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Monitoring guinea pig core temperature by telemetry during inhalation exposures
FUNDAMENTAL
AND
APPLIED
Monitoring
TOXICOLOGY
9,398-408
( 1987)
Guinea Pig Core Temperature during Inhalation Exposures
by Telemetry
PETER S. THORNE,’ CURTIS P. YESKE,* AND MERYL H. KAROL Department of Industrial Environmental Health Sciences, Graduate School ofPublic Health, University ofpittsburgh, Pittsburgh, Pennsylvania 15261
Monitoring
Guinea Pig Core Temperature by Telemetry during Inhalation Exposures. S., YESKE, C. P., AND KAROL, M. H. (1987). Fundam. Appl. Toxicol. 9, 39% 408. A temperature telemetry system was incorporated into an existing animal model for inhalation toxicology. This system facilitated continuous monitoring ofguinea pig temperature during inhalation exposures. Components of the system included Mini-Mitter temperature-controlled oscillators, AM receivers, and an IBM microcomputer. Software was developed to perform signal processing and filtering. Transmitters were calibrated and then sterilized and surgically implanted in the guinea pig peritoneum. Monitoring the baseline temperature of nine animals indicated a mean temperature of 38.6 f OYC. Guinea pigs were treated with agents to induce transient hypo- or hyperthermia. For the former, exposure for 3 hr to 7.8 ppm diphenylmethane+‘diisocyanate resulted in a 3°C temperature decrease. The temperature was determined from 230 readings per animal per hour. Inhalation of 9 or 44 pg/m3 endotoxin for 6 hr induced hyperthermia with a 1.5”C maximum increase in core temperature at 4.8 hr. With endotoxin, an increase in respiratory rate was also noted and followed the same pattern as temperature with a maximal increase of 52% occurring at 4.2 hr. The temperature telemetry system enabled continuous long-term monitoring of toxicant-induced hypothermia and pyrexia without interruption of inhalation exposure or measurements of respiratory parameters. Q 1987 THORNE,
P.
Society of Toxicology.
ter temperature transmitters (Mini-Mitter Co., Inc.) have been used in behavior studies in amphibians and rodents (Bradford, 1984; DeCastro, 1978; Pickard et al., 1984; fismiller and Heldmaier, 1985). We have previously reported development of an animal model for study of pulmonary sensitivity to industrial toxicants. The model demonstrates both immediate- and late-onset pulmonary responses. Fuller characterization of the syndrome would be achieved by inclusion of temperature monitoring to detect possible occurrence of hypersensitivity pneumonitis. This study describes incorporation of a temperature telemetry system into the animal model studies. Inhalation exposure of guinea pigs in whole body plethysmographs to diphenylmethane-4,4’-diisocyanate (MDI) and to endotoxin produced hypothermia and
Inhalation of numerous industrial compounds has been reported to affect body temperature. Mill fever is’ associated with exposure to endotoxin in cotton or flax dust; metal fume fever has been reported following exposure to metals such as zinc; and polymer fume fever results from inhalation of fumes from burning Teflon (Parkes, 1982). Hypersensitivity pneumonitis from exposure to isocyanates and anhydrides is characterized by fever which may reach 40°C (Fink, 1984). A “radio pill” was described by Fox et al. (196 1) for measurement of intragastric temperature in humans. Subsequently the device was used to study heat stress in Pittsburgh steelworkers (Minard et al., 197 1). Mini-Mit’ To whom correspondence should be addressed. 2 Present Address: Computing Center, Camegie-Mellon University, Pittsburgh, PA 152 13. 0272-0590/87 $3.00 Copyright 0 1987 by the Society of Toxicolc+ty. All rights of reproduclion in any form reserved.
398
CORE TEMPERATURE
MONITORING
hyperthermia, respectively. The responses were detected through use of continuous monitoring of temperature transmitter signals. MATERIALS
AND
METHODS
Temperature telemetry system. The transmitter, a dual thermistor, temperature-controlled oscillator (Mini-Mitter Model M-FH, Mini-Mitter Co., Inc., Sunriver, OR) powered by a replaceable 1.5 V silver oxide battery, was contained within a 1.1-cm-diameter, I .8-cm-long plastic capsule coated with a paraffin-resin mix (Elvax, E. I. DuPont DeNemours Co., Wilmington, DE) and weighed 2.7 g. At physiological temperatures the transmitters emitted pulses at 300 to 500 Hz on a broad-band AM carrier. The actual frequency emitted for a given temperature was different for each transmitter. The radio signal received from a transmitter was demodulated and amplified by an AM receiver with an external ferrite antenna. The transmission range was dependent upon both the amplifier gain and the orientation ofthe transmitter with respect to the antenna. The usable transmission range extended to about 30 cm. An oscilloscope display of the speaker output from the receiver is illustrated at the top of Fig. 1 and shows four peaks of a 3.0-V, 370-Hz signal. An analog to digital converter (A to D) board (DASH-g, MetraByte Corp., Taunton, MA) sampled the voltage of the input signal in a period of 250 msec for a given channel at a rate of 4200 samples/set with each sample hasting 55 nsec. This set of 1050 conversions was analyzed by determining the number of peaks detected in 250 msec (80 to 120 peaks) with amplitude exceeding the user-specified threshold value. Typically the threshold was set to 1 V to exclude lower amplitude interference accompanying the transmitter signal. The average period of all these peaks was then determined and compared with the period for each individual peak. Any that were not within 25% of the mean value were dropped from the data set and the frequency was determined from the new mean period. This value was then logged to a file on the IBM hard disk. Figure 1 summarizes the signal analysis scheme by illustrating the threshold value and two random noise peaks. Signals with positive or negative polarity could be processed. Although the wave pattern typically had a flat baseline and few noise peaks, interferences occasionally arose from electrical and radio frequency noise generated in adjacent laboratories. These were effectively filtered by the signalprocessing software. The monitoring program generated about 230 temperature determinations per animal per hour. When monitoring eight animals, a 20 Mbyte hard disk could hold 7 months of continuous temperature data. Transmitter calibration. Prior to calibration, each new transmitter was operated at room temperature for 2
BY TELEMETRY
399
months to stabilize the thermistors. Following that the batteries were replaced and the transmitters were recoated and calibrated in a thermostatically controlled, stirred water bath equipped with a precision thermometer (+O.O5”C). For calibration a transmitter was placed in the constant temperature bath and allowed to stabilize. Temperature readings were taken from the thermometer while the computer logged the emitted frequencies. The water bath temperature was then raised and the process was repeated. The temperature versus frequency data were fitted with a power series regression: Temperature (“C) = a X Frequency! Animals. Male, English shorthair guinea pigs weighed between 300 and 350 g at the time of transmitter implantation. Transmitters were coated, calibrated, and then sterilized in a laminar flow hood with alcohol and I5 min exposure to a 30-W ultraviolet germicidal lamp. They were then stitched to the inner wall of the peritoneum under ketamine (100 mg/kg) and acepromazine anesthesia (1 m&kg, intraperitoneally) through a 2-cm midsagittal incision. Chemicals. MD1 was a gift from Mobay Chemical Co. (Pittsburgh, PA). It was recrystallized from HPLC-grade hexane prior to use. Endotoxin, prepared from Enterobacter agglomerans (Abbott Laboratories, North Chicago, IL) had a potency of 100% based on the Limuluspotyphemus amebocyte lysate assay(LAL). It was ob tained from Dr. J. Fischer (University of North Carolina, Chapel Hill, NC). Aerosol generation and analysis. Aerosols of MD1 were generated using a modified Rapaport-Weinstock apparatus (Rapapott and Weinstock, 1955) in which dry nitrogen at 10092 was bubbled through a midget impinger containing 5 ml of MD1 maintained at IOO’C. The resulting MD1 vapor was directed into a stream of dried air at ambient temperature. Upon cooling a fine aerosol containing approximately 0.07 ppm residual vapor was produced. The low vapor pressure of MD1 at room temperature precluded exposure to vapors in excess of 0.07 ppm. The concentration of the MD1 was adjusted by dilution within a 2.7-liter glass mixing chamber. The MD1 atmosphere was then drawn into individual I.7-liter glass, whole-body plethysmographs which housed the animals (Thome et al., 1986). Chamber concentrations were monitored using a modification of an HPLC method described by Sang6 (1979). Air samples from the plethysmographs were collected onto I3-mm binder-free glass fiber filters (Gelman Sciences Inc., Ann Arbor, MI) which had previously been coated with pnitrobenzyl-N,n-propylamine hydrochloride (I&Us Chemical Co., Morton Grove, IL). Filters were desorbed with ,HPLC-grade acetonitrile (J. T. Baker, P-T NJ) anU ana&zed using a bondedphase RPLC column (10 pm, RP-18 Lichrosorb, E. Merck, West Germany) operated at 1.O ml/min with iSocratic ehltion Using a 3:1 KihItiOn of acetonitrile and HPLC water. The water phase contained 1% tfiethylamine and had been adjusted to pH 3 with phosphoric
400
THORNE, YESKE, AND KAROL
acid. MD1 concentration was determined from the absorbance at 254 nm. Endotoxin was generated using a Pitt No. 1 generator (Wong and Alarie, 1982) fed from a syringe pump (Harvard Apparatus, South Natick, MA) operated at 0.7 ml/ min. Solutions of endotoxin were prepared using pyrogen-free water. Concentrations were determined by collecting air samples onto 0.2-pm cellulose filters (Millipore Corp., Bedford, MA), followed by elution for 1 hr with pyrogen-free water and analysis using the LAL assay (Associates of Cape Cod, Inc., Woods Hole, MA) standardized with Escherichia coli endotoxin. Pulmonary responses. A differential pressure transducer (Gaeltec 8T-2, Medical Measurements Inc., Hackensack, NJ) was attached to each plethysmograph for monitoring of respiratory rate and plethysmograph pressure changes as previously described (Karol et al., 198 1). Voltage signals were amplified and input to the DASH-8 A to D board of a second IBM AT or XT microcomputer. The signals were digitized at a rate of 50 samples/set/ channel. Flow-volume loops were obtained using a head-only plethysmograph equipped with a pneumotachograph (Fleisch Type U-000, OEM Medical, Inc., Richmond, VA) and a Gaeltec 8T-2 transducer (Schaper et al., 1985). The transducer voltage signal was digitized as described above but at 250 samples/set/channel. The calibrated flow signal was digitally integrated to obtain the tidal volume and these were used to generate flow-volume loops. The loops, frequency, tidal volume, and minute volume were computed for the animals breathing air or breathing a mixture of 10% COz ,20% 02, and 70% N2 (Wong and Alarie, 1982). Statistical analysis. Analysis of variance (ANOVA) was performed to test for differences between group means for temperature maxima and minima. The analyseswere performed using the radio frequency data. Based on repeated long-term monitoring of guinea pigs in these chambers, temperature changes with periods greater than 12 min and in excess of 0.3-C were found to be meaningful. At least 4 hr of baseline data were selected for comparisons. All statistical analyses were performed using UNIX:STAT software (Gary Perlman, Wang Institute, Tyngsboro, MA) verified by BMDP (BMDP Statistical Software, University ofcalifornia, Berkeley, CA).
RESULTS
Temperature Telemetry System Software was developed which permitted collection and filtering of *I: T;frequency data from the Mini-Mitters. ?&se data were stored on hard disk for ‘l&ter retrieval and plotting. Temperature was calculated from frequency data using calibration parameters
stored in a file. As shown in Fig. 1 (inset), the signal had an amplitude of 3 V and very low noise. The lower hypothetical curve illustrates selection by the software of peaks with amplitudes exceeding the specified threshold. The program effectively removed further noise peaks by comparing their period to that of the rest of the data set. Frequency determinations based upon analysis of 80 to 120 peaks resulted in 230 temperature points/animal/hr. The sensitivity of the temperature telemetry system was found to be limited to 0.1 “C by the sensitivity of the transmitter. The response time of the transmitter was characterized. The time to reach 90% of a l.o”C step change to 37°C was 83 sec. Thus, the calibration technique which allowed lomin equilibration periods between incremental changes provided more than enough time for the Mini-Mitters to reach steady state. The rapid response time of the transmitters also assured that physiological temperature changes could be followed effectively. Calibration of a transmitter is illustrated in Fig. 2a. Frequencies were monitored as incremental changes were made in the water bath temperature. The stability of the water bath was such that the temperature fell at a rate of O.O2”C/min at 42S”C. This can be seen by examining the last temperature step shown in Fig. 2a. This decline could produce a maximum calibration error of 0.03”C and was considered negligible. Figure 2b demonstrates calibration data points from Fig. 2a. Several equations were tested to determine the best regression model to fit the calibration data. Included were linear, exponential, logarithmic, power series, and second-order polynomial functions. Correlation coefficients (R2) greater than 0.998 were obtained with the latter two. Power series was chosen to best express the data since it was simpler to use and required less microprocessor computation time. Three transmitters were recalibrated 1 year after their initial calibration. Each demonstrated a small increase in the frequency emitted at a given temperature. The calibration change corresponded to differences of
CORE TEMPERATURE
MONITORING
401
BY TELEMETRY
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if
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FIG. 1. Temperature telemetry signals. The inset displays an oscilloscope tracing of a typical transmitter signal with amplitude of 3.0 V. The peak frequency of 370 Hz corresponded to a temperature of 35.8’C for this transmitter. The wave enlarged from the diagrammatic tracing at the bottom illustrates 55 nsec sampling of the signal at a rate of 4200 conversions/set. At physiological temperatures the transmitter emitted 300 to 500 peaks/set and each wave was characterized by 10 to 15 digitizations. Peaks that exceed a user-specified “threshold value” (indicated on the bottom tracing) were identified and counted. This example indicates eight transmitter peaks and two noise peaks which exceeded the threshold. A filtering algorithm eliminated noise peaks such as these from the data set.
O.OTC/week. Over the 2 1-day course of a single experiment this change was within the sensitivity of the telemetry system. Baseline Monitoring
of Core Temperature
Guinea pigs monitored continuously for 15 hr demonstrated an average core temperature of 38.6 -+ 0.3”C (mean -t SD, N = 9). Figure 3 illustrates monitoring of baseline temperature for one guinea pig (No. T-004). A core temperature of 38.0 & O.l”C (mean f SD) was observed over 15 hr from 3500 data points. Upward excursions from the baseline (arrow) were occasionally observed
and attributed to activities such as chewing and grooming. Such periods of increased movement were confirmed by the plethysmograph pressure signals. For the exposures, guinea pigs were taken from a 12-hr light-dark cycle and monitored for 24 hr with continuous lighting. Under these conditions circadian rhythms which have been noted in free-roaming rats and hamsters (DeCastro, 1978; Pickard et al., 1984) were not apparent. Hypothermib
R&&se
to MDI Exposure
Temperature monitoring into studies of pulmonary
was incorporated irritation caused
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FIG. 2. Calibration curve for transmitter 3009. (A) Calibration was performed in a thermostatically controlled, stirred water bath in which the temperature was monitored with a precision thermometer. Incremental changes were made in temperature and resulting frequencies were monitored. (B) Correlation of frequency with temperature yielded the following power series regression curve with a correlation coefficient (I?) of 0.998: Temperature (“C) = 1.6422. Frequency0~52’5.
by inhalation of MDI. As shown in Fig. 4a, immediately upon exposure to 7.8 f 1.4 ppm MD1 the temperature began to drop and reached 35.1 “C by the end of the 3-hr exposure. The temperature within the plethysmograph, monitored throughout the experiment, remained at 25°C. Immediately following MD1 exposure, the core temperature of the guinea pig started to return toward baseline, a process which took 1.5 hr. Analysis of variance indicated that during the period
from 5 to 17.5 hr following the exposure, the animal’s temperature averaged 38.23 r+ 0.24”C and was significantly above the baseline value obtained during the 6 hr prior to exposure (F = 4680, p < 0.001). Respiratory frequency was also monitored during the exposure (see Fig. 4b). The respiratory rate demonstrated an immediate 28% increase, then fell to 36% below baseline. The latter level was maintained for the remainder of the 3-hr exposure. The pattern of breathing
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FIG. 3. Baseline monitoring of intraperitoneal temperature. Animal T-004 exhibited a baseline temperature of 38.0 + O.l”C averaged over the 15-hr period. The arrow marks a period of increased activity.
of pulmonary irritation. At the end of exposure, breathing frequency began to increase and continued so until 9 hr postexposure when it had attained a value of 102 breaths/min (26% increase). The respiratory rate was still elevated 24 hr postexposure. Breathing volume, measured from changes in plethysmograph pressure (Karol et al., 198 l), increased 40% during the exposure and returned to baseline by 12 hr postexposure (data not shown). was typical
Pyretic Response to Endotoxin
Exposure
In conjunction with studies of the acute respiratory effects of cotton dust, guinea pigs were exposed for 6 hr to atmospheres of E. agglomerans endotoxin at concentrations from 9 to 44 &m3. Temperature telemetry was incorporated into the study. Animals were exposed to 9.5 &m3 endotoxin on one occasion and then reexposed 12 days later to a concentration of 44 pg/m3. From temperature telemetry signals guinea pig T-O 10 registered a baseline temperature of 38.6”C determined over a 24-hr period. Figure 5 illustrates that upon first exposure to endotoxin there was an initial 0.6”C hyperthermic response (F = 2010, p < 0.001). Thereafter, a larger temperature increase oc-
curred which was maximal at 5 hr. Within 8 hr postexposure the temperature had returned to normal. It should be noted that temperature telemetry enabled detection of the maximum response which occurred during the exposure. At 19 to 23 hr an additional hyperthermic response was observed (F = 1160, p < 0.001). This delayed-onset increase was observed for other animals identically exposed. Respiratory measurements were also recorded throughout the exposure. Animal T010 demonstrated a 64% increase in respiratory rate at 3.7 hr. Inspiratory and expiratory air flows were measured at termination of the exposure. From integration of these values a 3 1% decrease in tidal volume was noted. Guinea pigs were reexposed to a higher concentration of endotoxin (44 &m3) 12 days later. Temperature, respiratory rate, and tidal volume were monitored continuously and flow-volume loops were obtained immediately before, and again 24 hr following exposure. The temperature and respiratory rate profiles for animal T-010 upon reexposure are shown in Fig. 6. After approximately 1 hr of exposure, the respiratory rate began to rise to a maximum 52% increase which occurred at 4.5 hr (upper curve). Core temperature (lower curve) felI insignificantly during the initial 2 hr of exposure and then began a rapid
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TIME (hours) FIG. 4. Hypothermic and respiratory responses of guinea pig T-004 to MD1 exposure. (A) Baseline temperature (“C) was monitored for 7.5 hr prior to 3 hr inhalation (indicated by arrowheads) of 7.8 ppm MDI. Maximum hypothermic response occurred at the end of exposure. Temperature returned to baseline within 1.5 hr following exposure. (B) Respiratory response measured during exposure (arrowheads) indicated an immediate 28% increase in respiratory frequency which preceded a 36% decrease that was maintained during the rest of exposure. Upon termination of exposure the respiratory rate increased and was 36% above baseline at 15 hr. By 24 hr postexposure the rate had fallen but was still above baseline (data not shown).
increase toward a maximum of 40.1”C, reached at 5 hr. The rate of increase during this period was O.g”C/hr. For both temperature and respiratory rate, decline from maximum responses began during the exposure period. In contrast to results following the first endotoxin exposure, the temperature had not returned to baseline during the 17 hr following this second exposure.
To further characterize the pulmonary response, lung function tests were performed prior to and 24 hr postexposure while the guinea pigs were breathing air and again while breathing air containing 10% C02. These measurements are presented in Table 1. During air breathing, respiratory rate was unchanged from the preexposure value, whereas tidal volume was decreased 45%
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TIME hcwr¶) FIG. 5. Temperature response of guinea pig T-010 during 6 hr inhalation (indicated by arrowheads) of 9.5 pg./m’ Enterobucter agglomerans endotoxin. At 5 hr the maximum temperature was obselved (4O.l”C). Although exposure continued, the temperature decreased. Baseline temperature was reached 8 hr postex-
from baseline. When breathing 10% C02, the respiratory rate was increased 24% above the preexposure value and tidal volume was 67% lower, resulting in a 60% decrease in minute volume. This pattern of response to 10% CO* was typical of that noted with other pulmonary irritants including cotton dust and methyl isocyanate (Alarie and Schaper, 1987). DISCUSSION Use of temperature telemetry in scientific investigations has been reported, most notably in behavioral studies. Several investigators have described construction and use of devices to monitor temperature of laboratory and feral rodents (DeCastro and Brower, 1977; Blatteis and Smith, 1980; Pickard et al., 1984; Tocco-Bradley et al., 1985). All of these studies relied on either manual counting of low frequency signals or use of custom-designed and built circuitry. In 1985, Gallaher and co-workers described use of a high-frequency transmitter to monitor core temperature (Gallaher et al., 1985). These investigators constructed a telemetry system using Model XM-PII Mini-Mitters, a modified mi-
crocomputer, and interfacing boards of their own design for signal processing. This system sampled, printed, and stored temperatures once every 10 min. The current study describes exclusive use of commercial components in a telemetry system that uses high-frequency transmitters and a high-digitization rate to record and plot temperatures every 12 set during inhalation exposures. Temperature telemetry offers several advantages when compared with other methods of temperature monitoring. Pii once the transmitter is implanted the measurement technique is completely passive. Therefore, the act of making the measurement does not influence the result. Gallaher and co-workers reported experiments in which core temperature of rats was monitored by telemetry while rectal temperatures were measured (Gallaher et al., 1985). The act of insertion of a rectal temperature probe was found to produce a 1°C hyperthermia that lasted for several hours. A second advantage of temperature telemetry is that restraint of animals is not required. Other techniques, such as the use of implanted thermocouples, necessitate restraining guinea pigs which may result in stress-induced disturbances of physiological homeostasis.
406
THORNE,
PULMONARY
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FUNCTION
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AND
TABLE
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MEASUREMENTS FOLLOWING 6 hr EXPOSURE (GUINEA PIG T-O 10)
Time of measurement
Respiratory rate (breaths/min)
Preexposure 24 hr Postexposure % Change
106 108 +2.5
Tidal
A further advantage of telemetry is that it provides continuous temperature monitoring ensuring that transient changes will be detected. During endotoxin exposure (Fig. 6) a temperature measurement taken only at the end of exposure would have indicated a maximum hyperthermic response of OXC, 50% less than that which occurred during the exposure (1 .YC increase at 5 hr). Surgical implantation of the transmitter was accomplished with little trauma to the
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TO 44 &m3
-67
guinea pig. Transmitters removed 6 months after implantation were found encased in a thin layer of tissue with no evidence of inflammation. The average battery life for an implanted transmitter was 4 months, a period of time sufficient for most toxicological experiments using rodents. Using the telemetry system described here, baseline temperatures of guinea pigs were in excellent agreement with those previously reported. An average core temperature of 38.6
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FIG. 6. Respiratory and temperature responses upon reexposure to endotoxin. Guinea pig T-010 was placed in the plethysmograph 6 hr prior to the 6-hr exposure to 44 Irplrn’ endotoxin. At 4.5 hr, respiratory rate had increased from 100 to 152 breaths/min (upper curve). By the end of exposure, breathing frequency had recovered by 47%. Baseline rate was reached 2.5 hr later. The pyretic response (lower curve) was first noted 3.5 hr into exposure. The temperature response mimicked the respiratory response in that it was maximal at 5 hr. Return to baseline was not completed by 17 hr postexposure.
CORE TEMPERATURE
MONITORING
h 0.3”C (mean + SD) was observed for nine guinea pigs kept at temperatures of 23 + 2°C. Riley et al. ( 1978), using a temperature transmitter of their own design, reported a baseline temperature of 39°C as typical for guinea pigs. Blatteis and Smith (1980) using commercially made low-frequency transmitters, reported the average core temperature of six guinea pigs to be 37.5 -+ 0.4”C at room temperature. For 56 eutherian mammalian species weighing from 0.00 1 to 1000 kg, Morrison and Rysen ( 1952) reported a mean body temperature of 37.9”C. Schmidt-Nielsen ( 1984) concluded that within a species, core temperature is independent of body size since heat production and heat loss scale together. Few reports of chemically induced hypothermia exist. High doses of ethanol were reported to induce hypothermia (Gallaher et al., 1985). Intraperitoneal injection of 4 g/kg ethanol (20%, v/v, in saline) into a single rat produced a maximal hypothermia of 3.o”C at 4 hr. Return to baseline was noted 9 hr after the injection. The hypothermia-inducing effect of diisopropyl fluorophosphate (DFP) has been investigated in rats and in mice. The magnitude of the observed decreases with DFP were similar to those seen in response to MD1 exposure in the present study. In rats, intramuscular injection of 1.0 mg/kg DFP produced a maximum decrease of 3.2”C at 6 hr and slow return to baseline over the next 15 hr (Kozar et al., 1976). Inhalation of DFP for 5 min in mice produced a maximum core temperature decrease of 3.5”C that occurred 30 min after exposure and returned to baseline by 10 hr (Scimeca et al., 1985). MD1 inhalation produced hypothermia of similar onset and degree to that following inhalation of DFP. However, recovery from MD1 hypothermia was more rapid and led to mild hyperthermia postexposure. Chemically induced hyperthermia has been more extensively observed and the mechanisms underlying febrile responses, in general, have been under investigation for more than 40 years. Atkins (1960) showed that a group of compounds, now known as
BY TELEMETRY
407
interleukin 1 (IL-l), was responsible for the fever that developed in response to exogenous pyrogens. Currently it is believed that for an animal to display a febrile response, the reticuloendothelial system must be capable of activation to release IL- 1, the thermoregulatory center of the hypothalamus must be intact, and the vascular and skeletal systems must be able to generate heat to obtain an elevated set point temperature (Dinarello, 1984). In the current study, inhalation of endotoxin induced a fever maximal at 4.5 to 5 hr. Temperature telemetry revealed recovery beginning before the end of exposure (Figs. 5 and 6). Inhalation of endotoxin also induced changes in pulmonary function marked by increased breathing frequency during exposure (Fig. 6, upper curve) and reduced minute volume (Table 1). Tocco-Bradley et al. (1985) reported a temperature response in rats to intravenous injection of endotoxin. Similar to our findings in guinea pigs, rats demonstrated a response beginning 2 hr after endotoxin injection that reached a. maximum increase of 1.7”C at 4.5 hr. In summary, we have described the use of a temperature telemetry system which is sensitive to both hypothermic and hyperthermic responses of guinea pigs which can occur during inhalation of airborne chemicals. The system consists of a microcomputer, an A to D board, AM receivers, temperature transmitters, and a water bath for transmitter calibration. It is sensitive to temperature changes of 0.1 “C. When using this system, there is no interruption of exposure or of monitoring other physiological measurements (i.e., respiratory frequency and volume). For these reasons, the system can be readily incorporated into existing experimental protocols for inhalation toxicology. ACKNOWLEDGMENTS We thank the following individuals for their contributions to this work: Dr. Robert Sandridge, Mobay Chemical Corp., for consultation regarding MD1 generation
408
THORNE,
YESK E, AND
and analysis; Meredith Karol, Andrea Shelton, and Barbara Stephens for help with transmitter calibrations; Dr. Adeniyi Ogundiran and Carolyn Catty for assistance with endotoxin exposures; and Julie Hillebrand for help with MD1 experiments. This work was supported by NIEHS
ES01 532.
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BRADFORD, D. T. ( 1984). Temperature modulation in a high-elevation amphibian, Rana muscosa. Copeia 4, 966-976.
DECASTRO, J. M. (1978). Diurnal rhythms of behavioral effects on core temperature. Physiol. Behav. 21,883-
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DINARELLO, C. A. ( 1984). Interleukin- 1. Rev. Inject. Dis. 6,5 l-95.
FINK, J. N. (1984). Hypersensitivity pneumonitis. J. Allergy Clin. Immunol.
74, 1-9.
Fox, R. H., GOLDSMITH, R., AND WOLFF, H. S. ( 196 1). The use of a radio pill to measure deep body temperature. J. Physiol. (London) 160,22-23. GALLAHER, E. J., EGNER, D. A., AND SWEN, J. W. ( 1985). Automated remote temperature measurement in small animals using a telemetry/microcomputer interface. Comput. Biol. Med. 15, 103-l 10. KAROL, M. H., STADLER, J., UNDERHILL, D., AND ALARIE, Y. (1981). Monitoring delayed-onset pulmonary hypersensitivity in guinea pigs. Toxicol. Appl. Pharmacol. 61,277-285.
KOZAR, M. D., OVERSTREET, D. H., CHIPPENDALE, T. C., AND RUSSELL, R. W. (1976). Changes of acetylcholinesterase activity in three major brain areas and related changes in behavior following acute treatment with diisopropyl fluorophosphate. Neuropharmacofogy 15.291-298.
Respir.
Environ.
Exercise
Physiol.
45,
1016-1018. RISMILLER, P. D., AND HELDMAIER, G. (1985). Thermal behavior as a function of the time of day: Heat exchange rates and oxygen consumption in the Lacertid lizard Lacert viridis. Physiol. Zool. 58,7 l-79. SANG& C. ( 1979). Improved method for determination of traces of isocyanates in working atmospheres by high-performance liquid chromatography. J. Liq. Chromatogr.
886.
DECASTRO, J. M., AND BROWER, E. (1977). Simple, reliable and inexpensive telemetry system for continuous monitoring of small animal core temperature. Physiol. Behav.
MINARD, D., GOLDSMITH, R., FARRIER, P. H., JR., AND LAMBIOTTE, B. J., JR. (1971). Physiological evaluation of industrial heat stress.Amer. Ind. Hyg. Assoc. J. 32, 17-28. MORRISON, P. R., AND RYSEN, F. A. (1952). Weight and body temperature in mammals. Science 116,23 l-232. PARKES, W. R. (1982). Occupational Lung Disorders. 2nd edition. Butterworth, Boston, MA. PICKARD, G. E., KAHN, R., AND SILVER, R. (1984). Splitting of the circadian rhythm of body temperature in the golden hamster. Physiol. Behav. 32,763-766. RAPAPORT, E., AND WEINSTOCK, S. (1955). A generator for homogeneous. Experientia 11,363-366. RILEY, J. L., THURSTON, J. R., EGEMO, C. L., AND ELLIOTT, H. L. (1978). A radiotelemetry transmitter for transmitting temperatures from small animals. J. Appl.
40,580-646.
BLATTEIS, C. M., AND SMITH, K. A. (1980). Behavioral fever induced in guinea pigs by intrapreoptic pyrogen.
KAROL
2,763-774.
SCHAPER, M., THOMPSON, R. D., AND ALARIE, Y. ( 1985). A method to classify airborne chemicals which alter the normal ventilatory response induced by COZ. Toxicol. Appl. Pharmacol. 79,332-34 1. SCHMIDT-NIELSEN, K. (1984). Scaling: Why Is Animal Sizeso Important?Cambridge Univ. Press, New York. SCIMECA, J. A., LITTLE, P. J., AND MARTIN, B. R. ( 1985). Relationship between the pharmacological effects and the biodisposition of [3H]diisopropylfluorophosphate in mice after inhalation. Toxicol. Appl. Pharmacol. 79,502-5 10. THORNE, P. S., HILLEBRAND, J., MAGRENI, C., RILEY, E. J., AND KAROL, M. H. (1986). Experimental sensitization to subtilisin. I. Production of immediate- and late-onset pulmonary reaction. Toxicol. Appl. Pharmacol. 86, 112-123. TOCCO-BRADLEY, R., KLUGER, M. J., AND KAUFFMAN, C. A. (1985). Effect of age on fever and acute-phase response of rats to endotoxin and Salmonella typhimurium.
Infect.
Immunol.
47, 106- 111.
WONG, K. L., AND ALARIE, Y. (1982). A method for repeated evaluation of pulmonary performance in unanesthetized, unrestrained guinea pigs and its application to detect effects of sulfuric acid mist inhalation. Toxicol.
Appl. Pharmacol.
63,72-90.
AND
APPLIED
Monitoring
TOXICOLOGY
9,398-408
( 1987)
Guinea Pig Core Temperature during Inhalation Exposures
by Telemetry
PETER S. THORNE,’ CURTIS P. YESKE,* AND MERYL H. KAROL Department of Industrial Environmental Health Sciences, Graduate School ofPublic Health, University ofpittsburgh, Pittsburgh, Pennsylvania 15261
Monitoring
Guinea Pig Core Temperature by Telemetry during Inhalation Exposures. S., YESKE, C. P., AND KAROL, M. H. (1987). Fundam. Appl. Toxicol. 9, 39% 408. A temperature telemetry system was incorporated into an existing animal model for inhalation toxicology. This system facilitated continuous monitoring ofguinea pig temperature during inhalation exposures. Components of the system included Mini-Mitter temperature-controlled oscillators, AM receivers, and an IBM microcomputer. Software was developed to perform signal processing and filtering. Transmitters were calibrated and then sterilized and surgically implanted in the guinea pig peritoneum. Monitoring the baseline temperature of nine animals indicated a mean temperature of 38.6 f OYC. Guinea pigs were treated with agents to induce transient hypo- or hyperthermia. For the former, exposure for 3 hr to 7.8 ppm diphenylmethane+‘diisocyanate resulted in a 3°C temperature decrease. The temperature was determined from 230 readings per animal per hour. Inhalation of 9 or 44 pg/m3 endotoxin for 6 hr induced hyperthermia with a 1.5”C maximum increase in core temperature at 4.8 hr. With endotoxin, an increase in respiratory rate was also noted and followed the same pattern as temperature with a maximal increase of 52% occurring at 4.2 hr. The temperature telemetry system enabled continuous long-term monitoring of toxicant-induced hypothermia and pyrexia without interruption of inhalation exposure or measurements of respiratory parameters. Q 1987 THORNE,
P.
Society of Toxicology.
ter temperature transmitters (Mini-Mitter Co., Inc.) have been used in behavior studies in amphibians and rodents (Bradford, 1984; DeCastro, 1978; Pickard et al., 1984; fismiller and Heldmaier, 1985). We have previously reported development of an animal model for study of pulmonary sensitivity to industrial toxicants. The model demonstrates both immediate- and late-onset pulmonary responses. Fuller characterization of the syndrome would be achieved by inclusion of temperature monitoring to detect possible occurrence of hypersensitivity pneumonitis. This study describes incorporation of a temperature telemetry system into the animal model studies. Inhalation exposure of guinea pigs in whole body plethysmographs to diphenylmethane-4,4’-diisocyanate (MDI) and to endotoxin produced hypothermia and
Inhalation of numerous industrial compounds has been reported to affect body temperature. Mill fever is’ associated with exposure to endotoxin in cotton or flax dust; metal fume fever has been reported following exposure to metals such as zinc; and polymer fume fever results from inhalation of fumes from burning Teflon (Parkes, 1982). Hypersensitivity pneumonitis from exposure to isocyanates and anhydrides is characterized by fever which may reach 40°C (Fink, 1984). A “radio pill” was described by Fox et al. (196 1) for measurement of intragastric temperature in humans. Subsequently the device was used to study heat stress in Pittsburgh steelworkers (Minard et al., 197 1). Mini-Mit’ To whom correspondence should be addressed. 2 Present Address: Computing Center, Camegie-Mellon University, Pittsburgh, PA 152 13. 0272-0590/87 $3.00 Copyright 0 1987 by the Society of Toxicolc+ty. All rights of reproduclion in any form reserved.
398
CORE TEMPERATURE
MONITORING
hyperthermia, respectively. The responses were detected through use of continuous monitoring of temperature transmitter signals. MATERIALS
AND
METHODS
Temperature telemetry system. The transmitter, a dual thermistor, temperature-controlled oscillator (Mini-Mitter Model M-FH, Mini-Mitter Co., Inc., Sunriver, OR) powered by a replaceable 1.5 V silver oxide battery, was contained within a 1.1-cm-diameter, I .8-cm-long plastic capsule coated with a paraffin-resin mix (Elvax, E. I. DuPont DeNemours Co., Wilmington, DE) and weighed 2.7 g. At physiological temperatures the transmitters emitted pulses at 300 to 500 Hz on a broad-band AM carrier. The actual frequency emitted for a given temperature was different for each transmitter. The radio signal received from a transmitter was demodulated and amplified by an AM receiver with an external ferrite antenna. The transmission range was dependent upon both the amplifier gain and the orientation ofthe transmitter with respect to the antenna. The usable transmission range extended to about 30 cm. An oscilloscope display of the speaker output from the receiver is illustrated at the top of Fig. 1 and shows four peaks of a 3.0-V, 370-Hz signal. An analog to digital converter (A to D) board (DASH-g, MetraByte Corp., Taunton, MA) sampled the voltage of the input signal in a period of 250 msec for a given channel at a rate of 4200 samples/set with each sample hasting 55 nsec. This set of 1050 conversions was analyzed by determining the number of peaks detected in 250 msec (80 to 120 peaks) with amplitude exceeding the user-specified threshold value. Typically the threshold was set to 1 V to exclude lower amplitude interference accompanying the transmitter signal. The average period of all these peaks was then determined and compared with the period for each individual peak. Any that were not within 25% of the mean value were dropped from the data set and the frequency was determined from the new mean period. This value was then logged to a file on the IBM hard disk. Figure 1 summarizes the signal analysis scheme by illustrating the threshold value and two random noise peaks. Signals with positive or negative polarity could be processed. Although the wave pattern typically had a flat baseline and few noise peaks, interferences occasionally arose from electrical and radio frequency noise generated in adjacent laboratories. These were effectively filtered by the signalprocessing software. The monitoring program generated about 230 temperature determinations per animal per hour. When monitoring eight animals, a 20 Mbyte hard disk could hold 7 months of continuous temperature data. Transmitter calibration. Prior to calibration, each new transmitter was operated at room temperature for 2
BY TELEMETRY
399
months to stabilize the thermistors. Following that the batteries were replaced and the transmitters were recoated and calibrated in a thermostatically controlled, stirred water bath equipped with a precision thermometer (+O.O5”C). For calibration a transmitter was placed in the constant temperature bath and allowed to stabilize. Temperature readings were taken from the thermometer while the computer logged the emitted frequencies. The water bath temperature was then raised and the process was repeated. The temperature versus frequency data were fitted with a power series regression: Temperature (“C) = a X Frequency! Animals. Male, English shorthair guinea pigs weighed between 300 and 350 g at the time of transmitter implantation. Transmitters were coated, calibrated, and then sterilized in a laminar flow hood with alcohol and I5 min exposure to a 30-W ultraviolet germicidal lamp. They were then stitched to the inner wall of the peritoneum under ketamine (100 mg/kg) and acepromazine anesthesia (1 m&kg, intraperitoneally) through a 2-cm midsagittal incision. Chemicals. MD1 was a gift from Mobay Chemical Co. (Pittsburgh, PA). It was recrystallized from HPLC-grade hexane prior to use. Endotoxin, prepared from Enterobacter agglomerans (Abbott Laboratories, North Chicago, IL) had a potency of 100% based on the Limuluspotyphemus amebocyte lysate assay(LAL). It was ob tained from Dr. J. Fischer (University of North Carolina, Chapel Hill, NC). Aerosol generation and analysis. Aerosols of MD1 were generated using a modified Rapaport-Weinstock apparatus (Rapapott and Weinstock, 1955) in which dry nitrogen at 10092 was bubbled through a midget impinger containing 5 ml of MD1 maintained at IOO’C. The resulting MD1 vapor was directed into a stream of dried air at ambient temperature. Upon cooling a fine aerosol containing approximately 0.07 ppm residual vapor was produced. The low vapor pressure of MD1 at room temperature precluded exposure to vapors in excess of 0.07 ppm. The concentration of the MD1 was adjusted by dilution within a 2.7-liter glass mixing chamber. The MD1 atmosphere was then drawn into individual I.7-liter glass, whole-body plethysmographs which housed the animals (Thome et al., 1986). Chamber concentrations were monitored using a modification of an HPLC method described by Sang6 (1979). Air samples from the plethysmographs were collected onto I3-mm binder-free glass fiber filters (Gelman Sciences Inc., Ann Arbor, MI) which had previously been coated with pnitrobenzyl-N,n-propylamine hydrochloride (I&Us Chemical Co., Morton Grove, IL). Filters were desorbed with ,HPLC-grade acetonitrile (J. T. Baker, P-T NJ) anU ana&zed using a bondedphase RPLC column (10 pm, RP-18 Lichrosorb, E. Merck, West Germany) operated at 1.O ml/min with iSocratic ehltion Using a 3:1 KihItiOn of acetonitrile and HPLC water. The water phase contained 1% tfiethylamine and had been adjusted to pH 3 with phosphoric
400
THORNE, YESKE, AND KAROL
acid. MD1 concentration was determined from the absorbance at 254 nm. Endotoxin was generated using a Pitt No. 1 generator (Wong and Alarie, 1982) fed from a syringe pump (Harvard Apparatus, South Natick, MA) operated at 0.7 ml/ min. Solutions of endotoxin were prepared using pyrogen-free water. Concentrations were determined by collecting air samples onto 0.2-pm cellulose filters (Millipore Corp., Bedford, MA), followed by elution for 1 hr with pyrogen-free water and analysis using the LAL assay (Associates of Cape Cod, Inc., Woods Hole, MA) standardized with Escherichia coli endotoxin. Pulmonary responses. A differential pressure transducer (Gaeltec 8T-2, Medical Measurements Inc., Hackensack, NJ) was attached to each plethysmograph for monitoring of respiratory rate and plethysmograph pressure changes as previously described (Karol et al., 198 1). Voltage signals were amplified and input to the DASH-8 A to D board of a second IBM AT or XT microcomputer. The signals were digitized at a rate of 50 samples/set/ channel. Flow-volume loops were obtained using a head-only plethysmograph equipped with a pneumotachograph (Fleisch Type U-000, OEM Medical, Inc., Richmond, VA) and a Gaeltec 8T-2 transducer (Schaper et al., 1985). The transducer voltage signal was digitized as described above but at 250 samples/set/channel. The calibrated flow signal was digitally integrated to obtain the tidal volume and these were used to generate flow-volume loops. The loops, frequency, tidal volume, and minute volume were computed for the animals breathing air or breathing a mixture of 10% COz ,20% 02, and 70% N2 (Wong and Alarie, 1982). Statistical analysis. Analysis of variance (ANOVA) was performed to test for differences between group means for temperature maxima and minima. The analyseswere performed using the radio frequency data. Based on repeated long-term monitoring of guinea pigs in these chambers, temperature changes with periods greater than 12 min and in excess of 0.3-C were found to be meaningful. At least 4 hr of baseline data were selected for comparisons. All statistical analyses were performed using UNIX:STAT software (Gary Perlman, Wang Institute, Tyngsboro, MA) verified by BMDP (BMDP Statistical Software, University ofcalifornia, Berkeley, CA).
RESULTS
Temperature Telemetry System Software was developed which permitted collection and filtering of *I: T;frequency data from the Mini-Mitters. ?&se data were stored on hard disk for ‘l&ter retrieval and plotting. Temperature was calculated from frequency data using calibration parameters
stored in a file. As shown in Fig. 1 (inset), the signal had an amplitude of 3 V and very low noise. The lower hypothetical curve illustrates selection by the software of peaks with amplitudes exceeding the specified threshold. The program effectively removed further noise peaks by comparing their period to that of the rest of the data set. Frequency determinations based upon analysis of 80 to 120 peaks resulted in 230 temperature points/animal/hr. The sensitivity of the temperature telemetry system was found to be limited to 0.1 “C by the sensitivity of the transmitter. The response time of the transmitter was characterized. The time to reach 90% of a l.o”C step change to 37°C was 83 sec. Thus, the calibration technique which allowed lomin equilibration periods between incremental changes provided more than enough time for the Mini-Mitters to reach steady state. The rapid response time of the transmitters also assured that physiological temperature changes could be followed effectively. Calibration of a transmitter is illustrated in Fig. 2a. Frequencies were monitored as incremental changes were made in the water bath temperature. The stability of the water bath was such that the temperature fell at a rate of O.O2”C/min at 42S”C. This can be seen by examining the last temperature step shown in Fig. 2a. This decline could produce a maximum calibration error of 0.03”C and was considered negligible. Figure 2b demonstrates calibration data points from Fig. 2a. Several equations were tested to determine the best regression model to fit the calibration data. Included were linear, exponential, logarithmic, power series, and second-order polynomial functions. Correlation coefficients (R2) greater than 0.998 were obtained with the latter two. Power series was chosen to best express the data since it was simpler to use and required less microprocessor computation time. Three transmitters were recalibrated 1 year after their initial calibration. Each demonstrated a small increase in the frequency emitted at a given temperature. The calibration change corresponded to differences of
CORE TEMPERATURE
MONITORING
401
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FIG. 1. Temperature telemetry signals. The inset displays an oscilloscope tracing of a typical transmitter signal with amplitude of 3.0 V. The peak frequency of 370 Hz corresponded to a temperature of 35.8’C for this transmitter. The wave enlarged from the diagrammatic tracing at the bottom illustrates 55 nsec sampling of the signal at a rate of 4200 conversions/set. At physiological temperatures the transmitter emitted 300 to 500 peaks/set and each wave was characterized by 10 to 15 digitizations. Peaks that exceed a user-specified “threshold value” (indicated on the bottom tracing) were identified and counted. This example indicates eight transmitter peaks and two noise peaks which exceeded the threshold. A filtering algorithm eliminated noise peaks such as these from the data set.
O.OTC/week. Over the 2 1-day course of a single experiment this change was within the sensitivity of the telemetry system. Baseline Monitoring
of Core Temperature
Guinea pigs monitored continuously for 15 hr demonstrated an average core temperature of 38.6 -+ 0.3”C (mean -t SD, N = 9). Figure 3 illustrates monitoring of baseline temperature for one guinea pig (No. T-004). A core temperature of 38.0 & O.l”C (mean f SD) was observed over 15 hr from 3500 data points. Upward excursions from the baseline (arrow) were occasionally observed
and attributed to activities such as chewing and grooming. Such periods of increased movement were confirmed by the plethysmograph pressure signals. For the exposures, guinea pigs were taken from a 12-hr light-dark cycle and monitored for 24 hr with continuous lighting. Under these conditions circadian rhythms which have been noted in free-roaming rats and hamsters (DeCastro, 1978; Pickard et al., 1984) were not apparent. Hypothermib
R&&se
to MDI Exposure
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by inhalation of MDI. As shown in Fig. 4a, immediately upon exposure to 7.8 f 1.4 ppm MD1 the temperature began to drop and reached 35.1 “C by the end of the 3-hr exposure. The temperature within the plethysmograph, monitored throughout the experiment, remained at 25°C. Immediately following MD1 exposure, the core temperature of the guinea pig started to return toward baseline, a process which took 1.5 hr. Analysis of variance indicated that during the period
from 5 to 17.5 hr following the exposure, the animal’s temperature averaged 38.23 r+ 0.24”C and was significantly above the baseline value obtained during the 6 hr prior to exposure (F = 4680, p < 0.001). Respiratory frequency was also monitored during the exposure (see Fig. 4b). The respiratory rate demonstrated an immediate 28% increase, then fell to 36% below baseline. The latter level was maintained for the remainder of the 3-hr exposure. The pattern of breathing
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of pulmonary irritation. At the end of exposure, breathing frequency began to increase and continued so until 9 hr postexposure when it had attained a value of 102 breaths/min (26% increase). The respiratory rate was still elevated 24 hr postexposure. Breathing volume, measured from changes in plethysmograph pressure (Karol et al., 198 l), increased 40% during the exposure and returned to baseline by 12 hr postexposure (data not shown). was typical
Pyretic Response to Endotoxin
Exposure
In conjunction with studies of the acute respiratory effects of cotton dust, guinea pigs were exposed for 6 hr to atmospheres of E. agglomerans endotoxin at concentrations from 9 to 44 &m3. Temperature telemetry was incorporated into the study. Animals were exposed to 9.5 &m3 endotoxin on one occasion and then reexposed 12 days later to a concentration of 44 pg/m3. From temperature telemetry signals guinea pig T-O 10 registered a baseline temperature of 38.6”C determined over a 24-hr period. Figure 5 illustrates that upon first exposure to endotoxin there was an initial 0.6”C hyperthermic response (F = 2010, p < 0.001). Thereafter, a larger temperature increase oc-
curred which was maximal at 5 hr. Within 8 hr postexposure the temperature had returned to normal. It should be noted that temperature telemetry enabled detection of the maximum response which occurred during the exposure. At 19 to 23 hr an additional hyperthermic response was observed (F = 1160, p < 0.001). This delayed-onset increase was observed for other animals identically exposed. Respiratory measurements were also recorded throughout the exposure. Animal T010 demonstrated a 64% increase in respiratory rate at 3.7 hr. Inspiratory and expiratory air flows were measured at termination of the exposure. From integration of these values a 3 1% decrease in tidal volume was noted. Guinea pigs were reexposed to a higher concentration of endotoxin (44 &m3) 12 days later. Temperature, respiratory rate, and tidal volume were monitored continuously and flow-volume loops were obtained immediately before, and again 24 hr following exposure. The temperature and respiratory rate profiles for animal T-010 upon reexposure are shown in Fig. 6. After approximately 1 hr of exposure, the respiratory rate began to rise to a maximum 52% increase which occurred at 4.5 hr (upper curve). Core temperature (lower curve) felI insignificantly during the initial 2 hr of exposure and then began a rapid
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TIME (hours) FIG. 4. Hypothermic and respiratory responses of guinea pig T-004 to MD1 exposure. (A) Baseline temperature (“C) was monitored for 7.5 hr prior to 3 hr inhalation (indicated by arrowheads) of 7.8 ppm MDI. Maximum hypothermic response occurred at the end of exposure. Temperature returned to baseline within 1.5 hr following exposure. (B) Respiratory response measured during exposure (arrowheads) indicated an immediate 28% increase in respiratory frequency which preceded a 36% decrease that was maintained during the rest of exposure. Upon termination of exposure the respiratory rate increased and was 36% above baseline at 15 hr. By 24 hr postexposure the rate had fallen but was still above baseline (data not shown).
increase toward a maximum of 40.1”C, reached at 5 hr. The rate of increase during this period was O.g”C/hr. For both temperature and respiratory rate, decline from maximum responses began during the exposure period. In contrast to results following the first endotoxin exposure, the temperature had not returned to baseline during the 17 hr following this second exposure.
To further characterize the pulmonary response, lung function tests were performed prior to and 24 hr postexposure while the guinea pigs were breathing air and again while breathing air containing 10% C02. These measurements are presented in Table 1. During air breathing, respiratory rate was unchanged from the preexposure value, whereas tidal volume was decreased 45%
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TIME hcwr¶) FIG. 5. Temperature response of guinea pig T-010 during 6 hr inhalation (indicated by arrowheads) of 9.5 pg./m’ Enterobucter agglomerans endotoxin. At 5 hr the maximum temperature was obselved (4O.l”C). Although exposure continued, the temperature decreased. Baseline temperature was reached 8 hr postex-
from baseline. When breathing 10% C02, the respiratory rate was increased 24% above the preexposure value and tidal volume was 67% lower, resulting in a 60% decrease in minute volume. This pattern of response to 10% CO* was typical of that noted with other pulmonary irritants including cotton dust and methyl isocyanate (Alarie and Schaper, 1987). DISCUSSION Use of temperature telemetry in scientific investigations has been reported, most notably in behavioral studies. Several investigators have described construction and use of devices to monitor temperature of laboratory and feral rodents (DeCastro and Brower, 1977; Blatteis and Smith, 1980; Pickard et al., 1984; Tocco-Bradley et al., 1985). All of these studies relied on either manual counting of low frequency signals or use of custom-designed and built circuitry. In 1985, Gallaher and co-workers described use of a high-frequency transmitter to monitor core temperature (Gallaher et al., 1985). These investigators constructed a telemetry system using Model XM-PII Mini-Mitters, a modified mi-
crocomputer, and interfacing boards of their own design for signal processing. This system sampled, printed, and stored temperatures once every 10 min. The current study describes exclusive use of commercial components in a telemetry system that uses high-frequency transmitters and a high-digitization rate to record and plot temperatures every 12 set during inhalation exposures. Temperature telemetry offers several advantages when compared with other methods of temperature monitoring. Pii once the transmitter is implanted the measurement technique is completely passive. Therefore, the act of making the measurement does not influence the result. Gallaher and co-workers reported experiments in which core temperature of rats was monitored by telemetry while rectal temperatures were measured (Gallaher et al., 1985). The act of insertion of a rectal temperature probe was found to produce a 1°C hyperthermia that lasted for several hours. A second advantage of temperature telemetry is that restraint of animals is not required. Other techniques, such as the use of implanted thermocouples, necessitate restraining guinea pigs which may result in stress-induced disturbances of physiological homeostasis.
406
THORNE,
PULMONARY
Breathing
FUNCTION
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YESKE,
AND
TABLE
1
KAROL
MEASUREMENTS FOLLOWING 6 hr EXPOSURE (GUINEA PIG T-O 10)
Time of measurement
Respiratory rate (breaths/min)
Preexposure 24 hr Postexposure % Change
106 108 +2.5
Tidal
A further advantage of telemetry is that it provides continuous temperature monitoring ensuring that transient changes will be detected. During endotoxin exposure (Fig. 6) a temperature measurement taken only at the end of exposure would have indicated a maximum hyperthermic response of OXC, 50% less than that which occurred during the exposure (1 .YC increase at 5 hr). Surgical implantation of the transmitter was accomplished with little trauma to the
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213 153 -44
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165 204 +24
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TO 44 &m3
-67
guinea pig. Transmitters removed 6 months after implantation were found encased in a thin layer of tissue with no evidence of inflammation. The average battery life for an implanted transmitter was 4 months, a period of time sufficient for most toxicological experiments using rodents. Using the telemetry system described here, baseline temperatures of guinea pigs were in excellent agreement with those previously reported. An average core temperature of 38.6
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FIG. 6. Respiratory and temperature responses upon reexposure to endotoxin. Guinea pig T-010 was placed in the plethysmograph 6 hr prior to the 6-hr exposure to 44 Irplrn’ endotoxin. At 4.5 hr, respiratory rate had increased from 100 to 152 breaths/min (upper curve). By the end of exposure, breathing frequency had recovered by 47%. Baseline rate was reached 2.5 hr later. The pyretic response (lower curve) was first noted 3.5 hr into exposure. The temperature response mimicked the respiratory response in that it was maximal at 5 hr. Return to baseline was not completed by 17 hr postexposure.
CORE TEMPERATURE
MONITORING
h 0.3”C (mean + SD) was observed for nine guinea pigs kept at temperatures of 23 + 2°C. Riley et al. ( 1978), using a temperature transmitter of their own design, reported a baseline temperature of 39°C as typical for guinea pigs. Blatteis and Smith (1980) using commercially made low-frequency transmitters, reported the average core temperature of six guinea pigs to be 37.5 -+ 0.4”C at room temperature. For 56 eutherian mammalian species weighing from 0.00 1 to 1000 kg, Morrison and Rysen ( 1952) reported a mean body temperature of 37.9”C. Schmidt-Nielsen ( 1984) concluded that within a species, core temperature is independent of body size since heat production and heat loss scale together. Few reports of chemically induced hypothermia exist. High doses of ethanol were reported to induce hypothermia (Gallaher et al., 1985). Intraperitoneal injection of 4 g/kg ethanol (20%, v/v, in saline) into a single rat produced a maximal hypothermia of 3.o”C at 4 hr. Return to baseline was noted 9 hr after the injection. The hypothermia-inducing effect of diisopropyl fluorophosphate (DFP) has been investigated in rats and in mice. The magnitude of the observed decreases with DFP were similar to those seen in response to MD1 exposure in the present study. In rats, intramuscular injection of 1.0 mg/kg DFP produced a maximum decrease of 3.2”C at 6 hr and slow return to baseline over the next 15 hr (Kozar et al., 1976). Inhalation of DFP for 5 min in mice produced a maximum core temperature decrease of 3.5”C that occurred 30 min after exposure and returned to baseline by 10 hr (Scimeca et al., 1985). MD1 inhalation produced hypothermia of similar onset and degree to that following inhalation of DFP. However, recovery from MD1 hypothermia was more rapid and led to mild hyperthermia postexposure. Chemically induced hyperthermia has been more extensively observed and the mechanisms underlying febrile responses, in general, have been under investigation for more than 40 years. Atkins (1960) showed that a group of compounds, now known as
BY TELEMETRY
407
interleukin 1 (IL-l), was responsible for the fever that developed in response to exogenous pyrogens. Currently it is believed that for an animal to display a febrile response, the reticuloendothelial system must be capable of activation to release IL- 1, the thermoregulatory center of the hypothalamus must be intact, and the vascular and skeletal systems must be able to generate heat to obtain an elevated set point temperature (Dinarello, 1984). In the current study, inhalation of endotoxin induced a fever maximal at 4.5 to 5 hr. Temperature telemetry revealed recovery beginning before the end of exposure (Figs. 5 and 6). Inhalation of endotoxin also induced changes in pulmonary function marked by increased breathing frequency during exposure (Fig. 6, upper curve) and reduced minute volume (Table 1). Tocco-Bradley et al. (1985) reported a temperature response in rats to intravenous injection of endotoxin. Similar to our findings in guinea pigs, rats demonstrated a response beginning 2 hr after endotoxin injection that reached a. maximum increase of 1.7”C at 4.5 hr. In summary, we have described the use of a temperature telemetry system which is sensitive to both hypothermic and hyperthermic responses of guinea pigs which can occur during inhalation of airborne chemicals. The system consists of a microcomputer, an A to D board, AM receivers, temperature transmitters, and a water bath for transmitter calibration. It is sensitive to temperature changes of 0.1 “C. When using this system, there is no interruption of exposure or of monitoring other physiological measurements (i.e., respiratory frequency and volume). For these reasons, the system can be readily incorporated into existing experimental protocols for inhalation toxicology. ACKNOWLEDGMENTS We thank the following individuals for their contributions to this work: Dr. Robert Sandridge, Mobay Chemical Corp., for consultation regarding MD1 generation
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THORNE,
YESK E, AND
and analysis; Meredith Karol, Andrea Shelton, and Barbara Stephens for help with transmitter calibrations; Dr. Adeniyi Ogundiran and Carolyn Catty for assistance with endotoxin exposures; and Julie Hillebrand for help with MD1 experiments. This work was supported by NIEHS
ES01 532.
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