Ventil is a device originally developed for differential mechanical lung ventilation and can be used in one-lung ventilation as well as in the case of asymmetric lung pathologies. It splits the flow according to the selected ratio (by a division knob). However, it must be emphasized that the Ventil is not a simple splitter of the flow from the ventilator. It is a device that can adjust minute ventilation, keeping the parameters stable regardless of changes in the air duct properties of the lungs. The division of the flow is stabilized by two flowmeters, continuously measuring the flows in output ports and sending these flow signals to the control system, which then corrects the splitting to maintain the flow division according to the selected ratio. External mechanical or electromagnetic (fixed or adjustable) positive end-expiratory pressure (PEEP) valves can be used with the Ventil. Therefore, the PEEP for both lungs can also be regulated. The Ventil requires one-way valves to separate tracks (particular lung circuits) and protect from gas mixing from these tracks. Instead of two lungs, Ventil can be technically used to ventilate two patients.
The test stand during experiments on animals
Two Puritan Bennett 980 and 840 ventilators (Medtronic, Minneapolis, MN) and two Anastazja 7700 anesthesia ventilators (Farum Ltd., Warsaw, Poland) were used with two Ventil devices (serial numbers SN0022 and SN0024). Four patient monitors (Datex-Ohmeda S/5, GE Healthcare, Boston, MA) were used with the E-PRESTN module (GE Healthcare, Boston, MA). Three of them were also equipped with an E-CAIOV module (GE Healthcare, Boston, MA). Data from patient monitors were collected by Windows 10 PC-based computers. We calculated the median and interquartile range (IQR) values for the following continuously recorded signals during the experiments: oxygen saturation (SpO2), oxygen inspiratory fraction (FiO2), end-tidal carbon dioxide partial pressure (EtCO2), peak inspiratory pressure (PIP), PEEP, respiratory rate (RR), tidal volume (TV) and calculated parameters: driving pressure (ΔP), minute ventilation (MV) and static compliance (Cst). The MV was an inspired tidal volume times RR. The driving pressure was calculated as the difference between a plateau pressure (Pplat) and positive end-expiratory pressure (Pplat – PEEP). The static compliance was the ratio of the expired TV and the driving pressure. These 3 parameters were calculated per recorded sample. The statistics were processed for all pigs used in the experiments. The spirometric variables were not recorded by the fourth patient monitor due to the E-CAIO (GE Healthcare, Boston, MA) instead of the E-CAIOV module implemented (the E-CAIO module does not measure spirometric variables, in contrast to the E-CAIOV module). All recorded data were postprocessed by MATLAB R2019b and MS Excel 2019 software.
Two Ventil flowmeters (Ventil outputs) were connected with inspiratory arms of two adult polypropylene (PP) anesthesia extendible 60/180 cm breathing circuits with Y-pieces (Medtronic, Minneapolis, MN) by Fixed elbow 22 M - 7.6 mm port - 22 M/15F connectors (Intersurgical, New York, NY) and electrostatic antibacterial and antiviral filters Barrierbac S 22 M/15F (Medtronic, Minneapolis, MN) in series. The Y-pieces were connected with animals by electrostatic filters (various vendors), and the patient monitored probes. The animals' expiratory limps were connected to electrostatic antibacterial and antiviral filters Barrierbac S 22 M/15F (Medtronic, Minneapolis, MN), one-way valves (Intersurgical, New York, NY), and 2.5 cmH2O PEEP valves (Intersurgical, New York, NY or Flexicare, Mountain Ash, Great Britain). The PEEP valve output was connected (through 30 M-22 M (Intersurgical, New York, NY) or 22 M-22 M/15F (R-Vent Medikal, İzmir, Turkey) connectors) by third adult PP anesthesia extensible 60/180 cm breathing circuits (Medtronic, Minneapolis, MN) to the ventilator (Y-piece site) by a 22F-22F connector (Intersurgical, New York, NY). The configuration is presented in Fig. 1A.
Animal use protocol
This animal study was approved by the First Bioethical Commission in the Ethics Committee for Animal Experiments in Warsaw (agreement no WAW2/047/2020). All methods were carried out in accordance with relevant guidelines and regulations and reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). After obtaining the approval, the animals (pigs) were classified for testing; males and females of the Great White Polish Breed, aged approximately 3-6 months. The choice of pigs was based on the assumption of similarity of body and lung mass and therefore ventilation parameters to humans, tidal volume 6-8 ml/kg. Preparation for qualified animals was carried out according to an established schedule. Prior to intubation and mechanical ventilation, animals received premedication: medetomidine 0.05-0.1 mg/kg (Cepetor 1 mg/ml, ScanVet Ltd., Warsaw, Poland), butorfanol 0.1-0.2 mg/kg (Butomidor 10 ml, Richter Pharma AG, Wels, Austria), ketamine 5-10 mg/kg (Bioketan, Vetoquinol Biowet Ltd., Gorzów Wielkopolski, Poland) as an intramuscular injection (buttock muscles). After securing an intravenous line (BD Venflon 1.2 mm, Becton Dickinson, Franklin Lakes, NJ) with access to the posterior ear vein (vena auricularis posteriori), the animals were induced into general anesthesia with propofol (Scanofol 10 mg/ml, ScanVet Ltd., Warsaw, Poland) at a dose based on body weight. After intubation (body weight-dependent tube number), general anesthesia was conducted with isoflurane (Aerrane Baxter, Healthcare Baxter Inc., Warsaw, Poland) in a volumetric percentage adequate to the induced clinical effect, starting with a concentration of 5 vol%, with a continuation of 2 vol%. Subsequently, the anesthetized animals underwent a computer tomography scan (lungs were inflated with air) and then were connected to the anesthesia machine or ventilator by the Ventil. All animals under this experiment had artery cannulas placed in the iliac artery and a central intravenous line in the iliac vein, and both were used to assess cardiovascular indices and to obtain blood samples for laboratory tests. For all objects, a bladder catheter was also placed to assess diuresis. Anesthesia was conducted with isoflurane during the experiment when animals were ventilated by an anesthetic machine, while propofol was applied in those experiments when the ventilator was used. Arterial blood gas (ABG) samples were collected and analyzed by a blood analyzer (epoc® Blood Analysis System, Siemens Healthineers, Erlangen, Germany) in terms of the activity of hydrogen ions (pH), carbon dioxide partial pressure (pCO2), oxygen partial pressure (pO2), bicarbonate concentration (HCO3 ̅), base excess in the extracellular fluid (BE (ecf)), oxygen saturation of hemoglobin (SaO2) and lactate level. ABG samples were collected at 0, 5, 12, 17, and 24 hours of the experiment from all objects (except experiment 3, when they were recorded at 0, 2, 4, 6, 8, and 10 hours). After the experiment, a computer tomography scan was repeated for all animals.
The Mann–Whitney U test was used to compare the differences between all values of ABG parameters in pairs with similar weights (WS) and different weights (WD) groups acquired across the experiments because the distribution of these parameters differed from the normal distribution and because for most of them, the homogeneity of variances was not fulfilled. The Friedman test and Spearman's rank correlation were performed to analyze the repeatable AGB parameters measured at fixed intervals of time. The analyses were performed separately for each of the parameters for the WS and WG groups separately. The value for a statistically significant difference was set at α = 0.05 for all statistical analyses. The Statistica v.13.3 software package was used for the calculations.
Laboratory test bench
A Ventil with serial number SN0003 with two flowmeters (SpiroQuant H, EnviteC, Germany) was used. Two elbow connectors with luer-lock ports (22 M/15F- and straight connectors (22 M-22 M and 22F-22F) connected the Ventil flowmeters and the air-gas flowmeters SFM3000 (Sensirion, Switzerland). Two adult PP anesthesia extendible 60/180 cm breathing circuits with Y-pieces (Medtronic, Minneapolis, MN) were connected to SFM 3000 (inspiratory arms) with artificial test lungs SmartLung 2000 (IMT Analytics, Switzerland) through HMEF filters. Expiratory limps were connected to electrostatic antibacterial and antiviral filters Barrierbac S 22 M/15F (Medtronic, Minneapolis, MN), one-way valves (Intersurgical, New York, NY), and 2.5 cmH2O PEEP valves (Intersurgical, New York, NY). The PEEP valve output was connected (through 30 M-22 M connectors (Intersurgical, New York, NY)) by third adult PP anesthesia extensible 60/108 cm breathing circuits (Medtronic, Minneapolis, MN) to the ventilator (Y-piece site) by a 22F-22F connector (Intersurgical, New York, NY). Luer-lock ports were connected with 143SC01D-PCB pressure sensors (Sensortechnics GmbH, Germany). These pressure sensors and SFM3000 flow sensors are part of the measurement system. Pressure signals were recorded by the real-time NI PXI-1042 system with a NI PXI-6289 data acquisition board installed (both of them National Instruments, Austin, TX). Flow data from SFM3000 flow sensors were recorded by the STM32VLDISCOVERY board (STMicroelectronics, France-Italy). All pressure and flow data were collected by a Windows 7 PC laptop with its own developed software in LabVIEW™ 2013 (National Instruments, Austin, TX) for data storage and visualization. The configuration is presented in Fig. S1. The picture with the given setup is shown in Fig. S2.
Laboratory experiments: Effects of changing resistance and compliance in one artificial lung ( AL ) on flow and pressure in the second lung
We changed the resistance (R, all values expressed in mbar/L/s) from the baseline value R = 5 to values 20 (R5 to R20), 50 (R5 to R50) and 200 (R5 to R200) and compliance (C, all values expressed in mL/mbar) from baseline value C = 75 to values 60 (C75 to C60) and 25 (C75 to C25) as well as from C = 60 (for both AL) to C = 75 (C60 to C75). We also performed breathing circuit disconnection (in the patient's filter point – P1 marker in Fig. S1) tests (R5 to R0) and replaced the first AL (R = 5, C=75) with the 2 liters respiratory bag (Medtronic, Minneapolis, MN) with the R ~0 and C ~ 15 (AL to Bag). The second AL parameters were not affected during the experiments. For the 'R5 to R20', 'R5 to R50', 'R5 to R200' and 'R5 to R0' events, a baseline C = 75 value was used. For the 'C75 to C60', 'C75 to C25' and 'C60 to C75' events, a baseline R = 5 value was used. Baseline R = 5 and C = 75 values were used for AL in the ‘AL to Bag’ event. We investigated how the pressure and delivered tidal volume were changed in the #2 AL (expressed by index 2 for pressures and volumes in Table 3) when the parameters of the #1 AL were changed (pressures and volumes in Table 3 for this AL are expressed by index 1). All tests were performed for three respiratory rate values of 12, 18, and 24 breaths/min.
Simulation of cross-contamination
The experimental measurement system and conditions are described in supplement R1 - Technical Report of IBBE PAS on the transmission of nanoparticles/solutions in two respiratory branches of the Ventil system for experiments carried out in the period April 4 - 29, 2020. Figure R1 (technical report R1) shows a diagram of the test system with the Ventil apparatus supplying the respiratory tract during independent ventilation of two artificial lungs (right and left). Left: Right flow ratio 6:4. To test the possibility of transmission between the two airways, the test solutions and suspensions of phantoms were administered to the expiratory tract of the artificial L lung using an Areogen nebulizer (N) in 3 mL portions repeated several times. The checkpoints for the presence of test fluorescent substances and nanoparticles were at test points T1, T2, and T3. In experiments, fluorescent compounds, such as sodium fluorescein (fluorescein) and methylene blue, purchased from Sigma and fluorescent red and green polystyrene nanospheres (red fluorescent polystyrene microspheres - EPRUI-RF-100C and green fluorescent polystyrene microspheres - EPRUI-GF-100C) with a diameter of 100 nm were used as virus phantoms. The stable fluorescence of the nanospheres was ensured by the incorporation of the dye inside them (so-called internal labeling). Fluorescein and methylene blue solutions of concentration of 1 mM were used. In contrast, fluorescent nanospheres were administered as a 60 µL suspension and 240 µL stock suspension (provided by the manufacturer) per 3 mL of deionized water. Then, the estimated number of nanospheres in the prepared suspensions was 6.5.1012 and 24.1012 particles per 3 mL, respectively.