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Print version ISSN 0102-7638
Rev Bras Cir Cardiovasc vol.27 no.1 São José do Rio Preto Jan./Mar. 2012
Estudo comparativo experimental da proteção miocárdica com soluções cristalóides para transplante cardíaco
Melchior Luiz LimaI; Alfredo Inácio FiorelliII; Dalton Valentim VassalloIII; Bruno Botelho PinheiroIV; Noedir Antonio Groppo StolfV; Otoni Moreira GomesVI
IPhD in Cardiovascular Surgery Cardiovascular Foundation for St. Francis of Assisi, Belo Horizonte, MG, Brazil; Cardiovascular Surgery Specialist by the AMBPresidente DEPEX / BSCVS, Cardiovascular Surgeon Vitória/ES)
IIPhD in Cardiovascular Surgery at the University of Sao Paulo-USP
IIIPhD in Biological Sciences from the Universidade Federal do Rio de Janeiro - UFRJ
IVMaster of Cardiovascular Surgery Cardiovascular Foundation for St. Francis of Assisi - BH / MG; Cardiovascular Surgeon - Goiânia GO
VPhD Faculty of Medicine, USP, the Incor Director - SP
VIPhD in Cardiovascular Surgery at the University São Paulo -USP, Professor of Surgery, Federal University of Minas Gerais (UFMG), Scientific Director of the Cardiovascular Foundation St. Francis of Assisi - Servcor-Belo Horizonte, MG, Brazil
BACKGROUND: There is a growing need to improve myocardial protection, which will lead to better performance of cardiac operations and reduce morbidity and mortality. Therefore, the objective of this study was to compare the efficacy of myocardial protection solution using both intracellular and extracellular crystalloid type regarding the performance of the electrical conduction system, left ventricular contractility and edema, after being subjected to ischemic arrest and reperfusion.
METHODS: Hearts isolated from male Wistar (n=32) rats were prepared using Langendorff method and randomly divided equally into four groups according the cardioprotective solutions used Krebs-Henseleit-Buffer (KHB), Bretschneider-HTK (HTK), St. Thomas-1 (STH-1) and Celsior (CEL). After stabilization with KHB at 37ºC, baseline values (control) were collected for heart rate (HR), left ventricle systolic pressure (LVSP), maximum first derivate of rise left ventricular pressure (+dP/dt), maximum first derivate of fall left ventricular pressure (-dP/dt) and coronary flow (CF). The hearts were then perfused at 10ºC for 5 min and kept for 2 h in static ischemia at 20ºC in each cardioprotective solution. Data evaluation was done using analysis of variance in completely randomized One-Way ANOVA and Tukey's test for multiple comparisons. The level of statistical significance chosen was P<0.05.
RESULTS: HR was restored with all the solutions used. The evaluation of left ventricular contractility (LVSP, +dP/ dt and -dP/dt) showed that treatment with CEL solution was better compared to other solutions. When analyzing the CF, the HTK solution showed better protection against edema.
CONCLUSION: Despite the cardioprotective crystalloid solutions studied are not fully able to suppress the deleterious effects of ischemia and reperfusion in the rat heart, the CEL solution had significantly higher results followed by HTK>KHB>STH-1.
Keywords: heart arrest, induced. myocardial reperfusion injury. cardioplegic solutions. ventricular function, left. rats, Wistar.
INTRODUÇÃO: Existe crescente necessidade de aprimorar a proteção miocárdica, para melhor desempenho das operações cardíacas e diminuição da morbimortalidade. Portanto, o objetivo deste estudo foi comparar a eficácia da proteção miocárdica usando tanto solução cristaloide tipo intracelular como extracelular quanto ao desempenho do sistema de condução elétrica, contratilidade do ventrículo esquerdo e edema, após parada isquêmica e posterior reperfusão.
MÉTODOS: Corações isolados de ratos Wistar foram montados em Langendorff e aleatoriamente divididos em quatro grupos. de acordo com as soluções cardioprotetoras utilizadas Krebs-Henseleit-Buffer (KHB), Bretschneider-HTK (HTK), St. Thomas-1(STH-1) e Celsior (CEL). Após a estabilização com KHB a 37ºC, valores basais (controle) foram coletados para frequência cardíaca (FC), pressão sistólica do ventrículo esquerdo (PSVE), derivada máxima de aumento da pressão ventricular esquerda (+dP/dt), derivada máxima de queda da pressão ventricular esquerda (-dP/dt) e fluxo coronariano (FCo). Os corações foram então perfundidos a 10ºC por 5 min e mantidos por 2 h em isquemia estática a 20ºC em cada solução cardioprotetora. Avaliação dos dados foi por análise de variância inteiramente casualizados em One-Way ANOVA e teste de Tukey para comparações múltiplas. O nível de significância estatística escolhido foi P<0,05.
RESULTADOS: Houve recuperação da FC com todas as soluções utilizadas. A avaliação da contratilidade ventricular esquerda (PSVE, +dP/dt e -dP/dt) demonstrou que o tratamento com a solução CEL foi melhor em comparação às outras soluções. Ao analisar o CF, a solução HTK indicou melhor proteção contra edema.
CONCLUSÃO: Apesar das soluções cristaloides cardioprotetoras estudadas não serem capazes de suprimir os efeitos deletérios da isquemia e reperfusão no coração de ratos, a solução CEL apresentou resultado superior seguido por HTK>KHB>STH-1.
Descritores: parada cardíaca induzida. traumatismo por reperfusão miocárdica. soluções cardioplégicas. função ventricular esquerda. ratos Wistar.
Currently, most heart surgeries are performed with anoxic arrest induced by using different cardioplegic solutions, suggesting the lack of a gold standard for myocardial protection . Procedures with short period of ischemia, preservation is simpler. However, procedures where long ischemic periods are common, myocardial viability may be compromised by the current methods of myocardial preservation . Thus, establishing satisfactory method of preservation is critical to ensure success in procedures with prolonged ischemic time, particularly in cardiac transplantation, which can also lead to expanding the pool of donors .
Due to the shortage of donated hearts, selection criteria are under constant review in order to increase the number of marginal donors [3,4]. Nevertheless, studies in the field of myocardial protection have great relevance for the advancement of heart transplantation . Prolonged myocardial ischemia is an independent risk factor for early and late survival of the patient .
The crystalloid cardioplegic solutions were initially idealized in order to depolarize the cell membrane. Thus, their initial formulations were basically ionic. The progress of research on myocardial protection showed the need for additives in the solutions to expand their effectiveness. The main actions of additives aimed at removal of free radicals, providing nutrients, prevention of intracellular acidosis and stabilization of cell membranes to minimize swelling .
Studies on myocardial protection with additives in the solutions associated with hypothermia, demonstrated improved contractile function after long periods of ischemia . Hypothermia protects cellular energy metabolism acting improving the resistance to ischemia in cardioplegic cardiac arrest . The increase in the ratio between supply and energy demand during ischemia is generally attributed to hypothermic protection. The hypothermia also combats oxidative stress induced by ischemia and reperfusion .
There is still growing need to further investigate and improve heart preservation methods, thus improving performance of cardiac operations, reducing morbidity, increasing the donor pool, and extending its indications and benefits .
The objective of this study was to compare the efficacy of myocardial protection solution using both intracellular and extracellular crystalloid type regarding the performance of the electrical conduction system, left ventricular contractility and edema, after being subjected to ischemic arrest and reperfusion.
The Brazilian College of Animal Experimentation (COBEA) and the Ethics Committee of the Fundação Cardiovascular São Francisco de Assis, Belo Horizonte, Minas Gerais, Brazil, approved all experiments. All experiments used an isolated isovolumetrically contracting rat heart. Male Wistar albino rats (n=32), 310 to 320 g, were anesthetized by intraperitoneal injection of a mixture of ketamine (50 mg/kg) and xilazine (10 mg/kg). After the chest was opened, heparin (500 IU) was injected into the left atrium. An aortic cannula filled with perfusate was rapidly inserted into the aorta, and retrograde perfusion was started with an oxygenated Krebs-Henseleit buffer at 37ºC and maintained at a constant pressure of 100 mmHg in a single pass way by the Langendorff technique . The pulmonary artery was incised to allow outflow of the perfusate. A latex balloon was placed in the left ventricle and connected to a pressure transducer line. The balloon was inflated with water to create a diastolic pressure of 7 to 9 mmHg. The hearts were beating spontaneously at an average rate of 300 beats/ min. After 15 min of perfusion at 37ºC with KHB solution for stabilization, we collected the values considered baseline (control) for the following parameters: heart rate (HR) to evaluate the electrical conduction system; left ventricle systolic pressure (LVSP), the maximum rate of rise in left ventricular (+dP/dt), the maximum rate of fall in left ventricular (-dP/dt) pressures to evaluate the ventricular contractility and coronary flow (CF) to evaluate the edema.
The hearts were randomly divided equally into four groups, as follows: Group 1 were treated with Krebs-Henseleit (KHB) solution (Research Laboratory of Fundação Cardiovascular São Francisco de Assis, Belo Horizonte, MG, Brazil), Group 2 with Bretschneider-HTK (HTK) solution (Dr. Franz Köhler Chemie GMBH - Germany), Group 3 with St. Thomas No. 1 (STH-1) solution (Braile Biomédica Industry, Sâo Paulo, SP, Brazil), and Group 4 with Celsior (CEL) solution (Genzyme Polyclonals S.A.S., France). Table 2 shows the chemical composition of the solutions studied.
The hearts were then perfused with their respective cardioprotective solutions for 5 min at 10ºC and kept for 2 h in static ischemia at 20ºC. Subsequently, the hearts were reperfused with KHB at 37ºC for 60 min and data were collected every 5 min. Data evaluation was based on analysis of variance in completely randomized One-Way ANOVA and Tukey's test for multiple comparisons. The criterion for significance was P<0.05 for all comparisons.
To evaluate myocardial protection, we first compared the effect of the solutions on HR. Figure 1 shows the trend in HR of the solutions used in the experiment at 10ºC, compared with the control, represented by a ratio of 1.0 (basal HR). CEL and KHB solutions provided a more stable HR throughout the length of the experiment. On the other hand, use of HTK and STH-1 solutions initially resulted in lower HR, which increased after 15 min and 30 min, respectively, and stabilized at a similar HR compared to the other solutions. These results indicated that all four solutions were able to recover the HR.
Left ventricular contractility was represented by the corresponding hemodynamic variables LVSP, (+dP/dt), and (-dP/dt) (Figures 2 to 4). These variables show similar trends with the different solutions. CEL solution was more stable and with higher rates compared to the other solutions. With the HTK solution, rates increased constantly throughout the 60 min period, and were higher compared to STH-1 and KHB. KHB solution resulted in higher rates for all variables compared to STH-1, but was still lower than HTK at LVSP and (-dP/dt). Despite these differences, KHB reached approximately the same rate of (+dP/dt) after 40 min, compared to HTK. The contractile performance of STH-1 was lower than the other solutions. Here, the data show that treatment with CEL is superior to the others solutions.
Because the occurrence of edema, which is a negative factor in the recovery of the heart, the CF was considered in the corresponding hemodynamics variables dynamics. All treatments showed a downward trend (Figure 5). However, treatment with HTK solution produced higher flow values compared to the others. Moreover, these treatments indicated a decreasing order of efficiency: HTK>CEL>KHB>STH-1. Together, these results indicate that performance on CF maintenance is time-dependent. However, use of HTK suggests better protection against development of tissue edema.
To better evaluate the efficiency of myocardial protection was made a study of multiple comparisons between treatments (Table 1). For HR, only CEL versus HTK were not significantly different. For LVSP, (+dP/dt), (dP/dt) and CF, all comparisons were significantly different. Overall, use of CEL resulted in significant improvement in hemodynamic variable outcome compared to the other solutions.
Clinical investigations on the comparative performance of the cardioplegic solutions offer the greatest difficulties on result interpretation and may bring false judgment. Langendorff system was chosen because it is well standardized in our laboratories about myocardial protection evaluation and is possible also analyzing the direct effects on the heart with systemic interferences exclusion .
Hypothermia was adopted in this study because it is a standard strategy of myocardial protection. Cleveland et al.  showed that hypothermia is the most important factor in myocardial protection. Studies on myocardial protection with cardioprotective additives, associated with hypothermia, demonstrated improved contractile function after long ischemia periods .
Pereda et al.  compared the performance of Celsior (CEL) versus St. Thomas No. 2 (STH-2) solutions, as blood cardioplegia, demonstrating that they were not significantly different.
Loganathan et al.  analyzed the effects of reperfusion up to 24 hours using Bretschneider-HTK (HTK) solution and modified Bretschneider-HTK (Custodiol-N). The last one improves myocardial and endothelial function during the critical phase of reperfusion after heart transplantation.
Lee et al. , contrariwise, found that Bretschneider-HTK (HTK) solution exhibited superior protective effects over CEL against prolonged cold ischemia in a syngeneic rat transplantation model.
The current clinical practice in the Brazilian myocardial protection in cardiac transplantation commonly uses the St. Thomas No. 1 (STH-1) crystalloid solution, extracellular type. Currently, other solutions were added to the therapeutic arsenal of myocardial protection, such as the Celsior solution (CEL), extracellular type, and Bretschneider-HTK solution (HTK), intracellular type (Table 2). Such solutions have been used increasingly in major transplant centers. Supported in the international literature we compared the ventricular performance experimentally using the above solutions in the myocardium of rats subjected to ischemia and reperfusion.
In the present investigation we adopted the absence of cardiac pacing to enhance the intrinsic rhythm of the heart. Additionally, it should be emphasized that the heart's conduction tissue is more sensitive to ischemia . Thus, heart rate is ultimately a variable capable of providing indirect information on the severity of injury caused by ischemia and reperfusion . All solutions provided preservation of the HR, but the results were below the baseline value for this variable. It was observed that after 30 min of reperfusion, all solutions were stable. Note that the STH-1 solution took 30 min for stabilization.
The myocardial contractility was assessed in an integrated manner by the following variables: LVSP, (+dP/ dt), and (-dP/dt). We observed that the effects of ischemia and reperfusion on the myocardium are extremely deleterious, producing a marked reduction in ventricular performance. We infer that the concentrations of K+ (15 mmol/L) and Ca2+ (0.25 mmol/L) of CEL solution can contribute to a better performance by promoting the depolarizing arrest without contributing to an overload of intracellular calcium during the ischemic period [17,18].
Considering that isolated hearts used in this study had a fixed pressure gradient, essentially the only factor responsible for decreased blood flow would be related to interstitial edema. Therefore, by analyzing the behavior of coronary flow, we aimed to relate it directly to myocardial edema. The results indicate that HTK solution were those that produced the highest flow values. The other solutions used showed a descending order of efficiency in maintaining coronary flow, as follows: CEL>KHB>STH-1. We suggest that each solution has an optimal preservation temperature, where hypothermia can facilitate or interfere with tissue edema, possibly by directly influencing membrane conductive properties in myocardial cells, as well as modifying the permeability of the endothelium [8,19,20]. Moreover, another antagonistic factor to edema development could be related to the osmotic properties of each solution used [18,21]. Relative to osmolarity, these solutions have the following decreasing order: KHB>STH-1>CEL>HTK. However, we did not observe this same order considering comparative performance. Additionally, Na+ is also an important variable in this process, and these solutions have the following decreasing order in concentration of this ion: STH-1>KHB>CEL>HTK. The comparative performances between them do not obey this order, indicating that Na+ is not solely responsible for the participation of edema .
This study is part of a line of research that includes endothelial dysfunction and apoptosis using different cardioprotective methods, and has inherent limitations. Perfusion of non-human isolated hearts with solutions without blood produces disturbances in cardiac performance. However, even though the data obtained cannot be translated directly to clinical application, one must consider that comparative studies with animal models have proven effective in research related to myocardial preservation [22,23].
Despite the cardioprotective crystalloid solutions studied are not fully able to suppress the deleterious effects of ischemia and reperfusion in the rat heart, the CEL solution had significantly higher results followed by HTK>KHB>STH-1. Other researches are still needed, considering different infusion temperatures and others cardioplegic solutions to extend the cardioprotective methods.
The authors would like to thank the Brazilian Association for Organ Transplant (ABTO) for their support and efforts in providing scientific writing workshops.
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Melchior Luiz Lima
Rua Benedito Mello Serrano, 165 - Mata da Praia
Vitória, ES, Brazil - ZIP Code: 29054-040
Article received on September 9th, 2011
Article accepted on January 8th, 2012
Work performed at Fundação Cardiovascular São Francisco de Assis, Belo Horizonte, MG, Brazil.