Appendix 1: DETAILED MATERIALS AND METHODS
Animal Model
Animal use adhered to principles stated in the Guide for Care and Use of Laboratory Animals and procedures were performed under protocols approved by the institutional committee for care and use of animals at the University of Michigan (USA). Twenty-three domestic cross breed swine weighing 50+/- 5 kg were prophylactically treated with tulathromycin (5mg/kg IM) by the supplier one week prior to shipment and pigs were given at least 48 hours prior to use for recovery from transport stress.
Food was withheld overnight prior to surgery but water was provided ad libitum. Sedation was achieved by intramuscular injection of 2.2 mg/kg xylazine hydrochloride and 6.6 mg/kg of tiletamine- zolazapam (Telazol® Zoetis Inc., Kalamazoo, MI) then pigs were transported to the surgical suite, orotracheally intubated and maintained under anesthesia by inhalation of 1-3% isoflurane in oxygen.
Volume controlled mechanical ventilation (Narkomed 2B - North American Drager, Telford, PA) was used throughout the experiment targeting PaCO2: 35-45mmHg, Tidal volume: 8-10 mL/Kg,
respiratory rate 8-10 breaths per minute, and SaO2 100%. Five lead electrocardiogram pads were placed for cardiac monitoring. Pigs received and 1 g of nafcillin sodium and 120 mg of gentamicin sulphate as antibiotic prophylaxis and lidocaine hydrochloride (1-2 mg/kg IV) was administered immediately prior to sternotomy to limit cardiac arrhythmias. Under sterile conditions pigs were instrumented for right atrial to aortic CPB and hemodynamic monitoring. A perivascular flow probe (COnfidence Flowprobes, Transonic Systems Inc.) was fitted to the main pulmonary artery to
measure cardiac output and 18g teflon catheters (Angiocath, Bekton Dickinson, USA) were secured in the femoral artery, pulmonary artery and left atrium and connected to pressure transducers to measure respective luminal pressures. Parameters were monitored with a Marquette Solar 8000 Patient Monitor (Marquette Medical Systems USA, Milwaukee, WI). The urinary bladder was
surgically accessed through a suprapubic incision and attached to a closed collection system. Core temperature was measured using a thermistor within the rectum.
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Cardiopulmonary bypass (CPB): The right atrium (RA) was cannulated with a two-stage venous cannula 32-36/40-46 Fr MC2® (Medtronic Inc. Minneapolis, MN), the distal ascending aorta with a DLP® 14-16 Fr arterial cannula (Medtronic Inc. Minneapolis, MN), and the aortic root with14 Ga Medtronic DLP® (Medtronic Inc. Minneapolis, MN) for cardioplegia delivery and left ventricular
venting. A clinical CPB circuit comprised of a 3.5 L Capiox Advance FX15 hard-shell venous reservoir (Sarns, Terumo Inc., Somerset, NJ), a multi-flow roller pump (Sarns, Terumo Inc., Somerset, NJ), a Capiox Advance FX15 hollow-fiber oxygenator with integrated arterial filter (Sarns, Terumo Inc., Somerset, NJ), and a heat exchanger (Sarns, 3M Inc., Minneapolis, MN), connected by Tygon 3/8 in ID poly-vinyl chloride tubing (Saint-Grobain Corporation, Malvern, PA) was primed with one liter of Lactated Ringer’s Solution (Baxter Healthcare Corporation, Deerfield, IL) supplemented with 50 mEq of sodium bicarbonate (Hospira Inc., Lake Forest, IL) and 10,000 units of heparin (Sargent
Pharmaceuticals Inc., Schaumberg, IL) and recirculated within the extracorporeal circuit to 39 °C.
After obtaining baseline measurements and samples, unfractionated sodium heparin (300 IU/kg IV) was administered to achieve an activated clotting time (ACT) >600 sec and CPB was initiated. Non- pulsatile perfusion was maintained at 2.4 -3.0 L/min/m2 body surface area with moderate hypothermia (33 ± 1○C) and a targeted mean arterial pressure of 50 ± 5 mmHg by modification of flow and/or infusion of phenylephrine up to 50 ug/min. During CPB, ventilation was reduced to 4 breaths per minute with a tidal volume of 2 ml/kg to prevent complete atelectasis. Sixty minutes into bypass, the ascending aorta was cross clamped and anterograde high potassium, cold blood cardioplegia
administered every 15 minutes. The cross clamp was removed after 45 minutes, pigs were warmed to 37○C and electrocardioversion was performed as needed to restore sinus rhythm. CPB continued for an additional 75 minutes with a gradual weaning off support then cannulas were removed, a
mediastinal drain placed and the chest wall was apposed around the monitoring equipment.
Replacement fluid was administered at 5ml/kg/h during instrumentation, CPB, and the post-operative observation period with additional boluses of 0.9% NaCl solution and up to total of 20mL/kg of 6%
hydroxyethlyltarch given as needed to maintain baseline cardiac filling pressures. Phenylephrine (50- 26
27 28 29 30 31 32 33 34 35 36 37
38 39 40 41 42 43 44 45 46 47 48 49 50 51
200ug/min) and/or norepinephrine (2-4 ug/min) were administered when required to try to maintain a mean arterial pressure of 60 ± 5 mmHg and cardiac output of ≥ 2.0 L/min post CPB. Glucose and electrolytes were supplemented based on arterial blood gas analysis of serum electrolytes. Pigs were monitored for 5 hours post CPB and then euthanized by intravenous overdose of pentobarbital (Euthasol®,100mg/kg, Virbac USA) for harvest of vital organs.
L-MOD Therapy: L-MOD therapy was accomplished using an SCD within a citrated extracorporeal blood circuit. The L-MOD device consisted of a 25 cm x 4 cm cylindrical polycarbonate shell
containing approximately 4,000 hollow porous polysulfone synthetic fibers.Blood flowed across the outer surface of the fibers providing contact with a fiber surface area of 1.4 m2. Blood flow rates of 100-200 mL/min were maintained in the 300 mL L-MOD circuit using a peristaltic blood pump (Fresenius 2008H, Walnut Creek, CA). A low ionized calcium (iCa) environment, essential to the leukocyte inhibitory action of the therapy, was achieved within the L-MOD using RCA. A 4% citrate solution (anticoagulant citrate dextrose solution formula A, Fenwal Inc., Lake Zurich, IL) was infused into the L-MOD circuit (200-400 mL/h), to reduce iCa to <0.40 mM. Per standard RCA protocol, a 2%
calcium chloride solution (American Reagent, Shirley, NY) was infused into the line returning blood to the general circulation. Systemic iCa levels were measured and maintained in the physiologic range (0.9-1.1 mmol/L) by adjusting the rate of calcium infusion.
Cardiovascular function was assessed using hemodynamic parameters, IV fluid requirements and dependence on inotropic and vasopressor medications. Data capture occurred using Microsoft Excel 2010 (Microsoft Inc., Redmond, Washington) and included; systemic systolic, diastolic, and mean arterial pressures; pulmonary systolic, diastolic, and mean pulmonary artery pressures; left atrial pressure; central venous pressure; cardiac output; flow and line pressure of the extracorporeal circuit while on bypass; tidal volume, respiratory rate, positive end expiratory pressure, peak airway
pressure, mean airway pressure, percentage of oxygen in the inspired gas, and end-tidal CO2;
diuresis; temperature; volume and type of intravenous fluids and all drugs administered.
52 53 54 55 56
57 58 59 60 61 62 63 64 65 66 67 68
69 70 71 72 73 74 75 76
Blood Sampling
All sample draws accessed systemic arterialized blood. During CPB, blood was sampled from the extracorporeal circuit post oxygenator. Arterial blood gas analysis included measurement of electrolytes (Radiometer ABL800FLEX, Copenhagen, Denmark) and was performed every 30-60 minutes. Total manual leukocyte counts were determined from whole blood using the Unopette®
system (BD Biosciences, Bedford MA) with manual differentials determined from blood smears after ethanol fixation and Wright staining, counting 300 cells per slide under oil immersion. Activated Clotting time (ACT) was measured hourly using a Hemochron 801 (International Technodyne Corp., Edison, NJ)
Neutrophil and Monocyte Acute Activation Analysis
Blood samples were obtained via catheter and immediately placed on ice. Cooled whole blood samples were incubated with fluorescein isothiocyantate (FITC)-conjugated anti-porcine CD11R3 (ABD Serotec, clone: 2F4/11) and Alexafluor 647 conjugated CD172a/SWC3 (BD 561499) for 15 minutes, followed by fixation/ red cell lysis with BD Facs Lyse solution. Within 24 hours, samples were rinsed, resuspended in PBS with 0.1% albumin and analyzed via flow cytometry (BD Accuri C6).
Values are presented as Mean Fluorescence Intensity (MFI) ± SE. Along with scatter profiles, CD172a was used to identify cells of the MO family.
Immediately after each study using an L-MOD, adherent cells were eluted and quantified as previous described [12]. Cells recovered from the L-MOD were processed for CD11R3 expression similar to blood. Additionally, to further assess the activation status of the cells associated with the membranes, eluted NE were evaluated for their ability to undergo apoptosis in vitro. The cell suspension was cultured in media containing fetal bovine serum for 36 hours prior to staining with FITC-conjugated Annexin V (AbCam, ab14085). Analysis by gated flow cytometry determined the percentage of Annexin V negative cells, which are resistant to apoptosis and reflective of activated cells.
77 78 79 80 81 82 83 84 85
86 87 88 89 90 91 92 93
94 95 96 97 98 99 100
Markers of Systemic Inflammation and End-Organ Injury
Measurement of systemic levels of representative pro- and anti-inflammatory cytokines, TNFα and IL- 10, respectively, and the myocardial injury marker, cardiac troponin-I, was performed on EDTA plasma using commercial porcine specific ELISA kits following the manufacturer’s directions (R&D Systems; TNFα, PTA00; and IL-10, P1000 and Life Diagnostics CTNI-9-US).
Cardiovascular function was assessed using hemodynamic parameters, IV fluid requirements and dependence on inotropic and vasopressor medications. Data capture occurred using Microsoft Excel 2010 (Microsoft Inc., Redmond, Washington) and included; systemic systolic, diastolic, and mean arterial pressures; pulmonary systolic, diastolic, and mean pulmonary artery pressures; left atrial pressure; central venous pressure; cardiac output; flow and line pressure of the extracorporeal circuit while on bypass; tidal volume, respiratory rate, positive end expiratory pressure, peak airway
pressure, mean airway pressure, percentage of oxygen in the inspired gas, and end-tidal CO2; diuresis; temperature; volume and type of intravenous fluids and all drugs administered.
The dose of vasoactive-vasopressor medications administered was used to generate an Ionotropic score using the modified formula: Ionotropic score = (noradrenaline dose x 100) + (phenylephrine dose x 100), wherein all doses are expressed as ug/kg/min. As described by Cruz et al, a dose response relationship was then calculated as the vasopressor dependency index, the ratio of inotropic score to mean arterial pressure (MAP); the higher the score, the greater the vasopressor requirement.
Urine output was recorded hourly. Samples were obtained from a 60-minute urine collection,
centrifuged to remove debris and frozen until assay. Urine neutrophil gelatinase-associated lipocalin (NGAL), an early marker of structural acute kidney injury, was measured by ELISA (Alpco BioPorto 54-044).
Partial pressures of oxygen and carbon dioxide in arterial blood and the reported annual atmospheric pressure at the study site were used to calculate the Alveolar to arterial gradient of oxygen (A:a 101
102 103 104 105
106 107 108 109 110 111 112 113
114 115 116 117 118 119
120 121 122 123
124 125
gradient) as an indicator of pulmonary function. Lung edema (based on water content) and
accumulation of activated leukocytes in pulmonary tissue were evaluated as indices of pulmonary injury. Leukocyte accumulation was assessed using immunohistochemistry. Two random sections from each lung lobe were obtained post mortem and cryosectioned into 5um slices that were placed on slides and incubated with goat serum in PBS for 2 hours to minimize nonspecific antibody
adsorption. Sections were then incubated with mouse anti-CD11R3(Manufacturer/# MCA2309 -manufacture?) at 4OC overnight, followed by incubation with 1:200 goat anti-mouse IgG
Alexafluor594 (Manufacturer/#) conjugate for 30 minutes and then counterstained with DAPI. Image J software was used to quantitate percent CD11R3+ area in 10 random 10x images taken with Zeiss Axiovision image capture and normalized to cell number by percent DAPI+ area. Water content of pulmonary tissue was determined by homogenizing the remainder of each lobe, obtaining wet weight of 3 aliquots per lobe then desiccating the samples in an oven at 70○C until no further decrease in weight was detected. Water content was calculated as (wet weight-dry weight)/dry weight.
126 127 128 129 130 131 132 133 134 135 136 137 138
139
140