Miniaturization of Respiratory Measurement System in Artificial

sensors

Article Miniaturization of Respiratory Measurement System in Artiﬁcial Ventilator for Small Animal Experiments to Reduce Dead Space and Its Application to Lung Elasticity Evaluation

Homare Yoshida 1,*, Yoshihiro Hasegawa 1, Miyoko Matsushima 2, Tomoshi Sugiyama 3, Tsutomu Kawabe 2 and Mitsuhiro Shikida 1

1 Department of Biomedical Information Sciences, Hiroshima City University, Hiroshima 731-3194, Japan; [email protected] (Y.H.); [email protected] (M.S.) 2 Division of Host Defense Sciences, Omics Health Sciences, Department of Integrated Health Sciences, Graduate School of Medicine, Nagoya University, Nagoya 461-0047, Japan; [email protected] (M.M.); [email protected] (T.K.) 3 Department of Thoracic Surgery, Graduate School of Medicine, Nagoya University, Nagoya 466-8560, Japan; [email protected] * Correspondence: [email protected]

Abstract: A respiratory measurement system composed of pressure and airflow sensors was introduced to precisely control the respiratory condition during animal experiments. The flow sensor was a hot-wire thermal airflow meter with a directional detection and airflow temperature change

compensation function based on MEMS technology, and the pressure sensor was a commercially available one also produced by MEMS. The artificial dead space in the system was minimized to × Citation: Yoshida, H.; Hasegawa, Y.; the value of 0.11 mL by integrating the two sensors on the same plate (26.0 mm 15.0 mm). A Matsushima, M.; Sugiyama, T.; Kawabe, balloon made of a silicone resin with a hardness of A30 was utilized as the simulated lung system T.; Shikida, M. Miniaturization of and applied to the elasticity evaluation of the respiratory system in a living rat. The inside of the Respiratory Measurement System in respiratory system was normally pressurized without damage, and we confirmed that the developed Artificial Ventilator for Small Animal system was able to evaluate the elasticity of the lung tissue in the rat by using the pressure value Experiments to Reduce Dead Space obtained at the quasi-static conditions in the case of the ventilation in the animal experiments. and Its Application to Lung Elasticity Evaluation. Sensors 2021, 21, 5123. Keywords: ventilator; barotrauma; dead space; miniaturization; lung elasticity evaluation https://doi.org/10.3390/s21155123

Academic Editor: Massimo Sacchetti 1. Introduction Received: 14 July 2021 Accepted: 25 July 2021 Ventilators [1–4] are used in the medical ﬁeld to assist with the maintenance of breath- Published: 28 July 2021 ing as a vital activity, and they are also applied to research on the respiratory system in animal experiments. Generally, the ventilator for small experimental animals consists of

Publisher’s Note: MDPI stays neutral a mechanically simple piston structure, and it generates a sinusoidal shaped respiration with regard to jurisdictional claims in airflow. As a result, the respiration airflow conditions, such as the tidal airflow volume and published maps and institutional affil- the frequency, are determined prior to the experiments, and these values are not normally iations. changed during the experiment [5,6]. However, the respiratory system properties, e.g., tissue elasticity, sometimes change during the experiments depending on what kind of experiment it is. This leads to various problems when the respiration airflow conditions do not follow along with the change of the respiratory system properties. For example, the

Copyright: © 2021 by the authors. lung tissue could be physically damaged when a positive excessive pressure airflow from Licensee MDPI, Basel, Switzerland. the ventilator is supplied [7–16]. Conversely, choking may occur if there is a respiration This article is an open access article airflow volume shortage caused by a poorly fitted tube in the ventilator system. In addi- distributed under the terms and tion, uneven ventilations between the alveoli will be unresolved by the plateau effect [17] conditions of the Creative Commons (stagnation) when the sinusoidal-waved respiration airflow from the ventilator is applied. Attribution (CC BY) license (https:// The first problem abovementioned, the ventilator barotrauma, is related to the incre- creativecommons.org/licenses/by/ ment of the pressure value inside the respiratory system, and the second one is caused by 4.0/).

Sensors 2021, 21, 5123. https://doi.org/10.3390/s21155123 https://www.mdpi.com/journal/sensors Sensors 2021, 21, 5123 2 of 17

the change of the airflow volume by the ventilation. To solve these problems, two measurements devices are required: a sensor for the pressure and another for the airflow volume. Pressure and airflow volume measurements devices for the ventilator have recently been developed as options for monitoring the respiratory system properties during experiments. However, these devices are generally large, heavy, and expensive. From the technical aspect of the sensors developments, various types of physical sensors, for example pressure [18,19], airflow [20,21], shear stress [22,23] acceleration [24–26], gyro [27,28], and force [29–31], have been miniaturized and integrated onto a chip for in- creasing the functional capability in a system by Micro Electro Mechanical Systems (MEMS) technologies. Both pressure and airflow sensors have been developed from the beginning of MEMS, and the small-sized sensors with a size of a few mm are now applied in automobile, health monitoring, and air-conditioning systems [32,33]. Thus, to overcome above-stated problems in the usage of the ventilator in the animal experiments, the miniaturized thermal flow and pressure sensors were applied to produce a cheap and light respiration monitoring system for ventilators in our previous studies [34,35]. A small balloon having a similar volume to that of the respiratory system of small experimental animals was used as the simulated lung, and it was confirmed that the developed respiration monitoring system could statically detect values of both the pressure and airflow volume inside the balloon supplied from the syringe pump, after which the balloon elasticity could be derived from these values. Additionally, a measurement method based on the quasi-static condition was introduced to derive the balloon elasticity in the case of the sinusoidal airflow condition supplied by the ventilator. The developed respiration monitoring system was finally applied to a rat, and values of both the pressure and the airflow volume in the respiratory system were successfully detected. However, each pressure and airflow sensor element was connected by a tube a few dozen mm long, which extended the artificial airway. Thus, the previous respiration monitoring system has a problem in that its dead space increment [36–38] by the tube connection leads to insufficient aeration. Thus, in this study, we newly integrated both the pressure and airflow sensors on a chip substrate to minimize the artificial dead space. We applied MEMS technology [39–42] to manufacture the integrated system and tackled the evaluation of lung tissue elasticity as one of the respiratory system properties in rats under the sinusoidal airflow condition in the ventilator.

2. Respiratory Measurement System 2.1. Dead Space at Lung System in Animal The normal tidal volume (airflow volume per cycle) is usually set to 6.0 mL/kg when small animals (mammals) are used in experiments, about one-third of which are called the physiological dead space volume. Therefore, the dead space ventilation ratio (VD/VT) between the tidal volume (VT) and the physiological dead space volume (VD) becomes about 0.3 in the animals. In the case of animal experiments, the ventilator and respiratory monitoring systems are connected to the airway of the experimental animals by means of a flexible tube of a certain length. This means the dead space ventilation ratio increases according to amount of tube space working as the artificial dead space, and insufficient aeration will occur when the ratio becomes high. The average body weight of the rats used in our study was roughly 0.3 kg, so the values of the tidal volume and the physiological dead space became 1.8 mL and 0.6 mL, respectively. In the previous study, the artificial dead space caused by the tubing in the respiratory measurement system was 0.47 mL, and thus the total dead space in the respiration became 1.07 mL, corresponding to 60% of the tidal volume. As a result, insufficient aeration was caused when the experiment reached the 2–3 min mark. To resolve this issue, we integrated the pressure and airflow sensors on a chip in order to decrease the artificial dead space. The detailed specifications are discussed in the following section. Sensors 2021, 21, x FOR PEER REVIEW 3 of 19

Sensors 2021, 21, 5123 3 of 17 we integrated the pressure and airflow sensors on a chip in order to decrease the artificial dead space. The detailed specifications are discussed in the following section.

2.2.2.2. Structure Structure and and Fabrication Fabrication TheThe structure of the respiratory measurement system for the animal ventilation is shownshown in in Figure Figure 1.1. It It had had a a rectangular rectangular outer outer shape shape of of 26.0 26.0 mm mm × 15.0× 15.0 mm mm × 3.5× mm,3.5 mm,and weand sandwiched we sandwiched the sensor-device the sensor-device area areawith with1.0-mm-thick 1.0-mm-thick glass glass plates. plates. Three Three layered layered pol- ystyrenepolystyrene plates plates with with a thickness a thickness of 0.5 of 0.5mm mm and and sensor sensor film film made made of ofpolyimide polyimide with with a thicknessa thickness of 7.5 of 7.5µm µwerem were used used to produce to produce the flow the channel flow channel and the and airflow the airflowsensor, respec- sensor, tively.respectively. Two-hole Two-hole patterns patterns were formed were formedon the bottom-layered on the bottom-layered plate for platethermal for isolation thermal fromisolation the fromsensor the film sensor and filmthe electrical and the electrical feedthrough feedthrough from the from sensor the element. sensor element. Two plates Two wereplates applied were applied as the middle-layered as the middle-layered plates and plates assembled and assembled on the film on the sensor film sensorwith a gap with of a 6.5gap mm, of 6.5 corresponding mm, corresponding to the flow to the channel flow channelwidth. The width. flow The channel flow channelwas formed was by formed plac- ingby placingthe top-layered the top-layered plate between plate between them. Thus, them. the Thus, thickness the thickness of the middle of the middle layer corre- layer spondedcorresponded to the to height the height of the of flow the channel. flow channel.

FigureFigure 1. 1. StructureStructure of of the the designed designed respiratory respiratory meas measurementurement system for the animal ventilator.

AA micro-machined thermal sensorsensor isis frequentlyfrequently applied applied in in airflow airflow measurement, measurement, espe- es- peciallycially in in the the small small flow flow rate rate region, region, because because it can it can provide provide a high a high sensitivity sensitivity measurement measure- mentwith awith simple a simple structure. structure. Three differentThree differen typest of types principles, of principles, a hot-wire, a hot-wire, a calorimetric, a calorimet- and a ric,time-of-flight and a time-of-flight airflow meter airflow were meter used aswere the used thermal as the sensors thermal [43– 45sensors]. A hot-wire [43–45]. thermal A hot- wireairflow thermal meter airflow with a directionalmeter with detectiona directional and detection airflow temperature and airflow change temperature compensation change compensationfunction [46,47 function] based on[46,47] MEMS based technology on MEMS was technology used in this was study, used as in itthis has study, a large as dy- it hasnamic a large range dynamic in the airflow range in rate the measurement. airflow rate measurement. The heater worked The heater as the worked hot-wire as thermalthe hot- wireairflow thermal sensor, airflow the meander-shaped sensor, the meander-sh metal patternsaped workedmetal patterns as the airflow worked direction as the airflow sensor, directionand the airflow sensor, temperature and the airflow compensation temperature sensor compensation was fabricated sensor in the was thin fabricated polyimide in filmthe with a thickness of 7.5 µm to reduce the thermal capacity. A photolithography, a thin metal film deposition by a sputtering, and a lift-off process were used in the fabrication. Two layered metals composed of Cr and Au were used as the sensor materials. Au was used

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Sensors 2021, 21, 5123 4 of 17 thin polyimide film with a thickness of 7.5 µm to reduce the thermal capacity. A photolithography, a thin metal film deposition by a sputtering, and a lift-off process were used in the fabrication. Two layered metals composed of Cr and Au were used as the sensor materials.as the sensor Au was material, used andas the Cr sensor was applied material, as theand adhesionCr was applied layer between as the adhesion the polyimide layer betweenand the the Au polyimide layer. The and sensor the film Au appliedlayer. The in sensor this study film isapplied shown in Figure this study2a (see is ref.shown [ 47 ] Figurefor a detailed 2a (see ref. fabrication [47] for process).a detailed The fabrication sensor film process). was elastically The sensor bent film to fitwas on elastically the inside benttube to surface, fit on the as inside in the tube case surface, of the previousas in the case study of [ the34,35 previous], however study the [34,35], planar however one was thesimply planar placed one was on the simply polystyrene placed plateon the in polyst this study,yrene as plate shown in this in Figure study,1 .as As shown stated in above, Fig- urethe 1. width As stated and heightabove, ofthe the width flow and channel height at of the the airflow flow channel sensing areaat the were airflow 6.5 mmsensing and area0.5 mm,were respectively,6.5 mm and so0.5 themm, airflow respectively, distribution so the became airflow a distribution plane Poiseuille became flow a plane at the Poiseuilleairflow sensing flow at area. the airflow The theoretical sensing area Reynolds. The theoretical number, expressed Reynolds by number, a kinetic expressed viscosity by(m a2 /s),kinetic an averageviscosity velocity (m2/s), (m/s),an average and a velocity characteristic (m/s), linear and a dimension characteristic (m), linear was estimated dimen- sionto confirm (m), was the estimated flow condition to confirm in the the system. flow condition The hydraulic in the diameter system. wasThe usedhydraulic as the diam- value eterof the was characteristic used as the value linear of dimension the characterist in theic case linear of thedimension rectangular-shaped in the case of flow the channelrectan- gular-shapedapplied in the flow airflow channel sensor applied area, and in the it approximately airflow sensor became area, and double it approximately the flow channel’s be- cameheight double due to the its flow wide channel’s shape. The height number due ofto theits wide order shape. of hundreds The number was derived of the order from theof −5 2 hundredsfollowing was experimental derived from conditions: the following the kinetic experimental viscosity conditions: value of air: the 1.5 kinetic× 10 viscositym /s at ◦ value20 C, of the air: average 1.5 × 10 velocity−5 m2/s at 0.77 20 °C, m/s the in average the case velocity of the tidal 0.77 volumem/s in the of 5.0case mL of driventhe tidal by volume0.5 Hz sinusoidal-shapedof 5.0 mL driven by oscillating 0.5 Hz sinusoidal-shaped airflow, and the oscillating hydraulic airflow, dimeter and 1.0 the mm hydrau- (double licthe dimeter flow channel 1.0 mm height). (double Thus, the flow we channel confirmed height). that the Thus, air flowwe confirmed in the system that becomesthe air flow the inlaminar the system flow becomes in our experimental the laminar conditions.flow in our experimental conditions. TheThe cavity cavity with with a aheight height of of0.5 0.5 mm mm on onthe the backside backside of the of thesensor sensor film film was wasformed formed for thefor thermal the thermal isolation, isolation, minimizing minimizing the thermal the thermal capacity. capacity. Elastic Elastic tubes with tubes a with2.0 mm a 2.0 inner mm diameterinner diameter deformed deformed to the toellipse the ellipse at the atcros thes-sectional cross-sectional direction direction were wereconnected connected to the to rectangularthe rectangular shaped shaped flow flow channel channel from from the the device device plate. plate. They They were were sandwiched sandwiched by by the the solidifiedsolidified dimethylpolysiloxane dimethylpolysiloxane (PDMS) (PDMS) sheets sheets in in the the lateral lateral direction, direction, and and liquid liquid PDMS PDMS was poured into the gap between them as the sealing material. One part of the side surface was poured into the gap between them as the sealing material. One part of the side surface body of one tube was opened to form a hole 0.1–0.2 mm in diameter, and another tube was body of one tube was opened to form a hole 0.1–0.2 mm in diameter, and another tube placed in the opened hole to access the pressure sensor. was placed in the opened hole to access the pressure sensor.

(a) (b) (c)

FigureFigure 2. 2. ThermalThermal airflow airflow and and pressure pressure sensors sensors used used in respiratory in respiratory measurement measurement system: system: (a) fabricated (a) fabricated sensor sensor film; film;(b) pattern(b) pattern of hot-wire of hot-wire thermal thermal airflow airflow meter; meter; (c) pressure (c) pressure sensor sensor used used in this in thisstudy. study.

For the pressure measurement, a commercial pressure sensor (Honeywell gauge pressure type ABPDLNN030PGAA5, Charlotte, NC, USA) produced by MEMS technology was used in this study (see Figure2b), because the small-sized sensor with a high sensitivity is now easily available in the market. Two types MEMS pressure sensors, absolute- and gauge- pressures, are generally provided. The former and the latter express the pressure values

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based on the vacuum and atmospheric pressure, respectively. Conversely, the needed specifications of the pressure sensor were decided as follows from the previous experiments: (1) Variation of pressure value; the pressure variation insides of the balloon and the respiratory in the rat caused by the airflow supply are assumed up to 10 kPa and 1.0 kPa, respectively. (2) Resolution; the pressure variation becomes less than 100 Pa under the small airflow volume condition in the case of the rat. Thus, we assumed that a few Pa of resolution was needed in the following animal experiments. On another front, the atmospheric pressure value is about 101 kPa, and thus the sensor has to detect the pressure variation from less than 100 Pa to 1.0 kPa with a few Pa resolutions in the case of the animal experiments. However, the atmospheric pressure value fluctuates according to the length of time and, for example, its value changed 140 Pa roughly during the previous animal experiments. Thus, the gauge-type pressure sensor was chosen in this study to eliminate the atmospheric pressure value fluctuation in the pressure measurements. The outer shape of the sensor having a single axial barbless port was also considered to facilitate its assembly into the system. The outer diameter and the length at the port were 2.7 mm and 4.8 mm, respectively. The sensor substrate and the weight were 8.0 mm × 11.0 mm and 0.67 g, respectively. The sensor was operated by the supply voltage of 5.0 V. The dead space in our newly developed respiratory measurement system became 0.11 mL thanks to the integration of the sensor devices, which was reduced to 23% com- pared with the devices used in the previous study. The absolute value of 0.11 mL was relatively small to the physiological dead space of 0.6 mL in the case of the rat, which demonstrates that our respiratory measurement system could improve the air ventilation in animal experiments where the breathing is controlled by an artificial ventilator.

2.3. Calibration Method A hot-wire thermal airflow sensor is operated by a constant-driving-voltage or constant- temperature-circuits, and they are controlled by an open- and a closed-loop, respectively. To shorten the response time, the thermal airflow sensor was operated at 76.5 ◦C by a constant temperature circuit, because it is applied to the oscillating airflow measurement by the ventilator. The experimental setup used for the calibration is shown in Figure3.A compressed gas cylinder was applied as an air source, and a commercially available mass flow controller (KOFLOC Model 3200, Kojima Instruments Inc., Kyoto, Japan) was used as a reference to calibrate the fabricated airflow sensor. A three-port solenoid valve was placed in between the mass flow controller and the airflow sensor for the evaluating the following response properties. The constant temperature circuit and the solenoid valve were operated by the function generators. The calibration curve showing the relationship between the airflow volume rate and the sensor output under both the forward and backward directions was obtained due to the oscillating airflow supply by the ventilator, as shown in Figure4. The calibration range was empirically determined to be 0–2000 ccm based on past small-animal experiments. The calibration curve in the case of the hot-wire thermal airflow sensor operated by the temperature constant circuit is theoretically expressed by King’s equation [48], but the experimental data at the low flow rate region often scattered from the equation. King’s equation derived from the forced convection from the heater to the airflow, and therefore it normally fits with the experimental results obtained under the high-flow-rate region, as the forced convection dominates the heat flow in this range. Conversely, it does not fit at the low flow rate region because the free convection dominates the heat flow phenomena in this case. Therefore, an approximate polynomial equation with six order was applied as the calibration curve in this study. The assembly of the pressure sensor does not significantly affect dead space, so we used the commercially available miniaturized dip-type pressure sensor. The measurement range and the sensitivity were 0–206 kPa and 0.038 mV/Pa, Sensors 2021, 21, 5123 6 of 17

Sensors 2021, 21, x FOR PEER REVIEW 6 of 19 respectively. The obtained sensor output voltage was translated into the pressure value by referring to the data sheet.

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Figure 3. Experimental setup for airﬂowairflow sensor calibration.

The calibration curve showing the relationship between the airflow volume rate and the sensor output under both the forward and backward directions was obtained due to the oscillating airflow supply by the ventilator, as shown in Figure 4. The calibration range was empirically determined to be 0–2000 ccm based on past small-animal experiments. The calibration curve in the case of the hot-wire thermal airflow sensor operated by the temperature constant circuit is theoretically expressed by King’s equation [48], but the experimental data at the low flow rate region often scattered from the equation. King’s equation derived from the forced convection from the heater to the airflow, and therefore it normally fits with the experimental results obtained under the high-flow-rate region, as the forced convection dominates the heat flow in this range. Conversely, it does not fit at the low flow rate region because the free convection dominates the heat flow phenomena in this case. Therefore, an approximate polynomial equation with six order was applied as the calibration curve in this study. The assembly of the pressure sensor does not significantly affect dead space, so we used the commercially available miniaturized dip-type pressure sensor. The measurement range and the sensitivity were 0–206 kPa and 0.038 mV/Pa, respectively. The obtained sensor output voltage was translated into the pressure value by referring to the data sheet.

FigureFigure 4. 4. C Calibrationalibration curve curve of of flow ﬂow sensor sensor under under bo bothth forward forward and and backward backward flow ﬂow directions. directions.

Additionally,Additionally, the the response response time time of of both both the the airflow airflow flow flow and and the the pressure pressure sensors sensors havehave to to be be shortened shortened sufficiently sufficiently to to follow follow the the airflow airflow rate rate and and th thee direction direction changes changes in in oscillatingoscillating airflow airflow measurements measurements supplied supplied by bythe the ventilator. ventilator. The Theresponse response property property of the of fabricatedthe fabricated airflow airflow sensor sensor was experimental was experimentallyly examined. examined. The step-shaped The step-shaped waveform waveform was conducted as the airflow input, and thus it was evaluated by a step response. The three- port valve was used to generate the step-shaped airflow waveform (see Figure 3). The response properties under both the forward and the backward flow directions were evaluated in the same way to the above-stated flow detection characteristics. The response waveforms obtained by the step airflow input at the airflow rate of 300 ccm are shown in Figure 5. The response time value was defined as the time period in which the sensor output reached to 90% of the steady state, and those values were derived from the response waveforms. The obtained response time values under four different conditions, raising and falling under the forward flow and raising and falling under the backward flow, were 63 ms, 65 ms, 64 ms, and 51 ms, respectively. In our study, the targeted respiratory frequency of a rat was a few Hz corresponding to several hundred ms in the cycle time, and thus we concluded that the produced airflow sensor had a sufficient response speed following the oscillating airflow supplied by the ventilator.

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was conducted as the airflow input, and thus it was evaluated by a step response. The three-port valve was used to generate the step-shaped airflow waveform (see Figure3). The response properties under both the forward and the backward flow directions were evaluated in the same way to the above-stated flow detection characteristics. The response waveforms obtained by the step airflow input at the airflow rate of 300 ccm are shown in Figure5. The response time value was defined as the time period in which the sensor output reached to 90% of the steady state, and those values were derived from the response waveforms. The obtained response time values under four different conditions, raising and falling under the forward flow and raising and falling under the backward flow, were 63 ms, 65 ms, 64 ms, and 51 ms, respectively. In our study, the targeted respiratory frequency of a rat was a few Hz corresponding to several hundred ms in the cycle time, and thus we concluded that the produced airflow sensor had a sufficient response speed following the oscillating airflow supplied by the ventilator. As stated above, we applied the commercially available miniaturized dip-type pres- Sensors 2021, 21, x FOR PEER REVIEW 8 of 19 sure sensor, which has a sufficiently high response speed of 1 ms for oscillating airflow measurement by the ventilator.

(a)

(b)

Figure 5. ResponseResponse waveform waveform of of the the airflow airflow sensor sensor (flow (flow rate: rate: 300 300 ccm): ccm): (a) forward (a) forward flow flow direction; direction; (b) backward (b) backward flow flowdirection. direction.

As stated above, we applied the commercially available miniaturized dip-type pressure sensor, which has a sufficiently high response speed of 1 ms for oscillating airflow measurement by the ventilator.

3. Experiments 3.1. Outline of Experiments The experimental setup is shown in Figure 6. A balloon made of a silicone resin with a hardness of A30 was first applied as the artificial lung system for the evaluation of the developed respiratory measurement system, prior to applying it to living respiratory evaluation. We used a sphere-shaped balloon with an inner volume of 0.68 mL, which is almost the same as the respiratory system in small experimental animals. The inner diameter and the thickness were 1.0 mm and 0.3 mm, respectively. It was attached at one end of the respiratory measurement system. The elasticity of the balloon was evaluated by supplying the airflow with a syringe and the ventilator (Harvard mouse ventilator (model 683), Holliston, MA, USA).

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3. Experiments 3.1. Outline of Experiments The experimental setup is shown in Figure6. A balloon made of a silicone resin with a hardness of A30 was first applied as the artificial lung system for the evaluation of the developed respiratory measurement system, prior to applying it to living respiratory evaluation. We used a sphere-shaped balloon with an inner volume of 0.68 mL, which is almost the same as the respiratory system in small experimental animals. The inner Sensors 2021, 21, x FOR PEER REVIEWdiameter and the thickness were 1.0 mm and 0.3 mm, respectively. It was attached at one9 of 19 end of the respiratory measurement system. The elasticity of the balloon was evaluated by supplying the airflow with a syringe and the ventilator (Harvard mouse ventilator (model 683), Holliston, MA, USA). AfterAfter that, that, the the airway airway of of the the rat rat (weight: (weight: 326 326 g) g) was was connected connected to to the the end end of of the the de- developedveloped respiratory respiratory measurement system, system, and and we we investigated investigated whether whether our systemour system could could detectdetect the the elasticity elasticity of theof livingthe living rat. The rat. supplied The supplied airflow volumeairflow was volume gradually was increased,gradually in- andcreased, we alternately and we alternately evaluated theevaluated results ofthe the resu syringelts of the and syringe the ventilator and the airflow ventilator supply airflow sosupply that we so could that we compare could them. compare The animalthem. The experiments animal experiments were conducted were in conducted accordance in ac- withcordance the Nagoya with the University Nagoya University Animal Experiment Animal Experiment Regulations Regulations with the approval with the of approval the Animalof the EthicsAnimal Committee Ethics Committee (Approved (Approved number: 20021, number: Date: 20021, 16 March Date: 2020). 16 March The detailed 2020). The experimentaldetailed experimental procedure andprocedure results and are discussed results are in discussed the following in the sections. following sections.

FigureFigure 6. 6.Experimental Experimental setup. setup.

3.2. Balloon Elasticity Evaluation The elasticity of the balloon was expressed by the ratio between the airflow volume and the pressure value inside the balloon and, therefore, the relationship between them was evaluated. First, we used the no-flow condition (static condition: Pressure was statically balanced) to precisely evaluate the pressure value inside the balloon. The syringe was connected to the other end of the respiratory measurement system, and the airflow was supplied from the syringe to the balloon from 0.2 mL to 0.8 mL in 0.2-mL increments. After the airflow supply, the three-way stopcock was switched and closed to make a statically pressure balanced condition in the system, and the pressure value was measured (static condition: no-flow and constant pressure). Then, the three-way stopcock was slowly opened and the pressurized air inside the balloon was exhausted to the atmosphere, and the airflow volume stored in the balloon was detected by the airflow sensor. The typical measured airflow and pressure waveforms when the airflow volumes were 0.2 mL and

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3.2. Balloon Elasticity Evaluation The elasticity of the balloon was expressed by the ratio between the airflow volume and the pressure value inside the balloon and, therefore, the relationship between them was evaluated. First, we used the no-flow condition (static condition: Pressure was statically balanced) to precisely evaluate the pressure value inside the balloon. The syringe was connected to the other end of the respiratory measurement system, and the airflow was supplied from the syringe to the balloon from 0.2 mL to 0.8 mL in 0.2-mL increments. After the airflow supply, the three-way stopcock was switched and closed to make a statically pressure balanced condition in the system, and the pressure value was measured (static condition: no-flow and constant pressure). Then, the three-way stopcock was slowly opened and Sensors 2021, 21, x FOR PEER REVIEW 10 of 19 the pressurized air inside the balloon was exhausted to the atmosphere, and the airflow volume stored in the balloon was detected by the airflow sensor. The typical measured airflow and pressure waveforms when the airflow volumes were 0.2 mL and 0.6 mL are 0.6shown mL are in Figure shown7 .in The Figure pressure 7. The value pressure became value a constant became a after constant the airflow after the supply airflow by sup- the plysyringe, by the as syringe, shown in as Figure shown7, in and Figure thus we7, an confirmedd thus we that confirmed the pressure that the inside pressure the balloon inside wasthe balloon statically was balanced. statically balanced.

(a) (b)

Figure 7. MeasuredMeasured both both airflow airflow and and pressure pressure waveforms waveforms wh whenen airflow airflow volume volume was was supplied supplied by syringe: by syringe: (a) airflow (a) airflow vol- volume:ume: 0.2 0.2mL; mL; (b) (airflowb) airflow volume: volume: 0.6 0.6mL. mL.

The relationship between the airflow airflow volume and the pressure value in the balloonballoon obtained under the statically pressure balanced condition is plotted by the open dots in Figure 88.. TheThe gradientgradient ofof thethe pressurepressure vs.vs. flowflow volumevolume curvecurve showsshows thethe elasticity,elasticity, andand thethe pressure non-linearly increased with the increase of the airflowairflow volume in the case of the balloon. Its value rapidly increased at the pressurepressure value of 107 kPa. Thus, we concluded that the balloon used in this experiment had the following characteristics: (1) TheThe balloon balloon elasticity waswas almostalmost constantconstant in in the the region region of of the the small small balloon balloon deforma- defor- mationtion by by the the airflow airflow inflating. inflating. (2) TheThe balloon balloon elasticity elasticity largely largely changed changed in inthe the large large deformation deformation region region when when the bal- the loonballoon was was inflated inflated over over the thepressure pressure of 107 of 107kPa. kPa.

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FigureFigure 8. 8.Relationship Relationship between between pressure pressure and and airﬂow airflow volume volume inside inside the balloon.the balloon.

Conversely,Conversely, the the sinusoidal-shaped sinusoidal-shaped oscillating oscillating airflow airflow was was basically basically supplied supplied by the by the ventilatorventilator in in the the case case of of the the animal animal experiments, experiment ands, and thus thus the balloonthe balloon elasticity elasticity evaluation evaluation underunder the the oscillating oscillating airflow airflow was was confirmed. confirmed. The The ventilator ventilator was was connected connected to the to system, the system, andand the the sinusoidal-shaped sinusoidal-shaped oscillating oscillating airflow airflow at aat fixed a fixed frequency frequency of 1.0 of Hz1.0 wasHz was applied. applied. TheThe airflow airflow volume volume (tidal (tidal airflow airflow volume) volume) was changedwas changed from 0.25from mL 0.25 to 1.75mL mLto 1.75 in 0.25-mL mL in 0.25- incrementsmL increments by switching by switching the control the control valve in valve the ventilator. in the ventilator. The air was The cyclically air was cyclically supplied sup- toplied the balloonto the balloon from the from ventilator, the ventilator, and the supplied and the airflow supplied was airflow then exhausted was then to exhausted outside to of the atmosphere. The obtained typical airflow and pressure waveforms when the airflow outside of the atmosphere. The obtained typical airflow and pressure waveforms when volumes were 1.0 mL and 1.5 mL in the case of the ventilator are shown in Figure9. As the airflow volumes were 1.0 mL and 1.5 mL in the case of the ventilator are shown in shown in the airflow and the pressure waveforms, the sinusoidal-shaped airflow was suppliedFigure 9. by As the shown motion in the of the airflow piston and in thethe ventilator,pressure waveforms, and the pressure the sinusoidal-shaped value inside the air- balloonflow was increased supplied according by the motion to the airflow of the inpiston the case in the ofthe ventilator, airflow supply.and the Conversely, pressure value theinside pressure the balloon value became increased to zeroaccording in a moment to the dueairflow to the in pressurizedthe case of airthe reliefairflow to thesupply. atmosphereConversely, in the the pressure case of the value air exhaust. became to zero in a moment due to the pressurized air reliefThe to airflowthe atmosphere became zero in the when case the of airflowthe air exhaust. direction was changed in the case of the sinusoidal-shapedThe airflow became oscillating zero airflow when supply,the airflow as shown direction in Figure was 9changed. Thus, thein the pressure case of the value,sinusoidal-shaped in which the airflowoscillating direction airflow was supply, changed as fromshown the in supply Figure to 9. the Thus, exhaust, the waspressure usedvalue, as in the which value the under airflow the quasi-static direction was condition changed in thefrom case the of supply the oscillating to the exhaust, airflow was supplyused as [18 the,19 ].value The relationshipunder the quasi-static between the cond airflowition volume in the case and theof the pressure oscillating value airflow in thesupply balloon [18,19]. obtained The relationship under the sinusoidal-shaped between the airflow oscillating volume airflow and the condition pressure is plottedvalue in the byballoon the solid obtained black circle under in the Figure sinusoidal-shaped8. The relationships oscillating obtained airflow under bothcondition the static is plotted and by quasi-staticthe solid black conditions circle were in Figure coincident, 8. The which relationships demonstrates obtained that we under were ableboth to the evaluate static and the balloon elasticity by using the pressure value under the quasi-static condition, even if quasi-static conditions were coincident, which demonstrates that we were able to evaluate we use the sinusoidal-shaped oscillating airflow as the air ventilation. the balloon elasticity by using the pressure value under the quasi-static condition, even if we use the sinusoidal-shaped oscillating airflow as the air ventilation.

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(a) (b)

FigureFigure 9. 9. BothBoth measured measured airflow airflow and and pressure pressure waveforms waveforms when when sinusoidal sinusoidal shaped shaped oscillating oscillating airflow airflow was supplied supplied by by the the ventilator:ventilator: ( (aa)) airflow airflow volume: volume: 1.0 1.0 mL; mL; ( (bb)) airflow airflow volume: volume: 1.5 1.5 mL. mL.

3.3.3.3. Elasticity Elasticity Evaluation Evaluation of of Lung Lung Tissue Tissue in in Living Living Rat Rat TheThe respiratory respiratory measurement measurement system system was was placed placed between between the the airway airway extracted extracted by by tracheostomytracheostomy from from the the rat’s rat’s pharynx pharynx and and the the syringe syringe or the or theventilator ventilator (see (seeFigure Figure 6). Fig-6). ureFigure 10 shows 10 shows the thephotograph photograph of the of the animal animal experiment experiment when when the the ventilator ventilator was was applied. applied. TheThe developed developed respiratory respiratory measurement measurement system system was was directly directly connected connected to tothe the airway airway of theof therat. rat. In Inthe the previous previous study, study, insufficient insufficient aeration aeration was was caused caused when when the the experiment reachedreached the the 2–3 2–3 min min mark. mark. Thus, Thus, the the system system was was frequently frequently disconnected disconnected from from the the airway airway toto resume resume thethe breathingbreathing in in the the measurements. measurements However,. However, thanks thanks to the to reductionthe reduction of the of dead the deadspace space in the in newly the newly developed developed system system by the by sensor the sensor integration integration onto theonto chip, the wechip, could we couldkeep itskeep connection its connection with thewith airway the airway up to theup to end the of end the followingof the following elasticity elasticity evaluation evalu- of ationthe lung of the tissue lung in tissue the rat. in The the biologicalrat. The biologic reactionsal reactions caused by caused the insufficient by the insufficient aeration did aera- not tionoccur did even not ifoccur the developed even if the systemdeveloped was syst connectedem was during connected the experimentsduring the experiments in this study. in this study.The tidal volume in the case of the rat (weight: 0.2–0.4 kg) corresponds to almost 2.0 mL,The so tidal we setvolume the maximum in the case ventilation of the rat volume(weight: to 0.2–0.4 3.0 mL kg) to avoidcorresponds damage to to almost the living 2.0 mL,body so and we theset the respiratory maximum system. ventilation Using volume the same to 3.0 approach mL to avoid as the damage balloon experimentsto the living bodyabove, and we the evaluated respiratory the elasticity system. Using of the lungthe same tissue approach in the living as the rat balloon under both experiments the static above,condition we evaluated for the syringe the elasticity airflow caseof the and lung the tissue quasi-static in the living one for rat the under sinusoidal-shaped both the static conditionoscillating for airflow. the syringe airflow case and the quasi-static one for the sinusoidal-shaped oscillatingIn the airflow. case of the static condition, the syringe was connected to the respiratory mea- surementIn the system,case of the and static the amountcondition, of the airflow syringe volume was connected from the syringe to the respiratory was increased meas- in urement0.5-mL increments.system, and Spontaneousthe amount of breathingairflow vo causeslume from backing, the syringe which was makes increased it difficult in 0.5- to mLmeasure increments. both the Spontaneous pressure and breathing the airflow causes volume, backing, so which we performed makes it difficult the experiments to meas- ureunder both deep the anesthesiapressure and (coma the condition)airflow volume, to avoid so spontaneouswe performed breathing. the experiments The ventilator under deepassistance anesthesia was incorporated(coma condition) to stabilize to avoid the spontaneous physical condition breathing. after The each ventilator measurement assis- tanceinterval. was Theincorporated airflow and to stabilize pressure the waveforms physical condition when the after airflow each volumes measurement were 2.0 inter- mL, val.2.5 mL,The andairflow 3.0 mLand arepressure shown waveforms in Figure 11 when. the airflow volumes were 2.0 mL, 2.5 mL, and 3.0As mL shown are shown in Figure in Figure 11, the 11. airflow was slowly supplied into the inside of the lung tissueAs with shown the in time Figure period 11, ofthe about airflow 1.0 was s over slowly in the supplied case of theinto rat,the andinside kept of forthe alung few tissueseconds; with then, the time the pressurized period of about air in 1.0 the s lungover in tissue the case was releasedof the rat, into and the kept atmosphere. for a few seconds;The air exhaust then, the was pressurized driven by air the in elasticitythe lung tissue of the was lung released tissue, andinto thusthe atmosphere. the airflow The was airgradually exhaust decreased was driven to by zero the over elasticity a time of period the lung of about tissue, 1.0 and s by thus the lungthe airflow elasticity. was It grad- takes more time if the lung tissue becomes stiff by the disease. ually decreased to zero over a time period of about 1.0 s by the lung elasticity. It takes more time if the lung tissue becomes stiff by the disease.

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FigureFigure 10.10. PhotographPhotograph ofof thethe experimentalexperimental setupsetup inin thethe casecase ofof animalanimal experiment experiment (ventilator (ventilator was was connected). connected).

OnOn anotheranother front,front, thethe pressurepressure valuevalue graduallygradually increasedincreased accordingaccording toto thethe airflow airflowairflow supplysupply byby thethe syringe,syringe, andand itit maximized maximized afterafter thethe airflow airflowairflow supply. supply. However,However, itit slightly slightly decreaseddecreased andand thenthen becamebecame aa constant,constant, whichwhich wawa wasss differentdifferent fromfrom the the resultsresults in in the the case case of of thethethe balloon.balloon. balloon. Generally,Generally, Generally, thethe the livingliving living tissuetissue tissue hashas has both both thethe elasticelastic andand thethe viscosityviscosity properties,properties, andand thusthus wewe nownow guessguess thatthat thethe viscoelasticviscoelastic prpr propertyopertyoperty inin thethe lunglung tissuetissue isis relatedrelated to to the the reasonreasonreason for for this this pressure pressure valuevaluevalue decrease, decrease,decrease, and andand the ththee pressure pressurepressure in inin the thethe lung lunglung tissue tissuetissue was waswas balanced balancedbalanced in inina delayed aa delayeddelayed way. way.way. As inAsAs the inin balloonthethe balloonballoon experiment, experiment,experiment, the obtained thethe obtainedobtained relationship relationshiprelationship between betweenbetween the airflow thethe airflowairflowvolume volumevolume and the andand pressure thethe pressurepressure value invaluevalue the inin respiratory thethe respiratoryrespiratory system systemsystem measured measuredmeasured under underunder the statically thethe stat-stat- icallyicallypressure-balanced pressure-balancedpressure-balanced condition conditioncondition is plotted isis plottedplotted by the byby open thethe dotopenopen in dotdot Figure inin FigureFigure 12. 12.12.

((aa)) ((bb))

Figure 11. Cont.

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((cc)) FigureFigure 11.11.11. Both BothBoth measured measuredmeasured airflow airflowairflow and andand pressure pressurepressure waveforms waveformswaveforms when whenwhen the airflow thethe airflowairflow volume volumevolume wassupplied waswas suppliedsupplied by the by syringe:by thethe syringe:syringe: (a) airflow ((aa)) airflow volume: 2.0 mL; (b) airflow volume: 2.5 mL; (c) airflow volume: 3.0 mL. volume:airflow volume: 2.0 mL; (2.0b) airflowmL; (b) volume:airflow volume: 2.5 mL; (2.5c) airflow mL; (c) volume:airflow volume: 3.0 mL. 3.0 mL.

Figure 12. Relationship between pressure and airflow volume in respiratory interior. FigureFigure 12.12. RelationshipRelationship betweenbetween pressurepressure andand airflowairflow volumevolume inin respiratoryrespiratory interior.interior. Next, we examined the elasticity of the lung tissue in the living rat under the oscillat- Next,Next, we examined the the elasticity elasticity of of the the lung lung tissue tissue in in the the living living rat rat under under the the oscillat- oscil- ing airflow. The sinusoidal-shaped oscillating airflow was applied and its volume was latinging airflow. airflow. The The sinusoidal-shaped sinusoidal-shaped oscillating oscillating airflow airflow was was applied applied and and its volumevolume waswas increased in 0.5-mL increments. In this study, the ventilation frequency was set to 1.67 Hz increasedincreased inin 0.5-mL0.5-mL increments.increments. InIn thisthis study,study, thethe ventilationventilation frequencyfrequency waswas setset toto 1.671.67 HzHz (100(100 breaths/min), breaths/min), which which is is the the respiratory respiratory frequency frequencyfrequency of ofof a aa typical typicaltypical rat, rat,rat, and andand the thethe tidal tidaltidal volume volume waswas changedchanged fromfrom 0.50.5 toto 2.52.5 mL.mL.

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The airflow and the pressure waveforms when the airflow volumes were 2.0 mL, 2.5 mL, andThe 3.0 airflow mL in andthe case the pressureof the ventilator waveforms are shown when in the Figure airflow 13. volumesThe sinusoidal-shaped were 2.0 mL, airflow2.5 mL, was and mechanically 3.0 mL in the supplied case of the into ventilator the inside are of shownthe lung in system Figure by13 .the The ventilator sinusoidal- by theshaped same airflow method was as in mechanically balloon experiments, supplied and into then the insidethe stored of the air lungwas expired system byby the lungventilator elasticity. by the Thus, same the method airflow gradually as in balloon decrea experiments,sed to zero andin the then case the of the stored air exhaust, air was expired by the lung elasticity. Thus, the airflow gradually decreased to zero in the case and its waveforms were almost coincident with those obtained in the above-stated static of the air exhaust, and its waveforms were almost coincident with those obtained in the experiments performed by the syringe. The pressure value increased with the increase of above-stated static experiments performed by the syringe. The pressure value increased the applied airflow volume. The pressure value, in which the airflow direction was with the increase of the applied airflow volume. The pressure value, in which the airflow changed from the supply to the exhaust, was used as the value under the quasi-static con- direction was changed from the supply to the exhaust, was used as the value under the dition. The relationship between the airflow volume and the pressure value in the lung quasi-static condition. The relationship between the airflow volume and the pressure value system in the living rat obtained under the sinusoidal-shaped oscillating airflow condition in the lung system in the living rat obtained under the sinusoidal-shaped oscillating airflow is plotted by a solid black circle in Figure 12. condition is plotted by a solid black circle in Figure 12. As we can see, the pressure value linearly increased with the increase of airflow vol- As we can see, the pressure value linearly increased with the increase of airflow ume up to 3.0 mL. Generally, the differential pressure value in the respiratory system, and volume up to 3.0 mL. Generally, the differential pressure value in the respiratory system, the critical differential pressure value at which damage of the respiratory system occurs, and the critical differential pressure value at which damage of the respiratory system areoccurs, 1.0–2.0 are 1.0–2.0kPa and kPa 3.4–5.9 and 3.4–5.9kPa, respectively. kPa, respectively. However, However, we found we found that the that maximum the maximum dif- ferentialdifferential pressure pressure between between the the respiratory respiratory in insideside and and the the atmosphere atmosphere in in our our experiments experiments was 0.35 kPa at the absolute pressure value of 100.15 kPa. Thus, we conclude that the lung tissuetissue was was normally pressurized without damage.damage. These results indicate the following: (1) TheThe variation variation of of the the pressure pressure value value by by the the airflow airflow volume volume depends on the lung tissue elasticity,elasticity, which demonstrates thatthat wewe are are able able to to estimate estimate its its elasticity elasticity by by the the gradient gradi- entof theof the pressure pressure vs. vs. flow flow volume volume curve. curve. In theIn the case case of our of our lung lung system system experiment, experiment, the thecurve curve showed showed the the linear linear relation relation in the in th pressuree pressure range range from from 100.15 100.15 kPa kPa to 100.50 to 100.50 kPa kPa(pressure (pressure difference: difference: 0.35 0.35 kPa), kPa), and and thus thus we conclude we conclude that thethat elasticity the elasticity of the of lung the lungtissue tissue is constant is constant in this in pressurethis pressure region. region. (2) TheThe experimental experimental results results obtained obtained under under both both the static and quasi-static conditions areare generally generally coincident. coincident. This This result result followed followed the the same same trend trend as as the the results of the balloonballoon experiment. experiment. Therefore, Therefore, we we demonstrated demonstrated that that the the developed respiratory measurementmeasurement system system is is able able to to evaluate evaluate the the el elasticityasticity of the lung tissue in a living rat byby using using the the pressure pressure value value under under the the quasi-static quasi-static condition in the case of ventilation (animal(animal experiments). experiments).

(a) (b)

Figure 13. Cont.

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(c)

Figure 13. Both measuredmeasured airflowairflow andand pressure pressure waveforms waveforms when when the the sinusoidal-shaped sinusoidal-shaped oscillating oscillating airflow airflow was was supplied supplied by by the ventilator: (a) airflow volume: 2.0 mL; (b) airflow volume: 2.5 mL; (c) airflow volume: 3.0 mL. the ventilator: (a) airflow volume: 2.0 mL; (b) airflow volume: 2.5 mL; (c) airflow volume: 3.0 mL.

4. Conclusions In this work, we newly integrated bothboth airflowairflow andand pressurepressure sensorssensors intointo ourour respira- respir- atorytory measurement measurement system system to to minimize minimize the the artificial artificial dead dead space space in the in the ventilation ventilation during during ani- animalmal experiments. experiments. A hot-wire A hot-wire thermal thermal airflow airflow meter meter with awith directional a directional detection detection and airflow and airflowtemperature temperature change compensationchange compensation function andfunction a commercially and a commercially available pressureavailable sensor pres- sureproduced sensor by produced MEMS technologies by MEMS technologies were assembled were onto assembled a chip sizedonto a 26.0 chip mm sized× 15.026.0 mm,mm ×and 15.0 the mm, dead and space the dead was reducedspace was to reduced the value to of the 0.11 value mL, of which 0.11 mL, is just which 23% is of just that 23% in the of thatprevious in the device. previous We alsodevice. evaluated We also the evaluated elasticity the of theelasticity lung tissue of the in lung a living tissue rat in under a living the ratoscillating under the airflow oscillating and obtained airflow and the followingobtained the results: following results: (1) TheThe elasticity elasticity of of the the lung lung tissue tissue in in a a li livingving rat can be evaluated by gradient of the pressurepressure vs. vs. flow flow volume volume curve. curve. (2) ItIt can can be be also also evaluated evaluated by by using using the pr pressureessure value obtained under the quasi-static conditioncondition in in the the case case of of the the sinusoidal-shaped oscillating airflow airflow ventilation used in thethe animal animal experiments. experiments. WeWe now now think think that that the the developed developed measuremen measurementt system composed of both airflow airflow and pressure sensorssensors hashas potential,potential, which which will will be be used used in in the the following following different different applications applications in inanimal animal experiments. experiments. (1) TheThe system system can can evaluate evaluate the the respiration properties in the case of the spontaneousspontaneous breathingbreathing by simply connectingconnecting itit to to the the airway airway of of the the experimental experimental animal animal (under (under no noventilation ventilation condition). condition). (2) ByBy integrating integrating aa COCO22 sensorsensorworking working as as a a capnometer capnometer onto onto the the system, system, it can it can detect detect the thevalue value of its of partial its partial pressure pressure in the breathing,in the breathing, and thus and we canthus estimate we can the estimate respiration the respirationconditions, conditions, such as the such arrested as the respiration, arrested respiration, and the partial and the CO partial2 pressure CO2 pressure value in valuearterial in blood.arterial blood. The system will also be applied to a bag-valve mask, and Jackson Rees used for resuscitation in medical emergency in the case of humans, because of itsits miniaturizationminiaturization and weight-saving potential. The structure of the system will be redesigned, in which the Reynolds number becomes the same.

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Author Contributions: Conceptualization, H.Y.; methodology, H.Y., Y.H. and M.S.; animal experiment, T.S., M.M. and T.K.; validation, H.Y., Y.H. and M.S.; writing—original draft preparation, H.Y.; writing—review and editing, Y.H., T.S., M.M., T.K., M.S.; supervision, T.K., M.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of Tokai National Higher Education and Research (Regulation No. 74 of 1 April 2020) System Regu- lations on Animal Care and Use in Research, and approved by the Ethics Committee of Nagoya University (protocol code: 20021 and date of approval: 16 March 2020). Informed Consent Statement: Not applicable. Data Availability Statement: No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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