All experimental protocols regarding the use and care of animals in the present study were approved by the Laboratory Animal Care Committee of the Faculty of Medicine at Tottori University (approval number: 10-Y-13). Adult female Japanese white rabbits (2.2–3.1 kg) were purchased from Oriental Yeast Co. (Tokyo, Japan). The rabbits were kept under standard housing conditions with free access to food and water.
Manufacture of the prototype ventilator
We created a prototype ventilator that can perform intermittent positive pressure ventilation and mimic the breathing cycle of the 2:2 breathing rhythm during a marathon (Fig. 1). The prototype ventilator is a combination of digital timers, solenoid valves, and existing respiratory equipment (ACOMA AR-300, Acoma Co., Tokyo, Japan). The ACOMA AR-300 is a volume-limited, time-cycling ventilator, in which the I:E ratio can be adjusted from 1:3 to 3:1, and the TV can be set from 20 to 300 mL. The ACOMA AR-300 is a piston-driven ventilator, designed to deliver a constant inspiratory flow rate. The I:E ratio of the ACOMA AR-300 was set to 1:1. The detector switch was attached to the piston rod of the ACOMA AR-300 and the switch was set to emit a signal each time it detected the start of the inspiratory period of the ventilator. The signal was sent to four digital timers (H5BR-B, Omron Co., Tokyo, Japan). Each of these timers was connected to a solenoid valve: three tri-directional valves (VDW350-6G-3-01, SMC Co., Tokyo, Japan) and one bidirectional valve (VDW31-6G-3-01, SMC Co.). These four solenoid valves were powered by 12-Volt DC supplied by the connected digital timer. All digital timers were reset as soon as the ACOMA AR-300 inspiratory start signal was received, at which point they started to measure elapsed time. The start time and duration of the 12-Volt DC output (controlling the solenoid valves) could be set individually for each of the four digital timers.
A schematic diagram of the prototype ventilator is shown in Fig. 1A, and Fig.1B is an example of the time allocation for the four solenoid valves with the RR set to 30 cycles/min. An example of the solenoid valve operation and the corresponding inspired and expired gas dynamics follow. One cycle was defined as the sum of eight separate actions: the 1st inspiratory period, a resting period, the 2nd inspiratory period, a resting period, the 1st expiratory period, a resting period, the 2nd expiratory period, and a final resting period. In this example, the respiration rate of the ACOMA AR 300 was set to 30 breaths/min. In response to the inspiratory start signal of the ACOMA AR 300, all digital timers were reset and began to record the time. First, the solenoid valve #1 opened for 0.3 s, and air from the ventilator flowed directly into the inspiratory circuit (1st inspiratory period: 0.3 s). When solenoid valve #1 closed, the air moved towards solenoid valve #2, but since solenoid valve #2 was closed, the air was released into the atmosphere from the exhaust pipe (resting period). After 0.5 s from the start signal, solenoid valve #2 was opened for 0.3 s, and the air from the ventilator went into the inspiratory circuit again (2nd inspiratory period: 0.3 s). When solenoid valve #2 was closed, the air was released into the atmosphere from the exhaust pipe of solenoid valve #2 (resting period). After 1.0 s from the start signal, solenoid valve #3 was opened for 0.3 s, and expired gas was released from the exhalation pipe of this valve into the atmosphere (1st expiratory period: 0.3 s). When solenoid valve #3 was closed, the expired gas moved towards solenoid valve #4, but since solenoid valve #4 was closed, the emission of the expired gas into the atmosphere was interrupted (resting period). After 1.5 s from the start signal, solenoid valve #4 was opened for 0.3 s, and the expired gas was released from the exhalation pipe of solenoid valve #4 into the atmosphere (2nd expiratory period: 0.3 s). When solenoid valve #4 was closed, the emission of the expired gas into the atmosphere was interrupted (resting period). Two seconds after the start signal, the detector switch detected the next inspiratory cycle of the ACOMA AR 300, the digital timer was reset, and the MBV breathing cycle was repeated. In this example, it did not matter if the open duration of solenoid valve #4 was set to values above 0.5 s, because the detector switch detected the start of the next inspiratory cycle in the ACOMA AR 300. This terminated the open duration of valve #4 since the reset of the digital timers was prioritized, thus the actual open duration of solenoid valve #4 was 0.5 s.
With this prototype ventilator, producing mechanical ventilation in a 2:2 breathing rhythm, the RR can be adjusted in 5 cycles/min increments from 10 cycles/min to 65 cycles/min. In addition, the positive end-expiratory pressure (PEEP) can be set at arbitrary pressure values independently of the exhaust pipes of the solenoid valves #3 and #4. The ventilation volume of one MBV cycle is the difference between the TV set in the ACOMA AR 300 and the exhaust volume from the exhaust pipe of solenoid #2. Since this ventilator is in the prototype stage, the ventilation volume of one MBV cycle has to be determined from the average gas volume of several expiratory cycles collected by the classical water displacement method.14
In rabbits in vivo (n = 9), a preliminary experiment was conducted on the physiological effects of the MBV method on intact lungs. The rabbits were tracheostomized, anesthetized with pentobarbiturate, and paralyzed with pancuronium bromide. We measured arterial blood gas values, TV, minute volume, RR, airway pressure, etc. during the MBV method and the IRV method (both I:E ratio of 1:1).
Figure 2 shows the above-mentioned outcomes of each parameter in a radar chart. Normalization was performed using each average value of the IRV method. The pH was expressed by the hydrogen ion concentration. Compared to the IRV method, the MBV method was expected to effectively eliminate carbon dioxide with a smaller amount of TV and minute volume. In this preliminary study each rabbit was assessed with both ventilation methods, however we did not test for significant differences in the mean values between the methods since the order of testing was not randomized.
Figure 3 displays typical waveforms of the airway pressure for both mechanical ventilation methods with a PEEP of 0 cmH2O in the same rabbit. The waveform in Fig. 3A describes the airway pressure in the IRV method, whereas Fig. 3B presents the airway pressure curve in the MBV method. In the MBV method, the airway pressure transiently decreased immediately after the end of the 1st inspiratory period and transiently increased immediately after the end of the 1st expiratory period.
Isolated perfused lung preparation
We compared the effect of MBV with IRV on pre-pulmonary edema in isolated perfused rabbit lungs. The isolated perfused rabbit lungs were prepared using the method described in detail by Liu et al.15 with minor modifications. Briefly, the rabbits were anesthetized with pentobarbital 30 mg/kg intravenously, followed by ketamine 25 mg/kg intramuscularly, and anticoagulation with heparin 500 u/kg intravenously. After local anesthesia of the anterior neck and the sternum region with 1% lidocaine, tracheal intubation was performed through a tracheostomy and the rabbits were ventilated mechanically. A median sternotomy was performed, and an incision was made into the right ventricle. The rabbits were euthanized by rapidly exsanguinating whole blood (70 mL) from the incision site in the right ventricle. The pulmonary artery and the left atrium were cannulated via the right and left ventriculotomies, respectively. Finally, the lungs were removed en bloc and enclosed in a humidified chamber.
The lungs were perfused with bicarbonate-buffered physiological salt solution (PSS), which comprised of NaCl, 119; KCl, 4.7; MgSO4, 1.17; NaHCO3, 22.61; KH2PO4, 1.18, and CaCl2, 3.2 mM in a recirculating manner. To every 100 mL of PSS stock solution, 100 mg dextrose, 20 mU insulin, 3 g Ficoll® PM70 (GE Healthcare Bio-Sciences, Little Chalfont, UK), and 2 mg indomethacin (Sigma Chemical, St. Louis, MO) were added. Ficoll® PM70 is a high molecular weight sucrose polymer with an average molecular weight of 70 000. If an isolated rabbit lung is perfused with Krebs Ringer solution supplemented with 4% (w/v) albumin without the addition of red blood cells to the perfusate, it will develop pulmonary edema within 2 h.16 Ficoll® PM70, as well as albumin, provide a normal colloidal osmotic pressure at 4% (w/v).17 If isolated murine lungs are perfused with Dulbecco's Modified Eagle's Medium containing 4% (w/v) Ficoll® PM70 without the addition of red blood cells to the perfusate, an edema will form in the lung interstitium within 1 h18. In the current study, a pulmonary pre-edema model was created in the isolated rabbit lung via perfusion with bicarbonate-buffered PSS containing 3% (w/v) Ficoll® PM70 without the addition of red blood cells to the perfusate.
The perfusate flow rate was gradually increased to 35 mL/kg/min, while the ventilation gas was changed from air to a mixed gas (O2, 21%; CO2, 5%; N2, 74%). The perfusate flow rate was continuously measured with an electromagnetic flowmeter (MF-1200, Nihon Kohden, Tokyo, Japan). The left atrial pressure was set to 4 mmHg by regulating the height of a reservoir connected to the venous circuit. The lungs were ventilated with a TV of 6 mL/kg and a respiratory frequency of 40 breaths/min with a PEEP of 2 cmH2O using a Harvard Ventilator 683. Blood gases were measured using iSTAT. The perfusate pH was adjusted to 7.40 using an appropriate amount of 1 mM NaHCO3 (Meylon, Otsuka Pharmaceutical, Tokyo, Japan), and the temperature of the perfusate was maintained at 38°C using a regulated heating system. Pulmonary arterial pressure, left atrial pressure, and airway pressure were recorded using a PowerLab system (AD Instruments, New South Wales, Australia; software, Chart ver. 5) with a transducer connected to amplifiers. The isolated perfused lungs were allowed to stabilize for 20 min before proceeding to the experimental protocol.
The fourteen isolated rabbit lung preparations were randomly divided into the IRV and MBV groups.
In the IRV group, rabbit lungs (n = 7; body weight, 2.5 ± 0.2 kg) were ventilated using a Harvard Ventilator 683 with a TV of 8 mL/kg, an RR of 30 cycles/min, and a PEEP of 2 cmH2O for 60 min.
In the MBV group, rabbit lungs (n = 7; body weight, 2.4 ± 0.2 kg) were ventilated using the prototype ventilator with a PEEP of 4 cmH2O (first step) and 2 cmH2O (second step) for 60 min. Before the experiment, the TV of the MBV was adjusted to 6 mL/kg using a test lung and measured by the water displacement method.14 The mixed gas containing 5% CO2 partially dissolves in water, so the volume of the gas decreases. Therefore, we measured in advance that the decrease in volume after exposure to water for 10 minutes was less than 0.05 mL per 10 mL. The MBV group had a RR of 30 cycles/min, and the time allocation for one cycle had the following pattern: 1st inspiratory period 0.3 s, resting period 0.2 s; 2nd inspiratory period 0.3 s, resting period 0.2 s; 1st expiratory period 0.3 s, resting period 0.2 s; and 2nd expiratory period 0.3 s, resting period 0.2 s.
Peak airway pressure and mean airway pressure
Peak airway pressure (pPaw) and mean airway pressure (mPaw) at the stabilization period (baseline) and after 60min ventilation were calculated based on the values recorded by the PowerLab system.
Pressure volume curve measurement
The inflation pressure volume (PV) curve was measured at the stabilization period (baseline) and after 60 min ventilation using the quasi-static method and a syringe containing mixed gas, while ventilation and perfusion were temporarily removed.4 This method involved the measurement of airway pressure as the lungs were gradually inflated in 5-mL steps, until a volume of 40 mL is reached. The total inhalation volume was assessed up to 40 mL to prevent injuries caused by the measurement method itself. Each inflation interval was set at 15 s to obtain a plateau pressure. The deflation PV curve was not measured.
Lung wet-to dry ratio
After all the measurements had been completed, the left lung was excised, and its wet weight was measured. It was dried at 60°C in an oven for two weeks, and its dry weight was measured to determine the lung wet-to-dry ratio (W/D) using the formula: W/D = wet weight / dry weight.15
Bronchoalveolar lavage fluid analysis
After all the measurements had been completed, the right lung was used for bronchoalveolar lavage fluid (BALF) preparation. Three aliquots (5 mL each) of sterile saline were instilled separately through the trachea and drained. The lavage fluid was centrifuged at 200 × g for 10 min at 4°C, and the cell-free supernatant was stored at -70°C as BALF for further chemical analyses.
The BALF was used to measure total protein concentration and myeloperoxidase (MPO) activity. Total protein concentration was measured using the BAC Protein Assay Reagent Kit (Pierce, Rockford, IL). MPO activity was measured using the method of o-dianisidine dihydrochloride oxidation.19 MPO activity was expressed as the change in optical density (∆OD) per min and per mL of BALF. W/D, total protein concentration in BALF, and MPO activity in BALF were used to determine histochemical lung injury.
All data are expressed as the mean ± standard deviation. Prism® ver. 4 (GraphPad Software, San Diego, CA) software was used for statistical calculations and figure preparations. Data were compared using Welch’s t-test; p-values < 0.05 were considered to be statistically significant.