| Mechanical trauma is the fifth cause of death in urban and rural populations. More than sixpercent of deaths each year are attributed to mechanical trauma. Recently published clinicalreports have indicated that traffic accidents, sports or blunt chest trauma causes myocardialinfarction a few days after trauma even if ECG, X-ray, myocardial enzymes and other testsshowed no obvious abnormalities within24hours after trauma.Acute myocardial infarction (AMI) is an important contributor to cardiovascular mortality.Prompt reperfusion is considered the optimal management for AMI. In recent years manystudies have shown that many risk factors like high-sugar and high-fat diets impact thereperfusion therapy and increase ischemia/reperfusion (I/R) injury, resulting in larger infarctsize and more severe heart failure. After being treated for myocardial I/R, it is still unknownwhether myocardial infarction patients with trauma history further aggravated the reperfusioninjury.Apoptosis, also known as programmed cell death, is a natural death process activated bystarting own internal mechanism and activating endogenous DNA endonuclease. Many studieshave confirmed that inhibition of apoptosis can significantly reduce myocardial infarct size.Our preliminary study confirmed that trauma caused myocardial apoptosis. However, theimpact of I/R injury in posttraumatic rats on myocardial apoptosis is unclear. This study aimedto explore whether the trauma rats that underwent myocardial I/R would further aggravatemyocardial apoptosis and thus increase the myocardial I/R injury.Objective:1. To investigate whether mechanical traumatic rats may increase myocardial I/Rinjury.2. To determine whether myocardial apoptosis may contribute to increasedmyocardial I/R injury in traumatic rats.Material and Methods:1. Animal and groupsHealthy male SD rats weighing180-220g were randomly divided into thefollowing four groups: sham trauma+sham operation group, sham trauma+I/R group,trauma+sham operation group, trauma+I/R group and trauma+I/R+Z-VAD-FMKgroup.2. Establishment of mechanical traumatic rat model Male SD rats were anesthetized with chloral hydrate (3ml/kg). The rats were thenplaced in a Noble-Collip drum, a plastic wheel with internal shelves on which a rat istraumatized as the wheel is rotated (40rpm,5min). Sham trauma rats were subjected tothe same revolutions but the animals were taped to the inner wall of the drum, thusavoiding traumatic injury.3. Establishment of myocardial I/R modelsRats were anesthetized, fixed, intubated and connected with ECG monitoringelectrodes. A left thoracotomy was performed and the heart was exposed fully throughthe second intercostal space. The pericardium was incised and the left anteriordescending artery was ligated by6-0silk. Ischemia was confirmed by the camponotusupward elevation of the ST segment. After30min ischemia, coronary blood flow wasreperfused (3h) by releasing the ligature. In sham-operated rats, the same procedurewas executed, without releasing the ligature.4. Detection of cardiac function in vivoHeart rate (HR), the average arterial blood pressure (MABP), left ventricular systolicpressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricularmaximum rate of contraction (+dp/dtmax) and left ventricular diastolic maximum rate(-dp/dtmax) were detected using the BL-410biological signal analysis system.5. Determination of creatine kinase isoenzyme MB (CK-MB) and cardiactroponin I (cTnI) contentCreatine kinase isoenzyme MB (CK-MB) and cardiac troponin I (cTnI) content inrat serum was determined by the ABC-ELISA method. After adding serum samplesand other reagents according to the manufacturer’s instructions, the optical density(OD) value was determined by the microplate reader seted the wavelength at450nm.Then the content of CK-MB and cTnI were calculated according to the standard curveand expressed as ng/ml.6. Assessment of myocardial infarct sizeAt the end of the3h reperfusion, the ligature around the coronary artery was retiedand2ml of1%Evans blue dye was injected into the left ventricular cavity. The dye wascirculated and uniformly distributed except in the region of the heart previously perfusedby the occluded coronary artery (AAR). The heart was quickly excised and the leftventricle was cut into six transverse slices after the atria and right ventricle were excised.In each slice, the AAR was separated from the non-ischemic zone and incubated in1%triphenyltetrazolium chloride (TTC) in phosphate buffer at37°C for15min todifferentiate necrotic (white) from non-necrotic (red) area at risk. Blue, red and white areas of the region were determined respectively by Image-Pro Plus6.0image analysissoftware. The AAR was expressed as a percentage of the left ventricular area(AAR/LV×100%), and the area of necrosis (AN) as a percentage of the AAR(AN/AAR×100%, infarct size).7. Determination of myocardial Caspase-3activityCaspase-3activity was detected by the Caspase-3fluorescent assay kit. Myocardialtissue was clearaged, centrifuged, and the supernatants were collected. Then proteinconcentrations were measured by the coomassie brilliant blue method. After adding thesample lysis buffer and substrate working solution to each well of a96-well plate, theabsorbance value (OD) was quantifed by using a microplate reader. The activity ofCaspase-3was indicated by the OD value of the well normalized with the total proteinconcentration.8. The detection of myocardium apoptosis by TUNEL assayMyocardial apoptotic index was analyzed by TUNEL assay. After myocardialtissue was made into paraffin sections, TUNEL staining was performed and TUNELstaining for apoptotic cell nuclei (green) and DAPI staining for all myocardial cellnuclei (blue). Twenty fields were selected randomly in each paraffin section, and100cells were counted in one field. The index of apoptosis was indicated by the number ofTUNEL-positive nuclei/total number of nuclei.9. Statistical analysisAll values are presented as means±SEM. Computations for the statistical methoddescribed were performed using SPSS13.0. Differences between treatment groups wereevaluated using Student’s t-test. One-way ANOVA was used to determine statisticaldifferences among groups. Probabilities of P<0.05were considered statisticallysignificant.Results:1. Sensitivity to myocardial I/R injury increased in mechanical trauma rats1.1Cardiac function significantly reduced in posttraumatic rats subjected toI/RThe results showed that+dp/dtmaxand LVSP in the trauma+I/R group were(1979±164) mmHg/sec and (62±5)mmHg. Compared with the sham trauma+I/R group,(3648±152) mmHg/sec and (93±4)mmHg, differences were statistically significant(P<0.01, Fig.).-dP/dtmaxand LVEDP in trauma+I/R group were (-1626±208)mmHg/sec and (26.5±1.9)mmHg. Compared with the sham trauma+I/R group, (-2546±224) mmHg/sec and (15.9±1.5)mmHg, the differences were statisticallysignificant (P<0.01).1.2Serum levels of CK-MB and cTnI were significantly increased inposttraumatic rats subjected to I/RCompared with the sham trauma+I/R group, the levels of CK-MB in thetrauma+I/R group remarkably increased (4920±326ng/ml vs.2933±369ng/ml, P<0.01).The levels of cTnI in trauma+I/R group were signifcantly higher than the shamtrauma+I/R group (17.84±2.06ng/ml vs.12.46±1.39ng/ml, P<0.01).1.3Myocardial infarct size significantly increased in posttraumatic ratssubjected to I/RCompared to the sham trauma+I/R group, infarct size was markedly increasedin the trauma+I/R group (40±3.9%vs.28±3.4%, P<0.01).2. The possible mechanism of increased sensitivity to myocardial I/R injury inmechanical trauma rats2.1Myocardial apoptosis significantly increased in posttraumatic ratssubjected to I/RCompared with the sham trauma+I/R group, a significant number ofTUNEL-positive cells were observed in the trauma+I/R group (AI:30.5±1.6%vs.22.6±1.5%, P<0.01), and Caspase-3activity in trauma+I/R group increasedsignificantly compared to the sham trauma+I/R group (78.9±8.1%vs.48.2±3.8%,P<0.01).2.2After being given Z-VAD-FMK, myocardial apoptosis markedly decreasedin post-traumatic rats subjected to I/R.Compared with the trauma+I/R group, myocardial apoptotic index andCaspase-3activity decreased significantly in trauma+I/R+Z-VAD-FMKgroup[Caspase-3activity：32.4±6%vs.78.9±8.1%，P<0.01；apoptotic index：11.2±1.4%vs.30.5±1.6%，P<0.01].2.3After being given Z-VAD-FMK, myocardial infarct size significantlyreduced in post-traumatic rats subjected to I/R.Compared with the trauma+I/R group, myocardial infarct size was decreasedmarkedly in the trauma+I/R+Z-VAD-FMK group[(22±2.2%)%：(40±3.9%)%]. Therewas a significant statistical difference (P<0.01).2.4After being given Z-VAD-FMK, serum CK-MB and cTnI weresubstantially reduced in the post-traumatic rats subjected to I/R. Compared with the trauma+I/R group, serum CK-MB and cTnI were markedly reduced in trauma+I/R+Z-VAD-FMK group[CK-MB:(1474±520)ng/ml vs.(4960±588)ng/ml，P<0.01；cTnI：(6.75±0.73)ng/ml vs.(18.10±3.06)ng/ml, P<0.01].2.5After being given Z-VAD-FMK, cardiac function in vivo was significantlyimproved in post-traumatic rats subjected to I/R.Compared with the trauma+I/R group, cardiac systolic function and diastolic function markedly improved in trauma+I/R+Z-VAD-FMK group[+dp/dtmax：(4217±115)mmHg/sec vs.(1988±182)mmHg/sec，P<0.01；-dp/dtmax:(-3623±134)mmHg/sec vs.(-1622±200)mmHg/sec, P<0.01]. There was a significant statistical difference (P<0.01).Conclusion:1. Mechanical trauma increased the sensitivity of the rat myocardium to myocardial I/Rinjury. The result suggested that clinicians, especially cardiovascular practitionersshould ask the history of trauma during the patient inquiry and pay more attention topatients with a history of trauma.2. Cardiomyocyte apoptosis is a reason for the increased sensitivity induced bymechanical trauma to myocardial I/R injury in the rat myocardium. The resultssuggested that if effective clinical treatment was used for myocardial infarct patientswith a history of trauma before reperfusion, the degree of I/R injury would bereduced by depressing the cadiomyocyte apoptosis. |
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