| Objective: The cases of sudden coronary death (SCD) caused by acute myocardial infarction(AMI) often occur in the forensic medicine practice. The morphplogical changes of cardiac tissue caused by ischemic injury were so limited that normal phathological examination could not detect manifest myocardial damage. Therefore the assessment of SCD became difficult and complex. It has been demonstrated that AMI could induce nitricoxide synthase(iNOS) expression in myocardia cell, resulted in the increase of NO; peroxynitrite formed from NO and O2ˉ, could led to nitration of protein tyrosine residues producting 3-nitrotyrosine (NT). Immunohistochemical technology, as a convenient and practical method, which can detect the changes of proteins in the ischemic myocardial cells, may be a possible method for the postmortem identification of AMI. In this study, we detected the expression of iNOS and NT in myocardium using IHC at different time after early myocardial ischemia, and observed the effect of postmortem changes and fixation time on the expression of iNOS and NT, in order to search an utility, sensitiveness immunohistochemical markers for assist postmortem diagnosis of SCD.1. Expression and sensitiveness of iNOS and NT in myocardium at different time after early myocardial ischemiaMethods: 50 SD rats weighting 250~300g of either sex, were randomly distributed into Sham group and ischema group. The rat model of early aute myocardial ischemia was established by ligating the left anterior descending branch of coronary artery. The ischemia group rats were killed at 0.5h, lh, 2h, 3h, 4h, 5h and 6h after ligation. Sham group rats underwent the same surgical protocol as the ischemia group except LAD ligation, and were killed at lh, 3h, and 6h after operation. Then all hearts were dissected and fixed, the tissue blocks were taken and embedded in paraffin and the tissue sections were prepared according to routine histological procedures. Then histological sections were dyed with HE staining, and the expression of iNOS and NT were detected by IHC. The data were analyzed using SPSS13.0 statistical program. A level of P<0.05 was considered as statistical significance.Results:1 Specific stain1.1 Stain of Evans Blue: Myocardium of ischemic area was unstained presenting red; in contrast, myocardium of non-ischemic area was stained with Evans Blue presenting blue.1.2 TTC staining: 0.5h, 1h and 2h groups, all around the myocardium presented red; 3h group, small pale area of left ventricle(LV); manifest pale area of LV compared obviously to other area during 4~6h.2 HE staining: Myocardium of control group and myocardial ischemia group within 2h was stained clearly, with very mild cellular swelling or/and interstitial edema in ischemic myocardium. During 3~5h after myocardial ischemia there were manipulus inflammatory cells in the matrix and cardiac wavy muscle fibers. In the 6h group which a few myocardial acidophiliar was reinforcing, and a few of myocardium necrosis.3 Immunohistochemical in ischemic myocardium:3.1 The change of the expression of iNOS at different time points after myocardial ischemia: In sham group there was little expression of iNOS in myocardium. In the ischemia groups, there was weakly positive expression of iNOS in the cardiac cytoplasm at 0.5h after myocardial ischemia, and the intensity of positive expression was reinforcing significantly along with ischemic time; there was strong positive expression in ischemic myocardium during 3~6h, having obvious difference compared to sham group's (P<0.05).3.2 The change of the expression of NT at different time points after myocardial ischemia: In sham group there was little expression of NT. In the ischemic groups, there was weakly positive expression of NT in the cardiac cytoplasm at 0.5h after myocardial ischemia, and the intensity of positive expression was reinforcing with ischemic time, having obvious difference compared to sham group's (P<0.05). .2.Study on the antigen stability of iNOS and NTMethods: 120 healthy adult rats weighting 250~300g of either sex, were randomly distributed into 4℃group, room temperature group and fixation group. 4℃group and room temperature group were distributed into 1d, 3d, 5d, 7d and 14d groups, which include ischemic group and sham group; fixation time group was distributed into 3d, 5d, 7d and 14d groups, using the S3h group in part one as control. The ischemic group rabbits were killed at 3h after ligation. Sham group rats underwent the same surgical protocol as the ischemic group except LAD ligation. The myocardium of 4℃groups and room temperature groups was fixed for 24h after 1d, 3d, 5d, 7d and 14d of postmortem stored at 4℃and room-temperature respectively. The myocardium of fixation time group was fixed in 10% neutral formalin immediately after killed for 3d, 5d, 7d and 14d. The tissue blocks were taken and embedded in paraffin and the tissue sections were prepared according to routine histological procedures. Then histological sections were dyed with HE staining, and the expression of iNOS and NT were detected by IHC. Observe the changes of ischemic myocardia as well as the expression of iNOS and NT stored at the same environment for different time intervals after death. The data were analyzed using SPSS13.0 statistical program. A level of P<0.05 was considered as statistical significance.Results:1.HE staining:Stored at 4℃: There were some pathological changes of early myocardial ischemia in ischemic myocardium, no definite indicators of myocardial infraction. At 3~5d, the cellular cloudy swelling blur or disapearanse of transverse striation in ischemic myocardium are similar to sham group's changes. While samples stored at 4℃for 7~14d after death, the two groups experienced similar changes in morphous of tissue.Stored at room-temperature: For 1d the autolysis is not obviously with partly plasmolysis and light-stained nucleus in both control and ischemic group's, and there were some pathological changes of early myocardial ischemia in ischemic myocardium. For 3d there were blue-stained cenobium, hypochromatosis, cellularity unclear and hyalomitome light-stained in part myocardial cell; while some changes in ischemic myocardium are similar to sham group's changes. While samples stored at room-temperature for 5~7d nuclei almost disappeared, cell spreaded looseing with outline of tissue kept only; the two groups experienced similar changes in morphous of tissue; For stored 14d after death the two groups experienced similar changes in morphous of tissue and in ischemic myocardial cell with nuclei disappeared and hyalomitome homogenized.Fixation: there were not obviously changes of HE staining for different fixation time.2 Immunochemistry in ischemic myocardium:2.1 The effect of stabilitu to iNOS and NT stored at 4℃:Sham group: there was little positive expression in myocardium.Ischemia group: intensity and extent of positive expression to iNOS and NDT in ischemic myocardial cell were decreasing along with stored time. The positive expression to iNOS of 7d and NT of 5d was decreasing obviously, but having obvious difference compared to sham group's(P<0.05). While iNOS stored for 14d and NT stored for 7d after death, there was little positive expression, having no difference compared to sham group's (P>0.05).2.2 The effect of stability to iNOS and NT stored at room temperature:Sham group: there was little positive expression in myocardium.Ischemia group: intensity and extent of positive expression to iNOS and NT in ischemic myocardial cell were decreasing along with stored time; at 3d after death the positive expression was decreasing obviously, but have difference compared to sham group's(P<0.05). For 5d-14d there was little positive expression having no difference compared to sham group's (P>0.05).2.3 The effect of fixation time to iNOS and NT stability:The positive expression of iNOS and NT decreased slowly in ischemic myocardial cell along with fixation time. At 14d there was still obvious difference compared to sham group's (P<0.05).Conclusions: iNOS and NT were sensitivity to early rat's AMI, and appeared positive expression 0.5h after AMI; the intensity of positive expression to iNOS and NT was increasing with ischemic time. The antigen stability of iNOS and NT was relatively small influenced of autolysis and fixation time; it can be conclude that the immunohistochemical detection of iNOS can be used in myocardia of rat stored at 4℃for 7d or less than 7d after death, stored at room temperature for 3d or less than 3d after death and fixed for 14d or less than 14d after death. It also can be conclude that the immunohistochemical detection of NT can be used in myocardia of rat stored at 4℃for 5d or less than 5d after death, stored at room temperature for 3d or less than 3d after death and fixed for 14d or less than 14d after death.
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