INTRODUCTION

Myocardial protection is one of the most important issues in cardiac surgery, and cardioplegic solutions improve the tolerance to ischemia and protect against the adverse effects of reperfusion. Custodiol (histidine-tryptophan-ketoglutarate) is an intracellular crystalloid cardioplegic solution that provides diastolic cardiac arrest via hyperpolarization of the myocyte plasma membrane. Consequently, Custodiol improves high energy production, stabilizes cell membranes, and maintains osmotic regulation of the cell membrane. In recent years, Custodiol has been used as an organ preservation solution for cardiac transplantation, as well as other organs^[1]. Custodiol was developed by Bretschneider in the 1970s and has been used as a cardioprotective solution for cardiac surgery and transplantation^[2,3]. The components of Custodiol act as a multifunctional protection mechanism; histidine buffers the acidosis accumulated by anaerobic metabolism, tryptophan stabilizes the cell membrane, ketoglutarate improves adenosine triphosphate (ATP) production during reperfusion, and mannitol decreases cellular edema^[2].

Blood cardioplegia is a routine and conventional solution that has been proposed as a safe and reliable technique for myocardial protection. Its high potassium content causes diastolic arrest via myocyte membrane depolarization. Blood, as a medium for cardioplegia delivery, has a greater oxygen-carrying capacity and is less associated with hemodilution^[4]. The composition of Custodiol and blood cardioplegia is listed in Table 1. Currently, there is no consensus as to the optimal cardioplegic solution delivery. Therefore, we aimed to compare the histopathological and clinical outcomes of Custodiol versus blood cardioplegia solutions.

Table 1 - Composition of Custodiol versus blood cardioplegia.

METHODS

This study was designed as a prospective study and performed on 20 patients who underwent mitral valve surgery. Patients were included in this study upon receipt of informed written consent. The study protocol was approved by the local ethics committee (Mehmet Akif Ersoy Clinical Research Ethics Committee). Demographic data and risk factors are listed in Table 2. Patients were divided into two groups according to the cardioplegia solution used. Cardioplegia solutions were designated as Custodiol in group 1 (n=10) and blood cardioplegia in group 2 (n=10). Patients were randomly included in the groups preoperatively. Group 1 received a single dose of antegrade cold (4°C) Custodiol solution and group 2 received repetitive antegrade isothermic (34°C) blood cardioplegic solution. No retrograde cardioplegia and additional topical cooling were used in any patient.

Table 2 - Demographic data and risk factors.

Conventional general anesthesia was used in all patients. Mitral valve surgery was performed via a left atriotomy with a median sternotomy. All surgeries were performed using cardiopulmonary bypass (CPB) with the use of a roller pump, ascending aortic cannulation, double venous cannulation, and moderate systemic hypothermia (32-34°C). Anticoagulation was provided through systemic heparinization and CPB was initiated in the process of activated clotting time over 400 seconds. Patients who required arrhythmia surgery were excluded from the study.

Isothermic blood cardioplegia was delivered antegradely through a cannula placed in the ascending aorta every 20 minutes with a pressure of 200-250 mmHg at the time of aortic cross-clamping. A total of 900 mL of blood cardioplegia was administered to the patient initially. Thereafter, 500 mL of blood cardioplegia was given antegradely every 20 minutes^[5]. During the delivery, aortic valve competence was checked through digital palpation of the pressure in the ascending aorta and volume overload at the left ventricle. Custodiol was administered antegradely through the ascending aorta at an initial perfusion pressure of 80-100 mmHg at the time of 6-8 minutes as described by the manufacturer. Each patient received 20-25 mL of Custodiol per kg of body weight, in accordance with previous studies^[6]. Atriotomies were performed after cardiac arrest was sustained. Therefore, Custodiol coming from the coronary sinus went directly to the CPB reservoir. Ultrafiltration was used for patients receiving Custodiol. Twenty percent of the patients had mild hyponatremia after using Custodiol. Postoperative 0, 1^st, 3^rd, and 6^th hours arterial blood gas analyses were routinely performed, and electrolyte values were closely followed. All electrolyte imbalances were normalized within the first 24 hours.

Continuous telemetry monitoring was used for every patient from the beginning of the surgery until discharge. Intra- and postoperative adverse events were recorded and listed in Tables 3 and 4.

Table 3 - Operative data.

Table 4 - Comparison of cardiac enzymes in the postoperative 6^th, 12^th and 24^th hours.

Troponin I (Tn I) and creatine kinase isoenzyme MB (CK-MB) were defined as equivalence in surgical outcomes between Custodiol and blood cardioplegia. Blood samples for determination of Tn I and CK-MB were obtained 6, 12, and 24 hours following the release of the cross-clamp (Table 4). These molecules were studied in the central laboratory (Cobas-c501 Roche, USA, 0-0.014 ng/mL) of the hospital.

RESULTS

Demographic data and risk factors are evaluated in Table 2 for blood cardioplegia and Custodiol groups. There was no significant difference between groups. Mitral valve surgery (Custodiol group, n=7, blood cardioplegia group, n=8) and mitral valve and concomitant tricuspid valve surgery (Custodiol group, n=3, blood cardioplegia group, n=2) procedures were performed. The mean CPB (101.9±12.05 min vs. 94.0±18.59; P=0.273 min) and cross-clamp times (83.30±11.08 min vs. 76.90±15.56; P=0.304 min) were analyzed and the groups were considered similar. After cross-clamp removal, bradycardia requiring temporary pacing was observed in 2 patients from the Custodiol group and 3 from the blood cardioplegia group. A normal rhythm was reestablished on the 2^nd postoperative day and pacing leads were extracted before discharge.

During the intensive care unit (ICU) follow-up, no acute myocardial infarction, ventricular fibrillation, new-onset left ventricular dysfunction, new-onset nephropathy, and prolonged ventilation occurred in any patient in either group. Only one patient from the blood cardioplegia group needed high-dose inotropic support for 3 days. Electrocardiography (ECG) studies were performed in all patients postoperatively and no ECG changes occurred in six patients from the Custodiol group and five from the blood cardioplegia group. At rest, atrial fibrillation was diagnosed in two patients from each group, and nonspecific changes were detected in two patients from the Custodiol group and three from the blood cardioplegia group.

The Tn I and CK-MB plasma levels obtained at the intervals are evaluated in Figures 1 and 2. Similar results were obtained in both groups; no superiority of any solution could be observed via released enzyme concentrations (P>0.05). Perioperatively and during ICU follow-up, there were no clinically and/or statistically discernible differences between the two groups.

Fig. 1 - Mean troponin I values for both Custodiol and blood cardioplegia groups in 6^th, 12^th and 24^th hours after the cross-clamp was removed.

Download

Fig. 1 - Mean troponin I values for both Custodiol and blood cardioplegia groups in 6^th, 12^th and 24^th hours after the cross-clamp was removed.

Fig. 2 - Mean CK-MB values for both Custodiol and blood cardioplegia groups in 6^th, 12^th and 24^th hours after the cross-clamp was removed.was removed.

Download

Fig. 2 - Mean CK-MB values for both Custodiol and blood cardioplegia groups in 6^th, 12^th and 24^th hours after the cross-clamp was removed.was removed.

Immunohistochemical studies showed moderate eNOS staining (+++), mild iNOS staining (++), mild VEGF staining (++) mild annexin staining (++), and moderate Bcl-2 staining (+++) in the blood cardioplegia group (Figure 3). However, severe eNOS staining (++++), mild iNOS staining (++), mild VEGF staining (++), mild annexin staining (++), and severe Bcl-2 staining (++++) were observed in the Custodiol group (Figure 3). The iNOS, VEGF, and annexin staining were similar in both groups. However, eNOS and Bcl-2 staining were more prominent in the Custodiol group than in the blood cardioplegia group (Figures 4 and 5) (P<0.01).

Fig. 3 - Immunohistochemical markers and median values of staining patterns for Custodiol and blood cardioplegia groups. C: Custodiol group; B: blood cardioplegia group.

Download

Fig. 3 - Immunohistochemical markers and median values of staining patterns for Custodiol and blood cardioplegia groups. C: Custodiol group; B: blood cardioplegia group.

Fig. 4 - eNOS immunostainings of Custodiol versus Blood cardioplegia samples. (A) Custodiol group. Moderate or intense eNOS immunostaining is seen in this section. (B) Blood cardioplegia group. In this section, mild or moderate eNOS immunostaining is seen. Arrows: eNOS stained cardiomyocytes.

Download

Fig. 4 - eNOS immunostainings of Custodiol versus Blood cardioplegia samples. (A) Custodiol group. Moderate or intense eNOS immunostaining is seen in this section. (B) Blood cardioplegia group. In this section, mild or moderate eNOS immunostaining is seen. Arrows: eNOS stained cardiomyocytes.

Fig. 5 - Bcl-2 immunostaining of Custodiol (A) versus blood cardioplegia (B) samples, 20X. (A) Bcl-2 immunostaining is very intense in this section. Custodiol group, 20X. (B) Bcl-2 immunostaining is weak or moderate in this section. Arrows: Bcl-2 stained cardiomyocytes.

Download

Fig. 5 - Bcl-2 immunostaining of Custodiol (A) versus blood cardioplegia (B) samples, 20X. (A) Bcl-2 immunostaining is very intense in this section. Custodiol group, 20X. (B) Bcl-2 immunostaining is weak or moderate in this section. Arrows: Bcl-2 stained cardiomyocytes.

DISCUSSION

Blood cardioplegia has been a standard solution for myocardial protection and diastolic arrest. However, Custodiol has gained popularity. These solutions have been compared for clinical outcomes and enzymatic changes; nevertheless, immunohistochemical studies have not been reported in the literature. The focus of this study was to analyze the cellular immunohistochemical results and clinical outcomes of these solutions.

Immunohistochemical studies are performed by analyzing molecules that are active in apoptosis, inflammation, and cell death during the ischemia-reperfusion processes. Inducible nitric oxide synthase (iNOS) is one of the enzymes that generate nitric oxide (NO) from the amino acid l-arginine. Expression of iNOS is induced in response to cytokines and other agents^[7]. Endothelial nitric oxide synthase (eNOS) is an enzyme that mediates oxidative stress in ischemia/reperfusion injury^[8]. Also, eNOS is a major isoform that regulates vascular function, and is a powerful vasodilator^[9]. Vascular endothelial growth factor (VEGF) is the molecule of response to ischemia generated by the cell^[10]. It is upregulated by hypoxia in cell lines. Bcl-2 is a proto-oncogene molecule that suppresses apoptotic cell death in a variety of in vitro systems and cell lines^[11]. Annexins are a family of phospholipid-binding proteins that prohibit apoptosis and inflammation to mitigate cellular damage^[12]. Annexin plasma levels increase during ischemia.

In our study, there was no significant difference between groups in demographic data and risk factors. Additionally, the procedures performed were also similar for both groups. As a consequence, clinical outcomes and postoperative adverse events were also similar (Table 3). However, an increase in ventricular fibrillation after cross-clamp removal in patients receiving Custodiol was reported by Braathen et al.^[5]. This might be a result of heterogeneous myocardial perfusion, oxidative stress, and electrolyte imbalance. The surgeries we performed were not complex and did not involve coronary artery occlusive disease, consequently, ventricular fibrillation (VF) did not occur in our study groups. Myocardial infarction and left ventricular (LV) systolic dysfunction also did not occur in any patient. In our opinion, this was a result of the homogenous distribution of cardioplegia solutions and electrolyte balance. Only one patient required high-dose inotropic support in the blood cardioplegia group, which was temporary. Hachida et al.^[13] reported that the promotion of anaerobic glycolysis during Custodiol ischemia resulted in superior prolonged preservation of myocardial contractile functions. The temperatures of cardioplegic solutions were different as 32°C (blood cardioplegia) versus 4°C (Custodiol). This is the debate in the study, because Custodiol should be infused at 4°C to prevent myocardial injury and results in different myocardial warming degrees between groups. Different temperatures and solutions were studied by von Oppell et al.^[14] and the best solution for myocardial preservation was presented as Custodiol.

Tn I and CK-MB levels and changes in time are good markers of ischemia induced by myocardial degeneration. In this study, myocardial injury biomarkers, Tn I and CK-MB, were analyzed at different time intervals and slight differences occurred between groups. Cross-clamp time, VF, ablation procedures, and myocardial hypothermia level are factors that change Tn I and CK-MB releases. In our study, these factors affecting myocardial viability were similar for both groups. However, various studies reported that both types of cardioplegia had equivalent efficacy as a method of myocardial protection during cardiac arrest^[5,15].

There was no significant difference regarding the immunoexpression of iNOS, VEGF, and annexin in the myocardium between the Custodiol and blood cardioplegia groups. Custodiol can prevent the inflammatory response of tissue via iNOS molecules because of its myocardial protective effect^[16]. Immunocytochemical analysis revealed that the cardioprotective and vasodilator molecule, eNOS, was preserved in the atrial tissue in Custodiol and blood cardioplegia groups, but, importantly, it was increased in the Custodiol group. Furthermore, the expression of the Bcl-2 protein in the prevention of apoptosis was also demonstrated in isolated rat myocytes^[17]. In this study, anti-apoptotic Bcl-2 molecules were well preserved in the myocardial tissue in Custodiol and blood cardioplegia groups, but they were increased in the Custodiol group. Custodiol may contribute to the anti-apoptotic response of tissue via Bcl-2 molecules because of the myocardial protective effect.

This study has potential limitations. First, immunohistochemical studies with myocardial biopsy have high costs; therefore, the sample size is relatively small. There is a lack of evidence in the literature on the subject of our study, so this is a preliminary study. Previously performed animal studies also have small sample sizes. Similar experiments with larger samples should be conducted to obtain more precise results.

CONCLUSION

Clinical outcomes and cardiac enzyme analysis demonstrated that a single dose of antegrade cold Custodiol cardioplegia and repetitive antegrade blood cardioplegia in elective cardiac surgery were equally effective in protecting the myocardium. However, immunohistochemical studies documented superior cellular oxidative stress resistance and cellular viability with Custodiol infusion. In our opinion, myocardial immunohistochemical analysis needs to be performed with larger series of patients because cardiac histochemical changes can be critical for long-term cardiac performance.