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© 2004 European Association of Cardio-Thoracic Surgery
Systemic ischemic preconditioning plus hemodilution enhanced early functional recovery of reperfused heart in the rabbits
a Department of Anesthesiology, Renmin Hospital, Wuhan University, Wuhan 430060, China
* Corresponding author. Tel.: +1-604-822-6980; fax: +1-604-822-6012 Received March 4, 2004; received in revised form May 20, 2004; accepted May 21, 2004
We investigated whether transient systemic ischemia can precondition the heart against the ensuing ischemic insult. Rabbits were randomly divided into systemic ischemic preconditioning (SIP), hemodilution (HD) and control (C) groups. SIP was induced by rapidly withdrawing blood from femoral artery and maintaining the mean artery pressure at 50 mmHg for 10 min. Afterwards, 50% of the withdrawn blood was re-infused with equal volumes of 6% deferoxamine-conjugated hydroxyethyl-starch (D-HES). Hemodilution in HD group was achieved by withdrawing blood at 8 ml/kg accompanied by concomitant infusion of equal volumes of D-HES. Animals were subjected to 30 min of coronary artery occlusion followed by 120 min of reperfusion. Cardiac output (CO) (thermodilution method), plasma malondialdehyde and nitric oxide content were measured at baseline and until 120  min of reperfusion. At  CO in group-C was significantly lower than its baseline value, accompanied by a significant reduction in nitric oxide production and increase in malondialdehyde concentration; CO in group-SIP and group-HD maintained at baseline levels throughout reperfusion. At CO in group-SIP, but not in group-HD, was significantly higher than that in group-C. It is concluded that transient SIP followed by hemodilution with D-HES facilitates early functional recovery of the ischemic-reperfused rabbit hearts.
Key Words: Systemic ischemic preconditioning; Myocardial ischemia; Rabbits; Hemodilution; Reperfusion
Ischemic preconditioning is a powerful mechanism in reducing post-ischemic myocardial injury. However, salutary early functional improvement of reperfused heart has not been observed following classic ischemic preconditioning or pharmacological preconditioning or acute systemic hypoxia [1–3], despite significant reduction in infarct size. We investigated whether transient systemic ischemia (i.e. systemic ischemic preconditioning, SIP), followed by hemodilution (HD) with deferoxamine-conjugated hydroxyethyl-starch, can precondition the heart against the ensuing ischemic insult and facilitate early cardiac functional recovery. Also, we investigated if SIP could stimulate the generation of nitric oxide (NO), a major signaling molecule of the vascular system, which has been shown to be a trigger and mediator of the late phase of ischemic preconditioning, in vivo in the rabbit [4,5].
The study was approved by the Committee of Animal Care of Wuhan University. Animals received humane care in compliance with the European Convention on Animal Care. 2.1. Anesthesia and surgical procedures Adult New Zealand White rabbits (2.2–3.2 kg) were fasted for 24 h with free access to water before anesthesia. Anesthesia was induced with intravenous pentobarbital 30 mg/kg and fentanyl 10 µg/kg and maintained with fentanyl and pancuronium. The rabbits were ventilated with 100% oxygen and end-tidal PCO2 were maintained between 35 and 45 mmHg. The right femoral artery and carotid vein were, respectively, cannulated and connected to a Life-Scope Physiological recorder (Japan) to monitor systemic arterial blood pressure (SBP) or central venous pressure. The heart was exposed via a left thoracotomy through the fourth intercostal space. A 4–0 silk suture with a taper needle was passed around the left anterior descending coronary artery (LAD). After the completion of thermistor catheter implantation (see below), the animals were stabilized for 10 min before recording baseline hemodynamic parameters. All animals received intravenous infusion of lactated Ringer's solution at a rate of 10 ml/kg/h throughout the experiment. 2.2. Experimental protocol After postsurgical stabilization, rabbits were randomly assigned into one of the three groups: (1) untreated control group (control, ): rabbits were further stabilized for 30 min prior to inducing 30 min LAD occlusion followed by 120 min of reperfusion. LAD was occluded by tightening on the snare and clamping the polyethylene sleeve with a hemostat to produce a zone of ischemia. (2) SIP group (SIP,  ): SIP was achieved by quickly (within 5 min) reducing mean arterial blood pressure (MAP) to and maintaining it at 50 mmHg for a period of 10 min via the withdrawal of blood from the femoral artery. Thereafter, 50% volume of the withdrawn blood was re-infused over a period of 5–10 min together with an equal volume of 6% deferoxamine-conjugated hydroxyethyl-starch (D-HES) through the carotid vein to restore the MAP to its baseline levels. The MAP was then maintained stabilized for another 5–10 min before inducing 30 min of LAD occlusion and 120 min of reperfusion; (3) HD group (HD, ): blood was withdrawn from the femoral artery at the speed of 8 ml/kg over a period of 10 min (amounting to about 10% of the total blood volume). In the meanwhile, 6% D-HES was infused at the same speed through the carotid vein. After 20 min of hemodynamic stabilization, rabbits were subjected to 30 min of LAD occlusion and 120 min of reperfusion. Hemodynamic data were measured and recorded at baseline, pre-ischemia, at 1 (Rep-1), 30 (Rep-30) and 120 (Rep-120) min after reperfusion. Arterial blood was sampled for the measurement of plasma levels of nitric oxide (NO), superoxide dismutase (SOD), malondialdehyde (MDA) and creatine kinase (CK). 2.3. Measurement of cardiac output by thermodilution A thermistor was connected to a Life-Scope Physiological recorder (Japan), which was programmed to calculate the cardiac output by integrating the area under the temperature (i.e. voltage) time curve, taking into account of biological constants [6]. The injectate and the thermistor catheter were implanted, respectively, into the left ventricle and the descending aorta through the carotid and femoral arteries. Repeated measurements of cardiac output (CO) were undertaken by the rapid injection of 1 ml physiological saline of known temperature (4 °C) into the left ventricle over a period of 2 s.2.4. Bio-assays Superoxide dismutase, MDA, LDH and CK were measured by chemical analysis as previously described [7]. The plasma concentration of nitrites and nitrates, stable end products of nitric oxide (NO), was determined by the Griess reaction. The total nitrite was measured at 540 nm absorbance by diazotization with Griess reagent.2.5. Determination of area at risk At Rep-120, the heart was excised and the aorta was cannulated and perfused with physiological saline to remove blood from the vasculature. The LAD was then reoccluded at the same site previously chosen for occlusion. Then, 5 ml of 2% Evan's blue was infused into the aortic root to demarcate the perfused myocardium and thus mark the non-blue perfusion field of the occluded artery (the risk area).The area at risk was expressed as a percentage weight of the non-blue area versus the weight of the whole left ventricular tissue.2.6. Statistical analysis All data are presented as means±SEM. Hemodynamic and bio-chemical data were compared by two-way ANOVA with Bonferroni's correction. The correlation relationships were evaluated by the Pearsons test. (two tailed) was considered statistically significant.
Rabbit body weights did not differ among groups. Total blood loss in the HD group was 8.04±0.68 ml/kg, which was replaced by equal volume of 6% D-HES. The total blood withdrawal in the SIP group was initially 15.56±0.84 ml/kg and half of the withdrawn blood was then re-infused together with equal volume of 6% D-HES. The volume of net total blood loss and the volume of 6% D-HES infusion as well as the ensuing haematocrit values (0.223±0.024,  mean±SD) did not differ between the HD and SIP groups  The areas at risk were similar in the control (28.2±2.1%), HD (27.7±1.8%) and SIP (29.1±2.6%) groups 3.1. Plasma NO (nitrite/nitrates) concentration during reperfusion Nitric oxide levels (Fig. 1A) in the control, but not in the HD group, decreased significantly after 120 min of reperfusion ( vs baseline). Interestingly, NO significantly increased upon reperfusion in the SIP group and maintained at higher levels than its baseline  and than the corresponding values in control group  and the HD group during reperfusion up to 120 min of the observation.
3.2. Plasma levels of SOD activity and MDA content during reperfusion Plasma SOD activity (Fig. 1B) in the control group decreased gradually during reperfusion and reached statistical significance at Rep-120 ( vs baseline). SOD activity in the SIP group increased gradually during reperfusion. At Rep-120, SOD activity in the SIP group, but not in the HD group, was higher than that in the control group.
Plasma MDA (Fig. 1C) increased significantly at Rep-1 and Rep-30 in all groups. Plasma MDA in the control group continued to increase at Rep-120 and was significantly higher than the corresponding values in the HD and SIP groups 3.3. Plasma CK concentration during reperfusion Plasma CK (Fig. 2) increased significantly in all groups at Rep-30 as compared to the corresponding baseline values At Rep-120, Plasma CK in the SIP group, but not in the HD group, was significantly lower than that in the control group.
3.4. Hemodynamic changes during reperfusion Heart rate (HR), SBP, diastolic blood pressure (DBP), MAP and CO did not differ among groups at baseline and before inducing regional myocardial ischemia (Table 1 and Fig. 3). Heart rate did not significantly change over time during reperfusion in the three groups. Significant decrease in blood pressure and CO was seen in all groups during 30 min of LAD occlusion (data not shown). Blood pressures (SBP, DBP and MAP) recovered to close to the corresponding baseline values at Rep-30 in all groups. The blood pressures in the control group were then significantly decreased at Rep-120. At Rep-120, the MAP value in SIP group, but not that in the HD group, was significantly higher than the corresponding value in the control group (Table 1). SBP in the SIP group was higher than corresponding values in both the control and the HD groups at Rep-120.
Cardiac output in the control group was significantly lower than its baseline value at Rep-120 (Fig. 3). Cardiac output in the HD and SIP groups maintained at baseline levels throughout the period of reperfusion. However, during later reperfusion (from Rep-30 to Rep-120), the CO in the SIP group increased (8.9% increase) while CO in the HD group was on the decline (6.1% decrease,  SIP vs HD, Fig. 3). At Rep-120, cardiac output in the SIP group  but not in the HD group  was significantly higher than that in the control group. 3.5. Correlation analysis A weak but statistically significant correlation existed between plasma levels of NO and CO at Rep-120 (  ).
The findings of the present study can be summarized as follows: (1) in anesthetized rabbits, transient systemic ischemia, followed by HD with D-HES, can enhance early cardiac functional recovery following coronary artery occlusion and reperfusion, functioning as a powerful mechanism of ischemic preconditioning; (2) Transient systemic ischemia is associated with increased endogenous NO production or release during reperfusion, which may have contributed to the improved early myocardial functional recovery in the rabbits. Study has shown that endogenous NO protects against ischemia-reperfusion injury in the anesthetized rabbits, since blockade of NO synthase activity with L-nitro arginine, a NO synthase inhibitor, increased heart infarct size as compared to untreated controls [8]. However, the vascular endothelium, the primary source of endogenous NO, is damaged by reperfusion following coronary occlusion [9,10], resulting in impaired release of NO. In our study, SIP not only significantly increased plasma levels of NO but also increased plasma SOD activity during reperfusion as compared to the control group (Fig. 1). SIP induced increase in SOD activity may have blocked or attenuated the noxious interplay between superoxide anion and NO (i.e. the formation of toxic peroxinitrite) during reperfusion, resulting in significantly reduced lipid peroxidation (Fig. 1C), reduced release of CK (Fig. 2) and improved CO (Fig. 3). It should be noted that the SIP cardioprotective effect seen in our study may attribute, in part, to haemodilution that reduces the availability of circulating neutrophils, and in part to the potential cardioprotective effects of D-HES [11]. Nevertheless, it is safe to say that it is primarily the SIP rather than the haemodilution and D-HES that have contributed mostly to the cardioprotection seen in the SIP group. The extent of haemodilution and volume of D-HES utilization are similar between the SIP and the HD groups. However, at Rep-120, plasma CK level in the HD group, but not in SIP group, remained significantly elevated as compared to its baseline value. Also, major haemodynamic parameters (MAP and CO) were decreasing in the HD and the control groups toward the end of experiment, but MAP and CO in the SIP group was increasing. Besides, arterial oxygen partial pressure (PaO2) in the SIP group, but not in the D-HES group, was significantly higher than that in the control group at Rep-120 (data not shown). This is similar in nature to our recent finding that remote organ ischemic preconditioning increased PaO2 following coronary occlusion and reperfusion in the sheep [12]. Study limitations: Low peri-operative hematocrit (<20%) may adversely affect post-operative outcome [13]. SIP should be applied with particular caution in patients with pre-existing borderline low hematocrit. Although no metabolic acidosis was manifested during the current study (data not shown), systemic (hypovolemic) preconditioning may pose risks to patients with severe other organ dysfunctions or marginal myocardial reserves. For those patients alternative means of cardioprotection might be preferential. In summary, our present study suggests that SIP as a potentially useful means to reduce myocardial ischemia-reperfusion injury under clinical conditions, in particular in patients undergoing cardiac surgery using cardiopulmonary bypass. Under the status of anesthesia and adequate ventilation with oxygen, transient systemic ischemia and haemodilution should be tolerable. Mild HD with D-HES before surgery may provide additional cardiac protection.
Supported in part by a Scientific Achievement Award (982038-5, to Zhengyuan Xia) from Hubei Province, China and in part by an internal research funding (to Xian-Yi Liu) from Renmin Hospital of Wuhan University, China.
Presented in part at the American Society of Anesthesiologists 2003 Annual Meeting, San Francisco, California, October 11–15, 2003 and published in abstract form in Anesthesiology 2003;99:A752. 1 Present address: Centre for Anesthesia and Analgesia, Department of Pharmacology and Therapeutics, The University of British Columbia, Vancouver, Canada V6T 1N6. 
2 Present address: Department of Anesthesia, First People's Hospital of Jingzhou, Jingzhou 434000, China.
doi:10.1016/j.icvts.2004.05.007
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Abstract
1. Introduction
or 
vs baseline; 
or 0.01 vs control group; 
vs HD group.
vs control group.