Life Sciences Vol. 17, pp . Printed in the U.S .A .
Pergamon Press
901-906
THERMOREGULATORY RESPONSES OF RESTRAINED VERSUS UNRESTRAINED RABBITSI G. N . McEwen, Jr . Ames Research Center, NASA, Moffett Field,
California 94035
(Received in final form Auqust 11, 1975) S~ary Two male and one female New Zealand white rabbits were used in this study. At ambient temperatures of 20, 10, and 0° C, the animals were either lightly restrained with a Plexiglas collar or were unre etrained . Heat balance was zero during these experiments, as indicated by a stable rectal temperature . Heat losses due to vasomotor state and respiratory evaporative water loss were not significantly different between the restrained and unrestrained animals, whereas metabolism and heart rate were sig nificantly higher in the restrained animals . Inappropriate posture, which is caused by the restraint, may be responsible for an increased energy expenditure at low ambient temperatures of as much as 32â of the resting heat production . The thermoregulatory system is currently being used as a tool to study central nervous control of physiological processes . The importance of central nervous control versus peripheral control, open loop gain, has been studied by changing the temperature of CNS areas at different ambient temperatures in dogs (1-4), cats (5, 6), and rabbits (7-11) . Effects of localized perfusion of inorganic and organic substances into the CNS have been studied in primates (12), and hibernators (13, 14) . Further, single unit recordings have been reported in response to CNS temperature changes in rabbits (15) . Many studies of thermoregulation have used restrained animals, apparently assuming that the various methods of restraint have little effect on thermoregulatory responses . We have previously stated that postural changes may be a major thermoregulatory response (10, 11) . Restraint, therefore, may lead to a biased determination of thermal response . In order to investigate the effects of restraint on thermoregulatory responses, metabolism, breathing rate, heart rate, and several body temperatures have been measured in rabbits which were restrained during some experiments and unrestrained during others . It has been determined that restraint has a significant effect on thermoregulation, due to interference with appropriate postural responses .
1This work was completed at Ames Research Center, NASA, under an NRC Postdoctoral fellowship, and supported in part by NSF Grant GB-13797 . 901
90 2
Restrained Va . Unrestrained Rabbits
Vol . 17, No . 6
Materials and Methods Two male and one female New Zealand white rabbits, Oryctolagus cuniculus , 3 .93-4.84 kg, were used in this study. Maintenance of animals, apparatus for measurement of thermoregulatory responses, and calculations of heat loss and heat production have been previously reported (10, 11) . The restraining device consisted of a Plexiglas collar, with an inside diameter of 7 .8 cm and an outside diameter of 11 .0 cm, supported on a Plexiglas pillar 5 .0 cm above a flat Plexiglas plate . The rabbit sat on the plate with its head through the collar . The collar was loose enough so that the animal could pull its head out . The rabbits accepted the restraining device and sat quietly in the chamber during the experiments . Each animal was trained to the restraining device a minimum of three 2-hr periods before data were collected . Data reported are based on (or derived from) a minimum of 25 experiments at ambient temperatures of 0, 10, and 20 ° C. Each experiment consisted of an acclimation period in the teat chamber to permit metabolism to reach its resting level, as indicated by stable metabolism, rectal temperature, and ear temperature, followed by a 30-min period of data collection . Only experiments which met the criteria stated above for resting metabolism are reported . Each animal was exposed to only one ambient temperature and one experimental period while either restrained or unrestrained on any given day. Mean values for the thermoregulatory responses were taken for each animal while restrained and unrestrained at each ambient temperature . These values were then averaged, the standard deviation taken with an N of 3, and reported in the results . The _t-test for paired samples was used to test for significant differences, and P < 0 .01 was considered significant . The heat loss coefficient (K) is calculated as the elope of increasing metabolism, on ambient temperature below the thermal neutral zone . This is the increase in metabolism necessary to balance an increase in heat lose in order to maintain a stable core temperature below the thermal neutral zone . This regression extrapolates to zero metabolism when core temperature equals ambient temperature . Taking advantage of this fact, only two points are necessary to determine _K ; one, the metabolism at some point below the thermal neutral zone, and two, zero metabolism at an ambient temperature equaling the core temperature . Weight changes in an animal can cause a small change in the absolute determination of _K, since _R is a measure of the total animal's response to its environment . A correction factor for this change in _K was obtained by repeated heat production experiments on the same rabbits under the same experimental conditions over a period of one year as their weights changed . _R may be corrected for weight changes of an individual rabbit by multiplying the weight change by 0 .07 W" ° 1 " kg and adding this value to K.
C
Results Heat conservation by vaeomotion in the ear pinnae, indicated by ear temperature, and respiratory evaporative heat lose, indicated by breathing rate, were not significantly different between the restrained and unrestrained animals (Table I) . Back akin temperature was significantly different at 20 and 10 ° C, between restrained and unrestrained animals, but this difference was slight in terms of total heat balance . Mean oxygen pulse (cm3 02 consumed heart beat-1 ) was not significantly different in the restrained versus unrestrained experiments, suggesting no change in heart stroke volume assuming a constant A-V P0 2 difference . Ear temperature decreased with ambient temperature under both conditions, indicating an increase in heat conservation by vasomotor changes in the ear pinnae . Metabolism and heart rate were significantly higher with restraint at all ambient temperatures (Table I) . The relative difference in weight-specific metabolism (W "kg-1 ) was greatest at 0° C, 24X higher in the
Vol . 17, No . 6
Restrained Vs . Unrestrained Rabbits
903
iABLB I iharroregulatory Responses to Ambient 1'eaipera[ure of 3 Rabbits While Raatralaed (R) AId Onraetraiaed
Semperature (°C) Amblent
20
10
0
Rectal
R
Far Pinne
Back Skin
(0)
Breathe/min
Hesrt Beats/sin
Weight-Specific Nataboliaa
watts/kg
total Netaboli® (velte /enLel)
39 .1 + .14
33 .5 + 1.96
36 .5 +
,12
63 .3 +
191.3 + 9.1
3.04 + .097
13 .83 +
.868
39 .1 + .09
29 .7 + 1.11
35 .0 +
,29w
fi9 .0 + 19 .3
152.7 + 9,1a
2.fi4 + ,0099+
10 .76 +
,254a
39 .3 + ,24
21 .5 + 1.04
35 .6 +
.34
61 .1 + 16 .1
205.7 + 8.2
3 .17 + .120
14 .34 +
.847
39 .1 + .12
23 .5 +
.67
32 .8 +
.54e
70 .0 +
6 .5
175.7 + 3.6+~
2.67 + .092e
10 .77 +
.4841
B
39 .0 + .31
15 .5 + 1.31
32 .3 +
.31
61 .0 +
5.1
227.3 + 9,0
3.51 + ,146
16 .19 + 1.367
0
39 .3 + .12
16 .6 +
30 .7 + 1.49
69 .0 +
7.8
205.7 + 4.5+
2.84 + ,165~
12 .15 +
R
.68
4.1
.286a
Values are mean valuaa +1 a[asdard dwL[ion, calculated vi[h sa N of 3. ~Bignificsnce at the P < ,O1 lwel by t-teat.
restrained condition. At 20 ° C, weight-specific metabolism was 15X higher in the restrained condition. Total metabolism was approximately 32X higher during restraint at all ambient temperatures in these rabbits . Calculation of the heat loss coefficient (R) for the whole animal gives a _R of 0 .31 ±0 .007 W" ° C -1 for the anitoals while unrestrained, and 0.42 ± 0 .039 W" ° C-1 for the animals while restrained (Fig . 1) . This is an increase in R of approximately 35X . 18
N
m Q H W
6
0
10
20 30 AMBIENT TEMPERATURE, ~C
40
50
FIG. 1 Whole animal metabolism of 3 rabbits while restrained (~~), and unrestrained (o~) . Bars indicate two standard deviations . Heat loss coefficient (K) is indicated for restrained (0 .42) and unrestrained (0 .31) animals . Tre indicates mean rectal temperature .
90 4
Restrained Vs . Unrestrained Rabbits
Vol . 17, No . 6
Discussion These experiments suggest that restraint causes an increase in energy expenditure of approximately 32X in rabbits at the ambient temperatures reported here . This increase might be due to a psychological factor, e .g ., fear . If this were the case, a change in heat balance would be expected during the experiment, and the rectal temperatures would be different in the two experimental states . This was not found to be the case . Further, rectal temperatures did not change significantly during any of the experiments reported, and common modes of heat exchange _i .e ., vasomotion and evaporative water loss, were not significantly different between the two experimental states . The postural change caused by the restraint was therefore implicated as the primary cause of the increased heat production . With more rigorous methods of restraint, the increase in heat production could be even more extreme than reported here . Birkebak _et _al . (16) calculated heat lose for shrews at two postures, one shortened, the other extended, and found a change in magnitude of _K approximately the same as reported here for restrained versus unrestrained rabbits . This indicates that the increase in _K found in these experiments, could be accounted for by a change in posture involving a greater surface-area-to-volume ratio. Some postural adjustments were observed being made by the restrained rabbits in response to lower ambient temperatures . These adjustments were indicated by the fact that the relative difference in mean metabolism between the restrained and unrestrained states changes . At lower temperatures, the relative difference is greater, indicating an increasing importance of posture at lower ambient temperatures (Fig . 1) . Amore rigorous restraint could therefore cause an even greater increase in this relative difference, leading to a higher _R and a higher determination of the lower critical temperature . The _R value reported here for unrestrained rabbits, (0 .31 W" °C-1 ) is slightly higher than 0.29 W" ° C-1 previously reported (10) . This increase is ascribed to an increase in size of the animals . Since the animals were heavier when restrained experiments were conducted, _R was corrected for the increase in weight during these experiments, and a R of 0.40 W" °C-1 was determined . This figure is still significantly higher (P < 0.01), by approximately 32X, than in the unrestrained condition, showing that the differences in weight are not the significant factors . Aa accurate determination of both R and low resting metabolism are not only important to the study of the energy balance of the animal, but are also important in modeling relative importance of central versus peripheral control sys tems . A high _R results in a lower open loop gain (11), which causes a biased determination of the total response of the animal to a thermal stimulus and This bias may increases the apparent importance of peripheral thermal input . also be important when dealing with nonthermoregulatory stimuli, if the responses measured involve heart rate or oxygen consumption, since these values would be abnormally high, and this might affect the elope of their response . Experiments attempting to quantify control systems should be designed so Further, the that major modes of normal response are not denied to the animals. animals should be thoroughly trained to the experimental environment prior to testing .
Vol . 17, No . 6
Restrained Va . Unrestrained Rabbits
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
M. J . FIISCO, J . D. HARDY, and H. T. HAMMEL, _Am. _J . Physiol . _200, 572-580 (1961) . H. T . HAMMEL, _Ann . _Rev . _of Physiol . _30, 641-710 (1968) . H. T . HAMMEL, J. D. HARDY, and M. M. FUSCO, Am . _J . Physiol . _198, 481-486 (1960) . B. HELLSTROM and H. T. HAMMEL, _Aa. _J . Physiol. 213, 547-556 (1967) . T. ADAMS, _J . Appl . Physiol . _18, 772-777 (1963) . F. H . JACOBSON and R. D. SQUIRES, _Am. _J . Physiol . _218, 1575-1582 (1970) . J. A. DOWNEY, R . F. MOTTRAM, and G. W . PICRERING, _J . Physiol . _170, 415-441 (1964) . J. D . GIIIEU and J. D . HARDY, _J . Appl . Physiol. _28, 540-542 (1970) . J. D . HARDY, Fed . Proc . 28, 713 (1969) . G. N . McSWEN, JR . and J. E. HEATH, J . Appl . Physiol. _35, 884-886 (1973) . G. N . McEWEN, JR . and J . E . HEATH, _Am. _J . Physiol. _227, 954-957 (1974) . R. D . MYERS and T. L . YARSA, J. Physiol. 218, 609-633 (1971) . J. L . HANEGAN and W. A. WILLIAMS, Science _181, 663-664 (1973) . R. D . MYERS and J. E . EUCRMAN, Am . J . Physiol. 223, 1313-1318 (1972) . T. NAKAYAMA and J. D . HARDY, _J . Âppl . Physiol. _27, 848-457 (1969) . R. C . BIRREBAR, C. J . CREMEAS, and E . A. LE FEBVRE, _J . Heat Transfer _2, 125-130 (1966) .
905
Pergamon Press
901-906
THERMOREGULATORY RESPONSES OF RESTRAINED VERSUS UNRESTRAINED RABBITSI G. N . McEwen, Jr . Ames Research Center, NASA, Moffett Field,
California 94035
(Received in final form Auqust 11, 1975) S~ary Two male and one female New Zealand white rabbits were used in this study. At ambient temperatures of 20, 10, and 0° C, the animals were either lightly restrained with a Plexiglas collar or were unre etrained . Heat balance was zero during these experiments, as indicated by a stable rectal temperature . Heat losses due to vasomotor state and respiratory evaporative water loss were not significantly different between the restrained and unrestrained animals, whereas metabolism and heart rate were sig nificantly higher in the restrained animals . Inappropriate posture, which is caused by the restraint, may be responsible for an increased energy expenditure at low ambient temperatures of as much as 32â of the resting heat production . The thermoregulatory system is currently being used as a tool to study central nervous control of physiological processes . The importance of central nervous control versus peripheral control, open loop gain, has been studied by changing the temperature of CNS areas at different ambient temperatures in dogs (1-4), cats (5, 6), and rabbits (7-11) . Effects of localized perfusion of inorganic and organic substances into the CNS have been studied in primates (12), and hibernators (13, 14) . Further, single unit recordings have been reported in response to CNS temperature changes in rabbits (15) . Many studies of thermoregulation have used restrained animals, apparently assuming that the various methods of restraint have little effect on thermoregulatory responses . We have previously stated that postural changes may be a major thermoregulatory response (10, 11) . Restraint, therefore, may lead to a biased determination of thermal response . In order to investigate the effects of restraint on thermoregulatory responses, metabolism, breathing rate, heart rate, and several body temperatures have been measured in rabbits which were restrained during some experiments and unrestrained during others . It has been determined that restraint has a significant effect on thermoregulation, due to interference with appropriate postural responses .
1This work was completed at Ames Research Center, NASA, under an NRC Postdoctoral fellowship, and supported in part by NSF Grant GB-13797 . 901
90 2
Restrained Va . Unrestrained Rabbits
Vol . 17, No . 6
Materials and Methods Two male and one female New Zealand white rabbits, Oryctolagus cuniculus , 3 .93-4.84 kg, were used in this study. Maintenance of animals, apparatus for measurement of thermoregulatory responses, and calculations of heat loss and heat production have been previously reported (10, 11) . The restraining device consisted of a Plexiglas collar, with an inside diameter of 7 .8 cm and an outside diameter of 11 .0 cm, supported on a Plexiglas pillar 5 .0 cm above a flat Plexiglas plate . The rabbit sat on the plate with its head through the collar . The collar was loose enough so that the animal could pull its head out . The rabbits accepted the restraining device and sat quietly in the chamber during the experiments . Each animal was trained to the restraining device a minimum of three 2-hr periods before data were collected . Data reported are based on (or derived from) a minimum of 25 experiments at ambient temperatures of 0, 10, and 20 ° C. Each experiment consisted of an acclimation period in the teat chamber to permit metabolism to reach its resting level, as indicated by stable metabolism, rectal temperature, and ear temperature, followed by a 30-min period of data collection . Only experiments which met the criteria stated above for resting metabolism are reported . Each animal was exposed to only one ambient temperature and one experimental period while either restrained or unrestrained on any given day. Mean values for the thermoregulatory responses were taken for each animal while restrained and unrestrained at each ambient temperature . These values were then averaged, the standard deviation taken with an N of 3, and reported in the results . The _t-test for paired samples was used to test for significant differences, and P < 0 .01 was considered significant . The heat loss coefficient (K) is calculated as the elope of increasing metabolism, on ambient temperature below the thermal neutral zone . This is the increase in metabolism necessary to balance an increase in heat lose in order to maintain a stable core temperature below the thermal neutral zone . This regression extrapolates to zero metabolism when core temperature equals ambient temperature . Taking advantage of this fact, only two points are necessary to determine _K ; one, the metabolism at some point below the thermal neutral zone, and two, zero metabolism at an ambient temperature equaling the core temperature . Weight changes in an animal can cause a small change in the absolute determination of _K, since _R is a measure of the total animal's response to its environment . A correction factor for this change in _K was obtained by repeated heat production experiments on the same rabbits under the same experimental conditions over a period of one year as their weights changed . _R may be corrected for weight changes of an individual rabbit by multiplying the weight change by 0 .07 W" ° 1 " kg and adding this value to K.
C
Results Heat conservation by vaeomotion in the ear pinnae, indicated by ear temperature, and respiratory evaporative heat lose, indicated by breathing rate, were not significantly different between the restrained and unrestrained animals (Table I) . Back akin temperature was significantly different at 20 and 10 ° C, between restrained and unrestrained animals, but this difference was slight in terms of total heat balance . Mean oxygen pulse (cm3 02 consumed heart beat-1 ) was not significantly different in the restrained versus unrestrained experiments, suggesting no change in heart stroke volume assuming a constant A-V P0 2 difference . Ear temperature decreased with ambient temperature under both conditions, indicating an increase in heat conservation by vasomotor changes in the ear pinnae . Metabolism and heart rate were significantly higher with restraint at all ambient temperatures (Table I) . The relative difference in weight-specific metabolism (W "kg-1 ) was greatest at 0° C, 24X higher in the
Vol . 17, No . 6
Restrained Vs . Unrestrained Rabbits
903
iABLB I iharroregulatory Responses to Ambient 1'eaipera[ure of 3 Rabbits While Raatralaed (R) AId Onraetraiaed
Semperature (°C) Amblent
20
10
0
Rectal
R
Far Pinne
Back Skin
(0)
Breathe/min
Hesrt Beats/sin
Weight-Specific Nataboliaa
watts/kg
total Netaboli® (velte /enLel)
39 .1 + .14
33 .5 + 1.96
36 .5 +
,12
63 .3 +
191.3 + 9.1
3.04 + .097
13 .83 +
.868
39 .1 + .09
29 .7 + 1.11
35 .0 +
,29w
fi9 .0 + 19 .3
152.7 + 9,1a
2.fi4 + ,0099+
10 .76 +
,254a
39 .3 + ,24
21 .5 + 1.04
35 .6 +
.34
61 .1 + 16 .1
205.7 + 8.2
3 .17 + .120
14 .34 +
.847
39 .1 + .12
23 .5 +
.67
32 .8 +
.54e
70 .0 +
6 .5
175.7 + 3.6+~
2.67 + .092e
10 .77 +
.4841
B
39 .0 + .31
15 .5 + 1.31
32 .3 +
.31
61 .0 +
5.1
227.3 + 9,0
3.51 + ,146
16 .19 + 1.367
0
39 .3 + .12
16 .6 +
30 .7 + 1.49
69 .0 +
7.8
205.7 + 4.5+
2.84 + ,165~
12 .15 +
R
.68
4.1
.286a
Values are mean valuaa +1 a[asdard dwL[ion, calculated vi[h sa N of 3. ~Bignificsnce at the P < ,O1 lwel by t-teat.
restrained condition. At 20 ° C, weight-specific metabolism was 15X higher in the restrained condition. Total metabolism was approximately 32X higher during restraint at all ambient temperatures in these rabbits . Calculation of the heat loss coefficient (R) for the whole animal gives a _R of 0 .31 ±0 .007 W" ° C -1 for the anitoals while unrestrained, and 0.42 ± 0 .039 W" ° C-1 for the animals while restrained (Fig . 1) . This is an increase in R of approximately 35X . 18
N
m Q H W
6
0
10
20 30 AMBIENT TEMPERATURE, ~C
40
50
FIG. 1 Whole animal metabolism of 3 rabbits while restrained (~~), and unrestrained (o~) . Bars indicate two standard deviations . Heat loss coefficient (K) is indicated for restrained (0 .42) and unrestrained (0 .31) animals . Tre indicates mean rectal temperature .
90 4
Restrained Vs . Unrestrained Rabbits
Vol . 17, No . 6
Discussion These experiments suggest that restraint causes an increase in energy expenditure of approximately 32X in rabbits at the ambient temperatures reported here . This increase might be due to a psychological factor, e .g ., fear . If this were the case, a change in heat balance would be expected during the experiment, and the rectal temperatures would be different in the two experimental states . This was not found to be the case . Further, rectal temperatures did not change significantly during any of the experiments reported, and common modes of heat exchange _i .e ., vasomotion and evaporative water loss, were not significantly different between the two experimental states . The postural change caused by the restraint was therefore implicated as the primary cause of the increased heat production . With more rigorous methods of restraint, the increase in heat production could be even more extreme than reported here . Birkebak _et _al . (16) calculated heat lose for shrews at two postures, one shortened, the other extended, and found a change in magnitude of _K approximately the same as reported here for restrained versus unrestrained rabbits . This indicates that the increase in _K found in these experiments, could be accounted for by a change in posture involving a greater surface-area-to-volume ratio. Some postural adjustments were observed being made by the restrained rabbits in response to lower ambient temperatures . These adjustments were indicated by the fact that the relative difference in mean metabolism between the restrained and unrestrained states changes . At lower temperatures, the relative difference is greater, indicating an increasing importance of posture at lower ambient temperatures (Fig . 1) . Amore rigorous restraint could therefore cause an even greater increase in this relative difference, leading to a higher _R and a higher determination of the lower critical temperature . The _R value reported here for unrestrained rabbits, (0 .31 W" °C-1 ) is slightly higher than 0.29 W" ° C-1 previously reported (10) . This increase is ascribed to an increase in size of the animals . Since the animals were heavier when restrained experiments were conducted, _R was corrected for the increase in weight during these experiments, and a R of 0.40 W" °C-1 was determined . This figure is still significantly higher (P < 0.01), by approximately 32X, than in the unrestrained condition, showing that the differences in weight are not the significant factors . Aa accurate determination of both R and low resting metabolism are not only important to the study of the energy balance of the animal, but are also important in modeling relative importance of central versus peripheral control sys tems . A high _R results in a lower open loop gain (11), which causes a biased determination of the total response of the animal to a thermal stimulus and This bias may increases the apparent importance of peripheral thermal input . also be important when dealing with nonthermoregulatory stimuli, if the responses measured involve heart rate or oxygen consumption, since these values would be abnormally high, and this might affect the elope of their response . Experiments attempting to quantify control systems should be designed so Further, the that major modes of normal response are not denied to the animals. animals should be thoroughly trained to the experimental environment prior to testing .
Vol . 17, No . 6
Restrained Va . Unrestrained Rabbits
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 .
M. J . FIISCO, J . D. HARDY, and H. T. HAMMEL, _Am. _J . Physiol . _200, 572-580 (1961) . H. T . HAMMEL, _Ann . _Rev . _of Physiol . _30, 641-710 (1968) . H. T . HAMMEL, J. D. HARDY, and M. M. FUSCO, Am . _J . Physiol . _198, 481-486 (1960) . B. HELLSTROM and H. T. HAMMEL, _Aa. _J . Physiol. 213, 547-556 (1967) . T. ADAMS, _J . Appl . Physiol . _18, 772-777 (1963) . F. H . JACOBSON and R. D. SQUIRES, _Am. _J . Physiol . _218, 1575-1582 (1970) . J. A. DOWNEY, R . F. MOTTRAM, and G. W . PICRERING, _J . Physiol . _170, 415-441 (1964) . J. D . GIIIEU and J. D . HARDY, _J . Appl . Physiol. _28, 540-542 (1970) . J. D . HARDY, Fed . Proc . 28, 713 (1969) . G. N . McSWEN, JR . and J. E. HEATH, J . Appl . Physiol. _35, 884-886 (1973) . G. N . McEWEN, JR . and J . E . HEATH, _Am. _J . Physiol. _227, 954-957 (1974) . R. D . MYERS and T. L . YARSA, J. Physiol. 218, 609-633 (1971) . J. L . HANEGAN and W. A. WILLIAMS, Science _181, 663-664 (1973) . R. D . MYERS and J. E . EUCRMAN, Am . J . Physiol. 223, 1313-1318 (1972) . T. NAKAYAMA and J. D . HARDY, _J . Âppl . Physiol. _27, 848-457 (1969) . R. C . BIRREBAR, C. J . CREMEAS, and E . A. LE FEBVRE, _J . Heat Transfer _2, 125-130 (1966) .
905
