Acute myocardial ischemia tissue inducible nitric oxide synthase and nitrotyrosine expression and antigenic variation stability
|School||Hebei Medical University|
|Keywords||Forensic Pathology Acute myocardial infarction Postmortem diagnosis Inducible nitric oxide synthase Nitrotyrosine|
Objective: Acute myocardial infarction is the most common cause of sudden death, sudden death type. Postmortem routine pathological examination methods often detect no obvious pathological changes in acute myocardial ischemia, making sudden coronary death (sudden coronary death, SCD) Case identification becomes difficult and complex. Studies have shown that acute myocardial ischemia in rat myocardial cells can be induced inducible nitric oxide synthase (induce nitricoxide synthase, iNOS) expression is increased, leading to increased production of NO, NO can react with O2 ˉ formation of peroxynitrite (peroxynitrite , ONOO ˉ), ONOO ˉ make protein nitration of tyrosine residues to form 3 - nitrotyrosine (3-nitrotyrosine, NT). Immunohistochemistry (immunohistochemistry, IHC) is a convenient and practical method that can detect ischemic changes in intracellular proteins, thus postmortem diagnosis of acute myocardial infarction provides a technical support. IHC technology to be adopted in this experiment, the early detection of acute myocardial ischemia ischemic myocardium at different times the expression of iNOS and NT, and to study the changes and postmortem tissue fixation time on the iNOS and NT-antigen stability in order to find sensitive , practical IHC indicators to assist postmortem diagnosis of SCD. A. INOS and NT expression in the ischemic myocardium and sensitivity Methods: 50 healthy SD rats, weighing 250 to 300 g, male or female, were randomly divided into sham group (S group) and ischemic group (A group). By ligating the left anterior descending coronary artery (LAD) method to establish rat model of acute myocardial ischemia; ischemic rats after LAD ligation were 0.5h, 1h, 2h, 3h, 4h, 5h and 6h sacrificed sham only in rats without LAD ligation suture passes under, respectively, after 1h, 3h and 6h killed, the other steps are the same as the ischemia group. After drawn Paraffin sections were observed by HE staining of pathological changes in rat heart and cardiac muscle were measured by IHC and NT expression of iNOS dynamic changes; SPSS13.0 statistical analysis software with experimental data were statistically analyzed to P lt; 0.05 was considered statistically significant. Result: a special staining 1.1 Evans Blue dye: S rats showed a blue heart; A rat anterior and lateral wall of the left ventricular myocardium is red, I myocardial blue. 1.2 TTC staining: acute myocardial ischemia 0.5h, 1h and 2h myocardium was uniform brick red, 3h group left ventricular lateral wall myocardial appear small area of pale color area, 4h, 5h and 6h group left ventricle front, side pale wall region increases, and the surrounding area contrasting red brick boundary clearer; endocardium was brick red. 2 HE staining: sham myocardial tissue structure is clear, acute myocardial ischemia and myocardial cells only partially within 2h and interstitial edema, 3 ~ 5h partial visible interstitial infiltration of inflammatory cells, and occasionally swelling and wave myocardial fiber degeneration , no significant necrosis; 6h local minority eosinophilic myocardial cells and individually enhanced myocardial necrosis. 3 IHC staining: 3.1 at different time points after ischemia iNOS expression in ischemic myocardium: sham operation group: Myocardial cytoplasmic expression of iNOS was negative; ischemia group: 0.5h after acute myocardial ischemia and myocardial ischemic cell pulp weakly positive expression of iNOS, 1h after the ischemic myocardium in each group cytoplasmic expression of iNOS prolonged ischemic time with gradually increased to 3 ~ 6h after ischemia myocardial cells and interstitial vascular endothelial cells of iNOS showed strong expression compared with the sham group were significantly different (P lt; 0.05). 3.2 at different time points after ischemia ischemic myocardium NT expression: sham operation group: Myocardial cytoplasmic expression of NT-negative; ischemia group: 0.5h after acute myocardial ischemia ischemic myocardium was NT cytoplasm weak expression, 1h after ischemic myocardial cells in each group of NT expression gradually increased with time, 6h visible expression of a wide range of NT-positive, compared with the sham group were significantly different (P lt; 0.05). Two. INOS and NT-antigen stability Experimental Methods: 120 healthy SD rats, weighing 250 to 300 g, male or female, were randomly divided into 3 ℃ group (D), at room temperature (20 ℃) group (E group) and a fixed group (F) three groups. D group and E group were divided into 1d, 3d, 5d, 7d and 14d five subgroups, each subgroup were divided into ischemic group and sham operation group; E group was divided into 3d, 5d, 7d and 14d four subgroup to the first part of S3h sham operation control group. The ischemic rats were sacrificed after 3h after LAD ligation, sham-operated rats only passes under the LAD ligation suture without the other steps are the same as the ischemia group. Group D and E, respectively, after the rats were sacrificed at 4 ℃ and room temperature place 1d, 3d, 5d, 7d and 14d fixed after 24h; F rats were sacrificed immediately after the heart was removed placed in 10% neutral formalin fixed respectively 3d, 5d, 7d and 14d; drawn after Paraffin sections were observed by HE staining of histological changes in rat heart and determination by IHC in different environments myocardial expression of iNOS and NT dynamic changes; using SPSS13.0 statistical analysis software for statistical analysis of the experimental data to P lt; 0.05 was considered statistically significant. Results: 1 HE staining: 1.1 4 ℃ placed 1d, myocardial tissue structure clear, no significant changes in autolysis; ischemic myocardial ischemia visible early nonspecific pathological changes. Place 3 ~ 5d, some changes, such as myocardial ischemia and myocardial cells cloudy swelling, stripes blur or disappear with normal myocardium postmortem changes similar and difficult to distinguish. Place 7 ~ 14d, fuzzy cellular structure, loosely arranged cytoplasm homogenization Iraqi red nucleus most of the disappeared, ischemic myocardial tissue and cell morphology similar to the control group. 1.2 at room temperature for 1d, myocardial mild autolysis, partial dissolution of the cytoplasm, the nucleus lightly stained, ischemic myocardium is still visible local infiltration of inflammatory cells and other pathological changes. Blue 3d partial visible contamination group, nuclear fusion, cell structure fuzzy focal lightly stained cytoplasm; ischemic myocardium after the death of some changes and change is not easy to distinguish between normal myocardium. 5 ~ 7d majority of myocardial cells disappear, cells arranged loosely organized outline only surviving, 14d nucleus disappeared completely homogenized cytoplasm Iraqi Red; 5 ~ 14d ischemic myocardial tissue and cell morphology similar to the control group. 1.3 Fixed 14d less myocardial tissue HE staining results are not significantly affected. 2 IHC staining: 2.1 4 ℃ to save the stability of iNOS and NT sham group: Myocardial cytoplasm of iNOS and NT always negative expression; ischemia group: With the extension of storage time of iNOS and NT-ischemic strength and range of expression are gradually weakened, iNOS placed 7d less, NT placed within 5d expression intensity compared with the sham group were significantly different (P lt; 0.05); iNOS placed 14d, NT placed 7d when compared with the sham group no significant difference compared (P gt; 0.05); 2.2 at room temperature on the stability of iNOS and NT sham group: Myocardial cytoplasm of iNOS and NT always negative expression; ischemia group: With the extension of storage time missing Blood expression of iNOS and NT were waning strength and scope, 3d when the positive expression of iNOS and NT strength and range has been significantly weakened, but there is still a difference for the sham (P lt; 0.05), 5 ~ 14d iNOS and NT expression compared with the sham group had no significant difference (P gt; 0.05); 2.3 fixed time on the stability of iNOS and NT ischemic expression of iNOS and NT as the extension of a fixed time decreased slowly to fixed at 14d with the sham group still has a significant difference between (P lt; 0.05). Conclusion: iNOS and NT on acute myocardial ischemia with high sensitivity, acute myocardial ischemic myocardium after ischemia 0.5h cells that have positive expression, expression intensity with the extension of ischemic time gradually. iNOS and NT have better antigen stability, iNOS can be used for death within 4 ℃ for 7d, 3d room temperature and fixed within less than 14d rat heart specimens; NT for death within 4 ℃ for 5d, at room temperature Within less than 14d 3d rat heart and fixed specimens.