Refinement of tube acclimatisation protocols for male Crl:WI(Han) rats using lung function data as a marker of activity
Creators: name: Paul, Graham
Publisher: name: F1000 Research Limited
Year: 2019

description: Laboratory species are not ‘dosed’ by inhalation <i>per se</i> but are exposed to a test article mixed with air, from which they breathe. Inhaled ‘doses’ are often calculated using an equation to estimate respired minute volume (MV) from body weight. This may overlook variability in respiratory parameters associated with a drug’s properties or habituation of animals to the ‘dosing’ technique. Maintaining animals calm with a consistent breathing pattern may potentially reduce variability in achieved lung doses. Head-out plethysmography (HOP) was used to investigate the effect of acclimatisation on activity and potential stress of rats in restraint tubes, using lung function as a surrogate marker of these variables. Results were expected to facilitate refinement of protocols.<br><br>Preliminary work indicated study-related activities (visual checks on rats and aerosol sampling) induced erratic breath-waves and increased breath frequency (BF) and MV. Checks were done less intrusively to limit such disturbance in two key experiments which are summarised as follows. Rats (n=16/group) were acclimatised to plethysmographs for 2 or 6 days by progressively increasing the period of restraint each day, up to 1 hour. Rats acclimatised over 6 days were first subjected to a diaphragm (neck seal to facilitate data capture) on the fourth day of restraint. Rats were then exposed to an anti-inflammatory drug for 1 hour. For each rat, mean MV was calculated for 5-minute periods ending 5, 15, 30, 45 and 60 minutes from the start of exposure and mean pooled data were then subjected to statistical analysis to compare acclimatisation periods. Rats (n=3/group) were acclimatised to restraint tubes for 2 days and administered an aerosol (nebulised solution or micronised crystals), 1 hour/day for 28 days. Rats were allowed to settle in tubes for 15 minutes before aerosol administration. MV was measured on Days -1 (pre-treatment), 1, 4, 14 and 26 to 28. The effect of conditioning the animals to the diaphragm was investigated by inclusion of the seal in the plethysmograph pre-treatment and Days 1 to 4, 14 and 26 to 28 of aerosol administration. Transient increases in MV evident at the start of inhalation exposure were more pronounced in rats acclimatised over 2 days. A stable baseline in MV and BF was established from 15 minutes of exposure, with no difference between rats acclimatised for 2 or 6 days.<br><br>When animals were allowed to settle in tubes for 15 minutes prior to the start of aerosol administration, lower MV and BF were achieved for the period of ‘dose’ administration; MV was not significantly affected by the aerosol form. MV was more variable than body weight-derived estimates (eRMV), especially when rats were exposed to an aerosol for the first time, and on Days 14 and 26 following a treatment period in which neck-seals were omitted from the plethysmographs. MV was similar to eRMV when the seal was used during days of restraint preceding data capture on Days 1, 4 and 28. Reintroduction of seals from Day 26 showed a progressive realignment of MV and eRMV with reconditioning of rats achieving parity on Day 28.<br><br>Two days of acclimatisation (or reacclimatisation after a reduced level of restraint) of rats in restraint tubes was sufficient to achieve parity of MV (measured) with eRMV (body weight-derived estimate). GSK procedures for acclimatisation of rodents to restraint tubes were refined, with animals restrained for 30 minutes on two consecutive days before the first dose, irrespective of exposure route or treatment period (single or repeated administrations); a 30-minute period was selected to ensure rats achieve a settled state before being returned to their holding cages. The refined acclimatisation protocol has been successfully applied in routine inhalation studies in rats and mice (30 or 60-minute exposures) and in a 7-day intravenous (4-hour) infusion study in rats, with no restlessness exhibited by rodents during restraint. This represented a significant reduction in pre-study training for a 4-hour infusion period, since previous protocols progressively increased restraint from 15 minutes up to 4 hours over a 5-day period. Head-out plethysmography thus facilitated refinement of inhaled ‘dose’ administration but also other procedures utilising tube restraint.