Imunohistochemical Study Of H-FABP On The Postmortemdiagnosis Of Early Myocardial Infraction

Background: Study of SCD is one of the most important fields and the postmortem diagnosis of EMI is a focus as well as difficult problem in forensic pathology .It is unlikely to find special changes with either unaided eye or light microscope for myocardial infarction less than 6 hours. Without the use of special methods, it is impossible to get the histological diagnosis of EMI as a cause of sudden death. A series of pathophysiologic changes have been taken place in the ischemic myocardial before the appearance of characteristic morphology on the routine histology stains. Although early ischemic myocardial injury can be detected biochemically, histochemically, enzyme histochemically, fluorescence histochemically, electronmicroscopicly. These methods are limited in forensic practice,either because they are too complexed to be operated, or because they are too unspecific,instabile,expensive or easy to be influenced by autolysis and putrefaction of tissue . However immunohistochemical technology as a swift convenient and practical method can show the qualitative and quantitative changes of proteins in the ischemic myocardial cell, which make it possible to diagnose EMI postmortem.Heart-type fatty acid binding protein (H-FABP) is a low molecular weight protein (14-15kDa) with special tissue distribution, which is abundant in the cytoplasm of myocardial cells. It may play an important role in the uptake and oxygenation of long-chain fatty acids in cardiac myocyte. As myocardial cell membrane damaged by ischemia, H-FABP leaks to the extracellular space and enters the blood circulation very easily and quickly due to its small size and water soluble. It has been demonstrated that H-FABP is more sensitive and specific than myoglobin (Mb) for clinical detection of AMI within 12 hours after the onset of symptoms by quantifying its plasma concentration, because its molecule is smaller than that of Mb (18kDa) and it presents mainly in cardiomyocyte. So it is proposed as an early indicator of AMI by World Health Organization recently. Whether an immunohistochemical marker can be used in forensic practice or not depends on not only its specificity and sensitivity, but also the influence of autolysis and putrefaction of tissue on antigen . At present, what needs studying further is postmortem stability of immunohistochemical markers used in forensic practice and the regularity of its expression alteration in different postmortem intervals.Objective: The present study used an animal model of early myocardial ischemia by ligating the left anterior descending branch of coronary artery of animals. In order to explore its postmortem sensitivity and stability , we detected the expression of immunohistochemical markers of H-FABP in myocardium at different interval. The applicability Value of the new immunohistochemical marker was evaluated for diagnosis of SCD in forensic practice.This study included two parts:1. Expression of heart-type fatty acid-binding protein in myocardial ischaemic and study on its sensitivity. Methods: The present study used an animal model of myocardial ischemia by ligating the left anterior descending branch of coronary artery of rats. we detected the expression of immunohistochemical markers H-FABP in myocardium at different interval . Briefly, 45 SD rats weighting 250-350g of either sex, were randomly distributed into8 study groups and 1 control group. Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation.The study group ratswere killed at 15min, 30min, l h, 2h, 3h,4h, 6h,8h after ligation and the control rats killed after operation. Then all hearts were dissected and fixed in 10% formalin over 24h. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. Then treated with HE staining and H-FABP immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the intensity of positive reaction and the depletion area of H-FABP in each sample of the two groups.Results:1. HE staining: The control group and myocardial ischemia group within 3h were stained clearly, with distinct cross striation and oval, dense nuclei. Mild cellular swelling or/and interstitial edema, increased eosinophilia were found in ischaemic zones after 4h's period of ischaemia. No typical necrotic myocardial cells and inflammatory cells were found in all groups. In 8h myocardial ischemia that myocardial eosinophilia has noticeably increased, and some of myocardial cells ischemic necrosis, inflammatory cells increased.2. IHC staining: In control group, cytoplasm of all cardiomyocytes were stained homogeneous brown (positive for H-FABP), after counterstaining with hematoxylin, the nuclei stained light blue. No positive expression of H-FABP was found in interstitial tissue or other cell component. In study groups, the depletion of H-FABP from cardiomyocytes in infarcted areas at varying post-infarction intervals exhibited dentical pattern. Briefly, partial depletion occurred in a few disseminated cells in the subendo cardial layer and papillary muscles in 15min group. In 30min group, diminished or absent immuno staining cells appeared, but still located in subendo cardial layer and papillary muscles. Patchy depletion happened in lh group, extended to the middle myocardium in 2h group and extended to the outer myocardium in 3h group. By 4h after ligation completely depletion involving almost all myocardium which was distinct from normal cell started and then the depletion area increased with the prolongation of infarcted time. The comparison and statistical analysis between the depletion areas of H-FABP in various ischemic time group show: the depletion area of H-FABP in every study group is significant different from that in their relative control group; there is no significant difference between the depletion area of H-FABP within 3hours; but the depletion area of H-FABP in 3h group is significant different from that in 4h group. The sensitivity of H-FABP manifested mainly in the 3h prior myocardial ischemia.2.Study on the postmortem stability of heart fatty-acid-binding protein for the diagnosis of the early myocardial infractionMethods:The present study used an 3h animal model of myocardial ischemia by ligating the left anterior descending branch of coronary artery of rabbits. We detected the expression of immunohistochemical markers H-FABP in myocardium at different postmortem interval. Briefly, 20 healthy adult rabbits weighting 2-2.5kg of either sex, were randomly distributed into study groups and control group. Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation. The study group rabbits were killed at 3h after ligation and the control rabbits killed were sacrificed directly without ligation. The myocardium was fixed after 1h, 2h,4h,8h,16h,1d,2d,3d,4d,5d,6d,7d,10d and 14d of postmortem stored at 4℃. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. Then treated with HE staining and H-FABP immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the intensity of positive reaction and the depletion area in each sample of the two groups stored at 4℃for different time intervals after death.Results:1. HE staining: There some pathological changes of early myocardial ischemia such as cellular cloudy swelling, hydropic degeneration, blur or disappearance of transverse striation, hypereosinophilia, nucleus contract with deeper staining, waviness of muscle fiber in ischemic myocardium, no definite indicators of myocardial infraction. During 3-4 days, the cellular cloudy swelling blur or disapearanse of transverse striation in ischemic myocardium are similar to control group's changes. While samples stored at 4℃for 7-14d after death, the two groups experienced similar changes in morphous of tissue and in ischemic myocardial cell were similar to control group's.2. IHC staining: Myocardial tissue in the control group 4℃for 3 d, H-FABP in myocardial cells of a strong expression, and there was no obvious loss of District 4℃for 4 d endometrial myocardial under some small pieces missing. It showed irregularity punctiform, focus, patch depletion regions with ununiform distribution in ischemic myocardial cytoplasm, and the positive reaction was weakened in partial regions. Compared with the serial paraffin section stained with HE, the endochylema of cadiocytes showed fortified eosnophilia, nucleus contract with deep staining. There is a clear boundary between the normal positive expression of H-FABP in normal myocardium and the weakened positive expression of H-FABP at the junction of infracted zone and non-infracted myocardium. The comparison and statistical analysis between the depletion areas of H-FABP in various postmortem interval group show: While samples stored at 4℃for 3 days after death, the intensity of positive expression without significant weakened and the depletion areas without increased of H-FABP in two groups. The results of image analysis indicate that the intensity of positive reaction trends weaker as well as bigger in the depletion area along with prolongation of postmortem interval. Stored at 4℃for 4 days after death, there were significant differences in the depletion areas of H-FABP between the ischemic groups and the control groups(p<0.05). After 5 day , the depletion area of H-FABP in every study group is not significant different from that in their relative control group(p>0.05).Conclusion:1. There can be loss of H-FABP in myocardial ischemia cells in rats after 15 minutes, and with the ischemia intervals prolonged, the deletion areas increased gradually of myocardial cells. The deletion areas of H-FABP reached a peak at four hours of myocardial ischemia. H-FABP is a sensitive detection of myocardial ischemia, and can be detected the deletion area in the ischemic or infracted myocardial cells during 3 hours.2. The stability of H-FABP was relatively small influenced of autolysis. While stored at 4℃within four days, the depletion areas of H-FABP in ischemic myocardial of rabbit is significant different from that in the normal myocardial. So it can be conclude that the immunohistochemical detection of H-FABP can be used in corpse stored at 4℃for 4d or less than 4d after death, and is helpful for postmortem diagnosis of AMI.