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Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament
BRAIN RESEARCH ELSEVIER
Brain Research 649 (1994) 253-259
Research Report
Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament Qi Zhao, Hajime Memezawa, Maj-Lis Smith *, Bo K. Siesj6 Laboratory for Experimental Brain Research, UniversityHospital, University of Lund, S-221 85 Lund, Sweden (Accepted 15 March 1994)
Abstract
The present experiments were designed to study under what circumstances middle cerebral artery (MCA) occlusion by an intraluminal filament technique leads to hyperthermia and what the mechanisms are. We found that permanent MCA occlusion by this technique lead to a rise in body (core) temperature to 39.0-39.5°C during the first 2-4 h, and to sustained hyperthermia thereafter (38.5-39.0°C). After 2 h of transient MCA occlusion hyperthermia could only be avoided if anesthesia (with control of temperature) was maintained for 2 h of ischemia and 1 h of recirculation or, in unanesthetized animals, if external cooling was maintained for 2 h of ischemia and 2 h of recirculation. Control of temperature only during ischemia did not prevent a postischemic rise in temperature. One hour of MCA occlusion had less effect on body temperature. Results are presented which suggest that the hyperthermia observed is due to an interference, by the intraluminal filament, of circulation to hypothalamic centers regulating body temperature. It is speculated that the hyperthermia induced may blunt or obliterate the effect of drugs, normally considered to ameliorate brain damage due to focal ischemia.
Key words: Focal ischemia; MCA occlusion; Hyperthermia; Temperature control; Hypothalamic damage; Rat
I. Introduction
It has been known for decades that hypothermia protects the brain against global ischemic/hypoxic insuits [5,9,15,29], and that even in the absence of ischemia, hyperthermia can cause brain damage [29]. During recent years, it has become increasingly clear that even small decreases in temperature can ameliorate damage due to global or forebrain ischemia [6,22,23], while equally moderate rises in body (and brain) t e m p e r a t u r e aggravates damage [6,12,23]. This means that accidental variation in temperature can become a confounding factor, complicating interpretations of experiments on drug treatment. For example, the ameliorating effects of dizocilpine maleate (MK801) in gerbil ischemia could be largely attributed to drug-induced hypothermia [4,10], and halothane anesthesia was found to ameliorate damage by preventing spontaneous postischemic hyperthermia [18].
* Corresponding author. Fax: (46) (46) 151480. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
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Recent data demonstrate that temperature influences focal ischemic damage due to middle cerebral artery (MCA) occlusion to a comparable degree [7,8,24,31]. In two studies, a rise in temperature to 39-40°C clearly increased infarct size [7,31]. In one of these, hyperthermia was detrimental even when instituted after transient ischemia [7]. A technique for inducing transient M C A occlusion by an intraluminal filament ('embolus') is now gaining increasing popularity [7,8,19,25]. This technique has the advantage that it does not require a craniectomy with the associated operative trauma. We have published a modification of this technique, describing focal and perifocal changes in blood flow and labile metabolites, as well as histopathologic changes after various occlusion times [13,20,21]. However, we failed to find that dimethylthiourea ( D M T U ) ameliorated tissue damage and observed a spontaneous rise in body temperature to 39.0-39.5°C due to transient (2 h) ischemia [16]. In this study, we have assessed t e m p e r a t u r e changes following transient or permanent M C A occlusion, ex-
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plored its possible cause(s), and devised a technique to curtail the hyperthermia.
2.2. Middle cerebral artery occlusion We used the intraluminal filament technique described by Koizumi et al. [17] to occlude the MCA. In the first part of the study, we employed the filament (nylon monofilament fishing thread, O.D. 0.28 mm) described by Memesawa et al. [20,21] but inserted the occluder from the external carotid artery according to Chen et al. [7]. In the latter part of the study, we used a modification of Koizumi's type of filament, i.e. a silicone rubber-coated nylon fishing line. This occluder was inserted from the common carotid artery, which was ligated proximal to the incision made for the occluder.
2. Materials and methods 2.1. Animal preparations Male Wistar rats (M¢llegaard's Breeding Center, Copenhagen, Denmark), weighing 310-340 g, were used. Animals were fasted overnight but had free access to water. After induction of anesthesia with 3-3.5% halothane, the rats were orally intubated. Operation was performed under mechanical ventilation with 1.0-1.5% halothane. A PE-50 polyethylene catheter was inserted into a tail artery for blood pressure recording and blood sampling. When the middle cerebral artery (MCA) occlusion (see below) had been performed, some animals were extubated and allowed to wake up. Others were maintained under continued anesthesia with 0.5% halothane. In these, Norcuron® (vecuronium bromide, 2 r a g . h - t ) was infused to maintain muscle relaxation.
2.3. Measurement and regulation of temperature During operation, an electrical temperature probe was inserted 5-6 cm into the rectum to record core temperature, which was regularly maintained at 37°C. In some of the animals a telemetric sensor (Data Sciences, St. Paul, Minnesota, USA) was placed intraperitoneally and secured by a suture to the gastric wall. In these animals, temperature was recorded telemetrically for 24 h. Some of
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Q. Zhao et al. / Brain Research 649 (1994) 253-259 42
damage to hypothalamic centers. In order to assess whether such damage could be verified we re-examined autoradiographic films from a previous study of cerebral blood flow (CBF) changes following M C A occlusion [20]. The CBF m e a s u r e m e n t s were made after 60 min of M C A occlusion (n = 6), and 15 min after the circulation had been restored after a period of 60 min of occlusion (n = 6). The technique of Sakurada et al. [27] was used, with [14C]iodoantipyrine as the diffusible tracer, for details see [20]. In addition, a series of animals with 2 h of M C A occlusion followed by 2 - 7 days of recirculation (n = 12) was examined by light microscopy for histological hypothalamic damage (Zhao et al., unpublished results). All sections were obtained from animals whose brains were fixed by perfusion with buffered formaldehyde, and the sections were stained with acid fuchsin and Celestine blue as previously described [1,30].
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There were three series of experimental animals, with either p e r m a n e n t M C A occlusion or transient occlusion of 1 or 2 h duration. In p e r m a n e n t ischemia (n = 4), the temperature was not regulated after M C A occlusion. In the 1 h ischemia group (n = 5), the animals were kept under continued anesthesia during ischemia and for the first hour of recirculation. In the experiments with 2 h ischemia, we had four subgroups: (i) the temperature was not regulated after M C A occlusion (n = 4), (ii) the animals were cooled by the air-cooling method only during ischemia (n = 8), (iii) the animals were kept under anesthesia during ischemia and for the first hour of recirculation (n = 4), and (iv) the animals were cooled by the aircooling method during ischemia and for the first 2 h of recirculation (n = 10). In all animals, temperature during anesthesia was main'tained at 37.0-37.5°C. Awake animals were either allowed to attain their spontaneous temperature, or were air-cooled for different periods (see above) in an attempt to maintain temperature close to normal levels also in the awake state.
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the animals were allowed to spontaneously adjust their temperature in cages at room temperature ( ~ 22°C), while others were cooled so that their core temperature was maintained close to normal levels. For the latter purpose, we developed an air cooling system to curtail the spontaneous rise in body temperature. The system consisted of three parts: a source of compressed air, a device to cool the air (a plastic tube spiral in ice water), and outlets in all four corners of the cage, allowing the cold air to circulate in the cage. The temperature inside the cage was regulated to between 6 and 15°C as required.
3. Results
3.1. Permanent M C A occlusion
2.4. Assessment of hypothalamic damage W h e n it was shown that MCA-occluded animals developed hyperthermia, the suspicion arose that the filament caused irritation or
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middle cerebral artery occlusion, but was then not regulated further. As Fig. 1 shows, temperature rose to 39.0-39.5°C during the first 2 h of ischemia. The observation confirms our previous data for 2 h of ischemia [16], but extend the obervation to 24 h of ischemia. As observed from the figure, mean core temperature was 38.5-39.0°C for the entire period of ischemia, the highest temperature being encountered during the first 7 h. Thus core temperature during occlusion is 0.51.0°C higher than that recorded in sham-operated animals. In two additional animals with telemetric recording, we kept the rats under anesthesia and maintained the temperature at 37-37.5°C for 2 and 3 h, respectively. In both cases, body temperature rose to 39.0-39.5°C when the anesthesia was discontinued. These results suggest that hyperthermia is a seemingly inevitable consequence of intraluminal filament occlusion of the MCA.
3.3. MCA occlusion for two hours
As already stated, 2 h of MCA occlusion in the awake and freely moving animal leads to an unacceptable rise in body temperature. Fig. 3 shows that the hyperthermia developing during the 2 h period of ischemia persists for, at least, the first 2 h of recirculation. Therefore, we next studied the effect of cooling the freely moving animals during the period of ischemia. As Fig. 3 shows, this prevented an intraischemic rise in temperature. However, when cooling was discontinued temperature rose to about 39.0°C. We found that two procedures prevented the postischemic hyperthermia. One was to keep the animals
3.2. MCA occlusion for one 1 hour
After 1 h transient ischemia, the rectal temperature rose to near 39°C at the end of ischemia if the animal was allowed to wake up after MCA occlusion. However, if the animal was kept under anesthesia and the temperature was maintained at ~ 37°C during the ischemia and for the first hour of recirculation, hyperthermia was not oberserved during the 24-h period of recirculation (Fig. 2). We concluded from this that MCA occlusion for 1 h, with the intraluminal filament technique, can be induced without a rise of temperature if the temperature is maintained constant ( ~ 37°C) during ischemia and the first hour of recirculation.
Fig. 6. A representative autoradiogram from a local cerebral blood flow experiment, showing ischemic areas (white) in an animal with 1 h of M C A occlusion. Note the hypothalamic ischemia (arrowhead).
Q. Zhao et al. / Brain Research 649 (1994) 253-259
anesthetized for the period of ischemia and the first hour of recirculation (Fig. 4), the other, applicable to unanesthetized animals, involved external cooling during ischemia and during the first 2 h of recirculation (Fig. 5). However, this procedure worked in only 7/10 animals; in the remainder (3/10), temperature rose to about 39°C following discontinuation of cooling.
257
3.4. Possible causes of ischemic /post ischemic hyperthermia As the hyperthermia developed rapidly (1-2 h), it seemed unlikely to reflect an inflammatory reaction. Since it was possible that the temperature regulating center in the hypothalamus was affected, we examined
Fig. 7. Representative photomicrograph from hypothalamus of an animal subjected to 2 h of M C A occlusion followed by 2 days of recovery before perfusion fixation with formaldehyde. A: shows a low magnification (bar = 500 ~zm) of hypothalamus at the level of bregma - 2 mm. Framed parts of the left and right ( M C A occluded side) anterior hypothalamus are shown below as (B) and (C), respectively. In (B) the tissue has a normal appearance, while in (C) all neurons are necrotic and the neuropil spongy (bars = 100/zm). Celestine b l u e / a c i d fuchsin.
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CBF autoradiograms and histological sections as described in section 2. Middle cerebral artery occlusion with the intraluminal filament led to a reduction of CBF in preoptic and hypothalamic areas, i.e. areas which are outside the distribution territory of the MCA. The blood flow in the preoptic area at 1 h of ischemia was 5.11 _+5.5% of that in the contralateral side (range 0-15c/~), and in the anterior hypothalamus (Fig. 6) corresponding values were 7.2 _+ 7.3% (range 0-19%). This reduction in blood flow during ischemia is of a similar magnitude as is seen in the ischemic focus, consisting of lateral caudoputamen (CBF 4.4%) and overlying cortex (CBF 7.2%) [20]. Recirculation was achieved in both preoptic area and hypothalamus when the filament was withdrawn after 60 rain of MCA occlusion, as evidenced from CBF values ranging between 0.50 to 1.42 m l . g l . m i n ~. These values were around 50-60% of contralateral CBF values, and similar to postischemic flow in other structures [20]. Histological examination of perfusionfixed brains revealed ischemic damage in preoptic area a n d / o r anterior hypothalamus in 10 of 12 animals subjected to 2 h of MCA occlusion followed by 2 - 7 days of reperfusion before fixation for histology (Fig. 7).
4. Discussion
The present results document a complication of the intraluminal filament technique for MCA occlusion, namely hyperthermia. This hyperthermia develops during the first 2 h of ischemia [16], and is sustained for at least 24 h if the ischemia lasts for 2 h or longer. Since we have not been able to find similar data in the literature, it seems that the hyperthermia is 'specific' for the intraluminal filament MCA occlusion technique. Accordingly, it appeares profitable to look for factors which are related to the introduction of the filament. Our results suggest that hyperthermia occurs in both types of insertion of occluder (e.g. from the external carotid artery and from the common carotid artery), and with both types of filament (e.g. a nylon monofilament fishing thread and a silicone rubbercoated nylon fishing line), because the filament interferes with the circulation to hypothalamic centers regulating temperature. The anterior hypothalamus and preoptic area often suffers ischemic damage with the intraluminal filament technique of MCA occlusion if the occlusion time is 2 h or more [8,20,21]. Lesions in this area are known to cause hyperthermia [2,14]. The anterior hypothalamus and preoptic area has its blood supply partly from arteries arising from the anterior cerebral artery, and partly from branches of the anterior choroidal artery, a ramification from the internal carotid artery [11,28]. The intraluminal filament in the internal carotid artery must block the ipsilateral ante-
rior choroidal artery, and create ischemia in its target areas (including also part of the amygdala). However, the filament may also compromise collateral flow from the anterior or posterior cerebral arteries. These factors must obviously be taken into account when this model is used to assess the efficacy of drugs in ameliorating damage due to MCA occlusion. We wish to emphasize that our observation is not unique. Thus, although Chen et al. [8] did not comment on the findings, their histopathological data suggest that cell necrosis occurred in the hypothalamic centers discussed presently. Since temperature results have not been reported by others, we do not know if our findings are applicable to filament occlusion techniques in general. Thus, other types of filaments may not interfere with blood flow to hypothalamic temperature-regulating centers, and it is not certain that all rat species have the vascular anatomy which may be a requisite for the effects observed. It is clear that continued anesthesia, with contol of temperature, prevents the rise in temperature observed in our model. Thus, if the experiments are confined to a 4 - 6 h observating period, and if anesthesia is maintained, temperature never becomes a problem [26]. However, since 'maturation' of ischemic damage may take a longer time, especially if the ischemia is transient, it is desirable to employ a long-term recovery period. Then, temperature control becomes an important issue. Our experience is that air cooling of awake animals is less labor-intensive than prolonged anesthesia and, besides, it avoids the potentially complicating factor of continued anesthesia. We have not measured brain temperature in the experiments in this study. However, MCA occlusion reduces CBF in a relatively small part of the brain, and the ischemic focus is surrounded by perfused tissue, hence we assume the brain temperature to reflect the changes in body temperature. As discussed in the introduction, hyperthermia is concievably a confounding factor in the analysis of factors contributing to ischemic brain damage. For example, hyperthermia may obliterate the effect of drugs, normally ameliorating ischemic brain damage. Such drugs encompass D M T U , an established free radical scanvenger [16] and MK-801, an N M D A antagonist which is known to ameliorate focal ischemic lesions induced by MCA occlusion [3,26]. Thus, although the effect of MK-801 is well documented, the drug fails to ameliorate the tissue lesion caused by the present type of MCA occlusion (Memezawa, Zhao et al., in preparation). It is tempting to conclude that this is because hyperthermia has an overriding influence, preventing the therapeutic potential of various drugs to be expressed. Clearly, although the intraluminal filament technique for MCA occlusion is attractive (it requires no craniectomy, and allows easy reperfusion), it may have
Q. Zhao et al. / Brain Research 649 (1994) 253-259
some unusual complications. One of these, described in the present article, is intra- and post-ischemic hyperthermia. This complication can be avoided by anesthesia a n d / o r cooling of awake animals. However, it remains to be assessed if other potential complications, such as endothelial cell damage, thrombocyte adhesion, and platelet embolization, can be as easily avoided.
Acknowledgements This study was supported by Swedish Medical Research Council (Grant No. B93-14X-00263-29C), the National Institute of Health, US Public Health Service (Grant No. 5R01 NS. 07838-23), and the Medical Faculty of Lund University.
References [1] Auer R.N., Wieloch, T., Olsson, Y. and Siesj6, B.K., Hypoglycemic brain injury in the rat: correlation of density of brain damage with the EEG isoelectric time. A quantitative study, Diabetes, 33 (1984) 1090-1098. [2] Barr M.L., The Human Nervous System. An Anatomical Vitewpoint, Harper & Row Inc., Hagerstown, Maryland, USA, 1974, p. 190. [3] Buchan, A.M., Do NMDA antagonists protect against cerebral ischemia: are clinical trials warranted? Cerebravasc. Brain Metab. ReL,., 2 (1990) 1-26. [4] Buchan, A. and Pulsinelli, W.A., Hypothermia but not the N-methyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia, J. Physiol., 253 (1990) H869-H873. [5] Busto, R., Dietrich, W.D., Globus, M.Y.T. and Ginsberg, M.D., Postischemic moderate hypothermia inhibits CA-I hippocampal ischemic neuronal injury, Neurosci. Lett., 101 (1989) 199-304. [6] Busto, R., Dietrich, W.D., Globus, M.Y.T., Valde's, I., Scheinberg, P. and Ginsberg, M.D., Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cereb. Blood Flow Metab., 7 (1987) 729-738. [7] Chen, H., Chopp, M. and Welch, K.M.A., Effect of mild hyperthermia on the ischemic infart volum after middle cerebral artery occlusion in the rat, Neurology, 41 (1991) 1133-1135. [8] Chen, H., Chopp, M., Zhang, Z.G. and Garcia, J.H., The effect of hypothermia on transient middle cerebral artery occlusion in the rat, J. Cereb. Blood Flow Metab., 12 (1992) 612-628. [9] Connolly, J.E., Boyd, R.J. and Calvin, J.W., The protective effect of hypothermia in cerebral ischemia: Experimental and clinical application by selective brain cooling in the human, Surgery, 52 (1962) 15-24. [10] Corbett, D., Evans, S., Thomas, C., Wang, D. and Jonas, R.A., MK-801 reduced cerebral ischemic injury by inducing hypothermia, Brain Res., 514 (1990) 300-304. [11] Coyle, P., Arterial patterns of the rat rhinencephalon and related structures, Exp. Neurol., 14 (1957) 671-690. [12] Dietrich, W.D., Busto, R., Valde's, I. and Loor, Y., Effects of normothermic versus mild hyperthermic forebrain ischemia in rats, Stroke, 21 (1990) 1318-1325. [13] Folbergrovfi, J., Memezawa, H., Smith, M.-L. and Siesj6, B.K.,
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Focal and perifocal changes in tissue energy state during middle cerebral artery occlusion in rats, J. Cereb. Blood Flow Metab., 12 (1992) 25-33. [14] Heimer, L., The Human Brain and Spinal Cord. Functional Neuroanatomy and Dissection Gguide, Springer Verlag, New York, 1983. [15] Hirsh, H.A., Bolte, A., Schandig, A. and T6nnis, D., 0 b e r die Wiederbelebung des Gehirns Bei Hypothermia, Pfliigers Arch., 265 (1957) 328-336. [16] Kiyota, Y., Pahlmark, K., Memezawa, H., Smith, M.-L. and Siesj6, B.K., Free radicals and brain damage due to transient middle cerebral artery occlusion: the effect of dimethylthiourea, Exp. Brain Res., in press. [17] Koizumi, J., Yoshida, Y., Nakazawa, T. and Ooneda, G., Experimental studies of ischemic brain edema: 1. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area, Jpn. J. Stroke, 8 (1986) 1-8. [18] Kuroiwa, T., Bonnekoh, P. and Hossmann, K.A., Prevention of postischemic hyperthermia prevents ischemic injury of CA-1 neurons in gerbils, J. Cereb. Blood Flow Metab., 10 (1990) 550-556. [19] Longa, E.L., Weinstein, P.R., Carlson, S. and Cummins, R., Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke, 20 (1989) 84-91. [20] Memezawa, H., Minamisawa, H., Smith, M.-L., Siesj6, B.K., Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat, Exp. Brain Res., 89 (1992) 67-78. [21] Memezawa, H., Smith, M.-L. and Siesj6, B.K., Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats, Stroke, 23 (1992) 552-559. [22] Minamisawa, H., Nordstr6m, C.H., Smith, M.-L. and Siesj6, B.K., The influence of mild body and brain hypothermia on ischemic brain damage, Z Cereb. Blood Flow Metab., 10 (1990) 365-374. [23] Minamisawa, H., Smith, M.-L. and Siesj6, B.K., The effect of mild hyperthermia and hypothermia on brain damage following 5, 10, and 15 minutes of forebrain ischemia, Ann. NeuroL, 28 (1990) 26-33. [24] Morikawa, E., Ginsberg, M.D., Dietrich, W.D., Duncan, S.K., Globus, M.Y.T. and Busto, R., The significance of brain temperature in focal cerebral ischemia: histopathological consequences of middle cerebral artery occlusion in the rat, .L Cereb. Blood Flow Metab., 12 (1992) 380-389. [25] Nagasawa, H. and Kogure, K., Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion, Stroke, 20 (1989) 1037-1043. [26] Park, C.K., Nehls, D.G., Graham, D.I., Teasdale, G.M. and McCulloch, J., The glutamate antagonist MK-801 reduces focal ischemic brain damage in the rat, Ann. Neurol., 24 (1988) 543-551. [27] Sakurada, O., Kennedy, C., Jehle, J., Brown, J.D., Carbin, G.L. and Sokoloff, L., Measurement of local cerebral blood flow with iodo-14C-antypyrine, Am. J. Physiol., 234 (1978) H59-H66. [28] Scremin, O.U., The vascular anatomy of the rat's hypothalamus in stereotaxic coordinates, J. Comp. Neurol., 139 (1970) 31-52. [29] Siesj6, B.K., Brain Energy Metabolism, John Wiley & Sons, Ltd., New York, 1978, pp. 324-344. [30] Smith, M.-L., Kalimo, H., Warner, D.S. and Siesj6, B.K., Morphological lesions in the brain preceding the development of postischemic seizure, Acta. Neuropathol. (Berl.), 76 (1988) 253264. [31] Xue, D., Huang, Z.G., Smith, K.E., Buchan, A.M., Immediate or delayed mild hypothermia prevents focal cerebral infarction, Brain Res., 587 (1992) 66-72.
Brain Research 649 (1994) 253-259
Research Report
Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament Qi Zhao, Hajime Memezawa, Maj-Lis Smith *, Bo K. Siesj6 Laboratory for Experimental Brain Research, UniversityHospital, University of Lund, S-221 85 Lund, Sweden (Accepted 15 March 1994)
Abstract
The present experiments were designed to study under what circumstances middle cerebral artery (MCA) occlusion by an intraluminal filament technique leads to hyperthermia and what the mechanisms are. We found that permanent MCA occlusion by this technique lead to a rise in body (core) temperature to 39.0-39.5°C during the first 2-4 h, and to sustained hyperthermia thereafter (38.5-39.0°C). After 2 h of transient MCA occlusion hyperthermia could only be avoided if anesthesia (with control of temperature) was maintained for 2 h of ischemia and 1 h of recirculation or, in unanesthetized animals, if external cooling was maintained for 2 h of ischemia and 2 h of recirculation. Control of temperature only during ischemia did not prevent a postischemic rise in temperature. One hour of MCA occlusion had less effect on body temperature. Results are presented which suggest that the hyperthermia observed is due to an interference, by the intraluminal filament, of circulation to hypothalamic centers regulating body temperature. It is speculated that the hyperthermia induced may blunt or obliterate the effect of drugs, normally considered to ameliorate brain damage due to focal ischemia.
Key words: Focal ischemia; MCA occlusion; Hyperthermia; Temperature control; Hypothalamic damage; Rat
I. Introduction
It has been known for decades that hypothermia protects the brain against global ischemic/hypoxic insuits [5,9,15,29], and that even in the absence of ischemia, hyperthermia can cause brain damage [29]. During recent years, it has become increasingly clear that even small decreases in temperature can ameliorate damage due to global or forebrain ischemia [6,22,23], while equally moderate rises in body (and brain) t e m p e r a t u r e aggravates damage [6,12,23]. This means that accidental variation in temperature can become a confounding factor, complicating interpretations of experiments on drug treatment. For example, the ameliorating effects of dizocilpine maleate (MK801) in gerbil ischemia could be largely attributed to drug-induced hypothermia [4,10], and halothane anesthesia was found to ameliorate damage by preventing spontaneous postischemic hyperthermia [18].
* Corresponding author. Fax: (46) (46) 151480. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
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Recent data demonstrate that temperature influences focal ischemic damage due to middle cerebral artery (MCA) occlusion to a comparable degree [7,8,24,31]. In two studies, a rise in temperature to 39-40°C clearly increased infarct size [7,31]. In one of these, hyperthermia was detrimental even when instituted after transient ischemia [7]. A technique for inducing transient M C A occlusion by an intraluminal filament ('embolus') is now gaining increasing popularity [7,8,19,25]. This technique has the advantage that it does not require a craniectomy with the associated operative trauma. We have published a modification of this technique, describing focal and perifocal changes in blood flow and labile metabolites, as well as histopathologic changes after various occlusion times [13,20,21]. However, we failed to find that dimethylthiourea ( D M T U ) ameliorated tissue damage and observed a spontaneous rise in body temperature to 39.0-39.5°C due to transient (2 h) ischemia [16]. In this study, we have assessed t e m p e r a t u r e changes following transient or permanent M C A occlusion, ex-
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plored its possible cause(s), and devised a technique to curtail the hyperthermia.
2.2. Middle cerebral artery occlusion We used the intraluminal filament technique described by Koizumi et al. [17] to occlude the MCA. In the first part of the study, we employed the filament (nylon monofilament fishing thread, O.D. 0.28 mm) described by Memesawa et al. [20,21] but inserted the occluder from the external carotid artery according to Chen et al. [7]. In the latter part of the study, we used a modification of Koizumi's type of filament, i.e. a silicone rubber-coated nylon fishing line. This occluder was inserted from the common carotid artery, which was ligated proximal to the incision made for the occluder.
2. Materials and methods 2.1. Animal preparations Male Wistar rats (M¢llegaard's Breeding Center, Copenhagen, Denmark), weighing 310-340 g, were used. Animals were fasted overnight but had free access to water. After induction of anesthesia with 3-3.5% halothane, the rats were orally intubated. Operation was performed under mechanical ventilation with 1.0-1.5% halothane. A PE-50 polyethylene catheter was inserted into a tail artery for blood pressure recording and blood sampling. When the middle cerebral artery (MCA) occlusion (see below) had been performed, some animals were extubated and allowed to wake up. Others were maintained under continued anesthesia with 0.5% halothane. In these, Norcuron® (vecuronium bromide, 2 r a g . h - t ) was infused to maintain muscle relaxation.
2.3. Measurement and regulation of temperature During operation, an electrical temperature probe was inserted 5-6 cm into the rectum to record core temperature, which was regularly maintained at 37°C. In some of the animals a telemetric sensor (Data Sciences, St. Paul, Minnesota, USA) was placed intraperitoneally and secured by a suture to the gastric wall. In these animals, temperature was recorded telemetrically for 24 h. Some of
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Q. Zhao et al. / Brain Research 649 (1994) 253-259 42
damage to hypothalamic centers. In order to assess whether such damage could be verified we re-examined autoradiographic films from a previous study of cerebral blood flow (CBF) changes following M C A occlusion [20]. The CBF m e a s u r e m e n t s were made after 60 min of M C A occlusion (n = 6), and 15 min after the circulation had been restored after a period of 60 min of occlusion (n = 6). The technique of Sakurada et al. [27] was used, with [14C]iodoantipyrine as the diffusible tracer, for details see [20]. In addition, a series of animals with 2 h of M C A occlusion followed by 2 - 7 days of recirculation (n = 12) was examined by light microscopy for histological hypothalamic damage (Zhao et al., unpublished results). All sections were obtained from animals whose brains were fixed by perfusion with buffered formaldehyde, and the sections were stained with acid fuchsin and Celestine blue as previously described [1,30].
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There were three series of experimental animals, with either p e r m a n e n t M C A occlusion or transient occlusion of 1 or 2 h duration. In p e r m a n e n t ischemia (n = 4), the temperature was not regulated after M C A occlusion. In the 1 h ischemia group (n = 5), the animals were kept under continued anesthesia during ischemia and for the first hour of recirculation. In the experiments with 2 h ischemia, we had four subgroups: (i) the temperature was not regulated after M C A occlusion (n = 4), (ii) the animals were cooled by the air-cooling method only during ischemia (n = 8), (iii) the animals were kept under anesthesia during ischemia and for the first hour of recirculation (n = 4), and (iv) the animals were cooled by the aircooling method during ischemia and for the first 2 h of recirculation (n = 10). In all animals, temperature during anesthesia was main'tained at 37.0-37.5°C. Awake animals were either allowed to attain their spontaneous temperature, or were air-cooled for different periods (see above) in an attempt to maintain temperature close to normal levels also in the awake state.
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the animals were allowed to spontaneously adjust their temperature in cages at room temperature ( ~ 22°C), while others were cooled so that their core temperature was maintained close to normal levels. For the latter purpose, we developed an air cooling system to curtail the spontaneous rise in body temperature. The system consisted of three parts: a source of compressed air, a device to cool the air (a plastic tube spiral in ice water), and outlets in all four corners of the cage, allowing the cold air to circulate in the cage. The temperature inside the cage was regulated to between 6 and 15°C as required.
3. Results
3.1. Permanent M C A occlusion
2.4. Assessment of hypothalamic damage W h e n it was shown that MCA-occluded animals developed hyperthermia, the suspicion arose that the filament caused irritation or
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middle cerebral artery occlusion, but was then not regulated further. As Fig. 1 shows, temperature rose to 39.0-39.5°C during the first 2 h of ischemia. The observation confirms our previous data for 2 h of ischemia [16], but extend the obervation to 24 h of ischemia. As observed from the figure, mean core temperature was 38.5-39.0°C for the entire period of ischemia, the highest temperature being encountered during the first 7 h. Thus core temperature during occlusion is 0.51.0°C higher than that recorded in sham-operated animals. In two additional animals with telemetric recording, we kept the rats under anesthesia and maintained the temperature at 37-37.5°C for 2 and 3 h, respectively. In both cases, body temperature rose to 39.0-39.5°C when the anesthesia was discontinued. These results suggest that hyperthermia is a seemingly inevitable consequence of intraluminal filament occlusion of the MCA.
3.3. MCA occlusion for two hours
As already stated, 2 h of MCA occlusion in the awake and freely moving animal leads to an unacceptable rise in body temperature. Fig. 3 shows that the hyperthermia developing during the 2 h period of ischemia persists for, at least, the first 2 h of recirculation. Therefore, we next studied the effect of cooling the freely moving animals during the period of ischemia. As Fig. 3 shows, this prevented an intraischemic rise in temperature. However, when cooling was discontinued temperature rose to about 39.0°C. We found that two procedures prevented the postischemic hyperthermia. One was to keep the animals
3.2. MCA occlusion for one 1 hour
After 1 h transient ischemia, the rectal temperature rose to near 39°C at the end of ischemia if the animal was allowed to wake up after MCA occlusion. However, if the animal was kept under anesthesia and the temperature was maintained at ~ 37°C during the ischemia and for the first hour of recirculation, hyperthermia was not oberserved during the 24-h period of recirculation (Fig. 2). We concluded from this that MCA occlusion for 1 h, with the intraluminal filament technique, can be induced without a rise of temperature if the temperature is maintained constant ( ~ 37°C) during ischemia and the first hour of recirculation.
Fig. 6. A representative autoradiogram from a local cerebral blood flow experiment, showing ischemic areas (white) in an animal with 1 h of M C A occlusion. Note the hypothalamic ischemia (arrowhead).
Q. Zhao et al. / Brain Research 649 (1994) 253-259
anesthetized for the period of ischemia and the first hour of recirculation (Fig. 4), the other, applicable to unanesthetized animals, involved external cooling during ischemia and during the first 2 h of recirculation (Fig. 5). However, this procedure worked in only 7/10 animals; in the remainder (3/10), temperature rose to about 39°C following discontinuation of cooling.
257
3.4. Possible causes of ischemic /post ischemic hyperthermia As the hyperthermia developed rapidly (1-2 h), it seemed unlikely to reflect an inflammatory reaction. Since it was possible that the temperature regulating center in the hypothalamus was affected, we examined
Fig. 7. Representative photomicrograph from hypothalamus of an animal subjected to 2 h of M C A occlusion followed by 2 days of recovery before perfusion fixation with formaldehyde. A: shows a low magnification (bar = 500 ~zm) of hypothalamus at the level of bregma - 2 mm. Framed parts of the left and right ( M C A occluded side) anterior hypothalamus are shown below as (B) and (C), respectively. In (B) the tissue has a normal appearance, while in (C) all neurons are necrotic and the neuropil spongy (bars = 100/zm). Celestine b l u e / a c i d fuchsin.
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CBF autoradiograms and histological sections as described in section 2. Middle cerebral artery occlusion with the intraluminal filament led to a reduction of CBF in preoptic and hypothalamic areas, i.e. areas which are outside the distribution territory of the MCA. The blood flow in the preoptic area at 1 h of ischemia was 5.11 _+5.5% of that in the contralateral side (range 0-15c/~), and in the anterior hypothalamus (Fig. 6) corresponding values were 7.2 _+ 7.3% (range 0-19%). This reduction in blood flow during ischemia is of a similar magnitude as is seen in the ischemic focus, consisting of lateral caudoputamen (CBF 4.4%) and overlying cortex (CBF 7.2%) [20]. Recirculation was achieved in both preoptic area and hypothalamus when the filament was withdrawn after 60 rain of MCA occlusion, as evidenced from CBF values ranging between 0.50 to 1.42 m l . g l . m i n ~. These values were around 50-60% of contralateral CBF values, and similar to postischemic flow in other structures [20]. Histological examination of perfusionfixed brains revealed ischemic damage in preoptic area a n d / o r anterior hypothalamus in 10 of 12 animals subjected to 2 h of MCA occlusion followed by 2 - 7 days of reperfusion before fixation for histology (Fig. 7).
4. Discussion
The present results document a complication of the intraluminal filament technique for MCA occlusion, namely hyperthermia. This hyperthermia develops during the first 2 h of ischemia [16], and is sustained for at least 24 h if the ischemia lasts for 2 h or longer. Since we have not been able to find similar data in the literature, it seems that the hyperthermia is 'specific' for the intraluminal filament MCA occlusion technique. Accordingly, it appeares profitable to look for factors which are related to the introduction of the filament. Our results suggest that hyperthermia occurs in both types of insertion of occluder (e.g. from the external carotid artery and from the common carotid artery), and with both types of filament (e.g. a nylon monofilament fishing thread and a silicone rubbercoated nylon fishing line), because the filament interferes with the circulation to hypothalamic centers regulating temperature. The anterior hypothalamus and preoptic area often suffers ischemic damage with the intraluminal filament technique of MCA occlusion if the occlusion time is 2 h or more [8,20,21]. Lesions in this area are known to cause hyperthermia [2,14]. The anterior hypothalamus and preoptic area has its blood supply partly from arteries arising from the anterior cerebral artery, and partly from branches of the anterior choroidal artery, a ramification from the internal carotid artery [11,28]. The intraluminal filament in the internal carotid artery must block the ipsilateral ante-
rior choroidal artery, and create ischemia in its target areas (including also part of the amygdala). However, the filament may also compromise collateral flow from the anterior or posterior cerebral arteries. These factors must obviously be taken into account when this model is used to assess the efficacy of drugs in ameliorating damage due to MCA occlusion. We wish to emphasize that our observation is not unique. Thus, although Chen et al. [8] did not comment on the findings, their histopathological data suggest that cell necrosis occurred in the hypothalamic centers discussed presently. Since temperature results have not been reported by others, we do not know if our findings are applicable to filament occlusion techniques in general. Thus, other types of filaments may not interfere with blood flow to hypothalamic temperature-regulating centers, and it is not certain that all rat species have the vascular anatomy which may be a requisite for the effects observed. It is clear that continued anesthesia, with contol of temperature, prevents the rise in temperature observed in our model. Thus, if the experiments are confined to a 4 - 6 h observating period, and if anesthesia is maintained, temperature never becomes a problem [26]. However, since 'maturation' of ischemic damage may take a longer time, especially if the ischemia is transient, it is desirable to employ a long-term recovery period. Then, temperature control becomes an important issue. Our experience is that air cooling of awake animals is less labor-intensive than prolonged anesthesia and, besides, it avoids the potentially complicating factor of continued anesthesia. We have not measured brain temperature in the experiments in this study. However, MCA occlusion reduces CBF in a relatively small part of the brain, and the ischemic focus is surrounded by perfused tissue, hence we assume the brain temperature to reflect the changes in body temperature. As discussed in the introduction, hyperthermia is concievably a confounding factor in the analysis of factors contributing to ischemic brain damage. For example, hyperthermia may obliterate the effect of drugs, normally ameliorating ischemic brain damage. Such drugs encompass D M T U , an established free radical scanvenger [16] and MK-801, an N M D A antagonist which is known to ameliorate focal ischemic lesions induced by MCA occlusion [3,26]. Thus, although the effect of MK-801 is well documented, the drug fails to ameliorate the tissue lesion caused by the present type of MCA occlusion (Memezawa, Zhao et al., in preparation). It is tempting to conclude that this is because hyperthermia has an overriding influence, preventing the therapeutic potential of various drugs to be expressed. Clearly, although the intraluminal filament technique for MCA occlusion is attractive (it requires no craniectomy, and allows easy reperfusion), it may have
Q. Zhao et al. / Brain Research 649 (1994) 253-259
some unusual complications. One of these, described in the present article, is intra- and post-ischemic hyperthermia. This complication can be avoided by anesthesia a n d / o r cooling of awake animals. However, it remains to be assessed if other potential complications, such as endothelial cell damage, thrombocyte adhesion, and platelet embolization, can be as easily avoided.
Acknowledgements This study was supported by Swedish Medical Research Council (Grant No. B93-14X-00263-29C), the National Institute of Health, US Public Health Service (Grant No. 5R01 NS. 07838-23), and the Medical Faculty of Lund University.
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