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A novel method for chronic measurement of pleural pressure in conscious rats
A Novel Method for Chronic Measurement of Pleural Pressure in Conscious Rats Dennis J. Murphy, Jonathan P. Renninger, and Kent A. Gossett Department of Toxicology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA
Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals. Previous attempts to measure pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal. These problems have been eliminated by developing a novel surgical procedure for placement of a pressure-sensitive catheter beneath the pleural surface. The catheter (attached to a radiotelemetry transmitter) is surgically implanted beneath the serosal layer of the esophagus within the thoracic cavity. This is accomplished by making a small incision in the serosal layer of the esophagus caudal to the diaphragm and advancing the catheter cranially into the thoracic cavity until pressure changes are maximal. The accuracy of these measurements was verified by comparison with direct pleural pressure measurements over the range of 23 to 234 cm H2O. The pleural pressure changes remained constant for at least 14 weeks following surgery, and there was no evidence of tissue damage or growth around the catheter. This novel method for measuring pleural pressure chronically in conscious rats will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory (breathing) patterns and lung function in conscious animals. © 1998 Elsevier Science Inc. Key Words: Pleural pressure; Rats; Lung function; Respiration
Introduction Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals (Costa, 1985; Murphy, 1994). The mechanical properties of the lung, which include compliance (or elasticity) and resistance to lung airflow, are critical measures of lung function (Amdur and Mead, 1958; Davidson et al., 1966; Diamond and O’Donnell, 1977). In a spontaneously breathing animal, lung compliance is determined by measuring the transpulmonary pressure (i.e., pleural pressure 2 mouth pressure) required to maximally inflate the lung, while resistance to lung airflow is generally determined by measuring the transpulmonary pressure required to generate maximal airflow during inspiration and/or expiration (Amdur and Address reprint requests to Dr. Dennis J. Murphy, UE0364, SmithKline Beecham Pharmaceuticals, Department of Toxicology, P.O. Box 1539, King of Prussia, PA 19406-0939, USA. Received November 12, 1997; revised and accepted March 3, 1998. Journal of Pharmacological and Toxicological Methods 39, 137–141 (1998) © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
Mead, 1958). These properties of the lung affect alveolar ventilation and gas exchange by controlling airflow to the alveoli during inspiration and the removal of alveolar gas during expiration. Pleural pressure measurements in animals have involved either inserting a pressure-sensitive probe through the thoracic wall directly into the pleural cavity (Amdur and Mead, 1958; Santing et al., 1992; Weissberg et al., 1976) or advancing a probe down the esophagus a distance sufficient to place it within the thoracic cavity (Davidson et al., 1966; Diamond and O’Donnell, 1977; Dorato et al., 1983; Koo et al., 1976; Lai, 1979; Lai and Hildebrandt, 1978; Palecek, 1969; Strope et al., 1980). Esophageal pressure changes within the thoracic cavity have been shown to be an accurate method for assessing pleural pressure changes (Palecek, 1969; Gillespie et al., 1973; Koo et al., 1976). Both of these procedures, however, are for acute measurements. Generally the animals are anesthetized, although modifications have been made for 1056-8719/98/$19.00 PII S1056-8719(98)00008-2
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short-term use in conscious animals (Amdur and Mead, 1958; Santing et al., 1992; Weissberg et al., 1976; Dorato et al., 1983). Attempts to develop a method for measuring pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage (Amdur and Mead, 1958) and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal (Amdur and Mead, 1958; Douglas et al., 1971; Kruger et al., 1961). To avoid catheter encapsulation, a fluid- or air-filled balloon attached to the end of a catheter has been used in conscious guinea pigs (Santing et al., 1992; Douglas et al., 1971; Kruger et al., 1961). The procedure, however, causes disruption of the pleural surface and could lead to lung damage. The successful use of this procedure beyond 11 days has not been reported. The objective of this study was to develop a method for measuring pleural pressure chronically in a conscious animal. This was achieved in the rat by developing a novel surgical procedure for implanting a pressuresensitive catheter beneath the pleural surface. This method will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory patterns and the mechanical properties of the lung in conscious animals.
Methods Animals Male albino Sprague–Dawley virus-antibody-free rats from Charles River Laboratories (Raleigh, NC, USA) were used in this study. The rats weighed between 340 and 609 g and were approximately 12–15 weeks of age. The rats were housed individually in stainless-steel cages in a controlled environment set to maintain a temperature range of 72° 6 4° F and a relative humidity of 40 –70%. The room was maintained on a 12-h light/dark cycle. Certified Rodent Diet no. 5002 (PMI Feeds, Inc., St. Louis, MO) and filtered tap water were available ad libitum.
Sciences International, St. Paul, MN) and the Data Sciences International data acquisition and analysis software system (LabPRO, Version 3.0) sampling at a rate of 500 Hz were used to analyze the telemetry signals. Signals were transmitted for 10 sec every 15 min during each data collection period. The transmitter and catheter were surgically implanted using aseptic surgical technique. Rats were anesthetized with inhaled isoflurane, and the abdominal wall was shaved and disinfected. An abdominal incision was made through the skin and musculature approximately 1 cm below the xyphoid process, continuing caudally approximately 5 cm along the linea alba. The lobes of the liver were retracted, and the esophagus was isolated approximately 2 cm from its junction with the diaphragm (Hiatus oesophagicus). A small incision was made through the serosal layer of the esophagus and a 22-gauge needle was inserted between the serosal and muscularis layers (Figure 1). The needle was tunneled cranially past the juncture with the diaphragm and into the thoracic cavity. The needle was removed and the transmitter catheter was threaded through the channel. Pressure was monitored continuously, and when maximal pleural pressure changes was attained (approximately 1–1.5 cm beyond the Hiatus oesophagicus), the catheter was secured in place at the entry point with medical grade tissue adhesive (Vetbond). The presence of the catheter beneath the serosal surface and above the muscularis layer was subsequently confirmed by histological evaluation (Figure 2). The body of the transmitter was secured to the abdominal wall during closure of the abdominal musculature. The skin incision was closed with suture and wound clips, and the animal was placed in a polycarbonate box with soft bedding during recovery.
Pleural Pressure Measurements Subpleural catheter. Pleural pressures were measured chronically in conscious rats by surgically implanting a fluid-filled polyurethane catheter (length 5 8 cm; O.D. 5 0.7 mm) attached to a pressure-sensitive radiotelemetry transmitter (Model TA11PA-C40, Data Sciences International, St. Paul, MN) beneath the pleural surface. A compatible receiver (Model RLA0120, Data
Figure 1. Schematic drawing of a rat showing placement of the pressure-sensitive subpleural catheter and radiotelemetry transmitter. The enlargement is a cross-section through the esophagus showing the position of the catheter between the serosal and muscularis layers.
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D.J. MURPHY ET AL. CHRONIC PLEURAL PRESSURE MEASUREMENT IN RATS
tween intrapleural and subpleural measures of pleural pressure was evaluated using linear regression and correlation analyses.
Animal Care and Use All animals used in this study were cared for and used humanely in accordance with the requirements specified by the SmithKline Beecham Animal Care and Use Committee (ACUC).
Figure 2. Photomicrograph of a formalin-fixed esophagus showing the placement of the subpleural catheter between the serosal and muscularis layers. A section of the esophagus within the thoracic cavity was embedded in hardened paraffin (EM-100, Surgipath Medical Industries, Richmond, IL) and stained with hematoxylin and eosin (magnification 318). (A) Site of the catheter (catheter is not present in the slide). (B) Serosal layer. (C) Muscularis layer. (D) Lumen of the esophagus.
Intrapleural catheter. Pleural pressures were measured acutely in anesthetized rats by inserting a catheter directly into the pleural cavity. Each rat was anesthetized (urethane, 1 g/kg, i.p.), the chest area was shaved, and a fluid-filled catheter with an 18-gauge needle at the tip was inserted into the pleural cavity at the fifth intercostal space approximately 2–3 cm from the sternum. The catheter was attached to a fluid-filled differential pressure transducer, and pressures were recorded using a blood pressure analyzer (Buxco Electronics, Inc, Sharon, CT) and a chart recorder.
Results A tracing typical of the pleural pressure changes measured in a conscious, spontaneously breathing rat with a surgically implanted subpleural catheter is shown in Figure 3. End-expiratory pressures were generally 0 (65) mmHg (relative to atmospheric pressure) and were reduced to approximately 25 to 215 mmHg (27 to 220 cm H2O) at end-inspiration. These values are similar to the direct (intrapleural) pleural pressure measurements obtained acutely in spontaneously breathing rats (Palecek, 1969), guinea pigs (Amdur and Mead, 1958) and hamsters (Koo et al., 1976; Lai, 1979). The ability of the subpleural catheter to monitor pleural pressure chronically is demonstrated in Figure 4. Pleural pressure changes associated with individual breaths were measured in five rats for a 24-h period at 1, 2, 3, 4, 6, 10, and 14 weeks after surgery. There was no evidence of damping or loss of the pressure signal in four of the five rats. In one rat, the pressure signal remained constant for 3 weeks, but was reduced by over 50% at
Comparison of Subpleural and Intrapleural Pressure Measurements The accuracy of the subpleural pressure measurements was determined by comparing the pressures obtained simultaneously with the intrapleural and subpleural catheters. Animals with a surgically implanted subpleural catheter were anesthetized with urethane (1 g/kg, i.p.) and were tracheotomized with a polyethylene tube (2.4 mm O.D.). The intrapleural pressure catheter was then inserted into the pleural cavity, and both measures of pleural pressure were monitored concurrently. Pleural pressures for individual breaths were maximized (to 234 cm H2O) by occluding the tracheotomy tube, while pressures were minimized (to 23 cm H2O) by giving phenobarbital intravenously (25–50 mg/ kg) via a catheter surgically implanted into the jugular vein.
Analysis of Data Group means and standard error of the means (SEM) are presented for all group data. The relationship be-
Figure 3. A tracing showing typical pleural pressure changes measured by the subpleural catheter in a conscious, nonrestrained rat. End-expiratory pressures were generally 0 (65) mmHg (relative to atmospheric pressure) and were reduced to approximately 25 to 215 mmHg (27 to 220 cm H2O) at end-inspiration.
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Figure 4. Pleural pressure changes in conscious, nonrestrained rats following implantation of the subpleural catheter. The subpleural catheter remained beneath the serosal surface of the esophagus, while the intrapleural catheter penetrated through the serosal surface into the pleural cavity. Each value for the subpleural catheter represents the mean of four rats and the error bars are 6 SEM. Values for the intrapleural catheter represent a single rat.
weeks 4 –14. Evaluation of the catheter implant sites at necropsy indicated that the catheters of the four rats with constant pressure changes were intact beneath the serosal surface, whereas the catheter of the rat with the reduced pressure changes had penetrated the serosal and pleural surfaces and was encapsulated. This confirms the conclusion that insertion of a pressure catheter into the pleural cavity will result in encapsulation and signal damping or loss. A comparison of the pleural pressure changes obtained simultaneously with the subpleural and intrapleural catheters demonstrated that the subpleural measurements are accurate and change in proportion to values obtained with direct measurements over the range of 23 to 234 cm H2O (Figure 5). Linear regression analysis of the data from two rats demonstrated that the slopes of the lines were not statistically difference from one (p . 0.05) and that the Y intercepts were not statistically different from zero. The correlation coefficients (r2) were $0.77. A comparison of the tracings of the intraand subpleural pressure signals obtained simultaneously from these rats showed that the shapes were similar and in phase (data not presented). This is expected since tracings of pressure signals obtained from within the esophagus of rats and hamsters have been shown to have similar shapes and be in phase with simultaneously recorded intrapleural pressures (Palecek, 1969; Koo et al., 1976).
Figure 5. Comparison of intrapleural and subpleural measures of pleural pressure. Intrapleural and subpleural pressure changes for individual breaths were obtained simultaneously in anesthetized, spontaneously breathing rats. The data points in each graph represent pressure changes of individual breaths obtained from a single rat. The straight line through the data points was calculated by least squares linear regression analysis, and the dashed lines are 95% confidence limits of the regression line. The equation describing the regression line and the correlation coefficient (r2) are given for each rat.
Discussion A novel procedure for chronically monitoring pleural pressure in conscious rats has been developed by surgically implanting a pressure-sensitive catheter beneath the serosal layer of the esophagus within the thoracic cavity. The use of telemetry allows these measurements to be conducted in nonrestrained animals. This procedure can be used to monitor breathing rates or assess (indirectly) airflow obstruction in conscious, nonre-
D.J. MURPHY ET AL. CHRONIC PLEURAL PRESSURE MEASUREMENT IN RATS
strained rats (Santing et al., 1992). When combined with lung airflow measurements, obtained using a plethysmograph chamber or a face mask and pneumotachometer, these pleural pressure measurements can be used to chronically monitor changes in lung compliance and airflow resistance—two critical parameters for assessing lung function. This procedure should be applicable to other species; however, the dimensions of the sensor may have to be adjusted, and the influence of esophageal wall thickness (in larger animals) on damping the pressure signal would have to be evaluated. The authors wish to thank Ms. Kathleen Rhodes for preparing the esophagus for histological evaluation and Mr. Thomas Covatta for producing the photomicrograph.
References Amdur MO, Mead J (1958) Mechanics of respiration in unanesthetized guinea pigs. Am J Physiol 192(2):364 –368. Costa DL (1985) Interpretation of new techniques used in the determination of pulmonary function in rodents. Fund Appl Toxicol 5:423– 434. Davidson JT, Wasserman K, Lillington GA, Schmidt RW (1966) Pulmonary function testing in the rabbit. J Appl Physiol 21(3):1094–1098. Diamond L, O’Donnell M (1977) Pulmonary mechanics in normal rats. J Appl Physiol: Respirat Environ Exercise Physiol 43(6):942–948.
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Dorato MA, Carlson KH, Copple DL (1983) Pulmonary mechanics in conscious Fisher 344 rats: multiple evaluations using nonsurgical techniques. Toxicol Appl Pharmacol 68:344 –353. Douglas JS, Dennis MW, Ridgeway P, Bouhuys A (1971) Airway dilation and constriction in spontaneously breathing guinea pigs. J Pharmacol Exp Ther 180:98 –109. Gillespie DJ, Lai YL, Hyatt RE (1973) Comparison of esophageal and pleural pressures in the anesthetized dog. J Appl Physiol 35(5):709–713. Koo KW, Leith DE, Sherter CB, Snider GL (1976) Respiratory mechanics in normal hamsters. J Appl Physiol 40(6):936 –942. Kruger JJ, Bain T, Patterson Jr JL (1961) Elevation gradient of intrathoracic pressure. J Appl Physiol 16:465– 468. Lai YL (1979) Lung volume and pleural pressure in the anesthetized hamster. J Appl Physiol: Respirat Environ Exercise Physiol 46(5): 927–931. Lai YL, Hildebrandt J (1978) Respiratory mechanics in the anesthetized rat. J Appl Physiol: Respirat Environ Exercise Physiol 45(2):255–260. Murphy DJ (1994) Safety pharmacology of the respiratory system: techniques and study design. Drug Dev Res 32:237–246. Palecek F (1969) Measurement of ventilatory mechanics in the rat. J Appl Physiol 27(1):149 –156. Santing RE, Meurs H, van der Mark TW, Remie R, Oosterom WC, Brouwer F, Zaagsma J (1992) A novel method to assess airway function parameters in chronically instrumented, unrestrained guinea-pigs. Pulm Pharmacol 5:265–272. Strope GL, Cox CL, Pimmel RL, Clyde Jr WA (1980) Dynamic respiratory mechanics in intact anesthetized hamsters. J Appl Physiol: Respirat Environ Exercise Physiol 49(2):197–203. Weissberg RM, Marian J, Bradshaw J (1976) Respiratory and cardiovascular effects of prostaglandins in the conscious guinea pig. J Pharmacol Exp Ther 198:197–208.
Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals. Previous attempts to measure pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal. These problems have been eliminated by developing a novel surgical procedure for placement of a pressure-sensitive catheter beneath the pleural surface. The catheter (attached to a radiotelemetry transmitter) is surgically implanted beneath the serosal layer of the esophagus within the thoracic cavity. This is accomplished by making a small incision in the serosal layer of the esophagus caudal to the diaphragm and advancing the catheter cranially into the thoracic cavity until pressure changes are maximal. The accuracy of these measurements was verified by comparison with direct pleural pressure measurements over the range of 23 to 234 cm H2O. The pleural pressure changes remained constant for at least 14 weeks following surgery, and there was no evidence of tissue damage or growth around the catheter. This novel method for measuring pleural pressure chronically in conscious rats will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory (breathing) patterns and lung function in conscious animals. © 1998 Elsevier Science Inc. Key Words: Pleural pressure; Rats; Lung function; Respiration
Introduction Pleural pressures are used to evaluate lung function and are generally measured acutely in anesthetized animals (Costa, 1985; Murphy, 1994). The mechanical properties of the lung, which include compliance (or elasticity) and resistance to lung airflow, are critical measures of lung function (Amdur and Mead, 1958; Davidson et al., 1966; Diamond and O’Donnell, 1977). In a spontaneously breathing animal, lung compliance is determined by measuring the transpulmonary pressure (i.e., pleural pressure 2 mouth pressure) required to maximally inflate the lung, while resistance to lung airflow is generally determined by measuring the transpulmonary pressure required to generate maximal airflow during inspiration and/or expiration (Amdur and Address reprint requests to Dr. Dennis J. Murphy, UE0364, SmithKline Beecham Pharmaceuticals, Department of Toxicology, P.O. Box 1539, King of Prussia, PA 19406-0939, USA. Received November 12, 1997; revised and accepted March 3, 1998. Journal of Pharmacological and Toxicological Methods 39, 137–141 (1998) © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
Mead, 1958). These properties of the lung affect alveolar ventilation and gas exchange by controlling airflow to the alveoli during inspiration and the removal of alveolar gas during expiration. Pleural pressure measurements in animals have involved either inserting a pressure-sensitive probe through the thoracic wall directly into the pleural cavity (Amdur and Mead, 1958; Santing et al., 1992; Weissberg et al., 1976) or advancing a probe down the esophagus a distance sufficient to place it within the thoracic cavity (Davidson et al., 1966; Diamond and O’Donnell, 1977; Dorato et al., 1983; Koo et al., 1976; Lai, 1979; Lai and Hildebrandt, 1978; Palecek, 1969; Strope et al., 1980). Esophageal pressure changes within the thoracic cavity have been shown to be an accurate method for assessing pleural pressure changes (Palecek, 1969; Gillespie et al., 1973; Koo et al., 1976). Both of these procedures, however, are for acute measurements. Generally the animals are anesthetized, although modifications have been made for 1056-8719/98/$19.00 PII S1056-8719(98)00008-2
138
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short-term use in conscious animals (Amdur and Mead, 1958; Santing et al., 1992; Weissberg et al., 1976; Dorato et al., 1983). Attempts to develop a method for measuring pleural pressure chronically in conscious animals have involved surgical implantation of pressure-sensitive catheters directly into the pleural cavity. The success of these techniques has been limited by lung damage (Amdur and Mead, 1958) and/or tissue growth and encapsulation of the pressure-sensitive catheter with damping or loss of the signal (Amdur and Mead, 1958; Douglas et al., 1971; Kruger et al., 1961). To avoid catheter encapsulation, a fluid- or air-filled balloon attached to the end of a catheter has been used in conscious guinea pigs (Santing et al., 1992; Douglas et al., 1971; Kruger et al., 1961). The procedure, however, causes disruption of the pleural surface and could lead to lung damage. The successful use of this procedure beyond 11 days has not been reported. The objective of this study was to develop a method for measuring pleural pressure chronically in a conscious animal. This was achieved in the rat by developing a novel surgical procedure for implanting a pressuresensitive catheter beneath the pleural surface. This method will facilitate evaluation of the effects of drugs, environmental agents, or disease on respiratory function by allowing repeated and simultaneous measurements of both ventilatory patterns and the mechanical properties of the lung in conscious animals.
Methods Animals Male albino Sprague–Dawley virus-antibody-free rats from Charles River Laboratories (Raleigh, NC, USA) were used in this study. The rats weighed between 340 and 609 g and were approximately 12–15 weeks of age. The rats were housed individually in stainless-steel cages in a controlled environment set to maintain a temperature range of 72° 6 4° F and a relative humidity of 40 –70%. The room was maintained on a 12-h light/dark cycle. Certified Rodent Diet no. 5002 (PMI Feeds, Inc., St. Louis, MO) and filtered tap water were available ad libitum.
Sciences International, St. Paul, MN) and the Data Sciences International data acquisition and analysis software system (LabPRO, Version 3.0) sampling at a rate of 500 Hz were used to analyze the telemetry signals. Signals were transmitted for 10 sec every 15 min during each data collection period. The transmitter and catheter were surgically implanted using aseptic surgical technique. Rats were anesthetized with inhaled isoflurane, and the abdominal wall was shaved and disinfected. An abdominal incision was made through the skin and musculature approximately 1 cm below the xyphoid process, continuing caudally approximately 5 cm along the linea alba. The lobes of the liver were retracted, and the esophagus was isolated approximately 2 cm from its junction with the diaphragm (Hiatus oesophagicus). A small incision was made through the serosal layer of the esophagus and a 22-gauge needle was inserted between the serosal and muscularis layers (Figure 1). The needle was tunneled cranially past the juncture with the diaphragm and into the thoracic cavity. The needle was removed and the transmitter catheter was threaded through the channel. Pressure was monitored continuously, and when maximal pleural pressure changes was attained (approximately 1–1.5 cm beyond the Hiatus oesophagicus), the catheter was secured in place at the entry point with medical grade tissue adhesive (Vetbond). The presence of the catheter beneath the serosal surface and above the muscularis layer was subsequently confirmed by histological evaluation (Figure 2). The body of the transmitter was secured to the abdominal wall during closure of the abdominal musculature. The skin incision was closed with suture and wound clips, and the animal was placed in a polycarbonate box with soft bedding during recovery.
Pleural Pressure Measurements Subpleural catheter. Pleural pressures were measured chronically in conscious rats by surgically implanting a fluid-filled polyurethane catheter (length 5 8 cm; O.D. 5 0.7 mm) attached to a pressure-sensitive radiotelemetry transmitter (Model TA11PA-C40, Data Sciences International, St. Paul, MN) beneath the pleural surface. A compatible receiver (Model RLA0120, Data
Figure 1. Schematic drawing of a rat showing placement of the pressure-sensitive subpleural catheter and radiotelemetry transmitter. The enlargement is a cross-section through the esophagus showing the position of the catheter between the serosal and muscularis layers.
139
D.J. MURPHY ET AL. CHRONIC PLEURAL PRESSURE MEASUREMENT IN RATS
tween intrapleural and subpleural measures of pleural pressure was evaluated using linear regression and correlation analyses.
Animal Care and Use All animals used in this study were cared for and used humanely in accordance with the requirements specified by the SmithKline Beecham Animal Care and Use Committee (ACUC).
Figure 2. Photomicrograph of a formalin-fixed esophagus showing the placement of the subpleural catheter between the serosal and muscularis layers. A section of the esophagus within the thoracic cavity was embedded in hardened paraffin (EM-100, Surgipath Medical Industries, Richmond, IL) and stained with hematoxylin and eosin (magnification 318). (A) Site of the catheter (catheter is not present in the slide). (B) Serosal layer. (C) Muscularis layer. (D) Lumen of the esophagus.
Intrapleural catheter. Pleural pressures were measured acutely in anesthetized rats by inserting a catheter directly into the pleural cavity. Each rat was anesthetized (urethane, 1 g/kg, i.p.), the chest area was shaved, and a fluid-filled catheter with an 18-gauge needle at the tip was inserted into the pleural cavity at the fifth intercostal space approximately 2–3 cm from the sternum. The catheter was attached to a fluid-filled differential pressure transducer, and pressures were recorded using a blood pressure analyzer (Buxco Electronics, Inc, Sharon, CT) and a chart recorder.
Results A tracing typical of the pleural pressure changes measured in a conscious, spontaneously breathing rat with a surgically implanted subpleural catheter is shown in Figure 3. End-expiratory pressures were generally 0 (65) mmHg (relative to atmospheric pressure) and were reduced to approximately 25 to 215 mmHg (27 to 220 cm H2O) at end-inspiration. These values are similar to the direct (intrapleural) pleural pressure measurements obtained acutely in spontaneously breathing rats (Palecek, 1969), guinea pigs (Amdur and Mead, 1958) and hamsters (Koo et al., 1976; Lai, 1979). The ability of the subpleural catheter to monitor pleural pressure chronically is demonstrated in Figure 4. Pleural pressure changes associated with individual breaths were measured in five rats for a 24-h period at 1, 2, 3, 4, 6, 10, and 14 weeks after surgery. There was no evidence of damping or loss of the pressure signal in four of the five rats. In one rat, the pressure signal remained constant for 3 weeks, but was reduced by over 50% at
Comparison of Subpleural and Intrapleural Pressure Measurements The accuracy of the subpleural pressure measurements was determined by comparing the pressures obtained simultaneously with the intrapleural and subpleural catheters. Animals with a surgically implanted subpleural catheter were anesthetized with urethane (1 g/kg, i.p.) and were tracheotomized with a polyethylene tube (2.4 mm O.D.). The intrapleural pressure catheter was then inserted into the pleural cavity, and both measures of pleural pressure were monitored concurrently. Pleural pressures for individual breaths were maximized (to 234 cm H2O) by occluding the tracheotomy tube, while pressures were minimized (to 23 cm H2O) by giving phenobarbital intravenously (25–50 mg/ kg) via a catheter surgically implanted into the jugular vein.
Analysis of Data Group means and standard error of the means (SEM) are presented for all group data. The relationship be-
Figure 3. A tracing showing typical pleural pressure changes measured by the subpleural catheter in a conscious, nonrestrained rat. End-expiratory pressures were generally 0 (65) mmHg (relative to atmospheric pressure) and were reduced to approximately 25 to 215 mmHg (27 to 220 cm H2O) at end-inspiration.
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Figure 4. Pleural pressure changes in conscious, nonrestrained rats following implantation of the subpleural catheter. The subpleural catheter remained beneath the serosal surface of the esophagus, while the intrapleural catheter penetrated through the serosal surface into the pleural cavity. Each value for the subpleural catheter represents the mean of four rats and the error bars are 6 SEM. Values for the intrapleural catheter represent a single rat.
weeks 4 –14. Evaluation of the catheter implant sites at necropsy indicated that the catheters of the four rats with constant pressure changes were intact beneath the serosal surface, whereas the catheter of the rat with the reduced pressure changes had penetrated the serosal and pleural surfaces and was encapsulated. This confirms the conclusion that insertion of a pressure catheter into the pleural cavity will result in encapsulation and signal damping or loss. A comparison of the pleural pressure changes obtained simultaneously with the subpleural and intrapleural catheters demonstrated that the subpleural measurements are accurate and change in proportion to values obtained with direct measurements over the range of 23 to 234 cm H2O (Figure 5). Linear regression analysis of the data from two rats demonstrated that the slopes of the lines were not statistically difference from one (p . 0.05) and that the Y intercepts were not statistically different from zero. The correlation coefficients (r2) were $0.77. A comparison of the tracings of the intraand subpleural pressure signals obtained simultaneously from these rats showed that the shapes were similar and in phase (data not presented). This is expected since tracings of pressure signals obtained from within the esophagus of rats and hamsters have been shown to have similar shapes and be in phase with simultaneously recorded intrapleural pressures (Palecek, 1969; Koo et al., 1976).
Figure 5. Comparison of intrapleural and subpleural measures of pleural pressure. Intrapleural and subpleural pressure changes for individual breaths were obtained simultaneously in anesthetized, spontaneously breathing rats. The data points in each graph represent pressure changes of individual breaths obtained from a single rat. The straight line through the data points was calculated by least squares linear regression analysis, and the dashed lines are 95% confidence limits of the regression line. The equation describing the regression line and the correlation coefficient (r2) are given for each rat.
Discussion A novel procedure for chronically monitoring pleural pressure in conscious rats has been developed by surgically implanting a pressure-sensitive catheter beneath the serosal layer of the esophagus within the thoracic cavity. The use of telemetry allows these measurements to be conducted in nonrestrained animals. This procedure can be used to monitor breathing rates or assess (indirectly) airflow obstruction in conscious, nonre-
D.J. MURPHY ET AL. CHRONIC PLEURAL PRESSURE MEASUREMENT IN RATS
strained rats (Santing et al., 1992). When combined with lung airflow measurements, obtained using a plethysmograph chamber or a face mask and pneumotachometer, these pleural pressure measurements can be used to chronically monitor changes in lung compliance and airflow resistance—two critical parameters for assessing lung function. This procedure should be applicable to other species; however, the dimensions of the sensor may have to be adjusted, and the influence of esophageal wall thickness (in larger animals) on damping the pressure signal would have to be evaluated. The authors wish to thank Ms. Kathleen Rhodes for preparing the esophagus for histological evaluation and Mr. Thomas Covatta for producing the photomicrograph.
References Amdur MO, Mead J (1958) Mechanics of respiration in unanesthetized guinea pigs. Am J Physiol 192(2):364 –368. Costa DL (1985) Interpretation of new techniques used in the determination of pulmonary function in rodents. Fund Appl Toxicol 5:423– 434. Davidson JT, Wasserman K, Lillington GA, Schmidt RW (1966) Pulmonary function testing in the rabbit. J Appl Physiol 21(3):1094–1098. Diamond L, O’Donnell M (1977) Pulmonary mechanics in normal rats. J Appl Physiol: Respirat Environ Exercise Physiol 43(6):942–948.
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Dorato MA, Carlson KH, Copple DL (1983) Pulmonary mechanics in conscious Fisher 344 rats: multiple evaluations using nonsurgical techniques. Toxicol Appl Pharmacol 68:344 –353. Douglas JS, Dennis MW, Ridgeway P, Bouhuys A (1971) Airway dilation and constriction in spontaneously breathing guinea pigs. J Pharmacol Exp Ther 180:98 –109. Gillespie DJ, Lai YL, Hyatt RE (1973) Comparison of esophageal and pleural pressures in the anesthetized dog. J Appl Physiol 35(5):709–713. Koo KW, Leith DE, Sherter CB, Snider GL (1976) Respiratory mechanics in normal hamsters. J Appl Physiol 40(6):936 –942. Kruger JJ, Bain T, Patterson Jr JL (1961) Elevation gradient of intrathoracic pressure. J Appl Physiol 16:465– 468. Lai YL (1979) Lung volume and pleural pressure in the anesthetized hamster. J Appl Physiol: Respirat Environ Exercise Physiol 46(5): 927–931. Lai YL, Hildebrandt J (1978) Respiratory mechanics in the anesthetized rat. J Appl Physiol: Respirat Environ Exercise Physiol 45(2):255–260. Murphy DJ (1994) Safety pharmacology of the respiratory system: techniques and study design. Drug Dev Res 32:237–246. Palecek F (1969) Measurement of ventilatory mechanics in the rat. J Appl Physiol 27(1):149 –156. Santing RE, Meurs H, van der Mark TW, Remie R, Oosterom WC, Brouwer F, Zaagsma J (1992) A novel method to assess airway function parameters in chronically instrumented, unrestrained guinea-pigs. Pulm Pharmacol 5:265–272. Strope GL, Cox CL, Pimmel RL, Clyde Jr WA (1980) Dynamic respiratory mechanics in intact anesthetized hamsters. J Appl Physiol: Respirat Environ Exercise Physiol 49(2):197–203. Weissberg RM, Marian J, Bradshaw J (1976) Respiratory and cardiovascular effects of prostaglandins in the conscious guinea pig. J Pharmacol Exp Ther 198:197–208.