Ir J Med Sci DOI 10.1007/s11845-013-1046-3
ORIGINAL ARTICLE
Protective effect of CDP-choline on ischemia-reperfusion-induced myocardial tissue injury in rats C. Coskun • B. Avci • M. Yalcin • A. Yermezler M. S. Yilmaz • V. Savci
•
Received: 16 January 2013 / Accepted: 19 November 2013 Ó Royal Academy of Medicine in Ireland 2013
Abstract Background CDP-choline exerts tissue protective effect in several ischemic conditions. Recently we have reported that the drug prevents cardiac arrhythmias and improves survival rate in short-term myocardial ischemia reperfusion in rats. Aim In the current study, we determined the effect of intravenously administered CDP-choline on myocardial tissue injury induced by 30-min ischemia followed by 3-h reperfusion in anesthetized rats. Methods Myocardial ischemia was produced by ligature of the left main coronary artery. CDP-choline (100–500 mg/kg) was intravenously injected in the middle of the ischemic period. Cardiovascular parameters were recorded through the experimental period. At the end of the reperfusion period, the hearts of the animals were removed and stained for the investigation of tissue necrosis and apoptosis. The infarct size was evaluated as the ratio of the infarct area to the risk area. Apoptotic activation was assessed by TUNEL assay. Also the blood samples of rats were collected for the measurement of M30–M65, ADMA, homocysteine, and lactate levels.
Results Ischemia/reperfusion caused serious injury in myocardium, increased blood ADMA and lactate levels without influencing other parameters. CDP-choline significantly reduced the infarct size and the number of apoptotic cells in the risk area. Blood pressure increased after CDPcholine injection; however, it returned back to the basal levels before the onset of reperfusion. CDP-choline failed to alter any other measured parameters. Conclusion The present results demonstrate that intravenously administered CDP-choline is able to protect myocardium from injury induced by long-term coronary occlusion–reperfusion in rats. The inhibition of apoptosis by the drug may contribute to its protective effect. But neither the increase in blood pressure in response to CDPcholine injection nor changes in plasma ADMA concentration appear to mediate the attenuation of the myocardial injury. Keywords CDP-choline Apoptosis Necrosis ADMA M30 M65 Coronary ischemia/reperfusion
Introduction
C. Coskun M. S. Yilmaz V. Savci Department of Medical Pharmacology, Faculty of Medicine, Uludag University, 16059 Bursa, Turkey B. Avci A. Yermezler Department of Histology and Embryology, Faculty of Medicine, Uludag University, 16059 Bursa, Turkey M. Yalcin (&) Department of Physiology, Faculty of Veterinary Medicine, Uludag University, Gorukle, 16059 Bursa, Turkey e-mail: [email protected]; [email protected]
Acute myocardial infarction continues to be one of the leading causes of death [1, 2]. Despite recent advances in our understanding of cellular and molecular aspects of myocardial injury, the cell death still remains to be the most important clinical consequence of this condition and to be the key of success of any therapeutic intervention which aims to prevent it. Necrosis has long been believed to be the only mode of myocardial cell death; however, recent studies support the idea that apoptosis contributes significantly to the myocardial ischemic injury [3–8]. Today we know that not only the ischemia but also the
123
Ir J Med Sci
reperfusion by re-establishing the coronary blood flow contributes to the tissue injury and cell death in this condition. Despite the importance and essential nature of early reperfusion for tissue salvage, it paradoxically increases myocardial injury possibly by increasing the inflammatory response and oxidative stress along with the acceleration the onset of apoptosis [3, 9]. CDP-choline (cytidine-5-diphosphate choline; citicoline) is an endogenous intermediate product of Kennedy pathway, which is the main synthetic route of membrane phosphatidylcholine [10]. Its physiological and pharmacological effects have been investigated in experimental and clinical studies for more than three decades [11–13]. Clinical studies mainly focused on drug’s anti-ischemic and tissue protective effects, because CDP-choline protects the membrane from ischemic insult through the prevention of fatty acids release [14], stimulation of phosphatidylcholine synthesis [15], preservation of cardiolipin and sphingomyelin levels [16]. The drug also decreases oxidative stress by increasing glutathione synthesis and glutathione reductase activity [17]. CDP-choline can also have the antiapoptotic activity by reducing the expression of all procaspases involved in apoptosis, particularly by inhibiting the caspase-3 activation [18]. When administered exogenously, i.e., orally or intravenously (i.v.), CDP-choline is rapidly hydrolyzed to choline and cytidine [19, 20]. These final metabolites increase in the circulation, cross blood–brain barrier, are taken up by the cells/neurons and mediate re-synthesis of the molecule in the cell [19]. We have been investigating its cholinergic nature and usefulness in several shock conditions for almost a decade. Our studies have shown that CDP-choline can exert significant hemodynamic and endocrine effects in normal and stimulated situations [21–25]. It increases blood pressure in normal conditions [25], restores hypotension and increases survival in hemorrhagic shock [22]. The activation of central cholinergic receptors through the increase in brain choline levels mediates these effects [22]. We also reported that CDP-choline is able to decrease neuronal injury in spinal-cord transected rats by limiting oxidative injury [26]. More recently we demonstrated that intravenously given CDP-choline exerts strong protection against cardiac arrhythmias and lowers mortality rates in short-term myocardial ischemia reperfusion (I/R) in rats by activating efferent vagal pathways followed by increased brainstem cholinergic transmission through the activation of central muscarinic receptors [27]. Taking these data into consideration together with the importance of cell loss in the myocardial infarction, we suggested that CDP-choline may protect myocardial tissue from the injury induced by long-term I/R. The present work aimed to investigate this hypothesis by determining the effect of intravenously injected CDP-choline on the
123
myocardial infarct size and apoptosis in I/R conditions. We recorded blood pressure and heart rate of animals throughout the experiments to determine if hemodynamic effects of CDP-choline are involved in its tissue protective effect. Moreover, plasma concentrations of asymmetrical dimethylarginine (ADMA), a circulating endogenous inhibitor of NO synthase, were determined since the elevations of plasma ADMA levels have been reported to be a predictive value for the worse clinical outcome of patients undergoing percutaneous coronary intervention [28].
Materials and methods Animals Adult male Wistar albino rats (250–300 g) were used throughout the study. The animals were obtained from Experimental Animals Breeding and Research Center, Uludag University, Bursa, Turkey. They were housed five per cage in a temperature (20–24 °C) and humidity (60–70 %) controlled room set to a 12-h light: 12-h dark cycle and had access to standard rat chow and water ad libitum. All procedures were approved by the Animal Care and Use Committee of Uludag University and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (http://oacu.od. nih.gov/regs/guide/guide.pdf). Surgical procedures Under urethane (1.25 g/kg intraperitoneally; Fluka; Buchs, Switzerland) anesthesia, the left common carotid artery for monitoring mean arterial pressure and the left jugular vein for drug administration were cannulated with PE 50 tubing filled with heparinized saline (100 U/ml) and the trachea of rats was catheterized with tracheal cannula. After intubation of the trachea, the animals were ventilated with room air via a respirator for small rodents (CWE model SAR830/AP, PA, USA) with a stroke volume of approximately 20 ml/kg and a rate of 70 strokes per minute. Following cannulation process, rats were placed in the supine position on a heated operating platform to maintain a rectal temperature of 37 ± 0.2 °C. Temperature of animals was monitored using a rectal probe throughout the study. For mean arterial pressure and heart rate monitoring, the arterial cannula was connected to a volumetric pressure transducer (BPT 300), which was attached to a DA100B general purpose transducer amplifier (Commat, Ankara, Turkey). Mean arterial pressures of rats were recorded and analyzed using the MP100 system and AcqKnowledge software (BIOPAC Systems, CA, USA). Mean arterial pressure is reported as mean arterial pressure (MAP;
Ir J Med Sci
mmHg), and heart rate is expressed as beats per minute (HR; bpm). Needle electrodes were placed subcutaneously on the limbs and an electrocardiogram (ECG) was continuously recorded. The chest was then opened by left thoracotomy, the pericardium was incised, the heart was exteriorized, and a loose loop (6/0 braided silk suture attached to a 10-mm micropoint reverse cutting needle) was placed around the left main coronary artery (LCA). The heart was replaced in the chest cavity with the ligature ends exteriorized, and any animal in which this procedure produced dysrhythmias or a sustained fall in mean arterial pressure to \60 mmHg was withdrawn from the study at this point. Ischemia/reperfusion procedure After an equilibration period of 20 min, the ligature was tied to start the ischemia; after a 30-min period of coronary occlusion, reperfusion was obtained by loosening the suture, and the animals were then monitored for a further 180 min. The MAP, HR, occurrence of dysrhythmias, and lethality were recorded. Each rat underwent a single coronary occlusion. Rats subjected to sham ischemia underwent all surgical procedures experienced by the ischemic rats, except coronary ligation. Saline (1.0 ml/kg) or CDPcholine (100, 250, and 500 mg/kg) were intravenously injected in the middle of the ischemia period (15th min of ischemia). Reperfusion period lasted 180 min. Evaluation of myocardial infarct size At the end of experiments, the heart was removed after thoracotomy and was immediately put in a closed heart chamber and antegradely perfused with isotonic saline at a constant flow of 10 ml/min to remove blood in the heart. To demarcate the area at risk, the left main coronary artery was re-occluded and 1 ml of acridine orange stain (1 mg/ ml in saline) (Sigma-Aldrich Chemie GmbH, Germany) was infused for 1 min via the aorta. At the end of the staining, the heart was immediately frozen at -30 °C for 10 min and then sliced into 2-mm thick slices perpendicular to the left main coronary artery up to the area of ligation. In order to delineate the risk area, which was perfused by the left main coronary artery, the slices were examined using a fluorescence microscope (Olympus BX 50, Japan). The areas, which are not stained by acridine orange, were described as the area at risk of the heart. To examine the myocardial necrotic area, the heart slices were each incubated for 30 min at 37 °C in 10 ml of 1 % 2,3,5-triphenyltetrazolium chloride (TTC) solution (pH 7.4). Sections were fixed overnight in 4 % paraformaldehyde for contrast enhancement between the stained and unstained tissues. Viable myocardium is stained red by
TTC while the necrotic, infarcted area remains unstained. The sections were then placed between two coverslips and digitally photographed using a fluorescence microscope (Olympus BX 50, Japan). The calculation of the volumes of each slices and quantification of the area at risk and the area of necrosis were done with planimetric method using Scion software (Maryland, USA). The size of necrosis was calculated using ‘‘total area of necrosis/total area at risk 9 100’’ formula. Evaluation of myocardial apoptosis After I/R, the hearts were perfused first 0.9 % NaCl for 5 min and then with 4 % paraformaldehyde in PBS (pH 7.4) for 20 min. Four transverse sections (1 mm each) of left ventricle wall were cut and further fixed in 4 % paraformaldehyde in PBS overnight at room temperature. Fixed tissues were immersed in increasing concentrations of sucrose solutions and 15 serial cryosections (20 lm) in certain intervals were cut from each tissue block. Apoptotic cardiomyocytes were detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) method. TUNEL staining was performed using an In situ Cell Death Detection Kit (Roche Molecular Biochemicals, USA) according to the protocol provided by the manufacturer. The digoxigenin-conjugated dUTP was incorporated to the ends of DNA fragments by terminal deoxynucleotidyl transferase (TdT). The cryosections were washed two times with PBS for 15 min and incubated in permeabilisation solution (0.1 % Triton X-100, 0.2 % sodium citrate, freshly prepared) for 2 min on ice (2–8 °C). Then, the sections were incubated in TUNEL reaction mixture (terminal deoxynucleotidyl transferase from thymus EC 2.7.7.31, recombinant in E. coli and label solution) for 60 min at 37 °C in a humidified atmosphere in the dark and rinsed three times with PBS for 10 min. The samples were analysed in a drop of PBS under a fluorescence microscope at this state (used an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nmgreen). The sections were incubated in Converter-POD (anti-fluorescein antibody, Fab fragment from sheep, conjugated with horse-radish peroxidase) for 10 min at room temperature, and then were rinsed three times with PBS and added DAB substrate on the sections for 10 min. After the DAB staining, slides were mounted under glass coverslip (with DPX) and analysed under photomicroscope (BX-50; Olympus). In the negative control experiment, sections were incubated in label solution (without terminal transferase) instead of TUNEL reaction mixture. Cardiomyocytes from four randomly selected slides per block were evaluated immunohistochemically to determine the number and percentage of cells exhibiting positive staining for apoptosis. For each slide, five microscopic fields were
123
Ir J Med Sci 50
Infarct Size (IA/RAx100)
randomly chosen. In each field, cells were calculated and the apoptotic cardiomyocytes counted (209 objective). To determine the total number of cardiomyocytes in these areas, serial sections were stained with H&E and total cardiomyocytes were counted in the same fields. The apoptotic index was determined (i.e., number of positively stained apoptotic myocytes/total number of myocytes counted 9 100 %). The assay was performed in a blinded manner.
40
30
*
*
*
100 mg/kg
250 mg/kg
500 mg/kg
20
10
Measurements At the end of each experiment, 2 ml of blood sample was collected through the arterial catheter (before the heart was removed) and their serum separated. Serum ADMA and serum M30/M65 levels were determined using commercially available ELISA kits which are manufactured by Immundiagnostic (Germany) and Peviva (Sweden), respectively. Serum lactate and homocysteine levels were studied using a DPC brand ‘‘Immulate 2000 Lactate and Homocysteine’’ kit on the Immulate 2000 apparatus (DPC, Los Angeles, CA, USA) using the immunoassay method. Drugs CDP-choline was purchased from Sigma-Aldrich Chemie GmbH (Germany). It was freshly dissolved in saline (0.9 % NaCl). Intravenous injection volume was 1.0 ml/kg. Each dose of CDP-choline was dissolved in one ml of saline solution (100, 250 or 500 mg/ml) and injected according to the animal’s body weight (100, 250 or 500 mg/ml/kg) in 30 s. Data and statistical analysis Results are expressed as mean ± SEM. Repeated measures analysis of variance (RM-ANOVA; two-way) and analysis of variance (ANOVA; one way) were performed for mean arterial pressure and heart rate results, and the area at risk and the area of necrosis, M30, M65, homocysteine and ADMA results. Tukey test was applied as a post hoc test when significant interactions were found. Blood lactate results were evaluated using Wilcoxon signed-rank test. A p value of \0.05 was considered significant.
Results Effect on myocardial infarct size The myocardial infarct size was determined by the ratio of necrotic or infarct area (IA) to the area at risk (RA) and expressed as percentage (i.e., IA/RA 9 100). Thirty
123
0
Saline
CDP-choline Fig. 1 Effect of CDP-choline on myocardial infarct size. Rats were subjected 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, hearts were removed and stained with acridine orange and TTC as described in ‘‘Materials and methods’’. The results are expressed as the percentage of the infarct area to the risk area. Values are mean ± SEM of 6–8 animals. Statistical analysis was performed using one-way ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from the saline-treated group
minutes’ coronary occlusion followed by the 3 h of reperfusion seriously injured the myocardial tissue perfused by left main coronary artery (risk area). Intravenous injection of CDP-choline attenuated the myocardial tissue injury at all doses compared with those observed in salinetreated rats (Fig. 1). The myocardial infarct size was 41 ± 3 % in saline group while it was 26.6 ± 3, 26.8 ± 4, 27.0 ± 3 % in 100, 250, 500 mg/kg CDP-choline groups, respectively (Fig. 1). Analysis of variance confirmed that all doses of CDP-choline significantly reduced the myocardial infarct size in similar way [f(3,21) = 5.079; p \ 0.001] (Fig. 1). Histopathological evaluation by H&E staining demonstrated that the injured area was much smaller in CDP-choline-treated (100 mg/kg; i.v.) animals than those observed in saline-treated rats (Fig. 2, top). Effect on apoptosis Since apoptosis may contribute significantly to the I/Rinduced myocardial tissue injury [3, 8] and CDP-choline has been shown to reduce apoptosis through the inhibition of caspase-3 activation in cerebral ischemic situation [18], we investigated if CDP-choline-treatment alters the apoptotic activation in these conditions. TUNEL assay was used for determination of the apoptotic activity in myocardial tissue. Quantitative analysis of DNA fragmentation was performed using the TUNEL method. TUNEL-positive cardiomyocyte nuclei are shown in Figs. 2, 3 and 4. We
Ir J Med Sci
Fig. 2 Effect of CDP-choline on histopathological changes and apoptotic activation induced by myocardial I/R injury. CDP-choline (100 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. Sham-operated animals underwent the same surgical procedures but without ligation of the LCA suture. Hematoxylin & Eosin staining indicates ischemic and normal areas at
myocardium (top). TUNEL-positive nuclei are shown as brown nuclei (bottom). CDP-choline significantly reduced the apoptotic activation and the number of TUNEL-positive myocytes versus the saline group after ischemia-reperfusion. Figures are representative of five separate experiments
observed significant number of apoptotic cell nuclei in ischemic/reperfused ventricles in saline-treated rats while a few apoptotic cell nuclei was detected in similar areas after CDP-choline (100 mg/kg, i.v.) administration (Figs. 2, 3 and 4). There is no TUNEL-positive cardiomyocyte in sham-operated control group (Fig. 2, left sections middle). The apoptotic index was reduced by almost 50 % (p \ 0.05) in CDP-choline-administrated group compared with saline-treated group (12.35 ± 0.7 versus 22.47 ± 3.3 %; CDP-choline versus saline group, respectively) (Fig. 5). We also measured plasma M30 and M65 levels at the end of the experiments to see if these levels can support the tissue findings. M30 levels help us to predict the apoptotic cell death by measuring caspase-cleaved CK18 produced during apoptosis and M65 measures the levels of both caspase-cleaved (apoptosis) and intact CK18 (necrosis). Plasma M30 and M65 levels did not significantly differ in saline or CDP-choline treated groups compared to values observed in sham-operated rats (Fig. 6).
(NO) synthase, were determined since the elevations of plasma ADMA levels have been reported to be a predictive value for the worse clinical outcome of patients undergoing percutaneous coronary intervention [28]. Plasma ADMA levels significantly increased after 30 min of coronary occlusion followed by 3 h reperfusion period (Fig. 7). CDP-choline treatment, at all doses, did not significantly alter the increased ADMA concentrations in myocardial I/R (Fig. 6). Although there was a tendency to decrease in ADMA levels in 100 mg/kg dose of CDP-choline-treated rats, these levels did not significantly different from those observed either saline-treated group or sham control animals (Fig. 7). Neither I/R conditions nor CDP-choline treatment affected plasma homocysteine concentrations (Table 1). Plasma lactate levels did not change in CDPcholine treated rats while the levels increased significantly after I/R itself (Table 1).
Effect on blood ADMA, homocysteine and lactate concentrations Plasma concentrations of asymmetrical dimethylarginine (ADMA), a circulating endogenous inhibitor of nitric oxide
Effect on blood pressure and heart rate After an equilibration period of 20 min following suture placing around the left main coronary artery, any animal in which this procedure produced dysrhythmias, such as ventricular tachycardia or ventricular fibrillation, or a sustained fall in mean arterial pressure to \60 mmHg was withdrawn from the study at this point. During the
123
Ir J Med Sci
Fig. 3 TUNEL assay in myocardial I/R. TUNEL assay was described in ‘‘Materials and methods’’. Figures are representative of five separate experiments. Sections were selected from apical region and same levels were used for both saline and CDP-choline-treated group.
Figure shows the apoptotic cells using TUNEL assay in fluorescence microscopy after ischemia-reperfusion. In TUNEL staining, sections were incubated with anti-BrdU-FITC antibody
Fig. 4 TUNEL immunostaining in myocardial I/R. Sections were selected same levels for sham, saline and CDP-choline treated groups. Figures demonstrate the apoptotic cells using TUNEL immunostaining method in light microscopy. Arrows show the TUNEL-positive myocytes
equilibration period, the mean arterial pressure and heart rate of animals were 77 ± 3 mmHg and 321 ± 15 BPM, respectively (Fig. 8). While blood pressure of rats decreased 8–10 mmHg with starting of ischemia, heart rate of animals did not change (Fig. 8). Intravenous injection of CDP-choline (100, 250, and 500 mg/kg) in the middle of
123
the ischemic period caused a prompt increase in arterial pressure (Fig. 8). Pressor effect was transient at each doses of the drug. Blood pressure of rats returned to pre-treatment levels within 15 min after CDP-choline (Fig. 8, top). CDPcholine (100, 250, and 500 mg/kg) did not alter the heart rate of the animals (Fig. 8, bottom).
Ir J Med Sci 30
A 1000
20 15
*
10 5 0 Saline
CDP-choline
Serum M30 Levels (U/L)
Apoptotic Activity (%)
25
Fig. 5 Effect of CDP-choline on apoptotic activity induced by myocardial I/R injury. The apoptotic activity was determined by the ratio of the number of TUNEL-positive cardiomyocytes to the total number of myocytes and expressed as the percentage. Bars represent the mean ± SEM of the apoptotic activity calculated from four tissue blocks of five rats for each experimental group (i.e., saline or CDPcholine 100 mg/kg). Statistical analysis was performed using one-way ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from the saline-treated group
The present data show that intravenously injected CDPcholine is able to decrease the myocardial infarct size caused by myocardial I/R. According to our TUNEL assay result, the drug strongly inhibited myocardial apoptosis induced by long-term myocardial I/R in rats. CDP-choline caused prompt but transient increase in blood pressure. Ischemia reperfusion itself increased blood ADMA levels significantly without changing M30/M65 and homocysteine levels. CDP-choline failed to affect any of these parameters in this condition. In the present study, 30-min occlusion of the LCA followed by a 3-h reperfusion seriously injured myocardial tissue. Infarct size, which was used as an index of myocardial injury, was evaluated as the ratio of necrotic (or infarct) area to determined risk area. All three doses of CDP-choline treatment exerted similar degree of protection. The doses of CDP-choline were chosen based on our previous observations in which the drug produced significant cardiovascular [22], endocrine [23, 24], and protective effects [26, 27] in several experimental conditions including shock. Our recent paper [27] which contains the most related data to this study reported that similar doses of i.v. injected CDP-choline was able to reduce the incidence of arrhythmias and mortality rate of rats in short-term myocardial I/R. In that study 250 mg/kg CDP-choline was as effective as lidocaine, which was used as a positive reference for an antiarrhythmic effect during I/R. We would like to mention that the present study was performed as an extension of that previous one, since we also wanted to investigate the tissue protective effect of the drug in similar
800
600
400
200
0
B 500
Serum M65 Levels (U/L)
Discussion
Sham Saline (ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
400
300
200
100
0
Fig. 6 Effect of CDP-choline on plasma M30 and M65 levels in myocardial I/R. Rats were subjected 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, blood samples of animals were taken from their arterial catheters and plasma M30 and M65 levels were measured using ELISA kit. Data are presented as the mean ± SEM of 5–7 animals. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test. No statistically significant interactions were found
cardiogenic shock situation. However, we preferred to apply the long-term myocardial I/R because the tissue injury cannot be observed in such a short period of I/R study. Although there is considerable evidence showing the tissue protective effect of CDP-choline in several cerebral ischemic conditions [12], head trauma [13], and spinal injury [26], this is the first study demonstrating the protective effect of CDP-choline in myocardial tissue injured by the 30 min of ischemia followed by 3 h reperfusion conditions. Studies investigating the mechanisms of the druginduced tissue protection in several ischemic conditions suggested that CDP-choline can decrease membrane-
123
Ir J Med Sci
Serum ADMA Levels (pg/ml)
4
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
3 *
*
*
2
1
0
Fig. 7 Effect of CDP-choline on plasma ADMA levels in myocardial I/R. Rats were subjected 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, blood samples of animals were taken from their arterial catheters and plasma ADMA levels were measured using ELISA kit. Data are presented as the mean ± SEM of 5–7 animals. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from sham control group. There is no any significance in CDP-choline treated groups compared with values observed in saline-treated rats
phospholipid breakdown/restore phosphatidylcholine levels by attenuating phospholipase A stimulation and loss of CTP: phosphocholine cytidylyltransferase activity and it can stabilize the membrane integrity [12, 13, 29]. The drug also has antioxidant effect by decreasing lipid peroxidation; therefore, some of its beneficial effects can be mediated by the decrease of oxidative stress induced by head trauma [30] or spinal injury [26]. Furthermore, we showed that CDP-choline could be considered as a cholinergic agent because it increases central cholinergic transmission when it is administered either centrally or peripherally [21– 25]. Our recent report not only supported these previous findings but also implicated that the activation of cholinergic anti-inflammatory pathway is involved in the protective effect of drug in short-term myocardial I/R study in rats [27]. According to these previous data we can suggest that CDP-choline can attenuate the myocardial injury by activating cholinergic anti-inflammatory pathway, by
attenuating oxidative stress and by decreasing membrane breakdown in the present conditions. Several studies show that in addition to necrosis, apoptosis may also contribute significantly to myocardial ischemic injury and cell death [3, 31]. Although apoptosis can be detected in either permanently ischemic or reperfused myocardium, the onset of apoptosis is accelerated by reperfusion possibly as a result of the reperfusion-associated inflammatory response [32, 33]. Apoptotic cell death is mediated by activation of caspases. CDP-choline has been shown to attenuate expression of pro-caspases, cleaved caspase-3, and nuclear DNA fragmentation after focal cerebral ischemia [18, 34]. Hence we wanted to investigate if the inhibition of apoptotic cell death contributes to the CDP-choline-induced myocardial protection. In order to accomplish this, we both performed immunohistochemistry to myocardial tissue by using TUNEL assay and also measured serum levels of full-length and caspasecleaved cytokeratin 18 (CK18), which are considered biomarker of apoptotic and necrotic cell death, using a combination of the M30 and M65 ELISAs. Although the levels of M30 and M65 tended to increase in I/R group they were not statistically different from those observed in sham control group. Also CDP-choline treatment did not change M30 and M65 levels compared with saline group. We did not use the ‘‘M30/M65 ratio’’ to interpret the death situations since we could not use any positive references for apoptosis or necrosis in our experimental conditions. On the other hand, our findings from TUNEL assay demonstrated that CDP-choline (100 mg/kg; i.v. injected 15 min after the onset of ischemia) significantly decreases the number of apoptotic cells and apoptotic activity in ischemic area (Figs. 2, 3). According to these immunohistochemistry data we can suggest that the inhibition of apoptosis can mediate the tissue protective effect of CDPcholine in myocardial I/R. However, the observation that there was no difference in M30 and M65 levels in both the control I/R animals and the drug-treated groups can be explained by the short time period of reperfusion (i.e., 3 h) which cannot be enough to determine the circulatory
Table 1 Effects of CDP-choline on serum plasma homocysteine and lactate levels Sham
Saline (ml/kg)
CDP-choline 100 (mg/kg)
250 (mg/kg)
500 (mg/kg)
Homocysteine (lmol/l)
6.2 ± 0.4
6.3 ± 1.3
6.3 ± 1.1
7.4 ± 0.7
6.7 ± 0.9
Lactate (mmol/l)
2.3 ± 0.3
3.8 ± 0.3*
3.6 ± 0.5*
3.8 ± 0.9*
4.1 ± 0.8*
Rats were subjected to 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, blood samples of animals were taken from their arterial catheters and plasma homocysteine and lactate levels were measured. Data are presented as the mean ± SEM of 5–7 animals. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test * p \ 0.05, significantly different from sham control group. There was no significant difference between CDP-choline and saline treated rats
123
Ir J Med Sci
A
Mean Arterial Pressure (mm Hg)
140 Treatment 120
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
*
100
*
80
60 Ischemia
Reperfusion
40 0
15 30 45 60 75 90 105 120 135 150 165 180 195 210 225
Time (minutes)
B 700
Heart Rate (beats/minute)
Treatment 600
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
500
400
300 Ischemia
Reperfusion
200 0
15 30 45 60 75 90 105 120 135 150 165 180 195 210 225
Time (minutes)
Fig. 8 Effects of CDP-choline on mean arterial pressure and heart rate in the myocardial I/R. Rats were subjected to myocardial ischemia for 30 min and reperfusion for 3 h. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. Arrows in the figures represent the treatment time point. Mean arterial pressure (a) and heart rates (b) of the animals were monitored during both ischemia/reperfusion periods. Data are presented as the mean ± SEM in mean arterial pressure and beats per minute in heart rate. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from its own previous value
changes reflecting the myocardial apoptosis. One of the recent articles supports our hypothesis by reporting that M30 levels peaked at 24 h in patients with acute coronary syndrome [35]. In the present study, we also determine whether plasma ADMA levels can increase in our experimental conditions and whether these levels can be affected by CDP-choline treatment. It has been known that ADMA is a circulating endogenous inhibitor of NO synthase [36, 37] and it can increase oxidative stress by uncoupling of electron transport between NO synthase and L-arginine
[36]. Elevated ADMA levels have been observed in patients with various risk factors of coronary artery disease and have been suggested to be an independent predictor of subsequent major adverse cardiovascular events after percutaneous coronary intervention [28, 37, 38]. We demonstrated that plasma ADMA levels significantly increased in myocardial I/R conditions. Showing the acute change of plasma ADMA levels in cardiogenic shock conditions is an interesting finding, because most of the literature reports the chronic or long-term changes of ADMA in plasma in several pathological situations [28, 37, 38]. On the other hand, we have another observation supporting this finding that plasma ADMA levels can acutely increase in hemorrhagic shock and immediately decrease after resuscitation with ringer lactate (unpublished data). Although the ADMA levels in 100 mg/kg CDP-choline given group were neither significantly higher than those observed in sham animals nor were lower than saline group, CDP-choline treatment failed to affect ADMA levels significantly (Fig. 6). These data suggest that ADMA may contribute to the myocardial injury under these experimental conditions but it may not be involved in the protective effect of CDPcholine. We also measured plasma homocysteine levels regarding its promoting effect on lipid peroxidation through free radical formation and contribution to cardiovascular disease by causing endothelial dysfunction [38]. Our results showed that myocardial I/R itself or CDP-choline treatment did not affect plasma homocysteine levels. In agreement with our data, in a previously reported study, the authors did not show any association between homocysteine and ADMA or between homocysteine and adverse cardiovascular events after percutaneous coronary intervention [28]. The pressor effect of CDP-choline lasted for a short period of time and blood pressure returned to pretreatment levels before the reperfusion; thus it cannot be responsible for the drug’s protective effect. This finding is in good accordance with those obtained from our previous studies in which the pressor effect was not suggested to mediate the cardio-protective and tissue protective effect of CDPcholine in short-term myocardial I/R injury [27] and spinal shock [26]. In conclusion, the present study shows that intravenously injected CDP-choline is able to protect tissue injury induced by long-term myocardial I/R in rats. The inhibition of apoptotic cell death by CDP-choline may mediate the drug’s protective effect. Acknowledgments This study was supported by The Commission of Scientific Research Projects of Uludag University, No. T-2008/43. Conflict of interest of interest.
The authors declare that there are no conflicts
123
Ir J Med Sci
References 1. Mathers CD, Boerma T, Ma Fat D (2009) Global and regional causes of death. Br Med Bull 92:7–32 2. WHO Fact Sheet (2011) Top ten causes of dead in The World 2008. 310 3. Fliss H, Gattinger D (1996) Apoptosis in ischemic and reperfused rat myocardium. Circ Res 79:949–956 4. Tanaka M, Ito H, Adachi S et al (1994) Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 75:426–433 5. James TN (1994) Normal and abnormal consequences of apoptosis in the human heart from postnatal morphogenesis to paroxysmal arrhythmias. Circulation 90:556–573 6. Gottlieb RA, Burleson KO, Kloner RA et al (1994) Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621–1628 7. Saraste A, Pulkki K, Kallajoki M et al (1997) Apoptosis in human acute myocardial infarction. Circulation 95:320–323 8. Yaoita H, Ogawa K, Maehara K et al (2000) Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. Cardiovasc Res 45:630–641 9. Yaoita H, Ogawa K, Maehara K et al (1998) Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation 97:276–281 10. Weiss GB (1995) Metabolism and actions of CDP-choline as an endogenous compound and administered exogenously as citicoline. Life Sci 56:637–660 11. Secades JJ (2011) Citicoline: pharmacological and clinical review, 2010 update. Rev Neurol 52:1–62 12. Adibhatla RM, Hatcher JF (2002) Citicoline mechanisms and clinical efficacy in cerebral ischemia. J Neurosci Res 70:133–139 13. Adibhatla RM, Hatcher JF (2005) Cytidine 50 -diphosphocholine (CDP-choline) in stroke and other CNS disorders. Neurochem Res 30:15–23 14. Dorman RV, Dabrowiecki Z, Horrocks LA (1983) Effects of CDP-choline and CDP-ethanolamine on the alterations in rat brain lipid metabolism induced by global ischemia. J Neurochem 40:276–279 15. Rao AM, Hatcher JF, Dempsey RJ (2001) Does CDP-choline modulate phospholipase activities after transient forebrain ischemia? Brain Res 893:268–272 16. Rao AM, Hatcher JF, Dempsey RJ (2000) Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDPcholine neuroprotection. J Neurochem 75:2528–2535 17. Adibhatla RM, Hatcher JF, Dempsey RJ (2001) Effects of citicoline on phospholipid and glutathione levels in transient cerebral ischemia. Stroke 32:2376–2381 18. Fiedorowicz M, Makarewicz D, Stan´czak-Mrozek KI et al (2008) CDP-choline (citicoline) attenuates brain damage in a rat model of birth asphyxia. Acta Neurobiol Exp 68:389–397 19. Savci V, Wurtman RJ (1995) Effect of cytidine on membrane phospholipid synthesis in rat striatal slices. J Neurochem 64:378–384 20. G-Coviella IL, Wurtman RJ (1992) Enhancement by cytidine of membrane phospholipids synthesis. J Neurochem 59:338–343 21. Savci V, Goktalay G, Ulus IH (2002) Intracerebroventricular choline increases plasma vasopressin and augments plasma vasopressin response to osmotic stimulation and hemorrhage. Brain Res 942:58–70
123
22. Savci V, Goktalay G, Cansev M et al (2003) Intravenously injected CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock: effect is mediated by central cholinergic activation. Eur J Pharmacol 468:129–139 23. Cavun S, Savci V, Ulus IH (2004) Centrally injected CDP-choline increases plasma vasopressin levels by central cholinergic activation. Fundam Clin Pharmacol 18:71–77 24. Cavun S, Savci V (2004) CDP-choline increases plasma ACTH and potentiates the stimulated release of GH, TSH and LH: the cholinergic involvement. Fundam Clin Pharmacol 18:513–523 25. Savci V, Cavun S, Goktalay G et al (2002) Cardiovascular effects of intracerebroventricularly injected CDP-choline in normotensive and hypotensive animals: the involvement of cholinergic system. Naunyn Schmiedebergs Arch Pharmacol 365:388–398 26. Coskun C, Avci B, Ocak N et al (2010) Effect of repeatedly given CDP-choline on cardiovascular and tissue injury in spinal shock conditions: investigation of the acute phase. J Pharm Pharmacol 62:497–506 27. Yilmaz MS, Coskun C, Yalcın M et al (2008) CDP-choline prevents cardiac arrhythmias and lethality induced by short-term myocardial ischemia-reperfusion injury in the rat: involvement of central muscarinic cholinergic mechanisms. Naunyn Schmiedebergs Arch Pharmacol 378:293–301 28. Lu TM, Ding YA, Lin SJ et al (2003) Plasma levels of asymmetrical dimethylarginine and adverse cardiovascular events after percutaneous coronary intervention. Eur Heart J 24:1912–1919 29. Adibhatla RM, Hatcher JF, Larsen EC et al (2006) CDP-choline significantly restores phosphatidylcholine levels by differentially affecting phospholipase A2 and CTP: phosphocholine cytidylyltransferase after stroke. J Biol Chem 281:6718–6725 30. Adibhatla RM, Hatcher JF, Dempsey RJ (2002) Citicoline: neuroprotective mechanisms in cerebral ischemia. J Neurochem 80:12–23 31. Moens AL, Claeys MJ, Timmermans JP et al (2005) Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int J Cardiol 100:179–190 32. Bazzani C, Guarini S, Botticelli AR et al (2001) Protective effect of melanocortin peptides in rat myocardial ischemia. J Pharmacol Exp Ther 297:1082–1087 33. Lee Y, Gustafsson AB (2009) Role of apoptosis in cardiovascular disease. Apoptosis 14:536–548 34. Krupinski J, Ferrer I, Barrachina M et al (2002) CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat. Neuropharmacology 42:846–854 35. Senturk T, Aydinlar A, Yilmaz Y et al (2009) Serial changes in circulating M30 antigen, a biomarker of apoptosis, in patients with acute coronary syndromes: relationship with the severity of coronary artery disease. Coron Artery Dis 20:494–498 36. Visser M, Paulus WJ, Vermeulen MA et al (2010) The role of asymmetric dimethylarginine and arginine in the failing heart and its vasculature. Eur J Heart Fail 12:1274–1281 37. Endemann DH, Schiffrin E (2004) Endothelial dysfunction. J Am Soc Nephrol 15:1983–1992 38. Bo¨ger RH (2005) Asymmetric dimethylarginine (ADMA) and cardiovascular disease: insights from prospective clinical trials. Vasc Med 10:19–25
ORIGINAL ARTICLE
Protective effect of CDP-choline on ischemia-reperfusion-induced myocardial tissue injury in rats C. Coskun • B. Avci • M. Yalcin • A. Yermezler M. S. Yilmaz • V. Savci
•
Received: 16 January 2013 / Accepted: 19 November 2013 Ó Royal Academy of Medicine in Ireland 2013
Abstract Background CDP-choline exerts tissue protective effect in several ischemic conditions. Recently we have reported that the drug prevents cardiac arrhythmias and improves survival rate in short-term myocardial ischemia reperfusion in rats. Aim In the current study, we determined the effect of intravenously administered CDP-choline on myocardial tissue injury induced by 30-min ischemia followed by 3-h reperfusion in anesthetized rats. Methods Myocardial ischemia was produced by ligature of the left main coronary artery. CDP-choline (100–500 mg/kg) was intravenously injected in the middle of the ischemic period. Cardiovascular parameters were recorded through the experimental period. At the end of the reperfusion period, the hearts of the animals were removed and stained for the investigation of tissue necrosis and apoptosis. The infarct size was evaluated as the ratio of the infarct area to the risk area. Apoptotic activation was assessed by TUNEL assay. Also the blood samples of rats were collected for the measurement of M30–M65, ADMA, homocysteine, and lactate levels.
Results Ischemia/reperfusion caused serious injury in myocardium, increased blood ADMA and lactate levels without influencing other parameters. CDP-choline significantly reduced the infarct size and the number of apoptotic cells in the risk area. Blood pressure increased after CDPcholine injection; however, it returned back to the basal levels before the onset of reperfusion. CDP-choline failed to alter any other measured parameters. Conclusion The present results demonstrate that intravenously administered CDP-choline is able to protect myocardium from injury induced by long-term coronary occlusion–reperfusion in rats. The inhibition of apoptosis by the drug may contribute to its protective effect. But neither the increase in blood pressure in response to CDPcholine injection nor changes in plasma ADMA concentration appear to mediate the attenuation of the myocardial injury. Keywords CDP-choline Apoptosis Necrosis ADMA M30 M65 Coronary ischemia/reperfusion
Introduction
C. Coskun M. S. Yilmaz V. Savci Department of Medical Pharmacology, Faculty of Medicine, Uludag University, 16059 Bursa, Turkey B. Avci A. Yermezler Department of Histology and Embryology, Faculty of Medicine, Uludag University, 16059 Bursa, Turkey M. Yalcin (&) Department of Physiology, Faculty of Veterinary Medicine, Uludag University, Gorukle, 16059 Bursa, Turkey e-mail: [email protected]; [email protected]
Acute myocardial infarction continues to be one of the leading causes of death [1, 2]. Despite recent advances in our understanding of cellular and molecular aspects of myocardial injury, the cell death still remains to be the most important clinical consequence of this condition and to be the key of success of any therapeutic intervention which aims to prevent it. Necrosis has long been believed to be the only mode of myocardial cell death; however, recent studies support the idea that apoptosis contributes significantly to the myocardial ischemic injury [3–8]. Today we know that not only the ischemia but also the
123
Ir J Med Sci
reperfusion by re-establishing the coronary blood flow contributes to the tissue injury and cell death in this condition. Despite the importance and essential nature of early reperfusion for tissue salvage, it paradoxically increases myocardial injury possibly by increasing the inflammatory response and oxidative stress along with the acceleration the onset of apoptosis [3, 9]. CDP-choline (cytidine-5-diphosphate choline; citicoline) is an endogenous intermediate product of Kennedy pathway, which is the main synthetic route of membrane phosphatidylcholine [10]. Its physiological and pharmacological effects have been investigated in experimental and clinical studies for more than three decades [11–13]. Clinical studies mainly focused on drug’s anti-ischemic and tissue protective effects, because CDP-choline protects the membrane from ischemic insult through the prevention of fatty acids release [14], stimulation of phosphatidylcholine synthesis [15], preservation of cardiolipin and sphingomyelin levels [16]. The drug also decreases oxidative stress by increasing glutathione synthesis and glutathione reductase activity [17]. CDP-choline can also have the antiapoptotic activity by reducing the expression of all procaspases involved in apoptosis, particularly by inhibiting the caspase-3 activation [18]. When administered exogenously, i.e., orally or intravenously (i.v.), CDP-choline is rapidly hydrolyzed to choline and cytidine [19, 20]. These final metabolites increase in the circulation, cross blood–brain barrier, are taken up by the cells/neurons and mediate re-synthesis of the molecule in the cell [19]. We have been investigating its cholinergic nature and usefulness in several shock conditions for almost a decade. Our studies have shown that CDP-choline can exert significant hemodynamic and endocrine effects in normal and stimulated situations [21–25]. It increases blood pressure in normal conditions [25], restores hypotension and increases survival in hemorrhagic shock [22]. The activation of central cholinergic receptors through the increase in brain choline levels mediates these effects [22]. We also reported that CDP-choline is able to decrease neuronal injury in spinal-cord transected rats by limiting oxidative injury [26]. More recently we demonstrated that intravenously given CDP-choline exerts strong protection against cardiac arrhythmias and lowers mortality rates in short-term myocardial ischemia reperfusion (I/R) in rats by activating efferent vagal pathways followed by increased brainstem cholinergic transmission through the activation of central muscarinic receptors [27]. Taking these data into consideration together with the importance of cell loss in the myocardial infarction, we suggested that CDP-choline may protect myocardial tissue from the injury induced by long-term I/R. The present work aimed to investigate this hypothesis by determining the effect of intravenously injected CDP-choline on the
123
myocardial infarct size and apoptosis in I/R conditions. We recorded blood pressure and heart rate of animals throughout the experiments to determine if hemodynamic effects of CDP-choline are involved in its tissue protective effect. Moreover, plasma concentrations of asymmetrical dimethylarginine (ADMA), a circulating endogenous inhibitor of NO synthase, were determined since the elevations of plasma ADMA levels have been reported to be a predictive value for the worse clinical outcome of patients undergoing percutaneous coronary intervention [28].
Materials and methods Animals Adult male Wistar albino rats (250–300 g) were used throughout the study. The animals were obtained from Experimental Animals Breeding and Research Center, Uludag University, Bursa, Turkey. They were housed five per cage in a temperature (20–24 °C) and humidity (60–70 %) controlled room set to a 12-h light: 12-h dark cycle and had access to standard rat chow and water ad libitum. All procedures were approved by the Animal Care and Use Committee of Uludag University and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (http://oacu.od. nih.gov/regs/guide/guide.pdf). Surgical procedures Under urethane (1.25 g/kg intraperitoneally; Fluka; Buchs, Switzerland) anesthesia, the left common carotid artery for monitoring mean arterial pressure and the left jugular vein for drug administration were cannulated with PE 50 tubing filled with heparinized saline (100 U/ml) and the trachea of rats was catheterized with tracheal cannula. After intubation of the trachea, the animals were ventilated with room air via a respirator for small rodents (CWE model SAR830/AP, PA, USA) with a stroke volume of approximately 20 ml/kg and a rate of 70 strokes per minute. Following cannulation process, rats were placed in the supine position on a heated operating platform to maintain a rectal temperature of 37 ± 0.2 °C. Temperature of animals was monitored using a rectal probe throughout the study. For mean arterial pressure and heart rate monitoring, the arterial cannula was connected to a volumetric pressure transducer (BPT 300), which was attached to a DA100B general purpose transducer amplifier (Commat, Ankara, Turkey). Mean arterial pressures of rats were recorded and analyzed using the MP100 system and AcqKnowledge software (BIOPAC Systems, CA, USA). Mean arterial pressure is reported as mean arterial pressure (MAP;
Ir J Med Sci
mmHg), and heart rate is expressed as beats per minute (HR; bpm). Needle electrodes were placed subcutaneously on the limbs and an electrocardiogram (ECG) was continuously recorded. The chest was then opened by left thoracotomy, the pericardium was incised, the heart was exteriorized, and a loose loop (6/0 braided silk suture attached to a 10-mm micropoint reverse cutting needle) was placed around the left main coronary artery (LCA). The heart was replaced in the chest cavity with the ligature ends exteriorized, and any animal in which this procedure produced dysrhythmias or a sustained fall in mean arterial pressure to \60 mmHg was withdrawn from the study at this point. Ischemia/reperfusion procedure After an equilibration period of 20 min, the ligature was tied to start the ischemia; after a 30-min period of coronary occlusion, reperfusion was obtained by loosening the suture, and the animals were then monitored for a further 180 min. The MAP, HR, occurrence of dysrhythmias, and lethality were recorded. Each rat underwent a single coronary occlusion. Rats subjected to sham ischemia underwent all surgical procedures experienced by the ischemic rats, except coronary ligation. Saline (1.0 ml/kg) or CDPcholine (100, 250, and 500 mg/kg) were intravenously injected in the middle of the ischemia period (15th min of ischemia). Reperfusion period lasted 180 min. Evaluation of myocardial infarct size At the end of experiments, the heart was removed after thoracotomy and was immediately put in a closed heart chamber and antegradely perfused with isotonic saline at a constant flow of 10 ml/min to remove blood in the heart. To demarcate the area at risk, the left main coronary artery was re-occluded and 1 ml of acridine orange stain (1 mg/ ml in saline) (Sigma-Aldrich Chemie GmbH, Germany) was infused for 1 min via the aorta. At the end of the staining, the heart was immediately frozen at -30 °C for 10 min and then sliced into 2-mm thick slices perpendicular to the left main coronary artery up to the area of ligation. In order to delineate the risk area, which was perfused by the left main coronary artery, the slices were examined using a fluorescence microscope (Olympus BX 50, Japan). The areas, which are not stained by acridine orange, were described as the area at risk of the heart. To examine the myocardial necrotic area, the heart slices were each incubated for 30 min at 37 °C in 10 ml of 1 % 2,3,5-triphenyltetrazolium chloride (TTC) solution (pH 7.4). Sections were fixed overnight in 4 % paraformaldehyde for contrast enhancement between the stained and unstained tissues. Viable myocardium is stained red by
TTC while the necrotic, infarcted area remains unstained. The sections were then placed between two coverslips and digitally photographed using a fluorescence microscope (Olympus BX 50, Japan). The calculation of the volumes of each slices and quantification of the area at risk and the area of necrosis were done with planimetric method using Scion software (Maryland, USA). The size of necrosis was calculated using ‘‘total area of necrosis/total area at risk 9 100’’ formula. Evaluation of myocardial apoptosis After I/R, the hearts were perfused first 0.9 % NaCl for 5 min and then with 4 % paraformaldehyde in PBS (pH 7.4) for 20 min. Four transverse sections (1 mm each) of left ventricle wall were cut and further fixed in 4 % paraformaldehyde in PBS overnight at room temperature. Fixed tissues were immersed in increasing concentrations of sucrose solutions and 15 serial cryosections (20 lm) in certain intervals were cut from each tissue block. Apoptotic cardiomyocytes were detected using terminal deoxynucleotidyl nick-end labeling (TUNEL) method. TUNEL staining was performed using an In situ Cell Death Detection Kit (Roche Molecular Biochemicals, USA) according to the protocol provided by the manufacturer. The digoxigenin-conjugated dUTP was incorporated to the ends of DNA fragments by terminal deoxynucleotidyl transferase (TdT). The cryosections were washed two times with PBS for 15 min and incubated in permeabilisation solution (0.1 % Triton X-100, 0.2 % sodium citrate, freshly prepared) for 2 min on ice (2–8 °C). Then, the sections were incubated in TUNEL reaction mixture (terminal deoxynucleotidyl transferase from thymus EC 2.7.7.31, recombinant in E. coli and label solution) for 60 min at 37 °C in a humidified atmosphere in the dark and rinsed three times with PBS for 10 min. The samples were analysed in a drop of PBS under a fluorescence microscope at this state (used an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nmgreen). The sections were incubated in Converter-POD (anti-fluorescein antibody, Fab fragment from sheep, conjugated with horse-radish peroxidase) for 10 min at room temperature, and then were rinsed three times with PBS and added DAB substrate on the sections for 10 min. After the DAB staining, slides were mounted under glass coverslip (with DPX) and analysed under photomicroscope (BX-50; Olympus). In the negative control experiment, sections were incubated in label solution (without terminal transferase) instead of TUNEL reaction mixture. Cardiomyocytes from four randomly selected slides per block were evaluated immunohistochemically to determine the number and percentage of cells exhibiting positive staining for apoptosis. For each slide, five microscopic fields were
123
Ir J Med Sci 50
Infarct Size (IA/RAx100)
randomly chosen. In each field, cells were calculated and the apoptotic cardiomyocytes counted (209 objective). To determine the total number of cardiomyocytes in these areas, serial sections were stained with H&E and total cardiomyocytes were counted in the same fields. The apoptotic index was determined (i.e., number of positively stained apoptotic myocytes/total number of myocytes counted 9 100 %). The assay was performed in a blinded manner.
40
30
*
*
*
100 mg/kg
250 mg/kg
500 mg/kg
20
10
Measurements At the end of each experiment, 2 ml of blood sample was collected through the arterial catheter (before the heart was removed) and their serum separated. Serum ADMA and serum M30/M65 levels were determined using commercially available ELISA kits which are manufactured by Immundiagnostic (Germany) and Peviva (Sweden), respectively. Serum lactate and homocysteine levels were studied using a DPC brand ‘‘Immulate 2000 Lactate and Homocysteine’’ kit on the Immulate 2000 apparatus (DPC, Los Angeles, CA, USA) using the immunoassay method. Drugs CDP-choline was purchased from Sigma-Aldrich Chemie GmbH (Germany). It was freshly dissolved in saline (0.9 % NaCl). Intravenous injection volume was 1.0 ml/kg. Each dose of CDP-choline was dissolved in one ml of saline solution (100, 250 or 500 mg/ml) and injected according to the animal’s body weight (100, 250 or 500 mg/ml/kg) in 30 s. Data and statistical analysis Results are expressed as mean ± SEM. Repeated measures analysis of variance (RM-ANOVA; two-way) and analysis of variance (ANOVA; one way) were performed for mean arterial pressure and heart rate results, and the area at risk and the area of necrosis, M30, M65, homocysteine and ADMA results. Tukey test was applied as a post hoc test when significant interactions were found. Blood lactate results were evaluated using Wilcoxon signed-rank test. A p value of \0.05 was considered significant.
Results Effect on myocardial infarct size The myocardial infarct size was determined by the ratio of necrotic or infarct area (IA) to the area at risk (RA) and expressed as percentage (i.e., IA/RA 9 100). Thirty
123
0
Saline
CDP-choline Fig. 1 Effect of CDP-choline on myocardial infarct size. Rats were subjected 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, hearts were removed and stained with acridine orange and TTC as described in ‘‘Materials and methods’’. The results are expressed as the percentage of the infarct area to the risk area. Values are mean ± SEM of 6–8 animals. Statistical analysis was performed using one-way ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from the saline-treated group
minutes’ coronary occlusion followed by the 3 h of reperfusion seriously injured the myocardial tissue perfused by left main coronary artery (risk area). Intravenous injection of CDP-choline attenuated the myocardial tissue injury at all doses compared with those observed in salinetreated rats (Fig. 1). The myocardial infarct size was 41 ± 3 % in saline group while it was 26.6 ± 3, 26.8 ± 4, 27.0 ± 3 % in 100, 250, 500 mg/kg CDP-choline groups, respectively (Fig. 1). Analysis of variance confirmed that all doses of CDP-choline significantly reduced the myocardial infarct size in similar way [f(3,21) = 5.079; p \ 0.001] (Fig. 1). Histopathological evaluation by H&E staining demonstrated that the injured area was much smaller in CDP-choline-treated (100 mg/kg; i.v.) animals than those observed in saline-treated rats (Fig. 2, top). Effect on apoptosis Since apoptosis may contribute significantly to the I/Rinduced myocardial tissue injury [3, 8] and CDP-choline has been shown to reduce apoptosis through the inhibition of caspase-3 activation in cerebral ischemic situation [18], we investigated if CDP-choline-treatment alters the apoptotic activation in these conditions. TUNEL assay was used for determination of the apoptotic activity in myocardial tissue. Quantitative analysis of DNA fragmentation was performed using the TUNEL method. TUNEL-positive cardiomyocyte nuclei are shown in Figs. 2, 3 and 4. We
Ir J Med Sci
Fig. 2 Effect of CDP-choline on histopathological changes and apoptotic activation induced by myocardial I/R injury. CDP-choline (100 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. Sham-operated animals underwent the same surgical procedures but without ligation of the LCA suture. Hematoxylin & Eosin staining indicates ischemic and normal areas at
myocardium (top). TUNEL-positive nuclei are shown as brown nuclei (bottom). CDP-choline significantly reduced the apoptotic activation and the number of TUNEL-positive myocytes versus the saline group after ischemia-reperfusion. Figures are representative of five separate experiments
observed significant number of apoptotic cell nuclei in ischemic/reperfused ventricles in saline-treated rats while a few apoptotic cell nuclei was detected in similar areas after CDP-choline (100 mg/kg, i.v.) administration (Figs. 2, 3 and 4). There is no TUNEL-positive cardiomyocyte in sham-operated control group (Fig. 2, left sections middle). The apoptotic index was reduced by almost 50 % (p \ 0.05) in CDP-choline-administrated group compared with saline-treated group (12.35 ± 0.7 versus 22.47 ± 3.3 %; CDP-choline versus saline group, respectively) (Fig. 5). We also measured plasma M30 and M65 levels at the end of the experiments to see if these levels can support the tissue findings. M30 levels help us to predict the apoptotic cell death by measuring caspase-cleaved CK18 produced during apoptosis and M65 measures the levels of both caspase-cleaved (apoptosis) and intact CK18 (necrosis). Plasma M30 and M65 levels did not significantly differ in saline or CDP-choline treated groups compared to values observed in sham-operated rats (Fig. 6).
(NO) synthase, were determined since the elevations of plasma ADMA levels have been reported to be a predictive value for the worse clinical outcome of patients undergoing percutaneous coronary intervention [28]. Plasma ADMA levels significantly increased after 30 min of coronary occlusion followed by 3 h reperfusion period (Fig. 7). CDP-choline treatment, at all doses, did not significantly alter the increased ADMA concentrations in myocardial I/R (Fig. 6). Although there was a tendency to decrease in ADMA levels in 100 mg/kg dose of CDP-choline-treated rats, these levels did not significantly different from those observed either saline-treated group or sham control animals (Fig. 7). Neither I/R conditions nor CDP-choline treatment affected plasma homocysteine concentrations (Table 1). Plasma lactate levels did not change in CDPcholine treated rats while the levels increased significantly after I/R itself (Table 1).
Effect on blood ADMA, homocysteine and lactate concentrations Plasma concentrations of asymmetrical dimethylarginine (ADMA), a circulating endogenous inhibitor of nitric oxide
Effect on blood pressure and heart rate After an equilibration period of 20 min following suture placing around the left main coronary artery, any animal in which this procedure produced dysrhythmias, such as ventricular tachycardia or ventricular fibrillation, or a sustained fall in mean arterial pressure to \60 mmHg was withdrawn from the study at this point. During the
123
Ir J Med Sci
Fig. 3 TUNEL assay in myocardial I/R. TUNEL assay was described in ‘‘Materials and methods’’. Figures are representative of five separate experiments. Sections were selected from apical region and same levels were used for both saline and CDP-choline-treated group.
Figure shows the apoptotic cells using TUNEL assay in fluorescence microscopy after ischemia-reperfusion. In TUNEL staining, sections were incubated with anti-BrdU-FITC antibody
Fig. 4 TUNEL immunostaining in myocardial I/R. Sections were selected same levels for sham, saline and CDP-choline treated groups. Figures demonstrate the apoptotic cells using TUNEL immunostaining method in light microscopy. Arrows show the TUNEL-positive myocytes
equilibration period, the mean arterial pressure and heart rate of animals were 77 ± 3 mmHg and 321 ± 15 BPM, respectively (Fig. 8). While blood pressure of rats decreased 8–10 mmHg with starting of ischemia, heart rate of animals did not change (Fig. 8). Intravenous injection of CDP-choline (100, 250, and 500 mg/kg) in the middle of
123
the ischemic period caused a prompt increase in arterial pressure (Fig. 8). Pressor effect was transient at each doses of the drug. Blood pressure of rats returned to pre-treatment levels within 15 min after CDP-choline (Fig. 8, top). CDPcholine (100, 250, and 500 mg/kg) did not alter the heart rate of the animals (Fig. 8, bottom).
Ir J Med Sci 30
A 1000
20 15
*
10 5 0 Saline
CDP-choline
Serum M30 Levels (U/L)
Apoptotic Activity (%)
25
Fig. 5 Effect of CDP-choline on apoptotic activity induced by myocardial I/R injury. The apoptotic activity was determined by the ratio of the number of TUNEL-positive cardiomyocytes to the total number of myocytes and expressed as the percentage. Bars represent the mean ± SEM of the apoptotic activity calculated from four tissue blocks of five rats for each experimental group (i.e., saline or CDPcholine 100 mg/kg). Statistical analysis was performed using one-way ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from the saline-treated group
The present data show that intravenously injected CDPcholine is able to decrease the myocardial infarct size caused by myocardial I/R. According to our TUNEL assay result, the drug strongly inhibited myocardial apoptosis induced by long-term myocardial I/R in rats. CDP-choline caused prompt but transient increase in blood pressure. Ischemia reperfusion itself increased blood ADMA levels significantly without changing M30/M65 and homocysteine levels. CDP-choline failed to affect any of these parameters in this condition. In the present study, 30-min occlusion of the LCA followed by a 3-h reperfusion seriously injured myocardial tissue. Infarct size, which was used as an index of myocardial injury, was evaluated as the ratio of necrotic (or infarct) area to determined risk area. All three doses of CDP-choline treatment exerted similar degree of protection. The doses of CDP-choline were chosen based on our previous observations in which the drug produced significant cardiovascular [22], endocrine [23, 24], and protective effects [26, 27] in several experimental conditions including shock. Our recent paper [27] which contains the most related data to this study reported that similar doses of i.v. injected CDP-choline was able to reduce the incidence of arrhythmias and mortality rate of rats in short-term myocardial I/R. In that study 250 mg/kg CDP-choline was as effective as lidocaine, which was used as a positive reference for an antiarrhythmic effect during I/R. We would like to mention that the present study was performed as an extension of that previous one, since we also wanted to investigate the tissue protective effect of the drug in similar
800
600
400
200
0
B 500
Serum M65 Levels (U/L)
Discussion
Sham Saline (ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
400
300
200
100
0
Fig. 6 Effect of CDP-choline on plasma M30 and M65 levels in myocardial I/R. Rats were subjected 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, blood samples of animals were taken from their arterial catheters and plasma M30 and M65 levels were measured using ELISA kit. Data are presented as the mean ± SEM of 5–7 animals. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test. No statistically significant interactions were found
cardiogenic shock situation. However, we preferred to apply the long-term myocardial I/R because the tissue injury cannot be observed in such a short period of I/R study. Although there is considerable evidence showing the tissue protective effect of CDP-choline in several cerebral ischemic conditions [12], head trauma [13], and spinal injury [26], this is the first study demonstrating the protective effect of CDP-choline in myocardial tissue injured by the 30 min of ischemia followed by 3 h reperfusion conditions. Studies investigating the mechanisms of the druginduced tissue protection in several ischemic conditions suggested that CDP-choline can decrease membrane-
123
Ir J Med Sci
Serum ADMA Levels (pg/ml)
4
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
3 *
*
*
2
1
0
Fig. 7 Effect of CDP-choline on plasma ADMA levels in myocardial I/R. Rats were subjected 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, blood samples of animals were taken from their arterial catheters and plasma ADMA levels were measured using ELISA kit. Data are presented as the mean ± SEM of 5–7 animals. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from sham control group. There is no any significance in CDP-choline treated groups compared with values observed in saline-treated rats
phospholipid breakdown/restore phosphatidylcholine levels by attenuating phospholipase A stimulation and loss of CTP: phosphocholine cytidylyltransferase activity and it can stabilize the membrane integrity [12, 13, 29]. The drug also has antioxidant effect by decreasing lipid peroxidation; therefore, some of its beneficial effects can be mediated by the decrease of oxidative stress induced by head trauma [30] or spinal injury [26]. Furthermore, we showed that CDP-choline could be considered as a cholinergic agent because it increases central cholinergic transmission when it is administered either centrally or peripherally [21– 25]. Our recent report not only supported these previous findings but also implicated that the activation of cholinergic anti-inflammatory pathway is involved in the protective effect of drug in short-term myocardial I/R study in rats [27]. According to these previous data we can suggest that CDP-choline can attenuate the myocardial injury by activating cholinergic anti-inflammatory pathway, by
attenuating oxidative stress and by decreasing membrane breakdown in the present conditions. Several studies show that in addition to necrosis, apoptosis may also contribute significantly to myocardial ischemic injury and cell death [3, 31]. Although apoptosis can be detected in either permanently ischemic or reperfused myocardium, the onset of apoptosis is accelerated by reperfusion possibly as a result of the reperfusion-associated inflammatory response [32, 33]. Apoptotic cell death is mediated by activation of caspases. CDP-choline has been shown to attenuate expression of pro-caspases, cleaved caspase-3, and nuclear DNA fragmentation after focal cerebral ischemia [18, 34]. Hence we wanted to investigate if the inhibition of apoptotic cell death contributes to the CDP-choline-induced myocardial protection. In order to accomplish this, we both performed immunohistochemistry to myocardial tissue by using TUNEL assay and also measured serum levels of full-length and caspasecleaved cytokeratin 18 (CK18), which are considered biomarker of apoptotic and necrotic cell death, using a combination of the M30 and M65 ELISAs. Although the levels of M30 and M65 tended to increase in I/R group they were not statistically different from those observed in sham control group. Also CDP-choline treatment did not change M30 and M65 levels compared with saline group. We did not use the ‘‘M30/M65 ratio’’ to interpret the death situations since we could not use any positive references for apoptosis or necrosis in our experimental conditions. On the other hand, our findings from TUNEL assay demonstrated that CDP-choline (100 mg/kg; i.v. injected 15 min after the onset of ischemia) significantly decreases the number of apoptotic cells and apoptotic activity in ischemic area (Figs. 2, 3). According to these immunohistochemistry data we can suggest that the inhibition of apoptosis can mediate the tissue protective effect of CDPcholine in myocardial I/R. However, the observation that there was no difference in M30 and M65 levels in both the control I/R animals and the drug-treated groups can be explained by the short time period of reperfusion (i.e., 3 h) which cannot be enough to determine the circulatory
Table 1 Effects of CDP-choline on serum plasma homocysteine and lactate levels Sham
Saline (ml/kg)
CDP-choline 100 (mg/kg)
250 (mg/kg)
500 (mg/kg)
Homocysteine (lmol/l)
6.2 ± 0.4
6.3 ± 1.3
6.3 ± 1.1
7.4 ± 0.7
6.7 ± 0.9
Lactate (mmol/l)
2.3 ± 0.3
3.8 ± 0.3*
3.6 ± 0.5*
3.8 ± 0.9*
4.1 ± 0.8*
Rats were subjected to 30 min of ischemia and 3 h of reperfusion. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. At the end of the experiment, blood samples of animals were taken from their arterial catheters and plasma homocysteine and lactate levels were measured. Data are presented as the mean ± SEM of 5–7 animals. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test * p \ 0.05, significantly different from sham control group. There was no significant difference between CDP-choline and saline treated rats
123
Ir J Med Sci
A
Mean Arterial Pressure (mm Hg)
140 Treatment 120
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
*
100
*
80
60 Ischemia
Reperfusion
40 0
15 30 45 60 75 90 105 120 135 150 165 180 195 210 225
Time (minutes)
B 700
Heart Rate (beats/minute)
Treatment 600
Sham Saline (1 ml/kg) CDP-choline (100 mg/kg) CDP-choline (250 mg/kg) CDP-choline (500 mg/kg)
500
400
300 Ischemia
Reperfusion
200 0
15 30 45 60 75 90 105 120 135 150 165 180 195 210 225
Time (minutes)
Fig. 8 Effects of CDP-choline on mean arterial pressure and heart rate in the myocardial I/R. Rats were subjected to myocardial ischemia for 30 min and reperfusion for 3 h. CDP-choline (100, 250, and 500 mg/kg; i.v.) or saline (1 ml/kg; i.v.) was injected in the middle of the ischemic period. Arrows in the figures represent the treatment time point. Mean arterial pressure (a) and heart rates (b) of the animals were monitored during both ischemia/reperfusion periods. Data are presented as the mean ± SEM in mean arterial pressure and beats per minute in heart rate. Statistical analysis was performed using two-way RM-ANOVA with a post hoc Tukey test. *p \ 0.05, significantly different from its own previous value
changes reflecting the myocardial apoptosis. One of the recent articles supports our hypothesis by reporting that M30 levels peaked at 24 h in patients with acute coronary syndrome [35]. In the present study, we also determine whether plasma ADMA levels can increase in our experimental conditions and whether these levels can be affected by CDP-choline treatment. It has been known that ADMA is a circulating endogenous inhibitor of NO synthase [36, 37] and it can increase oxidative stress by uncoupling of electron transport between NO synthase and L-arginine
[36]. Elevated ADMA levels have been observed in patients with various risk factors of coronary artery disease and have been suggested to be an independent predictor of subsequent major adverse cardiovascular events after percutaneous coronary intervention [28, 37, 38]. We demonstrated that plasma ADMA levels significantly increased in myocardial I/R conditions. Showing the acute change of plasma ADMA levels in cardiogenic shock conditions is an interesting finding, because most of the literature reports the chronic or long-term changes of ADMA in plasma in several pathological situations [28, 37, 38]. On the other hand, we have another observation supporting this finding that plasma ADMA levels can acutely increase in hemorrhagic shock and immediately decrease after resuscitation with ringer lactate (unpublished data). Although the ADMA levels in 100 mg/kg CDP-choline given group were neither significantly higher than those observed in sham animals nor were lower than saline group, CDP-choline treatment failed to affect ADMA levels significantly (Fig. 6). These data suggest that ADMA may contribute to the myocardial injury under these experimental conditions but it may not be involved in the protective effect of CDPcholine. We also measured plasma homocysteine levels regarding its promoting effect on lipid peroxidation through free radical formation and contribution to cardiovascular disease by causing endothelial dysfunction [38]. Our results showed that myocardial I/R itself or CDP-choline treatment did not affect plasma homocysteine levels. In agreement with our data, in a previously reported study, the authors did not show any association between homocysteine and ADMA or between homocysteine and adverse cardiovascular events after percutaneous coronary intervention [28]. The pressor effect of CDP-choline lasted for a short period of time and blood pressure returned to pretreatment levels before the reperfusion; thus it cannot be responsible for the drug’s protective effect. This finding is in good accordance with those obtained from our previous studies in which the pressor effect was not suggested to mediate the cardio-protective and tissue protective effect of CDPcholine in short-term myocardial I/R injury [27] and spinal shock [26]. In conclusion, the present study shows that intravenously injected CDP-choline is able to protect tissue injury induced by long-term myocardial I/R in rats. The inhibition of apoptotic cell death by CDP-choline may mediate the drug’s protective effect. Acknowledgments This study was supported by The Commission of Scientific Research Projects of Uludag University, No. T-2008/43. Conflict of interest of interest.
The authors declare that there are no conflicts
123
Ir J Med Sci
References 1. Mathers CD, Boerma T, Ma Fat D (2009) Global and regional causes of death. Br Med Bull 92:7–32 2. WHO Fact Sheet (2011) Top ten causes of dead in The World 2008. 310 3. Fliss H, Gattinger D (1996) Apoptosis in ischemic and reperfused rat myocardium. Circ Res 79:949–956 4. Tanaka M, Ito H, Adachi S et al (1994) Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res 75:426–433 5. James TN (1994) Normal and abnormal consequences of apoptosis in the human heart from postnatal morphogenesis to paroxysmal arrhythmias. Circulation 90:556–573 6. Gottlieb RA, Burleson KO, Kloner RA et al (1994) Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621–1628 7. Saraste A, Pulkki K, Kallajoki M et al (1997) Apoptosis in human acute myocardial infarction. Circulation 95:320–323 8. Yaoita H, Ogawa K, Maehara K et al (2000) Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. Cardiovasc Res 45:630–641 9. Yaoita H, Ogawa K, Maehara K et al (1998) Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation 97:276–281 10. Weiss GB (1995) Metabolism and actions of CDP-choline as an endogenous compound and administered exogenously as citicoline. Life Sci 56:637–660 11. Secades JJ (2011) Citicoline: pharmacological and clinical review, 2010 update. Rev Neurol 52:1–62 12. Adibhatla RM, Hatcher JF (2002) Citicoline mechanisms and clinical efficacy in cerebral ischemia. J Neurosci Res 70:133–139 13. Adibhatla RM, Hatcher JF (2005) Cytidine 50 -diphosphocholine (CDP-choline) in stroke and other CNS disorders. Neurochem Res 30:15–23 14. Dorman RV, Dabrowiecki Z, Horrocks LA (1983) Effects of CDP-choline and CDP-ethanolamine on the alterations in rat brain lipid metabolism induced by global ischemia. J Neurochem 40:276–279 15. Rao AM, Hatcher JF, Dempsey RJ (2001) Does CDP-choline modulate phospholipase activities after transient forebrain ischemia? Brain Res 893:268–272 16. Rao AM, Hatcher JF, Dempsey RJ (2000) Lipid alterations in transient forebrain ischemia: possible new mechanisms of CDPcholine neuroprotection. J Neurochem 75:2528–2535 17. Adibhatla RM, Hatcher JF, Dempsey RJ (2001) Effects of citicoline on phospholipid and glutathione levels in transient cerebral ischemia. Stroke 32:2376–2381 18. Fiedorowicz M, Makarewicz D, Stan´czak-Mrozek KI et al (2008) CDP-choline (citicoline) attenuates brain damage in a rat model of birth asphyxia. Acta Neurobiol Exp 68:389–397 19. Savci V, Wurtman RJ (1995) Effect of cytidine on membrane phospholipid synthesis in rat striatal slices. J Neurochem 64:378–384 20. G-Coviella IL, Wurtman RJ (1992) Enhancement by cytidine of membrane phospholipids synthesis. J Neurochem 59:338–343 21. Savci V, Goktalay G, Ulus IH (2002) Intracerebroventricular choline increases plasma vasopressin and augments plasma vasopressin response to osmotic stimulation and hemorrhage. Brain Res 942:58–70
123
22. Savci V, Goktalay G, Cansev M et al (2003) Intravenously injected CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock: effect is mediated by central cholinergic activation. Eur J Pharmacol 468:129–139 23. Cavun S, Savci V, Ulus IH (2004) Centrally injected CDP-choline increases plasma vasopressin levels by central cholinergic activation. Fundam Clin Pharmacol 18:71–77 24. Cavun S, Savci V (2004) CDP-choline increases plasma ACTH and potentiates the stimulated release of GH, TSH and LH: the cholinergic involvement. Fundam Clin Pharmacol 18:513–523 25. Savci V, Cavun S, Goktalay G et al (2002) Cardiovascular effects of intracerebroventricularly injected CDP-choline in normotensive and hypotensive animals: the involvement of cholinergic system. Naunyn Schmiedebergs Arch Pharmacol 365:388–398 26. Coskun C, Avci B, Ocak N et al (2010) Effect of repeatedly given CDP-choline on cardiovascular and tissue injury in spinal shock conditions: investigation of the acute phase. J Pharm Pharmacol 62:497–506 27. Yilmaz MS, Coskun C, Yalcın M et al (2008) CDP-choline prevents cardiac arrhythmias and lethality induced by short-term myocardial ischemia-reperfusion injury in the rat: involvement of central muscarinic cholinergic mechanisms. Naunyn Schmiedebergs Arch Pharmacol 378:293–301 28. Lu TM, Ding YA, Lin SJ et al (2003) Plasma levels of asymmetrical dimethylarginine and adverse cardiovascular events after percutaneous coronary intervention. Eur Heart J 24:1912–1919 29. Adibhatla RM, Hatcher JF, Larsen EC et al (2006) CDP-choline significantly restores phosphatidylcholine levels by differentially affecting phospholipase A2 and CTP: phosphocholine cytidylyltransferase after stroke. J Biol Chem 281:6718–6725 30. Adibhatla RM, Hatcher JF, Dempsey RJ (2002) Citicoline: neuroprotective mechanisms in cerebral ischemia. J Neurochem 80:12–23 31. Moens AL, Claeys MJ, Timmermans JP et al (2005) Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int J Cardiol 100:179–190 32. Bazzani C, Guarini S, Botticelli AR et al (2001) Protective effect of melanocortin peptides in rat myocardial ischemia. J Pharmacol Exp Ther 297:1082–1087 33. Lee Y, Gustafsson AB (2009) Role of apoptosis in cardiovascular disease. Apoptosis 14:536–548 34. Krupinski J, Ferrer I, Barrachina M et al (2002) CDP-choline reduces pro-caspase and cleaved caspase-3 expression, nuclear DNA fragmentation, and specific PARP-cleaved products of caspase activation following middle cerebral artery occlusion in the rat. Neuropharmacology 42:846–854 35. Senturk T, Aydinlar A, Yilmaz Y et al (2009) Serial changes in circulating M30 antigen, a biomarker of apoptosis, in patients with acute coronary syndromes: relationship with the severity of coronary artery disease. Coron Artery Dis 20:494–498 36. Visser M, Paulus WJ, Vermeulen MA et al (2010) The role of asymmetric dimethylarginine and arginine in the failing heart and its vasculature. Eur J Heart Fail 12:1274–1281 37. Endemann DH, Schiffrin E (2004) Endothelial dysfunction. J Am Soc Nephrol 15:1983–1992 38. Bo¨ger RH (2005) Asymmetric dimethylarginine (ADMA) and cardiovascular disease: insights from prospective clinical trials. Vasc Med 10:19–25
