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Ann Thorac Surg 2001;72:555-563
© 2001 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA
Accepted for publication March 24, 2001.
Address reprint requests to Dr McCully, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 77 Ave Louis Pasteur, Room 144, Boston, MA 02115
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Abstract |
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Methods. The effects of adenosine-enhanced ischemic preconditioning on necrosis and apoptosis were investigated in the sheep heart using models of stunning (15 minutes regional ischemia, 120 minutes reperfusion) and ischemia–reperfusion (30 and 60 minutes regional ischemia, 120 minutes reperfusion). Ischemic preconditioned hearts received 5 minutes regional ischemia, 5 minutes reperfusion before ischemia. Adenosine-enhanced ischemic preconditioned hearts received a 10 mmol/L adenosine bolus (10 mL) through the left atrium coincident with ischemic preconditioning. Adenosine hearts received a 10 mmol/L bolus (10 mL) of adenosine. Regional ischemic hearts received no pretreatment.
Results. Minimal apoptosis (< 45 per 3,000 myocytes) was observed in the stunning models but was significantly increased with ischemia–reperfusion in regional ischemic hearts after 30 minutes (p < 0.05 versus ischemic preconditioning, adenosine, or adenosine-enhanced ischemic preconditioning) and in adenosine and ischemic preconditioned hearts after 60 minutes ischemia (p < 0.05 versus adenosine-enhanced ischemic preconditioning). DNA laddering was apparent after 60 minutes ischemia in regional ischemia, adenosine, and ischemic preconditioning but not in adenosine-enhanced ischemic preconditioned hearts.
Conclusions. Adenosine-enhanced ischemic preconditioning significantly ameliorates necrosis and apoptosis in the regional ischemic blood-perfused heart.
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Introduction |
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Recently, we have investigated the mechanisms modulating APC cardioprotection, and we have shown that APC has both antiinfarct and antistunning effects in the ovine heart [5]. Our results indicated that APC-enhanced infarct size reduction occurred through the significant extension of antiinfarct effects, which were maintained for at least 60 minutes of regional ischemia [5].
Myocardial cellular injury can occur by at least two pathways, necrosis and apoptosis [6–9]. In the quiescent cardiac myocyte, both necrosis and apoptosis contribute to the reduction of contractile units and ultimately compromise myocardial function, dependent on severity and localization of the injury. Thus the development of strategies for the amelioration or prevention of myocardial ischemia–reperfusion injury resulting from either necrosis or apoptosis is central to development of cardioprotective protocols.
In this report we investigate the effect of APC on necrosis and apoptosis with direct comparison with IPC using a model of stunning and ischemia–reperfusion in the blood-perfused heart model.
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Material and methods |
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Surgical preparation
Dorset or Suffolk sheep of either sex (35 to 45 kg, n = 72) were sedated with ketamine hydrochloride (20 mg/kg, intramuscularly, Abbott Laboratories, North Chicago, IL) and anesthetized with sodium pentobarbital (25 mg/kg, intravenously, Abbott Laboratories). General anesthesia was maintained throughout the experiment with sodium pentobarbital. A tracheotomy was performed through a midline cervical incision (36F; Argyle-Sherwood Medical Co, St. Louis, MO), and ventilation was begun with a volume-cycled ventilator (model Narkomed II, North American Drager, Telford, PA; oxygen, 40%; tidal volume, 1,000 mL; ventilation rate, 12 breaths/min; positive end-expiratory pressure, 3 cm H2O; inspiratory-to-expiratory time ratio, 1:2). The right internal jugular vein was cannulated for intravenous access, and the right common carotid artery was cannulated for arterial blood sampling and intraarterial blood pressure monitoring (Millar Instruments, Houston, TX). Heparin sodium (Elkins-Sinn, Inc, Cherry Hill, NJ; 5000 IU intravenously) and 1% lidocaine (Elkins-Sinn, Inc; 5 mL intravenously) were given before thoracotomy. Heparin was administered at the same dose every 30 minutes to the end of the experiment. The pericardial sac was exposed through a median sternotomy and was opened to form a pericardial cradle. A catheter-tipped manometer (Millar Instruments) was introduced through the apex into the left ventricle (LV) to record LV pressure. A silk thread (0 silk, K834H, Ethicon, Inc, Somerville, NJ) was passed around the distal third of the left anterior descending coronary artery or its large diagonal branch with a taper needle, and both ends of the silk tie were threaded through a small vinyl tube to form a snare. The coronary artery was occluded by pulling the snare, which was then secured by clamping the tube with a mosquito clamp. Myocardial ischemia was confirmed visually by regional cyanosis of the myocardial surface [4].
Experimental protocol
Sheep were randomly divided and subjected to 15, 30, or 60 minutes of regional ischemia followed by 120 minutes of reperfusion. Fifteen minutes of regional ischemia and 120 minutes of reperfusion was used for investigation of the effects of stunning on the basis of previous results [5]. This regional ischemic time was chosen to allow for compliance with the strict definition of myocardial stunning in which mechanical dysfunction persists after reperfusion despite the absence of irreversible myocardial damage as indicated by infarct [10, 11]. IPC hearts (n = 18; 6 each time point) received a 10-mL saline bolus injection (vehicle control) at the immediate start of IPC, coincident with the tightening of the snare (5 minutes of zero flow regional ischemia followed by 5 minutes of reperfusion) before regional ischemia. APC hearts (n = 18; 6 each time point) received a 10-mL bolus injection of 10 mmol/L adenosine (Adenoscan; Medico, Inc, Research Triangle Park, NC) at the immediate start of IPC, coincident with the tightening of the snare (5 minutes of zero flow regional ischemia followed by 5 minutes of reperfusion) before regional ischemic. To separate the effects of adenosine from those of APC, adenosine only hearts (ADO; n = 18; 6 each time point) received a 10-mL bolus injection of 10 mmol/L adenosine 10 minutes before regional ischemia. Regional ischemic hearts (n = 18; 6 each time point) received no pretreatment. The bolus was injected into the left atrial appendage using a 19-gauge needle. Hemodynamic variables were continuously acquired throughout the experiment using a PO-NE-MAH digital data acquisition system (Gould, Valley View, OH), with an Acquire Plus processor board, LV pressure analysis software, and a Gould ECG/Biotach. Global hemodynamic measurements included heart rate, LV systolic pressure, LV end-diastolic pressure, LV peak developed pressure, the maximum positive value of the first derivative of the LV pressure, and mean arterial pressure [4].
Measurement of infarct size
The ischemic area at risk was delineated by monastral blue pigment injection into the aorta after ligation of the involved artery, at the end of the experiment. Infarct size was determined by triphenyl tetrazolium chloride staining (Sigma Chemical Co, St. Louis, MO), and was expressed as a percentage of the area at risk. The area at risk and the area of the infarcted zone were measured by computerized planimetry (Scion Image, Scion Corp, Frederick, MD) as previously described [4].
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, DNA isolation, and gel electrophoresis
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was performed using the ApopTag detection system (Intergen, Gaithersburg, MD). Myocardial tissue samples (approximately 3 mm x 5 mm each) from the area at risk (ischemic zone) and nonischemic area of LV (control zone) consisting of epicardial, myocardial, and endocardial tissue were removed at the end of each experiment and divided into two samples. Ischemic and nonischemic zone samples were confirmed by monastral blue pigment injection. The samples were snap-frozen, and one sample was sectioned (4 to 6 µm), mounted on glass slides, and used for in situ TUNEL. The remaining sample was used for DNA isolation and gel electrophoresis analysis. Both peroxidase and fluorescein staining were used for in situ TUNEL. Thirty to 35 slides were selected for each sample. Five to six slides from each sample were assayed using both TUNEL and propidium iodide staining. Indirect digoxigenin fluorescence labeling of DNA was visualized using an Axiovert 35 microscope (Zeiss, Oberkochen Germany) with propidium iodide and fluorescein isothiocyanate and dual propidium iodide/fluorescein isothiocyanate filters (Chroma Technologies, Battleboro, VT). Photomicrographs were taken in 10 to 15 random high-powered (20x) fields using a Zeiss MC80DX camera and exposure meter. Peroxidase TUNEL with methyl green counterstaining was performed on adjacent sequential serial slides (n = 5 to 6 for each sample) to allow for morphologic evaluation of specimens. All cells were counted on each slide, and TUNEL-positive cells were expressed per 3,000 myocardial cells [6]. Myocardial cell specificity was determined on opposite adjacent sequential serial slides (n = 5 to 6 for each sample) using the cardiac-specific monoclonal antibody for troponin I (Spectral Diagnostics Inc, Toronto, Ontario, Canada) labeled with antimouse IgG conjugated to Alexa 350 (Molecular Probes, Inc, Eugene, OR). Troponin I was visualized using a DAPI filter (Chroma Technologies). Only those cells that could be confirmed by both peroxidase staining and troponin I were classified as being of myocyte origin. Evaluation of TUNEL and myocyte morphology was performed by a blinded independent examiner. Control zone samples (nonischemic) were used to assess variability among individual animals. Mammary gland tissue obtained from rats at the fourth day of weaning was used as the positive control for TUNEL reactions (Intergen).
Confirmation of apoptosis was obtained by gel electrophoresis. DNA was isolated from the second frozen sample as previously described [12, 13]. DNA (5 µg) was fractionated on a 1.8% agarose gel in 40 mmol/L Tris-acetate and 2 mmol/L EDTA, using modified Tris acetate-EDTA loading buffer that excluded dye markers, and then stained with ethidium bromide [12].
Statistical analysis
Statistical analysis was performed using the SAS (version 6.12) software package (SAS Institute, Inc, Cary, NC). The mean ± standard error of the mean is shown for all variables. Statistical significance was determined by repeated measures analysis of variance with the group as a "between subjects" factor and time as a "within subjects" factor. Post hoc comparisons among groups for both the average effect and at individual times were made with the use of a Bonferroni correction to adjust for the multiplicity of tests. Statistical differences among groups in infarct size, TUNEL, and area at risk were evaluated by one-way analysis of variance. Linear regression analysis was performed to determine the relation between infarct size and TUNEL-positive nuclei per 3,000 myocytes in each group. Differences in regression lines among groups were compared using the general linear model. The general linear model was also used to test for significant nonlinear effects. Statistical significance was claimed at p less than 0.05.
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Results |
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Hemodynamics
No significant differences in heart rate, LV systolic pressure, LV end-diastolic pressure, LV peak developed pressure, the maximum positive value of the first derivative of the LV pressure, or mean arterial pressure were observed within or among groups before or during 15, 30, or 60 minutes of regional ischemia, or after 120 minutes of reperfusion (results not shown).
Area at risk and myocardial infarct size
No difference in area at risk was observed within or among groups. Infarct size expressed as a percentage of area at risk after 15, 30, and 60 minutes of regional ischemia and 120 minutes of reperfusion is shown for each group in Figure 1 (n = 6 for each group at each time). The was no observable infarct in any heart in any experimental group after 15 minutes of regional ischemia and 120 minutes of reperfusion. Infarct size after 30 minutes of regional ischemia and 120 minutes of reperfusion was 25.8% ± 5.7% in RI hearts, 12.9% ± 3.0% in ADO hearts (p < 0.05 versus RI), 11.6% ± 2.4% in IPC hearts (p < 0.05 versus RI), and 5.1% ± 1.6% in APC hearts (p < 0.05 versus RI, ADO, or IPC). Infarct size after 60 minutes of regional ischemia and 120 minutes of reperfusion was 49.8% ± 6.0% in RI hearts, 29.2% ± 5.0% in ADO hearts (p < 0.05 versus RI), 24.6% ± 2.7% in IPC hearts (p < 0.05 versus RI), and 12.4% ± 2.0% in APC hearts (p < 0.05 versus RI, ADO, or IPC). Infarct size was significantly increased (p < 0.05) in all groups after 60 minutes as compared with 30 minutes of regional ischemia and 120 minutes of reperfusion but was significantly decreased (p < 0.05) in APC hearts as compared with all other groups.
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Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling–positive cells per 3,000 myocytes were significantly increased to 711 ± 39/3,000 myocytes in RI; 829 ± 48/3,000 myocytes in ADO; 249 ± 39/3,000 myocytes in IPC, and 93 ± 17/3,000 myocytes in APC hearts after 60 minutes of regional ischemic and 120 minutes of reperfusion (p < 0.05 versus 30 minutes of RI). TUNEL–positive cells were significantly increased in IPC and ADO hearts as compared with APC hearts (p < 0.05). No TUNEL-positive cells were found in any control zone sample from any heart at any regional ischemic time (Figs 2, 3).
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Linear regression analysis was performed to determine the relation between infarct size and TUNEL-positive nuclei per 3,000 myocytes for each group. Linear regression analysis indicated that there was a linear effect between infarct size and TUNEL-positive nuclei per 3,000 myocytes in each group. The linear regression equations were y = 13.78x - 106.36 for RI hearts; y = 20.83x - 34.15 for ADO hearts; y = 7.19x - 13.26 for IPC hearts; and y = 6.09x - 5.97 for APC hearts. The correlation coefficient was 0.77 for RI, 0.63 for ADO, 0.81 for IPC, and 0.81 for APC. There was no evidence for a quadratic effect in any group.
DNA gel electrophoresis
Gel electrophoresis of ischemic and control zone DNA is shown in Figure 4. After 15 and 30 minutes of regional ischemia and 120 minutes of reperfusion, only minimal DNA fragmentation was observed in samples obtained from ischemic zones in RI, ADO, IPC, or APC hearts. However; after 60 minutes of regional ischemia and 120 minutes of reperfusion, DNA ladders of approximately 200, 400, 600, and 800 base pairs were observed in ischemic zone samples in RI, ADO, and IPC hearts. No oligomeric DNA pattern was observed in any DNA sample obtained from any ischemic zone sample in APC hearts or in any DNA sample obtained from any control zone sample from any experimental group. Radiolabeling of DNA using terminal deoxynucleotide transferase did not reveal any ladder formation in APC hearts (results not shown).
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Comment |
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Apoptosis is an evolutionarily conserved mode of cell death characterized by a discrete set of biochemical and morphologic events resulting in the ordered disassembly of the cell, distinct from cell death provoked by external injury [15]. Apoptosis is associated with cellular shrinkage, which may not be readily apparent, and is characterized by the fragmentation of nuclear DNA resulting in the generation of internucleosomal fragments (monomers and oligomers of 180 to 200 base pairs; DNA ladders), whereas DNA degradation in necrosis is random and nonspecific [6, 7, 9, 14].
DNA fragmentation can be detected using TUNEL. TUNEL is a sensitive but nonspecific methodology for the identification of apoptotic nuclei that uses the 3' hydroxyl termini present at DNA strand breaks for the identification of DNA degradation by enzymatic labeling of termini with modified nucleotides. The significance of TUNEL as an indicator of myocyte apoptosis has recently been challenged as terminal deoxynucleotide transferase allows for the labeling of both single-stranded (necrosis) and double-stranded (apoptosis) DNA breaks with free 3' hydroxyl termini. In a recent report Kanoh and associates [16] have shown that TUNEL-positive staining can occur in myocytes exhibiting neither apoptotic nor necrotic ultrastructure. These data suggest that TUNEL may not be a specific pathologic indicator and that further substantive analysis may be required for verification of apoptosis. To distinguish between apoptosis and necrosis we have used DNA gel electrophoresis, a specific but nonsensitive technique that allows for the visualization of DNA laddering, characteristic of apoptosis (for review see references 9, 15, 17).
It has been previously noted by others that to limit myocardial necrosis after ischemia, reperfusion must be reinstated, and that reperfusion itself contributes to cell death [18, 19]. The contributions of ischemia and ischemia–reperfusion to the mechanisms inducing apoptosis remain controversial as it has been shown that both ischemia and reperfusion and perhaps ischemia–reperfusion accelerate apoptosis [20–22].
In our experiments, we have used two clinically relevant models of stunning and ischemia–reperfusion injury. Sheep were chosen to elucidate the effects of APC as the ovine heart is known to be free of cardiac diseases, including hypertrophy, dilatation, fibrosis, parasites, cardiac storage diseases, atherosclerotic plaque, and infarction in species commercially available in the United States [23]. In addition the ovine model has limited native collateral coronary circulation to allow for amelioration of infarct size [24, 25].
In the in situ blood-perfused sheep heart, myocardial stunning was induced by 15 minutes of regional ischemia and 120 minutes of reperfusion, on the basis of preliminary experiments that indicated that this period decreased regional segmental shortening without inducing myocardial infarct as detected by triphenyl tetrazolium chloride staining [5]. Two hours of reperfusion was used based on previous reports in which we have investigated 2 hours as compared with 3 hours of reperfusion on infarct size as determined by triphenyl tetrazolium chloride staining and found no significant difference could be observed [1].
Our results indicate that using this model, less than 45 TUNEL-positive cells per 3,000 myocytes could be detected in the ischemic zone of the myocardium with no TUNEL-positive cells being evident in the nonischemic (control) zone. In addition we show that myocardial stunning does not induce DNA fragmentation or ladder formation in any experimental group in this model of stunning.
In our ischemia–reperfusion model in which regional ischemia times were extended to 30 and 60 minutes followed by 120 minutes of reperfusion, the number of TUNEL-positive cells per 3,000 myocytes was increased significantly (p < 0.05 versus 15 minutes of regional ischemia) in regional ischemic and ADO hearts. Ischemic preconditioning significantly decreased (p < 0.05 versus regional ischemia) the number of TUNEL-positive cells/3,000 myocytes after 30 and 60 minutes of regional ischemia and 120 minutes of reperfusion, in agreement with previous reports [19, 26, 27]. Only minimal DNA fragmentation was observed in samples obtained from ischemic zone in RI, ADO, IPC, or APC hearts. However, after 60 minutes of regional ischemia and 120 minutes of reperfusion, DNA fragmentation (observable as smearing) and oligonucleotide laddering consisting of approximately 200, 400, 600, and 800 base pairs were observed in ischemic zone samples in RI, ADO, and IPC hearts.
Of clinical significance our results indicate that APC significantly decreased the number of TUNEL-positive cells such that only 93 ± 17 per 3,000 myocytes were observed after 60 minutes of regional ischemia and 120 minutes of reperfusion (p < 0.05 versus IPC, ADO, or RI). No DNA fragmentation or oligonucleotide laddering was observable in any APC samples or in any samples from control (nonischemic) region in any experimental group.
Previous reports have indicated that oligonucleotide laddering is apparent 2 to 4 hours after reperfusion [19, 20]. Oligonucleotide laddering has been reported to occur from the initial cleavage of nuclear DNA into larger nucleosomal fragments (50 to 300 kb) before degradation into polynucleosomal (multimers of approximately 200 base pairs) [28]. In our experiments we have used 2 hours of reperfusion, after which oligonucleotide laddering was apparent in RI, ADO, and IPC samples but not in APC samples. To allow for verification of these results, APC samples were end-labeled with 
[32P]dATP using terminal deoxynucleotidyl transferase to allow for enhanced detection of any oligonucleotide ladders [29]. However, no oligonucleotide ladder formation was apparent in any APC or control sample using this method (results not shown).
Apoptosis and necrosis are two distinct mechanisms, that have been shown to independently contribute to cell death [30, 31]. Ohno and coworkers [20] have suggested that oncotic alterations may also occur with characteristics similar to that of apoptosis. Light microscopy of samples from ischemic and control (nonischemic) zones, however, did not reveal any ultrastructural differences, and there was no evidence in any experimental group of apoptotic bodies (Fig 5), which have been previously noted to occur in cells classified as apoptotic [18]. We were unable to determine the presence of oncosis in any of the samples using light microscopy; however, we can not rule out this process as a possible mechanism.
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It has also been suggested that the degree of apoptosis varies both within the ischemic region (central versus border zone) and within the myocardial sublayers in rats [26, 34]. In our investigations all ischemic samples were obtained within the ischemic zone and included tissue from all sublayers of the left ventricle. Triphenyl tetrazolium chloride analysis of the ischemic zone indicated diffuse necrosis with no discernible pattern of necrosis. Examination of TUNEL-positive photomicrographs also indicates no discernible pattern of apoptosis.
Previously, we have shown that the cardioprotection afforded by APC (significantly decreased infarct size and significantly enhanced postischemic functional recovery; p < 0.05 versus ADO or IPC) occurs by means of the additive actions of ADO and IPC, when used coincidentally, in both the isolated perfused rabbit heart and the in situ blood-perfused sheep heart [1–4]. Recently we have investigated the antistunning and antiinfarct effects of APC and have shown that segment shortening after 15 minutes of ischemia (stunning), in the area at risk, in which no infarct was incurred, was significantly decreased to approximately 32% of equilibrium value in both RI and ADO hearts but was significantly preserved in IPC and APC hearts [5]. When ischemic time was increased to 30 minutes, segment shortening was significantly preserved only in APC hearts (p < 0.05 versus RI, ADO, or IPC) but was less than 37% in IPC, ADO, and RI hearts. Extension of ischemic time to 60 minutes significantly decreased segmental shortening in all experimental groups. These results indicated that both IPC and APC have antistunning effects in the in situ blood-perfused heart whereas ADO has no antistunning effects. Adenosine-enhanced ischemic preconditioning significantly extends "antistunning" effects (p < 0.05 versus IPC); however, these effects are transient, being obviated as the regional ischemic time is increased to 60 minutes [5].
Our results also indicate that infarct size is significantly decreased at each regional ischemic time with APC (p < 0.05 versus RI, ADO, and IPC). Whereas ADO and IPC were able to decrease infarct size to a similar extent (p < 0.05 versus RI), the relative infarct levels in these groups was approximately two times greater (p < 0.05 versus APC) than that in APC hearts at each regional ischemic time. Thus, the use of APC allows for the significant extension of regional ischemic time providing equal infarct size reduction at 60 minutes of regional ischemia as that afforded by ADO or IPC at 30 minutes of RI. These data suggest that APC extends the "antiinfarct" effects of both ADO and IPC.
Our data shown herein suggest that the additive effects of IPC and ADO when used coincidentally also act to decrease TUNEL-positive cell number and to delay DNA ladder formation. The mechanism by which this enhanced cardioprotection occurs remains to be elucidated; however, recent investigation by us has indicated that the ATP-sensitive potassium channels and, in particular, the mitochondrial ATP-sensitive potassium channels are directly involved in APC myocardial infarct size reduction [35]. We speculate that the early, preischemia opening of these channels by APC allows for the preservation of mitochondrial function and the reduction of apoptotic signaling. The inability of ADO alone to decrease TUNEL would appear to involve a modifying action of IPC on ADO when used coincidentally.
Recently Zhao and coworkers [36] have shown that adenosine attenuates apoptosis by increasing Bcl-2 and Bax proteins in the regional ischemic dog heart. However, previous investigations have also indicated that adenosine is associated with increased apoptosis in cultured myocytes and carcinoma cells [37, 38]. In our report adenosine alone was found to increase apoptosis, and the differences observed may be related to the concentrations used and the time of infusion.
We have recently found that APC acts through adenosine A3 receptors [39]. Jacobson [40] has suggested that intense, acute activation of adenosine A3 receptors acts as a lethal input to cells, but that low concentrations of A3 receptor agonists protect against apoptosis. In the report of Zhao and colleagues [36], adenosine was infused at 140 µg/kg during more than 6 hours of reperfusion (3 to 7 mmol/L for 6 hours). In our study we have delivered a bolus injection of adenosine (10 mmol/L) before ischemia. In previous reports we have shown that 10 mmol/L of adenosine injected directly into the myocardium by means of the aortic root induces only a transient nonsignificant decrease in LV systolic pressure, LV developed pressure, and mean arterial pressure and that these hemodynamic alterations are eliminated in 1.0 ± 0.3 minutes [4]. We speculate that this high concentration, although not significantly affecting hemodynamics, may result in increased TUNEL when used alone and would account for the increased TUNEL-positive cell number seen in these experiments.
The ability to significantly decrease myocyte necrosis and apoptosis and to extend regional ischemic time in a blood-perfused heart model is of significance to myocardial cellular preservation and function. Our results indicate that when ischemia–reperfusion is extended to 60 minutes, apoptosis is evident by oligonucleotide ladder formation. Our results also indicate that both APC and IPC significantly decrease TUNEL-positive cells (p < 0.05 versus RI), but that APC is superior (p < 0.05 versus IPC). Our results further show that APC significantly delays the occurrence of TUNEL-positive cells with fewer than 100 TUNEL-positive cells per 3,000 myocytes, being evident after 60 minutes of regional ischemia and 120 minutes of reperfusion with no oligonucleotide ladders being evident.
In summary our results indicate that APC significantly decreases infarct size and significantly decreases TUNEL in the regional ischemic zone with no DNA laddering being evident after 60 minutes of regional ischemia and 120 minutes of reperfusion in the in situ blood-perfused heart. In contrast IPC and ADO were associated with significantly increased infarct size and significantly increased TUNEL in the regional ischemic zone in both IPC and ADO (p < 0.05 versus APC) and with DNA laddering apparent in both IPC and ADO after 60 minutes of regional ischemia and 120 minutes of reperfusion. These results indicate that the cardioprotection afforded by APC occurs at least in part by significantly decreasing necrosis and apoptosis as compared with ADO or IPC in the in situ blood-perfused heart. The classification of TUNEL-positive cells remains to be elucidated; however, the lack of oligonucleotide ladder formation suggests that apoptosis may not be present or is at least delayed with the use of APC. The benefits of APC in a human surgical model remain to be investigated, but our results described in this report and previous reports [1, 4, 5] using the surgically relevant in situ blood-perfused heart model suggest that the use of APC would allow for enhanced myocardial tissue salvage providing superior cardioprotection by significantly decreasing myocardial cell apoptosis and infarct size (p < 0.05 versus IPC, ADO).
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