Head‐Out Plethysmography in Safety Pharmacology Assessment

Jonathan P. Renninger1

1 GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 10.11
DOI:  10.1002/0471141755.ph1011s33
Online Posting Date:  July, 2006
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Abstract

The importance of the ability to accurately evaluate respiratory function in animals has been underscored by the classification of the respiratory system as a vital organ system by regulatory agencies in the United States, the European Union, and Japan. A comprehensive assessment of respiratory function should include an evaluation of ventilatory function, including rate and volume measurements, overall pulmonary ventilation (i.e., minute volume), and lung function, including resistance to lung airflow. A volume‐displacement, head‐out, plethysmograph chamber is widely considered the most accurate method for measuring respiratory flows and volumes in small animals because it allows direct assessment of both volume and rate as well as resistance to lung airflow, a predictor of airway constriction or obstruction. Direct assessment of airway resistance is important because mild to moderate increases are generally not detected as changes in ventilatory patterns. This unit describes a method for evaluating ventilatory and lung function in conscious rats.

Keywords: plethysmography; safety pharmacology; respiratory function; pleural pressure

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Sealant (e.g., silicone), optional
  • Male or Female Sprague‐Dawley rats, 250 to 500 g (Charles River Laboratories)
  • Anesthesia: isoflurane (1% to 4% in 100% O 2) by inhalation
  • Topical disinfectant (e.g., Betadine)
  • 70% ethanol
  • Medical grade adhesive (Vetbond or equivalent) and a cellulose patch
  • Test compounds in appropriate vehicles for oral or parenteral administration
  • Plethysmograph device (manufactured locally; see Fig. ) consisting of:
    • flow‐measurement chamber
    • head‐out animal‐holding chamber, 3‐liter internal volume with pneumotach port (six layers of 325 mesh stainless steel wire cloth; Small Parts) and rubber collar (1/8‐in. to 3/16‐in. thick, e.g., Neoprene; McMaster‐Carr)
  • Antivibrational table (e.g., marble)
  • Differential pressure transducer (Validyne Engineering MP‐45‐14; http://www.validyne.com)
  • 1/8‐in. copper threaded fitting, ∼3 cm
  • Flexible tubing (0.48‐cm o.d., 0.16‐cm i.d.)
  • Ambient reference chamber, ∼2‐liter internal volume (VWR Scientific Products)
  • Amplifier: differential pressure demodulator/amplifier/signal conditioner specifically designed for the Validyne 1/2 bridge transducer and recommended for low‐pressure applications (LDS Life Science VP‐800; http://www.lds‐group.com)
  • C12V analog voltage‐to‐frequency converter (DSI, http://www.datasci.com)
  • Data interface system and cable: Dataquest Advanced Research Technology (A.R.T.) or Dataquest OpenART system (DSI, http://www.datasci.com)
  • Telemetry receiver unit (DSI RLA1020 or RPC‐1; http://www.datasci.com)
  • Data acquisition system (pulmonary compliance and resistance analysis module of the PONEMAH Physiology Platform; http://www.ponemah.com)
  • Telemetry transmitter unit (DSI TA11PA‐C40 or TL11M2‐C50‐PXT; http://www.datasci.com)
  • 60‐ml plastic syringe
  • 3‐way stopcock
  • LCD digital pressure indicator (e.g., Heise Model PPM2; http://www.heise.com)
  • Surgical clippers
  • Surgical equipment
  • 2 × 2–in. Versalon sponges, sterile (or equivalent): moisten with sterile 0.9% NaCl
  • 22‐G needle, 1 in. long with ∼90° bend
  • 5 1/8‐in. cannulation forceps (Roboz Surgical Instruments)
  • Nonabsorbable and absorbable sutures or surgical wound clips
  • Polycarbonate box with bedding, clean
  • Animal balance accurate to 1 g (e.g., Mettler PE2000)
  • Continuous airflow source (e.g., house air or tank of breathable air)
  • Digital flow meter with minimum range of 1 to 1000 ml/min (e.g., J & W Scientific ADM 2000)
  • Plethysmograph calibration chamber: head‐out animal‐holding chamber, sealed and with no hole for the animal's head (manufactured locally)
  • Calibrated voltage generator with output of 0 to 1 V (e.g., Bio‐System Calibrator, Coulbourn Instruments)
  • Automated data analysis program (e.g., SAS, http://www.sas.com; or custom written programs, e.g., in Microsoft Excel, http://www.microsoft.com)
NOTE: Connect and test all equipment prior to conducting the first measurements (see Fig. for a schematic of the complete system).
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Figures

Videos

Literature Cited

Literature Cited
   Bargeton, D. and Barres, G. 1968. Time characteristics and frequency response of body plethysmograph. In Progress in Respiration Research, Body Plethysmography (A.B. DuBois, K.P. Van de Woestijne, and H. Herzog, eds.) pp. 2‐23. S. Karger, New York.
   Boggs, D.F. 1992. Comparative control of respiration. In Comparative Biology of the Normal Lung, Vol. 1 (R.A. Parent, ed.) pp. 309‐350. CRC Press, Boca Raton, Fla.
   Chapin, J.L. 1954. Ventilatory response of the unrestrained un‐anesthetized hamster to CO2. Am. J. Physiol. 179:146‐148.
   Diehl, K.‐H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.M., van de Vorstenbosch, C., European Federation of Pharmaceutical Industries Association, and European Centre for the Validation of Alternative Methods. 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21:15‐23.
   Drorbaugh, J.E. and Fenn, W.O. 1955. A barometric method for measuring ventilation in newborn infants. Pediatrics 16:81‐87.
   Dubois, A.B., Botelho, S.Y., and Comroe, J.H. Jr. 1956. A new method for measuring airway resistance in man using a body plethysmograph: Values in normal subjects and in patients with respiratory disease. J. Clin. Invest. 35:327‐335.
   Enhorning, G., Schaik, S., Lundgren, C., and Vargas, I. 1998. Whole‐body plethysmography, does it measure tidal volume of small animals? Can. J. Physiol. Pharmacol. 76:945‐951.
   Harkness, J.F., and Ridgway, M.D. 1980. Chromodacryorrhea in laboratory rats (Rattus norvegicus): Etiologic considerations. Lab. Anim. Sci. 30:841‐844.
   Murphy, D.J. 2002. Assessment of respiratory function in safety pharmacology. Fundam. Clin. Pharmacol. 16:183‐196.
   Murphy, D.J., Renninger, J.P., and Gossett, K.A. 1998. A novel method for chronic measurement of pleural pressure in conscious rats. J. Pharmacol. Toxicol. Methods 39:137‐141.
   Murphy, D.J., Renninger, J.P., and Coatney, R.W. 2001. A novel method for chronic measurement of respiratory function in the conscious monkey. J. Pharmacol. Toxicol. Methods 46:13‐20.
   Neter, J., Kutner, M.H., Nachtsheim, C.J., and Wasserman, W. 1996. Latin square and related designs In Applied Linear Statistical Models, Fourth Edition, pp. 1207‐1233. McGraw Hill, Boston, Mass.
   Pennock, B.E., Cox, C.P., Rogers, R.M., Cain, W.A., and Wells, J.H. 1979. A noninvasive technique for measurement of changes in specific airway resistance. J. Appl. Physiol. 46:399‐406.
   Peslin, R., Duvivier, C., Vassiliou, M., and Gallina, C. 1995. Thermal artifacts in plethysmographic airway resistance measurements. J. Appl. Physiol. 79:1958‐1965.
   Sinnett, E.E., Jackson, A.C., Leith, D.E., and Butler, J.P. 1981. Fast integrated flow plethysmograph for small animals. J. Appl. Physiol. 50:1104‐1110.
   Stocks, J., Marshal, F., Kraemer, R., Gutkowski, P., Yishay, E.B., and Godfrey, S. 1996. Plethysmographic assessment of functional residual capacity and airway resistance. In Infant Respiratory Function Testing (J. Stocks, P.D. Sly, R.S. Teeper, and W.J. Morgan, eds.) pp. 190‐240. Wiley‐Liss, New York.
   U.S. Food and Drug Administration. 2001. ICH guidance for industry: S7A safety pharmacology studies for human pharmaceuticals. U.S. Food and Drug Administration, Rockville, Md.
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