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1Department of Cardiovascular Dynamics, Advanced Medical Engineering Center, and 2Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Osaka, Japan
Submitted 4 June 2006 ; accepted in final form 1 November 2006
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ABSTRACT |
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vagal nerve; sympathetic nerve; cardiac microdialysis; cats
In previous studies from our laboratory, Kitagawa et al. (16) demonstrated that hypothermia attenuated the nonexocytotic NE release induced pharmacologically by ouabain, tyramine, or cyanide. Kitagawa et al. (15) also demonstrated that hypothermia attenuated the exocytotic NE release in response to vena cava occlusion or to local administration of high K+. The effects of hypothermia on the ischemia-induced myocardial interstitial NE release, however, were not examined in those studies. In addition, the effects of hypothermia on the ischemia-induced myocardial interstitial ACh release have never been examined. Because both sympathetic and parasympathetic nerves control the heart, simultaneous monitoring of the myocardial interstitial releases of NE and ACh (14, 31) would help integrative understanding of the autonomic nerve terminal function under hypothermia in conjunction with acute myocardial ischemia.
In the present study, the effects of hypothermia on the ischemia-induced and nerve stimulation-induced myocardial interstitial neurotransmitter releases were examined. We implanted a dialysis probe into the left ventricular free wall of anesthetized cats and measured dialysate NE and ACh levels as indexes of neurotransmitter outputs from the cardiac sympathetic and vagal nerve terminals, respectively. Based on our laboratory's previous results (15, 16), we hypothesized that hypothermia would attenuate the neurotransmitter releases in response to acute myocardial ischemia and to electrical nerve stimulation.
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MATERIALS AND METHODS |
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Animals were cared for in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, approved by the Physiological Society of Japan. All protocols were reviewed and approved by the Animal Subjects Committee of National Cardiovascular Center. Adult cats were anesthetized via an intraperitoneal injection of pentobarbital sodium (30–35 mg/kg) and ventilated mechanically through an endotracheal tube with oxygen-enriched room air. The level of anesthesia was maintained with a continuous intravenous infusion of pentobarbital sodium (1–2 mg·kg–1·h–1) through a catheter inserted from the right femoral vein. Mean arterial pressure (MAP) was measured using a pressure transducer connected to a catheter inserted from the right femoral artery. Heart rate (HR) was determined from an electrocardiogram.
Protocol 1: acute myocardial ischemia.
We examined the effects of hypothermia on the ischemia-induced myocardial interstitial releases of NE and ACh. The heart was exposed by partially removing the left fifth and/or sixth rib. A dialysis probe was implanted transversely into the anterolateral free wall of the left ventricle perfused by the left anterior descending coronary artery (LAD) to monitor myocardial interstitial NE and ACh levels in the ischemic region during occlusion of the LAD (13). Another dialysis probe was implanted transversely into the posterior free wall of the left ventricle perfused by the left circumflex coronary artery to monitor myocardial interstitial NE and ACh levels in a nonischemic region. Heparin sodium (100 U/kg) was administered intravenously to prevent blood coagulation. Animals were divided into a normothermic group (n = 8) and a hypothermic group (n = 6). In the hypothermic group, surface cooling with ice bags was performed until the esophageal temperature decreased to 33°C (15, 16). A stable hypothermic condition was obtained within 
2 h. In each group, we occluded the LAD for 60 min and examined changes in the myocardial interstitial NE and ACh levels in the ischemic region (i.e., the LAD region) and nonischemic region (i.e., the left circumflex coronary artery region). Fifteen-minute dialysate samples were obtained during the preocclusion baseline condition and during the periods of 0–15, 15–30, 30–45, and 45–60 min of the LAD occlusion.
Protocol 2: sympathetic stimulation. We examined the effects of hypothermia on the sympathetic nerve stimulation-induced myocardial interstitial NE release (n = 6). A dialysis probe was implanted transversely into the anterolateral free wall of the left ventricle. The bilateral cardiac sympathetic nerves originating from the stellate ganglia were exposed through a second intercostal space and sectioned. The cardiac end of each sectioned nerve was placed on a bipolar platinum electrode for sympathetic stimulation (5 Hz, 10 V, 1-ms pulse duration). The electrodes and nerves were covered with mineral oil to provide insulation and prevent desiccation. A 4-min dialysate sample was obtained during the sympathetic stimulation under the normothermic condition. Thereafter, hypothermia was introduced using the same cooling procedure as in protocol 1, and a second 4-min dialysate sample was obtained during the sympathetic stimulation.
Protocol 3: vagal stimulation. We examined the effects of hypothermia on the vagal nerve stimulation-induced ACh release (n = 5). A dialysis probe was implanted transversely into the anterolateral free wall of the left ventricle. The bilateral vagi were exposed through a midline cervical incision and sectioned at the neck. The cardiac end of each sectioned nerve was placed on a bipolar platinum electrode for vagal stimulation (20 Hz, 10 V, 1-ms pulse duration). To prevent severe bradycardia and cardiac arrest, which can be induced by the vagal stimulation, the heart was paced at 200 beats/min using pacing wires attached to the apex of the heart during the stimulation period. A 4-min dialysate sample was obtained during the vagal stimulation under the normothermic condition. Thereafter, hypothermia was introduced using the same cooling procedure as in protocol 1, and a second 4-min dialysate sample was obtained during the vagal stimulation.
Because of the relatively intense stimulation of the sympathetic or vagal nerve, the stimulation period in protocols 2 and 3 was limited to 4 min to minimize gradual waning of the stimulation effects. At the end of the experiment, the animals were killed by increasing the depth of anesthesia with an overdose of pentobarbital sodium. We then confirmed that the dialysis probes had been threaded in the middle layer of the left ventricular myocardium.
Dialysis Technique
The dialysate NE and ACh concentrations were measured as indexes of myocardial interstitial NE and ACh levels, respectively. The materials and properties of the dialysis probe have been described previously (2, 3). Briefly, we designed a transverse dialysis probe. A dialysis fiber (13-mm length, 310-µm outer diameter, 200-µm inner diameter; PAN-1200, 50,000 molecular weight cutoff; Asahi Chemical) was connected at both ends to polyethylene tubes (25-cm length, 500-µm outer diameter, 200-µm inner diameter). The dialysis probe was perfused with Ringer solution containing a cholinesterase inhibitor eserine (10–4 M) at a rate of 2 µl/min. We started dialysate sampling from 2 h after the implantation of the dialysis probe(s), when the dialysate NE and ACh concentrations had reached steady states. The actual dialysate sampling was delayed by 5 min from the collection period to account for the dead space volume between the semipermeable membrane and the sample tube. Each sample was collected in a microtube containing 3 µl of HCl to prevent amine oxidation. The dialysate ACh concentration was measured directly by HPLC with electrochemical detection (Eicom). The in vitro recovery rate of ACh was 70%. With the use of a criterion of signal-to-noise ratio of higher than three, the detection limit for ACh was 3 pg per injection. The dialysate NE concentration was measured by another HPLC-electrochemical detection system after the removal of interfering compounds by an alumina procedure. The in vitro recovery rate of NE was 55%. With the use of a criterion of signal-to-noise ratio of higher than three, the detection limit for NE was 200 fg per injection.
Statistical Analysis
All data are presented as means and SD values. For protocol 1, we performed two-way repeated-measures ANOVA using hypothermia as one factor and the dialysate sampling periods (the effects of ischemia) as the other factor. For protocols 2 and 3, we compared stimulation-induced releases of NE and ACh before and during hypothermia using a paired t-test. For all of the statistics, the difference was considered significant when P < 0.05.
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RESULTS |
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94-fold increase in the NE level during the 45- to 60-min interval. In the hypothermic group (solid bars), the LAD occlusion caused an 45-fold increase in the NE level during the 45- to 60-min interval. Compared with normothermia, hypothermia suppressed the baseline NE level to 59% and the NE level during the 45- to 60-min period to 29%. Statistical analysis indicated that the effects of both hypothermia and ischemia on the NE release were significant, and the interaction between hypothermia and ischemia was also significant.
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20%. Statistical analysis indicated that the effects of both hypothermia and ischemia on the ACh release were significant, and the interaction between hypothermia and ischemia was also significant.
Figure 2A illustrates changes in myocardial interstitial NE levels in the nonischemic region during the LAD occlusion. Note that scale of the ordinate is only one-hundredth of that in Fig. 1A. The LAD occlusion decreased the NE level in the normothermic group (open bars); the NE level during the 45- to 60-min interval was 59% of the baseline level. The LAD occlusion also decreased the NE level in the hypothermic group (solid bars); the NE level during the 45- to 60-min interval was 64% of the baseline level. Although the LAD occlusion resulted in a decrease in the NE level under both conditions, the NE level under hypothermia was nearly twice that measured under normothermia. The statistical analysis indicated that the effects of both hypothermia and ischemia on the NE release were significant, whereas the interaction between hypothermia and ischemia was not significant.
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3.4-fold increase in the ACh level during the 0- to 15-min interval in the normothermic group (open bars). The LAD occlusion caused an approximately ninefold increase in the ACh level during the 0- to 15-min interval in the hypothermic group (solid bars). These effects of ischemia on the ACh release were statistically significant. Although hypothermia seemed to attenuate the baseline ACh level, the overall effects of hypothermia on the ACh level were insignificant. Tables 1 and 2 summarize the MAP and HR data, respectively, obtained in protocol 1. Acute myocardial ischemia significantly reduced MAP (P < 0.01) and HR (P < 0.01). Hypothermia did not affect MAP but did decrease HR (P < 0.01). The interaction between ischemia and hypothermia was significant for MAP but not for HR by the two-way repeated-measures ANOVA.
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70% of the level observed during normothermia (Fig. 3A). Under normothermia, the sympathetic stimulation increased MAP from 114 mmHg (SD 27) to 134 mmHg (SD 33) (P < 0.01) and HR from 147 beats/min (SD 9) to 207 beats/min (SD 5) (P < 0.01). Under hypothermia, the sympathetic stimulation increased MAP from 117 mmHg (SD 11) to 136 mmHg (SD 22) (P < 0.05) and HR from 125 beats/min (SD 16) to 164 beats/min (SD 10) (P < 0.01).
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70% of the level observed during normothermia (Fig. 3B). Hypothermia did not change MAP [117 mmHg (SD 18) vs. 118 mmHg (SD 27)] but did decrease HR from 202 beats/min (SD 24) to 179 beats/min (SD 15) (P < 0.05) during the prestimulation, unpaced condition. MAP during the stimulation was 105 mmHg (SD 19) under normothermia and 93 mmHg (SD 33) under hypothermia. |
DISCUSSION |
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Effects of Hypothermia on Ischemia-induced NE and ACh Releases in the Ischemic Region
Acute myocardial ischemia causes energy depletion, which leads to myocardial interstitial NE release in the ischemic region (Fig. 1A). The NE release can be classified as exocytotic or nonexocytotic (18, 24). Exocytotic release indicates NE release from synaptic vesicles, which normally occurs in response to nerve discharge and subsequent Ca2+ influx through voltage-dependent Ca2+ channels. On the other hand, nonexocytotic release indicates NE release from the axoplasm, such as that mediated by a reverse transport through the NE transporter. A neuronal uptake blocker, desipramine, can suppress the ischemia-induced NE release (19, 24). Whereas exocytotic release contributes to the ischemia-induced NE release in the initial phase of ischemia (within 20 min), carrier-mediated nonexocytotic release becomes predominant as the ischemic period is prolonged (1). Hypothermia significantly attenuated the ischemia-induced NE release (Fig. 1A). The NE level during the 45- to 60-min period of ischemia under hypothermia was 20% of that obtained under normothermia. The NE uptake transporter is driven by the Na+ gradient across the cell membrane (23). The loss of the Na+ gradient due to ischemia causes NE to be transported out of the cell by reversing the action of the NE transporter. Hypothermia inhibits the action of the NE transporter and also suppresses the intracellular Na+ accumulation (8), thereby reducing nonexocytotic NE release during ischemia. The present results are in line with an in vitro study that showed hypothermia suppressed nonexocytotic NE release induced by deprivation of oxygen and glucose (30). The present results are also consistent with a previous study from our laboratory that showed hypothermia attenuated the nonexocytotic NE release induced by ouabain, tyramine, or cyanide (16).
Acute myocardial ischemia increases myocardial interstitial ACh level in the ischemic region, as reported previously (Fig. 1B) (13). The level of ischemia-induced ACh release during 0- to 15-, 15- to 30-, 30- to 45-, or 45- to 60-min period of ischemia is comparable to that evoked by 4-min electrical stimulation of the bilateral vagi (Fig. 3B). Compared with the normothermic condition, hypothermia significantly attenuated the ischemia-induced myocardial interstitial release of ACh in the ischemic region. Our laboratory's previous study indicated that intracellular Ca2+ mobilization is essential for the ischemia-induced release of ACh (13). Hypothermia may have prevented the Ca2+ overload, thereby reducing the ischemia-induced ACh release. Alternatively, hypothermia may reduce the extent of the ischemic injury, which in turn suppressed the ischemia-induced ACh release. Because ACh has protective effects on the cardiomyocytes against ischemia (11), the suppression of ischemia-induced ACh release during hypothermia itself may be unfavorable for cardioprotection.
There is considerable controversy regarding the cardioprotective effects of 
-adrenergic blockade during severe ischemia, with studies demonstrating a reduction of infarct size (10, 17) or no effects (7, 27). The -adrenergic blockade seems effective to protect the heart only when the heart is reperfused within a certain period after the coronary occlusion. The -adrenergic blockade would reduce the myocardial oxygen consumption through the reduction of HR and ventricular contractility and delay the progression of ischemic injury. Hence the infarct size might be reduced when the heart is reperfused before the ischemic damage becomes irreversible. The ischemia-induced NE release reached nearly 100 times the baseline NE level under normothermia (Fig. 1A), which by far exceeded the NE level attained by electrical stimulation of the bilateral stellate ganglia (Fig. 3A). Because high NE levels have cardiotoxic effects (22), ischemia-induced NE release might aggravate the ischemic injury. However, catecholamine depletion by a reserpine treatment fails to reduce the infarct size (26, 29), throwing a doubt on the involvement of catecholamine toxicity in the progression of myocardial damage during ischemia. It is, therefore, most likely that the hypothermia-induced reductions in NE and ACh are the result of reduced myocardial damage or a direct effect on nerve endings.
Van den Doel et al. (28) showed that hypothermia does not abolish necrosis, but rather delays necrosis during sustained ischemia, so that hypothermia protected against infarction produced by a 30-min occlusion but not against infarction produced by a 60-min occlusion in the rat heart. At the same time, they mentioned that hypothermia was able to reduce the infarct size after a 60-min coronary occlusion in the dog, possibly because of the significant collateral flow in the canine hearts. Because the feline hearts are similar to the canine hearts in that they have considerable collateral flow compared with the rat hearts (21), hypothermia should have protected the feline heart against the 60-min coronary occlusion in the present study.
Effects of Hypothermia on the NE and ACh Releases in the Nonischemic Region and on the Electrical Stimulation-induced NE and ACh Releases
The NE and ACh levels in the nonischemic region may reflect the sympathetic and parasympathetic drives to this region. As an example, myocardial interstitial ACh levels increase during activations of the arterial baroreflex and the Bezold-Jarisch reflex (14). In the present study, acute myocardial ischemia decreased the NE level from its baseline level, whereas it increased the ACh level from its baseline level (Fig. 2). Ischemia also decreased MAP and HR (Tables 1 and 2), suggesting that the Bezold-Jarisch reflex was induced by the LAD occlusion under both normothermia and hypothermia. Taking into account the fact that electrical stimulation-induced ACh release was attenuated to 70% (Fig. 3), similar ACh levels during ischemia imply the enhancement of the parasympathetic outflow via the Bezold-Jarisch reflex under hypothermia. These results are in line with the study by Zheng et al. (32), where pulmonary chemoreflex-induced bradycardia was maintained under hypothermia. Hypothermia increased the NE level in the nonischemic region, suggesting that sympathetic drive to this region also increased. Hypothermic stress is known to cause sympathetic activation, accompanying increases in MAP, HR, plasma NE, and epinephrine levels (4). In the present study, because the effect of hypothermia on MAP was insignificant (Table 1) and HR decreased under hypothermia (Table 2), the sympathetic activation observed in the nonischemic region might have been regional and not systemic.
Hypothermia attenuated the releases of NE and ACh in response to respective nerve stimulation to 70% of that observed under normothermia (Fig. 3). The suppression of the exocytotic NE release by hypothermia is consistent with a previous study from our laboratory, where hypothermia attenuated the myocardial interstitial NE release in response to vena cava occlusion or to a local high K+ administration (15). The suppression of NE release by hypothermia is consistent with an in vitro study by Kao and Westhead (12) in which catecholamine secretion from adrenal chromaffin cells induced by elevated K+ levels increased as the temperature increased from 4 to 37°C. On the other hand, because hypothermia inhibits the neuronal NE uptake, the NE concentration at the synaptic cleft is expected to be increased if the level of NE release remains unchanged. Actually, Vizi (30) demonstrated that hypothermia increased NE release in response to field stimulation in vitro. In the present study, however, the suppression of NE release might have canceled the potential accumulation of NE due to NE uptake inhibition. The present study also demonstrated that the ACh release was suppressed by hypothermia. In the rat striatum, hypothermia decreases the extracellular ACh concentration and increases the choline concentration (5). Hypothermia may inhibit a choline uptake transporter in the same manner as it inhibits a NE uptake transporter. The inhibition of the choline transporter by hypothermia may have hampered the replenishment of the available pool of ACh and thereby contributed to the suppression of the stimulation-induced ACh release.
Limitations
In protocol 1, because we did not measure the infarct size in the present study, the degree of myocardial protection by hypothermia was undetermined. Whether the reduction of ischemia-induced neurotransmitter release correlates with the reduction of infact size requires further investigations. In protocols 2 and 3, baseline NE and ACh levels were not measured. The reduction of stimulation-induced NE and ACh release by hypothermia might be partly due to the reduction of baseline NE and ACh levels. However, because transection of the stellate ganglia (31) or vagi (3) reduces the baseline NE and ACh levels, changes in the baseline NE and ACh levels by hypothermia in protocols 2 and 3 could not be as large as those observed under innervated conditions in protocol 1 (Figs. 1 and 2).
In conclusion, hypothermia attenuated the ischemia-induced releases of NE and ACh in the ischemic region to 30 and 20% of those observed under normothermia, respectively. Hypothermia also attenuated the nerve stimulation-induced releases of NE and ACh to 70% of those observed during normothermia. In contrast, hypothermia did not affect the decreasing response in the NE level and the increasing response in the ACh level in the nonischemic region, suggesting that the Bezold-Jarisch reflex evoked by the LAD occlusion was maintained.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-adrenergic blockade on infarct size following experimental coronary occlusion. Basic Res Cardiol 76: 144–151, 1981.[CrossRef][ISI][Medline]
. FEBS Lett 579: 2111–2118, 2005.[CrossRef][ISI][Medline]-blockade and calcium antagonists. Basic Res Cardiol 84: 564–582, 1989.[CrossRef][ISI][Medline]
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Hypothermia significantly attenuated the stimulation-induced NE release. B: efferent vagal nerve stimulation-induced release of myocardial interstitial ACh before and during hypothermia.