Preparation of the adsorption column
A polystyrene-based composite fiber reinforced with polypropylene was prepared and chemically modified as described previously [16]. Briefly, the fiber was treated with N-methylol-α-chloroacetamide in a mixture of sulfuric acid and nitrobenzene on ice for 2 h in the presence of paraformaldehyde; then reacted with tetraethylenepentamine in dimethyl sulfoxide (DMSO) for 3 h at 40°C; and then with 4-chlorophenylisocyanate in DMSO for 1 h at 30°C. After thorough washing with DMSO followed by pyrogen-free water, the column was packed with fibers and filled with pyrogen-free saline. The column was then sterilized using gamma ray irradiation (25,000 Gy).
In vitro adsorption of cytokines
Chemically modified fiber of 0.055 cm3 was incubated with 2000 pg/mL IL-8, 2000 pg/mL IL-6 and 100 ng/mL HMGB-1 in 1.65 mL fetal bovine serum (FBS) at 37°C for 2 h with gentle rotation. Concentrations of IL-8 and IL-6 in the supernatant were measured using ELISA (R & D Systems, Minneapolis, MN, USA). HMGB-1 was measured by Shino-Test Corp. (Tokyo, Japan). The adsorption ratio (%) was calculated as [(concentration of blank sample – concentration of test sample) / concentration of blank sample] × 100.
In vitro adsorption of activated inflammatory cells
Lipopolysaccharide (LPS) (E.coli O55-B5, Sigma-Aldrich Co. LLC, St. Louis, MO, USA) was added at 70 EU/mL to heparinized human whole blood samples from a healthy volunteer. After 30 min incubation, the blood was passed at a flow rate of 0.63 mL/min through the adsorption column filled with 0.86 cm3 of fiber. Blood samples were collected at the column inlet and outlet. Activated granulocytes and NPCs were analyzed by flow cytometry. Blood samples were incubated for 15 min at r.t. with APC-anti CD11b (activated) (CBRM1/5, Biolegend, San Diego, CA, USA), BV421-anti-CD62P (AK4, Biolegend), BV510-anti-CD45 (HI30, Biolegend) and FITC-anti CD66b (G10F5, BD Biosciences, Franklin Lakes, NJ, USA). Other blood samples were incubated with isotype controls (Biolegend or BD Biosciences). Red blood cells were lysed and cells were rinsed using PBS(-) and resuspended in buffer. After adding 7-AAD reagent (Biolegend) and Flow-Count (Beckman Coulter, Brea, CA, USA), cells were characterized by BD FACSCanto II (BD Biosciences) and data were analyzed using FlowJo (FlowJo, LLC., Ashland, OR, USA). Neutrophils, the most abundant type of granulocytes, were identified as CD45+ / CD66b+ cells, and NPCs were identified as CD11b (activated)+ / CD62P+ cells among the neutrophils. Because almost all the cells in LPS-treated blood were CD11b (activated)+, blood without LPS was also processed in the same way to show elimination of CD11b (activated)− cells. The absolute number of cells was calculated using Flow-Count. The adsorption ratio (%) was calculated as [(absolute cell number of inlet sample – absolute cell number of outlet sample) / absolute cell number of inlet sample] × 100. For histopathological observation of neutrophils and NPCs, the fiber after contact with blood was fixed in 50% Karnovsky fixative and osmic acid. After imbedding in resin, fiber was sectioned and stained with toluidine blue.
Animal model with lung-protective ventilation
Male New Zealand white rabbits (2.8–3.4 kg) were obtained from Kitayama Labes (Nagano, JP) and fed with a standard diet and water ad libitum. Rabbits were anesthetized by intravenous administration of sodium pentobarbital (30 mg/kg) and anesthesia was maintained by continuous infusion of sodium pentobarbital (8.2 mg/kg/h) and vecuronium bromide (0.06 mg/kg/h) through the jugular vein. After placement in the supine position and local anesthesia with 0.5% lidocaine, tracheotomy was performed and a 16 Fr endotracheal tube was intubated. Rabbits were ventilated using a mechanical ventilator (EVITA300, Drager, Lübeck, DE) in VC-AC mode with FIO2 of 1.0 and PEEP of 2.5 cmH2O. After alveolar recruitment, the respiration rate was adjusted to maintain a tidal volume of 7–8 mL/kg to achieve a blood pH of 7.35–7.45 at baseline. The animals were maintained at a constant body fluid balance by continuous infusion of physiological saline at 2 mL/kg/h from the jugular vein. The carotid artery was cannulated for monitoring arterial blood pressure and heart rate (HR) and for sampling of arterial blood for blood gas analysis, hematology and cytokine measurement. Lung injury was induced by intratracheal (i.t.) administration of 0.04 N HCl followed by 0.05 mg/kg LPS (E.coli O55-B5, Sigma-Aldrich) after a 30 min interval. In the 2 h after LPS administration, arterial blood pH was adjusted and maintained at 7.35 to 7.45 by changing the respiration rate. If end-inspiratory pause was observed, the inspiratory time was adjusted throughout the experiment.
Direct hemoperfusion
Rabbits were randomized in two groups after lung injury: the NOA-001 treated group (n = 7) and a sham group (n = 6) treated with an empty column with the same priming volume as that of NOA-001. The column was connected to the circuit and rinsed with heparinized saline. Unfractionated heparin was injected into the circuit at 60 IU/kg at the beginning of hemoperfusion, and continuously infused at 30–60 IU/kg/h from the anticoagulant injection port. The time of LPS administration was considered to be 0 h. Direct hemoperfusion was started at 15 min and continued for 8 h after LPS administration. Blood access was from the femoral artery to femoral vein and the flow rate was maintained at 5 mL/min using a peristaltic pump. Blood gas parameters (PaO2, PaCO2, pH and lactate) were measured using an i-STAT 200 portable clinical analyzer (Abbott, Chicago, IL, USA) and i-STAT cartridge CG4+ (Abbott) immediately after blood sampling. Hematological parameters (neutrophils, platelets) were measured using an XT-2000i analyzer (Sysmex, Hyogo, JP). Arterial blood pressure and HR were recorded and mean arterial pressure (MAP) was calculated using PowerLab systems (PowerLab8/30, LabChart7, AD Instruments, Sydney, Australia). Ventilation parameters were automatically saved in the ventilator. The oxygenation index (OI) was calculated as mean airway pressure / P/F ratio × 100. The plasma concentration of IL-8 was measured using an ELISA for rabbit IL-8 (Raybio, Norcross, GA, USA), and that of LPS was measured using Limulus ES-II Single Test Wako (Wako, Osaka, JP) and Toxinometer ET-5000 (Wako).
Histopathological analysis
Rabbits were euthanized by exsanguination under anesthesia with pentobarbital at 8 h. The whole lung was removed and inflated with 10% formaldehyde neutral buffer solution to a pressure of 20 cmH2O via the trachea, and then fixed in the same buffer solution. After fixation, the lung was divided into 4 sections and each section was stained with hematoxylin-eosin (HE).
Statistical analyses
All data are presented as the mean ± SEM. Two rabbits in the NOA-001 group and two in the sham group died between 7 and 8 h. The 8 h values of one rabbit in the NOA-001 group were treated as defective data because we were unable to collect a blood sample just before death. The 8 h values of the other three rabbits were substituted by the value measured just before death. Statistical analysis was performed using GraphPad Prism v.8.2.0 (GraphPad Prism Software, Inc., San Diego, CA). For in vitro adsorption of activated inflammatory cells, data were analyzed by one way ANOVA followed by a Dunnett post hoc test. In the in vivo study, baseline values in the NOA-001 and sham groups were compared by unpaired t test. Blood gas parameters, plasma IL-8, blood neutrophil counts, MAP, HR and plasma LPS in the two groups were compared by two way ANOVA with a Sidak post hoc test or Tukey post hoc test. Inlet and outlet levels of plasma IL-8 and blood neutrophils were compared by paired t test.