Respiratory Physiology & Neurobiology 206 (2015) 41–44
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Repetitive measurements of enhanced pause (Penh) Wei-hua Xu ∗ Respiration Department of Tongde Hospital of Zhejiang Province, Gucui Road #234, 310012 Hangzhou, China
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Article history: Accepted 6 November 2014 Available online 13 November 2014 Keywords: Airway resistance Plethysmography Methacholine Mice
a b s t r a c t Enhanced pause (Penh) was ﬁrst proposed in allergic mice and appeared to be correlated with airway responsiveness. However, some investigators have suggested that there is no theoretical basis for a correlation between Penh and airway resistance. Because the measurement of Penh is a noninvasive procedure, this value may be useful in repetitive measurements, but few researchers have emphasized this aspect. This study aimed to assess the validity of Penh values derived through repetitive measurements of both absolute and ratio Penh values in 10 male C57BL/6 mice on days 0, 3, 7, and 15. Tests of within-subject effects revealed signiﬁcant differences in both the absolute and ratio Penh values across the different time points. The administration of challenge aerosolized methacholine concentrations of 25, 50, and 100 mg/ml resulted in signiﬁcant differences in the ratio Penh values across the various time points. The ﬁndings of the present study indicate that Penh is not a good index for repetitive measurement because the Penh values are signiﬁcantly inﬂuenced by the time at which they are measured. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Enhanced pause (Penh) was ﬁrst proposed in allergic mice (Hamelmann et al., 1997), and some investigators regard Penh as a substitute parameter for airway responsiveness (Siebeneicher et al., 2014; Song et al., 2012) or as a surrogate for speciﬁc airway resistance (sRaw) in guinea pigs (Bergren, 2001; Chong et al., 1998; DeLorme and Moss, 2002). Although previous studies have provided evidence that Penh and airway responsiveness are correlated in pigs (Halloy et al., 2004) and BALBc mice (Hamelmann et al., 1997), other investigators have criticized the theoretical and experimental validity of Penh (Bates et al., 2004; Sly et al., 2005; Lundblad et al., 2007; Kirschvink, 2008). Interestingly, few investigators have focused on the fact that in the study by Hamelmann et al. (1997), airway responsiveness was not tested immediately, but 24 h after Penh values were tested and measured. The authors did not report whether the Penh measurements varied with time. Furthermore, because Penh is measured noninvasively, it is thought to be useful for repetitive measurements in animals (Berndt et al., 2011; Zhang et al., 2009). Although some investigators have questioned the theoretical basis of Penh (Bates et al., 2004; Sly et al., 2005; Lundblad et al., 2007; Kirschvink, 2008), few studies have investigated how Penh measurements in animals vary with time.
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http://dx.doi.org/10.1016/j.resp.2014.11.006 1569-9048/© 2014 Elsevier B.V. All rights reserved.
The objective of the present study was to evaluate the reliability and usefulness of Penh in a repetitive measurement experiment across different time points. 2. Materials and methods 2.1. Animals This study was performed with the approval of the Ethical Council Guide of Tongde Hospital of Zhejiang Province (Hangzhou, China). Ten 6–8-week-old pathogen-free male C57BL/6 mice (Experimental Animal Center, Tongde Hospital of Zhejiang Province, China) weighing 18–22 g were used in this experiment. The animals were maintained under standard housing conditions and given free access to food and water. Before the experimental measurements, the mice were acclimated to the temperature of the laboratory (27.0 ± 0.5 ◦ C) and a humidity of 50% for 1 h. This study was performed in accordance with the Ethical Council Guide of Tongde Hospital of Zhejiang University (Hangzhou, China). The mice were killed at the end of the experiment through an overdose of intraperitoneally injected sodium pentobarbital. 2.2. Effects of inhaled methacholine (Mch) Penh measurements The effects of inhaled Mch on Penh measurements from conscious, spontaneously breathing animals were determined by barometric plethysmography, as previously described (Song et al., 2008). Five whole-body plethysmographs (PLY 4211, Buxco
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Electronics) were used, and each chamber contained one mouse. The mice were placed in the plethysmographs and allowed to acclimate for 10 min. When the mice breathed, the changes in the pressure inside the chambers were measured by transducers (TRD5700, Buxco Electronics), and the signals were processed with an ampliﬁer (MAX1420, MAX2270, and MAX4371, Buxco Electronics). A monitor displayed the respiratory curves of the mice, and the data were displayed every 5 s. The Penh algorithm was based on the procedure developed by Hamelmann et al. (1997) and performed using the SFT3450 and SFT3812 software (Buxco Electronics) with the following equations: Te − Tr Tr
Penh = Pause ×
Day 0 Day 3
Day 7 Day 15
4 2 0 BS
where Te represents the expiratory time (i.e., the time from the end of inspiration to the beginning of the next inspiration), Tr represents the relaxation time (i.e., the time at which the pressure decayed to 36% of the total box pressure during expiration), PEP represents the peak expiratory pressure (i.e., the maximal positive box pressure that occurred in one breath), and PIP represents the peak inspiratory pressure (i.e., the maximal negative box pressure that occurred in one breath). Upon the initiation of the Penh measurements, baseline values were ﬁrst recorded for 3 min. Each mouse was subsequently challenged with aerosolized saline or Mch (3.125, 6.25, 12.5, 25, 50, and 100 mg/ml) for 3 min at each concentration, and the Penh readings were averaged over 3 min following each nebulization. Penh values used in this study are absolute Penh values and ratio Penh values, the latter of which are calculated as the percent ratios of the average Penh value for each aerosolized saline or Mch dose to the average baseline Penh value. 2.3. Repetitive measurements of Penh values across time After testing the Penh values on day 0, the same procedures were performed in the same 10 mice on days 3, 7, and 15. All Penh values are expressed as absolute values, and the ratio values are expressed as percent increases in the average Penh following each saline or Mch dose over the average baseline Penh value.
Fig. 1. Absolute Penh values recorded from male C57BL/6 mice exposed to increasing concentrations of nebulized methacholine at four observation time points. The absolute Penh values varied signiﬁcantly with time. BS, baseline and NS, saline.
10 Ratio Value of Penh
Absolute Value of Penh
Day 0 Day 3
Day 7 Day 15
4 2 0 BS
MCh mg/mL Fig. 2. Ratio Penh values recorded from male C57BL/6 mice exposed to increasing concentrations of nebulized methacholine at four observation time points. At the concentrations of 25, 50, and 100 mg/L, signiﬁcant differences across different time points were noted (P < 0.05). BS, baseline and NS, saline.
The means ± the standard errors of each parameter at each time point were evaluated. All of the data were analyzed with the Statistical Package for the Social Sciences (SPSS for MS Windows, Ver. 13.0; SPSS Inc., Chicago, IL, USA). Two-factor repeated-measures analyses of variance were performed to evaluate the differences in the Penh values at the different time points and different Mch doses. For each challenge concentration of Mch, one-way analysis of variance was used to compare the differences between the Penh values at the different observation time points. Differences were considered to be statistically signiﬁcant if P < 0.05.
According to the two-factor repeated-measures analysis of variance of the absolute Penh values (F = 7.777, P = 0.001) and the ratio Penh values (F = 7.432, P = 0.001), the within-subject results varied signiﬁcantly across the time points, i.e., both the absolute Penh values and the ratio Penh values of the same group of mice were signiﬁcantly different at various time points. We then examined the Penh values of the mice at each aerosolized dose. At 25 mg/ml Mch, the ratio values (F = 3.739, P = 0.019) but not the absolute values (F = 2.528, P = 0.073) were signiﬁcantly different between different time points. Similarly, at 50 mg/ml, there were signiﬁcantly different ratio values between different time points (F = 3.015, P = 0.042), but no signiﬁcant difference was found between the absolute values (F = 1.558, P = 0.216). The same trend was observed when the mice were challenged with 100 mg/ml: there was a signiﬁcant difference between the ratio values (F = 2.994, P = 0.043) but not between the absolute values (F = 1.620, P = 0.202).
The dose–response curves of the absolute Penh values and the ratio Penh values following the nebulization challenges at four observation time points are shown in Figs. 1 and 2. After inhaling a small dose of Mch ( 0.05). With increasing concentrations of Mch, the absolute Penh values and the ratio Penh values increased signiﬁcantly (P < 0.01). The aerosolized Mch challenge led to strongly dose-dependent responses in the mice.
Airway hyperresponsiveness is one characteristic of asthmatic animal models (Gil and Lauzon, 2007). Measurements of airway responsiveness include both invasive and noninvasive methods (Glaab et al., 2007). The invasive airway responsiveness methods, such as airway resistance (Adam et al., 2013; Gong et al., 2013), are direct and precise measurements. Noninvasive methods are indirect, although the animals remain alive, which allows researchers to perform repeated measurements. Hamelmann et al. (1997) ﬁrst introduced the Penh measurement as a noninvasive
2.4. Statistical analyses
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airway responsiveness index and concluded that Penh values are related to airway responsiveness. Following this initial studies, Penh measurements have been widely used in allergy experiments in animals other than allergic mice, including rats (Liu et al., 2013), dogs (Manens et al., 2012), guinea pigs (Olea et al., 2011), cats (Leemans et al., 2012), and pigs (Halloy et al., 2005). Based on the idea that Penh can be used as a substitute parameter for airway responsiveness in challenged animals, some researchers (Chuang et al., 2013; García-Guasch et al., 2013; Kim et al., 2013) have used Penh measurements alone to evaluate the airway responsiveness of rodents rather than using the traditional invasive methods of airway resistance. However, other researchers (Bates et al., 2004; Sly et al., 2005; Lundblad et al., 2007; Kirschvink, 2008) have clearly emphasized the theoretical and practical inability of such Penh measurements to reﬂect airway resistance. Furthermore, several other researchers have shown that changes in Penh and respiratory resistance are not correlated (Adler et al., 2004; Pauluhn, 2004) despite the empirical correlations observed by Hamelmann et al. (1997). Due to these theoretical issues and opposite results, the Penh value has become a highly controversial index. Although these contrasting ﬁndings have provided strong evidence against the use of Penh, previous studies have not focused on the fact that Hamelmann et al. (1997) did not test airway responsiveness immediately after measuring the Penh values but rather 24 h later. Zhang et al. (2009) compared the responsiveness of Penh and airway resistance measurements to methacholine in mice that were challenged with a single dose of ovalbumin on the same day, and the results revealed a signiﬁcant increase in the Penh measurement group that was not observed in the airway resistance group. In addition to the evidence against Penh (Bates et al., 2004; Sly et al., 2005; Lundblad et al., 2007; Kirschvink, 2008; Adler et al., 2004; Pauluhn, 2004; Zhang et al., 2009), the results of the present study revealed that the Penh values at different time points are signiﬁcantly different. In the present study, we not only measured the ratio Penh values (which were used in the study by Hamelmann et al. (1997)) but also simultaneously measured the absolute Penh values. The data showed that both the absolute Penh values and the ratio Penh values obtained for the same group of mice were signiﬁcantly different at various time points, although the absolute Penh values and the ratio Penh values exhibited different responses to Mch inhalation at doses of 25, 50, and 100 mg/ml. These ﬁndings provide further evidence that the conclusion drawn by Hamelmann et al. (1997) cannot be extrapolated beyond their own study. The close correlation between Penh and airway responsiveness is more likely a fortuitous relationship under the special circumstances of their study, in which increased Penh values coincided with increased airway responsiveness. Although reproducible measurements of airway resistance can be performed in intubated and anesthetized mice (Brown et al., 1999), researchers of animal physiology have been searching for a noninvasive index that can provide results comparable to those of airway resistance to evaluate airway responsiveness because noninvasive methods are more suitable for repeated pulmonary measurements. Due to the noninvasive nature of the method, Penh measurements are used in long-term follow-up studies and are thought to be indices that can be tested repeatedly and used to compare the variance of the same group of subjects at different time points. However, the present study measured the Penh values of 10 mice on days 0, 3, 7, and 15 and noted signiﬁcant differences in the Penh values across the different observation time points. Thus, Penh values are dependent on when they are measured. This result indicates that Penh values are unreliable indices for long-term repetitive measurements. The result also diminishes the signiﬁcance of Penh as a noninvasive index.
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