Abstract
BACKGROUND:
Recent studies have shown that α2-adrenergic agonists can reduce postresuscitation myocardial injury. This study was undertaken to observe changes of hemodynamics, myocardial injury markers cTnT and cardiac morphology by establishing a cardiopulmonary resuscitation model with rabbits, and to detect whether α-methyl norepinephrine (α-MNE) can reduce the myocardial injury after CPR and improve cardiac function.
METHODS:
Eighteen health rabbits, weighing 2.5-3.5 kg, both male and female, were provided by the Lanzhou Institute of Veterinary Medicine. After setting up a rabbit model of cardiopulmonary resuscitation, 18 rabbits were randomly divided into three groups. The rabbits in group A as an operation-control group were subjected to anesthesia, endotracheal intubation, and surgery without induction of ventricular fibrillation. The rabbits in group B as an epinephrine group were administered with 30 μg/kg epinephrineduring CPR. The rabbits in group C as a MNE group were administered with 100 μg/kg a-MNE during CPR. The left ventricular end-diastolic pressure (LVEDP), left ventricular pressure rise and fall rate (±dp/dt) and serum concentrations of BNP were measured. Statistical package of SPSS 10.0 was used for data analysis and significant differences between means were evaluated by ANOVA.
RESULTS:
Compared to group A, the LVEDP of other two groups increased respectively (P<0.01 all), and peak±dp/dt decreased in the other two groups (P<0.01). The increase of LVEDP was lower in group C than in group B (P<0.05), whereas peak±dp/dt was higher in group C than in group B (P<0.05) at the same stage. Compared to group A, the cTnT of the remaining two groups increased, respectively (P<0.01), and peaked at 30 minutes. cTnT was less elevated in group C than in group B (P<0.05) during the same period. In groups B and C, myocardial injury was seen under a light microscope, but the injury in group C was lighter than that in group B.
CONCLUSION:
Methylnorepinephrine can lessen myocardial dysfunction after CPR.
KEY WORDS: Cardiopulmonary resuscitation;, α2-adrenergic agonist;, Post-resuscitation myocardial dysfunction
INTRODUCTION
After cardiopulmonary resuscitation (CPR), myocardial dysfunction is the main reason for in-hospital death at the early stage. Although the initial success rate of CPR can reach 40%, most of these patients may die from heart failure and/or ventricular fibrillation within 72 hours. Finally, CPR itself can only save the lives of 1.4%-17.0% of the patients. Therefore, how to protect the myocardium, how to reduce myocardial damage, how to improve the stability of postresuscitation spontaneous circulation, and how to improve survival rate have been hot topics in this field. Recent studies[1-4] have shown that α2-adrenergic agonists can reduce post-resuscitation myocardial injury. The present study aimed to observe changes of hemodynamics, myocardial injury markers cTnT and cardiac morphology through establishing a cardiopulmonary resuscitation model of rabbits, and to detect whether α-methyl norepinephrine (α-MNE) can reduce myocardial injury after CPR and improve cardiac function.
METHODS
Experimental animals and grouping
Eighteen health rabbits, weighing 2.5-3.5 kg, both male and female, were provided by the Lanzhou Institute of Veterinary Medicine, China. After setting up a rabbit model of cardiopulmonary resuscitation, 18 rabbits were randomly divided into three groups: group A as an operation-control group; group B as an epinephrine group; and group C as α-MNE group.
Agents and equipments
The following agents and equipments were used in the sudy: α-methyl norepinephrine lyophilized powder for injection (USA Sigma Company, 1.0 g/support, batch number: RG-1083); adrenal hydrochloride injection (Amino Acid Co., Ltd. Tianjin Jin Yao, 1.0 mg/ 1 ml, batch number 0611011); heparin (Tianjin Biochemical Pharmaceutical Factory, batch number 20070304) with normal saline with 1.25 million and 1.25 million U/250 ml U/100 ml concentration of standby; cTnT kit products for the U.S. ADL (Lot QRCT-239652EIA\UTL, 96tests); a HX-200 animal respirator (Chengdu Thai Union Technology Co., Ltd.); BL-420E biological and functional experiment recorder (Chengdu Thai Union Technology Co., Ltd.); an animal heart catheter (diameter 2 mm, Chengdu TaimenUnion Technology Company Limited); an ECG monitor defibrillator machine (TEC 1 762lC, Japan); a high-speed desktop refrigerated centrifuge (TGL a 16G, Shanghai Anting Scientific Instrument Factory); an AC voltage converter (Shanghai Transformer Works).
Animal models and treatment
Rabbits were fasted for 12 hours before surgery but accessible to water. The rabbits were anesthetized with injection of 25% urethane (4 ml/kg) via the ear vein, and then fixed on the operating table and monitored with standard ECG limb lead ECG II. Under the aseptic conditions, a 2.0 cm incision in the midline was made along the neck, the subcutaneous tissue and muscle were separated, and the trachea was exposed. The trachea was cut between the tracheal rings below 1 cm of the larynx, and a t-tracheal tube was used for intubation and fixed with silk suture, and the rabbits breathed freely. After the separation of the right carotid artery, PE was inserted into the left ventricular catheter. PE was used to detect LVEDP and the left ventricular maximum rate of pressure rise (peak+dp/dt), and the maximal rate of left ventricular pressure drop (peak-dp/dt) using a pressure transducer and a multichannel physiologic monitor. The catheter was inserted in the left femoral artery to measure the mean arterial pressure (MAP) and collect blood samples for blood gas analysis. The trocar was used for jugular vein puncture for the delivery of agents. If necessary, normal saline (10-15 ml/h) was infused intravenously to maintain MAP≥80 mmHg for at least 10 minutes. Electrodes were inserted into the apex beating spot and right subclavian point in the chest wall to connect the loop to an AC adapter. The loop was connected to 50V AC and continuously discharged for 3 minutes to prevent the automatic recovery of ventricular fibrillation.
The criteria of successful ventricular fibrillation included that ECG showed the ventricular fibrillation and the arterial pressure was less than 20 mmHg. After the ventricular fibrillation lasted for 3 minutes, chest compressions were made by hand (200 times per minute, compression: ventilation ratio 15:2; the interval of compression and decompression was equal and the depth of compression was about the thoracic anteroposterior diameter of 1/3), and at the same time mechanical ventilation was provided (FiO2 1.0, tidal volume 15 ml/kg). The agent for resuscitation was administered according to the groups. The rabbits in group A were subjected to anesthesia, endotracheal intubation, and surgery without induction of ventricular fibrillation. The rabbits in group B were administered with 30 μg/kg epinephrine during CPR. The rabbits in group C were administered with 100 μg/kg α-MNE during CPR. After the chest compression lasted for 2 minutes, the rabbits received defibrillation with single-phase wave of 30 J for 3 times. The rabbits, which didn’t have successful resuscitation at the first time, received chest compression for 2 minutes before the second round of defibrillation. The criteria for restoration of spontaneous circulation were recovery of supraventricular rhythm, and MAP more than 60 mmHg lasting for 5 minutes. When the rabbits demonstrated spontaneous breathing, mechanical ventilation stopped or cancelled and 100% oxygen was given continuously through a T tube. After resuscitation, the rabbits were monitored for 4 hours, and no other agents were given. After 4 hours, all catheters including that for endotracheal intubation were removed. The rabbits were killed by intraperitoneal injection of 150 mg/kg of pentobarbital. Organ abnormalities were observed with naked eyes, including part of tube intubation, airway management, or part of secondary injury caused by compression. The experimental procedure was performed according to the Utstein-style guidelines.[5,6]
Detection of left ventricular end diastolic pressure (LVEDP)
The rise and fall of left ventricular pressure maximal rate (peak±dp/dt) were measured with the BL-420E biological and functional experimental system. Data were collected at 15 minutes before fibrillation and at 30, 60, 120, 180, and 240 minutes after recovery respectively.
Detection of serum cardiac troponin T (cTnT)
Two ml of blood was collected from the central vein at 15 minutes before VF and at 30, 60, 120, 180 and 240 minutes after restoration of spontaneous circulation. The blood was reserved in a free anti-condensing tube and kept for 20 minutes, and then the supernatant was transferred to a 3 ml centrifuge tube with a cover. At 4 °C, the blood was centrifugated at 3000 r/min for 20 minutes, and the serum was kept for the detection of cTnT concentration. Before the detection, the sample was dissolved again, and 1 ml serum was collected and measured by enzyme-linked immunosorbent assay (ELISA).
Myocardial morphology
Two rabbits from each group, which survived for 4 hours after successful resuscitation, were selected randomly. The hearts were removed rapidly, and the left ventricular myocardium was cut near the apex. The myocardial sections were fixed with formalin and glutaraldehyde, embedded by paraffin, cut into slices of 4 μm thickness, and stained with hematoxylin-eosin (HE) and dehydration fengpian. Morphological structure and organization of myocardial inflammation reaction were observed using an OLYMPUS optical microscope (200 times).
Statistical analysis
Data were analyzed by using SPSS10.0 software. The data were expressed as mean±standard deviation. The groups were compared with one-way analysis of variance (ANOVA) and multiple samples between mean numbers were compared with the SNK-q test. All data based on time were measured by repeated ANOVA.
RESULTS
Results of resuscitation
One rabbit in group A and one rabbit in group B died from operation, and others survived and were observed for 240 minutes.
Changes of hemodynamics
Before inducing ventricular fibrillation, no significances in hemodynamics were observed between the three groups (P>0.05). In group A, there were no significant differences during the observation period (P>0.05). Before inducing ventricular fibrillation, LVEDP in groups B and C increased significantly at 30 minutes after resuscitation (in group B, 290%, 2.68 ±0.44 mmHg vs. 7.76±0.68 mmHg; in group C, 262%, 2.59 ±0.53 mmHg vs. 6.80±0.54 mmHg, P<0.01); LVEDP was increased more significantly in group B than in group C (P<0.05).
At 60 minutes after successful CPR, LVEDP decreased in groups B and C, and it was less decreased in group B than in group C (P<0.05) (Figures 1).
Figure 1.
Changes of LVEDP in the three groups after resuscitation. BL: baseline before inducing ventricular fibrillation; compared with group B, *P<0.05, **P<0.01.
Peak+dp/dt and peak-dp/dt decreased after resuscitation, and peak+dp/dt and peak-dp/dt were decreased more significantly in group B than in group C (P<0.05); and compared to group A, there were significant differences in groups B and C (P<0.01) (Figures 2-3).
Figure 2.
Changes of peak + dp/dt in the three groups after resuscitation; BL: baseline before inducing ventricular fibrillation; compared with group B, *P<0.05.
Figure 3.
Changes of peak-dp/dt in the three groups after resuscitation. BL: baseline before inducing ventricular fibrillation; compared with group B, *P<0.05
Changes of cTnT
Before inducing ventricular fibrillation, there were no significances in cTnT between the three groups (P>0.05). Compared to group A, cTnT was significantly higher in groups B and C (P<0.05); cTnT was increased more significantly in group B than in group C, especially at 60 minutes (P<0.01) (Table 1, Figure 4).
Table 1.
Changes of serum cTnT before and after CPR (SD, ng/ml)
Figure 4.
Changes of cTnT in the three groups after resuscitation. BL: baseline before inducing ventricular fibrillation at 15 minutes; compared with group B, *P<0.05, **P<0.01.
Myocardium under a light microscope in the three groups
Myocardial cells in group A lined in order and sarcolemma kept integrity (Figure 5). In group B, myocardial fiber structure in the adrenaline group was disordered and some nuclei dissolved, disappeared and even muscle fibers dissolved. Some myocardial cells suffered from granular degeneration and vacuolar degeneration (Figure 5). In group C, there were mild lesion morphology, neatly arranged myocardial fibers, integrity of cell membrane, and slight edema of myocardial interstitia. The nucleus was non-soluble, cytoplasm was not vacuolized, myocardial stripes were present (Figure 5).
Figure 5.
Pathological changes of the myocardium.
DISSCUSSION
The nature of CPR is ischemia/reperfusion injury, and myocardial dysfunction is the main reason for death at early stage. According to the American Heart Association, if myocardial dysfunction after successful resuscitation could be treated, about 25000 patients per year would be saved. Therefore, how to protect the myocardium, how to reduce myocardial damage, how to improve the stability of postresuscitation spontaneous circulation, and how to improve survival rate have been the hot topics of investigation. Through establishing a cardiopulmonary resuscitation model in rabbits, we observed changes of hemodynamics, myocardial injury markers cTnT and cardiac morphology and detected whether α-methyl norepinephrine (α-MNE) can reduce myocardial injury after CPR and improve cardiac function.
Before inducing ventricular fibrillation, LVEDP in groups B and C increased significantly at 30 minutes after resuscitation (in group B, 290%, 2.68±0.44 mmHg vs. 7.76±0.68 mmHg; in group C, 262%, 2.59±0.53 mmHg vs. 6.80 ±0.54 mmHg, P<0.01); LVEDP was increased more significantly in group B than in group C (P<0.05). At 60 minutes after successful CPR, LVEDP gradually decreased in groups B and C, and LVEDP was less decreased in group B than in group C (P<0.05). Peak +dp/dt and peak-dp/dt decreased after resuscitation, and Peak+dp/dt and peak-dp/dt decreased more significantly in group B than in group C (P<0.05). Compared with group A, significant differences were observed in groups B and C (P<0.01). The results showed that α-MNE as a selective α2-adrenergic receptor agonist may not prevent the development of post-resuscitation myocardial dysfunction, but can improve the myocardial dysfunction. This finding is consistent with that reported elsewhere.[1-3]
Studies showed that after resuscitation for cardiac arrest, epinephrine significantly increased the severity of post-resuscitation myocardial dysfunction and decreased the duration of survival. Selective α-adrenergic agonists or blockade of ß1-adrenergic actions of epinephrine reduced post-resuscitation myocardial impairment and prolonged survival.[7,8]
Li et al[9] reported that peak+dp/dt and peak-dp/dt were less decreased and LVEDP and BNP were less increased in the epinephrine with esmolol (epinephrine and short acting ß1-adrenergic receptor blocker) group than those in the epinephrine group. Therefore, epinephrine increased post-resuscitation myocardial dysfunction. Pellis et al[10] found that epinephrine, epinephrine after alpha1- and beta-adrenergic blockade, and vasopressin were equally effective in restoring spontaneous circulation after prolonged ventricular fibrillation. However, combined alpha1- and beta-adrenergic blockade, which represented a predominantly selective alpha 2-vasopressor effect, resulted in improved post-resuscitation cardiac and neurological recovery.
CTnT is a heart-specific antigen. As long as myocardial injury happens, cTnT escapes rapidly from sarcoplasm, and the level of cTnT is consistent with the degree of pathological changes. cTnT has been a sensitive and specific marker of myocardial injury. cTnT concentration and morphological characteristics of myocardial cells under a light microscope can sensitively reflect the myocardial injury at its early phase.[11] The present study showed that compared to group A, cTnT was significantly higher in groups B and C (P<0.05) and it was increased more significantly in group B than in group C, especially at 60 minutes (P<0.01). Myocardial morphology showed that the rabbits in group B were more injured than those in group C, and myocardial ischemia-reperfusion injury existed continuously. α-MNE significantly reduced the myocardial ischemia-reperfusion injury after CPR, which was helpful to improve post-resuscitation myocardial dysfunction.
α-MNE as a selective α2-adrenergic receptor agonist activates α2-adrenergic receptors in the peripheral vascular smooth muscle. As a member of G protein-coupled receptors, α2-adrenergic receptor inhibits the activity of adenylate cyclase through Gi protein, and then reduces the level of intracellular cAMP. Eventually, α2-adrenergic receptor inhibits the protein kinase K (PKK) and the protein phosphorylation regulated by PKK. In the recent years, research on α2-adrenergic receptor agonists has concentrated on the central nervous system and its use in the field of anesthesia. Presently some scholars found that compared with epinephrine, α2-adrenergic receptor agonists may have more advantages in cardiopulmonary resuscitation. Sun et al[1] reported that the selective alpha 2-adrenergic agonist, alpha-MNE, was as effective as epinephrine for initial cardiac resuscitation but provided strikingly better post-resuscitation myocardial function and survival. Klouche et al[2] showed that α-MNE, a selective α-adrenergic agonist, was as effective as epinephrine in restoring spontaneous circulation after 7 minutes of untreated VF in a porcine model for CPR and demonstrated lesser post-resuscitation myocardial injury. Another study[3] showed that both post-resuscitation myocardial function and survival were improved significantly after administration of selective alpha 2-adrenergic receptor agonist, less significantly after administration of vasopressin, and least significantly after administration of epinephrine and saline placebo. Xu et al[12] found that α2-adrenergic receptor agonist has a direct anti-ischemic effect. Finally, compared with other selective α2-adrenergic receptor agonists such as cocaine and mifaxichun, α-methyl norepinephrine doesn’t pass through the blood-brain barrier or produce central level negative chronotropic action and inotropic action. Hence it is not effective in lowering the myocardial contractility and arterial blood pressure, but may exert an effect on post-resuscitation ischemia-reperfusion injury.
The present study showed that α2-adrenergic receptor agonists have the same effect as adrenaline in improving the development of post-resuscitation myocardial dysfunction, reducing the incidence of malignant arrhythmia, reducing the number of defibrillation energy and intrapulmonary shunt, and increasing the survival time.[13,14] In addition, α2-adrenergic receptor agonist also has direct anti-ischemic effect, and it is a potential agent for CPR. Limitations of the study include use of a small number of animals and no explanation of protection mechanisms at the molecular level.
Footnotes
Funding: None.
Ethical approval: Not needed.
Conflicts of interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Contributors: Li PJ proposed and wrote the main body of the paper. All authors contributed to the design and interpretation of the study and to further drafts.
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