A rodent treadmill for \inhalation toxicological studies and respirometry

J. Appl. Phys- iol. W(2): 673-679,1985.-A lo-runway treadmill was enclosed for inhalation toxicological studies of rodents under exercise exposure to environmental pollutants. The exposure system was lined with sheet stainless steel to minimize scrubbing of charged particles and reactive gases. Average metabolic gas exchange of exercising animals was derived from measurements of inlet or outlet airflow and data from an O2 analyzer in conjunction with either a CO,! or NP analyzer. An airflow rate of 400 Lmin-l ensured a response time of 1 min to reach 95% of a step change in metabolic rate and held scrubbing losses of an O3 test atmosphere to less than 2% of treadmill inlet concentration. Gas exchange averaged for 10 rats during incre- mental exercise up to their highest collective performance was similar to published data for rats tested individually.

inhalation toxicological studies and respirometry. J. Appl. Physiol. W(2): 673-679,1985.-A lo-runway treadmill was enclosed for inhalation toxicological studies of rodents under exercise exposure to environmental pollutants. The exposure system was lined with sheet stainless steel to minimize scrubbing of charged particles and reactive gases. Average metabolic gas exchange of exercising animals was derived from measurements of inlet or outlet airflow and data from an O2 analyzer in conjunction with either a CO,! or NP analyzer. An airflow rate of 400 Lmin-l ensured a response time of 1 min to reach 95% of a step change in metabolic rate and held scrubbing losses of an O3 test atmosphere to less than 2% of treadmill inlet concentration. Gas exchange averaged for 10 rats during incremental exercise up to their highest collective performance was similar to published data for rats tested individually. inhalation exposure apparatus; air pollutant; metabolic gas exchange; rodent exercise ALTHOUGH INHALATION EXPOSURES of laboratory animals are commonly performed at rest, there are good reasons to conduct studies during exercise. In many occupational and environmental human exposures, people working or playing have significantly elevated ventilation rates. In contrast, it is not uncommon to observe experimental animals maintaining minimal states of activity, in many cases apparently sleeping, during exposures in inhalation chambers. In studies using animals as models of human inhalation exposure the lack of similarity in such cases is striking.
Several events occur during exercise that are known or suspected to alter the biological effects of a given concentration of an airborne toxin. Increased minute ventilation will lead to greater total internal exposure doses per unit time. For the average adult human, resting minute ventilation is about 7 1. min? During brief heavy exercise an increase to over 100 1. min-' has been observed (3). Thus exercise can increase the rate of delivery of an inhaled toxin by more than lo-fold. Other events occurring during exercise that may modify response in-0161-7567/85 $1.50 Copyright 0 1985 the American Physiological Society elude greater depth of breathing, decreased contact times between inspired air and upper airways, an increase in the fraction of air drawn through the mouth (rather than the nose), and the potential overriding of reflex-mediated protective breathing patterns (e.g., shallow breathing) that are normally stimulated by irritants (2,6,28). In general, higher flow rates will change the deposition efficiencies for gases and particles in all regions of the respiratory tract, leading to a redistribution of the dose pattern and possibly a change in the total combined dose delivered to all regions. In many cases these factors would be expected to lead to increased responses to inhaled pollutants. Indeed, inhalation studies with both humans and laboratory animals have typically demonstrated greater responses during exposures at exercise than were seen at rest (9,12,(22)(23)(24).
For the purpose of examining the effect of exercise as a modifier of the toxic response to air pollutant compounds, an enclosed treadmill exposure system was developed for use with rats as an exercising animal model. The treadmill was designed to simultaneously expose 10 running animals to test gases or aerosols and to permit simultaneous measurement of group respiratory gas exchange as an index of metabolic work load during exposure. The treadmill was used to determine the relationship between running speed and metabolic gas exchange at two elevation grades (0 and 20%). In addition, procedures were tested for measuring 03 concentration in the treadmill with rats present.

METHODS
Treadmill design. The treadmill exposure system was constructed from a Quinton 42-15 rodent treadmill (Fig.  1). The treadmill contained 10 individual runways and controls for varying belt speed and inclination.
An electrically charged grid at the rear of the belts motivated the animals to continue exercise. The treadmill was enclosed with polycarbonate plastic (6 mm) and sealed against significant leaks with neoprene gaskets. The drive motor was external to the enclosure, and a seal on the belt drive shaft was effected with a pair of semicir- cular Teflon bushings mounted in an aluminum panel. centered at the front of each runway. One-centimeter-Inner surfaces of the runways were lined with a stainless diameter stainless steel disk baffles mounted 1 cm behind steel sheet to minimize both electrostatic charge effects the ports and stainless steel screens (2 X 2 mesh) posiand reaction with corrosive atmospheres. Inlet ports were tioned at 10 cm distributed the atmosphere uniformly in the runways and prevented rats from contacting inlet ports.
Removable panels above the charged grid provided access to individual runways. Another removable panel spanning the front of the treadmill below the inlet ports gave access to belt tension adjustment screws and to the enclosure floor removable tray beneath the belts. Excreta fell on the enclosure floor and fecal onto a masses clinging to the be1 .ts were brushed loose with w ire wipers mounted to press lightly on the belts. During operation the belts became wet with urine and spotted with smeared fecal matter. This raised concern about the possible development of significant ammonia levels in the treadmill although continuous inflow of exposure atmosphere made this unlikely. NH3 concentration was measured from the treadmill while rats performed intermittent exercise and rest. Atmosphere was sampled from a runway, and NH3 was collected into 0.05 M HzS04. The solution was adjusted to pH 13, and NH3 concentration was measured with an ion-selective electrode (HNU Systems, Newton, MA). Airborne NH, concentration was found to be less than 0.1 ppm.
A pair of exhaust lines at the rear of the treadmill conducted the atmosphere to a 30-liter baffled mixing chamber, then through a flowmeter to sample ports for humidity and metabolic gas fraction measurement and to an outdoor vent blower. Air inflow and outflow rates were adjusted to regulate the treadmill at ambient pressure.
Exposure atmospheres were produced by mixing test compounds or aerosols with purified air and delivering the mixture to each runway of the treadmill. Supply air was initially compressed and purified by passage through Purafil (Atlanta, GA) and Del-Monix (Deltech, Newcastle, DE) gas scrubbing filters, then decompressed, passed through a high-efficiency particulate absolute (HEPA) filter, and humidified by controlled injection of water vapor. The mixed atmosphere was passed to a l-m3 stainless steel chamber containing a temperature and humidity sensor (Hygrodynamics, Silver Spring, MD), then through paired Teflon-lined flexible ducts to a stainless steel manifold that di 10 treadmill runways. A pair of stributed the flow to the atmosphere sample ports s 3 cm upstream steel screen bar-was located in each ru nway a t position and 3 cm downstream of the stainless les the of the inlet atmosphere atin the immediate vicinity of the rats.
Metabolic rate measurement.
Average metabolic rate of 10 rats exercising in the treadmill was determined by riers. The mosphere first port withdrew samp and the second withdrew analysis of input and output fraction .a1 gas composition, water vapor content, and flow rate (see A .PPENDIX for details.) Measurements were made by alternately sampling upstream 'and downstream air with a mass spectrometer (Perkin-Elmer model 1100, Pomona, CA) calibrated with gravimetric standard gases (Liquid Carbonits, Los Angeles, CA). Atmosphere flow rates were measured either with a single Fleisch no. 4 pneumotachograph (Dynascience, Blue Bell, PA) in the exhaust stream or paired Fleisch no. 1 pneumotachographs on the input manifold in conjunction with differential pressure transducers (Validyne MP-45 Northridge, CA). Hu-midity and temperature were measured with a dew point sensor (EG&G model 911, Waltham, MA). Output of all instruments was displayed on a chart recorder (Gould model 2800, Cleveland, OH) and digitized at 40 Hz for calculation of respiratory gas exchange with a PDP ll-10 computer system (Digital Equipment, Maynard, MA). To perform a measurement under steady-state conditions, downstream and upstream gas fractions, along with atmosphere flow rate, temperature, and humidity, were sampled five times over a 30-s period. Gas exchange was then calculated by computer from mean values of the variables and expressed as the mean value per individual rat based on the number of animals in the treadmill.
Gas turnover kinetics of the treadmill atmosphere were evaluated by analyzing the response to step changes in a calibration gas input to treadmill runways. An exponential first-order relation between outflow gas fraction and time provided an adequate fit to the treadmill response. Logarithmic transformation of gas fraction data yielded a straight-line relation to time with slope l/t and inter- where t is the time constant and d is the delay time (19).
Correlation coefficients for repeated trials (n = 8) ranged from 0.97 to 0.99 for atmosphere flow rates of 400 or 100 1. min.
Time constants at these flow rates were 10 and 51 s, respectively, and corresponding delay times were 29 and 81 s. These values resulted in an expected time to reach 95% of steady-state gas fraction following a step change of 59 s at 400 1. mine1 input flow and 233 s at 100 1. min-' input flow.
For gas exchange measurements, the system was tested before and after an experiment with gravimetric standard calibration gas elevated in COZ and depressed in O2 composition. Calibration gas was bled into the treadmill runways, and expected values of O2 consumption (Vo2) and COZ production (VCO,) were calculated from Eqs. 5 and 6 or 7 and 8 (APPENDIX) using calibration gas composition as output fractions, treadmill inlet gas composition as input fractions, and metered flow rate (Bubble Meter, SKC West, Fullerton, CA) of calibration gas into the treadmill.
Animals, training, and exercise protocol. The relation between metabolic rate and running speed was determined for Sprague-Dawley rats (Hilltop Lab Animals, Chatsworth, CA) with mean weight at testing of 272 t 21 g. The animals were trained to run at level grade in the treadmill over a 3-day period commencing with 15 min of running at 8 momin-' alternating with 5 min of rest for 1.6 h. On the second day rats alternated 30 min of running with 10 min of rest for 3.2 h, and on the third day they ran continuously for 3 h. The purpose of the training protocol was to acquaint the rats with the treadmill and shock grid and to identify individuals that would not run readily in the apparatus. About 3% of the animals could not be trained to run reliably and were excluded from the experiment. Metabolic rate was measured from groups of 10 rats at an airflow through the treadmill of 400 1 l min.
Following initial measurements at rest, treadmill speed was set at 10 m*min-' at level grade, and speed was increased in step changes of 5 rn. min-' at 6min intervals. Metabolic rate was measured at each step MAUTZ, PHALEN, MCCLURE, AND BUFALINO after output flow gas fractions stabilized, and the test was terminated when rats failed to maintain continuous exercise. Additional sets of measurements were made at 20% grade with 10 momin-' step increases over the lower range of speeds. 120 1 PoZZutant atmosphere. An ozone (03) atmosphere was used to test treadmill distribution of a pollutant compound and assess possible scrubbing by reactive components of the treadmill inhalation system. O3 was produced by passing medical-grade O2 through an electrostatic discharge 03 generator (Sander Ozonizer, type III, Osterberg, FRG) and diluted into the humidified airstream. Samples of the exposure atmosphere were drawn in succession from three positions in the atmosphere stream: 1) in a l-m3 stainless steel chamber just before the atmosphere entered the treadmill manifold, 2) in the treadmill runways between the inlet ports and stainless steel screens, and 3) in the treadmill runways behind the stainless steel screen. Samples were drawn through Teflon tubing, and 03 concentration was measured by ultraviolet spectrophotometry in a Dasibi model 1003-AH 03 analyzer. The third position most closely approximated the rat breathing zone. However, because the rats had direct access to these sample tubes, samples could contain expired respiratory air or air aspirated through fur. The relation between running speed and metabolic gas exchange for groups of 10 rats running at level grade and 20% grade is shown in Fig. 2. On level grade, Vo2 and %ko2 increased linearly between 10 and 20 m*min? At higher speeds the response was attenuated as the rats approached exhaustion, and the experiment was terminated at 40 rn. min-' as animals became unable to continue running. Rats running at 20% grade exhibited only slightly higher rates of metabolic gas exchange at speeds < 30 momin-'; however, the response was linear over the entire range of speeds at which they were able to perform.
Distributions of 03 concentrations in parts per million (ppm) in the treadmill exposure system are shown in Table 1. O3 losses between the l-m3 chamber and the second sample port behind the screens. When-rats were present in the treadmill losses measured in the runways were greater. With animals running, the measured loss between sample ports was 5%, and when the animals were at rest in the treadmill the loss was 20%. However, mass spectrometer analysis of air samples from the second sample ports behind the screens revealed elevated and highly variable CO2 fractions which indicated that these samples contained expired respiratory air. Variation in 03 concentration among runways was statistically significant (F = 7.3, P < 0.001) for samples upstream of screens when rats were present, however the runway differences were not large. Mean concentrations in in-DISCUSS1oN dividual runways measured at upstream sample ports Exercise is expected to be an important modifier of with rats present ranged from 0.372 to 0.382 ppm with the toxic effects of inhaled pollutant compounds and a RODENT TREADMILL FOR TOXICOLOGY number of investigations have shown that exercise exposure to pollutant compounds exacerbates induced changes in pulmonary function of humans (1,9,22).
With the development of exercising animal models, inhalation toxicological studies may address questions that cannot be approached with human subjects, such as exercise effects on histopathology and biochemistry. The enclosed multichannel treadmill system described here provided an efficient means for exposing up to 10 exercising rats simultaneously and uniformly to a controlled atmosphere. A preliminary experiment with the apparatus (16) demonstrated exercise enhancement of ozoneinduced lung lesions. Fulfilling a basic requirement to contain exposure atmosphere in the treadmill serves in adapting the system for open flow respirometry and quantifying metabolic work load of the exercising animals. The choice of computational method for measuring metabolic gas exchange is governed by available instrumentation and practical considerations of exposure system design. The method presented (APPENDIX) is based on water vapor free gas fractions and correction of atmosphere flow to STPD. Techniques with single gas analyzers with and without water vapor and CO2 absorption have been described (10,11,14,27), and other sampling arrangements and equations appropriately derived from mixed and component gas flows (Eqs. l-4, APPENDIX) may be used. High flow rates in inhalation exposure systems make gas scrubbing of the total flow impractical; however, samples may be subjected to differential absorption before analysis. One may choose to measure input flow or output flow and apply different gas exchange equations for the two cases. If there are any leaks in the treadmill enclosure, an output flow measurement is preferable. The treadmill may then be operated under slightly negative pressure so that any leakage of air is directed inward, mixed with respired gas, and included in the measurements of total flow and output metabolic gas fractions. There will be no error in metabolic rate estimates unless respiratory gas fractions of the inlet exposure atmosphere differ from room air. If input flow is measured, any leakage (outward before complete mixing or inward) will alter output metabolic gas fractions and not be accounted in the flow measurement. Airflow rate through the treadmill must be set sufficiently high to ensure uniform exposure atmosphere distribution and an adequate response time to changes in metabolic rate; however, the flow must not be so great as to compromise resolution of metabolic gas fractions. We found a flow of 400 1. min-' appropriate for exposing 10 rats and measuring gas exchange in the treadmill system.
The relation between running speed and metabolic gas exchange for rats running in the exposure system was in agreement with data reported in other studies of exercise metabolism of laboratory rats (4,5,7,13,18,20,21).
There is a large degree of variation in results of these separate studies, and most investigators believe that regression slopes, presence of metabolic plateaus, and the values of maximum 02 consumption (To2 max) and its corresponding running speed depend on animal training, exercise protocols, and selection criteria for performance (4,13). Data for exercising Sprague-Dawlev rats of mass 677 similar to our animals (4,5,13,21) fall within the range of results reported here (Fig. 2). Gleeson and Baldwin (13) reported a sharp plateau in Tjoz of untrained rats performing incremental exercise, and others (5,7,18,20,21) have observed attenuation of metabolic response or plateaus with increasing running speed. In our experiments VO, continued to increase with increments of running speed, but while the response remained linear for running at 20% grade, an attenuated response and lower peak Vo2 were observed for level running. It is possible that attenuation would have been observed at 20% grade if additional measurements were made between 30 and 35 m l mine1 . However, there was an important difference between level and 20% grade protocols; rats running uphill had larger speed increments and shorter total exercise duration to reach peak sustained speed. Thus rats running at 20% grade became exhausted after four speed steps (18-24 min of running), whereas rats on level grade attained seven speed steps (36-42 min of running). Extended incremental exercise protocols may induce exhaustion and refusal to run at submaximal exercise levels. In another study using rats trained for several weeks, selected for superior running performance, and tested directly at each speed without progressive incremental increase (4), metabolic plateaus were not observed and the animals were capable of performing at speeds up to 50 rn. min-' at 17.8% grade. The diversity of metabolic responses and exhaustion behavior of rats under conditions of maximal exercise has made it difficult to precisely and reproducibly characterize VO, m8X. The highest vo2 measured in the present study (103 t 14 ml. kg-' l min-') fell within the range of VO, max values reported in other studies using both incremental and single speed protocols (5,7,13,20,21), although our values were based on repeated measurements of the collective sustained performance of 10 animals and might be expected to underestimate an average of individual animal measurements.
In view of the variability of . vo 2 max determinations and frequent absence of definitive physiological indicators such as metabolic plateaus and continuous lactate accumulation (4,5,25), any investigation of the effects of toxic substances on maximal metabolic rate must use control groups strictly matched in age, size, and exercise experience and must consider the influence of running behavior on the end point.
Characterization of inhaled pollutant concentration is an important problem in inhalation toxicology, as particles and gases may be scrubbed from the test atmosphere along the delivery path. In addition, atmosphere sampling in the breathing zone of test animals introduces the possibility of contaminating the sample with expired respiratory gas. Ideally one would like samples of inhaled concentration at the mouth and nose such as might be obtained with a respiratory mask and valve. For exposures of animals in chambers, however, inhaled concentration is usually assumed to be equivalent to that of inlet air. With sufficient input flow, losses to the chamber structure can be minimized, but evasive maneuvers by test subjects such as tucking the head in the fur remain a potential problem. Rats exercising in the enclosed treadmill system were advantageously exposed head-on to a continuous flow of test atmosphere. Samnles drawn directly from the runway in the breathing zone of the exercising animals were often spoiled by collection of expired respiratory gas, and this problem was greatest when the animals were at rest. Resting animals spent considerable time in close proximity to the sample line. The most representative atmosphere samples were obtained upstream, separated from the animals by coarse mesh stainless steel screens. Exhalant gas was not present in these atmosphere samples and with no animals present in the treadmill, losses of 03 flowing down the runway were minimal (Table 1).

APPENDIX
Methods for measuring metabolic gas exchange in open flow respirometers have been developed for several particular cases (7,8,10,11,14,15,26,27). All these methods can be related by a set of equations describing the balance flow of each component gas in question and the total flow rate of mixed atmosphere into and out of the respirometer. By substitution, i.70~ and h02 may be expressed in a minimum number of variables appropriate to available instruments. Symbol terminology follows earlier analyses (10,11,14,27). The relation between total input and output volume flow rates is TjE = TjI -vo2 + vco2 (4) Equations 1 and 2 contain both input and output airflow terms, VI and VE. By rearranging Eq. 4 and substituting for either VI or VE in Eqs. 1 and 2, respiratory gas exchange may be expressed in terms of either the input or the output flow, whichever is more conveniently measured. Both terms iJo and b02 are introduced into each of the equations by this substitution, but by simultaneous solution of the two equations for these two variables, expressions for vo2 and ho2 are obtained in terms of input and output fractional gas concentrations and a total airflow rate, be it either input or output flow. For the case in which VE is measured, these expressions are Alternatively, VI or VE may be eliminated from Eq. 1 and 2 by rearrangement and substitution of Eq. 3. This procedure yields expressions with the Haldane transformation using FINz and FEDS (8