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Experimental Physiology 89.5 pp 623-628
DOI: 10.1113/expphysiol.2004.027706
© The Physiological Society 2004
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Spontaneous changes in guinea-pig respiratory pattern during barometric plethysmography: role of catecholamines and nitric oxide

Blanca Bazán-Perkins1, Mario H. Vargas1, Edgar Sánchez-Guerrero1, Jaime Chávez1 and Luis M. Montaño2

1 Departamento de Investigación en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, México DF, México2 Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510, México DF, México


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Barometric plethysmography for unrestrained animals is a non-invasive method that allows repetitive measurements of pulmonary function, but habituation of the conscious animal to this technique has not been explored. Respiratory frequency (fR) and ‘enhanced pause’ (Penh) were measured by barometric plethysmography for a period of 8 h in guinea-pigs. Compared with basal values, during the first hour of recording a progressive increase in Penh (up to 25–50%) and a corresponding decrease in fR were recorded, followed by a relative plateau in each for up to 8 h. These changes were avoided by a 30-min pretreatment with propranolol and L-NAME (nitric oxide synthase inhibitor), with Penh values as high as this plateau phase since the beginning of recording. Atropine, salbutamol or budesonide did not modify the progressive increment in Penh. We concluded that catecholamines and nitric oxide are released when guinea-pigs are introduced into the plethysmographic chamber, leading to initial low Penh values. These mediators probably diminish owing to habituation of the animal to the new environment, with an apparent progressive increment in Penh. These spontaneous changes in Penh and fR must be taken into account during barometric plethysmography in order to avoid misinterpretation of the results.

(Received 25 March 2004; accepted after revision 8 July 2004; first published online 15 July 2004)
Corresponding author M. H. Vargas: Departamento de Investigación en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, Tlalpan 4502, CP 14080, México DF, México. Email: mhvargasb{at}yahoo.com.mx


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Barometric plethysmography for freely moving animals is an increasingly used method to assess pulmonary function (Chávez et al. 1996; Chong et al. 1998; Hamelmann et al. 1997; Sommer et al. 1998). The main variable in this method is Penh, which has been shown to be correlated with total lung resistance (RL) in BALBc mice (Hamelmann et al. 1997; Adler et al. 2004), though not in C57BL6 mice (Adler et al. 2004). Other authors have suggested that Penh is a surrogate of specific airway resistance (sRaw) in guinea-pigs (Bergren, 2001; Chong et al. 1998; DeLorme & Moss, 2002).

Major advantages associated with barometric plethysmography are the absence of animal restriction, the lack of pharmacological interference by anaesthetic agents, and the possibility to make repetitive or prolonged studies without animal suffering. However, when experiments are made in conscious animals, other factors must be taken into account. Most animal species become anxious when they are introduced to a new environment, and this is followed by a progressive habituation process (Bolivar et al. 2000). This period of habituation is clearly important in behavioural research (O'Keefe & Nadel, 1978), but it is rarely mentioned in experiments on respiratory physiology. In this context, it is reasonable to speculate that the respiratory pattern, which is highly influenced by stress or emotions (Rietveld et al. 1999), might vary during the habituation processes. As far as we are aware, there are no published studies documenting whether this habituation process can occur during barometric plethysmography. Here we evaluated changes in fR and Penh spontaneously occurring during barometric plethysmography in freely moving guinea-pigs.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
We used healthy male Hartley guinea-pigs (500.3 ± 10.9 g), bred in conventional conditions in our institutional laboratory animal facilities (12/12 h light–dark cycles, 21 ± 1°C, 50–70% humidity) and fed ad libitum with Harlan pellets for guinea-pig and sterilized water. The protocol was revised and approved by the Scientific and Ethics Committees of the Instituto Nacional de Enfermedades Respiratorias.

Study design

Because our first experiments showed that spontaneous changes in Penh and fR were fully developed after several plethysmographic sessions, all guinea-pigs were studied when they had at least five sessions, each one on different consecutive days. On the day of the study, guinea-pigs were introduced in the body plethysmograph and a basal recording for a period of 5 min was obtained. They then received a pharmacological pretreatment or sham manoeuvre and 30 min later were reintroduced into the plethysmographic chamber in order to reinitiate the recording for 8 h. The pharmacological pretreatment consisted of I.P. administration of atropine (1 mg kg–1), salbutamol (30 µg kg–1), L-NAME [an inhibitor of nitric oxide (NO) biosynthesis, 0.5 mg kg–1] or propranolol (3.1 mg kg–1). An additional group received the combination of L-NAME plus propranolol. In a separate group, budesonide (0.25 mg ml–1) was nebulized for 5 min. We corroborated that the selected doses of atropine and salbutamol were sufficient to cause a statistically significant rightward displacement of the concentration–response curve to inhaled acetylcholine. Doses of remaining drugs were selected according to published reports (Shindoh et al. 1998; Montaño et al. 1987; Wennergren et al. 1996). Each experimental group comprised four guinea-pigs. Control guinea-pigs (n= 4–8 in each group) received a sham administration of intraperitoneal or nebulized saline, as required.

Plethysmography

Every guinea-pig was placed in a whole body plethysmographic chamber for freely moving animals (Buxco Electronics Inc., Troy, NY, USA). A constant air flow (10 ml s–1) was delivered to this chamber throughout the experiments. The underlying principles of this technique have been previously described (Drorbaugh & Fenn, 1955; Epstein & Epstein, 1978; Hamelmann et al. 1997). Briefly, the pressure inside the barometric plethysmographic chamber is measured through a differential pressure transducer connected to a preamplifier. Because the air is heated and humidified in the lungs, during the inspiratory phase the volume of air inside the thorax is larger than the volume of air drawn by the animal from the plethysmographic chamber. This larger volume of air inside the thorax produces an increase in the pressure of the plethysmographic chamber. Thus, although the transducer does not directly measure inspiratory or expiratory flows, it senses the pressure changes inside the plethysmographic chamber caused by the addition of heat and water vapour to the inhaled air as it enters the respiratory system of the animal (DeLorme & Moss, 2002). This pressure signal was then processed with Buxco Biosystem XA v1.1 software to calculate several respiratory parameters, including Penh. This index was obtained from the following equation (Hamelmann et al. 1997):


{eph_066_m1}

where Rt is the expiratory time (s), Tr is the relaxation time (s), PEP is the peak expiratory pressure (cmH2O) and PIP is the peak inspiratory pressure (cmH2O). The software was adjusted to include only breaths with a tidal volume of 1 ml or more, with a minimal inspiratory time of 0.15 s, a maximal inspiratory time of 3 s and a maximal difference between inspiratory and expiratory volumes of 10%. After the guinea-pigs were placed inside the plethysmographic chamber, recording was initiated 5 min later and, from this point onwards, respiratory parameters were recorded at 5 and 10 min, and every 15 min thereafter. Because respiratory parameters were calculated in each breath, adjustments were made to the software in order to average values from all breaths occurring over a period of 15 s, and then to average those values during the last 5 min of each period.

Temperature and CO2 concentration inside the plethysmographic chamber were continuously monitored with a digital thermometer and a capnograph (Novametrix, Medical Systems Inc., CT, USA), respectively.

Statistical analysis

Paired Student's t test was used to evaluate changes in Penh and fR as compared with their respective basal values. ANOVA followed by Tukey or Dunnett tests were used for multiple comparisons. Statistical significance was set at two-tailed P < 0.05. Data in the text and figures are expressed as mean ±S.E.M.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Baseline Penh and fR values in the pooled group of animals were 0.268 ± 0.007 and 114.4 ± 3.1 breaths min–1, respectively. After introducing guinea-pigs to the plethysmographic chamber, all control animals showed a gradual increase in Penh values until reaching about 25–50% increment, with a corresponding progressive decrease in fR. These changes mainly occurred during the first hour of recording, with a relative steady-state thereafter until the end of the 8-h period of recording (Fig. 1). Changes observed in both variables tended to mirror each other. Although changes in fR and Penh were documented from the time animals were first submitted to a plethysmography session, they became progressively more noticeable in subsequent sessions, and were fully manifested after five or six sessions (Fig. 2). Therefore, all pharmacological experiments were performed in animals with at least five plethysmography sessions.



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  		Figure 1.  Changes in Penh and fR during an 8 h barometric plethysmographic recording in guinea-pigs
Symbols correspond to the mean of n= 19 animals, and vertical lines to S.E.M.*P < 0.05 with respect to basal value (time 0) (paired Student's t test). n.s., non-significant differences among the averaged 1 h intervals (ANOVA and Tukey's test).

 


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  		Figure 2.  Penh and fR averaged from the last 5 h of barometric plethysmography in guinea-pigs (n= 4) repetitively submitted (up to six times) to such study
*P < 0.05 and **P < 0.01 as compared with its respective basal value (paired Student's t test). {dagger}P < 0.05 as compared with session 1 (ANOVA and Dunnett's test). Bars correspond to mean and vertical lines to S.E.M.

 
Intraperitoneal administration of atropine or salbutamol did not modify changes in Penh and fR occurring during the first 2 h of recording, as compared with the control group receiving intraperitoneal saline alone (Fig. 3A). Similarly, inhaled budesonide did not modify these variables, when compared with the control group aerosolized with saline (Fig. 3B). Either propranolol or L-NAME alone caused a slight increase in Penh values since the beginning of the recording compared with control animals, though this did not reach statistical significance. However, a combination of L-NAME and propranolol increased Penh values above basal levels during the first 20 min of the recording (Fig. 3C). Owing to the progressive increase in Penh in control guinea-pigs, at later stages Penh values were similar in treated and control groups.



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  		Figure 3.  Effect of different pharmacological pretreatment on Penh and fR during the first 2 h of barometric plethysmography
Experimental animals received: A, 30 µg·kg–1I.P. salbutamol ({circ}), 1 mg kg–1I.P. atropine ({square}); B, 0.25 mg ml–1 aerosolized budesonide ({circ}); and C, 0.5 mg kg–1I.P.L-NAME ({circ}), 1.3 mg kg–1I.P. propranolol ({square}) and the combination of both ({blacktriangledown}). Control groups (•) received corresponding sham pretreatments. Symbols correspond to mean and vertical lines to S.E.M.*P < 0.05 as compared with control group (ANOVA and Dunnett's test).

 
CO2 concentration within the plethysmographic chamber did not change throughout the study (n= 4). Although temperature inside the chamber increased slightly (~1–2°C) when the guinea-pig was introduced, this variation occurred in the first few minutes (~5 min), and thereafter remained constant.


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our results showed that guinea-pigs submitted to a repetitive barometric plethysmography in conscious and unrestrained conditions experience acute changes in pulmonary function during the first hour after the animal enters the plethysmographic chamber. A possible explanation for this phenomenon is that the stress produced by factors such as handling and encountering a novel environment after introduction of the animal to the plethysmographic chamber caused an initial abnormal respiratory pattern, which progressively returned to a more physiological state with habituation. This type of modification of the normal respiratory function after acute stressing factors has been previously described in humans (Han et al. 1997; Rietveld et al. 1999).

A number of neural and hormonal changes have been associated with stressful situations. These include sympathetic nervous system activation, glucocorticoid release and NO production (Li & Quock, 2002; Sanchez et al. 2003; Shalev, 2002). In this context, catecholamines and NO are two well-known relaxing agents of the airway smooth muscle (Hamad et al. 2003; Sommer et al. 1997). Because the combination of propranolol and L-NAME abolished the low basal airway tone at the beginning of the plethysmographic session, it is reasonable to suggest that this phenomenon was mediated by the stress-induced release of catecholamines and NO.

Additionally, it has been described that once a stressful situation ends, a vagal rebound ensues (Mezzacappa et al. 2001). It is therefore possible that the progressive increase in Penh occurring during the first hour of the plethysmography session was due to increasing vagal tone as guinea-pigs became adapted to the chamber. However, we found that atropine was unable to prevent this increase in Penh, discounting the possibility of cholinergic rebound. On the other hand, we also discounted the involvement of other mediators such as eicosanoids, because budesonide was also unable to prevent the progressive increase in Penh.

There has been some uncertainty as to the precise physiological function measured by Penh or what the interpretation of this index should be (Mitzner & Tankersley, 2003). Penh is an index of several independent factors, including Te, Rt, PEP and PIP, and the way in which it correlates in a ‘mirror’ fashion with changes in fR prompts the question of whether lowering of fR could induce the increase measured in Penh. Nevertheless, independent of the physiological function measured by Penh, we found that the combination of propranolol and L-NAME did not modify the fR changes occurring in the first hour of plethysmography, in spite this combination having a notable effect on Penh. Thus, it seems that changes in Penh do not necessarily correspond to changes in fR.

Barometric plethysmography is an increasingly used method to assess Penh in several animal species. Although the habituation phenomenon was clearly observed in guinea-pigs, its presence awaits to be demonstrated in other animal species such as mouse, rat or rabbit.

In conclusion, our results suggested that catecholamines and NO are being released when guinea-pigs are introduced into the plethysmographic chamber, leading to an initial low basal Penh, followed by a progressive increase in this parameter during the first hour as the influence of these mediators gradually disappears. The spontaneous changes in Penh and fR must be taken into account during barometric plethysmography in order to avoid misinterpretation of the results, particularly when subtle modifications of Penh are the main objective of the study. Furthermore, we suggest that if repetitive plethysmographic sessions are to be performed, a habituation process of at least five plethysmographic sessions should be undertaken prior to obtaining repetitive measurements of Penh.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Adler A, Cieslewicz G & Irvin CG (2004). Unrestrained plethysmography is an unreliable measure of airways responsiveness in BALB/c and C57BL6 mice. J Appl Physiol 97, 286–292.[Abstract/Free Full Text]

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   Acknowledgements
 
This study was partially supported by a grant from PUIS (394–446/17-X-94), and DGAPA-UNAM (IN203502).





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