Lung function measurements in rodents in safety pharmacology studies

Abstract

The ICH guideline S7A requires safety pharmacology tests including measurements of pulmonary function. In the first step - as part of the "core battery" - lung function tests in conscious animals are requested. If potential adverse effects raise concern for human safety, these should be explored in a second step as a "follow-up study." For these two stages of safety pharmacology testing, both non-invasive and invasive techniques are needed which should be as precise and reliable as possible. A short overview of typical in vivo measurement techniques is given, their advantages and disadvantages are discussed and out of these the non-invasive head-out body plethysmography and the invasive but repeatable body plethysmography in orotracheally intubated rodents are presented in detail. For validation purposes the changes in the respective parameters such as tidal midexpiratory flow (EF(50)) or lung resistance have been recorded in the same animals in typical bronchoconstriction models and compared. In addition, the technique of head-out body plethysmography has been shown to be useful to measure lung function in juvenile rats starting from day two of age. This allows safety pharmacology testing and toxicological studies in juvenile animals as a model for the young developing organism as requested by the regulatory authorities (e.g., EMEA Guideline 1/2008). It is concluded that both invasive and non-invasive pulmonary function tests are capable of detecting effects and alterations on the respiratory system with different selectivity and area of operation. The use of both techniques in a large number of studies in mice and rats in the last years have demonstrated that they provide useful and reliable information on pulmonary mechanics in safety pharmacology and toxicology testing, in investigations of respiratory disorders, and in pharmacological efficacy studies.

Keywords: experimental animal models; irritant potential; juvenile models; lung function test; mouse; pharmacology; rat; safety pharmacology.

Figure 1

Head-out plethysmography system. (A) Schematic drawing of rodent head-out plethysmograph [reprinted with permission from (Glaab et al., 2001), modified] made of glass or Plexiglas^®. The plethysmographs are attached to a head exposure chamber. Respiratory flow is measured by means of pneumotachograph tube connected to a pressure transducer (see text for details). (B) Photos of head-out plethysmography systems for four mice (left) or four rats (right; only one of two systems shown).

Figure 2

Definition of midexpiratory flow (EF₅₀). Left: normal breathing pattern of an anesthetized, orotracheally intubated, spontaneously breathing BN rat. Right: breathing pattern during bronchoconstriction due to inhalation of ca. 15 μg ACh aerosol, illustrating the simultaneous decreases in EF₅₀ and G_L. Upper tracing (V_T): tidal volume (V_T) obtained from the integrated airflow signal over time, a vertical line indicates the value of EF₅₀ at midexpiratory V_T. Middle tracing: corresponding airflow signal from the pneumotachograph during expiration (above zero) and inspiration (below zero). Lower tracing (G_L): corresponding lung conductance G_L = 1/R_L [reprinted with permission from Glaab et al. (2002)].

Figure 3

Head-out plethysmography in a safety pharmacology core battery study: (A) Tidal midexpiratory flow (EF₅₀) and (B) respiratory frequency measured after a single intragastric treatment with a pharmacologically active test compound [mean ± SEM; +/#/* = low/medium/high dose p < 0.05, ^** = high dose p < 0.01 vs. control group; reprinted with permission from Hoymann (2007)].

Figure 4

Dose-response relationship to aerosolized acetylcholine chloride (ACh; 20–160 mg/m³) in naive Brown Norway rats. Non-invasive determination of the decline in EF₅₀ to ACh was followed by invasive recording of simultaneously measured decreases in EF₅₀ and G_L (G_L = 1/R_L) to ACh exposure in the same animals 24 h later. EF₅₀ and G_L were allowed to return to baseline before each subsequent challenge. Results are means ± SD (n = 8 rats) of percent changes to corresponding baseline values, which were taken as 0%. No significant differences in dose-related changes were observed between non-invasively and invasively measured EF₅₀. [reprinted with permission from Glaab et al. (2002)].

Figure 5

Invasive body plethysmography system. (A) Diagram of a plethysmograph used for pulmonary function testing of anesthetized, orotracheally intubated rodents [shown is a unit for a mouse, reprinted with permission from Glaab et al. (2004)]. A thermostat-controlled water basin (37°C) is built in the plethysmograph chamber to avoid decrease in body temperature. *: adaptor connected to a pressure control unit that maintains constant pressure conditions during measurements. (B) Photo of a plethysmograph unit for a rat. For the calculation of R_L, P_TP was recorded via an esophageal tube and tidal flow was determined by a pneumotachograph tube attached directly to the orotracheal tube.

Figure 6

Characteristic time course of an early airway response in an anesthetized, orotracheally intubated Brown Norway rat during and after inhalational challenge with ovalbumin. Marked increase in lung resistance (R_L) is shown and a decrease in dynamic compliance (C_dyn) is paralleled by decreases in lung conductance (G_L), midexpiratory flow (EF₅₀) and tidal volume (V_T). Also a slight increase in respiratory frequency (f) was observed. The x-axis represents the experimental time (unit = 1 min).

Figure 7

Development of respiratory parameters in non-anesthetized juvenile Wistar rats. (A) Tidal volume; (B) tidal midexpiratory flow; *p < 0.05 male vs. female; mean values ± SEM, measured values of n = 8/sex (except males on post-natal day (PND) seven and females on PND 21 and 45: n = 7). Closed symbols represent male animals (♦) and open symbols represent female animals (○). [reprinted with permission from Lewin et al. (2010)]; see there for additional data).

Figure 8

No impact of hyperoxia shows good correlation of invasively and non-invasively measured respiratory parameters in mice (Glaab et al., 2005). Lung function was measured in head-out plethysmographs and subsequently the mice were anesthetized, orotracheally intubated, and lung function was measured invasively. Values are means ± SD of 8 C57BL/6 mice per group exposed to 100% oxygen for 48 h or clean air (control). NS, not significant (vs. control group). EF_{50 conscious}: tidal midexpiratory flow of conscious mice, EF_50 anesth.: tidal midexpiratory flow of anesthetized mice, R_L: lung resistance.