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From the Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina
Address correspondence and reprint requests to Leanne Groban, MD, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157–1009. Address email to lgroban{at}wfubmc.edu
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Abstract |
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IMPLICATIONS: Our findings suggest that spinally-administered morphine provides a previously unrecognized cardioprotective benefit. In anesthetized rats subjected to ischemia-reperfusion injury, we show that very small doses of intrathecal morphine reduce infarct size in rats, and this benefit is as great as that provided by much larger doses of IV morphine.
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Introduction |
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-opioid receptor-K+ATP channel interaction within the myocardium is one presumed mode of cardioprotection (3,4), and this interaction may be further facilitated by adenosine (5) and protein kinase C (6). Since the mid-1990s, however, the use of large-dose opioid anesthesia for cardiac surgery has fallen into disfavor because of an association with extended duration of ventilator dependence and intensive care length of stay (7,8). Some clinicians have substituted intrathecal opioids (0.25 mg to 2.0 mg) for IV opioids in the hope of facilitating earlier tracheal extubation (9,10). Intrathecal (IT) dosing reduces the total amount of opioid required (11,12). Even though opioid receptor subtypes have been identified within the spinal cord and that spinal injection of opioids activates inhibitory pathways that can modulate neuronal trafficking from nociceptive stimuli (13), opioids administered spinally before an ischemia-reperfusion event may or may not afford the same degree of cardioprotection that is observed with systemic opioid "pretreatment." Given that morphine is highly ionized and hydrophilic, and that doses as small as 1/1000th those used IV are effective in modifying pain behavior in rats (14), studies have shown much smaller plasma concentrations of morphine after IT administration as compared with the plasma levels achieved after IV morphine (15,16). Likewise, IT injections of morphine produce larger concentrations of the opioid in the cerebral spinal fluid and brain than are measured after systemic administration (17). Accordingly, this study tests the hypotheses that small doses of IT morphine reduce infarct size in rats and that this benefit is as significant as that provided by much larger doses of IV morphine.
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Methods |
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Surgical Preparation
After male Wistar rats (250–350 g) were anesthetized with 2.5% halothane (Halocarbon Laboratories, River Edge, NJ), in a room air-oxygen mixture (1:1). Thoracic IT catheters were inserted using a modification of Yaksh and Rudy’s technique (18). Briefly, the polyethylene catheter (Intramedic, PE-10) was inserted, under direct vision, 4 cm caudally through the cisternal membrane into the IT space leaving the catheter near the origin of the upper thoracic nerve roots. Animals recovered for a minimum of 3 days thereafter. Rats with forelimb or hindlimb motor deficits after recovery were euthanized.
No sooner than the third postimplantation day, rats were instrumented for the acute ischemia-reperfusion experiment. Anesthesia was induced by intraperitoneal administration of thiobutabarbital sodium diluted in saline (Sigma Research Biochemicals Incorporated, Natick, MA) (100 mg/kg) followed by additional IV injections of thiobutabarbital (50–100 mg/kg) to maintain an anesthetic depth sufficient to eliminate both corneal response to touch and withdrawal to paw pinch. A tracheostomy tube was inserted, and each rat was artificially ventilated (rodent ventilator model 806; New England Medical Instruments Inc., Medway, Mass) with a room air-oxygen mixture (1:1). Blood samples for arterial blood gas determination were monitored at regular intervals and ventilation was adjusted to keep PaCO2 between 25 and 40 mm Hg, and PaO2 was kept between 90 and 150 mm Hg throughout the experimental protocol. Body temperature was monitored with a rectal temperature probe and maintained at 37°C with a heating pad. The right internal jugular vein was cannulated with a 0.9% saline-filled polyethylene tube (Intramedic, PE-50) for IV drug administration. The left common carotid artery was cannulated with a 20-gauge, 2-cm angiocatheter attached to a 0.9% saline/heparin-filled pressure transducer (1 U/mL) for arterial blood pressure recording and blood sampling.
A left thoracotomy was performed at the fifth intercostal space. The pericardium was opened, and the left coronary artery and vein were identified just under the left atrial appendage. A 6–0 polypropylene ligature was placed around the left descending coronary artery close to its origin and the ends of the suture were threaded through a propylene tube to form a snare.
After a 15-min stabilization period, rats were randomly assigned to receive IV morphine (Abbott Laboratories, Chicago, IL) (300 µg/kg) plus IT vehicle (10 µL normal saline), IT vehicle (10 µL normal saline), IT small-dose morphine (0.3 µg/kg 
0.1 µg), or IT large-dose morphine (3 µg/kg 1.0 µg). Morphine was diluted in normal saline. The IT morphine injected volume was 10 µL. Another 10 µL of saline was injected after each morphine injection to clear the catheter dead space. IT and IV drugs were administered over a period of 2 min 20 min before coronary occlusion. All rats were subjected to 30 min of coronary occlusion followed by 90 min of reperfusion. Coronary artery occlusion was produced by clamping the snare onto the epicardial surface of the heart with a hemostat and was verified by epicardial cyanosis accompanied in some cases by ventricular arrhythmias. Reperfusion was achieved by unclamping the hemostat and loosening the snare and was verified by observing an epicardial hyperemic response.
A schematic illustration of the experimental design is illustrated in Figure 1. Systemic arterial pressures and heart rate (HR) were continuously monitored on an oscilloscope. Recorded hemodynamics were acquired during a 6-s period using a computer (Dell OptiPlex GXMT 5133 pentium, Dell Computer, Austin, TX) interfaced with an analog-to-digital converter (model DT 2821; Data Translation Devices, Marlborough, MA). The average of three acquisitions of data was obtained with the coronary circulation intact at basal anesthetized conditions (Baseline 1), and 20 min after IT or IV drug pretreatment, just before coronary occlusion (Baseline 2), at min 30 of coronary occlusion (Ischemia), and at 15, 45, and 90 min of reperfusion.
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Animals were excluded from data analysis if 1) coronary artery occlusion did not produce severe ischemia, i.e., area-at-risk (AAR) expressed as a percentage of the LV was 
20%, or 2) if either ventricular fibrillation or severe hypotension (mean arterial blood pressure [MAP] below 40 mm Hg) were observed, or 3) inadequate IT drug administration was evident after intrathecal dye injection and cord extrusion.
Infarct Size Determination
After the heart was excised, a second investigator unaware of the randomization determined infarct size. The LV was isolated and sliced into 5 cross-sectional pieces. The unstained region of the myocardium (the AAR) was separated from the blue, nonischemic zone using a dissecting microscope. The AAR was incubated for 15 min in a 1% solution of triphenyltetrazolium chloride (TTC) in 100 mM phosphate buffer (pH 7.4) at 37°C. In the presence of intact dehydrogenase enzyme systems or NAD+/NADH, TTC dye forms red precipitates that identify non-necrotic tissue. Necrotic tissue, lacking these enzyme systems, remains a pale color. After TTC incubation, tissue samples were refrigerated overnight in a 10% solution of buffered formalin. The AAR was subdivided into non-necrotic (TTC-positive, red) and necrotic areas (AN) (TTC negative, pale) the next day using a dissecting microscope. The AAR and AN were determined gravimetrically. Areas of necrosis were expressed as a percentage of the AAR (AN/AAR).
Power calculations for infarct size were based on the two-sample Student’s t-test with standard deviation of 7.5%. Power analyses showed that 10 animals per treatment group would be sufficient to detect a 15% reduction in infarct size with a power of 90%.
Statistical Analysis
Statistical analyses were performed with PC SAS (SAS Institute, Cary, NC). Time-related differences and group-time interactions were analyzed by two-way analysis of variance for repeated measures adjusted for baseline values. One-way analysis of variance was used to determine the effects of treatment on gravimetric values of LV regions of risk and necrosis. Post hoc comparisons were performed with Student-Newman-Keuls test. Analysis of covariance was used to determine the effects of treatment on AN/AAR with covariate adjustments for LV and values for the "adjusted" AN/AAR are reported as least square means ± SE. All other values are reported as mean ± SEM. Statistical significance was defined as P < 0.05.
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Results |
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Figure 2 shows the HR and MAP at baseline, after 20 min of either IV or IT morphine or saline (vehicle) pretreatment (Baseline 2), after 30 min of coronary occlusion, and at 15, 45, and 90 min of reperfusion. HR and MAP were not different among groups at baseline. Twenty min after IT (both doses) and IV morphine, 5% to 8% reductions from baseline HR were observed. During ischemia-reperfusion, heart rate was unchanged from baseline conditions (baseline 1 and 2) in the "large" dose IT morphine group whereas in the "small" dose IT morphine, IV morphine, and vehicle groups, significant declines in HR from Baseline 1 were observed. In the vehicle and "small" dose IT groups, significant declines in HR from Baseline 2 were also noted. MAP declined 14% from Baseline 1 after both IV and IT morphine treatments (Baseline 2) (P < 0.05). During coronary occlusion and reperfusion MAP further declined 27 to 41% from Baseline 1 (P < 0.05) and 11 to 34% from Baseline 2 (P < 0.05). These responses in MAP were not different among morphine treatments or saline.
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Discussion |
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-opioids (e.g., morphine) administered IV before ischemia-reperfusion injury provide direct cardioprotection marked by a reduction in infarct size (3,4). Our findings further suggest that spinally-administered morphine may provide a previously unrecognized cardioprotective benefit. In anesthetized rats subjected to ischemia-reperfusion injury, we found that IT morphine pretreatment reduces infarct size (adjusted for LV mass) as compared with saline-treated control rats. Neuroaxial opioids produce antinociception in humans and have become an important technique in the management of postoperative pain. IT morphine administered to cardiac surgical patients at doses ranging between 0.25 mg and 2.0 mg produces more intense and prolonged postoperative analgesia than systemically-administered morphine and generally facilitates earlier extubation (5–10 hours) (9,11). In patients with medically intractable angina pectoris, small-dose IT morphine (0.6 mcg to 1.0 mg) can relieve anginal pain with minimal circulatory or respiratory side effects (19).
In rats, IT morphine produces antinociception (20) and reduces postoperative pain behaviors (14). Although effective doses depend on the specific nociceptive tests used, the minimum IT morphine doses reported to modify pain behavior in rats range between 0.3 and 2.0 µg (14). Because there is no recognized IT morphine dose for cardiogenic pain in rats subjected to experimental ischemia-reperfusion injury, we extrapolated our IT morphine dose from other models of nociceptive, visceral, and postoperative pain (14,20,21). In the present study we used the smallest neuraxial morphine dose that modified pain responses in these other models because our rats were also receiving general anesthesia. The IT morphine doses, 0.1 (0.3 µg/kg) and 1.0 µg (3 µg/kg), effective in our study were 1000-fold and 100-fold smaller than the cardioprotective, systemic morphine dose, 300 µg/kg, used by others (3,4) and ourselves. Interestingly, the IT morphine doses required to modify pain behavior in rats subjected to noxious, nociceptive stimuli have also been approximately 1000-fold smaller than the effective systemic morphine dose (14). In humans, the therapeutic ratio for single doses of systemic morphine and IT morphine that produce antinociception and postoperative pain relief is approximately 10:1. Specifically, with regard to analgesic potential in humans, 0.05–0.3 mg/kg IV morphine is comparable to 0.2–0.5 mg IT morphine. As a result of these markedly differing doses in both humans and rats, IT morphine results in much smaller plasma concentrations than IV morphine.
IT morphine can have activity through mechanisms other than relief of pain. Other potential mechanisms of cardioprotection from IT morphine include stress response attenuation and cardiac sympathectomy. Opioid receptor subtypes, predominately µ and , have been identified on pre- and postsynaptic neurons and in the spinal intermediolateral cell column where their activation can modulate autonomic responses to nociceptive stimuli (13). Opioids administered spinally activate bulbospinal inhibitory neurons, which can attenuate excitatory neuronal trafficking within the dorsal horn as well (13). In the anesthetized cat, neuraxial morphine decreased cardiac efferent sympathetic nerve activity presumably through direct inhibition of the sympathetic nervous system at the level of the brainstem and spinal cord (22). Interestingly, in anesthetized rats, IT µ agonists did not change resting HR and MAP but attenuated hemodynamic responses evoked by noxious thermal stimuli, suggesting that opioids attenuate the afferent limb of the pain-evoked autonomic reflex (23). In humans, spinal opioid receptor activation may attenuate increased sympathetic nerve activity elicited by myocardial ischemia; however, local anesthetics administered either spinally or epidurally possess greater efficacy at blocking sympathetic afferent and efferent fibers (24). Correspondingly, IT opioids may only partially attenuate the stress response in patients undergoing cardiac surgery (25). Additional studies will be required to determine which of these several mechanisms played a role in the cardioprotection observed in the current study.
We doubt that reduction in infarct size observed in the IT morphine groups was attributable to hemodynamic effects. The hemodynamic picture that accompanies acute myocardial ischemia is the result of a complex interaction between ischemic myocardium and excitatory and inhibitory reflexes arising from the heart. Occlusion of the left main coronary artery can produce LV dysfunction and dilation, conditions that provoke vagally-mediated reflexes. However, cardiac sympathetic afferents, activated with lesser degrees of ischemia or before maximal ventricular dilation, elicit dominant pressor sympathetic reflexes. In this study, occlusion and reperfusion of the left coronary artery elicited an inhibitory, depressor response similar to that reported by Sole et al. (26). However, in our rats treated with "large" dose IT morphine, HR was not decreased by coronary occlusion. Whether the "stable" heart rate response during ischemia and reperfusion influenced the reduced infarct size observed in this group is unclear. Opioid receptor stimulation induces multiple cardiovascular effects, including alterations in arterial tone, arterial blood pressure, HR, and contractility. In our model, these effects were further modulated by the underlying myocardial injury. It seems unreasonable to attribute a causal relationship between hemodynamics and ischemic injury because infarct size was also reduced in the IV and "small" dose IT morphine-treated rats (as compared with vehicle), despite similar declines in HR and blood pressure in these and the control animals.
The clinical implications of this study should be interpreted with caution. First, coronary occlusion of the main artery in the rat usually leaves the subendocardial and subepicardial muscle layers intact (27). In human infarcts, the subendocardial layers are often affected. Nonetheless, the rodent model has been extensively used to determine the role of systemic opioids and K+ATP channel agonists in myocardial protection (3–6) and has been used to establish the role of spinally-administered opioids in limiting nociceptive and visceral pain responses (14,20,21). Second, the choice of basal anesthetic used may have influenced the ischemia-induced, cardio-cardiac reflexes or the hemodynamic picture that accompanied the acute myocardial ischemia. For instance, pentobarbital depresses the effect of morphine on the cardiac acceleration responses to somatic noxious stimulation and this morphine-pentobarbital interaction is presumed to occur at the supraspinal level (28). In the current study, thiobutabarbital sodium, a long-acting barbiturate, was used as the basal anesthetic because of its frequent use in other rat studies of myocardial preconditioning and because of its stable hemodynamic profile (3–6).
Additionally, it cannot be deduced from this study whether this central pathway of myocardial protection is exclusively opioid-mediated. Similar to the cardioprotective K+ATP channel-opioid receptor interaction within the myocardium, it is reasonable to expect that there may also be opioid receptors activating K+ATP channels in the central nervous system (CNS). The central administration of K+ATP channel agonists, such as diazoxide, minoxidil, and lemakalim, has resulted in analgesia and antinociception in mice (29). This antinociceptive effect is blocked by both K+ATP channel antagonists and opiate receptor antagonists. Correspondingly, IT morphine-induced antinociception is blocked by K+ATP channel antagonists (29). Besides K+ATP channels, adenosine and protein kinase C have been implicated in the signal transduction pathway of peripheral opioid-mediated cardioprotection (5,6). Although there is evidence to suggest that adenosine and opioid receptors are tightly coupled within the CNS (30), it remains unclear whether this interaction is involved in a central mechanism of cardioprotection. Accordingly, future studies using central and peripheral-acting, selective opioid receptor and adenosine receptor antagonists, K+ATP channel blockers, and protein kinase C inhibitors will be required to determine the mechanism of cardioprotection by IT morphine. Also, it is not known whether IT morphine confers cardioprotection by limiting leu-kocyte activation at the injury site similar to that reported by Wang et al. (31) in morphine-preconditioned rats subjected to experimental myocardial infarction. Furthermore, the determination of plasma and cerebral spinal fluid concentrations of morphine may help distinguish whether the effect is independent of the route of opioid administration. Specifically, as the plasma concentrations from very small doses of IT morphine should be far smaller than that achieved from IV dosing (15,16), it is unlikely that IT dosing confers cardioprotection via a peripheral mechanism.
In summary, pretreatment with IT morphine protects the heart from subsequent ischemia-reperfusion injury by limiting the magnitude of infarction. The optimal dose, the maximal protective effect and the cellular mechanisms of IT opioid cardioprotection remain unknown. Our findings suggest that the CNS may serve as an alternative site of action for morphine-mediated cardioprotection. This could be singularly useful when intraoperative myocardial ischemia is expected, e.g., during off-pump coronary artery surgery.
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Acknowledgments |
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Footnotes |
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P < 0.05 significantly different from IV, IT small, VEH responses.
