| Background and ObjectiveTime of death (Postmortem Interval, PMI) inference is the focus of the study for forensic science. Indeed, it is important to the administration of justice such as the confirmation of the perpetrators, the detection of criminal cases and death medical disputes, inheritance and even the practice of arbitration.There are many studies on the traditional methods of estimation of time since death, including judging through the phenomenon of early body and the digestion of gastric content or other methods to infer the early PMI, or according to the level of corruption body and the growth of myiasis or other methods to infer the late PMI. However, there are unavoidably some errors in these methods because of many factors, multifarious detection methods and all kinds of complex problem.In recent years, many researchers use DNA and protein degradation in molecular biology techniques to infer PMI, which are more accurate and reliable, but they are still affected by its own and outside influential factors, and complicated to operate, thus remaining at a research stage. It is very important for PMI inference to control the experimental conditions.On the basis of studies from the other members of the task about rats PMI in the air, this experiment study the underwater corpses prior to controlling experimental conditions, such as the temperature, air circulation. Since underwater corpses are as the objects, we hope to probe into the relations between sequential changes that have parenchyma biomechanical traits with the law of PMI, so as to bring forensic biomechanical theory and technology into inferring PMI, to further enriching the content of forensic biomechanics, and lastly to develop new technical methods for PMI inference. At the same time, the experimental use of CDIA technology, observing the conventional HE staining of liver cells as pathological computer digital image analysis and quantification, together with systematic observation of the main viscera of the underwater bodies in the pathological form, with the expectations of inference for PMI in an effort to provide new ideas and reference.Materials and Methods90 clean healthy male SD rats, 3 to 3.5 months of age and weighing 250-300g, were needed. They were killed by cervical dislocation and were put into a large plastic barrel,which were 2 / 3 full of water, and then the barrel should be installed into the GXZ intelligent illumination incubators in 25±0.5℃constant temperature, so that each barrel of water could keep at constant temperature after the state of warming up.Placed the SD rats in barrels; used the wide fundus plastic rods to stress the rats into the water completely underneath the surface to about 5 cm, so that they could naturally corrupt. In 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 60h, 72h, 96h, 120h, 144h, 168h, 192h,distill skin 1×4cm2 from the center of the chest, abdominal muscle 1×4cm2 from abdominal white line near 1cm right, colon into 4~10cm where was part of the ileum as the large intestine, stomach below 6~12cm as the small intestine to detect biological mechanics.It was necessary to picked five rats each time and to test those soft tissue biomechanical targets: max load, tensile stress, Strain and other indicators. Meanwhile, it was also essential to select the HE stained brain, heart, liver, and kidney and lung biopsy under the microscope at a different time to observe. The corruption of the organizations should be observed since the dissolution of the morphological changes, and inspected rat liver cells pathological images and analysis as well as giving them quantized treatment. Moreover, the karyoplasmic proportion, karyoplasmic ratio, the karyoplasmic density radio and karyoplasmic index of density of liver cells should be detected by using SPSS statistical software for processing and analysis, in order to explore the relationship between PMI and the changes by the law of PMI.Results1 Main organs organizations morphological changesIt took SD rat about 8 days from death to cadaveric lividity, to cadaveric rigidity, to vein network corruption, to abdominal distention, and lastly to collapse (Fig.ure1).Through the observation with HE staining lens, it showed from each viscera sequential changes. There was karyopyknosis in the in the neurons cells in 0-24h, and there was karyoplasmic amplification to the brain liquefaction in 24-48h. There was sarcoplasm swelling, muscle gap widened of the heart in 0-24h; there was sarcoplasm blurring,fracture, muscle cells aryolysis,fragmentation and disappearance in 24-48 h, The outline of tissues gradually cleared out in 48-72h; in muscle cells, there was engorgement, and the gap lessening till no boundaries in 0-24h, and there was liver cells pyknosis, fracture and dissolution till disappearance in 24-48h. The outline of the tissues blurred in 48-72h. The acidophilia of proximal renal tubular epithelial cells enhanced in 0-24h, the cell nucleus of epithelium and the glomerulus had pyknosis, fragmentation and dissolution till disappearance in 24-72h, while nucleus completely disappeared in 96h. It showed from lung that alveolus lung epithelial cytochylema became hypochromatic in 0-24h, and alveolar epithelial cells had shrinkage, fragmentation, dissolution until disappearance in 24-48h. The alveolar liquid deposited in 0-24h till the lung tissue completely broken, structure unclear in 48-72h (Table 1).After compared with various viscera, it was found that the brain autolyzed and corrupted fastest, then came the liver, the heart, and the lung, and the kidney autolysis ranked slowest. Thereinto, the brain has been liquefied, so it could not already be cut into slices at 48h. The liver, heart and the lung had lost their organizational structure in 72h. The kidney autolysis corruption became slower, and cell nucleus totally disappeared in 96h, and the organizational outline became ill-defined in 120h.2 Biomechanical testing and analysis of results2.1 Max loadThe means of five organizations'Max load sorting from high to low were as follows: skin (14.71N)> colon (3.96N)> muscle (3.85N)> vascular (1.97N)> small intestine (1.27N). It showed from the whole organizations after death an overall downward trend while there were differences among the rate of declining points in time.It showed from skin a rapid decline in 0-18 h (the linear regression equation was YPMI=33.087-1.3121XML, r= 0.999), and adjacent points were significantly different (P<0.01), then, it showed a slow downward trend in 18-36h,96-168h(YPMI=120.153-9.009XML, r=0.965;YPMI=13.822-0.0485XML,r=0.941,P<0.05), 36-96h,168-192h, the adjacent points in time was not obviously different (P>0.05).It has showed from muscle a clear downward trend in 0-24h, 96-168h trend (the linear regression equations were YPMI=42.914-4.9861XML,r=-0.989;YPMI=177.6-24.289XML,r=-0.970).It was "platform period" in 24-96h, and during 168-192h, the difference was not obvious (P>0.05).It showed from colon an evident downward trend in 0-36h, 72-192h (YPMI=66.238-8.6983XML,r=-0.981;YPMI=225.14-41.488XML,r=-0.978), and it was "platform period" in 36-72 h. It has showed from the small intestine an obvious downward trend in 0-18h (YPMI=53.903-27.422XML,r=-0.968), 120-192h (YPMI=350.55-200.57XML,r=-0.986). It was the "platform period"(P>0.05). it didn't showed from vascular an obvious trend in the whole process.2.2 StrainIt showed from vascular Strain that the largest was 3.96, but it was not obvious trend. It showed from small intestine that its Strain had reached the minimum, which was 0.34. It showed from skin, muscle, large intestine, and small intestine that it was an overall downward trend.It showed from skin that it decreased rapidly in 0-24h (YPMI=88.894-77.665XSR ,r=-0.962), but decreased slowly in 60-120h(YPMI=52.112-41.544 XSR,r=-0.970), and it was"platform period"in 120-192h.It showed from muscle that it decreased rapidly in 0-30h(YPMI=59.106-55.031XSR,r=-0.970), and was the"platform period"in 30-192h.It showed from colon that it decreased rapidly in 0-24h(P<0.01,YPMI =307.03-346.59 XSR,r=-0.962), and it was the "platform period" in 24-72h, and it was an obvious downward trend(P<0.01)It showed from small intestine that it declined rapidly in 0-24h (YPMI =56.758-87.079 XSR,r=-0.949), and was the "platform period" in 24h-192h.3 Liver cells pathological image quantification by computer digitalIt showed from liver cells that there was no statistically significance in different time(P>0.05). It was in an upward trend in 24-48h, and there was no statistically significance in 48h and 24h (P>0.05).It showed from liver cells that the karyoplasmic ratio was a declining trend (the linear regression equation: YPMI=0.3683-0.0045XRKC,r=-0.9361), which the rate of decreasing ranked the most in 12-24h;it slowed down in 0-12h,24-72h. Pair wise time point comparisons in each time period were statistically significant (P<0.05).It showed from liver cells that the karyoplasmic density ratio was a gradual upward trend in 0-36h ( Y=69.751X-78.117,r=0.982 ) , and was a gradual downward trend in 36-72h(Y=198.44-97.676X,r=0.999), an upward trend in 12-36h, and it decreased in nearly a straight line in 36-72h. The pair wise time point comparison in each time period were statistically significant (P<0.05).It showed that the index of density was an upward trend(YPMI=30.177XRKC-31.707,r=0.989), it sped up the fastest in 12-36h, in the next was 0-12h(P<0.05), and there was no statistically significance in 48-72h (P>0.05).. Conclusion1 It had been shown there existed obvious relevance between the PMI itself and morphological changes happened in different PMI from different underwater corpses'organs, which was mainly the result of autocytolysis and corruption. The results suggested that the daily forensic pathology tests should pay attention to the identification of diffuse disease before death and the change of autolytic corruption after death, on the basis of which could make an objective scientific diagnosis. The underwater corpses reached its peak at 72h of its dissolution and corruption, so it's better to freeze the body or to have autopsy after death in 72h.2 There was obvious changes in the sequential changes in each tissues'and organs'biomechanical character, in particular there was obvious linear pertinence with PMI, which was called "window period"; what's more, a different "window period" could make up for each other and suggest that the parenchyma biomechanical indicators and method could be a simple, objective, quantitative test facility to infer the time of death.3 The technology of computer digital pathological image analysis could be used to detect described various indicators of conventional HE cells, which could quantitatively analyze cells'disease before death and reflect the level of morphological changes of autolytic corruption after death. In particular, some relative compared indicators of the histiocyte itself could have been avoided the effect from different producers, staining effect , as a result, it could more objectively response to the condition of the disease and the classification standards, thus providing a new way of thinking for quantization pathology.
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