| Sudden cardiac death (SCD) is most commonly defined as unexpecteddeath from a cardiac cause within a limited time period, which ranks aboveother causes of adult sudden death. Among the causes of SCD, acutemyocardial ischemia (AMI) is the major one. Due to transitory(coronarospasm) or constant (occlusive coronary stenosis) acute myocardialischemia, blood flow in coronary artery decreases, such supplies asmyocardial oxygen reduces and metabolite clearance becomes slow, resultingin the disorder of cardiac electrophysiological activities and/or the dysfunctionmyocardial mechanical contraction, which can cause most of the patients todie within6h, even1hour, of onset. Morphological changes (acidophilicdegeneration necrotic clotting, multifocal patches of wavy fibers,karyopyknosis, contraction band necrosis, marginal rearrangement reactionswith early inflammatory infiltrate, etc.) under light microscope with HE stainshall occur at least6h after myocardial ischemia. However, in the practice offorensic medicine, most victims of early myocardial ischemia (EMI) diewithin6h of the onset of myocardial ischemia. In routine examination, exceptfor coronary atherosclerosis of various degrees, the myocardial morphologicalchanges resulting from ischemic injuries are extremely difficult to detect. Inthe absence of convictive objective evidence, forensic expertise may bechallenged of postmortem diagnosis of EMI.Over many years, Chinese and foreign researchers have been seekingsensitive, stable and practical methods and indicators for diagnosis of EMI,ranging from HE stain, histochemical stain, immunohistochemistry (IHC),ultrastructure observation, change of ion content, myocardial injury marker inthe blood and liquor pericardii, postmortem imaging examination, etc. A greatmany of active explorations have been made and all of them enrich the diagnosis of myocardial ischemia from various perspectives and to variousextent. However, the application of above-mentioned techniques are quitelimited due to complicated operation, less practical utility, absence ofdistinctive characteristics or excessive cost. The experts hold various views totheir practical values. There haven’t been a sensitive and practical methods orindicators to solve the diagnosis problems in reality.Among the researching techniques, IHC has been employed in theresearch and application of EMI because it’s high sensitive, practical anddistinctive. In recent years, many scholars have found the phenomenon thatprotein molecule, such as actin, cardiac troponin, heart type fatty acid bindingprotein (H-FABP), dystrophin, etc, disappear from cardiomyocytes duringacute myocardial ischemia; that such proteins as complements and fibronectin(Fn) in blood distribute abnormally in ischemic myocardia; that ischemiccardiomyocytes express some sensitive proteins, such as hypoxia-induciblefactor-1alpha (HIF-1α), vascular endothelial growth factor, etc. Theseresearches have observed various changes from multiple angels when acutemyocardial ischemia occurs. Some indicators are tested in the heart specimensof sudden death from EMI. Considering the development features of EMI andthe complexity and specificity of forensic medicine practice, the application ofsolo method and solo testing indicator can hardly show their diagnostic valueeffectively for being less convictive. However, these researches have laid atheoretical basis for the combined application of multiple indicators.Meanwhile, considering the specificity of forensic investigation, such as theonset time of EMI, preserving condition of body, fixed time, etc., thecombination of different testing indicators for different cases is expected toachieve most satisfactory results.Therefore, this project will take the rat model of AMI and human autopsyhearts as researching objects and is to①observe comparatively the stainingabilities to ischemic cardiomyocytes of Heidenhain stain, chrometrope2R-brilliant green stain and HBFP stain, as well as the effects resulting fromdifferent preserving conditions and10%formalin at fixed time;②choose testing marker from various perspectives: structural protein or cytoplasmicprotein in the cardiomyocytes: actin (HHF35)、H-FABP、S100A1；abnormalaccunmlation of plasma proteins: C5b-9、Fn；inducible expression of proteinmolecule in the ischemic cardiomyocytes: clusterin (CLU)、HIF-1α、HIF-2α、a disintegrin and metalloproteinase with thrombospondin type1motifs(ADAMTS-1), and observe the expression changes in the acute ischemiccardiomyocytes of rat through IHC;③observe the effects to theimmunohistochemical testing marker resulting from different preservingconditions and fixed in10%formalin for different time;④to verify withhuman autopsy hearts, observe the testing marker expression and specificity,and provide objective foundation for the postmortem diagnosis of EMI.Part I: Application and comparison of three histochemical stains inthe postmortem diagnosis of early myocardial ischemiaObjective: To observe comparatively the applications of Heidenhainstain, chrometrope2R-brilliant green stain and HBFP stain for ischemiccardiomyocytes, as well as the effects resulting from different preservingconditions and fixed in10%formalin for different time.Methods:The rat model of AMI was constructed through permanent ligation of theleft anterior descending coronary artery (LAD). One hundred and ninety SDrats either sexes weighing250-300g were randomly distributed into7groups:control group (n=5), sham operated control group(n=5), acute myocardialischemia group (15min,30min,1h,2h,3h,4h,5h,6h after LADocclusion respectively, n=5), preservation at-20℃group, preservation at4℃group, preservation at room temperature group (The rat hearts, which weresubjected to6hours’ period of myocardial ischemia, were preserved at-20℃,4℃and room temperature for1d,3d,5d,7d,14d,21d,28d, n=5), fixed timegroup (The rat hearts, which were subjected to6hours’ period of myocardialischemia, were fixed in10%formalin solution for3d,5d,7d,14d,21d,28d,42d, n=5). The three histochemical stainings, i.e. Heidenhain stain,chrometrope2R-brilliant green stain and HBFP stain were used to observe ischemic cardiomyocytes.Results:1The punctate positive staining was seen in the subendocardialmyocardium and papillary muscle of left ventricular15min after acutemyocardial ischemia with Heidenhain stain, chrometrope2R-brilliant greenstain and HBFP stain, and the positive stained areas expanded as the ischemiacontinued. The positively stained areas of three special stains were concordant.2After the hearts, which suffered acute myocardial ischemia for6hours,were preserved at-20℃,4℃and room temperature, the coloration ability andpositive stained areas of the three histochemical stains tended to decrease withthe preservation time elapsed. Preserved for28days at-20℃and4℃,21daysat room temperature separately, the Heidenhain stain was the more stable thanchrometrope2R-brilliant green stain and HBFP stain, especially for displaycontraction band necrosis.3For the heart fixed for42d in10%formalin, the Heidenhain stain wasthe more stable than chrometrope2R-brilliant green stain and HBFP stain. Theischemic cardiomyocytes were stained positively, and the borderlines betweenischemic areas and non-ischemic areas were distinctive.Summary: Comparing with chrometrope2R-brilliant green stain andHBFP stain, Heidenhain stain is more operable, sensitive and less liable tochange after death and be affected by the fixation time in10%formalin.Heidenhain staining can be the first choice for the postmortem diagnosis ofEMI in the practice of forensic medicine.Part II: Detection of multiple immunohistochemical markers in acuteischemic myocardium of rat.Objective: To observe the change of plasma S100A1concentrations andthe expression and change pattern of actin (HHF35), H-FABP, S100A1, C5b-9,Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1in the cardiomyocytes at differenttime after LAD occlusion of rats.Methods:1The plasma S100A1concentrations in different groups (sham operated group,15min,30min,1h,2h,4h,6h after LAD occlusion) were tested byELISA.2The protein levels of ADAMTS-1in different groups (sham-operatedgroup;15min,30min,1h,2h,4h,6h after LAD occlusion) were examinedby western blotting.3. IHC was used to observe the expression and change pattern of actin(HHF35), H-FABP, S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1in the cardiomyocytes at different time after acute myocardialischemia of rat.Results:1At15min after LAD occlusion, the S100A1concentration wassignificantly higher than that of the sham-operated group (P<0.001). With thecontinuation of occlusion time, plasma S100A1concentrations furtherincreased, and peaked at6h after LAD occlusion.2At15min after LAD occlusion, the protein level of ADAMTS-1detected by western blotting was significantly elevated than that of thesham-operated group (P<0.001). With the continuation of occlusion time, theprotein levels of ADAMTS-1further increased, and peaked at6h after LADocclusion.3Immunohistochemical results at different time for rats after acutemyocardial ischemia①Actin (HHF35), H-FABP, S100A1: All cardiomyocytes of rats incontrol group are evenly stained positively brown-yellow. At15min afterLAD occlusion, the depletion of actin (HHF35), H-FABP, S100A1occurred inthe cardiomyocytes of the subendocardial layer and papillary muscles inischemic area. With the ischemic time prolonged, the depletion of stained areaextended from the endocardial to the epicardium. By4h after LAD occlusion,this area extended to all myocardial tissue in the ischemic area. A cleardividing line is drawn between the stained area and unstained area. Thepositions of unstained area of three proteins are basically the same in the eachgroup. ②ADAMTS-1, CLU: Negative expression occurred in thecardiomyocytes of rats in control group, sham operated control group. At15min after LAD occlusion, the positive expression of ADAMTS-1and CLUoccurred in the cardiomyocytes of the subendocardial layer and papillarymuscles in ischemic area. With the ischemic time prolonged, the positive areaextended. By4h after LAD occlusion, the sheet positive expression occurredin the cardiomyocytes of ischemic area. The positively stained area extendedto epicardium. Expression of CLU and ADAMTS-1reached the peak by4hand6h after LAD occlusion respectively.③C5b-9, HIF-1α：The negative expression occurred in cardiomyocytesof rats in control group, sham operated control group and15min after LADocclusion group. At30min after LAD occlusion, the positive expression ofC5b-9and HIF-1α occurred in the cardiomyocytes of the subendocardial layerand papillary muscles in ischemic area. With the ischemic time prolonged, thepositive area gradually extended. By4h after LAD occlusion, the sheetpositive expression occurred in cardiomyocytes in ischemic area and itspositive stained area extended to the epicardium. Expression of C5b-9andHIF-1α reached the peak by6h after LAD occlusion.④HIF-2α: Negative expression occurred in the cardiomyocytes of rats incontrol group, sham operated control group,15min and30min after LADocclusion group. By1h after LAD occlusion, the positive expression ofHIF-2α occurred in the cardiomyocytes of the subendocardial layer andpapillary muscles in ischemic area. The positive area extended with theprolongation of ischemic time. By5h after LAD occlusion, the positiveexpression of HIF-2α occurred in the majority of cardiomyocytes in ischemicarea and extended to epicardium, reached the peak.⑤Fn: Negative expression occurred in the cardiomyocytes of rats incontrol group,15min and30min after LAD occlusion group. The positiveexpression of Fn was shown in the serum of capillary vessel of thesubendocardial layer and papillary muscles in ischemic area. By2h LADocclusion, the positive expression occurred in the cardiomyocytes of the subendocardial layer and papillary muscles in ischemic area. The serum ofcapillary vessel and vascular wall are stained positively. With the ischemictime prolonged, the positive area extended gradually and reached the peak by6h after LAD occlusion.Summary: The diminished or absent actin (HHF35), H-FABP andS100A1occurs in rats at15min after LAD occlusion, and the positiveexpression occurs in ADAMTS-1and CLU. The positive expression of C5b-9and HIF-1α occurs at30min after LAD occlusion. The positive expression ofHIF-2α occurs at1h after LAD occlusion. The positive expression of Fnoccurs at2h after LAD occlusion. With the ischemic time prolongs, thedepletion of actin (HHF35), H-FABP and S100A1becomes more evident,while the protein expression of C5b-9、Fn、CLU、HIF-1α、HIF-2α andADAMTS-1become strong gradually.Part III: Influence of different preservation conditions and fixationtime in10%formalin on the immunohistochemistrical diagnosticmarkers of acute myocardial ischemia.Objective: To observe the influence of three preservation conditions:-20℃,4℃and room temperature, and fixation time in10%formalin on thenine stained immunohistochemistrical diagnostic markers observed in thesecond part.Methods:Influence of three preservation conditions:-20℃,4℃and roomtemperature, and fixation time in10%formalin on the changed expression ofthe nine diagnostic markers was observed in the ischemic cardiomyocytes byIHC.Results:1Influence of three preservation conditions on the nineimmunohistochemistrical diagnostic markers:①The target protein of IHC was most serious seriously affected in roomtemperature preservation, less seriously affected by4℃preservation, andmost slightly affected by-20℃preservation. ②When the rat heart, which was harvested at6h after LAD, preservedat-20℃,4℃and room temperature, actin (HHF35), H-FABP and S100A1were worst in terms stability. Only when they were preserved in-20℃, roomtemperature for1day and in4℃temperature for3days can be meaningful forpostmortem diagnosis.③C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all showed aexcellent feature of stability, among which CLU was most stable and can beapplicable to detect ischemic heart for28days preserved under threetemperature conditions. C5b-9was most unstable and can be used to detectischemic heart in-20℃temperature for28days, in4℃temperature for21days, and in room temperature for14days. After preserved for a differentperiod of time, no false positive result appeared in the cardiomyocytes of thenon-ischemic area that interrupted the estimation.2Influence of fixation time in10%formalin on the nineimmunohistochemistrical diagnostic markers:With the fixed time being prolonged, IHC-positive stained intensityshowed a tendency of decline, especially in the central part of ischemic area.When fixed for42days, all the diagnostic markers of IHC were still legibleand clear, and ischemic area and non-ischemic area was clearly demarked.Summary: Actin (HHF35), H-FABP, S100A1are worst in terms stability,and only when the ischemic heart is preserved in-20℃temperature, in roomtemperature for1day, and in4℃temperature for3days can be meaningfulfor postmortem diagnosis. C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all show a excellent feature of stability, among which CLU is most stable andcan be applicable to detect of ischemic heart for28days preserved under threetemperature conditions. C5b-9is most unstable and can be used to detect theischemic heart preserved in-20℃temperature for28days, in4℃temperaturefor21days, and in room temperature for14days. All the nine diagnosticmarkers can be used to detect the heart samples which are fixed in10%formalin for42days. Part IV: An immunohistochemical study on the postmortemdiagnosis with human autopsy hearts.Objective: The nine diagnostic markers are used to detect human autopsyhearts with the purpose of testing its application value and specificity.Methods:The human autopsy hearts were divided into four groups：①normalcontrol group (n=10);②definite myocardial infarction group (n=10);③suspected myocardial infarction group(n=10);④other cases group (n=23,including mechanical asphyxia, electrocution, hemorrhagic shock, carbonmonoxide poisoning, viral myocarditis). The expression of all the ninediagnostic markers was observed in human autopsy hearts by IHC.Results:1Normal control group showed homogenous brown reactions for actin(HHF35), H-FABP and S100A1. Diffuse or focal depletion of actin (HHF35),H-FABP and S100A1could be found in the definite myocardial infarctiongroup, suspected myocardial infarction group and other cases group. Thepositive rate of actin (HHF35), H-FABP and S100A1in the definitemyocardial infarction group, suspected myocardial infarction group and othercases group was significantly higher than that in the other cases group, butthere was not significant difference between the three groups.2Normal control group and hemorrhagic shock group showed negativestaining for C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1. The positiveexpression of C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1occurred inthe definite myocardial infarction group and most cases of suspectedmyocardial infarction group. The positive expression of HIF-1α and HIF-2αoccurred in some cases of mechanical asphyxia. The Fn positive expressionoccurred in some cases of electrocution. The positive expression of C5b-9,CLU, HIF-1α, HIF-2α and ADAMTS-1occurred in some cases of carbonmonoxide poisoning case. The positive expression of C5b-9, Fn, CLU andADAMTS-1occurred in viral myocarditis cases. The positive rate was higherin definite myocardial infarction group and suspected myocardial infarctiongroup than that in normal control group and other cases group. Summary: The nine diagnostic markers, namely actin (HHF35), H-FABP,S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1, are verified onthe EMI human autopsy hearts. Among them, actin (HHF35), H-FABP andS100A1are least in diagnostic specificity, which are not suitable to be appliedalone to postmortem diagnosis of EMI. C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1are more useful in postmortem diagnosis of EMI, but theirexpressions are also found in some cases of non-infarctive direct or indirectmyocardial injury (mechanical asphyxia, electrocution, hemorrhagic shock,carbon monoxide poisoning, viral myocarditis). The combined application ofmultiple diagnostic markers will improve the accuracy of the postmortemdiagnosis of EMI.Conclusion：From a practical perspective of forensic pathology, thisproject took the rat model of AMI and human autopsy hearts as researchingobjects and observed the sensitivity, practicability and specificity of threehistochemical stains and nine immunohistochemistrical diagnostic markers forthe postmortem diagnosis of EMI. Main conclusions are reached as follows:①Comparing with chrometrope2R-brilliant green stain and HBFP stain,Heidenhain stain is more operable, sensitive and less liable to change afterdeath and be affected by the fixation time in10%formalin. Heidenhainstaining can be the first choice for the postmortem diagnosis of EMI in thepractice of forensic medicine.②The diminished or absent actin (HHF35), H-FABP and S100A1occurs in rats at15min after LAD occlusion, and the positive expressionoccurs in ADAMTS-1and CLU. The positive expression of C5b-9and HIF-1αoccurs at30min after LAD occlusion. The positive expression of HIF-2αoccurs at1h after LAD occlusion. The positive expression of Fn occurs at2hafter LAD occlusion. With the ischemic time prolongs, the depletion of actin(HHF35), H-FABP and S100A1becomes more evident, while the proteinexpression of C5b-9、Fn、CLU、HIF-1α、HIF-2α and ADAMTS-1becomestrong gradually.③Actin (HHF35), H-FABP, S100A1are worst in terms stability, and only when the ischemic heart is preserved in-20℃temperature, in roomtemperature for1day, and in4℃temperature for3days can be meaningfulfor postmortem diagnosis. C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all show a excellent feature of stability, among which CLU is most stable andcan be applicable to detect of ischemic heart for28days preserved under threetemperature conditions. C5b-9is most unstable and can be used to detect theischemic heart preserved in-20℃temperature for28days, in4℃temperaturefor21days, and in room temperature for14days. All the nine diagnosticmarkers can be used to detect the heart samples which are fixed in10%formalin for42days.④The nine diagnostic markers, namely actin (HHF35), H-FABP,S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1, are verified onthe EMI human autopsy hearts. Among them, actin (HHF35), H-FABP andS100A1are least in diagnostic specificity, which are not suitable to be appliedalone to postmortem diagnosis of EMI. C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1are more useful in postmortem diagnosis of EMI, but theirexpressions are also found in some cases of non-infarctive direct or indirectmyocardial injury (mechanical asphyxia, electrocution, hemorrhagic shock,carbon monoxide poisoning, viral myocarditis). The combined application ofmultiple diagnostic markers will improve the accuracy of the postmortemdiagnosis of EMI.To sum up, this research studies systematically the application ofhistochemical staining and the nine diagnostic markers of IHC, whichprovides theoretical basis for the forensic experts of the postmortem diagnosisof EMI. |
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