Skip to main content

In vivo MRI and ex vivo histological assessment of the cardioprotection induced by ischemic preconditioning, postconditioning and remote conditioning in a closed-chest porcine model of reperfused acute myocardial infarction: importance of microvasculature

Abstract

Background

Cardioprotective value of ischemic post- (IPostC), remote (RIC) conditioning in acute myocardial infarction (AMI) is unclear in clinical trials. To evaluate cardioprotection, most translational animal studies and clinical trials utilize necrotic tissue referred to the area at risk (AAR) by magnetic resonance imaging (MRI). However, determination of AAR by MRI‚ may not be accurate, since MRI-indices of microvascular damage, i.e., myocardial edema and microvascular obstruction (MVO), may be affected by cardioprotection independently from myocardial necrosis. Therefore, we assessed the effect of IPostC, RIC conditioning and ischemic preconditioning (IPreC; positive control) on myocardial necrosis, edema and MVO in a clinically relevant, closed-chest pig model of AMI.

Methods and results

Acute myocardial infarction was induced by a 90-min balloon occlusion of the left anterior descending coronary artery (LAD) in domestic juvenile female pigs. IPostC (6 × 30 s ischemia/reperfusion after 90-min occlusion) and RIC (4 × 5 min hind limb ischemia/reperfusion during 90-min LAD occlusion) did not reduce myocardial necrosis as assessed by late gadolinium enhancement 3 days after reperfusion and by ex vivo triphenyltetrazolium chloride staining 3 h after reperfusion, however, the positive control, IPreC (3 × 5 min ischemia/reperfusion before 90-min LAD occlusion) did. IPostC and RIC attenuated myocardial edema as measured by cardiac T2-weighted MRI 3 days after reperfusion, however, AAR measured by Evans blue staining was not different among groups, which confirms that myocardial edema is not a measure of AAR, IPostC and IPreC but not RIC decreased MVO.

Conclusion

We conclude that IPostC and RIC interventions may protect the coronary microvasculature even without reducing myocardial necrosis.

Background

Despite the wide availability of advanced revascularization techniques, acute myocardial infarction (AMI) is one of the leading causes of mortality and morbidity in developed countries [1]. Preclinical studies of various ischemic conditioning techniques have shown promising results in the reduction of AMI-related cardiac damage (see for an extensive recent review: [2]). Ischemic preconditioning (i.e., brief cycles of ischemia and reperfusion of the involved coronary artery before sustained ischemia; IPreC) was shown first to attenuate subsequent myocardial damage [3]. Since then, it has been demonstrated that ischemic conditioning reduces myocardial ischemia/reperfusion injury (IRI) if applied after cardiac ischemia (ischemic postconditioning; IPostC), which is more feasible for clinical application [4]. Moreover, cardioprotection could be elicited by applying ischemic stimuli on a distant area of the heart or on a remote organ (e.g., kidney, skeletal muscle) termed remote ischemic conditioning (RIC) [5]. Both IPostC and RIC have been reported to be cardioprotective in preclinical [5] and clinical [69] settings. However, to date, the largest clinical trials in IPostC did not show any benefit of IPostC on ST-elevation myocardial infarction patients after primary percutaneous intervention (POST and DANAMI 3-iPOST trial) in terms of long-term outcome [10, 11]. So far, a small-scale clinical trial involving ST-elevation myocardial infarction (STEMI) patients has been completed and demonstrated long-term efficacy of RIC as assessed by significant improvement of major adverse cardiac and cerebrovascular events [12]. Nevertheless, two large clinical trials reported that RIC did not improve the long-term outcome after cardiac surgery (ERICCA [13] and RIPHEART [14] trials).

The cardioprotective efficacy of an intervention is assessed by different methods in preclinical settings and in clinical trials. For instance, there is no accurate and widely available method to measure the area at risk (AAR; the area excluded from the coronary circulation), which is the basis of preclinical cardioprotective studies. Although it has been proposed that myocardial edema by T2-weighted magnetic resonance imaging (MRI) correlates well with histopathology-based evaluation of AAR [15], it has been demonstrated that myocardial edema correlates better with the myocardial necrosis than with the AAR [16]. Furthermore, efficacy of interventions in (pre)clinical studies are almost exclusively based on the measurement of myocardial necrosis by assessing infarct size or necroenzyme release, which primarily represents the decay of cardiac myocytes, and which are major determinants of the outcome of AMI according to clinical studies [17]. However, damage to other cell types of the heart, such as the coronary microvasculature may also contribute to the IRI [2, 18]. For instance, myocardial edema [19], microvascular obstruction (MVO; ischemic area, where myocardial perfusion is not restored despite successful revascularization) [20], endothelial dysfunction, and microembolisation are major signs of microvascular damage [21].

Therefore, we studied the effects of IPostC and RIC by comparing them to the effects of the positive control (IPreC) on measures of IRI (myocardial necrosis, edema and MVO) as determined by in vivo MRI and ex vivo histological staining in a clinically relevant, closed-chest swine model of AMI.

Methods

Experimental design (Fig. 1)

Domestic female pigs (25–35 kg; genotype: DanBred hybrid, purchased from the University of Kaposvár, Hungary) were kept according to the Big Dutchman principles. The animals were fed with a pregnant sow diet containing a low energy and balanced protein level produced by Dalmand co. Ltd. They further underwent regular veterinarian check-ups, and only healthy animals were selected for the study. Furthermore, before percutaneous intervention, transthoracic echocardiography, and angiography was served as a baseline screening method to exclude animals with abnormal coronary anatomy or myocardial disease.

Fig. 1
figure 1

Experimental protocol. Isch ischemia only, IPreC ischemic preconditioning, IPostC ischemic postconditioning, RIC remote ischemic conditioning, LAD left anterior descendent coronary artery, TTC triphenyl tetrazolium chloride, MRI magnetic resonance imaging

The pigs were block randomized [22] into four groups: ischemia only (Isch; n = 17), IPreC (n = 12), IPostC (n = 14) and RIC (n = 17). Three animals died during myocardial ischemia (Isch: 1; IPreC: 1; IPostC: 0; RIC: 1) and 4 during reperfusion due to therapy resistant malignant ventricular rhythm disturbances (Isch: 2; IPreC: 1; IPostC: 1; RIC: 0). Additionally, 3 animals were excluded due to procedural technical reasons. The final case numbers were 14, 9, 12 and 14 in Isch, IPreC, IPostC and RIC groups, respectively. According to the current ESC STEMI guidelines [23, 24], the pigs were pretreated with loading doses of 250 mg acetyl salicylic acid and 300 mg clopidogrel with maintenance doses of 100 and 75 mg pro day, respectively. Although 600 mg dose of clopidogrel has been shown to superior over a 300 mg dose in myocardial necrosis reducing effects [25], our experimental protocol has been planned to minimize the effect of possible confounding factors such as clopidogrel [26]. Animals were sedated with 12 mg/kg ketamine hydrochloride, 1 mg/kg xylazine, and 0.04 mg/kg atropine intramuscularly after an overnight fast. Anesthesia was induced by inhalation of isoflurane (2–2.5 vol%). Animals were intubated endotracheally and anesthesia was maintained by inhalation of an isoflurane oxygen mix (2–2.5 vol% and 3 L/min). Magnesium sulphate (4.06 mEq diluted in 10 mL, in every 60 min) and a continuous amiodarone infusion (300 mg diluted in 500 mL saline) were being administered throughout the procedure via an ear vein. Sheaths were inserted into femoral artery and femoral vein to further have entry routes for the catheterization, and 5000–5000 IU heparin was administered via each sheath. Cardiac function was assessed with echocardiography. Baseline hemodynamics were recorded, and selective angiography of the left coronary artery was performed. After the analysis of the baseline angiogram, a balloon catheter (2.75 mm diameter, 8 mm length) (Abbott Vascular) was placed in the mid part of the left anterior descending coronary artery (LAD) after the origin of the 2nd diagonal branch. For induction of AMI, the intracoronary balloon was inflated with 5 atm for 90 min, followed by deflation of the balloon, resulting in reperfusion (3 h or 3 days) which was confirmed by coronarography. In IPreC group, LAD was occluded by the inflation of the balloon at 5 atm 3 times for 5 min followed by 5 min of reperfusion, while in other animals the balloon was left deflated for 30 min [27]. Then LAD was occluded by inflating coronary balloon, which was confirmed by coronarography. RIC was performed by 4 cycles of 5 min occlusion and 5 min reperfusion of the femoral vessels by tightening and releasing of a snare around the right hind limb starting at the 50th min of LAD occlusion [9]. We verified the hind limb ischemia in three ways: (1) Apparent lividity during ischemia and pronounced hyperemia during reperfusion was observed distal to the occlusion. (2) In each animal, a superficial femoral artery was cannulated distal to the occlusion, and blood pressure was measured during the intervention. The minimum of 30/30 mmHg blood pressure was achieved while the wire was tightened around the hind limb. (3) In one particular animal, we performed a femoral angiography before and during hind limb ischemia as well [Additional file 1: Video_S1.avi (before), Additional file 2: Video_S2.avi (during hind limb ischemia)]. IPostC was initiated within 1 min after the termination of 90-min index ischemia and was performed by applying 6 cycles of 30/30 s LAD occlusion and reperfusion [28]. The interventional cardiologist was not blinded during the investigation due to the nature of the procedure: IPreC and IPostC were achieved by inflating a balloon in the LAD, which required an unblinded interventional cardiologist. Nonetheless, the interventional cardiologist was not aware of the allocation until the initiation of the experimental intervention (e.g. right after balloon deflation if the animal was in IPostC group). After reperfusion was initiated 5000 IU heparin was given intracoronary. Final reperfusion was confirmed with coronarography and the catheters were removed. Ten min after the initiation of reperfusion, the hemodynamic data was recorded again. Anesthesia was either maintained for 3 h or in case of 3 days reperfusion, wounds were closed and anesthesia was terminated by the withdrawal of isoflurane. Analgesia was applied by intramuscular injections of 1 g metamizole. An antibiotic cocktail (100 mg benzathine benzylpenicillin, 100 mg procaine benzylpenicillin, 200 mg dihydrostreptomycin-sulphate) was given i.m. before recovery.

Measurement of myocardial necrosis, edema and MVO by cardiac MRI

The amount of myocardial necrosis and MVO were determined by acquiring late gadolinium-enhanced images (gold standard method) [9, 29]. Dark blood prepared IR-TSE sequence single slice breath-hold acquisition T2w protocol was used to detect myocardial edema [9, 29]. 3 days (70–78 h) after the deflation of the balloon in LAD, anesthesia was induced by inhalation of an isoflurane-oxygen mix. Prior to the cardiac MRI, atracurium was administered and ventilation was maintained with mechanical ventilation. For the assessment of myocardial necrosis and edema, cardiac MRI was performed using a 1.5T clinical scanner (Avanto, Siemens) using a phased array coil and a vector ECG system. Cine MRI images was acquired using a retrospectively ECG-gated, steady-state free precession cine MRI technique (Cinetruefisp sequence) in short-axis and long-axis views of the heart using 1.2 ms echo time, 40 ms repetition time, 65° flip angle, 15 segments, 360 mm field-of-view, 8 mm slice thickness, and 256 × 256 image matrix. The image resolution was 1.4 × 1.4 × 8 mm. T2-weighted cardiac MRI was performed by short inversion time inversion recovery dark blood technique with single slice breath-hold acquisition and inversion recovery preparation (TI = 170 ms; 15 segments; every second trigger pulse; TE 74 ms, flip angle 180°). The slice position and resolution was identical as cine images. The late gadolinium-enhanced images were acquired to determine the amount of myocardial necrosis and MVO. A 2-dimensional single shot Truefisp sequence with non-selective IR pulse shift acquisition to a diastolic phase of the cardiac cycle by adjusting the TR was used 12–15 min after administration of a gadolinium-based contrast agent (0.13 mmol/kg gadobutrol, Gadovist 1.0 mmol, Bayer), with slice positions identical to the cine images. Typical in-plane resolution was 1.4 × 1.4 × 8 mm (echo time 1.2 ms, flip angle 50°, triggering to every other heart beat). The inversion time was set to null the signal of viable myocardium and ranged from 280–320 ms. Left and right ventricular end-diastolic and end-systolic volumes, stroke volumes, ejection fractions, and masses were quantified using manual planimetry of end-diastolic and end-systolic short-axis SSFP cine images with MASS 7.6 analysis software (Magnetic Resonance Analytical Software System, Medis Medical Imaging Software, Leiden, The Netherlands). Myocardial necrosis and edema were quantified after manual planimetry both on the delayed contrast enhancement and T2-weighted images by delineation of myocardium with signal intensity 4 SDs above the mean signal obtained in the remote noninfarcted myocardium using MASS 7.6 analysis software. If present, the hypointense zone in the center of the hyperenhancement (MVO) was quantified and added to the infarct volume as previously described [30]. Values were expressed relative to the left ventricular mass. The measurement of MVO with late gadolinium enhancement closely correlates with myocardial contrast echocardiography, angiographic and invasive indices used for the assessment of MVO [31]. Moreover, late-gadolinium enhancement correlates with the histological measurement of MVO as demonstrated by Zalewski et al. [29].

Myocardial necrosis and AAR measurement by ex vivo staining

Hearts were removed from the chest, and placed immediately in ice-cold saline 3 h after reperfusion. LAD was reoccluded, ex vivo at the same place as in vivo (prior to the obduction, coronary angiography of the pig was reviewed, and the occlusion site was identified.), and Evans blue (Sigma) was injected into the coronary arteries through their orifices to negatively stain the AAR [29, 32]. 10 mm slices were then cut and incubated in 1% triphenyltetrazolium chloride (TTC, Sigma) at 37 °C for 15 min to stain viable areas. After overnight fixation with 4% formalin, slices were weighed and scanned for blind planimetric analysis (InfarctSize 2.4b software; Pharmahungary Group). AAR was expressed relative to the left ventricular (LV) mass, and myocardial necrosis relative to the AAR mass.

Coronary angiography and AAR calculation

All animals underwent coronary angiography according to the protocol established by the catheterization laboratory. Anterograde flow in the artery before and after balloon inflation was characterized using the TIMI (Thrombolysis in Myocardial Infarction) system [33]. TIMI myocardial perfusion grade and myocardial blush grade were assessed visually on the angiogram and made by expert interventional cardiologist, and all data were entered prospectively into a database. Myocardial blush grade has been defined as follows: 0, no myocardial blush or contrast density; 1, minimal myocardial blush or contrast density; 2, moderate myocardial blush or contrast density but less than that obtained during angiography of a contralateral or ipsilateral non–infarct-related coronary artery; and 3, normal myocardial blush or contrast density, comparable with that obtained during angiography of a contralateral or ipsilateral non–infarct-related coronary artery. When myocardial blush persisted (“staining”), this phenomenon suggested leakage of contrast medium into the extravascular space and was graded 0 [34, 35]. No digital techniques were used. The AAR was established by using the modified APPROACH score [36].

Transthoracic echocardiography

Two-dimensional, M-mode and Doppler echocardiographic examinations were performed in accordance with the criteria of the American Society of Echocardiography with a Vivid i portable ultrasound system (General Electric Medical Systems) using a phased array 2.7–8 MHz transducer (6S-RS probe). Data of three consecutive heart cycles were analysed (EchoPac Dimension software; General Electric Medical Systems) in a blinded manner. The mean values of three measurements were calculated and used for statistical evaluation.

Statistics

Values were expressed as mean ± standard error of mean. Statistical analyses were done by using one way or repeated measures ANOVA with LSD or Dunnett’s post hoc test and KruskalWallis test as indicated (IBM SPSS Statistics, Version 19). To limit the case numbers in the cardiac MRI study, one-way ANOVA was performed by using bootstrapping with 1000-sample replacement [37], as frequently used in clinical studies [3840] and recommended for preclinical studies as well [41]. Statistical significance was accepted if the p value was below 0.05.

Results

Myocardial necrosis, edema and MVO by cardiac MRI

To quantify myocardial necrosis, edema and MVO, cardiac MRI was performed 3 days after coronary occlusion and reperfusion. Myocardial necrosis (% of LV) was not affected by IPostC and RIC (n = 7, and 8, respectively), however, IPreC (n = 4) attenuated it significantly as compared to the Isch group (n = 7), although only 4 IPreC cases were studied with MRI (Fig. 2a). Myocardial edema (% of LV) was significantly decreased by IPostC and RIC (n = 9, and 7, respectively) as compared to the Isch group (n = 7), however, only a tendency of decrease was observed by IPreC (n = 4; p = 0.06; Fig. 2b). At baseline, hemodynamic parameters were not significantly different between groups, whereas at reperfusion heart rate was significantly lower in IPreC and RIC groups as compared to the Isch group (Table 1). AAR based on angiographic score was not different between groups (Table 2). Furthermore, angiographic measures of reperfusion, i.e., TIMI score, myocardial blush grade, and TIMI myocardial perfusion grade were not affected by conditioning stimuli (Table 2). MVO volume (% of LV) was significantly decreased by IPreC and IPostC (n = 4, and 7, respectively), but not by RIC (n = 8) as compared to the Isch group (n = 7) (Fig. 2c).

Fig. 2
figure 2

The effect of IPreC, IPostC and RIC on myocardial necrosis (a; n = 4–8/group), edema (b; n = 4–9/group) and MVO (c; n = 4–8/group) size as evaluated with cardiac MRI. d-e Representative MRI images of myocardial necrosis and edema. Green line: apicardial outline, red line: endocardial outline, red area: myocardial necrosis, dark blue area: MVO, light blue area: myocardial edema. *p < 0.05 vs. Isch. Isch ischemia only, IPreC ischemic preconditioning, IPostC ischemic postconditioning, RIC remote ischemic conditioning, MRI magnetic resonance imaging, LV left ventricle, MVO microvascular obrstruction

Table 1 Hemodynamic data
Table 2 Angiographic evaluation

Myocardial necrosis and AAR evaluated by ex vivo staining

To assess myocardial necrosis and AAR by an ex vivo histological method, the gold standard TTC and Evans blue staining were applied after 3 h of reperfusion. IPostC and RIC did not decrease myocardial necrosis (n = 5, and 5, respectively) (% of AAR), however, IPreC (n = 6), significantly decreased it as compared to the Isch group (n = 5) (Fig. 3a). There was no difference in AARs between the experimental groups (% of LV) as evaluated by Evans blue staining (Fig. 3b).

Fig. 3
figure 3

The effect of IPreC, IPostC and RIC on myocardial necrosis (a; n = 5–6/group) and AAR (b; n = 5–6/group) as evaluated with conventional TTC and Evans blue staining. c Representative images of Evans blue/TTC-stained heart sections from three different hearts indicating myocardial necrosis and AAR (Each image was taken from the apical side of the third 1-cm slice. Images are optimized for visualization of TTC staining in the InfarctSize software.). Orange slice outline, green ventricular chamber, purple AAR, yellow myocardial necrosis. *p < 0.05 vs. Isch. Isch ischemia only, IPreC ischemic preconditioning, IPostC ischemic postconditioning, RIC remote ischemic conditioning, AAR area at risk, TTC triphenyl tetrazolium chloride, MRI LV left ventricle

Myocardial function by cardiac MRI and echocardiography

Myocardial function was analyzed by cardiac MRI and echocardiography. Myocardial function was not different between groups after either 3 h or 3 days of reperfusion (Tables 3, 4, 5).

Table 3 Myocardial function as assessed by MRI
Table 4 Cardiac function parameters measured by echocardiography before interventions and after 3 h of reperfusion
Table 5 Cardiac function parameters measured by echocardiography before interventions and after 3 days of reperfusion

Discussion

Here we studied the effect of IPostC and RIC on IRI-related MRI parameters in a clinically relevant, closed-chest porcine model of reperfused AMI and demonstrated that IPostC and RIC protected the microvasculature against IRI, as they reduced myocardial edema, and IPostC decreased MVO. However, IPostC or RIC did not reduce myocardial necrosis, although the positive control IPreC reduced it. This is the first comparative demonstration of the variable effects of different conditioning maneuvres on IRI-related MRI parameters, and that myocardial edema and MVO change independently from myocardial necrosis. Our results support findings of clinical trials that myocardial edema may change due to various interventions and it does not represent AAR.

The translation of cardioprotective conditioning stimuli into the clinical practice has been proven difficult and disappointing despite numerous positive proof-of-concept clinical trials [18]. The neutral results have been attributed to many factors, such as recruitment of inadequate patient population, type of revascularization, inclusion of late revascularizations, comorbidities and comedications [18]. Moreover, the strict adherence to certain endpoints, such as myocardial infarct size and the neglect of the microvasculature might also hinder the successful translation of various cardioprotective strategies [21]. Here we demonstrated with ex vivo Evans blue staining and in vivo angiography scoring that AAR was not affected by conditioning stimuli, while myocardial edema was significantly decreased by both IPostC and RIC indicating that the extensive damage of the cardiac microvasculature was prevented. Similarly, two clinical trials showed that myocardial edema is attenuated by IPostC [6] or RIC [9], however, others reported otherwise (IPostC: [42, 43]; RIC: [8, 43]). These results indicate that edema might be independent from AAR in cardioprotection studies, therefore, applying myocardial edema as AAR may lead to false conclusions in clinical trials. To date, no clinical trial has been conducted to reveal the prognostic role of myocardial edema in ST-segment elevation AMI, although it has been shown that non-STEMI patients with myocardial edema had higher mortality [44]. However, it is well-established that the volume or even the presence of MVO, another clinically detectable marker of microvascular injury, correlates with long-term outcome of AMI (see for review [20]). In our present experimental model, IPostC, but not RIC, reduced MVO. Similarly to our findings, it has been reported in a clinical trial that IPostC reduces MVO [45], however, no other clinical trials confirmed the MVO-reducing ability of either IPostC [42, 43] or RIC [9, 43]. Nevertheless, the assessment of myocardial edema and MVO in preclinical and clinical studies may provide additional valuable indices. In our present study, we observed that myocardial function was not different between groups after reperfusion, although IPreC reduced myocardial necrosis. Our results are in line with a number of ischemic conditioning studies in translational models or in clinical trials. IPostC/RIC has been shown to reduce myocardial necrosis after AMI in porcine models, although myocardial function was not different between groups [29, 46, 47]. Similarly, White et al. demonstrated in a clinical trial that RIC did not improve early cardiac function after STEMI, while myocardial necrosis was significantly attenuated by RIC [9]. Furthermore, it seems that early post-AMI cardiac function is not necessarily a good predictor for the future outcome in ischemic conditioning translational studies: For instance, Munk et al. showed that RIC does not influence cardiac function at day 1 after AMI, however, cardiac function was improved at day 30 [48]. In summary, acute post-AMI myocardial function may be determined by many other facts than myocardial necrosis, such as, e.g., actual sympathetic tone, regional wall motion [49], collateral flow [50], transmurality of infarction [50].

Translational models of AMI have a major importance to develop interventions for the clinical practice [18, 51, 52]. For this purpose, pigs are excellent model animals, since their cardiac anatomy and cardiovascular physiology exhibit similarities to the human heart [53]. Although the pig is suitable for closed-chest experimentation, the majority of the studies on the effect of ischemic conditioning on AMI was performed by using open-chest models [32, 54], and relatively few studies are available in closed-chest models. Previously, IPostC [55, 56] and RIC [57, 58] have been shown to reduce myocardial necrosis in closed-chest swine models of AMI. However, in our present experimental conditions, IPostC and RIC did not reduce myocardial necrosis, but the positive control IPreC did. The discrepancy between our results and those of others might be explained by the significantly different perioperative medication and experimental design. The abovementioned studies applied medications required only for the perioperative procedures (e.g. anesthesia, pain control), however, the therapeutical management of AMI consists of other drugs as well. Here we treated animals with acetylsalicylic acid and clopidogrel according to the clinical guidelines [59, 60]. However, it has been shown that COX-2 is an essential mediator of IPreC [61] and of IPostC [62], and that its blockade neutralizes the cardioprotective effect of late IPreC [63]. Similarly, clopidogrel, a P2Y12 antagonist, has been retrospectively shown to reduce cardiac necrosis and to decrease cardiovascular events after AMI in clinical trials [64, 65], which might be attributed to its antiplatelet activity and a direct cardioprotective effect. Yang et al. demonstrated that IPostC did not further reduce infarct size when the P2Y12 antagonist, cangrelor-pretreatment was applied in rabbits [26]. However, cardioprotection could be elicited by an extended, 8-cycle-long IPostC in closed-chest pigs pretreated with acetylsalicylic acid and clopidogrel [29]. These data indicate that to ensure translational value of animal studies it is essential to apply perioperative medication according to clinical guidelines [66].

In our present study, anesthesia was maintained by isoflurane. It is well-documented that certain anaesthetics, such as fluranes, induce cardioprotection [67] and/or interfere with cardioprotective interventions [68] making the assessment of the effect of conditioning stimuli more difficult. In our present study, we observed a decrease in myocardial necrosis by IPreC but not by IPostC and RIC with isoflurane anesthesia. Although studies showed cardioprotective efficacy of remote ischemic postconditioning with the use of isoflurane [58], and of IPostC after a low-flow index ischemia with enflurane anesthesia in closed-chest porcine models [54], our results show no cardioprotection by IPostC and RIC. This discrepancy might be explained by significant differences between experimental protocols. In conclusion, there is a huge body of evidences that the application of antiplatelet drugs and inhalative anesthetics may interfere with various conditioning maneuvres, although we did not specifically investigate them in our present study.

Although here we assessed the cardioprotective effect of IPostC and RIC as well as the positive control IPreC in a clinically relevant closed-chest pig model of AMI, this study has some limitations. It has been recently shown that the extent of myocardial edema has two peaks over time in swine [69], but not in humans [70]. Therefore, in this study alteration in myocardial edema at day 3 might not necessarily reflect attenuation of edema by conditioning, but the altered dynamics of edema. A long-term follow up would provide data more relevant to the design of clinical studies. Therefore, here we could only speculate whether attenuation of myocardial edema by IPostC and RIC could be interpreted as a valid marker of the measure of IRI, or whether improved long-term outcome depends only on the reduction of myocardial necrosis. Although we did not measure myocardial edema directly (e.g., freeze-dry method), it is generally accepted that T2-hyperintensity correlates well with the myocardial water content, and T2-hyperintensity is excellent for tracking the differences in myocardial edema [19]. Furthermore, according to the clinical routine, cardiac MRI was performed at day 3 [9], whereas ex vivo histopathological staining at 3 h of reperfusion according to the preclinical standards [28]. Therefore, myocardial edema and definite AAR, i.e., Evans blue staining, were evaluated in separate groups. However, we calculated APPROACH score to estimate AAR in the same cohort, in which cardiac MRI was performed. Although APPROACH score system has not been validated in pigs, it is most plausible that it is suitable for the estimation of AAR in pig AMI experiments as well, since several anatomical studies demonstrated that the coronary anatomy of the pig shares a high similarity with that of the human heart (e.g., anastomoses, blood supply territory etc.) [53, 71].

Conclusions

We compared the cardioprotective efficacy of IPostC and RIC and the positive control IPreC in a clinically relevant, closed-chest porcine model of reperfused AMI for the first time in the literature by using in vivo MRI imaging and ex vivo histology methods. IPostC and RIC did not decrease myocardial necrosis in our model, however, the positive control IPreC reduced it. The coronary microvascular system has been protected by both IPostC and RIC as they attenuated myocardial edema, and IPostC reduced MVO. Our results indicate that parameters of microvascular protection may be important to assess IRI and these parameters might change independently from that of myocardial necrosis. Furthermore, since the intact microcirculation projects improved long-term outcome, its careful evaluation might help to avoid false negative results in preclinical or clinical studies of cardioprotection.

Abbreviations

AAR:

area at risk

AMI:

acute myocardial infarction

IPostC:

ischemic postconditioning

IPreC:

ischemic preconditioning

IRI:

ischemia/reperfusion injury

LAD:

left descending coronary artery

LV:

left ventricle

MRI:

magnetic resonance imaging

MVO:

microvascular obstruction

RIC:

remote ischemic conditioning

STEMI:

ST-elevation myocardial infarction

TTC:

triphenyltetrazolium chloride

References

  1. Moran AE, Forouzanfar MH, Roth GA, Mensah GA, Ezzati M, Flaxman A, et al. The global burden of ischemic heart disease in 1990 and 2010: the global burden of disease 2010 study. Circulation. 2014;129(14):1493–501.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Hausenloy DJ, Garcia-Dorado D, Bøtker HE, Davidson SM, Downey J, Engel FB, et al. Novel targets and future strategies for acute cardioprotection: position paper of the European Society of cardiology working group on cellular biology of the heart. Cardiovasc Res. 2017. In Press.

  3. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74(5):1124–36.

    Article  CAS  PubMed  Google Scholar 

  4. Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP, Guyton RA, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol. 2003;285(2):H579–88.

    Article  CAS  PubMed  Google Scholar 

  5. Heusch G, Botker HE, Przyklenk K, Redington A, Yellon D. Remote ischemic conditioning. J Am Coll Cardiol. 2015;65(2):177–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Thuny F, Lairez O, Roubille F, Mewton N, Rioufol G, Sportouch C, et al. Post-conditioning reduces infarct size and edema in patients with ST-segment elevation myocardial infarction. J Am Coll Cardiol. 2012;59(24):2175–81.

    Article  PubMed  Google Scholar 

  7. Botker HE, Kharbanda R, Schmidt MR, Bottcher M, Kaltoft AK, Terkelsen CJ, et al. Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet. 2010;375(9716):727–34.

    Article  PubMed  Google Scholar 

  8. Crimi G, Pica S, Raineri C, Bramucci E, De Ferrari GM, Klersy C, et al. Remote ischemic post-conditioning of the lower limb during primary percutaneous coronary intervention safely reduces enzymatic infarct size in anterior myocardial infarction: a randomized controlled trial. JACC Cardiovasc Interv. 2013;6(10):1055–63.

    Article  PubMed  Google Scholar 

  9. White SK, Frohlich GM, Sado DM, Maestrini V, Fontana M, Treibel TA, et al. Remote ischemic conditioning reduces myocardial infarct size and edema in patients with ST-segment elevation myocardial infarction. JACC Cardiovasc Interv. 2015;8(1 Pt B):178–88.

    Article  PubMed  Google Scholar 

  10. Hahn JY, Yu CW, Park HS, Song YB, Kim EK, Lee HJ, et al. Long-term effects of ischemic postconditioning on clinical outcomes: 1-year follow-up of the POST randomized trial. Am Heart J. 2015;169(5):639–46.

    Article  PubMed  Google Scholar 

  11. The third DANish study of optimal acute treatment of patients with ST-segment elevation myocardial infarction: ischemic postconditioning during Primary PCI - DANAMI 3-iPOST http://www.acc.org/latest-in-cardiology/clinical-trials/2016/03/29/22/46/danami-3-ipost.

  12. Sloth AD, Schmidt MR, Munk K, Kharbanda RK, Redington AN, Schmidt M, et al. Improved long-term clinical outcomes in patients with ST-elevation myocardial infarction undergoing remote ischaemic conditioning as an adjunct to primary percutaneous coronary intervention. Eur Heart J. 2014;35(3):168–75.

    Article  PubMed  Google Scholar 

  13. Hausenloy DJ, Candilio L, Evans R, Ariti C, Jenkins DP, Kolvekar S, et al. Remote ischemic preconditioning and outcomes of cardiac surgery. N Engl J Med. 2015;373(15):1408–17.

    Article  CAS  PubMed  Google Scholar 

  14. Meybohm P, Bein B, Brosteanu O, Cremer J, Gruenewald M, Stoppe C, et al. A multicenter trial of remote ischemic preconditioning for heart surgery. N Engl J Med. 2015;373(15):1397–407.

    Article  CAS  PubMed  Google Scholar 

  15. Aletras AH, Tilak GS, Natanzon A, Hsu LY, Gonzalez FM, Hoyt RF Jr, et al. Retrospective determination of the area at risk for reperfused acute myocardial infarction with T2-weighted cardiac magnetic resonance imaging: histopathological and displacement encoding with stimulated echoes (DENSE) functional validations. Circulation. 2006;113(15):1865–70.

    Article  PubMed  Google Scholar 

  16. Kim HW, Van Assche L, Jennings RB, Wince WB, Jensen CJ, Rehwald WG, et al. Relationship of T2-weighted MRI myocardial hyperintensity and the ischemic area-at-risk. Circ Res. 2015;117(3):254–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Gibbons RJ, Valeti US, Araoz PA, Jaffe AS. The quantification of infarct size. J Am Coll Cardiol. 2004;44(8):1533–42.

    Article  PubMed  Google Scholar 

  18. Hausenloy DJ, Erik Botker H, Condorelli G, Ferdinandy P, Garcia-Dorado D, Heusch G, et al. Translating cardioprotection for patient benefit: position paper from the working group of cellular biology of the heart of the European Society of cardiology. Cardiovasc Res. 2013;98(1):7–27.

    Article  CAS  PubMed  Google Scholar 

  19. Garcia-Dorado D, Andres-Villarreal M, Ruiz-Meana M, Inserte J, Barba I. Myocardial edema: a translational view. J Mol Cell Cardiol. 2012;52(5):931–9.

    Article  CAS  PubMed  Google Scholar 

  20. Niccoli G, Scalone G, Lerman A, Crea F. Coronary microvascular obstruction in acute myocardial infarction. Eur Heart J. 2016;37(13):1024–33.

    Article  PubMed  Google Scholar 

  21. Heusch G. The coronary circulation as a target of cardioprotection. Circ Res. 2016;118(10):1643–58.

    Article  CAS  PubMed  Google Scholar 

  22. Efird J. Blocked randomization with randomly selected block sizes. Int J Environ Res Public Health. 2011;8(1):15–20.

    Article  PubMed  Google Scholar 

  23. Task Force on the management of STseamiotESoC, Steg PG, James SK, Atar D, Badano LP, Lundqvist CB, Borger MA, Di Mario C, Dickstein K, Ducrocq G, Fernandez-Aviles F, Gershlick AH. ESC Guidelines for the management of acute myocardial infarction in patients presenting with ST-segment elevation. Eur Heart J. 2012;33(20):2569–619.

    Article  Google Scholar 

  24. Roffi M, Patrono C, Collet JP, Mueller C, Valgimigli M, Andreotti F, et al. 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: task force for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation of the European Society of cardiology (ESC). Eur Heart J. 2016;37(3):267–315.

    Article  PubMed  Google Scholar 

  25. Patti G, Barczi G, Orlic D, Mangiacapra F, Colonna G, Pasceri V, et al. Outcome comparison of 600- and 300-mg loading doses of clopidogrel in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: results from the ARMYDA-6 MI (antiplatelet therapy for reduction of myocardial damage during angioplasty-myocardial infarction) randomized study. J Am Coll Cardiol. 2011;58(15):1592–9.

    Article  CAS  PubMed  Google Scholar 

  26. Yang XM, Liu Y, Cui L, Yang X, Liu Y, Tandon N, et al. Platelet P2Y(1)(2) blockers confer direct postconditioning-like protection in reperfused rabbit hearts. J Cardiovasc Pharmacol Ther. 2013;18(3):251–62.

    Article  CAS  PubMed  Google Scholar 

  27. Ovize M, Aupetit JF, Rioufol G, Loufoua J, Andre-Fouet X, Minaire Y, et al. Preconditioning reduces infarct size but accelerates time to ventricular fibrillation in ischemic pig heart. Am J Physiol. 1995;269(1 Pt 2):H72–9.

    CAS  PubMed  Google Scholar 

  28. Iliodromitis EK, Georgiadis M, Cohen MV, Downey JM, Bofilis E, Kremastinos DT. Protection from post-conditioning depends on the number of short ischemic insults in anesthetized pigs. Basic Res Cardiol. 2006;101(6):502–7.

    Article  PubMed  Google Scholar 

  29. Zalewski J, Claus P, Bogaert J, Driessche NV, Driesen RB, Galan DT, et al. Cyclosporine A reduces microvascular obstruction and preserves left ventricular function deterioration following myocardial ischemia and reperfusion. Basic Res Cardiol. 2015;110(2):18.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Wu KC, Kim RJ, Bluemke DA, Rochitte CE, Zerhouni EA, Becker LC, et al. Quantification and time course of microvascular obstruction by contrast-enhanced echocardiography and magnetic resonance imaging following acute myocardial infarction and reperfusion. J Am Coll Cardiol. 1998;32(6):1756–64.

    Article  CAS  PubMed  Google Scholar 

  31. Nijveldt R, Beek AM, Hirsch A, Stoel MG, Hofman MB, Umans VA, et al. Functional recovery after acute myocardial infarction: comparison between angiography, electrocardiography, and cardiovascular magnetic resonance measures of microvascular injury. J Am Coll Cardiol. 2008;52(3):181–9.

    Article  PubMed  Google Scholar 

  32. Hausenloy DJ, Iliodromitis EK, Andreadou I, Papalois A, Gritsopoulos G, Anastasiou-Nana M, et al. Investigating the signal transduction pathways underlying remote ischemic conditioning in the porcine heart. Cardiovasc Drugs Ther. 2012;26(2):87–93.

    Article  CAS  PubMed  Google Scholar 

  33. Brandt PW, Partridge JB, Wattie WJ. Coronary arteriography; method of presentation of the arteriogram report and a scoring system. Clin Radiol. 1977;28(4):361–5.

    Article  CAS  PubMed  Google Scholar 

  34. Henriques JP, Zijlstra F, van ‘t Hof AW, de Boer MJ, Dambrink JH, Gosselink M, Hoorntje JC, Suryapranata H. Angiographic assessment of reperfusion in acute myocardial infarction by myocardial blush grade. Circulation. 2003;107(16):2115–9.

    Article  PubMed  Google Scholar 

  35. Gibson CM, de Lemos JA, Murphy SA, Marble SJ, Dauterman KW, Michaels A, et al. Methodologic and clinical validation of the TIMI myocardial perfusion grade in acute myocardial infarction. J Thromb Thrombolysis. 2002;14(3):233–7.

    Article  PubMed  Google Scholar 

  36. Graham MM, Faris PD, Ghali WA, Galbraith PD, Norris CM, Badry JT, et al. Validation of three myocardial jeopardy scores in a population-based cardiac catheterization cohort. Am Heart J. 2001;142(2):254–61.

    Article  CAS  PubMed  Google Scholar 

  37. Briggs AH, Wonderling DE, Mooney CZ. Pulling cost-effectiveness analysis up by its bootstraps: a non-parametric approach to confidence interval estimation. Health Econ. 1997;6(4):327–40.

    Article  CAS  PubMed  Google Scholar 

  38. Moscucci M, Kline-Rogers E, Share D, O’Donnell M, Maxwell-Eward A, Meengs WL, et al. Simple bedside additive tool for prediction of in-hospital mortality after percutaneous coronary interventions. Circulation. 2001;104(3):263–8.

    Article  CAS  PubMed  Google Scholar 

  39. Yotti R, Bermejo J, Benito Y, Antoranz JC, Desco MM, Rodriguez-Perez D, et al. Noninvasive estimation of the rate of relaxation by the analysis of intraventricular pressure gradients. Circ Cardiovasc Imaging. 2011;4(2):94–104.

    Article  PubMed  Google Scholar 

  40. Campos CM, van Klaveren D, Farooq V, Simonton CA, Kappetein AP, Sabik JF, et al. Long-term forecasting and comparison of mortality in the evaluation of the xience everolimus eluting stent vs. coronary artery bypass surgery for effectiveness of left main revascularization (EXCEL) trial: prospective validation of the SYNTAX score II. Eur Heart J. 2015;36(20):1231–41.

    Article  PubMed  Google Scholar 

  41. Laajala TD, Jumppanen M, Huhtaniemi R, Fey V, Kaur A, Knuuttila M, et al. Optimized design and analysis of preclinical intervention studies in vivo. Sci Rep. 2016;6:30723.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kim EK, Hahn JY, Song YB, Lee SC, Choi JH, Choi SH, et al. Effect of ischemic postconditioning on myocardial salvage in patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction: cardiac magnetic resonance substudy of the POST randomized trial. Int J Cardiovasc Imaging. 2015;31(3):629–37.

    Article  PubMed  Google Scholar 

  43. Eitel I, Stiermaier T, Rommel KP, Fuernau G, Sandri M, Mangner N, et al. Cardioprotection by combined intrahospital remote ischaemic perconditioning and postconditioning in ST-elevation myocardial infarction: the randomized LIPSIA CONDITIONING trial. Eur Heart J. 2015;36(44):3049–57.

    Article  PubMed  Google Scholar 

  44. Raman SV, Simonetti OP, Winner MW 3rd, Dickerson JA, He X, Mazzaferri EL Jr, et al. Cardiac magnetic resonance with edema imaging identifies myocardium at risk and predicts worse outcome in patients with non-ST-segment elevation acute coronary syndrome. J Am Coll Cardiol. 2010;55(22):2480–8.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Mewton N, Thibault H, Roubille F, Lairez O, Rioufol G, Sportouch C, et al. Postconditioning attenuates no-reflow in STEMI patients. Basic Res Cardiol. 2013;108(6):383.

    Article  PubMed  Google Scholar 

  46. Rodriguez-Sinovas A, Cabestrero A, Garcia del Blanco B, Inserte J, Garcia A, Garcia-Dorado D. Intracoronary acid infusion as an alternative to ischemic postconditioning in pigs. Basic Res Cardiol. 2009;104(6):761–71.

    Article  CAS  PubMed  Google Scholar 

  47. Schmidt MR, Smerup M, Konstantinov IE, Shimizu M, Li J, Cheung M, et al. Intermittent peripheral tissue ischemia during coronary ischemia reduces myocardial infarction through a KATP-dependent mechanism: first demonstration of remote ischemic perconditioning. Am J Physiol Heart Circ Physiol. 2007;292(4):H1883–90.

    Article  CAS  PubMed  Google Scholar 

  48. Munk K, Andersen NH, Schmidt MR, Nielsen SS, Terkelsen CJ, Sloth E, et al. Remote ischemic conditioning in patients with myocardial infarction treated with primary angioplasty: impact on left ventricular function assessed by comprehensive echocardiography and gated single-photon emission CT. Circ Cardiovasc Imaging. 2010;3(6):656–62.

    Article  PubMed  Google Scholar 

  49. Halkin A, Stone GW, Dixon SR, Grines CL, Tcheng JE, Cox DA, et al. Impact and determinants of left ventricular function in patients undergoing primary percutaneous coronary intervention in acute myocardial infarction. Am J Cardiol. 2005;96(3):325–31.

    Article  PubMed  Google Scholar 

  50. Ortiz-Perez JT, Lee DC, Meyers SN, Davidson CJ, Bonow RO, Wu E. Determinants of myocardial salvage during acute myocardial infarction: evaluation with a combined angiographic and CMR myocardial salvage index. JACC Cardiovasc Imaging. 2010;3(5):491–500.

    Article  PubMed  Google Scholar 

  51. de Waard GA, Hollander MR, Teunissen PF, Jansen MF, Eerenberg ES, Beek AM, et al. Changes in coronary blood flow after acute myocardial infarction: insights from a patient study and an experimental porcine model. JACC Cardiovasc Interv. 2016;9(6):602–13.

    Article  PubMed  Google Scholar 

  52. Kern KB, Hanna JM, Young HN, Ellingson CJ, White JJ, Heller B, et al. Importance of both early reperfusion and therapeutic hypothermia in limiting myocardial infarct size post-cardiac arrest in a porcine model. JACC Cardiovasc Interv. 2016;9(23):2403–12.

    Article  PubMed  Google Scholar 

  53. Heusch G, Skyschally A, Schulz R. The in situ pig heart with regional ischemia/reperfusion - ready for translation. J Mol Cell Cardiol. 2011;50(6):951–63.

    Article  CAS  PubMed  Google Scholar 

  54. Heusch G, Musiolik J, Gedik N, Skyschally A. Mitochondrial STAT3 activation and cardioprotection by ischemic postconditioning in pigs with regional myocardial ischemia/reperfusion. Circ Res. 2011;109(11):1302–8.

    Article  CAS  PubMed  Google Scholar 

  55. Sun H, Guo T, Liu L, Yu Z, Xu W, Chen W, et al. Ischemic postconditioning inhibits apoptosis after acute myocardial infarction in pigs. Heart Surg Forum. 2010;13(5):E305–10.

    Article  PubMed  Google Scholar 

  56. Ma XJ, Yin SJ, Jin JC, Wu CF, Huang Y, Shi DZ, et al. Synergistic protection of Danhong injection and ischemic postconditioning on myocardial reperfusion injury in minipigs. Chin J Integr Med. 2010;16(6):531–6.

    Article  PubMed  Google Scholar 

  57. Kharbanda RK, Mortensen UM, White PA, Kristiansen SB, Schmidt MR, Hoschtitzky JA, et al. Transient limb ischemia induces remote ischemic preconditioning in vivo. Circulation. 2002;106(23):2881–3.

    Article  CAS  PubMed  Google Scholar 

  58. Andreka G, Vertesaljai M, Szantho G, Font G, Piroth Z, Fontos G, et al. Remote ischaemic postconditioning protects the heart during acute myocardial infarction in pigs. Heart. 2007;93(6):749–52.

    Article  PubMed  PubMed Central  Google Scholar 

  59. O’Connor RE, Al Ali AS, Brady WJ, Ghaemmaghami CA, Menon V, Welsford M, et al. Part 9: acute coronary syndromes: 2015 American heart association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation. 2015;132(18 Suppl 2):S483–500.

    Article  PubMed  Google Scholar 

  60. Windecker S, Kolh P, Alfonso F, Collet JP, Cremer J, 2014 ESC/EACTS Guidelines on myocardial revascularization, et al. The task force on myocardial revascularization of the european society of cardiology (ESC) and the european association for cardio-thoracic surgery (EACTS) developed with the special contribution of the European Association of Percutaneous Cardiovascular Interventions (EAPCI). Eur Heart J. 2014;35(37):2541–619.

    Article  PubMed  Google Scholar 

  61. Alcindor D, Krolikowski JG, Pagel PS, Warltier DC, Kersten JR. Cyclooxygenase-2 mediates ischemic, anesthetic, and pharmacologic preconditioning in vivo. Anesthesiology. 2004;100(3):547–54.

    Article  CAS  PubMed  Google Scholar 

  62. Penna C, Mancardi D, Tullio F, Pagliaro P. Postconditioning and intermittent bradykinin induced cardioprotection require cyclooxygenase activation and prostacyclin release during reperfusion. Basic Res Cardiol. 2008;103(4):368–77.

    Article  PubMed  Google Scholar 

  63. Guo Y, Tukaye DN, Wu WJ, Zhu X, Book M, Tan W, et al. The COX-2/PGI2 receptor axis plays an obligatory role in mediating the cardioprotection conferred by the late phase of ischemic preconditioning. PLoS ONE. 2012;7(7):e41178.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Yusuf S, Zhao F, Mehta SR, Chrolavicius S, Tognoni G, Fox KK, et al. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med. 2001;345(7):494–502.

    Article  CAS  PubMed  Google Scholar 

  65. Roubille F, Lairez O, Mewton N, Rioufol G, Ranc S, Sanchez I, et al. Cardioprotection by clopidogrel in acute ST-elevated myocardial infarction patients: a retrospective analysis. Basic Res Cardiol. 2012;107(4):275.

    Article  PubMed  Google Scholar 

  66. Ferdinandy P, Hausenloy DJ, Heusch G, Baxter GF, Schulz R. Interaction of risk factors, comorbidities, and comedications with ischemia/reperfusion injury and cardioprotection by preconditioning, postconditioning, and remote conditioning. Pharmacol Rev. 2014;66(4):1142–74.

    Article  CAS  PubMed  Google Scholar 

  67. Alvarez P, Tapia L, Mardones LA, Pedemonte JC, Farias JG, Castillo RL. Cellular mechanisms against ischemia reperfusion injury induced by the use of anesthetic pharmacological agents. Chem Biol Interact. 2014;218:89–98.

    Article  CAS  PubMed  Google Scholar 

  68. Kottenberg E, Thielmann M, Bergmann L, Heine T, Jakob H, Heusch G, et al. Protection by remote ischemic preconditioning during coronary artery bypass graft surgery with isoflurane but not propofol-a clinical trial. Acta Anaesthesiol Scand. 2012;56(1):30–8.

    Article  CAS  PubMed  Google Scholar 

  69. Fernandez-Jimenez R, Garcia-Prieto J, Sanchez-Gonzalez J, Aguero J, Lopez-Martin GJ, Galan-Arriola C, et al. Pathophysiology underlying the bimodal edema phenomenon after myocardial ischemia/reperfusion. J Am Coll Cardiol. 2015;66(7):816–28.

    Article  PubMed  Google Scholar 

  70. Nordlund D, Klug G, Heiberg E, Koul S, Larsen TH, Hoffmann P, et al. Multi-vendor, multicentre comparison of contrast-enhanced SSFP and T2-STIR CMR for determining myocardium at risk in ST-elevation myocardial infarction. Eur Heart J Cardiovasc Imaging. 2016;17(7):744–53.

    Article  PubMed  PubMed Central  Google Scholar 

  71. Lelovas PP, Kostomitsopoulos NG, Xanthos TT. A comparative anatomic and physiologic overview of the porcine heart. J Am Assoc Lab Anim Sci. 2014;53(5):432–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010;8(6):e1000412.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Authors’ contributions

TB, ZG, ZVV, RS, MG and PF designed the experiments. TB, ZG, ZVV, GK, DL, AM, RG performed the experiments. MG conducted the cardiac catheterization. LT performed the cardiac MRI. AJ, CC, HV analyzed the cardiac MRI recordings. MS performed and analyzed echocardiography. ZR analyzed the coronarography. TB, ZG, ZVV, NP, BM, RS, MG and PF prepared the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Competing interests

PF is a founder and CEO of Pharmahungary, a group of R&D companies.

Availability of data and materials

The datasets analysed during the current study available from the corresponding author on reasonable request.

Ethics approval

This investigation was carried out according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and according to the ARRIVE guidelines [72], to the EU Directive (2010/63/EU) and was approved by the animal ethics committee of Hungarian National Food Chain Safety Office (SOI/31/26-11/2014).

Funding

This work was supported by the Hungarian Scientific Research Fund (OTKA ANN 107803, OTKA K-105555) Austrian-Hungarian Action Scholarship (88öu1), and R&D competitiveness and excellence cooperations program of the National Research, Development and Innovation Office of Hungary (NVKP_16-1-2016-0017), as well as by the Pharmahungary Group. TB supported by the ÚNKP-16-3 New National Excellence Program of The Ministry Of Human Capacities. MS was supported by the UNKP-UNKP-6-4 IKT/147-1787/8/2016-ÖSZT-120 New National Excellence Program of the Ministry of Human Capacities. ZG holds a “János Bolyai Fellowship” from the Hungarian Academy of Sciences.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Péter Ferdinandy.

Additional information

Tamás Baranyai and Zoltán Giricz contributed equally to this work

Mariann Gyöngyösi and Péter Ferdinandy contributed equally to this work

Additional files

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Baranyai, T., Giricz, Z., Varga, Z.V. et al. In vivo MRI and ex vivo histological assessment of the cardioprotection induced by ischemic preconditioning, postconditioning and remote conditioning in a closed-chest porcine model of reperfused acute myocardial infarction: importance of microvasculature. J Transl Med 15, 67 (2017). https://doi.org/10.1186/s12967-017-1166-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12967-017-1166-z

Keywords