Biomedicines | Free Full-Text | Potential Mechanisms for Organoprotective Effects of Exogenous Nitric Oxide in an Experimental Study
A single-center prospective randomized controlled study was conducted to identify the probable mechanisms of the organoprotective effects of exogenous NO in modeling operations with CPB, CPB, and CA (CPB + CA). The work was carried out on the basis of the laboratory of critical care medicine, the department of cardiovascular surgery, and the department of anesthesiology and critical care of the Federal State Budgetary Scientific Institution “Tomsk National Research Medical Center of the Russian Academy of Sciences”, Cardiology Research Institute. This study was approved by the Ethics Committee of the Cardiology Research Institute, Tomsk NRMC, Protocol No. 230, dated 28 June 2022.
The study methodology was developed in accordance with international guidelines for randomized controlled trials. The sequence of events and study methodology are presented below.
Randomization was carried out by the method of sealed opaque envelopes. The number of envelopes was equal to the estimated sample size, n = 24. The processing distribution was prepared by an independent operator (a researcher not involved in this study) and hidden in opaque sealed envelopes that were consecutively numbered. Each envelope contained one code name: “CPB”, “CPB + NO”, “CPB + CA”, or “CPB + CA + NO”. On the morning of the experiment, when an envelope was opened, its contents were not disclosed. Thus, the animals were assigned to the group of perioperative NO conditioning with CPB or CPB + CA or to the group of the standard protocol CPB or CPB + CA. All animals were randomized into 4 equal groups of 6 sheep in each group.
2.1. Method of Anesthesia and Cardiopulmonary Bypass
The experiment began with the sevoflurane mask induction of anesthesia in all groups. After reaching the target level of anesthesia, preoperative preparation of shaving and processing of the surgical area was performed. To carry out the induction of anesthesia with the aseptic technique, catheterization of the saphenous vein of the hind limb with an 18 G catheter was performed. General anesthesia was induced by fractional administration of propofol 1% at a dose of 5 mg/kg. While maintaining spontaneous breathing, direct laryngoscopy followed by orotracheal intubation with an endotracheal tube 6.5 mm with an introducer was performed. The endotracheal tube was fixed. Then, mechanical ventilation was performed with a 760 ventilator (Puritan Bennett, KY, USA).
In the CPB + NO and CPB + CA + NO groups, immediately after tracheal intubation, the delivery of nitric oxide was initiated through a modified breathing circuit at a dose of 80 ppm. Throughout the experiment, in order to maintain anesthesia, a continuous intravenous infusion of propofol 1% at a dose of 5 mg/kg/h was performed. Neuromuscular blockade was achieved with pipecuronium bromide at a dose of 0.1 mg/kg. Throughout the experiment, extensive monitoring during anesthesia was used, including electrocardiogram monitoring, invasive blood pressure monitoring, pulse oximetry, continuous monitoring of end-tidal carbon dioxide (etCO2), and thermometry using a BSM-4104A bedside patient monitor (Nihon Kohden, Tokyo, Japan); the urine flow rate was also taken into account. The temperature sensor was placed in the esophagus. The common carotid artery was surgically harvested and catheterized with a 20F catheter. The internal jugular vein underwent cannulation with a double-lumen 7F catheter. Surgical access was performed with right 4th–5th intercostal space thoracotomy.
A cardiopulmonary bypass was performed using anHL20 CPB machine (Maquet, Hechingen, Germany). The CPB machine was connected according to the “aorta—superior vena cava—inferior vena cava” scheme. Mean arterial pressure during CPB was maintained at 50–60 mm Hg.
In groups of animals that did not simulate CA, CPB was performed under normothermic conditions, and esophageal temperature was maintained at a level of 36–36.6 °C. In two other groups of animals with simulated CA, CPB was carried out with hypothermia. After reaching the target esophageal temperature of 30 °C, the descending aorta was occluded and, thus, non-perfusion CA was simulated for 15 min. Next, the descending aorta was unclamped, and then reperfusion and warming to 36.6 °C were carried out. The cumulative duration of CPB in all groups was 90 min. To ensure hypocoagulability during CPB, heparin at a dose of 300 U/kg was used, maintaining the activated clotting time > 450 s.
2.2. Nitric Oxide Conditioning
For nitric oxide delivery, a special device for plasma–chemical synthesis of nitric oxide was used. To ensure safety, NO/NO2 concentrations, as well as the level of methemoglobin (MetHb) in the blood, were continuously monitored throughout the entire experiment. An increase in NO2 to 2 ppm or more was considered critical; when the mentioned concentration was reached, the delivery of NO was supposed to be discontinued.
In the CPB + NO group, NO was delivered immediately after tracheal intubation through the circuit of the ventilator at a dose of 80 ppm, and then at the initiation of CPB, NO was delivered to the extracorporeal circulation circuit at a dose of 80 ppm throughout the entire period of CPB (90 min), and after the restoration of blood flow at the reperfusion stage, NO supply continued through the circuit of the ventilator at a dose of 80 ppm for 1 h (Figure 1).
In the CPB + CA + NO group, NO was delivered immediately after tracheal intubation through the ventilator circuit at a dose of 80 ppm throughout the entire experiment and then, at the start of CPB, NO was delivered to the extracorporeal circulation circuit for 90 min until hypothermic CA was initiated. During CA, when the target body temperature, an esophageal temperature of 30 °C, was reached, the perfusion index decreased to 1 L/min/m2, and the descending aorta was occluded for 15 min, and NO delivery was not performed. When completing CA, NO delivery to the extracorporeal circuit at a dose of 80 ppm was resumed and maintained until normothermia was achieved (the cumulative duration of CPB and CA was 90 min). After weaning from CPB, NO was supplied again through the ventilator circuit at a dose of 80 ppm for 1 h.
NO was delivered through the ventilator circuit into the inhale tube consisting of two circuit tubes connected by an absorber, which was filled with soda lime. The first tube was joined to the absorber with an angled Luer connector. NO was supplied to this connector. Next, the second tube was attached to the absorber. The other end of the second tube was connected to a Y- adapter with a straight Luer connector, from where gas was taken for continuous monitoring of NO and NO2 levels. The inhale and exhale tubes were connected with a Y- adapter. The inhale tube was equipped with a hydrophobic virus–bacterial filter with a Luer connector (Figure 1A).
NO was delivered to the CPB machine through the gas–air line. A gas flow regulating line consisting of two parts connected by two ¼ Luer-lock adapters was attached to the gas blender. The NO supply line was joined to the Luer connector located closer to the gas blender, and the gas sample intake line was connected to the Luer connector located closer to the oxygenator. The other end of the gas flow regulating line was connected to the oxygenator. In the oxygenator, venous blood was saturated with oxygen and NO. Next, the obtained arterial blood was supplied through the arterial line to the aorta (Figure 1B).
Collecting blood samples for biochemical analysis was carried out at the following stages: 1—before the initiation of CPB; 2—at the initiation of CPB; 3—after weaning from CPB.
To measure the glycocalyx degradation marker, heparan sulfate proteoglycan (HSPG), venous blood was collected in BD Vakuteiner tubes (BD, New Jersey, USA), and then centrifugation was performed for 10 min at 3500 rpm. Next, an enzyme-linked immunosorbent assay (ELISA) was performed to quantitatively measure HSPG in vitro in plasma using an ELISA kit (Cloud-Clone Corp, Houston, TX, USA).
To measure the endothelial dysfunction marker, asymmetric dimethylarginine (ADMA), venous blood was collected in BD Vakuteiner (BD, Bergen, NJ, USA) tubes and then centrifuged for 10 min at 3500 rpm. Next, the serum was frozen and stored at −20 °C. Quantitative determination of ADMA was carried out with ELISA using an ADMA Xpress ELISA KR7860 (Immundiagnostik AG, Bensheim, Germany). This assay is based on the competitive enzyme immunoassay technique.
To study erythrocyte deformability, the coefficient of microviscosity and polarity in the areas of lipid–lipid (CMLLI; CPLLI) and protein–lipid interactions (CMPLI; CPPLI) were assessed. To determine the coefficient of microviscosity and polarity of erythrocyte membranes, venous blood was collected immediately after intubation before the initiation of CPB and after weaning from CPB. Blood was collected into vacutainer tubes containing lithium heparin (17 IU/mL) sprayed on the walls. Blood samples were centrifuged at 1500 rpm for 10 min. After removing the plasma, erythrocytes were washed 3 times with cooled saline; each time the erythrocytes were sedimented at 1500 rpm for 10 min. Erythrocyte membranes were obtained by hypoosmotic hemolysis. The amount of total protein in the suspension of erythrocyte shadows was determined with the Micro–Lowry method and Ohnishi S.T. modification using Sigma-Aldrich reagents (Sigma-Aldrich, St. Louis, MO, USA). To assess spectral characteristics, samples of erythrocyte membranes were diluted in 10 mM Tris-HCl buffer (pH = 7.4) to a final protein concentration of 0.3 mg/mL.
To study the structural properties of the lipid phase of erythrocyte membranes, spectral characteristics of the interactions between membrane and pyrene fluorescent probes (Sigma-Aldrich, St. Louis, MO, USA) were assessed on a Cary Eclipse fluorescence spectrometer (Varian, Inc., Palo Alto, CA, USA). A total of 20 μL of a 10 μM alcohol solution of the pyrene probe was added to 2 mL of erythrocyte membrane suspension. Membrane microviscosity of annular and total lipids was assessed by the degree of pyrene excimerization calculating the excimer-to-monomer fluorescence intensity ratio (J470/J370) at an excitation wavelength (λEx) of 285 and 340 nm, respectively. Polarity was analyzed by the excimer-to-monomer vibration peak amplitude ratio (J390/J370) at excitation wavelengths (λEx) of 285 and 340 nm, respectively.
To measure the adenosine triphosphate (ATP) concentration in the heart and lung tissues, biopsy samples were taken 1 h after the completion of CPB and the restoration of spontaneous circulation. The biopsies were then frozen in liquid nitrogen. The obtained samples were homogenized in liquid nitrogen and centrifuged for 10 min at 3000 rpm and 2 °C. Next, a supernatant liquid was obtained, which was collected and neutralized, and the sample volume was adjusted to 2 mL. ATP measurement was carried out using the ATP Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) on a Lucy-2 luminometer (Anthos Labtec Instruments GmbH, Salzburg, Austria).
The lactate concentration measurement was carried out by enzyme immunoassay using the L-Lactate Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) on a multifunctional microplate reader Infinite 200 (Tecan Austria GmbH, Grödig, Salzburg, Austria). The principle of this assay is based on lactate oxidation, catalyzed by lactate dehydrogenase. Nicotinamide adenine dinucleotide (NAD) + hydrogen (NADH) formed during this reaction reduced a formazan reagent, and the color intensity of the resulting solution was proportional to the lactate concentration in the sample.
The normal distribution of quantitative variables was examined using the Shapiro–Wilk test. If the variables had a normal distribution, they were described by the mean value and standard deviation, mean ± SD; otherwise, by the median (Me) and interquartile range median (Q1; Q3). Significant differences in quantitative variables for independent and dependent samples were analyzed using Student’s t-test in case of the normal distribution of the variable in all compared groups, or otherwise using the Mann–Whitney U test for independent samples and the Wilcoxon signed-rank test for dependent samples. The significance threshold for testing hypotheses was p = 0.05.
In this study, statistical intragroup and intergroup analysis was performed. The “CPB” and “CPB + NO” groups, as well as the “CPB + CA” and “CPB + CA + NO” groups, were compared, which made it possible to assess the effect of NO on organs and systems when performing different models of mechanical perfusion.
