 20: 422–9. 6. Kobbe P, Stoffels B, Schmidt J, Tsukamoto T, Gutkin DW, Bauer AJ, Pape HC. IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock. Cytokine 2009; 45: 26–31. 7. Vincenzi R, Cepeda LA, Pirani WM, Sannomyia P, Rocha ESM, Cruz RJ Jr. Small volume resuscitation with 3% hypertonic saline solution decrease inflammatory response and attenuates end organ damage after controlled hemorrhagic shock. Am J Surg 2009; 198: 407–14. 8. Kanagawa F, Takahashi T, Inoue K, Shimizu H, Omori E, Morimatsu H, Maeda S, Katayama H, Nakao A, Morita K. Protective effect of carbon monoxide inhalation on lung injury after hemorrhagic shock/resuscitation in rats. J Trauma 2010; 69: 185–94. 9. Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS, Kuebler WM. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 2011; 44: 725–38. 10. Roesner JP, Petzelbauer P, Koch A, Tran N, Iber T, Vagts DA, Scheeren TW, Vollmar B, Noldge-Schomburg GE, Zacharowski K. Bbeta15-42 (FX06) reduces pulmonary, myocardial, liver, and small intestine damage in a pig model of hemorrhagic shock and reperfusion. Crit Care Med 2009; 37: 598–605. 11. Moosa HH, Peitzman AB, Borovetz HS, Steed DL, Webster MW. Pulmonary function after hemorrhagic shock in pigs. Curr Surg 1987; 44: 199–201.
Hemorrhagic shock and acute lung injury 12. Dobson GP. Membrane polarity: a target for myocardial protection and reduced inflammation in adult and pediatric cardiothoracic surgery. J Thorac Cardiovasc Surg 2010; 140: 1213–7. 13. Onorati F, Santini F, Dandale R, Ucci G, Pechlivanidis K, Menon T, Chiominto B, Mazzucco A, Faggian G. ‘Polarizing’ microplegia improves cardiac cycle efficiency after CABG for unstable angina. Int J Cardiol 2013; 167: 2739–46. 14. Shi W, Jiang R, Dobson GP, Granfeldt A, Vinten-Johansen J. The nondepolarizing, normokalemic cardioplegia formulation adenosine-lidocaine (adenocaine) exerts anti-neutrophil effects by synergistic actions of its components. J Thorac Cardiovasc Surg 2012; 143: 1167–75. 15. Letson HL, Dobson GP. Ultra-small intravenous bolus of 7.5% NaCl/Mg2+ with adenosine and lidocaine improves early resuscitation outcome in the rat after severe hemorrhagic shock in vivo. J Trauma 2011; 71: 708–19. 16. Letson HL, Pecheniuk NM, Mhango LP, Dobson GP. Reversal of acute coagulopathy during hypotensive resuscitation using small-volume 7.5% NaCl adenocaine and Mg2+ in the rat model of severe hemorrhagic shock. Crit Care Med 2012; 40: 2417–22. 17. Granfeldt A, Nielsen TK, Solling C, Hyldebrandt JA, Frokiaer J, Wogensen L, Dobson GP, Vinten-Johansen J, Tonnesen E. Adenocaine and Mg(2+) reduce fluid requirement to maintain hypotensive resuscitation and improve cardiac and renal function in a porcine model of severe hemorrhagic shock. Crit Care Med 2012; 40: 3013–25. 18. Clark JD, Gebhart GF, Gonder JC, Keeling ME, Kohn DF. Special report: the 1996 guide for the care and use of laboratory animals. ILAR J 1997; 38: 41–8. 19. Frankel DA, Acosta JA, Anjaria DJ, Porcides RD, Wolf PL, Coimbra R, Hoyt DB. Physiologic response to hemorrhagic shock depends on rate and means of hemorrhage. J Surg Res 2007; 143: 276–80. 20. Zhao ZQ, Nakamura M, Wang NP, Wilcox JN, Shearer S, Guyton RA, Vinten-Johansen J. Administration of adenosine during reperfusion reduces injury of vascular endothelium and death of myocytes. Coron Artery Dis 1999; 10: 617–28. 21. Reinhardt CP, Dalhberg S, Tries MA, Marcel R, Leppo JA. Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique. Am J Physiol Heart Circ Physiol 2001; 280: H108–16. 22. Peterson BT, Griffith DE, Tate RW, Clancy SJ. Single-cycle bronchoalveolar lavage to determine solute concentrations in epithelial lining fluid. Am Rev Respir Dis 1993; 147: 1216–22. 23. Solling C, Christensen AT, Krag S, Frokiaer J, Wogensen L, Krog J, Tonnesen EK. Erythropoietin administration is associated with short-term improvement in glomerular filtration rate after ischemia-reperfusion injury. Acta Anaesthesiol Scand 2011; 55: 185–95. 24. Granfeldt A, Hvas CL, Graversen JH, Christensen PA, Petersen MD, Anton G, Svendsen P, Solling C, Etzerodt A, Tonnesen E, Moestrup SK, Moller HJ. Targeting dexamethasone to macrophages in a porcine endotoxemic model. Crit Care Med 2013; 41: e309–18. 25. Brix-Christensen V, Gjedsted J, Andersen SK, Vestergaard C, Nielsen J, Rix T, Nyboe R, Andersen NT, Larsson A, Schmitz O, Tonnesen E. Inflammatory response during hyperglycemia and hyperinsulinemia in a porcine endotoxemic model: the contribution of essential organs. Acta Anaesthesiol Scand 2005; 49: 991–8. 26. Solling C, Christensen AT, Nygaard U, Krag S, Frokiaer J, Wogensen L, Krog J, Tonnesen EK. Erythropoietin does not attenuate renal dysfunction or inflammation in a porcine model of endotoxemia. Acta Anaesthesiol Scand 2011; 55: 411–21.
27. Hannon JP, Bossone CA, Rodkey WG. Splenic red cell sequestration and blood volume measurements in conscious pigs. Am J Physiol 1985; 248: R293–301. 28. Xanthos TT, Balkamou XA, Stroumpoulis KI, Pantazopoulos IN, Rokas GI, Agrogiannis GD, Troupis GT, Demestiha TD, Skandalakis PN. A model of hemorrhagic shock and acute lung injury in Landrace-Large White Swine. Comp Med 2011; 61: 158–62. 29. Peitzman AB. Hemorrhagic shock. Curr Probl Surg 1995; 32: 925–1002. 30. Sato H, Tanaka T, Kita T, Tanaka N. A quantitative study of lung dysfunction following haemorrhagic shock in rats. Int J Exp Pathol 2010; 91: 267–75. 31. Yang CH, Tsai PS, Wang TY, Huang CJ. Dexmedetomidineketamine combination mitigates acute lung injury in haemorrhagic shock rats. Resuscitation 2009; 80: 1204– 10. 32. Tsukamoto T, Pape HC. Animal models for trauma research: what are the options? Shock 2009; 31: 3–10. 33. Lomas-Niera JL, Perl M, Chung CS, Ayala A. Shock and hemorrhage: an overview of animal models. Shock 2005; 24 (Suppl. 1): 33–9. 34. Hauser CJ. Preclinical models of traumatic, hemorrhagic shock. Shock 2005; 24 (Suppl. 1): 24–32. 35. Rossaint R, Bouillon B, Cerny V, Coats TJ, Duranteau J, Fernandez-Mondejar E, Hunt BJ, Komadina R, Nardi G, Neugebauer E, Ozier Y, Riddez L, Schultz A, Stahel PF, Vincent JL, Spahn DR. Management of bleeding following major trauma: an updated European guideline. Crit Care 2010; 14: R52. 36. Bickell WH, Wall MJ Jr, Pepe PE, Martin RR, Ginger VF, Allen MK, Mattox KL. Immediate versus delayed fluid resuscitation for hypotensive patients with penetrating torso injuries. N Engl J Med 1994; 331: 1105–9. 37. West JB, Dollery CT, Naimark A. Distribution of blood flow in isolated lung; relation to vascular and alveolar pressures. J Appl Physiol 1964; 19: 713–24. 38. Claridge JA, Weed AC, Enelow R, Young JS. Laparotomy potentiates cytokine release and impairs pulmonary function after hemorrhage and resuscitation in mice. J Trauma 2001; 50: 244–52. 39. Kilicoglu B, Eroglu E, Kilicoglu SS, Kismet K, Eroglu F. Effect of abdominal trauma on hemorrhagic shock-induced acute lung injury in rats. World J Gastroenterol 2006; 12: 3593–6. 40. Bredenberg CE, Nomoto S, Webb WR. Pulmonary and systemic hemodynamics during hemorrhagic shock in baboons. Ann Surg 1980; 192: 86–94. 41. Buckberg GD, Lipman CA, Hahn JA, Smith MJ, Hennessen JA. Pulmonary changes following hemorrhagic shock and resuscitation in baboons. J Thorac Cardiovasc Surg 1970; 59: 450–60. 42. Thomas T, Marshall JM. The role of adenosine in hypoxic pulmonary vasoconstriction in the anaesthetized rat. Exp Physiol 1993; 78: 541–3. 43. Kucukhuseyin C, Silan C, Akbas N, Payat M, Oncel H, Barlas A. On the mechanisms of adenosine induced pulmonary vasoconstriction in rats. J Basic Clin Physiol Pharmacol 1997; 8: 287–99. 44. Brown RA, Lever R, Jones NA, Page CP. Effects of heparin and related molecules upon neutrophil aggregation and elastase release in vitro. Br J Pharmacol 2003; 139: 845– 53. 45. Lever R, Hoult JR, Page CP. The effects of heparin and related molecules upon the adhesion of human polymorphonuclear leucocytes to vascular endothelium in vitro. Br J Pharmacol 2000; 129: 533–40.
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© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd ACTA ANAESTHESIOLOGICA SCANDINAVICA
doi: 10.1111/aas.12355
Pulmonary function after hemorrhagic shock and resuscitation in a porcine model T. K. Nielsen1,2, C. L. Hvas1, G. P. Dobson3, E. Tønnesen1 and A. Granfeldt1,4
1 Department of Anesthesiology and Intensive Care Medicine, Aarhus University Hospital, Aarhus, Departments of 2Anesthesiology, 4Internal Medicine, Regional Hospital of Randers, Randers, Denmark and 3Heart and Trauma Research Laboratory, Department of Physiology and Pharmacology, James Cook University, Townsville, QLD, Australia
Background: Hemorrhagic shock may trigger an inflammatory response and acute lung injury. The combination adenosine, lidocaine (AL) plus Mg2+ (ALM) has organ-protective and anti-inflammatory properties with potential benefits in resuscitation.The aims of this study were to investigate: (1) pulmonary function and inflammation after hemorrhagic shock; (2) the effects of ALM/AL on pulmonary function and inflammation. Methods: Pigs (38 kg) were randomized to: sham + saline (n = 5); sham + ALM/AL (n = 5); hemorrhage control (n = 11); and hemorrhage + ALM/AL (n = 9). Hemorrhage animals bled to a mean arterial pressure (MAP) of 35 mmHg for 90 min, received resuscitation with Ringer’s acetate and 20 ml of 7.5% NaCl with ALM to a minimum MAP of 50 mmHg, after 30 min shed blood and 0.9% NaCl with AL were infused. Hemorrhage controls did not receive ALM/AL. Primary endpoints were pulmonary wet/dry ratio, PaO2/FiO2 ratio (partial pressure of arterial oxygen to the fraction of inspired oxygen), cytokine and protein measurements in bronchoalveolar lavage fluid (BALF)
and lung tissue, neutrophil invasion and blood flow in lung tissue. Results: In the hemorrhage groups, wet/dry ratio increased significantly compared with the sham groups. PaO2/FiO2 ratio decreased during shock but normalized after resuscitation. BALF did not indicate significant pulmonary inflammation, oxidative stress or increased permeability. Intervention with ALM caused a temporary increase in pulmonary vascular resistance and reduced urea diffusion across the alveolar epithelia, but had no effect on wet/dry ratio. Conclusion: Hemorrhagic shock and resuscitation did not cause acute lung injury or pulmonary inflammation. The question whether ALM/AL has the potential to attenuate acute lung injury is unanswered.
H
cardioplegic agent to arrest the heart and protect the myocardium during cardiac surgery.12,13 The combination of adenosine and lidocaine (AL) reduces activation and priming of neutrophils.14 Furthermore, in a small-animal model of hemorrhagic shock and hypotensive resuscitation, a bolus administration of 7.5% NaCl with ALM improved survival, hemodynamic function and reversed acute coagulopathy.15,16 This study is based on material from an animal series, from which results on cardiac and renal functioning after hemorrhagic shock and resuscitation have previously been published.17 The aims of this study were to investigate: (1) pulmonary function and inflammation after hemorrhagic shock and resuscitation in a largeanimal model; and (2) the effects of ALM/AL on pulmonary function and inflammation.
emorrhage is a major cause of trauma deaths.1,2 Resuscitation with fluid and blood restores blood pressure but leaves organs susceptible to reperfusion injury. This may result in multiple organ failure (MOF), the leading cause of late postinjury mortality.3,4 Acute lung injury (ALI) is often the first stage of MOF.5 Previous small-animal studies have described the presence of pulmonary inflammation following hemorrhagic shock alone and subsequently investigated the effects of anti-inflammatory interventions.6–8 Few studies have been carried out in large-animal models, in which the relevant pathophysiology and hemodynamic variables can be described in detail,9 and they have revealed conflicting results.10,11 The combination of adenosine, lidocaine and magnesium (ALM) is currently in use as a
Accepted for publication 25 May 2014 © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
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Methods The study was approved by the National Committee on Animal Research Ethics and conducted in accordance with the ‘Principles of Laboratory Animal Care’.18
Animal preparation Thirty-eight female Danish Landrace (crossbred Yorkshire/Duroc) pigs, 35–42 kg, were fasted overnight, but allowed free access to water. Anesthesia was induced with 20 mg intravenous (i.v.) midazolam and 250 mg i.v. s-ketamine, and maintained with a continuous infusion of fentanyl 60 μg/ kg/h and midazolam 6 mg/kg/h. The animals were mechanically ventilated with a positive endexpiratory pressure of 5 cm H2O, FiO2 0.35 and tidal volume 10 ml/kg. The ventilation rate was adjusted to maintain PaCO2 between 5.0–6.0 kPa. Body temperature was maintained at 38.5°C. All animals received a bolus of Ringer’s acetate (20 ml/kg) before surgical procedures. Sheaths were inserted into the carotid artery, the external jugular vein and the femoral vessels. A pulmonary artery catheter (CCOmbo; Edwards Lifesciences, Irvine, CA, USA) was inserted to monitor cardiac output (CO), core temperature, mean pulmonary artery pressure (MPAP), central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP) (Vigilance Monitor; Edwards Lifesciences). Pulmonary vascular resistance (PVR) (dyn s/cm5) was calculated using the equation: 80 × (MPAP − PCWP) / (CO). A pigtail catheter (Medtronic, Minneapolis, MN, USA) was placed in the left ventricle via the femoral artery sheath for injection of neutron-activated microspheres. After surgery, all animals were given boluses of heparin 200 U/kg and 100 U/kg hourly to prevent blood clotting.
Arterial blood gases were analyzed every 0.5 h (ABL 725, Radiometer, Brønshøj, Denmark). All animals rested for 1 h before the experiment started.
Experimental protocol The animals were randomly assigned to four groups: sham + saline (n = 5), sham + ALM/AL (n = 5), hemorrhage control (n = 11), hemorrhage + ALM/AL (n = 9) (Fig. 1). Sham animals underwent anesthesia, surgery and instrumentation only. Hemorrhage was performed by drawing blood via the femoral artery catheter at a rate of 2.15 ml/ kg/min for 7 min, and then 1.15 ml/kg/min, until target MAP of 35 mmHg was reached.19 A MAP of 30–35 mmHg was maintained for 90 min by withdrawing or infusing shed blood. The shed blood was stored in a citrated glucose solution at 37°C. After hemorrhagic shock, pre-hospital resuscitation and transport was simulated by resuscitating the hemorrhage control and hemorrhage + ALM/AL animals with Ringer’s acetate at an infusion rate of 120 ml/min to reach a target MAP of 50 mmHg for 30 min. The hemorrhage control group recieved 20 ml of 7.5% NaCl, while the hemorrhage + ALM/AL group received an infusion of 20 ml 7.5% NaCl supplemented with ALM/AL (Fig. 1). The ALM contained adenosine (0.23 mg/kg), lidocaine (0.64 mg/kg) and MgCl2 (0.41 mg/kg). If MAP decreased below 50 mmHg, 30-ml boluses of Ringer’s acetate were infused. After 30 min of hypotensive resuscitation, 75% of the shed blood volume was re-infused at a rate of 60 ml/min, and the pigs were observed for 6 h after reperfusion. During infusion of shed blood, 10 ml of 0.9% NaCl containing a higher concentration of adenosine (0.82 mg/ kg) and lidocaine (1.66 mg/kg) was given as a bolus injection in the hemorrhage + ALM/AL group, while hemorrhage control pigs received 10 ml of
Sham + saline (n = 5) Hemorrhage control (n = 11) HS
NS
Sham + ALM/AL (n = 5) Hemorrhage + ALM/AL (n = 9) ALM –60 min Baseline
1016
0
45
90 Fluid
AL 0 3060 120 180 240 300360 min
Shock Fluid Permissive hypotension
Reinfusion of shed blood + observation
Fig. 1. Experimental protocol. HS (hypertonic saline, 7.5% NaCl, 20 ml); NS (normal saline, 10 ml); adenosine, lidocaine and magnesium [ALM; 20 ml of 7.5% NaCl + adenosine (0.23 mg/kg), lidocaine (0.64 mg/kg) and MgCl2 (0.41 mg/kg)]; adenosine and lidocaine [AL; 10 ml 0.9% NaCl + adenosine (0.82 mg/kg), lidocaine (1.66 mg/kg)].
Hemorrhagic shock and acute lung injury
0.9% NaCl. All four groups received a maintenance infusion of Ringer’s acetate at a rate of 10 ml/kg/h starting 1 h after return of shed blood.
Lung tissue blood flow Lung tissue blood flow was measured using 15-μm neutron-activated microspheres (BioPhysics Assay Laboratory, Inc, Worcester, MA, USA) as previously described.20,21 Measurements were at baseline, during hemorrhage, during fluid resuscitation with Ringer’s acetate and 1.5 h after reperfusion (resuscitation with blood). Lung tissue blood flow is presented as an average of the upper and lower lobes in ml/min/g of tissue.
PaO2/FiO2 ratio PaO2/FiO2 ratio was calculated based on PaO2 and FiO2.
Wet/dry lung tissue weight ratio Representative samples of the right upper and lower lung lobes were weighed and placed in an oven at 70°C until no further weight loss. Ratios are presented as a mean of the upper and lower lobes.
Bronchoalveolar lavage (BAL) Performed as a single-cycle procedure with a fiberoptic bronchoscope (Olympus Medical Systems Corp, Tokyo, Japan).22 By visual guidance, 60 ml of 37°C saline was instilled into the left lower lobe over 15 s and immediately withdrawn. The last 5 ml of the withdrawn BAL fluid (BALF) was spun at 440 g for 10 min at 4°C. The supernatant was frozen at −80°C. BALF was analyzed for total protein (biuret method), urea, lactate dehydrogenase (LDH) and microalbumin according to Siemens® Clinical Methods for ADVIA 1650 (Tarrytown, NY, USA), and for malondialdehyde (MDA) by a colorimetric assay (Northwest Life Science Specialties, Vancouver, WA, USA). Standards and samples were determined at 532 nm, corrected for both substrate (substrate blind) and unspecific background at 570 nm. Intra-assay precision was 10.7% (coefficient of variance, CV). BALF cytokine analysis (IL-1β, IL-6, IL-8, IL-10 and TNF-α) was performed with a multiplex assay (Procarta® Porcine Cytokine Assay Kit; Panomics, Fremont, CA, USA), according to the manufacturer’s instructions and as previously reported.23,24 Detection limits (pg/ml) were IL-1β (3.14), IL-6 (2.28), IL-8 (34.4), IL-10 (102.36) and TNF-α (2.75). Inter-assay variations averaged 10–19%, and intra-assay variations averaged 3–9%.
Lung tissue cytokines IL-6, IL-8, IL-10 and TNF-α from the right middle lobe were analyzed using an in-house time-resolved immunofluorometric assay (TRIFMA) based on porcine-specific matched pairs of anti-cytokine antibodies in combination with recombinant cytokine standards (R&D Systems, Minneapolis, MN, USA).25 Detection limits (pg/mg of protein) were IL-6 (19.2), IL-8 (18), IL-10 (20.4) and TNF-α (80.8).
Immunohistochemistry Quantification of neutrophil infiltration and apoptosis in pulmonary tissue was performed. Neutrophil infiltration was evaluated using a rabbit monoclonal myeloperoxidase (MPO) antibody (Clone sp 72, Cell Marque Corp, Rocklin, CA, USA). Amplification was carried out using ultraView Universal DAB amplication system (Ventana Medical Systems, Inc, Tucson, AZ, USA). Apoptosis was quantified using an antibody for activated caspase 3 as described previously.26 Sections from porcine spleen served as positive controls. Stained cells were quantified using light microscopy by an examiner blinded to group assignment. Cells were counted in random non-overlapping microscope fields using 400× magnification (500 × 500 μm). MPO and caspase 3 are presented as cells per 10 and 15 fields of vision, respectively.
Statistics It was predetermined to analyze the temporal data in three phases: (1) the entire study, (2) the fluid resuscitation phase and (3) the blood resuscitation phase (reperfusion). All data were included in the statistical analyses, while graphs and tables were simplified with less time points presented. For continuous variables, a repeated two-way ANOVA was used to analyze data for time-dependency and between-group differences. The assumptions of the models were verified by inspecting scatter plots of the residuals vs. fitted values and normal quantile plots of the residuals. The following variables needed logarithmic transformation: heart rate (HR), MAP, PVR, MPAP and tissue blood flow. All variables were presented on the original scale of measurement. Single endpoint values were compared using a two-way ANOVA. Data not normally distributed were square root transformed to ensure normality. Variables were presented as medians with 95% confidence intervals (CI). Measurements considered not to be normally distributed even after transformation were analyzed by Kruskal–Wallis one-way ANOVA, and presented as median and
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25% and 75% interquartile range (IQR). P-values less than 0.05 were considered statistically significant. The analyses were performed using Sigmaplot 11 (Systat Software, Inc, San Jose, CA, USA) and Stata 11.0 (StataCorp LP, College Station, TX, USA). This study represents an analysis of pulmonaryrelated data collected as part of another study focused on the cardiovascular and renal effects of ALM and AL on hemorrhagic shock.17 For completeness, descriptive data on hemodynamic variables were presented from that study. Data on PVR, pulmonary blood flow, PaO2/FiO2 ratio, wet/dry ratio, BAL, lung cytokines and lung immunohistochemistry have not been published before. Hence, the number of animals included in the present study was based on an a priori power analysis of the primary endpoint from the previous study: Absolute difference in fluid treatment during hypotensive resuscitation between controls and ALM group (Diff: 9 ml/kg; SD = 5.8; α = 0.05 and β = 0.1, n = 9). Power calculation was also performed with respect to the primary outcome of this study’s wet/dry ratio (Diff: 0.3 g; SD = 0.17; α = 0.05 and β = 0.1, n = 7).
Results Blood loss and mortality Blood loss was equal in the hemorrhage groups; mean [95%CI] (ml/kg), hemorrhage control group: 49 [43–56] vs. hemorrhage + ALM/AL group: 49 [44–56], amounting to a total blood loss of 74% of total blood volume.27 Eight pigs were excluded as they developed ventricular fibrillation during the shock phase prior to randomization. Two pigs in the hemorrhage control group developed irreversible shock during the fluid resuscitation phase and could not be resuscitated with Ringer’s acetate.
Hemodynamic variables Less crystalloid fluid was necessary to maintain the target MAP in the hemorrhage + ALM/AL group (median [95%CI] (ml/kg): 24.7 [19.4–31.5]) compared with the hemorrhage control group (41.5 [27.7–61.8]). After re-infusion of shed blood, both hemorrhage groups showed an equal decline in MAP over time (Table 1). During hemorrhage, MPAP decreased in both groups. Infusion of Ringer’s acetate and shed blood resulted in a substantial increase in MPAP without group differences. MPAP returned to baseline value after resuscitation. Sham animals were hemodynamically stable throughout the study (Table 1).
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PVR increased in the hemorrhage groups prior to randomization without group differences; median [95%CI] (dyn s/cm5), sham + saline: 164 [124–215], sham + ALM/AL: 186 [142–245], hemorrhage control: 524 [424–646], hemorrhage + ALM/AL: 461 [376–565], (P < 0.0001). During fluid resuscitation and permissive hypotension, PVR was significantly higher in the hemorrhage + ALM/AL group compared with the hemorrhage control group; hemorrhage + ALM/AL: 1291 [1053–1583] vs. hemorrhage control: 1052 [838–1321], (P = 0.04), (Fig. 2). The higher PVR was related to a higher MPAP and lower cardiac index in the hemorrhage + ALM/AL group. No difference in PVR existed between hemorrhage groups after resuscitation with blood.
PaO2/FiO2 ratio PaO2/FiO2 ratio decreased significantly during the 90 min shock phase in both hemorrhage groups compared with sham groups (P < 0.0001). There was no difference between hemorrhage groups. PaO2/ FiO2 returned to sham group levels after blood resuscitation. All four groups showed a progressive decrease in PaO2/FiO2 ratio during the reperfusion phase with no group differences (P = 0.28, Fig. 3).
Pulmonary blood flow Lung parenchymal tissue blood flow decreased by 90% in the hemorrhage control group and by 94% in the hemorrhage + ALM/AL group during hemorrhage (no significant group difference). In both hemorrhage groups, blood flow was decreased 1.5 h after reperfusion (blood resuscitation) compared with baseline levels (P < 0.05). Treatment with ALM and AL had no effect on pulmonary blood flow (Table 1).
Wet/dry lung tissue ratio In the two hemorrhage groups, wet/dry tissue ratio increased significantly compared with the sham groups (P = 0.011).There was no difference between the two hemorrhage groups (P = 0.311, Fig. 4).
Bronchoalveolar lavage In all four groups, 45–65% of infused volumes were recovered, with no difference between groups (P = 0.46, Table 2). Urea BAL concentration was significantly lower in the two ALM/AL-treated groups as opposed to the non-treated groups (P = 0.04, Table 2). No effect of ALM/AL was observed on plasma urea levels.
Median [CI]
Median [CI]
Median [CI]
Statistics
1.09 [0.52–2.28] 0.56 [0.29–1.11]
17 [14–20] 20 [17–23] 18 [16–21] 18 [16–21]
54 [46–63] 59 [50–68] 57 [51–64] 58 [52–65]
94 [86–104] 98 [89–107] 90 [84–96] 91 [85–98]
Baseline
0.11 [0.05–0.24] 0.03 [0.02–0.07]
17 [14–20] 18 [16–22] 15 [13–17] 14 [12–15]
53 [45–62] 57 [49–67] 138 [123–155] 138 [123–155]
91 [83–100] 97 [88–106] 34 [32–37] 34 [31–36]
45 min
Shock
17 [14–20] 19 [16–22] 18 [16–20] 19 [16–21]
55 [47–64] 57 [49–67] 176 [156–198] 178 [159–201]
89 [81–98] 92 [83–101] 31 [29–34] 33 [31–35]
90 min
0.11 [0.05–0.24] 0.08 [0.04–0.16]
18 [16–22] 21 [18–24] 33 [29–37] 35 [31–39]
55 [47–65] 60 [51–70] 150 [133–168] 155 [137–174]
87 [79–96] 94 [86–103] 52 [48–56] 55 [51–59]
Fluid
Resuscitation
18 [15–21] 19 [16–23] 24 [22–28] 23 [21–27]
55 [47–64] 60 [51–70] 161 [143–181] 169 [150–190]
85 [78–94] 90 [82–99] 51 [47–54] 54 [50–58]
Blood
0.30 [0.14–0.62] 0.21 [0.11–0.41]
17 [15–21] 18 [16–22] 32 [28–36] 30 [27–34]
53 [46–63] 59 [50–69] 109 [97–122] 105 [93–118]
83 [75–91] 90 [82–99] 78 [73–84] 81 [76–87]
0.5 h
17 [15–20] 19 [16–22] 24 [21–27] 23 [20–26]
54 [46–63] 60 [52–71] 90 [80–101] 89 [79–100]
82 [75–90] 89 [81–98] 70 [65–75] 75 [70–80]
1h
After reperfusion
17 [15–21] 18 [16–23] 20 [17–22] 18 [16–20]
54 [46–64] 56 [48–65] 73 [65–82] 78 [70–88]
77 [70–85] 82 [72–87] 67 [62–72] 68 [63–73]
3h
19 [16–22] 21 [17–25] 20 [17–22] 18 [16–21]
52 [45–61] 57 [49–67] 73 [65–82] 72 [64–81]
73 [66–80] 76 [86–83] 58 [54–62] 62 [58–67]
6h
AL, adenosine and lidocaine; ALM, adenosine, lidocaine and magnesium; CI, confidence interval; HR, heart rate; MAP, mean arterial pressure; MPAP, mean pulmonary artery pressure.
Blood flow (ml/min/g of tissue) Hemorrhage control Median [CI] Hemorrhage + ALM/AL
Hemorrhage + ALM/AL
Hemorrhage control
Sham + ALM/AL
MPAP (mmHg) Sham + saline
Hemorrhage + ALM/AL
Hemorrhage control
Sham + ALM/AL
HR (bpm) Sham + saline
Hemorrhage + ALM/AL
Hemorrhage control
Sham + ALM/AL
MAP (mmHg) Sham + saline
Hemodynamic variables.
Table 1
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Fig. 2. Pulmonary vascular resistance (PVR) shown as median and 95%CI of groups. Adenosine, lidocaine and magnesium (ALM) treatment during fluid resuscitation augmented the increase in PVR caused by hemorrhagic shock #P < 0.05 for hemorrhage control vs. sham + saline, ¤P < 0.05 for hemorrhage + ALM/AL vs. hemorrhage control (analysis of variance).
Fig. 4. Wet/dry ratio shown as mean of upper and lower lobe wet/dry ratio for each individual, together with group mean. Ratio was higher in hemorrhage groups compared with sham groups; ¤P < 0.05 (two-way analysis of variance). Mean [95% confidence interval], sham + saline: 5.95 [5.70–6.19], sham + ALM/AL: 5.81 [5.56–6.06] vs. hemorrhage control: 6.06 [5.94–6.19], hemorrhage + ALM/AL: 6.07 [5.95–6.19]; (P = 0.011). AL, adenosine and lidocaine; ALM, adenosine, lidocaine and magnesium.
Protein, albumin, LDH and MDA concentrations in BALF were not significantly different between groups (Table 2). The levels of IL-1β and IL-8 were not significantly different between groups. The cytokines IL-6, IL-10 and TNF-α in BALF were below the detection limits.
Tissue cytokines
Fig. 3. Partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2)/FiO2 ratio shown as median and 95%CI of groups. PaO2/FiO2 ratio decreased significantly during the 90 min shock phase in both hemorrhage groups compared with sham groups (P < 0.0001). All four groups decreased in PaO2/FiO2 ratio from baseline to end of observation. For both non-treated groups, this reached statistical significance (P < 0.05 for time group effect, paired t-test), whereas ALM/AL-treated groups tended only to decrease.
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Shock and resuscitation were not associated with increased lung tissue levels of cytokines in the sham groups. IL-8 was significantly lower in the hemorrhage control group; median [25–75%IQR] (pg/mg of protein), 61.64 [48.3–73.0] compared with sham + saline 119.51 [90.0–281.4]. There was no difference in IL-6, IL-10 and TNF-α.
Immunohistochemistry MPO-stained cell count tended to be lower in the ALM/AL-treated groups; mean [95%CI] (cells/10 fields of vision), hemorrhage + ALM/AL: 173 [90– 256], hemorrhage control: 212 [131–293], sham + ALM/AL: 103 [39–167], sham + saline: 176 [87–265]; (P = 0.13). Activated caspase 3 was not significantly different among groups; (P = 0.28).
*Statistical significant lower urea levels i ALM/AL treated groups. ANOVA, analysis of variance; AL, adenosine and lidocaine; ALM, adenosine, lidocaine and magnesium; CI, confidence interval.
0.41
0.45
0.04*
0.65
0.12
0.26
0.44
0.63
0.37
Median [CI]
0.32 [0.11–0.53] 15.50 [–1.07–32.07] 0.65 [0.26–1.04] 0.14 [0.01–0.28] 5.00 [–0.66–10.66]
0.28 [–0.03–0.59] 17.20 [4.90–29.50] 0.45* [0.32–0.58] 0.14 [0.01–0.27] 10.00 [4.59–15.41]
0.42 [0.21–0.63] 26.30 [11.13–41.47] 0.63 [0.35–0.91] 0.26 [0.12–0.41] 12.00 [5.03–18.97]
0.35 [0.21–0.49] 20.60 [8.71–32.49] 0.44* [0.26–0.63] 0.093 [–0.06–0.24] 13.00 [1.06–24.94]
0.35
Discussion
Protein (g/l) Albumin (mg/l) Urea (mM) MDA (μM) LDH (U/l)
ALM/AL (two-way ANOVA)
0.46 (one-way ANOVA) 28.2 [16.7–39.6]
Hemorrhage + ALM/AL Hemorrhage control
34.3 [27.6–41.1] 31.8 [16.9–46.7]
Sham + ALM/AL Sham + saline
Mean [CI] BAL fluid (ml)
Bronchoalveolar lavage (BAL) fluid analysis.
Table 2
38.7 [24.7–52.7]
P-value
Hemorrhage (two-way ANOVA)
Hemorrhagic shock and acute lung injury
In the present study, resuscitation after hemorrhage did not cause ALI or significant pulmonary inflammation. There was a small but significant increase in lung water (wet/dry weight ratio) that did not affect oxygenation. Treatment with ALM and AL during fluid resuscitation and return of shed blood, respectively, were associated with a significant increase in PVR. Furthermore, in both ALM/AL groups, urea diffusion across the alveolar epithelia decreased. However, because hemorrhagic shock and resuscitation caused only minor alterations in pulmonary function in the current study, the effect of ALM/AL on ALI remains unanswered. Few studies have investigated the impact of hemorrhagic shock without concomitant tissue injury in a large-animal model. Regarding pulmonary function and inflammation, results are conflicting.11,28–31 The inconsistent findings may relate to the different animal models of hemorrhage and resuscitation.32–34 Moreover, there is a lack of a consensus of which features or outcome variables define ALI in the experimental animal model.9 In the present study of hemorrhage and resuscitation, we evaluated ALI with an extensive palette of features characterizing ALI, according to an international consensus workshop9 encompassing histology, alveolar capillary barrier permeability, inflammatory response and physiological dysfunction evidence. Furthermore, we used a model of severe hemorrhage with a realistic resuscitation protocol with permissive hypotension. Permissive hypotensive resuscitation is gaining acceptance in the initial phase after severe hemorrhage.35,36 The decrease in the PaO2/FiO2 ratio during hemorrhage can be explained by a change in the distribution of blood within the lung and a mismatch between ventilation and perfusion.37 Roesner et al.10 found a marked decrease in PaO2/ FiO2 ratio 3–5 h after hemorrhagic shock in a pressure-controlled pig model, indicating ALI. This is in contrast to our study, despite an average blood loss of 74% in our study compared with 60% in the study by Roesner et al. The difference may relate to the more extensive surgery for instrumentation (laparotomy). Although not intended, the surgical trauma may have acted as a second hit in addition to reperfusion injury.38,39 Another explanation for the lack of an effect of hemorrhage on pulmonary function could be the protective effect of hypotensive resuscitation, reducing volume overloading and subsequent develop-
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ment of ALI. Treatment with ALM/AL reduced epithelial leakage (BALF urea concentration) regardless of hemorrhagic shock because the reduction was also present in the sham ALM/AL group. This indicates that in the present study the impact of anesthesia and mechanical ventilation exceeds the impact of hemorrhage and resuscitation. This observation is substantiated by the decrease in PaO2/FiO2 ratio seen in all four groups during the experiment. PVR increased significantly when pigs were subjected to hemorrhagic shock, as found by other investigators,11,40,41 and decreased to baseline levels after resuscitation. The increase in PVR was due to a lower CO and PCWP and a higher MPAP. The more pronounced incline in PVR in the hemorrhage + ALM/AL is in accordance with the known vasoconstricting effects of adenosine in the pulmonary circulation,42,43 but the increase in PVR could also be related to the lower volume of crystalloid fluid during resuscitation in the ALM/AL group. The augmented increase in PVR in the hemorrhage + ALM/AL group did not affect oxygenation (PaO2/FiO2 ratio) or wet/dry ratio and was only temporary.
Limitations This study used pressure-controlled bleeding without tissue injury, which is clearly different from the clinical setting. The lack of concomitant tissue trauma avoids the triggering and elaboration of local and systemic inflammatory responses that may remotely prime the pulmonary inflammatory state of the lungs or cause direct injury to the lungs. In addition, the 6 h observation period after reperfusion might be too short to for fulminant ALI to develop, although other studies have demonstrated the presence of ALI early after hemorrhage.10 Heparin has been reported to possess antiinflammatory properties;44,45 hence, we cannot rule out that heparin may have reduced the inflammatory response in the current model. In conclusion, the minimal degree pulmonary damage found in this study questions whether global ischemia–reperfusion injury induced by hemorrhage without concomitant trauma and resuscitation is sufficient to elicit ALI in a large-animal model. Whether treatment with ALM/AL can prevent pulmonary inflammation and subsequent ALI will require adding a second hit, e.g. tissue trauma. Conflicts of interests: Geoffrey P. Dobson is the inventor of the ALM technology in cardiac surgery and preservation including trauma and infection;
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PCT patents pending. The remaining authors declare no competing interests. Funding: This work was supported by the Tryg Foundation, Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis’ Foundation, Aase and Ejner Danielsens Foundation, Holger and Ruth Hesses Foundation, the Danish Society of Anesthesiology and Intensive Care Medicine Foundation, A. P. Moeller Foundation for the Advancement of Medical Science, The Danish Medical Association Research Fund / The Søren Segels & Johanne Wiibroe Segels Fund, Helga and Peter Kornings Foundation and Health Research Fund of Central Denmark Region, Viborg, Denmark.
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T. K. Nielsen et al. Address: Torben Kær Nielsen Department of Anesthesiology and Intensive Care Medicine Aarhus University Hospital Nørrebrogade 44, Building 1C 1st Floor DK-8000 Aarhus Denmark e-mail: [email protected]
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