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Effects of induced hypothermia on organ blood flow in a hibernator and a nonhibernator
CRYBIOLOGY
23, 440-446 (1986)
ElIfects of Induced
Hypothermia on Organ Blood Flow in a Hibernator and a Nonhibernator
PER-OVE SJGQUIST, GGRAN DUKER, AND BENGT W. JOHANSSON” Department of Pharmacology and Biochemistry, S-431 83 MBlndal, and *Section of Cardiology,
Hiissle Cardiovascalar Research Laboratories, General Hospital, S-214 01 MalmB, Sweden
Regional blood flow and hemodynamic variables during induced hypothermia were compared in six guinea pigs and four hedgehogs. Tracer microspheres were used for blood flow measurements, since this technique is more accurate than the earlier method (86Rb+ distribution) used for cardiac output distribution measurements in hibernators. Heart rate and blood pressure decreased with reduced temperature in a comparable fashion in the two species, while cardiac output was less affected in the hedgehogs than in the guinea pigs. Total peripheral resistance increased in both species. At 34°C the hedgehogs had a higher myocardial blood flow per gram tissue than the guinea pigs and it was not reduced in the hedgehogs when the body temperature was lowered to 22”C, whereas in the guinea pigs it was markedly reduced. The brown adipose tissue of the hedgehogs showed a fourfold increase in blood perfusion at 22°C when compared with 34°C. In the hedgehogs the fractional distribution of cardiac output to the myocardium increased with decreasing body temperature, while the renal fraction decreased. In the guinea pigs, on the other hand, the fractional distribution of cardiac output to the myocardium remained unchanged but increased to the kidneys. o 1986 Academic press. IIIC.
Entering hibernation involves coordination between metabolic and neurogenic control. The primary result is a decrease in metabolism and heart rate, and a fall in body temperature is secondary (13). Arousal from hibernation initiates cardiovascular adjustments, the circulating blood is concentrated in the forepart of the animal, while the hindpart of the animal is to a great extent deprived of blood (IO). Earlier studies on cardiovascular characteristics in hibernators including regional blood flow measurements during arousal (4, 19) were mostly made with the rubidium technique (18). However, this technique has been questioned (1, 15, 16). Foster and Frydman (6) critically evaluated its reliability while investigating the cardiac output distribution in cold-acclimated rats. They concluded that the uptake of 86Rb+ by a tissue frequently does not provide a valid measurement of the fractional distribution of cardiac output. The method
based on tracer microspheres (8) has been reported to be more accurate (6). With the aim to study whether any blood flow characteristics could contribute to the explanation of why hibernators, in contrast to nonhibernators, do not suffer from fatal cardiac disorders during severe hypothermia (1 l), the microsphere method was used to compare the regional blood flow alterations induced by hypothermia in the hedgehog with those induced in the guinea pk.
Received November 25, 1985; accepted May 21, 1986 440 001 l-2240/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
MATERIAL
AND METHODS
Six male guinea pigs (&via porcellus), weighing between 940 and 1042 g, and four hedgehogs (Erinaceus europaeus), two males and two females weighing between 1200 and 1620 g, were included in this study. The hedgehogs were trappped in the south of Sweden during the spring. Until September all animals were kept in a photoperiod LD 12:12 with an ambient temperature equalling the outdoor temperature. Thereafter, for 4-6 weeks before the day of the experiment, the temperature in the animal room was kept between 18 and 20°C.
HYPOTHERMIA-ORGAN
BLOOD
To reduce excitability in the hedgehogs, they were premeditated with diazepam 0.1 mg/kg (ip). Hedgehogs are difficult to handle and anesthetize safely when they are in an anxious state. Safer and more reproducible anesthesia is produced after medication with diazepam. The half-life of diazepam is so short, l-2 hr in rabbits, guinea pigs, hamsters, rats, and mice (7), that we are certain that no diazepam effect is present in the hedgehogs at the time of the measurements. All animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (guinea pig 30 mg/kg; hedgehog 15 mg/kg). No supplementary anesthesia was needed during the hypothermic state. A tracheotomy was performed and the animals were ventilated mechanically with room air. The respiratory rate and tidal volume were adjusted to keep the measured arterial blood gases close to normal. To avoid over-oxygenization of the blood during hypothermia, the ventilation of the lungs was gradually decreased. The degree of reduction in the respiratory rate during hypothermia was mathematically derived from the arterial blood values obtained at 37°C (blood gas analyzer radiometer ABL 2, Copenhagen) using an experimental Q,,, of 2.0. In the hedgehogs, this method was checked measuring the pH, PO,, and PCO, of the arterial blood using thermostatic electrodes G297-G2, E5046, and E5036, respectively, regulated to the actual temperature of the animals (blood gas analyser radiometer PHM73, Copenhagen). The metabolic acidosis observed in the hedgehogs, but not in the guinea pigs, was counterbalanced throughout the experiment using an iv infusion (0.01 ml/min) of sodium bicarbonate solution (-2.5 mgimin). The body temperature was measured and recorded continuously throughout the experiment, using one thermistor probe in the rectum and another in the esophagus. Polyethylene catheters were placed in a carotid artery for blood pressure measure-
FLOW
IN A HIBERNATOR
441
ments and blood sampling and in a jugular vein for fluid replacement. After thoracotomy a catheter was placed in the left atrium for administration of microspheres. The radioactive microspheres used (8) were labeled with 141Ce,85Sr,W-, and 46Sc and had a diameter of 9 + 1 pm (3 M Co; St Paul, Minn.). They were suspended in physiological saline, with addition of Tween 80 (0.01%). Before injection the microspheres were dispersed mechanically and ultrasonically. About two million microspheres (1.5 ml saline) of each label were injected in the left atrium over a period of 30-45 sec. During the microsphere infusion a reference sample of blood was withdrawn from the carotid artery at a constant rate (0.55 ml/min). For each isotope the total amount of radioactivity injected was used for the cardiac output measurements (20). Organ blood flow measurements by means of the differently labeled microspheres were performed while cooling the animals in an ice-bath. The organ blood flow in the guinea pigs was measured at rectal and esophageal temperatures of 37, 34, 26, and 22°C (within a range of I’C). Asystole appeared at a body temperature of around 20°C. The blood flow in the hedgehogs was measured at body temperatures of 34, 26, 22, and 15°C (within a range of 1’C). At the end of the experiments the animals were killed, and organs and tissues of interest were dissected free. The wet tissues were cut into suitably sized pieces and blotted on paper and weighed. Together with the reference samples of blood, the tissues were counted for radioactivity in a y-counter (Packard Autogamma Scintillation spectrometer). The y-counter was connected to a computer, which performed the standard calculations, background subtraction, and correction for nuclide interaction. The blood flow to each tissue sample was calculated as ml*minl* 100 g-r tissue. Since the size of the organs differed between the two species studied, and samples of different
442
SJijQLJIST,
DLJKER,
AND JOHANSSON
size were obtained from some tissues, the fractional distribution of cardiac output to the selected organs was calculated in relation to the weight of tissue divided by the total weight of the animal (ml * min-l * 100 g - * organ weight/ml * min- l * 100 g-l body wt). In the text and tables, means are given together with their standard errors. The statistical significance of the difference in results obtained at the same body temperature in the two groups was assessed by the Mann-Whitney U-test.
cially in the interval during which body temperature has decreased from 34 to 22°C. Peripheral vascular resistance increased with decreasing body temperatures in both species. There were no measurable differences between rectal and esophageal temperatures during the normothermic or the hypothermic situation in either species. The organ blood flows during hypothermia are shown in Table 2. At 34°C the hedgehogs had a higher blood flow in the myocardium than the guinea pigs. Furthermore, the myocardial blood flow was not reduced in the hedgehogs when the body RESULTS temperature was lowered to 22°C. In the At the start of the experiments the body guinea pigs, myocardial blood flow at 26 temperature was 37°C in the guinea pigs and 22°C was reduced to about 50% when and 35°C in the hedgehogs. The hemody- compared to that at body temperatures of namic effects of hypothermia are presented 34°C. The brown fat of the hedgehogs in Table 1. From initially similar values ob- showed a fourfold increase in blood perfutained close to the normal body tempera- sion when the body temperature was retures (34°C for the hedgehogs and 37°C for duced from 34°C to 22°C. No further inthe guinea pigs), the heart rate decreased crease in blood flow was observed at 15°C. The blood flow of the small intestine was with reduced temperature in a comparable fashion in the two species studied. In con- lower in the hedgehogs than in the guinea interval trast, cardiac output and the calculated pigs within the temperature stroke volume were less affected in the studied. However, blood perfusion of the hedgehogs than in the guinea pigs, espe- large intestine was similar in the two
Hemodynamics
TABLE I of Hedgehogs and Guinea Pigs at Different
Body Temperatures
Hemodynamics Guinea pigs
Hedgehogs 26°C
22°C
15°C
37°C
34°C
26°C
22°C
13*
226 AZ17**
130 t 13*
62 -c 12
253 5 23
209 5 14
130 c 10
98 f 5
13
100 ? 15
82 e 4*
52 k I
50 + 3
65 2 4
6023
45 k 5
21
82 -t 5*
60 2 4
31 k 4
85 2 12
92 k 14
42 + 10
31 -c 4
34°C Heart rate (beats/mitt) 265 + Mean arterial blood pressure (mm Hd 86 k Cardiac output (ml/mm) 103 2 Stroke volume (ml) 0.38 t Peripheral vascular resistance (units) 96 +
0.07 0.36 k 0.05 0.46 e 0.05 0.55 e O.iO 0.34 r 0.06 0.48 r 0.10 0.33 -c 0.08 0.30 * 0.08
22
136 + 32
160 k 18
178 2 33
Note. Values given are the means ? SE. Hedgehogs, n = 4; guinea pig value. * P < 0.05.
** P < 0.01, Mann-Whitney
U test.
guinea
64 * 12
81 e 17
217 k 81
200 If- 47
pigs, n = 6. Significantly different from corresponding
HYPOTHERMIA-ORGAN
443
BLOOD FLOW IN A HIBERNATOR
TABLE 2 Organ Blood Flows in Hedgehogs and Guinea Pigs at Different Body Temperatures Blood flow (ml. min-’ . 100 g-l) Guinea pigs
Hedgehogs 34°C
26°C
22°C
15°C
37°C
34°C
26°C
22°C
38 -c 3 124 + 9 176 t 16* 271 IT 72* 203 f 14 100 f 17 12 + 5 11 + 2 23 f 4* 30 + 3* 35 c 2 5+2 4*1 4il 221
30 c 6 140 2 12* 230 e 32* 55 -c 15 153 -’ 24 120 ?I 45 26 t 10 14 2 2 25 -t 7* 29 t 6* 42 2 10 2 -t 1* 12 1 2*1 15 1
14 + 5 136 t 58* 198 + 29* 89 + 13 70 + 12 47 k 18 17 k 8 12 * 2 17 f 6* 14 5 3* 30 i 10 3t1 2+1 221 121
522 112 -c 20 121 t 22 32 k 7 37?17 16 k 6 721 6-cl 11 it_ 6 62 1 21 + 7 2+1 I?1 l?l 121
25 + 4 54 2 10 56 ? 8 63 + 11 160~30 50 k 4 2&l 37 ‘- 5 91 2 17 81 + 9 56 + 15 11 + 3 422 5+2 2+1 1+ I -
20 k 6 57 f 10 83 e 20 85 f 31 210260 66 + 16 2+1 46 + 8 75 2 7 72 f 10 45 i 6 13 % 4 421 13 + 5 l?l 221 -
8~2 32 + 7 41 e 13 52 it_ 29 116+32 22 + 6 lil 25 k 7 44 + 10 35 -t 2 23 + 5 823 121 l-+1 1+1 l-t1
10 5 2 38 -c 12 41 + 3 34 2 17 90 + 20 21 + 3 151 13 -c 3 45 5 12 24 2 5 17 F 5 6-t2 121 il * 1 l-cl 121 -
Organ Brain Right ventricle Left ventricle Lungs Kidney Adrenal gland Liver Stomach Duodenum Small intestine Large intestine Urinary bladder Muscle: hind leg psoas Skin White fat Brown fat
N&e. Values given are the means -c SE. Hedgehogs, n = 4; guinea pigs, PI = 6. Significantly different from corresponding guinea pig value. * P < 0.05, Mann-Whitney U test.
species. In both species the intestinal blood flow decreased with reduced body temperature. Figure 1 shows the fractional distribution of cardiac output (ml . min-’ * 100 g-l organ weight/ml . min-’ * 100 g-i body weight) to the left ventricle of the heart, the LEFT Fraction cardia
kidneys and the duodenum. In the hedgehogs, the fractional distribution of cardiac output to the myocardium increased with decreasing body temperature, while the renal fraction of the cardiac output decreased and the duodenal fraction was unchanged when the body temperature
VENTRICLE
DUODENUM
KIDNEY
of
60
60
45
45
30
30
15
15
0 I
1
35
I
1
30
25
1
20
I
I
15
OC
I
I
I
I
I
35
30
25
20
15
oc
35
io
is
2b
1’5 oc
FIG. 1. Fraction of cardiac output distributed to the organs at different body temperatures calcu-
lated as ml * mini . 100-i organ weight/ml. min-* . 100-i bod y wt. (0) Hedgehogs (n = 4), (0) guinea pigs (n = 6). Statistical significance between the two groups was assessed using the MannWhitney U test. *P < 0.05. ***p < 0.001.
444
SJijQUIST,
DUKER, AND JOHANSSON
was lowered. In contrast, in the guinea temperatures in the same animal (8); this pigs, the fractional distribution of cardiac experimental design was not possible with output to the myocardium was unchanged, the rubidium technique. However, the miwhile it increased to the kidneys and duo- crosphere technique has methodological denum. There was a significant increase in limitations. For instance, in an organ blood the brown fat fraction of cardiac output in flow encompassing both capillary flow, the hedgehogs, from 14 & 3 at 34°C to 74 which will trap the microspheres, and flow t 10 at 22°C (P < 0.02 Student’s t test for through A-V shunts, which will not trap paired observations). the microspheres, only the capillary fraction of the blood flow will be measurable. A validation and comparison between the rubidium and microsphere methods has DISCUSSION been made in cold-acclimated rats (6). The In the present study, we have used the validity of the microsphere technique was tracer microsphere technique to study the reasonable according to Foster and regional blood flow effects of induced hy- Frydman (6), whereas 86Rb+ measurepothermia in anesthetized hedgehogs and ments, in addition to fractional distribution guinea pigs. Earlier, cardiac output distri- of CO, could be influenced by resistances bution in hibernators was preferably of the capillary walls, cell membrane transstudied during arousal using the rubidium port of 8aRb+, and differences in the exmethod (4, 9, 17). It was supposed that the traction ratio for 86Rb+ between tissues. It distribution, and subsequently the organ must be concluded from a comparison of uptake, of 86Rb+ followed the distribution the advantages and the limitations of the of blood flow (18). The passage of rubidium microsphere method and the rubidium from the blood to the tissues is a question method that values of regional blood flows of establishing an equilibrium, which is a obtained with the two techniques should be rather slow diffusion process. This means compared with great care. that tissues through which blood passes relThe results of the present study show atively slowly will show a higher rubidium that under anesthetized conditions the efuptake than those where blood passes fects of acutely induced hypothermia on through quickly. The advent of labeled mi- regional blood flow distribution are compacrospheres has provided an alternative rable between the hibernator (hedgehog) method for measuring regional blood flow. and the nonhibernator (guinea pig). For The nuclide-labeled microspheres are of some organs such as the heart, kidneys, such a size (in the present study 9 pm) that and duodenum, cardiac output distribution during hypothermia was found to be estithey penetrate into the precapillary vessels, where they are trapped. This mated as significantly different between the causes embolism of a small fraction of the guinea pig and the hedgehog, with a higher vessels without increasing peripheral resis- proportion going to the heart at all tempertance, and the distribution of the blood atures in the hibernator, the reverse being flow can then be estimated (8). In this case, found for the kidney and duodenum. It is no diffusion is needed, and organs having a possible that more dramatic differences in high blood flow may be measured without the cardiac output distribution between the this systematical error. Another advantage species would have been revealed if the anof using tracer microspheres is that dif- imals had not been anesthetized. It cannot ferent nuclides can be used for labeling the be excluded that the anesthetic was able to spheres. This means that the regional superimpose hypothermically induced reblood flow can be determined at different flexes in either the guinea pig or the
HYPOTHERMIA-ORGAN
BLOOD
hedgehog or both. Sodium pentobarbital, a widely used anesthetic in experimental cardiovascular investigations, was used in the present study. We have not found any studies on how this barbiturate influences the cardiovascular response to hypothermia, but it is known that during normothermia pentobarbital affects systemic and regional hemodynamics only slightly, but depresses the myocardium markedly (14). It has been suggested that the tachycardia associated with pentobarbital anesthesia depends on a vagolytic effect and/or is mediated through the baroreceptor reflex (14). The regional blood flow distributions in the hedgehog during the acutely induced hypothermia show a different pattern from those reported earlier for other hibernating species in a comparable degree of hypothermia during natural arousal from hibernation. During arousal from hibernation, organs in the forepart of the hibernator are highly blood perfused, while in the hindpart of the body the flow is very restricted (4,9, 17). In the present study of induced hypothermia, any redistribution of blood flow between different parts of the body is much less pronounced than during arousal. We suggest that, in this respect, induced hypothermia seems to be similar to natural entrance into hibernation. In support of this theory are the results obtained by Lyman and O’Brien (12) in their study of the thirteen-lined ground squirrel. They found that the temperature in the heart region and abdomen was the same in the animals entering hibernation, while during arousal there was a pronounced difference between temperature values obtained from the esophagus or rectum at any body temperature studied. Difficulties in predicting natural entrances and problems with measuring regional blood flow at certain time intervals during the entrance into hibernation without disturbing the animals may be some of the reasons why so few attempts
FLOW
IN A HIBERNATOR
445
have been made to study the cardiac output distribution during the entrance period of the hibernating cycle. Soivio (19) was able to determine the distribution of blood, in a small number of animals, during entrance into hibernation using the rubidium technique. His results pointed to an even distribution with no specific areas of vasoconstriction. The changes in blood pressure and peripheral vascular resistance during induced hypothermia in the hedgehogs are similar to those recorded in the ground squirrel during entrance into hibernation. In the ground squirrel, blood pressure first remains within the range of the active animal, then decreases. The peripheral resistance increases continuously as the body temperature falls (12). The increase in peripheral vascular resistance could partly be explained by the effect of cold on the blood viscosity and on the elasticity of the vascular wall. However, it has also been suggested that there is an increase in vascular tone (13). Hibernators are characterized by their ability to tolerate low body temperatures, and they have good control of the physiological mechanisms at low temperatures. Included in this control is tolerance to cardiac arrhythmias, especially ventricular fibrillation, when entering and during arousal from hibernation and when subjected to experimentally induced myocardial ischemia (11). Various mechanisms have been proposed to account for this resistance to ventricular fibrillation (5). In the present study, the higher myocardial blood flow measured in the hedgehog compared with the guinea pig at all temperatures during induced hypothermia may be one contributing factor explaining the differences in resistance to cardiac arrhythmias between hibernators and nonhibernators. It must be stressed, however, that the present findings were obtained under anesthetic conditions, and that extrapolation of the results to conscious unrestrained animals living in their
446
SJ6QUIST, DUKER, AND JOHANSSON
natural habitat should be done with great caution. ACKNOWLEDGMENTS We thank Ms. G. Thylander for revising the English text and Ms. R. Jorgensen for secretarial support. REFERENCES 1. Alexander, G., Bell, A. W., and Hales, J. R. S. Effects of cold exposure on tissue blood flow in the newborn lamb. J. Physiol. (London) 234, 65-77 (1973). 2. Budden, R., Detweiler, D. K., and Zbinden, G. (Eds.) “The Rat Electrocardiogram in Pharmacology and Toxicology.” Pergamon, Elmsford, N.Y., 1981. 3. Bullard, R. W. Cardiac output of the hypothermic rat. Amer. J. Physiol. 196, 415-419 (1959). 4. Bullard, R. W., and Funkhouser, G. E. Estimated regional blood flow by rubidium 86 distribution during arousal from hibernation. Amer. J. Physiol. 203, 266-270 (1962). 5. Duker, G., Olsson, S. -O., Hecht, N. H., Senturia, J. B., and Johansson, B. W. Ventricular fibrillation in hibernators and nonhibernators. Cryobiology
20, 407-420 (1983).
6. Foster, 0. E, and Frydman, M. L. Comparison of microspheres and %Rb+ as tracers of the distribution of cardiac output in rats indicates invalidity of 86Rb-based measurements. Cunad. J. Physiol. Pharmacol. 56, 97-109 (1978). 7. Green, C. J. “Animal Anesthesia.” Laboratory Animal Handbooks 8, Spottiswoode Ballantyne Ltd., Colchester and London, 1979. 8. Heymann, M. A., Bruce, D. P., Hoffman, J. I. E., and Rudholph, A. M. Blood flow measurements with radionuclide-labeled particles. Prog. Cardiovasc. Dis. 20, 55-79 (1977). 9. Johansen, K., Distribution of blood in the arousing hibernator. Acta Physiol. Stand. 52, 379-386 (1961). 10. Johansson, B. W. Heart and circulation in hibernators. In “Mammalian Hibernation III.”
(R. C. Fisher, A. R. Dawe, L. C. Lyman, E. Schonbaum, and F. E. South, Eds.), pp. 199-218. New York, Elsevier, 1967. 11. Johansson, B. W. Cardiac responses in relation to heart size. Cryobiology 21, 627-636 (1984). 12. Lyman, C. P., and O’Brien, R. C. Circulatory changes in the thirteen-line ground squirrel during the hibernating cycle. Mus. Camp. 2001. Harvard Coil. 124, 353-372 (1960). 13. Lyman, C. P., Willis, J. S., Malan, A., and Wang, L. C. H. “Hibernation and Torpor in Mammals and Birds.” Academic Press, New York, 1982. 14. Manders, T. W., and Vatner, S. F. Effects of sodium pentobarbital anesthesia on left ventricular function and distribution of cardiac output in dogs, with particular reference to the mechanism for tachycardia. Circ. Res. 39, 512-517 (1976). 15. Mendel, P. L., and Hollenberg, N. K. Cardiac output distribution in the rat: Comparison of rubidium and microsphere methods. Amer. J. Physiol. 221, 1617-1620 (1971). 16. Rakusan, K., and Blahitka, J. Cardiac output distribution in rats measured by injection of radioactive microspheres via cardiac puncture. Canad.
J. Physiol.
Pharmacol.
52, 230-235
(1974). 17. Rauch, J. C. Sequential changes in regional distribution of blood in Eptesicus fuscus (big brown bat) during arousal from hibernation. Canad. J. Zool. 51, 973-981 (1973). 18. Sapirstein, L. A. Regional blood flow by fractional distribution of indicators. Amer. J. Physiol. 193, 161-168 (1958). 19. Soivio, A. Hibernation in the Hedgehog (Erinaceus europaeus L.): The Distribution of Blood, the Size of the Spleen, and the Hematocrit and Hemoglobin Values during the Annual and Hibernating Cycles. Annales Academiae Scientiarium Fennicae, Suomalainen Tiedeakatemia, Helsinki, 1967. 20. Towell, M. E., and Liggins, G. C. The effect of labour on uterine blood flow in the pregnant ewe. Q. J. Exp. Physiol. 61, 23-33 (1976).
23, 440-446 (1986)
ElIfects of Induced
Hypothermia on Organ Blood Flow in a Hibernator and a Nonhibernator
PER-OVE SJGQUIST, GGRAN DUKER, AND BENGT W. JOHANSSON” Department of Pharmacology and Biochemistry, S-431 83 MBlndal, and *Section of Cardiology,
Hiissle Cardiovascalar Research Laboratories, General Hospital, S-214 01 MalmB, Sweden
Regional blood flow and hemodynamic variables during induced hypothermia were compared in six guinea pigs and four hedgehogs. Tracer microspheres were used for blood flow measurements, since this technique is more accurate than the earlier method (86Rb+ distribution) used for cardiac output distribution measurements in hibernators. Heart rate and blood pressure decreased with reduced temperature in a comparable fashion in the two species, while cardiac output was less affected in the hedgehogs than in the guinea pigs. Total peripheral resistance increased in both species. At 34°C the hedgehogs had a higher myocardial blood flow per gram tissue than the guinea pigs and it was not reduced in the hedgehogs when the body temperature was lowered to 22”C, whereas in the guinea pigs it was markedly reduced. The brown adipose tissue of the hedgehogs showed a fourfold increase in blood perfusion at 22°C when compared with 34°C. In the hedgehogs the fractional distribution of cardiac output to the myocardium increased with decreasing body temperature, while the renal fraction decreased. In the guinea pigs, on the other hand, the fractional distribution of cardiac output to the myocardium remained unchanged but increased to the kidneys. o 1986 Academic press. IIIC.
Entering hibernation involves coordination between metabolic and neurogenic control. The primary result is a decrease in metabolism and heart rate, and a fall in body temperature is secondary (13). Arousal from hibernation initiates cardiovascular adjustments, the circulating blood is concentrated in the forepart of the animal, while the hindpart of the animal is to a great extent deprived of blood (IO). Earlier studies on cardiovascular characteristics in hibernators including regional blood flow measurements during arousal (4, 19) were mostly made with the rubidium technique (18). However, this technique has been questioned (1, 15, 16). Foster and Frydman (6) critically evaluated its reliability while investigating the cardiac output distribution in cold-acclimated rats. They concluded that the uptake of 86Rb+ by a tissue frequently does not provide a valid measurement of the fractional distribution of cardiac output. The method
based on tracer microspheres (8) has been reported to be more accurate (6). With the aim to study whether any blood flow characteristics could contribute to the explanation of why hibernators, in contrast to nonhibernators, do not suffer from fatal cardiac disorders during severe hypothermia (1 l), the microsphere method was used to compare the regional blood flow alterations induced by hypothermia in the hedgehog with those induced in the guinea pk.
Received November 25, 1985; accepted May 21, 1986 440 001 l-2240/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
MATERIAL
AND METHODS
Six male guinea pigs (&via porcellus), weighing between 940 and 1042 g, and four hedgehogs (Erinaceus europaeus), two males and two females weighing between 1200 and 1620 g, were included in this study. The hedgehogs were trappped in the south of Sweden during the spring. Until September all animals were kept in a photoperiod LD 12:12 with an ambient temperature equalling the outdoor temperature. Thereafter, for 4-6 weeks before the day of the experiment, the temperature in the animal room was kept between 18 and 20°C.
HYPOTHERMIA-ORGAN
BLOOD
To reduce excitability in the hedgehogs, they were premeditated with diazepam 0.1 mg/kg (ip). Hedgehogs are difficult to handle and anesthetize safely when they are in an anxious state. Safer and more reproducible anesthesia is produced after medication with diazepam. The half-life of diazepam is so short, l-2 hr in rabbits, guinea pigs, hamsters, rats, and mice (7), that we are certain that no diazepam effect is present in the hedgehogs at the time of the measurements. All animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (guinea pig 30 mg/kg; hedgehog 15 mg/kg). No supplementary anesthesia was needed during the hypothermic state. A tracheotomy was performed and the animals were ventilated mechanically with room air. The respiratory rate and tidal volume were adjusted to keep the measured arterial blood gases close to normal. To avoid over-oxygenization of the blood during hypothermia, the ventilation of the lungs was gradually decreased. The degree of reduction in the respiratory rate during hypothermia was mathematically derived from the arterial blood values obtained at 37°C (blood gas analyzer radiometer ABL 2, Copenhagen) using an experimental Q,,, of 2.0. In the hedgehogs, this method was checked measuring the pH, PO,, and PCO, of the arterial blood using thermostatic electrodes G297-G2, E5046, and E5036, respectively, regulated to the actual temperature of the animals (blood gas analyser radiometer PHM73, Copenhagen). The metabolic acidosis observed in the hedgehogs, but not in the guinea pigs, was counterbalanced throughout the experiment using an iv infusion (0.01 ml/min) of sodium bicarbonate solution (-2.5 mgimin). The body temperature was measured and recorded continuously throughout the experiment, using one thermistor probe in the rectum and another in the esophagus. Polyethylene catheters were placed in a carotid artery for blood pressure measure-
FLOW
IN A HIBERNATOR
441
ments and blood sampling and in a jugular vein for fluid replacement. After thoracotomy a catheter was placed in the left atrium for administration of microspheres. The radioactive microspheres used (8) were labeled with 141Ce,85Sr,W-, and 46Sc and had a diameter of 9 + 1 pm (3 M Co; St Paul, Minn.). They were suspended in physiological saline, with addition of Tween 80 (0.01%). Before injection the microspheres were dispersed mechanically and ultrasonically. About two million microspheres (1.5 ml saline) of each label were injected in the left atrium over a period of 30-45 sec. During the microsphere infusion a reference sample of blood was withdrawn from the carotid artery at a constant rate (0.55 ml/min). For each isotope the total amount of radioactivity injected was used for the cardiac output measurements (20). Organ blood flow measurements by means of the differently labeled microspheres were performed while cooling the animals in an ice-bath. The organ blood flow in the guinea pigs was measured at rectal and esophageal temperatures of 37, 34, 26, and 22°C (within a range of I’C). Asystole appeared at a body temperature of around 20°C. The blood flow in the hedgehogs was measured at body temperatures of 34, 26, 22, and 15°C (within a range of 1’C). At the end of the experiments the animals were killed, and organs and tissues of interest were dissected free. The wet tissues were cut into suitably sized pieces and blotted on paper and weighed. Together with the reference samples of blood, the tissues were counted for radioactivity in a y-counter (Packard Autogamma Scintillation spectrometer). The y-counter was connected to a computer, which performed the standard calculations, background subtraction, and correction for nuclide interaction. The blood flow to each tissue sample was calculated as ml*minl* 100 g-r tissue. Since the size of the organs differed between the two species studied, and samples of different
442
SJijQLJIST,
DLJKER,
AND JOHANSSON
size were obtained from some tissues, the fractional distribution of cardiac output to the selected organs was calculated in relation to the weight of tissue divided by the total weight of the animal (ml * min-l * 100 g - * organ weight/ml * min- l * 100 g-l body wt). In the text and tables, means are given together with their standard errors. The statistical significance of the difference in results obtained at the same body temperature in the two groups was assessed by the Mann-Whitney U-test.
cially in the interval during which body temperature has decreased from 34 to 22°C. Peripheral vascular resistance increased with decreasing body temperatures in both species. There were no measurable differences between rectal and esophageal temperatures during the normothermic or the hypothermic situation in either species. The organ blood flows during hypothermia are shown in Table 2. At 34°C the hedgehogs had a higher blood flow in the myocardium than the guinea pigs. Furthermore, the myocardial blood flow was not reduced in the hedgehogs when the body RESULTS temperature was lowered to 22°C. In the At the start of the experiments the body guinea pigs, myocardial blood flow at 26 temperature was 37°C in the guinea pigs and 22°C was reduced to about 50% when and 35°C in the hedgehogs. The hemody- compared to that at body temperatures of namic effects of hypothermia are presented 34°C. The brown fat of the hedgehogs in Table 1. From initially similar values ob- showed a fourfold increase in blood perfutained close to the normal body tempera- sion when the body temperature was retures (34°C for the hedgehogs and 37°C for duced from 34°C to 22°C. No further inthe guinea pigs), the heart rate decreased crease in blood flow was observed at 15°C. The blood flow of the small intestine was with reduced temperature in a comparable fashion in the two species studied. In con- lower in the hedgehogs than in the guinea interval trast, cardiac output and the calculated pigs within the temperature stroke volume were less affected in the studied. However, blood perfusion of the hedgehogs than in the guinea pigs, espe- large intestine was similar in the two
Hemodynamics
TABLE I of Hedgehogs and Guinea Pigs at Different
Body Temperatures
Hemodynamics Guinea pigs
Hedgehogs 26°C
22°C
15°C
37°C
34°C
26°C
22°C
13*
226 AZ17**
130 t 13*
62 -c 12
253 5 23
209 5 14
130 c 10
98 f 5
13
100 ? 15
82 e 4*
52 k I
50 + 3
65 2 4
6023
45 k 5
21
82 -t 5*
60 2 4
31 k 4
85 2 12
92 k 14
42 + 10
31 -c 4
34°C Heart rate (beats/mitt) 265 + Mean arterial blood pressure (mm Hd 86 k Cardiac output (ml/mm) 103 2 Stroke volume (ml) 0.38 t Peripheral vascular resistance (units) 96 +
0.07 0.36 k 0.05 0.46 e 0.05 0.55 e O.iO 0.34 r 0.06 0.48 r 0.10 0.33 -c 0.08 0.30 * 0.08
22
136 + 32
160 k 18
178 2 33
Note. Values given are the means ? SE. Hedgehogs, n = 4; guinea pig value. * P < 0.05.
** P < 0.01, Mann-Whitney
U test.
guinea
64 * 12
81 e 17
217 k 81
200 If- 47
pigs, n = 6. Significantly different from corresponding
HYPOTHERMIA-ORGAN
443
BLOOD FLOW IN A HIBERNATOR
TABLE 2 Organ Blood Flows in Hedgehogs and Guinea Pigs at Different Body Temperatures Blood flow (ml. min-’ . 100 g-l) Guinea pigs
Hedgehogs 34°C
26°C
22°C
15°C
37°C
34°C
26°C
22°C
38 -c 3 124 + 9 176 t 16* 271 IT 72* 203 f 14 100 f 17 12 + 5 11 + 2 23 f 4* 30 + 3* 35 c 2 5+2 4*1 4il 221
30 c 6 140 2 12* 230 e 32* 55 -c 15 153 -’ 24 120 ?I 45 26 t 10 14 2 2 25 -t 7* 29 t 6* 42 2 10 2 -t 1* 12 1 2*1 15 1
14 + 5 136 t 58* 198 + 29* 89 + 13 70 + 12 47 k 18 17 k 8 12 * 2 17 f 6* 14 5 3* 30 i 10 3t1 2+1 221 121
522 112 -c 20 121 t 22 32 k 7 37?17 16 k 6 721 6-cl 11 it_ 6 62 1 21 + 7 2+1 I?1 l?l 121
25 + 4 54 2 10 56 ? 8 63 + 11 160~30 50 k 4 2&l 37 ‘- 5 91 2 17 81 + 9 56 + 15 11 + 3 422 5+2 2+1 1+ I -
20 k 6 57 f 10 83 e 20 85 f 31 210260 66 + 16 2+1 46 + 8 75 2 7 72 f 10 45 i 6 13 % 4 421 13 + 5 l?l 221 -
8~2 32 + 7 41 e 13 52 it_ 29 116+32 22 + 6 lil 25 k 7 44 + 10 35 -t 2 23 + 5 823 121 l-+1 1+1 l-t1
10 5 2 38 -c 12 41 + 3 34 2 17 90 + 20 21 + 3 151 13 -c 3 45 5 12 24 2 5 17 F 5 6-t2 121 il * 1 l-cl 121 -
Organ Brain Right ventricle Left ventricle Lungs Kidney Adrenal gland Liver Stomach Duodenum Small intestine Large intestine Urinary bladder Muscle: hind leg psoas Skin White fat Brown fat
N&e. Values given are the means -c SE. Hedgehogs, n = 4; guinea pigs, PI = 6. Significantly different from corresponding guinea pig value. * P < 0.05, Mann-Whitney U test.
species. In both species the intestinal blood flow decreased with reduced body temperature. Figure 1 shows the fractional distribution of cardiac output (ml . min-’ * 100 g-l organ weight/ml . min-’ * 100 g-i body weight) to the left ventricle of the heart, the LEFT Fraction cardia
kidneys and the duodenum. In the hedgehogs, the fractional distribution of cardiac output to the myocardium increased with decreasing body temperature, while the renal fraction of the cardiac output decreased and the duodenal fraction was unchanged when the body temperature
VENTRICLE
DUODENUM
KIDNEY
of
60
60
45
45
30
30
15
15
0 I
1
35
I
1
30
25
1
20
I
I
15
OC
I
I
I
I
I
35
30
25
20
15
oc
35
io
is
2b
1’5 oc
FIG. 1. Fraction of cardiac output distributed to the organs at different body temperatures calcu-
lated as ml * mini . 100-i organ weight/ml. min-* . 100-i bod y wt. (0) Hedgehogs (n = 4), (0) guinea pigs (n = 6). Statistical significance between the two groups was assessed using the MannWhitney U test. *P < 0.05. ***p < 0.001.
444
SJijQUIST,
DUKER, AND JOHANSSON
was lowered. In contrast, in the guinea temperatures in the same animal (8); this pigs, the fractional distribution of cardiac experimental design was not possible with output to the myocardium was unchanged, the rubidium technique. However, the miwhile it increased to the kidneys and duo- crosphere technique has methodological denum. There was a significant increase in limitations. For instance, in an organ blood the brown fat fraction of cardiac output in flow encompassing both capillary flow, the hedgehogs, from 14 & 3 at 34°C to 74 which will trap the microspheres, and flow t 10 at 22°C (P < 0.02 Student’s t test for through A-V shunts, which will not trap paired observations). the microspheres, only the capillary fraction of the blood flow will be measurable. A validation and comparison between the rubidium and microsphere methods has DISCUSSION been made in cold-acclimated rats (6). The In the present study, we have used the validity of the microsphere technique was tracer microsphere technique to study the reasonable according to Foster and regional blood flow effects of induced hy- Frydman (6), whereas 86Rb+ measurepothermia in anesthetized hedgehogs and ments, in addition to fractional distribution guinea pigs. Earlier, cardiac output distri- of CO, could be influenced by resistances bution in hibernators was preferably of the capillary walls, cell membrane transstudied during arousal using the rubidium port of 8aRb+, and differences in the exmethod (4, 9, 17). It was supposed that the traction ratio for 86Rb+ between tissues. It distribution, and subsequently the organ must be concluded from a comparison of uptake, of 86Rb+ followed the distribution the advantages and the limitations of the of blood flow (18). The passage of rubidium microsphere method and the rubidium from the blood to the tissues is a question method that values of regional blood flows of establishing an equilibrium, which is a obtained with the two techniques should be rather slow diffusion process. This means compared with great care. that tissues through which blood passes relThe results of the present study show atively slowly will show a higher rubidium that under anesthetized conditions the efuptake than those where blood passes fects of acutely induced hypothermia on through quickly. The advent of labeled mi- regional blood flow distribution are compacrospheres has provided an alternative rable between the hibernator (hedgehog) method for measuring regional blood flow. and the nonhibernator (guinea pig). For The nuclide-labeled microspheres are of some organs such as the heart, kidneys, such a size (in the present study 9 pm) that and duodenum, cardiac output distribution during hypothermia was found to be estithey penetrate into the precapillary vessels, where they are trapped. This mated as significantly different between the causes embolism of a small fraction of the guinea pig and the hedgehog, with a higher vessels without increasing peripheral resis- proportion going to the heart at all tempertance, and the distribution of the blood atures in the hibernator, the reverse being flow can then be estimated (8). In this case, found for the kidney and duodenum. It is no diffusion is needed, and organs having a possible that more dramatic differences in high blood flow may be measured without the cardiac output distribution between the this systematical error. Another advantage species would have been revealed if the anof using tracer microspheres is that dif- imals had not been anesthetized. It cannot ferent nuclides can be used for labeling the be excluded that the anesthetic was able to spheres. This means that the regional superimpose hypothermically induced reblood flow can be determined at different flexes in either the guinea pig or the
HYPOTHERMIA-ORGAN
BLOOD
hedgehog or both. Sodium pentobarbital, a widely used anesthetic in experimental cardiovascular investigations, was used in the present study. We have not found any studies on how this barbiturate influences the cardiovascular response to hypothermia, but it is known that during normothermia pentobarbital affects systemic and regional hemodynamics only slightly, but depresses the myocardium markedly (14). It has been suggested that the tachycardia associated with pentobarbital anesthesia depends on a vagolytic effect and/or is mediated through the baroreceptor reflex (14). The regional blood flow distributions in the hedgehog during the acutely induced hypothermia show a different pattern from those reported earlier for other hibernating species in a comparable degree of hypothermia during natural arousal from hibernation. During arousal from hibernation, organs in the forepart of the hibernator are highly blood perfused, while in the hindpart of the body the flow is very restricted (4,9, 17). In the present study of induced hypothermia, any redistribution of blood flow between different parts of the body is much less pronounced than during arousal. We suggest that, in this respect, induced hypothermia seems to be similar to natural entrance into hibernation. In support of this theory are the results obtained by Lyman and O’Brien (12) in their study of the thirteen-lined ground squirrel. They found that the temperature in the heart region and abdomen was the same in the animals entering hibernation, while during arousal there was a pronounced difference between temperature values obtained from the esophagus or rectum at any body temperature studied. Difficulties in predicting natural entrances and problems with measuring regional blood flow at certain time intervals during the entrance into hibernation without disturbing the animals may be some of the reasons why so few attempts
FLOW
IN A HIBERNATOR
445
have been made to study the cardiac output distribution during the entrance period of the hibernating cycle. Soivio (19) was able to determine the distribution of blood, in a small number of animals, during entrance into hibernation using the rubidium technique. His results pointed to an even distribution with no specific areas of vasoconstriction. The changes in blood pressure and peripheral vascular resistance during induced hypothermia in the hedgehogs are similar to those recorded in the ground squirrel during entrance into hibernation. In the ground squirrel, blood pressure first remains within the range of the active animal, then decreases. The peripheral resistance increases continuously as the body temperature falls (12). The increase in peripheral vascular resistance could partly be explained by the effect of cold on the blood viscosity and on the elasticity of the vascular wall. However, it has also been suggested that there is an increase in vascular tone (13). Hibernators are characterized by their ability to tolerate low body temperatures, and they have good control of the physiological mechanisms at low temperatures. Included in this control is tolerance to cardiac arrhythmias, especially ventricular fibrillation, when entering and during arousal from hibernation and when subjected to experimentally induced myocardial ischemia (11). Various mechanisms have been proposed to account for this resistance to ventricular fibrillation (5). In the present study, the higher myocardial blood flow measured in the hedgehog compared with the guinea pig at all temperatures during induced hypothermia may be one contributing factor explaining the differences in resistance to cardiac arrhythmias between hibernators and nonhibernators. It must be stressed, however, that the present findings were obtained under anesthetic conditions, and that extrapolation of the results to conscious unrestrained animals living in their
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natural habitat should be done with great caution. ACKNOWLEDGMENTS We thank Ms. G. Thylander for revising the English text and Ms. R. Jorgensen for secretarial support. REFERENCES 1. Alexander, G., Bell, A. W., and Hales, J. R. S. Effects of cold exposure on tissue blood flow in the newborn lamb. J. Physiol. (London) 234, 65-77 (1973). 2. Budden, R., Detweiler, D. K., and Zbinden, G. (Eds.) “The Rat Electrocardiogram in Pharmacology and Toxicology.” Pergamon, Elmsford, N.Y., 1981. 3. Bullard, R. W. Cardiac output of the hypothermic rat. Amer. J. Physiol. 196, 415-419 (1959). 4. Bullard, R. W., and Funkhouser, G. E. Estimated regional blood flow by rubidium 86 distribution during arousal from hibernation. Amer. J. Physiol. 203, 266-270 (1962). 5. Duker, G., Olsson, S. -O., Hecht, N. H., Senturia, J. B., and Johansson, B. W. Ventricular fibrillation in hibernators and nonhibernators. Cryobiology
20, 407-420 (1983).
6. Foster, 0. E, and Frydman, M. L. Comparison of microspheres and %Rb+ as tracers of the distribution of cardiac output in rats indicates invalidity of 86Rb-based measurements. Cunad. J. Physiol. Pharmacol. 56, 97-109 (1978). 7. Green, C. J. “Animal Anesthesia.” Laboratory Animal Handbooks 8, Spottiswoode Ballantyne Ltd., Colchester and London, 1979. 8. Heymann, M. A., Bruce, D. P., Hoffman, J. I. E., and Rudholph, A. M. Blood flow measurements with radionuclide-labeled particles. Prog. Cardiovasc. Dis. 20, 55-79 (1977). 9. Johansen, K., Distribution of blood in the arousing hibernator. Acta Physiol. Stand. 52, 379-386 (1961). 10. Johansson, B. W. Heart and circulation in hibernators. In “Mammalian Hibernation III.”
(R. C. Fisher, A. R. Dawe, L. C. Lyman, E. Schonbaum, and F. E. South, Eds.), pp. 199-218. New York, Elsevier, 1967. 11. Johansson, B. W. Cardiac responses in relation to heart size. Cryobiology 21, 627-636 (1984). 12. Lyman, C. P., and O’Brien, R. C. Circulatory changes in the thirteen-line ground squirrel during the hibernating cycle. Mus. Camp. 2001. Harvard Coil. 124, 353-372 (1960). 13. Lyman, C. P., Willis, J. S., Malan, A., and Wang, L. C. H. “Hibernation and Torpor in Mammals and Birds.” Academic Press, New York, 1982. 14. Manders, T. W., and Vatner, S. F. Effects of sodium pentobarbital anesthesia on left ventricular function and distribution of cardiac output in dogs, with particular reference to the mechanism for tachycardia. Circ. Res. 39, 512-517 (1976). 15. Mendel, P. L., and Hollenberg, N. K. Cardiac output distribution in the rat: Comparison of rubidium and microsphere methods. Amer. J. Physiol. 221, 1617-1620 (1971). 16. Rakusan, K., and Blahitka, J. Cardiac output distribution in rats measured by injection of radioactive microspheres via cardiac puncture. Canad.
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(1974). 17. Rauch, J. C. Sequential changes in regional distribution of blood in Eptesicus fuscus (big brown bat) during arousal from hibernation. Canad. J. Zool. 51, 973-981 (1973). 18. Sapirstein, L. A. Regional blood flow by fractional distribution of indicators. Amer. J. Physiol. 193, 161-168 (1958). 19. Soivio, A. Hibernation in the Hedgehog (Erinaceus europaeus L.): The Distribution of Blood, the Size of the Spleen, and the Hematocrit and Hemoglobin Values during the Annual and Hibernating Cycles. Annales Academiae Scientiarium Fennicae, Suomalainen Tiedeakatemia, Helsinki, 1967. 20. Towell, M. E., and Liggins, G. C. The effect of labour on uterine blood flow in the pregnant ewe. Q. J. Exp. Physiol. 61, 23-33 (1976).