Asian Cardiovasc Thorac Ann 1999;7:267-271
© 1999 Asia Publishing EXchange Pte Ltd
Taurine Potentiates the Efficacy of Hypothermia
Nobuhisa Ohno, MD,
Koho-Julio Miyamoto, MD, PhD,
Tadaomi-Alfonso Miyamoto, MD
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Research Department Kokura Memorial Hospital Kitakyushu-shi, Fukuoka, Japan
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For reprint information contact: Tadaomi-Alfonso Miyamoto, MD Tel: 81 93 951 6582 Fax: 81 93 921 8497 Research Department, Kokura Memorial Hospital, 1-1 Kifune-cho, Kokura-kitaku, Kitakyushu-shi, Fukuoka 802, Japan.
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Abstract
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To assess the protective effect of exogenous taurine on the central nervous system, spinal cord ischemia was induced for 60 minutes in 16 rabbits randomized into 3 groups. Group 1 (n = 5) had hypothermia targeted to 30.5°C and 10 mmol•kg–1 taurine, group 2 (n = 5) had hypothermia targeted to 29.5°C, and group 3 (n = 6) had hypothermia targeted to 30°C. Group 1 was cooled to 30.6 ± 0.07°C and group 2 was cooled to 29.4 ± 0.07°C; both had total functional recovery (rabbits able to keep normal posture, to walk, and to hop) within 6 hours of reperfusion. None of the group 3 animals that were cooled to 29.9 ± 0.05°C recovered function. It was concluded that taurine combined with hypothermia protected the spinal cord from 60 minutes of ischemia at a temperature that could not otherwise ensure protection. The protective effect contributed by taurine was equivalent to 1.2°C.
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Introduction
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Despite significant advances in surgery, paraparesis or paraplegia due to concomitant spinal cord ischemia remains a serious complication of treatment of descending thoracic and thoracoabdominal aortic diseases. A number of investigators have explored the mechanisms of spinal cord injury; several methods of physical and pharma-cological protection have been proposed accordingly.1 Rokkas and colleagues2 proposed profound hypothermic circulatory arrest. Herold and colleagues3 reported complete protection by regional infusion of hypothermic saline and adenosine. Adenosine and taurine have been found to flood any area of the central nervous system during ischemia.4,5 Taurine, only present in the animal kingdom, is the most abundant amino acid in spite of not being a component of any protein and it is well known for its anticonvulsant, calcium-modulating, and antioxidant effects.6,7 Taurine is released by activation of adenosine receptors.8,9 Although it has not been demonstrated in in-vivo preparations, protective effects of exogenous taurine have been shown in hippocampal slices.10 To test the hypothesis that systemic intravenous administration of exogenous taurine could be used as an innocuous reliable agent to potentiate any protective strategy, the well-known protective properties of hypothermia were compared to hypothermia combined with taurine in a rabbit model of spinal cord ischemia for a practically meaningful period (60 minutes) that was long enough to make definitive observations.
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Materials and Methods
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Experimental protocols were approved by the institution's Animal Care Committee. Animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals approved by the Council of the Japanese Physiologic Society. Experiments were per-formed on 16 male Japanese White rabbits (2.7 to 3 kg). Volatile anesthesia (65% nitrous oxide with 30% to 35% oxygen and 1.5% to 2% isoflurane) was used without muscle relaxants. Spinal cord ischemia in rabbits is a well-established model.3,11,12 All animals were subjected to a 60-minute period of aortic occlusion by temporary placement of vascular clamps on the abdominal aorta distal to the left renal vein level and on the posterior mesenteric artery, to prevent collateral circulation, through midline laparotomy. Heparin sulfate (500 units•kg–1) was administered intravenously prior to aortic occlusion. Systemic (esophageal and rectal) temperatures via a thermocouple probe (Mon-a-therm, Model 6500; Mallinckrodt, St Louis, MO, USA) and the proximal aortic pressures (Polygraph system; Nihon Kohden, Tokyo, Japan) were monitored. The cannula (18F polyethylene tubing) introduced into the central stump of the left carotid artery also served to obtain blood samples for arterial blood gases. A venous access line (16F polyethylene tubing) advanced into the right atrium via the right jugular vein was secured. Metabolic acidosis was corrected with infusion of sodium bicarbonate only after declamping the aorta.
Rabbits were placed supine on a specially designed table equipped with a heat exchanger system that allowed temperature control to within 0.2 to 0.3°C of the target temperature. The spinal cord temperature (measured by placing a thermocouple probe into the epidural space in the ischemic region) in 2 animals was found to be stable during the entire ischemic period and closer to the esophageal temperature (measured 2 cm above the diaphragm) at the time the aorta was clamped (at the nadir of the after-drop) rather than the rectal temperature (Figure 1
). Thus, for the purpose of this study, only esophageal temperatures were used in the remaining animals to avoid compounding interpretation of neurologic function by hematomas or compression that could potentially be induced by the introduction of a thermo-couple probe into the epidural space. To minimize thermocouple variability (± 0.2°C), the same temperature probe was used for all experiments.
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Figure 1. Temperatures recorded during cooling, aortic crossclamping, and rewarming. Although the spinal cord temperature during cooling and rewarming lags slightly behind the esophageal temperature, it closely reflects the spinal cord temperature during aortic crossclamping if the crossclamp is applied at the nadir of the esophageal temperature curve.
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After anesthetic induction, the trachea was secured (tracheotomy) to maintain general anesthesia at the concentration used in humans. The respiratory rate was controlled with a mechanical ventilator (SN-490-5-T2; Shinano, Tokyo, Japan) to maintain end-expiratory carbon dioxide concentrations of 4.5% to 5% (Respina IH26; San-ei, Tokyo, Japan) during normothermia and closer to 5.5% to 6% (equivalent to pH-stat management) during hypothermia, especially below 32°C, which allowed blood pressure control without the need of vasopressors. Surface cooling was induced with bags of iced water placed on the chest and abdomen after all neck surgical preparations were completed. Allowing for the after-drop, the ice-water bags were sequentially removed (abdominal bags first) at a temperature 1.5°C to 1.8°C above the target temperature so that the nadir of the after-drop would coincide with the target temperature when the aorta was clamped. The target temperature was maintained during the ischemic period by controlling the room and table temperatures. Rewarming by means of the heat-exchanger table that excluded a 5-cm width of the paraspinal region, followed the release of the vascular clamps and abdominal closure.
In early studies, recovery beyond 360 minutes after reperfusion could not be documented, therefore, neurologic function was assessed 360 minutes after reperfusion or 270 minutes after termination of anesthesia. Since any strategy to be used in humans should ideally be 100% reliable, for the purposes of this study, a simple assessment in terms of whether the rabbit was totally recovered or protected (able to keep the normal posture, to walk and to hop, an objective endpoint easy to evaluate without observer bias), or unprotected (anything less than full recovery) was considered to be most appropriate, rather than trying to assess less than perfect clinical recovery using scores that are subjective.
To determine the temperature required to protect during 60 minutes of ischemia with taurine supplementation, pilot studies with various temperatures demonstrated that taurine (10 mmol•kg–1) administered with an esophageal (but not rectal) temperature of 30.5°C was consistently protective, whereas hypothermia of 30°C without taurine would uniformly fail to protect spinal cord function. Therefore in group 1 animals (n = 5), the temperature was targeted to 30.5°C and taurine 10 mmol•kg–1 was administered by a drip, one third of it as soon as the venous line was available, one third during the cooling period (30 to 40 minutes), and the remaining one third 15 minutes before declamping.
It was felt that if 30°C was not protective, it would be a futile exercise to cool only to 30.5°C just to make statistical comparison easy. Therefore, animals with hypothermia alone were given an equivalent volume of Ringer's lactate solution in a similar schedule and randomly cooled to various levels of hypothermia (29°C to 30.1°C) that resulted in 2 groups with a totally different neurologic outcome (group 2 with full recovery and group 3 that failed to recover) but were otherwise identically managed.
All values were expressed as mean ± standard deviation and analyzed using the commercially available Stat View Version 4 software (Abacus Concepts, Inc., Berkeley, CA, USA) by Bonferroni-Dunn's test or one-way analysis of variance for repeated measurements. Values of p less than 0.05 were considered to be significant.
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Results
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All animals tolerated the period of aortic occlusion and awoke from anesthesia to allow postoperative neurologic evaluation. No significant differences in blood pressure were observed during the ischemic period. In spite of the generally lower (close to 20 mm Hg) mean blood pressure in group 1 animals compared to the other groups during the cooling and rewarming phases (reaching statistical significance at some points) or after termination of anesthesia, all recovered neurologically.
All group 1 and group 2 rabbits recovered completely although group 1 were subjected to a 1.2°C-higher temperature than group 2 (p < 0.0001 ). In contrast, all group 3 animals had less than full recovery of neurologic function (Table 1). Two animals in each group were allowed to survive for 24 hours; those that were functionally recovered 6 hours after reperfusion, regardless of whether they had received taurine, remained recovered 24 hours later, whereas those with obvious neurological impairment at 6 hours did not show further improvement 24 hours later and changes, if any, were observed to be for the worse. No attempts were made to analyze 24-hour survival data since not all animals were observed for 24 hours.
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Discussion
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Because hypothermia reduces energy consumption of neural tissue in direct proportion to the degree of cooling, profound hypothermic circulatory arrest has been used as a very effective neurologic protective measure.2,3 In an effort to obtain protective effects similar to profound hypothermia but at a higher temperature, a number of pharmacologic agents such as vasodilators (intrathecal papaverine), cell membrane stabilizers (chlorpromazine, local anesthetics, and steroids), calcium channel blockers, excitatory amino-acid antagonists, mannitol, magnesium, and other free-radical scavengers have been investigated with variable results.11,13,14 However, in many studies the temperatures have been poorly controlled.
A ventilation strategy close to pH-stat management of cardiopulmonary bypass was used in this study because pH-stat acid-base management during hypothermic perfusion was recently reappraised in children and found to be superior.15 This is not surprising in view of the improved oxygen delivery over the widely used alpha-stat strategies that impair oxygen delivery. Whether alpha-stat management of the acid-base balance and cardio-pulmonary bypass using nonpulsatile pumps would reproduce these results obtained in a pulsatile model was not addressed in this study.
Adenosine offers the possibilities of activating mainly A1 receptors when given in small doses or mainly A2 receptors when given in large doses. A1 receptors decrease the release of ischemia-induced excitatory neurotransmitters by decreasing the activity-evoked membrane depolari-zation. A2 receptors release taurine, induce vasodilation, inhibit platelet aggregation, neutrophil activation, and subsequent free radical production.8,16,17 However, when administered in sufficient doses to be neurologically protective, severe hypotension is induced. For this reason, use of adenosine has not met with general acceptance. To circumvent the vascular effects of systemic administration, adenosine was regionally infused with hypothermic saline and although effective, hypotension could not be averted.3 Although taurine was found to have a mild vasodilating effect at the dose used, the slight hypotension did not prevent recovery of spinal cord function.
The assertion of Rokkas and colleagues2 that "the role of taurine in the spinal cord is not clear but it does not appear that taurine plays any significant role in modulating the ischemic injury" seems to be unfounded, since taurine was never administered to actually prove or disprove such an effect. Our results indicate otherwise; indeed they confirm the widely accepted in vitro protective effects of taurine in an in-vivo animal model of spinal cord ischemia.
Although observations are limited to a very small number, none of the fully recovered animals at 6 hours after reperfusion that were allowed to survive to 24 hours had evidence of neurologic worsening when observed the next day, whether given taurine or not. Therefore, the sole functional clinical evaluation as reported by Vacanti and Ames14 even 6 hours after reperfusion seems to be justified as a screening method to acutely assess the adequacy of protection strategies. Whether late improvement could be expected in animals that had not recovered by 6 hours and whether late (3 to 4 days) neuronal death could be prevented were not addressed in this study. Although apoptosis has been identified as the mechanism of delayed neuronal death in gerbil hippocampus and more recently, in rabbit spinal cord, late neuronal death may not occur if there is adequate protection in the acute phase.12,18 Apoptosis may be activated to get rid of the elements surviving subliminal injury but damaged enough to result in abnormal function in the long term.
The contribution of taurine could be expressed in terms of a temperature equivalent or the temperature difference between that required with taurine and that required during hypothermia without taurine. In this study, although incomplete (since the upper temperature limits compatible with full recovery for both groups 1 and 2 were not explored), the temperature equivalent was 1.2°C. This is a remarkable effect considering the fact that a temperature difference of only 0.5°C sharply divided the protected and unprotected animals by hypothermia alone. Although it has not been scrutinized in detail, if very rigid criteria of full recovery from a rather long period of ischemia are adopted to assess protection, a very narrow critical temperature threshold might result, dividing clearly protected from unprotected animals. It is in agreement with the concept that modest and small temperature differences induced by magnesium and the N-methyl-D-aspartate antagonist MK-801, result in different neurologic outcomes.14,19
It should also be stressed that in any study of a neurologic protective strategy, to meaningfully evaluate the true role played by the strategy per se, not only ischemic time but also temperature must be specified and rigidly controlled during the experiment. This is particularly relevant when shorter (40 minutes) ischemic times are studied, for which even minimal hypothermia alone, often induced by seasonal conditions, could be protective by itself or could potentiate the protective effects of the strategy being studied. Care must be exercised in interpreting studies involving N-methyl-D-aspartate and calcium antagonists that are known to promote a decrease in body temperature, or regional hypothermic perfusion in which the tempera-ture is difficult to control, resulting in uneven temperature distribution and making the results unpredictable and variable.3,11,14,19 The advantages of systemic adminis-tration of taurine in combination with only moderate hypothermia seem to be practical given the milder hematological effects compared to profound levels of hypothermia, as well as potentially offering protection of vital organs other than the central nervous system, such as the heart and liver.20
This study did not address the mechanism, timing (during ischemia or reperfusion), or optimal concentration of taurine. However, our results confirmed that profound hypothermia is not absolutely essential to protect the spinal cord against 60 minutes of ischemia (29.4°C), that taurine supplementation allows protection at an otherwise inadequate temperature (30.6°C), and that the protective effect of taurine was equivalent to at least 1.2°C. This suggests that it might be useful in emergency situations to give taurine intravenously and affords the possibility of extending such protective principles to the entire central nervous system and other vital organs with the same strategy.
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Acknowledgments
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We thank Dr. Hiroshi Takeshita and Dr. Toshihiko Ban, the former and current hospital directors, for their continuous encouragement and support of the research department, and the clinical laboratory staff for blood-gas measurements.
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Abstract
Introduction
