NONINVASIVE MEASUREMENT OF AIRWAY RESPONSIVENESS IN ALLERGIC MICE USING BAROMETRIC PLETHYSMOGRAPHY

To the Editor:

It was with great interest that we read the recent paper by Hamelmann and colleagues describing noninvasive measurement of airway responsiveness (1). The ability to monitor this responsiveness without sacrificing the mice allows for obvious advantages in a variety of experimental protocols. However, the paper raises several issues that the authors may wish to address. A general one relates to the meaning of the chosen index, i.e., Penh. This seems to be a somewhat whimsical calculation based on a combination of expected and observed changes in the shape of the box pressure waveform. That it increases with MCh challenge is not too surprising, since almost all respiratory mechanics variables show qualitative correlations (1). The concern with Penh is that there does not seem to be any way to interpret quantitative changes. What does a doubling of Penh mean? A tenfold increase in Penh clearly indicates more smooth muscle contraction than a doubling, but there is no way to know how much more.

As discussed in the paper, the pressure signal from an animal breathing in a box occurs because of multiple causes. Specifically, on inspiration the box pressure can rise because of either expansion (decompression) of the alveolar gas or heating and humidification of the inspired tidal volume. The decompression component is related to the airway resistance, and the heating and humidification component is related to the tidal volume. In previous work we have assumed the decompression to be small, and then used changes in box pressure to quantify changes in respiratory frequency and tidal volume in awake unrestrained mice (3, 4). Hamelmannn and colleagues (1) assumed the tidal volume component to be small, and then used this same box pressure to measure changes in airway resistance. Thus, it appears that this box pressure represents the ultimate physiologic variable, one which can be used to measure whatever one wants to know. The magnitudes of minute ventilation we determined from box pressure are consistent with other independent measurements and allometric extrapolations, and the changes in Penh seem to reflect changes in airway smooth muscle constriction. How this can occur is not entirely clear, but there are several considerations regarding Penh that remain to be clarified.

Figures 2 and 3 in the paper by Hamelmann and colleagues (1) are confusing because the inspiratory pressure changes are shown to be negative. Unless the box ambient temperature was greater than the animals' body temperature, there is no reason for the box pressure to become negative on inspiration. Additionally, in Figure 3 the breath-to-breath variability seems rather large. If the record shown was the standard "typical" experiment, then the normal variability must be even greater. It would be nice to know the breath-to-breath variability in Penh. Lastly, several of the experimental validations of the methodology seem inappropriate. That is, with the validations using mechanically ventilated mice, the physics of the system are entirely different. With a ventilated mouse in the box, the box pressure changes with ventilation do not arise from the same factors that occur when the animal is enclosed and breathing spontaneously. Most importantly, there is no expansion of alveolar gas with inspiration, so there can be no component related to airway resistance. One way to separate the tidal volume from resistive component would be to eliminate the tidal volume component by acutely raising the ambient temperature to equal body temperature. If the ambient air is also fully humidified, then the inspiratory box pressure signal will result just from alveolar gas decompression.

In summary, it seems that, although there may be some value in using Penh to repeatedly assess bronchoconstriction in individual animals, the quantitative interpretation is presently quite limited. I realize such a criticism coming from a coinventor of the APTI (5) may not be taken too seriously, but given the great ease with which Penh can be measured, the potential for misleading interpretations and conclusions seems more substantial.

Division of Physiology
Department of Environmental Health Sciences
The Johns Hopkins University
Baltimore, Maryland

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1. Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin, and E. W. Gelfand. 1997. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156: 766-775 [Abstract/Free Full Text].

2. Ewart, S., R. Levitt, and W. Mitzner. 1995. Respiratory system mechanics in mice measured by end-inflation occlusion. J. Appl. Physiol. 79: 560-566 [Abstract/Free Full Text].

3. Tankersley, C. G., R. S. Fitzgerald, R. C. Levitt, W. Mitzner, S. L. Ewart, and S. R. Kleeberger. 1997. Genetic control of differential baseline breathing pattern. J. Appl. Physiol. 82: 874-881 [Abstract/Free Full Text].

4. Tankersley, C. G., R. S. Fitzgerald, W. Mitzner, and S. R. Kleeberger. 1993. Hypercapnic ventilatory responses in mice differentially susceptible to acute ozone exposure. J. Appl. Physiol. 75: 2613-2619 [Abstract/Free Full Text].

5. Levitt, R. C., and W. Mitzner. 1988. Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice. FASEB J. 2: 2605-2608 [Abstract].

	From the Authors:

We thank Drs. Mitzner and Tankersley for the interest they have taken in our work. The questions and concerns they raise are important, as we have been asked or have asked these questions ourselves.

It is important to note that Penh, or enhanced pause, is not an index that we developed. It was only our purpose in this study to investigate in an empirical fashion whether this index might relate in some useful way to the monitoring of bronchospasm in response to agonists in the mouse. Our results clearly show that under the conditions of the experiments described there is a direct correlation between bronchospasm as measured with both RL and Penh. Accordingly, a doubling of Penh relates to a doubling of RL. We are surprised that Drs. Mitzner and Tankersley would think that the cause of such changes are solely due to smooth muscle contraction per se and not to other mechanisms.

We agree that the crux of the issue is understanding the physiologic factors which control pressure changes within the plethysmograph. Careful examination of the studies quoted in their letter (their References 3 and 4) show that in these investigations pressure measurements were made in an essentially closed system, whereas in our study, there was a mesh screen pneumotachograph between the chamber and the room. Hence, in our investigations, flow between the room and plethysmograph is the parameter of interest. Adibatic gas expansion requires a finite period of time; moreover, the mouse breathes even when bronchoconstricted at a rapid (3-6 Hz) breathing frequency. Accordingly, basic laws of physics dictate that there is insufficient time for heating and humidification to occur on a breath-by-breath basis. We believe, as Drs. Mitzner and Tankersley have eluded to, that the major proportion of the box pressure signal is due to gas compression and/or thoracic cage motion. This impression is supported by experiments in which volume was delivered via computer-controlled ventilation, where the delivered volume is accurately recorded by the box flow/pressure measurements. We disagree that the validations were not appropriate. Certainly, mechanical ventilation is not exactly equivalent to spontaneous respiration, but as was stated, it was only one piece of evidence we provided validating the approach that the measurements are likely to be reasonable. Clearly, the most convincing data to us was the close correlation between RL measured with a traditional approach and Penh.

We apologize for any confusion having depicted inspiration descending in a negative direction but are unclear why that should be confusing, as flow-volume or volume-time data are often plotted in a similar fashion. The breath-by-variability of Penh is dependent on many factors, including the individual mouse, ambient noise, time of day, etc. As shown in the several data sets throughout our paper, Penh variability was consistently ± 20%. The statement that "there is no component related to airway resistance" seems unclear since it is known that there is, in fact, significant inspiratory resistance whether under conditions of mechanical ventilation or spontaneous respiration. We are currently performing additional studies where the warming of the box to body temperature is but one of several experiments being conducted, and we hope to report on these results at a later time.

We concur that more investigation is clearly needed into the physiologic meaning, physics, and quantitative interpretations of measurements made during eupnic respiration in the mouse. We would be the first to agree there well may be many other more useful parameters besides Penh; however, it is useful to remember that the use of FEV₁ started in a similar fashionand we all know what has happened to that whimsical parameter.