repeat measurement of respiratory mechanics using the forced oscillation technique in non-paralysed...

Pulmonary Pharmacology & Therapeutics (1999) 12, 173–183

Article No. pupt.1999.0198, available online at http://www.idealibrary.com on

PULMONARYPHARMACOLOGY& THERAPEUTICS

Repeat Measurement of Respiratory Mechanics using the ForcedOscillation Technique in Non-paralysed Rats

J. M. H. Preuss∗, G. L. Hall, P. D. Sly

Clinical Sciences Division, TVW Telethon Institute for Child Health Research, PO Box 855,West Perth 6872, Western Australia, Australia

SUMMARY: The present study has established a method for obtaining low-frequency forced oscillation meas-urements of lung mechanics in the absence of neuromuscular blockade in the rat. Increasing the ventilation rateof the animals inhibited the spontaneous breathing of the animals for a short period of time; enough to make thelow-frequency forced oscillation measurements of lung mechanics without the need for paralysis of the animals.Using this technique, it was possible to show that neuromuscular blockade with pancuronium bromide (0.4 mg/kgiv) resulted in a significant inhibition of methacholine responses in the parenchymal, but not the airway componentsof the rat lung. In studies where the animals were examined in a repeated manner, there was no significantdifference in methacholine responses on day 3 compared with those obtained on day 1. Similarly, in animals thatwere both challenged with methacholine and lavaged, there was no significant difference in the methacholineresponses or in the total and differential cell numbers obtained from the bronchoalveolar lavage fluid. Thus, thisstudy presents a technique for obtaining low-frequency forced oscillation estimates of lung mechanics in non-paralysed rats and allows for repeated measures to be made in the same animals. In addition, this study hasdemonstrated that neuromuscular blockade has differential effects on methacholine responses in different parts ofthe lung.

1999 Academic Press

KEY WORDS: Low-frequency forced oscillation, Neuromuscular blockade, Bronchoalveolar lavage.

INTRODUCTION of days in an experimental protocol provides forstatistical comparisons that are more robust andresults in a smaller number of animals being requiredThe use of the forced oscillation technique (FOT) forfor the study because the same group of animals canmeasuring pulmonary function allows the partitioningbe examined more than once instead of using moreof lung function mechanics into airway and par-groups of different animals. A previous study ex-enchymal components.1–3 Previously, the major dis-amining the effects of repeated measurements of air-advantage in using the low-frequency forcedway and parenchymal mechanics on three occasionsoscillation technique was the requirement for animalsover a period of 14 days in paralysed rats dem-to be paralysed. This requirement has made the designonstrated that baseline lung mechanics did not sig-of repeated studies in the same animal difficult, espe-nificantly change over the period examined.6cially within a short period, as full recovery from

In order to develop experimental protocols over aparalysis can take some time and often requires thetime period of only a few days, similar to that pre-use of further drug treatment to reverse the effects ofviously used in the rabbit,7–9 it was necessary to developthe neuromuscular blockers. In addition, recent stud-a technique whereby forced oscillation measurementsies have suggested that neuromuscular blockade withof lung function mechanics could be made in theagents such as pancuronium bromide may inhibit theabsence of neuromuscular blockade. Thus, this studycontractile responses of muscarinic agonists such asdescribes the effects of increasing the animal’s vent-methacholine.4,5

ilation rate to induce a temporary apnoeic periodThe ability to study the same animal over a numberduring which forced oscillation measurements may be

∗Author for correspondence. made in the absence of neuromuscular blockade. In

1094–5539/99/030173+11 $30.00 1999 Academic Press173

174 J. M. H. Preuss et al

addition, this technique was conducted over a 3- By using the transmission line theory,11,12 Zrs wascalculated from the Pbox(x)/Ptr(x) spectra, as the loadday period to determine whether both baseline and

methacholine challenge data remained consistent with impedance of the wave-tube, i.e.:repeated experiments and with the additional insult

Zrs=Zo sinh (cL)/[(Pbox(x)/Ptr(x))−cosh (cL)]of bronchoalveolar lavage.

Where Zrs represents the impedance of the respiratoryMATERIALS AND METHODS system, L is the length of the wave-tube and Zo and

c are the characteristic impedance and the complexAnimal preparation propagation wave number respectively, both de-

termined by geometrical data and material constantsA total of 75 male Brown–Norway rats 12 weeks of of the tube and air. Chest wall impedance (ZW) wasage were used in this study. Prior to the experiment, calculated by assuming no flow loss to airway wall oranimals were anaesthetized with ketamine/xylazine alveolar gas compression as ZW=Zrs.(Pes(x)/Ptr(x)) and(50/5 mg/kg body weight) and intubated with an endo- lung impedance (ZL) was obtained by subtractiontracheal tube (6 cm long, 2 mm id). The rats were (ZL=Zrs−ZW).placed in the supine position and mechanically ventil-ated (model 683, Harvard Apparatus, South Natick,

Parameter estimationMassachusetts, USA) with a tidal volume of 9 ml/kgand frequency of 90 or 120 breaths/min. The tail and ZL data were evaluated in terms of a linear modelfemoral veins were cannulated to allow maintenance consisting of a frequency-independent Raw (airwaydoses of anaesthetic mixture to be administered and/ resistance) and Iaw (airway inertance) and a constant-or for intravenous drug delivery. phase tissue component:

ZL=Raw+jxIaw+(G−jH)/xa

Measurement of lung mechanics

Lung mechanics were measured using a low-frequency where G and H are coefficients for tissue dampingforced oscillation technique in which input impedance and elastance respectively, x is angular frequency and(ZIN) was measured from 0.5 to 20 Hz using the wave- a determines the frequency dependance of the realtube technique.6,10 A 114 cm polyethylene tube (2.0 mm and imaginary parts of the impedance.13–16

id) was connected to the tracheal cannula at oneend via a three-way tap and to a loudspeaker-in-box

Aerosol delivery of methacholinesystem at the other end. The wave-tube had sidearmsto measure lateral pressures (ICS model 33NA002D For all studies, baseline measurements were made totransducers) at the loudspeaker end (Pbox) and at the establish the reproducibility of the data followed bycannula end (Ptr). The loudspeaker was driven by a administration of aerosolized saline and further meas-computer-generated small amplitude pseudorandom urements of lung mechanics. For both baseline andsignal containing frequencies in the range 0.5–21 Hz, saline measurements, five measurements each wereaccording to the ‘non-integer multiple’ principle, with made and averaged to obtain data for each condition.<2 cm H2O peak-to-peak excursion in Pbox. The forcing For both baseline and saline conditions, the five meas-function was applied to the animal during a brief urements made were generally found to be highlyperiod (6 s) of apnoea, i.e. with the ventilator dis- reproducible within 5–10% of the average. Aerosolsconnected. The signals from Pbox(t) and Ptr(t) were low- of methacholine were generated by a jet nebulizer (LCpass filtered (fifth order Butterworth, 25 Hz corner Plus, Pari-Werk GmbH, Starnberg, Germany) drivenfrequency) and digitized at a sampling rate of 128 Hz. by 5 l/min air attached to the inspiratory port of theOesophageal pressure (Pes(t)) was measured using a ventilator and delivered to the animal via the trachealminiature solid-state catheter-tipped manometer cannula. Aerosolized methacholine was given in doub-(MTC 3F, Drager Medical Electronics, Best, The ling, cumulative concentrations of 1–16 mg/ml forNetherlands). Transpulmonary pressure (Ptp(t)) was a period of 90 s/concentration, with lung functioncalculated by subtracting Pes(t) from airway opening measurements taken at 1-min intervals for 4 min be-pressure (Ptr(t) in our set-up). The pressure transfer tween each dose with the first being made 30 s afterfunctions Pbox(x)/Ptr(x) and Pes(x)/Ptr(x) were computed completion of each aerosol challenge.by fast Fourier transformation of the 6 s recordings,by using 4 s time windows and 95% overlap (where

Intravenous delivery of methacholineP(t) represents the data obtained in the time domainand P(x) is the fast Fourier transform of the time For all studies, baseline measurements were made to

establish the reproducibility of the data followed bydomain data).

Measurement of Respiratory Mechanics using FOT 175

administration of intravenous saline and further meas- preparations of cells were stained with Leishman’sstain and differential cell counts of neutrophils, eos-urements of lung mechanics. For both baseline andinophils, lymphocytes and macrophages were alsosaline measurements, five measurements each weredetermined.made and averaged to obtain data for each condition.

For both baseline and saline conditions, the five meas-urements made were generally found to be highly

Statistical analysisreproducible within 5–10% of the average. Intravenousmethacholine was administered via a femoral vein The potency of methacholine in the presence or ab-catheter in doubling, cumulative doses of 1–16 lg/kg sence of treatment drugs was determined from theper min for a period of 3 min/concentration, prior to equation: PCX=−log10(ECX), where ECX is the con-measurements being taken and continuing for the centration of methacholine eliciting X% of the saline4 min during the measurement period. Subsequent response. Comparison of data obtained betweendoses were administered without any break in the groups was undertaken using one-way analysis ofintravenous infusion of methacholine. variance or paired Student’s t-tests as appropriate.

Cumulative concentration-effect curves were com-pared using two-way analysis of variance. In-

Experiment protocol: effects of neuromuscular flammatory cell numbers determined fromblockade bronchoalveolar lavage fluid samples obtained on

days 1 and 3 were analysed using paired or non-pairedAnimals were ventilated at a rate of 90 breaths/minStudent’s t-tests as appropriate. P values less thanin the presence of pancuronium bromide (0.4 mg/kg0.05 were deemed to be statistically significant.iv) or at a rate of 120 breaths/min in the presence

or absence of pancuronium bromide (0.4 mg/kg iv).Different groups of animals were used for each treat-

Drugsment protocol. Following baseline measurements oflung mechanics, responsiveness to methacholine was Drugs used in this study included methacholine chlor-determined. ide (acetyl b-methyl choline chloride) (Sigma Chemical

Company, St Louis, Missouri, USA), ketamine hydro-chloride (KETAMIL; Parnell, Sydney, Australia),

Experiment protocol: repeat measurements xylazine hydrochloride (ROMPUN; Bayer, Sydney,Australia) and pancuronium bromide (Astra, Sydney,Animals were ventilated at a rate of 120 breaths/min,Australia).which was shown to inhibit spontaneous breathing of

the animals for the time period (6 s) required to enablethe forced oscillation measurements to be made. Atthe conclusion of the experiment, the animals resumed RESULTSspontaneous breathing within a minute or two ofbeing removed from the ventilator and recovered Influence of ventilation rate on spontaneous breathingquickly with no adverse effects. Experiments wereconducted over a period of 3 consecutive days. On day In non-paralysed animals ventilated at 120 breaths/1, following baseline measurements of lung mechanics, min, apnoeic periods, long enough to make 6 s meas-responsiveness to methacholine was determined and urements, were easily and consistently obtained. Inanimals allowed to recover. In some studies, broncho- non-paralysed animals ventilated at 90 breaths/min,alveolar lavage (BAL) was undertaken following the spontaneous breathing was not inhibited and it waschallenge with methacholine. On day 3, methacholine thus impossible to make low-frequency forced os-challenge and BAL were repeated as for day 1. cillation measurements of lung function mechanics

in these animals in the absence of neuromuscularblockade.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) was performed on twoInfluence of ventilation rate on baseline mechanicsdifferent groups of animals: those that were lavaged

only on days 1 and 3 and those that were lavaged Baseline measurements for Raw and G were not sig-after methacholine challenge on days 1 and 3. The nificantly (P>0.05) different in animals ventilated atlavage involved the administration of 2×0.5 ml sterile 120 breaths/min compared to those ventilated atsaline via the endotracheal tube with immediate as- 90 breaths/min in the presence of pancuronium brom-piration and collection into a sterile tube. Total cell ide. In contrast, H showed a significant (P<0.05)counts were determined under light microscopy using decline in those animals ventilated at the greater rate

(Table 1).an improved Neubauer haemocytometer. Cytospin


Table 1 Baseline data for animals ventilated at 90 or 120 breaths/min in the presence or absence of pancuroniumbromide (0.4 mg/ml iv). Data presented as the mean±SEM for n animals in each group. Raw represents airwayresistance, G tissue damping and H tissue elastance.

Treatment n Raw (cmH2O.s/l) G (cmH2O/l) H (cmH2O/l)

+ Panc at 90 breaths/min 13 216.36±12.07 819.23±45.15 3334.37±228.12+ Panc at 120 breaths/min 12 205.44±5.49 774.42±127.65 2108.72±236.71∗− Panc at 120 breaths/min 12 202.30±10.33 652.62±37.76 2606.70±156.70

∗ Indicates data significantly different (P<0.05) from the corresponding data for animals examined in the presenceof pancuronium at 90 breaths/min.

Influence of ventilation rate on methacholine Influence of neuromuscular blockade on methacholineresponsesresponses

In animals ventilated at 120 breaths/min in the absenceChallenge with aerosolized methacholine producedof pancuronium bromide, there was a significantlyconcentration-dependent increases in tissue damping(P<0.05, two-way ANOVA) greater Raw response to(G) and elastance (H) with only small increases inmethacholine than the neuromuscularly blockedairway resistance (Raw) in all groups of animals. Theregroup (Fig. 2A). Similarly, a significant (P<0.05) in-was a small but significant (P<0.05, two-way-crease in tissue damping (G) was observed in animalsANOVA) decrease in Raw responses in paralysed an-ventilated at 120 breaths/min in the absence of neuro-imals ventilated at 120 breaths/min compared withmuscular blockade compared to those that werethose ventilated at 90 breaths/min (Fig. 1A). Similarly,neuromuscularly blocked (Fig. 2B) which was in-the tissue damping parameter G decreased sig-dicated by a 3.3-fold decrease in PC200 for G (Tablenificantly (P<0.05) when the ventilation rate was in-2). Tissue elastance (H) also showed a significantcreased from 90 to 120 breaths/min in(P<0.05) increase in animals ventilated at 120 breaths/neuromuscularly blocked animals (Fig. 1B), with themin in the absence of neuromuscular blockade com-PC200 for methacholine increasing 2.7-fold in animalspared to those that were neuromuscularly blockedventilated at 120 breaths/min compared with those atwith pancuronium bromide (Fig. 2C) and this increase90 breaths/min (Table 2). Tissue elastance (H) re-was observed primarily at the top end of the metha-sponses to aerosolized methacholine challenge dem-choline dose-response curve. Methacholine PC150 re-onstrated no significant (P>0.05) difference betweensponses of H were not significantly different for anyneuromuscularly blocked animals ventilated at 90 orof the animals examined (Table 2).120 breaths/min (Fig. 1C).

The dose-dependent increases in Raw observed fol-In line with previous findings,15 intravenously ad-lowing intravenously administered methacholine wereministered methacholine resulted in a more prominentnot significantly (P>0.05) different between the an-airway resistance (Raw) response and a less well definedimals ventilated at 120 breaths/min in the absence ortissue elastance (H) response than observed for metha-presence of neuromuscular blockade (Fig. 2D). Incholine administered via the aerosol route.contrast, there was a significant (P<0.05) shift to theThe dose-dependent increases in Raw and G ob-left of the dose-response curve for G in animalsserved following intravenously administered metha-ventilated at 120 breaths/min in the absence of neuro-choline were not significantly (P>0.05) differentmuscular blockade compared to those that werebetween the neuromuscularly blocked animals ventil-neuromuscularly blocked with pancuronium bromideated at 90 or 120 breaths/min (Fig. 1D,E). There(Fig. 2E). This was also observed by a 2.8-fold decreasewas no significant difference in tissue elastance (H)in the PC120 for G in these animals (Table 2). Thereresponses to intravenous methacholine between thewas no significant difference in tissue elastance (H)two groups (Fig. 1F).responses to intravenous methacholine between thetwo groups (Fig. 2F).

Influence of neuromuscular blockade on baselinemechanics Repeat measurements

In animals ventilated at a rate of 120 breaths/min in Measurement of lung mechanics and subsequent cal-the presence or absence of pancuronium bromide, culation of lung function parameters obtained frombaseline Raw, G and H parameters were not sig- animals on days 1 and 3 of the experiment protocolnificantly (P>0.05) different in either the aerosol or were compared in two groups of animals: those that

were exposed to methacholine challenge only, andthe intravenous groups (Table 1).


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Fig. 1 Lung function parameters determined in response to cumulative additions of aerosolized methacholine (A) Raw, (B) G and (C)H and intravenously administered methacholine (D) Raw, (E) G and (F) H in animals ventilated at 90 breaths/min (Β) or 120 breaths/min (Ε) in the presence of pancuronium bromide (0.4 mg/kg iv). Vertical lines represent the SEM of n=6 (aerosol) and 7 (intravenous)animals.

those that were lavaged to obtain BAL fluid following not significantly different on day 3 compared withthose obtained on day 1 for either of the treatmentthe methacholine challenge.

Baseline measurements of lung mechanics derived groups (Table 3).In animals that received aerosolized methacholinelung function parameters for baseline data that were


Table 2 Methacholine PC values for airway resistance (Raw), tissue damping (G) and tissue elastance (H) in animals ventilated at 90 or120 breaths/min in the presence of neuromuscular blockade with pancuronium bromide and those ventilated at 120 breaths/min in theabsence of neuromuscular blockade.

Aerosol methacholine Intravenous methacholine

Treatment n Log PC200 G Log PC150 H n Log PC150 Raw Log PC120 G(cmH2O/l) (cmH2O/l) (cmH2O.s/l) (cmH2O/l)

+ Panc at 90 breaths/min 6 0.79±0.09 0.80±0.17 7 0.92±0.09 0.74±0.18+ Panc at 120 breaths/min 6 1.16±0.07∗ 0.81±0.20 6 1.05±0.09 0.96±0.06− Panc at 120 breaths/min 6 0.64±0.11† 0.56±0.10 6 1.03±0.08 0.51±0.18†

∗ Indicates data significantly different (P<0.05) from the corresponding data for animals examined in the presence of pancuronium at90 breaths/min; † indicates data significantly different (P<0.05) from the corresponding data for animals examined in the presence ofpancuronium at 120 breaths/min.

challenge only, there was no significant difference forced oscillation technique in non-paralysed rats.Previously, low-frequency forced oscillation meas-(P>0.05) between responses to methacholine on days

1 and 3 for any of the lung parameters measured (Fig. urements in rats required the inhibition of spon-taneous breathing through neuromuscular blockade.3). In animals that were lavaged following metha-

choline challenge, there were similarly no significant This, however, presented numerous difficulties in un-dertaking repeated measurements in the same animaldifferences in lung function parameters between days

1 and 3 (Fig. 4). due to the need for recovery from the paralysis, oftenrequiring the administration of a cocktail of drugs toreverse the actions of the neuromuscular blockingBronchoalveolar lavageagents. In addition, recent evidence in guinea-pigs has

Bronchoalveolar lavage (BAL) samples were obtained suggested that some of the more commonly usedon days 1 and 3 of the experimental protocol. In neuromuscular blocking agents such as pancuroniumanimals that were lavaged in the absence of metha- bromide have inhibitory effects on methacholine-in-choline challenge, the yield of lavage fluid following duced bronchoconstriction.4 It has further been dem-aspiration was 73±2.0% on day 1 and 70±4.0% on onstrated in radioligand binding studies in Chineseday 3. Total and differential cell counts were not hamster ovary cells stably transfected with either thesignificantly (P>0.05; paired Student’s t-test) different M2- or the M3-muscarinic cholinoceptor, that someon day 3 compared with those obtained on day 1, of the neuromuscular blocking agents have affinitiesalthough a clear trend towards increased numbers for both M2- and M3-muscarinic cholinoceptors.5 Thewas observed in the total cell counts, primarily due effects of these neuromuscular blocking agents onto an apparent (though not statistically significant) muscarinic cholinoceptor responses in the rat haveincrease in neutrophil numbers (Table 4). not previously been examined. Thus, the present study

In animals that were lavaged following metha- was undertaken in order to design a methodologycholine challenge, the yield of lavage fluid was 81±5% whereby forced oscillation measurements of lung func-on day 1 and 81±4% on day 3. Total cell counts from tion in the rat could be undertaken without the needthe BAL fluid were significantly increased on day 3 for paralysis and thereby facilitating repeated meas-compared with those obtained on day 1 and this was urements of the same animal. The advantage in beingprimarily due to a small but significant (P<0.05; paired able to conduct repeat experiments in the same animalStudent’s t-test) increase in macrophage numbers. lies in the fact that each animal then acts as its ownOther inflammatory cell numbers did not significantly control and abolishes the need for a separate group(P>0.05; paired Student’s t-test) change from those of animals to be used as controls. In addition, theobtained on day 1 (Table 4). statistical analysis of data from within the same animal

Comparison of day 1 and day 3 cell counts from is based on paired comparisons and is therefore moreanimals subjected to methacholine challenge and BAL robust.were generally lower than that observed for animals In the first instance, we have demonstrated that bythat were treated with BAL only. This was only simply increasing the mechanical ventilation rate ofstatistically significant (P<0.05; non-paired Student’s the rat from 90 to 120 breaths/min, we can inhibit thet-test) for total and lymphocyte counts on day 3 (Table spontaneous breathing of the animal for the 6 s that4). is required to make the forced oscillation meas-

urements. At the conclusion of the experiment, theanimals began to take spontaneous breaths within halfDISCUSSIONa minute, resumed a regular pattern of spontaneousbreathing within 1–2 min and recovered quickly withThe present study has demonstrated a technique for

measuring lung function using the low-frequency no apparent adverse effects. Baseline measurements


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Fig. 2 Lung function parameters determined in response to cumulative additions of aerosolized methacholine (A) Raw, (B) G and (C)H and intravenously administered methacholine (D) Raw, (E) G and (F) H in animals ventilated at 120 breaths/min in the absence (Φ)or presence (Ε) of pancuronium bromide (0.4 mg/kg iv). Vertical lines represent the SEM of n=6 (aerosol) and 7 (intravenous) animals.

for airway resistance (Raw) and tissue damping (G) has demonstrated that challenge with inhaled, aero-solized methacholine resulted in significant dose-de-were not significantly altered in animals with the

increased ventilation rate, whereas tissue elastance pendent increases in tissue damping and tissueelastance with little change in airway resistance. Con-(H) decreased.

In line with previous studies,10,17 the current study versely, intravenously administered methacholine


Table 3 Baseline data for animals studied on days 1 and 3. Data presented as the mean±SEM for n animals ineach group. Raw represents airway resistance, G tissue damping and H tissue elastance.

Treatment n Raw (cmH2O.s/l) G (cmH2O/l) H (cmH2O/l)

MCh (no BAL)day 1 6 220.80±6.56 648.65±51.13 2999.62±193.15day 3 6 180.37±13.97 697.29±41.34 2508.88±328.52

MCh and BALday 1 6 208.54±10.92 786.57±78.47 3318.35±604.96day 3 6 195.95±7.60 663.74±47.35 2953.33±291.93

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Fig. 3 Lung function parameters determined in response to cumulative additions of aerosolized methacholine in animals ventilated at120 breaths/min. Data are from experiments carried out in the same animals on days 1 (Ε) and 3 (Φ). Raw=airway resistance, Iaw=airway inertance, G=tissue damping, H=tissue elastance.

produced significant increases in tissue damping and subtype and approximately 70% of the muscariniccholinoceptors present in the central airways of theairway resistance with little effect on tissue elastance.

The reasons for the different responses to aerosolized rat of the M2-muscarinic cholinoceptor subtype.19 Inrat peripheral lung tissue, M2-muscarinic cholino-and intravenously administered methacholine are un-

clear, although it has been suggested,10 that it is due ceptors predominate although both M1- and M3-mus-carinic cholinoceptors are also present.19 M3-to methacholine acting on different structures within

the lungs rather than inhomogeneities within the lung. muscarinic cholinoceptors are believed to be re-sponsible for the functional responses of the rat peri-It has previously been shown that contraction of

airway smooth muscle in response to muscarinic cho- pheral lung to muscarinic cholinoceptor agoniststimulation.20,21linoceptor agonists is mediated via the M3-muscarinic

cholinoceptor,18 although only 30% of the muscarinic Increasing the ventilation rate from 90 to120 breaths/min had a significant effect on Raw and Gpopulation are of the M3-muscarinic cholinoceptor


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Fig. 4 Lung function parameters determined in response to cumulative additions of aerosolized methacholine in animals ventilated at120 breaths/min followed by bronchoalveolar lavage. Data are from experiments carried out in the same animals on days 1 (Ε) and 3(Φ). Raw=airway resistance, Iaw=airway inertance, G=tissue damping, H=tissue elastance.

Table 4 Total and differential cell counts from bronchoalveolar lavage fluid obtained on days 1 and 3 from animals receivingbronchoalveolar lavage only and from animals who were exposed to methacholine prior to BAL fluid being taken on both days 1 and3. Data presented as the mean of n animals±SEM.

n Total Macrophages Lymphocytes Neutrophils Eosinophils(×104 cells/ml) (×104 cells/ml) (×104 cells/ml) (×104 cells/ml) (×104 cells/ml)

BAL only 7 day 1 24.18±6.60 22.68±5.93 1.46±0.73 0.01±0.01 0.03±0.037 day 3 45.18±8.94 34.55±7.78 2.60±0.54 6.46±3.78 0.52±0.30

BAL following 6 day 1 12.21±1.51 10.83±1.75 0.44±0.10 0.90±0.75 0.04±0.04methacholine 6 day 3 21.71±1.50∗† 17.44±1.34∗ 1.10±0.31† 2.90±0.62 0.27±0.14

∗Represents values different to that for the equivalent values from day 1; † represents data different from the BAL only data for theequivalent day.

following aerosolized methacholine and a significant that 30–60 s are needed to eliminate the effects of pre-vious ventilation from lung tissue mechanics.27 This iseffect on G in animals challenged with intravenous

methacholine. These results are not unexpected, how- commonly done by giving the animal a large breath,with or without an airway occlusion, and waitingever, as it is well known that an increase in the breathing

frequency results in decreased dynamic compliance 30–60 s before making a measurement. In the currentprotocol, this procedure could not be followed. Thus,and resistance.22,23 This has been demonstrated in the

cat,24 dog,25 and in patients with obstructive lung dis- measurements made during periods of mechanicalventilation at 90 or 120 breaths/min have differentease.26 This phenomenon is likely to be due to the

different ventilation (or volume) history of the lung ventilation histories and these differences are reflectedin the tissue mechanics.tissues. We, and others, have previously demonstrated


In animals ventilated at 120 breaths/min and ex- with methacholine and lavaged on both days, therewas no significant change in the lung function para-posed to aerosolized methacholine, there was a sig-

nificant leftward shift of the methacholine dose- meters determined. These results suggest that con-ducting repeated measures of lung function in ratsresponse curves for both tissue parameters (G and H)over this time period is possible without altering theand for airway resistance (Raw) in the absence ofresponses of the airways and lung tissue to metha-neuromuscular blockade compared to that in thecholine challenge. The inflammatory cell numbers inpresence of neuromuscular blockade. In animals giventhe lavage fluid were not statistically different on dayintravenous methacholine, only tissue damping (G)3 compared with that of day 1 in animals receiving onlywas altered by the presence of neuromuscular block-bronchoalveolar lavage. However, a trend towards anade with pancuronium bromide. This alteration, how-increase in total and neutrophil cell numbers wasever, is more likely due to inhomogeneities in theapparent. Similar results were obtained from animalsperipheral lung tissue. This is suggested by the factthat underwent BAL following methacholine chal-that the increase in G was not accompanied by anlenge with only a statistically significant increase inincrease in H, indicating that the increase in G is duetotal and macrophage cell numbers being evident.to the effects derived from an increase in Raw. InThese results therefore indicate that previous metha-addition, whilst an increase in Raw was observed incholine challenge and/or BAL do not alter the lungresponse to methacholine challenge by the intravenousfunction parameters obtained subsequently. In ad-route, this was not altered in the presence of pan-dition, only small and largely insignificant increasescuronium, suggesting that with respect to these re-in some inflammatory cell numbers were observed insponses, pancuronium was without inhibitory effectthe BAL fluid from animals having previously beenon the methacholine-induced responses.exposed to methacholine challenge and/or BAL.These results confirm a previous study by Okanlami

The cell counts from animals lavaged followinget al,4 demonstrating that pancuronium bromide in-methacholine challenge were generally smaller thanhibited acetylcholine-induced bronchoconstriction inthat from animals that received the lavage only. How-the guinea-pig through inhibition of M2 and M3 mus-ever, only the total and lymphocyte counts on day 3carinic cholinoceptors. The disparity of results fromreached statistical significance. The reason for theseaerosolized and intravenously administered metha-differences most likely lies in the fact that in thecholine in the current study suggests a possible differ-animals lavaged following methacholine challenge,ence in mechanism of action of methacholine by thethere is a greater yield of lavage fluid and this istwo routes of administration and confirms the findingsmost likely due to the airways, particularly the lowerof a previous study.10

airways, remaining a little constricted. The lowerIn the studies examining the effects of a repeat metha-cellular yield may reflect the constriction-induced in-choline challenge in the animals 48 h after the first chal-ability of the lavage fluid to penetrate as deeply intolenge, lung function parameters derived from lungthe lungs as in those animals that were not challengedmechanics demonstrated that there was no change inwith methacholine prior to the lavage.baselineRaw,GorHonday3comparedto thatobtained

In conclusion, this study has demonstrated that low-on day 1. This was true of the animals that receivedfrequency forced oscillation measurements of lungmethacholine challenge only and those that also hadfunction parameters can be obtained in non-paralysedbronchoalveolar lavage samples taken following therats. In addition, neuromuscular blockade with pan-methacholine challenge. These results suggest thatcuronium bromide inhibits methacholine-induced re-methacholine challenge and/or bronchoalveolar lavagesponses of both the airways and parenchymal tissuedoes not significantly alter the baseline mechanics ofof the lungs. Finally, this methodology for conductingthe lungs in this animal model over this short period oflow-frequency forced oscillation studies of lung func-time. In a previous study,6 it was shown that baselinetion in non-paralysed rats enables repeat meas-measurements of lung mechanics were unaltered whenurements to be made in the same animal andrepeat measurements in rats were made at weekly in-demonstrates that repeated methacholine challengetervals. The advantage of using a smaller time interval,and/or bronchoalveolar lavage do not alter subsequentas in the current study, lies in the ability to examine thelung function parameters and have only small butresponses of the lungs of sensitized animals 24 h priorlargely insignificant effects on inflammatory cellto and 24 h following allergen challenge. These timecounts in the lavage fluid.periods are significant as it is believed that the majority

of the important inflammatory responses in the lungsreach a peak 24 h following allergen challenge.

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Date accepted: 9 April.15. Oostveen E, Zwart A, Peslin R, Duvivier C. Respiratory

repeat measurement of respiratory mechanics using the forced oscillation technique in non-paralysed...

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