Ann Thorac Surg 2001;71:1591-1595
© 2001 The Society of Thoracic Surgeons
Original article: cardiovascular
Protection of the spinal cord with pentobarbital and hypothermia
Shigeru Kazama, MDa,
Yutaka Miyoshi, MDa,
Masaki Nie, MDa,
Hiroshi Imai, MDa,
Zong Bo Lin, MDa,
Atsushi Kurata, MDa,
Masato Machii, MDa
a Department of Cardiothoracic Surgery, Kitasato University School of Medicine, Kitasato, Sagamihara, Japan
Accepted for publication September 6, 2000.
Address reprint requests to Dr Kazama, Department of Cardiothoracic Surgery, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara 228-8555, Japan
e-mail: ig2s-kzm{at}asahi-net.or.jp
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Abstract
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Background. Ischemic spinal cord damage during thoracic aortic operations has not been eliminated despite application of various adjuncts. We experimentally investigated the protective effects of pentobarbital and hypothermia on the spinal cord subjected to ischemia.
Methods. Among nine groups of 6 rabbits each, groups AI to AIII underwent 20-minute infrarenal aortic occlusion, and groups BI to BVI underwent 40-minute occlusion. Five milligrams per kilogram of pentobarbital was administered to groups AII and BII; 10 mg/kg in groups AIII, BIII, and BVI; 20 mg/kg in group BIV; and none in groups AI, BI, and BV. In groups BV and BVI, hypothermia was induced. Forty-eight hours postoperatively, the motor function of the lower limbs was evaluated.
Results. Statistically significant recovery of motor function was observed in animals in groups AII, AIII, BIII, BIV, BV, and BVI.
Conclusions. Pentobarbital showed dose-dependent protective effects of the spinal cord. Moderate hypothermia alone also showed protective effects. Combined use of pentobarbital and hypothermia resulted in highly significant recovery of spinal cord function.
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Introduction
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Paraplegia that results from spinal cord ischemia remains a serious complication of operations on the thoracic and thoracoabdominal aorta. Various adjuncts have been investigated both experimentally and clinically to minimize this devastating complication. Adjuncts that have been investigated include distal perfusion, hypothermia [1], cerebrospinal fluid drainage [1], reimplantation of major intercostal and lumbar arteries, and administration of various pharmacological agents. However, postoperative spinal cord dysfunction has not been eliminated by application of these techniques. There is still ample need for continuing investigations to further reduce this complication rate.
Barbiturates have been known to protect the brain against ischemic injury. They decrease oxygen consumption, diminish attrition of ATP and phosphocreatine, and limit the extent of edema, and lessen infarction development in the brain subjected to ischemia. Recognition of this has caused us to postulate that barbiturates may exert a protective effect of the spinal cord. To date, there have been only eight sporadic reports in the literature dealing with the effects of barbiturates on spinal cord protection [2–9]. Among the many barbiturates, only thiopental and methohexital were investigated and controversial results have been reported. In our pilot study, pentobarbital, one of the most ubiquitous barbiturates, has emerged as a potent agent for protecting the ischemic spinal cord. In addition, hypothermia has been consistently shown to protect the spinal cord subjected to ischemia [1]. We therefore hypothesized that the combined use of pentobarbital and hypothermia might exert a significant protective effect on the ischemic spinal cord. In this investigation, we intended to test the hypothesis that: (1) pentobarbital has a dose-dependent protective effect of the spinal cord, and (2) combined use of pentobarbital and hypothermia provides significant protection of the spinal cord subjected to prolonged ischemia.
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Material and methods
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Fifty-four Japanese white rabbits weighing between 2.8 and 3.5 kg were premedicated with intramuscular ketamine hydrochloride (30 mg/kg) and anesthetized with GOF (1.5% halothane in a mixture of 1:1 oxygen and nitrous oxide) delivered by a nose cone. The animals maintained spontaneous breathing. The ear artery was cannulated for pressure monitoring and blood sampling. The proximal systemic arterial pressure was recorded at the beginning of the experiment (baseline) and 10 minutes after institution of cross-clamping (during aortic occlusion) with a transducer-recorder system (Nihon Kohden, Tokyo, Japan). The ear vein was also cannulated for administration of fluids and pentobarbital. The base deficit was corrected with administration of sodium bicarbonate. A thermister-mounted probe was inserted into the rectum to monitor the core temperature. After sterilization, the abdomen was entered through a median laparotomy and the abdominal aorta was exposed inferiorly to the left renal artery and down to the bifurcation. The aorta was encircled with a silk ligature distal to the renal artery and proximal to the bifurcation to facilitate secure occlusion. The posterior mesenteric artery was also encircled with a silk ligature. For animals that were to maintain normothermia, a heating pad was employed to keep the core temperature at 38°C to 39°C during the aortic occlusion period. For animals that were to undergo hypothermia, cooling was achieved by external application of ice bags to keep the core temperature at 34°C to 35°C. Each rabbit was anticoagulated with 200 units of heparin before aortic cross-clamping. The aorta was occluded distal to the left renal artery with a pediatric vascular clamp and proximal to the bifurcation with a similar clamp. The posterior mesenteric artery was also occluded with a clamp. After releasing aortic occlusion, the abdomen was closed in two layers and the animals were allowed to recover. Heating pads were used to restore normothermia for the groups of animals that underwent the hypothermic condition. Forty-eight hours postoperatively, the motor function of the lower limbs was evaluated in each animal according to the Tarlov Scale [10] (Table 1) by an independent observer who was unaware of the treatment modality for each animal. The animals were then sacrificed. The lumbar spinal cord was harvested and fixed in a 10% phosphate-buffered formalin solution. Sections were obtained from lower lumbar regions, stained with hematoxylin and eosin, and were studied for ischemic injury using standard light microscopy.
The animals were randomly divided into nine groups with 6 animals in each group (Table 2). Groups AI to AIII underwent 20 minutes of aortic occlusion, and groups BI to BVI underwent 40 minutes of aortic occlusion. Groups AI and BI served as the control, ie, the aorta was occluded under normothermia and with no pentobarbital administration. In groups AII and BII, 5 mg/kg of pentobarbital was administered intravenously over a several-minute period 20 minutes before aortic occlusion. In the same manner, 10 mg/kg of pentobarbital was given in groups AIII, BIII, and BVI, and 20 mg/kg of pentobarbital in group BIV. Groups BV and BVI underwent hypothermia. Group BV did not receive pentobarbital. The treatment modality for each group is presented in Table 3.
The arterial pressure and Tarlov scale data of experimental groups were compared with the Kruskal-Wallis one-way analysis of variance for difference among groups and with the Bonferroni modified LSD test for difference between groups. All analyses were performed using the SPSS software package (SPSS, Tokyo, Japan), and p values less than 0.05 were considered significant.
The experimental protocol was approved by the Kitasato University Institutional Animal Care and Use Committee. All experimental animals received humane care and treatment in accordance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, revised 1996.
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Results
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All animals tolerated the procedure well and were in a stable condition at the time of evaluating motor function of the lower limbs. There was no statistical difference in the baseline proximal arterial pressure in group A nor in B. During aortic cross-clamping, there was no statistical difference in the proximal arterial pressure in group A. There was a statistically significant difference in the proximal aortic pressure during aortic cross-clamping in group B (p = 0.0043). The proximal arterial pressure was significantly lower in groups BV and BVI than in group BI during aortic occlusion (p < 0.05). The lower arterial pressure in groups BV and BVI during aortic cross-clamping was considered to be from hypothermia (Table 3). The Tarlov scales of each experimental group is presented in Table 2.
There was a highly significant difference in motor function outcome in group A (p = 0.0127). Administration of 5 mg/kg of pentobarbital (group AII) resulted in a reduced incidence of severe motor dysfunction (p < 0.05), and administration of 10 mg/kg of pentobarbital (group AIII) resulted in complete recovery (p < 0.05). There was a highly significant difference as well in postischemic motor function in group B (p = 0.0013). Administration of 5 mg/kg of pentobarbital (group BII) did not show significant reduction of motor dysfunction. However, both administration of 10 mg/kg of pentobarbital (group BIII) and 20 mg/kg of pentobarbital (group BIV) resulted in significant reduction of motor dysfunction (p < 0.05). Application of hypothermia alone (group BV) showed significant recovery of motor function (p < 0.05), and combination of hypothermia and 10 mg/kg of pentobarbital (group BVI) showed complete recovery of lower limb motor function (p < 0.05).
Histological study of the spinal cords from animals that suffered paraplegia showed marked coagulative necrosis with macrophage infiltration of the central gray matter (Fig 1). The cords of animals with no motor function deficits showed only minimal cellular infiltrates in the gray matter (Fig 2). These findings were invariable among the same Tarlov scales groups irrespective of treatment modality.
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Fig 1. Histologic section of the lower lumber spinal cord in a rabbit with paraplegia. Extensive central gray matter coagulative necrosis is noted (original magnification x100).
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Fig 2. Central gray matter of the spinal cord taken from an animal in group BVI that showed complete recovery of the lower limb motor function. Neuronal cells are well preserved and there is only minimal cellular infiltration (original magnification x40).
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Comment
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Postoperative paraplegia is caused by the ischemic injury of the spinal cord and is related to thoracic aortic cross-clamping during operations, because critical intercostal arteries supplying the anterior spinal artery may be occluded by this technique. Once paraplegia develops, it is rarely curable; hence, its prevention far outweighs its diagnosis and treatment.
Barbiturates have been studied extensively for their protective effects on the brain. They are known to reduce ischemic damage of the brain by: (1) reducing metabolic rate, (2) edema prevention, (3) free radical scavenging, and (4) vascular smooth muscle relaxation [11, 12]. However, barbiturates’ effects on the spinal cord have been evaluated only sporadically and the results remain contradictory. Although protective effects of barbiturates on the ischemic spinal cord were observed by Naylander [2], Oldfield [3], Cheng [4], and Robertson [5] and their colleagues, their protective effects were denied by Kirshner [6], Naslund [7], Mutch [8], and Matsumoto [9] and their colleagues. These authors examined only thiopental and methohexital, the ultra-short-acting barbiturates, and used relatively short ischemic intervals (only up to 30 minutes) for producing ischemic damage. The dose-effect relationship was not investigated either. We had decided that a more critical experimental protocol was required for evaluating the effects of barbiturates on the spinal cord. We selected pentobarbital as a protective agent, because it is one of the most ubiquitous barbiturates and is longer acting than thiopental. We chose rabbits for this investigation, because the spinal cord of the rabbit is supplied by segmental arterial branches of the aorta; therefore, reproducible ischemic damage is caused by obstructing blood flow at a given segment of the aorta [4]. Infrarenal occlusion of the aorta results in neurological deficits of the lower extremities, but the systemic effect is not severe enough to prevent the survival of most animals. However, it should be noted that this rabbit model of infrarenal aortic clamping cannot be reliably reproduced in other mammalian species. We chose laparotomy and application of proximal and distal occluding clamps as well as posterior mesenteric artery occlusion to mimic clinical aortic operations for producing spinal cord ischemia. The 20-minute aortic occlusion period was chosen because this time interval appears to be the shortest interval at which severe ischemic damage of the spinal cord begins to ensue. An occlusion period of less than 15 minutes was well tolerated by all rabbits, whereas 30-minute occlusion invariably resulted in paraplegia [5, 13]. Rabbits subjected to 20- to 25-minute aortic occlusion variably showed postoperative paraplegia; therefore, this time interval appears to be an appropriate subset to test the effectiveness of a protective measure under a relatively mild ischemic insult. However, most of operations on the thoracic aorta require aortic occlusion of 40 minutes or longer. Therefore, any protective techniques must provide data that they can protect the spinal cord under a 40-minute ischemia condition if they are to be of value in managing patients undergoing thoracic aortic operations. In this investigation, motor function status of the hind limbs was determined at 48 hours postoperatively. In our other studies (not published), it had been observed that motor function of hind limbs seen at 48 hours postoperatively did not change for another 48 hours. Also, we thought it unethical to let the paralyzed animals live for a prolonged period.
The results of the present investigation appeared to demonstrate the effectiveness of pentobarbital and moderate hypothermia as protective measures of the spinal cord subjected to ischemia. Pentobarbital showed dose-dependent protective effects both in the 20-minute and 40-minute aortic occlusion conditions. However, it should be pointed out that the effectiveness of pentobarbital is limited in terms of practical value. Administration of 10 mg/kg pentobarbital resulted in complete neurological recovery when the aorta was occluded for 20 minutes. However, 20 minutes is not long enough for thoracic aortic operations. When the aorta was clamped for 40 minutes, pentobarbital protected the spinal cord at both 10- and 20-mg/kg doses with a statistical significance. But actually, only 4 of 6 animals recovered without neurologic deficits. Hypothermia alone also showed a statistically significant protective effect of the spinal cord subjected to 40 minutes of ischemia. However, the actual number of animals that recovered without neurological deficits was only 2 of 6. Accordingly, hypothermia alone cannot be relied upon to eliminate postoperative paraplegia. When administration of 10 mg/kg of pentobarbital and hypothermia were combined to protect the spinal cord subjected to 40-minute ischemia, all 6 animals recovered without neurological deficits. From the standpoint of clinical applicability, this normal recovery rate is noteworthy.
The proximal arterial pressure during aortic cross-clamping, the major determinant of the spinal cord perfusion pressure, was lower in groups BV and BVI than in controls (group BI). Therefore, improved motor function outcome in groups BV and BVI could not be attributed to increased spinal cord perfusion pressure, which was not the case in this investigation.
Hypothermia has been established as an important adjunct in preserving function of the spinal cord subjected to ischemia. Recently, it was shown that hypothermia prevented release of excitatory amino acids in the ischemic spinal cord [14]. Also, the mechanisms of pentobarbital action are only recently beginning to be understood. The principal effects appear to be on ligand-gated ion channels and interference with excitatory amino acids. Hetzler and Krekow [15] showed in their study using rats that pentobarbital suppression of visual-evoked potentials was augmented by hypothermia. This potentiation of the effects of pentobarbital by hypothermia is in agreement with the results of the present investigation. Inhibition of amino acid release by hypothermia and interference with excitatory amino acids by pentobarbital may combine to effect spinal cord protection subjected to ischemia.
Finally, caution is required concerning the use of barbiturate anesthesia for animals in experiments on spinal cord protection. A number of experimental studies have been done under barbiturate anesthesia [4, 5, 8–10, 14, 16–21]. The results of these studies should be interpreted cautiously because barbiturate anesthesia can modify the outcome of neurological function.
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References
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Abstract
Introduction
