CRYOBIOLOGY
13, 80-83 (1976)
Further
Studies on Hypothermia and the Blood-Brain Barrier System LLOYD
ALLAN
WELLS 1
Depaztment of Anatomy, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 INTRODUCTION
Profound hypothermia has been shown to disrupt the blood-brain barrier system of several homeothermic species to some tracers, although not to others (6, 7). Previous studies from this laboratory have demonstrated that the disruption of the barrier system to rubidium tracer occurs at a core temperature near 16°C and is related to this “critical” temperature (5), rather than to respiratory depression or the total length of hypothermia (1). The present investigations were undertaken to determine whether several episodes of profound hypothermia in the same animal might affect the extent of bloodbrain barrier system disruption to rubidium tracer at 16”C, and whether changes in the permeability of the blood-brain barrier system to rubidium tracer persist when the animal is allowed to rewarm after reaching the “critical” temperature range for barrier disruption. MATERIALS
AND
METHODS
Thirty-eight female white rats of the Sprague-Dawley strain were used in these experiments. On 5 consecutive days, eight rats were made hypothermic by immersion in an ice-and-water bath following the administration of ether anesthesia, as previously described (6). Core temperature Received February 1, 1974. 1 Present address: Mayo Foundation, Rochester, Minn.
COPyright 0 1976 by Academic Press, Inc. All rights of reproduction in my form +eservd.
was measured by the rectal insertion of an accurate thermometer with a precision of *O.O5”C, until the bulb could be felt in apposition to the animal’s diaphragm. On the first and second days, the rats were removed from the bath and allowed to rewarm when a temperature of 25°C had been attained in the cooling process. On the third and fourth days, the rats were ahowed to reach 20°C before they were removed from the bath, and an incandescent lamp was provided as an exogenous source of heat during the initial phase of rewarming. On the fifth day, the rats were divided into two groups and were again subjected to hypothermia. Rats in the first group were allowed to reach 16°C and were then injected with an aliquot of 8”RbC1 via the external jugular vein (6). Two hundred heart beats after termination of the injection, the animals were sacrificed by cervical dislocation followed by immediate decapitation and brain excision. Brains were placed in tared vials and weighed. Vials were counted for gamma radioactivity in a Beckman scintihation counter. Rats in the second group were injected with 86RbC1 when a 1-min period of apnea had occurred. They were sacrificed, and their brains processed, in a manner identical to that used for the first group. Thirty rats were made hypothermic as described above. They were then divided into five groups of six rats each. Animals in the first group were injected with 86RbCl
HYPOTHERMIA
AND
BLOOD-BRAIN
tracer at 16°C and were sacrificed as described above. Rats in the other groups were removed from the ice-and-water bath when core temperature was between 16 and 17°C; they were allowed to rewarm. An incandescent lamp was used as a source of exogenous heat, and ventilation was assisted with a tube where necessary. Groups were injected when rats had reached a temperature of 20, 25, 30, and 37°C. They were sacrificed 200 heart beats after termination of the injection, and brains were processed as described above. The relative activity level (RAL) was determined as 100 times the percentage of total radioactivity injected which was recovered from the brain, divided by the weight of the brain in grams, as previously described (6, 7).
81
BARRIER
CEREBRAL Rb
OEPDSlTlON
DURING REWARMING II-25]1GG
(1972)
n-5
(166
n-5 (ZOC n-4 125C -
n-5 13OC
11-337c n-22 37C $2, ?5
FIG. 2. hypothermia the brain. at various hypothermic compared euthermic
so
RAL
Effects of rewarming from profound on the deposition of rubidium in Shown are mean RAL for rats injected temperatures after reaching an initial temperature of 16-17°C. They are with the mean RAL of a series of rats (6, 7).
RESULTS
injected after a I-min period of apnea, with final body temperatures of 15.7, 15.3, and 16.O”C. The mean RAL for these rats was 55 4 9.1. The difference in RAL between these two groups was not significant by t test, nor was there any significant difference between brain RAL in these rats and those of a large series sacrificed at CEREBRAL Rb DEPOSITION AFTER RECURRENT 16°C after only one episode of hypothermia HYPOTHERMIA (6). These results are depicted in Fig. 1. Of the 30 rats which were cooled to 1617°C and subsequently allowed to rewarm to various temperatures; 27 survived until sacrifice. Mean RAL for the rats sacrificed at 16°C was 64 * 11.6; for the group sacrificed at 20°C 26 t 11.4; at 25”C, 23 + 3.5; at 30°C 17 t 1.7; and at 37°C 9 * 1.7. The difference in mean RAL be(111 2'5 n-25 5,0 1 'O::; tween the rats sacrificed at 16°C and the group at 20°C was significant at the 0.025 level by t-test. There was no significant difference in RAL value between the FIG. 1. Effects of single and recurrent hypogroups sacrificed at 20 and 25°C. RAL of thermia on the deposition of rubidium in the rat brain. Shown are mean RAL for rats sacrificed the group sacrificed at 25°C was signifiat 16°C and after a l-min period of apnea, followcantly greater than that of the group sacriing four artificial test drops” in the previous 4 ficed at 30°C at the 0.05 level, while the days. They are compared with the mean RAL 30°C group showed a significantly greater for a series of rats injected at 1617°C in their initial episode of hypothermia ( 6. 7). RAL than the 37°C group at the 0.05 level.
Of the eight rats subjected to recurrent, progressively deeper hypothermia (artificial “test drops”), five survived until the final episode of hypothermia. Two of these rats were injected at 16°C; their mean RAL was 48 + 2.5 (SE). Three rats were
LLOYD
82 The group injected 37°C did not show a in RAL from a large rats injected without hypothermia (6, 7). depicted in Fig. 2.
ALLAN
after rewarming to significant difference group of euthermic being subjected to These results are
DISCUSSION
Previous studies have shown that in contrast to the artificiaIIy hypothermic rat at l6-17°C actively hibernating mammals at temperatures as low as 3°C are able to restrict the cerebral deposition of rubidium and other tracers (6, 7). Strumwasser (3) and others have suggested that the “test drops” shown by some but not all (2, 4) hibernators prior to hibernation may have a preparatory effect on those homeostatic mechanisms of the animal necessary for prolonged survival during hibernation. In decrease these “test drops,” hibernators their temperatures to progressively lower Ievels and then compIetely rewarm, during the period shortly prior to hibernation. The first experiment described in this paper was an attempt to screen the hypothesis that artificial “test drops” in the rat might protect the blood-brain barrier system from the disruption to rubidium tracer normally observed at 1617°C. The sample size for this experiment was very small and was further diminished by the death of three of the animals during the five episodes of hypothermia. The experimental animaIs showed an increased deposition of rubidium tracer at 16”C, which was not significantly different from that of a large series of animals injected at the termination of a sole hypothermic episode (6, 7). Despite the small sample size, it is tentatively concluded that the artificial “test drops” in the rat had no effect on the aIteration of the blood-brain barrier system to rubidium in hypothermia. Clearly, these results have no bearing on the possible effects of naturally occurring “test drops” in hibernators. It has been hypothesized that the altered rate of entry of small molecules into the
WELLS
hypothermic brain at a critical temperature might interfere with hypothalamic and medullary mechanisms of heat gain and ventilation (7). In the second group of experiments reported here, rats were rewarmed from 16°C and were injected with rubidium and sacrificed at various rewarming temperatures. Most of these animals required artificial ventilation until a temperature of 17-19°C had been attained, and most required an exogenous source of heat until the temperature was near 25°C. There was a marked reduction in rubidium deposition in the brain between 16 and 20°C but nevertheless, nearly four times as much tracer entered the brains of rats at 20°C as those of euthermic controls. Since rats injected with rubidium at an initial minimal hypothermic temperature of 20°C show no increase in rubidium deposition over euthermic controls (5)) it seems probable that the disruption which occurs at 1617°C is responsible for the increased permeability found in the rewarmed rats of this experiment at 20°C. In rats sacrificed between rewarming temperatures of 20 to 37°C there was a gradual reduction in cerebral deposition of rubidium tracer, and at 37°C there was no significant difference in RAL from that of euthermic animals never subjected to hypothermia. This experiment suggests that the alteration of the blood-brain barrier system associated with hypothermia is reversible. This suggestion is compatible with the hypothesis that the alteration is caused by a disruption of ionic pumping mechanisms by enzyme inhibition at a critical temperature, rather than by a morphological alteration (7). While previous suggestions that death in hypothermia may be an indirect result of the disruption of the blood-brain barrier system are not directly affected by this experiment (6, 7), it is apparent that the disruption of the barrier system in the artificially hypothermic rat is compatible with life if artificial ventila-
HYPOTHERMIA
AND
BLOOD-BRAIN
tion and an exogenous source of heat are supplied. Whether the failures of ventilation and thermogenesis which occur in profound hypothermia are a resdt of alterations in the blood-brain barrier system or some other homeostatic system is unresolved.
2.
3.
SUMMARY
Rats subjected to five episodes of recurrent, progressively deeper hypothermia showed no difference in cerebral deposition of rubidium tracer at 16°C and/or apnea from animals lowered to this temperature and/or
condition
4.
only once. Rats allowed
to rewarm from 16°C showed persisting increased cerebral deposition of tracer at 20°C with gradual diminution at higher temperatures; at 37”C, deposition of rubidium tracer in brains of rewarmed rats was not different from that of euthermic rats which were not subjected to hypothermia. REFERENCES 1. Baldwin, M., Galindo, A., and R. Farrier. Cerebral reaction to sodium fluorescein dur-
5.
BARRIER
83
ing profound hypothermia. Neurology 12, 193 ( 1962). Lyman, C. P. In “Discussion following Strumwasser’s paper. In “Mammalian Hibernation,” C. P. Lyman and A. R. Dawe, (Ed.) Bull. Mus. Comp. 2001. (Harvard) 124, 319, (1960). Strumwasser, F. Some physiological principles governing hibernation. In “Citellus beecheyi” C. P. Lyman and A. Ft. Dawe (Eds. ), Bull. Mus. Comp. Zool. (Harvard) 124, 285, ( 1960). Twente, J, W., and J. A. Twente. Seasonal variation in the hibernating behavior of Cite&s Zateralis. In “Mammalian Hibernation III” K. C. Fisher, A. R. Dawe, C. P. Lyman, E. Schonbaum, and F. E. South, Jr. (Eds.), p. 47, Oliver and Boyd, 1967. Wells, L. A. Alteration of the blood-brain barrier system by hypothermia: Critical time period versus critical temperature, Camp. Biochem. Physiol. 44A: 293 ( 1973).
6. Wells, L. A. Permeability of the blood-brain barrier system to rubidium in euthermia, hibernation, Comp. and hypothermia. Biochem. Physiol. 42A: 551 (1972). 7. Wells, L. A. The effects of low body temperatures on deposition of tracers in the mammalian brain. CryobioZogy 9, 367 ( 1972).
13, 80-83 (1976)
Further
Studies on Hypothermia and the Blood-Brain Barrier System LLOYD
ALLAN
WELLS 1
Depaztment of Anatomy, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 INTRODUCTION
Profound hypothermia has been shown to disrupt the blood-brain barrier system of several homeothermic species to some tracers, although not to others (6, 7). Previous studies from this laboratory have demonstrated that the disruption of the barrier system to rubidium tracer occurs at a core temperature near 16°C and is related to this “critical” temperature (5), rather than to respiratory depression or the total length of hypothermia (1). The present investigations were undertaken to determine whether several episodes of profound hypothermia in the same animal might affect the extent of bloodbrain barrier system disruption to rubidium tracer at 16”C, and whether changes in the permeability of the blood-brain barrier system to rubidium tracer persist when the animal is allowed to rewarm after reaching the “critical” temperature range for barrier disruption. MATERIALS
AND
METHODS
Thirty-eight female white rats of the Sprague-Dawley strain were used in these experiments. On 5 consecutive days, eight rats were made hypothermic by immersion in an ice-and-water bath following the administration of ether anesthesia, as previously described (6). Core temperature Received February 1, 1974. 1 Present address: Mayo Foundation, Rochester, Minn.
COPyright 0 1976 by Academic Press, Inc. All rights of reproduction in my form +eservd.
was measured by the rectal insertion of an accurate thermometer with a precision of *O.O5”C, until the bulb could be felt in apposition to the animal’s diaphragm. On the first and second days, the rats were removed from the bath and allowed to rewarm when a temperature of 25°C had been attained in the cooling process. On the third and fourth days, the rats were ahowed to reach 20°C before they were removed from the bath, and an incandescent lamp was provided as an exogenous source of heat during the initial phase of rewarming. On the fifth day, the rats were divided into two groups and were again subjected to hypothermia. Rats in the first group were allowed to reach 16°C and were then injected with an aliquot of 8”RbC1 via the external jugular vein (6). Two hundred heart beats after termination of the injection, the animals were sacrificed by cervical dislocation followed by immediate decapitation and brain excision. Brains were placed in tared vials and weighed. Vials were counted for gamma radioactivity in a Beckman scintihation counter. Rats in the second group were injected with 86RbC1 when a 1-min period of apnea had occurred. They were sacrificed, and their brains processed, in a manner identical to that used for the first group. Thirty rats were made hypothermic as described above. They were then divided into five groups of six rats each. Animals in the first group were injected with 86RbCl
HYPOTHERMIA
AND
BLOOD-BRAIN
tracer at 16°C and were sacrificed as described above. Rats in the other groups were removed from the ice-and-water bath when core temperature was between 16 and 17°C; they were allowed to rewarm. An incandescent lamp was used as a source of exogenous heat, and ventilation was assisted with a tube where necessary. Groups were injected when rats had reached a temperature of 20, 25, 30, and 37°C. They were sacrificed 200 heart beats after termination of the injection, and brains were processed as described above. The relative activity level (RAL) was determined as 100 times the percentage of total radioactivity injected which was recovered from the brain, divided by the weight of the brain in grams, as previously described (6, 7).
81
BARRIER
CEREBRAL Rb
OEPDSlTlON
DURING REWARMING II-25]1GG
(1972)
n-5
(166
n-5 (ZOC n-4 125C -
n-5 13OC
11-337c n-22 37C $2, ?5
FIG. 2. hypothermia the brain. at various hypothermic compared euthermic
so
RAL
Effects of rewarming from profound on the deposition of rubidium in Shown are mean RAL for rats injected temperatures after reaching an initial temperature of 16-17°C. They are with the mean RAL of a series of rats (6, 7).
RESULTS
injected after a I-min period of apnea, with final body temperatures of 15.7, 15.3, and 16.O”C. The mean RAL for these rats was 55 4 9.1. The difference in RAL between these two groups was not significant by t test, nor was there any significant difference between brain RAL in these rats and those of a large series sacrificed at CEREBRAL Rb DEPOSITION AFTER RECURRENT 16°C after only one episode of hypothermia HYPOTHERMIA (6). These results are depicted in Fig. 1. Of the 30 rats which were cooled to 1617°C and subsequently allowed to rewarm to various temperatures; 27 survived until sacrifice. Mean RAL for the rats sacrificed at 16°C was 64 * 11.6; for the group sacrificed at 20°C 26 t 11.4; at 25”C, 23 + 3.5; at 30°C 17 t 1.7; and at 37°C 9 * 1.7. The difference in mean RAL be(111 2'5 n-25 5,0 1 'O::; tween the rats sacrificed at 16°C and the group at 20°C was significant at the 0.025 level by t-test. There was no significant difference in RAL value between the FIG. 1. Effects of single and recurrent hypogroups sacrificed at 20 and 25°C. RAL of thermia on the deposition of rubidium in the rat brain. Shown are mean RAL for rats sacrificed the group sacrificed at 25°C was signifiat 16°C and after a l-min period of apnea, followcantly greater than that of the group sacriing four artificial test drops” in the previous 4 ficed at 30°C at the 0.05 level, while the days. They are compared with the mean RAL 30°C group showed a significantly greater for a series of rats injected at 1617°C in their initial episode of hypothermia ( 6. 7). RAL than the 37°C group at the 0.05 level.
Of the eight rats subjected to recurrent, progressively deeper hypothermia (artificial “test drops”), five survived until the final episode of hypothermia. Two of these rats were injected at 16°C; their mean RAL was 48 + 2.5 (SE). Three rats were
LLOYD
82 The group injected 37°C did not show a in RAL from a large rats injected without hypothermia (6, 7). depicted in Fig. 2.
ALLAN
after rewarming to significant difference group of euthermic being subjected to These results are
DISCUSSION
Previous studies have shown that in contrast to the artificiaIIy hypothermic rat at l6-17°C actively hibernating mammals at temperatures as low as 3°C are able to restrict the cerebral deposition of rubidium and other tracers (6, 7). Strumwasser (3) and others have suggested that the “test drops” shown by some but not all (2, 4) hibernators prior to hibernation may have a preparatory effect on those homeostatic mechanisms of the animal necessary for prolonged survival during hibernation. In decrease these “test drops,” hibernators their temperatures to progressively lower Ievels and then compIetely rewarm, during the period shortly prior to hibernation. The first experiment described in this paper was an attempt to screen the hypothesis that artificial “test drops” in the rat might protect the blood-brain barrier system from the disruption to rubidium tracer normally observed at 1617°C. The sample size for this experiment was very small and was further diminished by the death of three of the animals during the five episodes of hypothermia. The experimental animaIs showed an increased deposition of rubidium tracer at 16”C, which was not significantly different from that of a large series of animals injected at the termination of a sole hypothermic episode (6, 7). Despite the small sample size, it is tentatively concluded that the artificial “test drops” in the rat had no effect on the aIteration of the blood-brain barrier system to rubidium in hypothermia. Clearly, these results have no bearing on the possible effects of naturally occurring “test drops” in hibernators. It has been hypothesized that the altered rate of entry of small molecules into the
WELLS
hypothermic brain at a critical temperature might interfere with hypothalamic and medullary mechanisms of heat gain and ventilation (7). In the second group of experiments reported here, rats were rewarmed from 16°C and were injected with rubidium and sacrificed at various rewarming temperatures. Most of these animals required artificial ventilation until a temperature of 17-19°C had been attained, and most required an exogenous source of heat until the temperature was near 25°C. There was a marked reduction in rubidium deposition in the brain between 16 and 20°C but nevertheless, nearly four times as much tracer entered the brains of rats at 20°C as those of euthermic controls. Since rats injected with rubidium at an initial minimal hypothermic temperature of 20°C show no increase in rubidium deposition over euthermic controls (5)) it seems probable that the disruption which occurs at 1617°C is responsible for the increased permeability found in the rewarmed rats of this experiment at 20°C. In rats sacrificed between rewarming temperatures of 20 to 37°C there was a gradual reduction in cerebral deposition of rubidium tracer, and at 37°C there was no significant difference in RAL from that of euthermic animals never subjected to hypothermia. This experiment suggests that the alteration of the blood-brain barrier system associated with hypothermia is reversible. This suggestion is compatible with the hypothesis that the alteration is caused by a disruption of ionic pumping mechanisms by enzyme inhibition at a critical temperature, rather than by a morphological alteration (7). While previous suggestions that death in hypothermia may be an indirect result of the disruption of the blood-brain barrier system are not directly affected by this experiment (6, 7), it is apparent that the disruption of the barrier system in the artificially hypothermic rat is compatible with life if artificial ventila-
HYPOTHERMIA
AND
BLOOD-BRAIN
tion and an exogenous source of heat are supplied. Whether the failures of ventilation and thermogenesis which occur in profound hypothermia are a resdt of alterations in the blood-brain barrier system or some other homeostatic system is unresolved.
2.
3.
SUMMARY
Rats subjected to five episodes of recurrent, progressively deeper hypothermia showed no difference in cerebral deposition of rubidium tracer at 16°C and/or apnea from animals lowered to this temperature and/or
condition
4.
only once. Rats allowed
to rewarm from 16°C showed persisting increased cerebral deposition of tracer at 20°C with gradual diminution at higher temperatures; at 37”C, deposition of rubidium tracer in brains of rewarmed rats was not different from that of euthermic rats which were not subjected to hypothermia. REFERENCES 1. Baldwin, M., Galindo, A., and R. Farrier. Cerebral reaction to sodium fluorescein dur-
5.
BARRIER
83
ing profound hypothermia. Neurology 12, 193 ( 1962). Lyman, C. P. In “Discussion following Strumwasser’s paper. In “Mammalian Hibernation,” C. P. Lyman and A. R. Dawe, (Ed.) Bull. Mus. Comp. 2001. (Harvard) 124, 319, (1960). Strumwasser, F. Some physiological principles governing hibernation. In “Citellus beecheyi” C. P. Lyman and A. Ft. Dawe (Eds. ), Bull. Mus. Comp. Zool. (Harvard) 124, 285, ( 1960). Twente, J, W., and J. A. Twente. Seasonal variation in the hibernating behavior of Cite&s Zateralis. In “Mammalian Hibernation III” K. C. Fisher, A. R. Dawe, C. P. Lyman, E. Schonbaum, and F. E. South, Jr. (Eds.), p. 47, Oliver and Boyd, 1967. Wells, L. A. Alteration of the blood-brain barrier system by hypothermia: Critical time period versus critical temperature, Camp. Biochem. Physiol. 44A: 293 ( 1973).
6. Wells, L. A. Permeability of the blood-brain barrier system to rubidium in euthermia, hibernation, Comp. and hypothermia. Biochem. Physiol. 42A: 551 (1972). 7. Wells, L. A. The effects of low body temperatures on deposition of tracers in the mammalian brain. CryobioZogy 9, 367 ( 1972).
