Bronchial provocation tests in small animals: a quantified and automated procedure M. CHAUVEAU,

M. LEROY,

G. LE GALL,

AND

A. LOCKHART

Laboratoire de Physiologie Respiratoire, Unite’ de Formation Cochin Port Royal, 75014 Paris, France

et de Recherche,

CHAUVEAU,M.,M. LEROY,G.LEGALL,AND A. LOCKHART. ing the airways. Whether Bronchial provocation automated procedure.

tests in small animals:

a quantified

and

J. Appl. Physiol. 73(2): 410-419, 1992.-Bronchial provocation tests using aerosols in laboratory animals are difficult to standardize and quantify, because the amount of drug actually reaching the airways is unknown. To improve the quantification of aerosolized inhaled stimuli, we designedan apparatus that allows, in anesthetized intubated ventilated animals, control of temperature and hygrometry of inspired air, computerized measurement of pulmonary resistance, and fully automated delivery of a known amount of aerosolizeddrug directly into the trachea. Calibration of the aerosoldelivery involved direct measurementof liquid delivered at the tip of the tracheal cannula. Despite all our efforts at standardization and full automation of all steps, reproducibility of aerosol delivery was poor, ‘with stroke-bystroke differences of 26 or 42%, according to whether an air-jet or an ultrasonic nebulizer was used. Histamine dose-response curves performed in 15 guinea pigs with this device confirmed marked differences amonganimals and also disclosedlarge intraindividual changesin bronchial responsiveness.

direct injection of aerosol in the respiratory circuit of a ventilated animal or aerosol inhalation by a spontaneously breathing animal (10) is used, the dosage is estimated from the concentration of the nebulized solution and the volume insufflated or the number of inhalations (7-9,1&l@. To our knowledge no such dosage has ever actually been measured. Furthermore it is likely that deposition of aerosol in the ventilatory circuit and upper airways varies from one experiment to another. Breathing dry and/or cool air may damage the airways (4, 13) and influence the bronchial responsiveness (19). Therefore it is important that both temperature and humidity of inspired air be controlled in mechanically ventilated animals. We therefore designed an experimental setup that permits control of the temperature and hygrometry of inspired air, automated measurement of resistance of the respiratory system, and insufflation of a known amount of aerosol directly into the trachea in mechanically ventilated small laboratory animals.

airway sensitivity; aerosol; guinea pig; methodology METHODS BRONCHIAL PROVOCATION TESTS in laboratory

animals are widely used for studying the mechanisms of bronchial hyperresponsiveness to various provocative agents. However, contrary to investigations in humans, bronchial provocation tests performed in laboratory animals are not standardized, which makes comparison of studies from different sources difficult. Bronchoactive agents may be administered either by intravenous injection or by aerosol. With the former but not the latter (8, l4), the administered dose is exactly known; however, aerosol administration has the advantage of reducing undesirable side effects, such as cardiovascular consequences that may modify the direct bronchial effects of the agent used through reflex mechanisms. The interindividual scatter of responses is very wide, particularly in guinea pigs. According to Hulbert et al. (9), there is a threefold variation of sensitivity and a 13fold variation of reactivity to histamine among animals; according to Agrawal and Hyatt (l), the sensitivity to histamine varies over a lo-fold range. The variability seems even higher with rabbits (17) but lesser with dogs (5, 6, 15). Such scatter of bronchial responsiveness results from both biological variability and inaccuracies in the determination of the dose of aerosol actually reach410

0161~X67/92

Apparatus

The apparatus is designed for small animals (rat or guinea pig) and is comprised of three subunits (Fig. 1): a thermostated plethysmographic box, a ventilatory pump with a saturator to ensure warming and humidification of inspired air, and an automated nebulizing system. Plethysmographic box. The plethysmographic box is made of methacrylate (5 mm thick), and its internal size is 34 cm long, 16 cm wide, and 13.5 cm high. The removable cover is fitted with a Fleisch pneumotachograph. Three holes are drilled in the front wall to connect inspiratory and expiratory lines to the ventilator and the tubing to the nebulizer. This tubing is connected by a Y piece to the expiratory line, and its patency is controlled by an electrovalve (NV) located inside the plethysmographic box. A thermostated floor prevents cooling of the animal. Ventilation circuit and air conditioning (Fig. 1). The anesthetized and tracheotomized animal is ventilated through a tracheal cannula (Butterfly 16 ST needle, 1.4 mm ID) attached through a Y piece to the inspiratory and expiratory lines of a Harvard pump (model 683), the frequency and tidal volume of which are adjusted according to the animal’s weight. The resistance of the tracheal cannula is 0.11 cmH,O . s ml? Both inspiratory and expiratory lines are equipped with electrovalves (IV and

$2.00 Copyright 0 1992 the American Physiological

l

Society

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QUANTIFICATION

Compressed

Compressed

OF BRONCHIAL

PROVOCATION

411

TESTS

air

air

FIG. 1. Compressed air nebulizer system. Functions of 6 electrovalves: AV, admission; EV, expiratory; IV, inspiratory; LV, leak; NV, nebulizing; PV, purge; SC, screw-clamp. See Fig. 3 and METHODS for position of valves at each step of experiment.

EV, respectively) located outside the plethysmographic box. A vent opened to the atmosphere branches from the expiratory line between EV and the box and is controlled by an electrovalve (PV). It allows free expiration when both EV and IV are closed. A 0.4.l/min airflow from a compressed dry medical air tank flows through the saturator to the ventilator. The saturator is a 32 X 9 X l&cm tank supplied with sprinkling water at 38OC from a thermostated water bath. Jets of water from 100 small holes regularly arranged along two longitudinal pipes abut against the upper wall. The level of water is maintained 2 cm below the upper wall. Because of a central partition, air flows through sprinkling water over a distance of 64 cm, with a mean transit time of 86 s. The hygrometry measured at the outlet of the saturator is between 98 and 100%. The saturator, ventilatory pump, and the whole circuitry up to the plethysmographic box are enclosed in a 38OC thermostated 60 X 55 X 24-cm chamber. Ventilatory Measurements (Fig. 2)

Tracheal pressure (Ptr) was measured with a 5O-mbar pressure transducer (Schlumberger model 5020/5 and CA 9036/5 preamplifier) connected via a catheter (20 cm long, 2 mm ID) to the inspiratory line. The natural frequency of the system, measured by the pop-test method, was 120 Hz. Ventilatory flow (V) was measured with a no. 000 Fleisch pneumotachograph and a l-mbar differential pressure transducer (model CH 5105/5, Schlumberger) and preamplifier (model CA 9036/5). Tidal volume (VT) was obtained by electrical integration (model 13-4615-70, Gould) of the flow signal. Ptr, V, and VT were displayed

on a strip chart recorder (model 2600, Gould) for graphical analysis and monitoring. Ptr and V were fed in parallel into an Apple 2e microcomputer, which calculated resistance of the respiratory system (Rrs) by means of a program developed in our laboratory (Fig. 2). The computer samples and stores Ptr and V at a 204-Hz frequency during an adjustable time ranging from 1 to 9 s. First, V values are integrated, yielding volumes. Then a correction (Vq) proportional to V is added to compensate for the phase lag between measured (Fleisch pneumotachograph) and true (animal) flows. Corrected VT is then differentiated to yield the true V curve. Rrs is calculated for each ventilatory cycle with the isovolume method (2) and printed Rrs = APIAV

= (PI - PE)/(%

- VE)

where VI is the highest flow during inspiration, PI is.Ptr, and isoV is the pulmonary volume at the same time. VE is the flow recorded during the following expiration, when the pulmonary volume is again equal to isoV, and PE is Ptr at the same time point. The mean difference between automatically and manually computed Rrs was 0.1 t 4% (SD) . Aerosol Delivery: Three Different Methods System A (Fig. 1). The aerosol is generated by a nebulizer (model 646, DeVilbiss) driven by compressed air (1.5 atm). The delivery of compressed air to the nebulizer is controlled by an electrovalve (AV). The nebulizer, which is located inside the thermostated chamber next to the front wall of the plethysmographic box, has two outlets: One is connected by a thin Silastic tube (75 mm long, 1

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412

QUANTIFICATION

OF

BRONCHIAL

PROVOCATION

TESTS

FIG. 2. Computerized calculation of resistance of respiratory system. R,, Fleisch head resistance; T, temperature in “C; Pb, pressure inside box; PB, barometric

pressure;

r

dbcb--

Pdif,

pressure

drop

across

Fleisch

head;

Cb,

compliance of gas in box; Vb, difference between box and animal volum,es; Ptr, tracheal pressure; VI, highest

vq

inspiratory

flow;

VE, expiratory

flow;

VT, tidal

volume;

isoV, pulmonary volume during inspiration; &‘&, time derivative of tidal volume. Rs = 0.168 X [ 1 - 0.0028 X (37 - T)] mmH,O. se ml-‘; K = Pb/Pdif = 1.45; Cb = -Vb/l.4 PB. Vs, tidal volume actually detected by Fleisch pneumotachograph; Vq, difference between Vs and true VT of animal. Vq is proportional to Pb, which is computed from Pdif, because calibration revealed that Pb = Pdif X 1.45. K had been measured in a preliminary experiment by simultaneously recording Pdif (with Fleisch head) and Pb (with water manometer)

. I Pdrf

A-D

converter

while

box

was ventihted

with

steady

airflows,

inward

and outward. Over a Pb range of -12 to +10.2 mmH,O, Pdif was proportional to Pb (Pdif = Pb X 0.68) with a negligible scattering around regression line (r = 0.999). Fleisch

pneumotachograph

output

See text

fracheal pressure

mm ID, 0.06 ml dead space) to the expiratory line. Its opening is controlled by the electrovalve (NV) located in the plethysmographic box. The other outlet is an adjustable leak, which divides into two branches opening to the air. One branch is wide (8 mm ID), and its opening is controlled by the electrovalve LV. The other branch may be narrowed by means of a screw clamp and represents an adjustable resistance that allows control of the pressure in the nebulizer when LV is closed; i.e., the pressure under which the aerosol is injected into the tracheal cannula is adjustable. The nebulizing sequence includes six main steps, corresponding to ten different states of the operating logic (Fig. 3). Step 0: basal state, normal ventilation; only IV, EV, and LV are open. Sighs are periodically achieved by closing EV during two pump cycles (the frequency and dura-

for further

explanations.

relays with adequate cut-off ability, ensuring galvanic insulation of each unit (miniature relays Elesta SGR 282 with x10-ms response time) and operated by logic switch relays (subminiature relays Siemens V23042, with