An isolated perfused lung model with real time data collection and analysis of lung function

ELSEVIER

An Isolated Perfused Lung Model With Real Time Data Collection and Analysis of Lung Function Craig E. Bernard, Ray Dahlby, and Betty-ann Hoener Department

of Biopharmaceutical

Sciences, School of Pharmacy, University of California, Instruments, Vancouver, BC, Canada

San Francisco,

CA, USA and Raytech

Our primary purpose in making this report has been to describe an isolated perfused lung system which permits the real time collection and analysisof lung mechanical functioning. The distinct advantage of our system lies in its capacity for breath by breath data acquisition and analysis. In addition, because of the modular nature of the components, the system can be readily expanded or contracted depending on the type of experiment being conducted. As configured, the lung mechanic parameters of air flow, lung volume, transpulmonary pressure, pulmonary artery pressure, weight, resistance, elastance (inverse of compliance), and positive end expiratory pressure were monitored, recorded, and evaluated simultaneously throughout the experimental period. We present the results of a 3-h study with control lungs illustrating the stability of these measurements throughout the entire period. Also included is a brief discussion of 3-h studies which show a progressive loss of viability in lungs treated with the redox cycler nitrofurantoin. 0 1997 Elsevier Science Inc. Key Words:

Isolated lung perfusion; Lung mechanics; Real time data analysis

Introduction The lung communicates with itself, the rest of the body, and the environment via the circulation and/or inspired air. Because the isolated perfused lung (IPL) maintains the integrity of these natural physiological functions, it has become a popular and powerful tool for investigating the structure and function of the lung (Levey and Gast, 1966; Dunbar et al., 1984; Niemeier, 1984; Rhoades, 1984; Bates et al., 1989; Joad et al., 1994; Uhlig and Wollin, 1994; Kennedy et al., 1995; Merker and Dawson, 1995; Weksler et al., 1995). In comparison to in vivo studies, the IPL offers the distinct advantage that the lung is isolated from other organs and their possible effects. In comparison to in vitro procedures where individual cells or subcellular components may be isolated and examined, cells in the IPL remain part of an intact organ permitting the investigation of their interaction with each other. Additionally, the IPL permits the Address reprint requests to Betty-arm Hoener, Ph.D., Department of Biopharmaceutical Sciences, School of Pharmacy, Box 0446, University of California, San Francisco, CA 94143-0446, USA. e-mail: [email protected] Received May 2, 1997; accepted June 16, 1997. Journal of Pharmacological and Toxicological Methods 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York. NY 10010

38, 41-46

delivery of endogenous and exogenous material from the capillary side of the lung as would occur in vivo (Dunbar et al., 1984; Niemeier, 1984; Uhlig and Wollin, 1994; Merker and Dawson, 1995; Weksler et al., 1995). Alternatively, the IPL may be used to deliver substances by way of the inspired air (Niemeier, 1984; Rhoades, 1984; Joad et al., 1994; Kennedy et al., 1995; Weksler et al., 1995). As a result, a wide array of different types of studies using the IPL have been performed. Selecting positive or negative ventilation pressure, the rate and volume of respiration, the type of gas used for respiration, the type of perfusate, and the decision of whether to use an open or closed circuit are but a few of the many choices investigators must make when conducting their experiments. While attention has been focused on defining these variables, less attention has been placed on acquiring and analyzing the data collected in these studies. However, the need to monitor changes in the physiological conditions that result from the choice of experimental design remains a crucial, integral component of any system. In this report we discuss a modular system which accomplishes this task in real time on a breath by breath basis.

(1997) 1056-8719/97/$17.00 PII S1056-8719(97)00047-6

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Methods Isolated Perfused Lungs The isolated perfused lungs were prepared from male Sprague-Dawley rats weighing 300 g + 40 g (Charles River, Hollister, CA). Each rat was anesthetized with 130 mg/kg of intraperitoneal pentobarbital. The trachea was cannulated with a modified 14 GA X 3” animal feeding needle (Biomedical Needles, New Hyde Park, NY) cut 40 mm from the base, and then ventilated with 5% CO, and 95% air at a rate of 60 breaths/min and a tidal volume of 2.5 ml with a Harvard Rodent Ventilator (Harvard, South Natick, MA). An injection of 650-700 units/kg of heparin was made into the right ventricle which was immediately incised. A cannula composed of a 25-mm long 14 GA animal feeding tube with a 4-mm ball (Biomedical Needles) attached to a 15 GA Intramedic Luer Stub Adapter (Becton Dickinson, Parsippany, NJ) was placed into the main pulmonary artery. The left ventricle was incised, and the lungs were washed free of blood [via a Masterflex easy load head model 7518-10 attached to a Cole-Parmer motor model 7553-80 and controlled by a Masterflex speed controller (Cole-Parmer, Niles, IL)] at a rate of 6-8 ml/min/kg with

a warmed

(37°C)

Krebs-Henseleit

bicarbonate

buffer with 4.5% bovine serum albumin, and 0.1% glucose, pH 7.30-7.40. Prior to use, the perfusate was filtered through a 0.45 uM Cellulose Acetate filter (Corning, Corning, NY) followed by an additional filtration through a 0.22+M Cellulose Acetate filter (Corning). The left atrium was then cannulated with a 20-mm long PE-240 Intramedic polyethylene (Becton Dickinson) exit tube to allow for outflow of perfusate. The lung was then removed, suspended in the chamber for perfusion, and sighed for 10 sec. Once the lung was suspended, the flow rate of the perfusate was adjusted to S-10 ml/min/kg. Ventilation was maintained at 60 breaths/min, with humidified and warmed (37°C) gas (5% COd95%). The lung was allowed to recover for 15 min at which time the lung mechanic parameters of flow (F), volume (V), transpulmonary pressure (Ptp), pulmonary artery pressure (PA), weight (W), resistance (R), elastance (E, inverse of compliance), and positive end expiratory pressure (PEEP) had stabilized, and the clock was reset to 0. A 15-min baseline was then established. Following this initial period, an exposure period of 60 min of drug treatment (or blank control) occurred, followed by an additional 105 min of blank perfusate for a total of 3 h. The lung was sighed with 20 cm H,O pressure for 5 set every 5 min and for 10 set every 10 min to prevent and reverse atelectasis. At the end of each perfusion, the lung was lavaged with three 5-ml aliquots of saline. The lavage fluids were collected and centrifuged for cell counts and determination of the lavage protein levels (BCA Protein Assay, Pierce, Rock-

ford, IL). A portion of the lung was removed and placed in buffered formalin for histological examination, and the remainder was frozen in liquid nitrogen. Appar-atus The lung and most of the pumping/monitoring equipment were housed in a customized environmental control box (Figure 1) (Air Control Inc., Huntingdon Valley, PA). The pH was measured using a Chemcadet pH meter (Fisher, Pittsburgh, PA) with an electrode (Accumet 13-620-252, Fisher) placed into the perfusate reservoir. The pH was manually maintained between 7.30 and 7.40 by bubbling CO, gas directly into the reservoir. The perfusate was pumped from the perfusate reservoir to the upper level of the environmental control box by the Cole-Parmer pump. All perfusate was transported in Tygon tubing (Fisher) R-3603 ID l/8” OD 3/16” wall thickness l/32”. In line, the perfusate first entered a Y-connector where the temperature of the perfusate was measured with a probe connected to a monitoring thermometer (Fisher). The perfusate then entered a T-connector containing a temperature control probe wired to a Thermistemp Temperature Controller (Pamotor, Burlingame, CA). The temperature within the box was maintained between 35” and 37°C by convection through the use of two 15-watt fans (Pamotor) circulating heat generated by two heating tapes (Fisher) located in the rear of the box and regulated by the controller. From here the perfusate entered a 30-ml inverted syringe that acted as a bubble trap. After the perfusate left the bubble trap, it was monitored by a Ohmeda (Madison, WI) DTX disposable blood pressure transducer that measured the PA. The perfusate then entered the right atrium of the lung and exited via the

1. Schematic representation of the isolated lung perfusion apparatus showing the pathways for the perfusate, air flow to the lung, and air flow to the pressure transducers. See the text for a more detailed description of the components.

Figure

43

C.E. BERNARD ET AL. ISOLATED PERFUSED LUNG

left ventricle. The exiting perfusate was collected by a funnel that fed into a collection reservoir located on the bottom level of the environment control box. The funnel rested in a shallow dish filled with saline and surrounded by a 314 round plastic shield which was closed with plastic wrap once the lung was in position. The 5% COd95% air was delivered to and from the lung by Tygon tubing (R-3603 ID 13/16” OD l/4” wall thickness l/32”). To ensure a continuous supply of gas to the Harvard ventilator, a latex air reservoir bag was placed in line 12 inches in front of the ventilator. The ventilator pumped the gas into the box where the gas entered a humidifier. From here the warmed, humidified gas entered a T-connector. The top of the T-connector was attached to a strain-gauge force transducer (Raytech Instruments, Vancouver, B.C.) that measured total weight. The bottom of the T-connector was connected to a pneumotach (Hans Rudolph model 8430, Kansas City, MO). The pneumotach was heated by a pneumotach heater model HR 8430 (Raytech Instruments). The cannulated trachea of the lung was attached directly to the bottom of the pneumotach. Both the T-connector and the pneumotach were a common pathway for inspired and expired gas depending the breathing cycle of the lung. All expired gas exited the lung via the Tconnector through Tygon tubing (R-3603 ID 13/16” OD l/4” wall thickness l/32”). The gas returned to the ventilator where it was bubbled into a 1” diameter reservoir filled with 2 cm of water. This provided the lung with continual back pressure. The lung was sighed by switching a stopcock from this 2-cm reservoir to a 1”-diameter, 20-cm reservoir. The pneumotach was connected to its transducers by Tygon tubing (R-3603 ID 13/16” OD l/4” wall thickness l/32”). A differential pressure transducer (Validyne DP45-32, Northridge, CA) measured transpulmonary pressure. The airflow and lung volume were measured by another pressure transducer (Validyne DP45-16). Data Sampling All data were transmitted to a Darcom 486 DX2 66 MHZ computer (Darcom, Foster City, CA) via a Raytech Instruments DIREC physiological recording system which included a caseframe containing 2 ACB modules and 2 DCB modules. These modules transmitted information via the caseframe to a 16-bit A/D conversion data acquisition card. Both the Validyne DP4516 and DP45-32 plugged into an ACB module, while the Ohmeda blood transducer and the Raytech force transducer were each connected to a DCB module. The resistance and elastance (reciprocal of compliance) were tabulated on a breath-by-breath basis. All data were continuously recorded directly to disk, while a 14-inch color monitor (Microscan 4GP, ADI, Taipei, Taiwan)

simultaneously displayed the maximum, mean, and minimum of each waveform trace. Data Analysis The lung version of the DIREC data acquisition system is based on a recursive least square algorithm which continually fits the data points for flow, volume, and pressure to the equation of motion for the lung: Pressure = E *Volume + R *Flow + PO Pressure for our purpose was a measure of transpulmonary pressure (the difference between the airway opening pressure and the esophageal pressure). The R and E are the resistance and elastance of the lung alone, while the PO estimates the difference between PEEP and the resting pleural pressure. For the calculation of R and E, the technique incorporated was developed by Bates and has proven to be superior to the method of Amdur and Mead (Bates et al., 1989; Lauzon and Bates, 1991; Ludwig et al., 1991; Sato et al., 1991).

Results Typical lung mechanic printouts of a 180-min IPL are shown for a control and for a nitrofurantoin-treated lung (Figures 2, 3). Nitrofurantoin is an antimicrobial agent which causes a lung toxicity believed to be caused by oxidative stress generated during the redox cycling of its nitro group and its radical anion (Dunbar et al., 1984). In addition, the lung mechanic printouts for the first and last 30 set of a 180-min nitrofurantoin-treated lung are shown (Figures 4, 5). The lung mechanics presented in the figures from top to bottom are F, V, Ptp, PA W, R, E, and PEEP. While both the control and drug-treated lungs lasted a full 180 min, the differences in final viability between the two conditions can be seen by comparing the two preparations. For example, noticeable changes to the drug-treated lung begin to manifest themselves after 90 min of perfusion time. An increase in amplitude of the Ptp trace is noticeable while the Ptp trace for the control has a uniform width through the entire 180-min perfusion experiment. The R and E show a change in both the shape of their curves and a divergence from their horizontal path. This indicates that an increase in E has occurred simultaneously with a decrease in R. However, the traces of R and E for control IPL remained constant throughout the entire 180-min experiment. A decrease in weight for both the control and drug-treated preparations occurs during the first 15 min. This occurred uniformly throughout all lung preparations as the lung reached a final recovery from the surgery. However, while the weight gain for the control lungs, 0.63 f: 0.56 g (n = 4), remained negligible throughout the experiment, the drug-treated lungs be-

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Figure 2. Printout of the computer recorded and generated traces of lung mechanical function parameters over a 3-h period for a control lung. The X-axis ranges from 0 to 3:00:00 h. The tracings are from top to bottom: Flow (ml/set); Volume (ml); Transpulmonary pressure (Ptp in cm H,O); Pulmonary artery pressure (PA in mmHg); Weight (g); Resistance (cm Elastance (cm H,ONsec); H,O/l); and positive end expiratory pressure (Peep in cm HP).

Figure 3. Printout of the computer recorded and generated traces of lung mechanical function parameters over a 3-h period for a nitrofurantointreated lung. See Figure 2 for further details.

gan accumulating weight, 2.71 k 0.56 g (n = 4), soon after the 60-min drug treatment was begun and continued to increase throughout the remainder of the experiment. These differences in weight gain were mirrored in the accumulation of lavage proteins of 16 2 9 mg and 85 k 15 mg for the control and treated preparations, respectively. A least squares linear regression analysis of the weight gain versus total lavage proteins had a correlation coefficient, r’ = 0.90.

Discussion The IPL is well-suited for evaluating the physiological functioning and viability of the lung as it responds to external stimuli. In addition to the post facto determination of the dry/wet weight ratio, quantitation of the composition of the perfusate and histological examination of the lungs cell, many investigators have relied upon the real time assessment of physical parameters of

4.5

C.E. BERNARD ET AL. ISOLATED PERFUSED LUNG

Figure 4. Printout of the computer recorded and generated traces of lung mechanical function parameters for the first 30 s of the same nitrofurantointreated lung seen in Figure 3. The X-axis ranges from 0 to 0:00:30 h. See Figure 2 for further details.

w

Figure 5. Printout of the cornputer recorded and generated traces of lung mechanical function parameters for the last 30 set of the same nitrofurantointreated lung seen in Figure 3. The X-axis ranges from 2:59:30 to 3:00:00 h. See Figure 2 for further details.

-,o,DD 5 20.0~ pl w o,w 75.00 ue,*t ti 55.00 0.20 Res

perfusion or transpulmonary pressure and air flow rates to determine lung viability (Levey and Gast, 1966; Dunbar et al., 1984; Niemeier, 1984; Rhoades, 1984; Bates et al., 1989; Joad et al., 1994; Uhlig and Wollin, 1994; Kennedy et al., 1995; Merker and Dawson, 1995; Weksler et al., 1995). Monitoring systems vary greatly in accomplishing this task from the simple to the complex. For example, Uhlig and Wollin have developed an IPL

-

system that is fully automated and allows for the measurement of lung mechanics, gas exchange, segmental vascular resistance, and the capillary filtration coefficient (Uhlig and Wollin, 1994). However, while this system focuses considerable attention on the automated control of the experimental conditions, data acquisition remains problematic. All data is transmitted to a Compaq 286 computer via a DASH16 AD-Converter for

46

analysis of compliance, resistance, and tidal volume but two chart recorders separate from the computer are required to record lung mechanic parameters. The result is a system where data analysis and recording occur in two separate locations. Additionally, the computer requires 10 set to determine compliance and resistance resulting in a final resolution for plotting data of 1 point/min. Joad et al. use pressure transducers and a Modular Instrument Data Acquisition System to calculate the mean value over a 5-set period of resistance, compliance, and lung volume (Joad et al., 1994). While both of these systems are useful, our system (Figure 1) is capable of recording all lung mechanic traces side by side and performing data analysis on a breath by breath basis (Figures 2-5). We were successful in adapting a commercially available computer-based, physiological animal monitoring system for the acquisition of data in the IPL. The system records all lung mechanics in real time and displays all traces in a rolling waveform side-by-side on up to 16 channels along with all calculated lung mechanic parameters data on a color monitor. Not only is this system capable of recording the lung mechanics of tidal volume, air flow, transpuimonary pressure, arterial pressure, and weight gain (a measure of edema) at a frequency of 120 samples/set, but it is also able to accurately compute pulmonary resistance, pulmonary elastance (the inverse of compliance), and positive end expiratory pressure at an equally high frequency (Figures 2-5) (Bates et al., 1989; Lauzon and Bates, 1991; Ludwig et al., 1991; Sato et al., 1991). This permits instant assessment of the lungs and allows the user to make adjustments as needed. In addition, all data are recorded continuously on a hard drive during the experiment. This method of data storage permits easy archiving and retrieval of the entire experiment or specific parts during the playback mode. We used this system to demonstrate the stability of control IPL perfused with a modified Krebs-Henseleit buffer containing 4.5% bovine serum albumin for 3 h. These preparations were compared to IPL treated with nitrofurantoin (Figures 2 and 3). As the lung became edematous as measured by a weight gain, Ptp increased while V and F decreased. In addition, the calculated values of R and E showed dramatic changes in both shape and amplitude. A comparison of the first and last 30 set of the nitrofurantoin-treated lung (Figures 4 and 5) reinforces these observations. In both figures, the 30 breaths in these 30 set are apparent in the F, V and Ptp curves. Also notable in Figure 4 are the changes in these tracings when the lung was hyperinflated during the initial sigh. A comparison of these tracings to those obtained during the last 30 set (Figure 5) shows the dramatic reduction in F and V and the increase in Ptp in these edematous lungs. The total weight gain correlated well with the total protein recovered in the lavage fluids

JPM Vol. 38, No. 1 September 1997:41-46

(2 = 0.90). Th us, we have developed an IPL system capable of monitoring the mechanical functioning of the lung on a breath by breath basis over the entire experimental period. Because of the modular nature of the components, the system can be readily expanded or contracted depending on the type of experiment being conducted. For example, the perfusate could be easily sampled as it enters and exits the lung, the perfusate could be recirculated, or exogenous substances could be added to the inspired gas. The pH and temperature of the perfusate could be monitored and/or controlled continuously. Additional probes capable of monitoring CO, or 0, could be easily incorporated. While our system is not fully automated (Uhlig and Wollin, 1994), it does offer an unrivaled data acquisition and analysis system. We thank Jesse Joad and John Brie of the University of California at Davis for sharing the details of their lung perfusion system with us and allowing us to observe their experiments. This work was supported by a grant AI 27796 from the National Institutes of Health.

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JP, McDonald RJ, Giri SN, Brie JM (1994) Ozone effects on mechanics and arachidonic acid metabolite concentrations in isolated rat lungs. Environ Res 66:186-97.

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