Expression of Autophagy-Associated Protein in Rat Muscle Tissues after Antemortem and Postmortem Injury / 法医学杂志

Objective To study the expression of the three autophagy-associated proteins, BECN1, LC3 and p62, after the injury of the skeletal muscle of rats and to explore its application in differentiation between antemortem and postmortem injury. Methods The 72 healthy Sprague-Dawley rats were randomly divided into the undamaged control group, the antemortem injury group （0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h） and postmortem injury group （0.5 h, 1 h, 2 h and 4 h）. A model of the injured right hind limb of rats was constructed. The expressions of the autophagy-associated proteins, BECN1, LC3-2/LC3-1 and p62, in the control group, the antemortem injury group and postmortem injury group were detected by Western blotting method. The data were respectively centralized and standardized and the orthogonal partial least square-discrimination analysis （OPLS-DA） identification model of antemortem and postmortem injury groups was constructed. Results The expression of BECN1, p62 protein and LC3-2/LC3-1 after the injury of the skeletal muscle of the rats showed different degrees of changes, but the differences among the 3 groups had no statistical significance. Antemortem and postmortem injury groups can be distinguished by centralizing and standardizing the expression levels of autophagy protein BECN1 and the ratio of LC3-2/LC3-1. The principal components extracted from OPLS-DA model of antemortem injury and postmortem injury had a relatively good interpretation of the model （Rx2=0.563, Ry2=0.439）, but it were less predictive （Q2=0.366）. Conclusion The expression of BECN1 and the ratio of LC3-2/LC3-1 in injured local tissue of the rat skeletal muscle can be used for the differentiation of antemortem injury group and postmortem injury group.

Wound Age Estimation by Neutrophil Migration Distance / 法医学杂志

Objective To explore the application of neutrophil migration distance for wound age estimation of skeletal muscles in rats, and to provide methodological basis for follow-up study in future. Methods The skeletal muscle contusion model was established in rats, and the control group and the 2, 4, 6 h post-traumatic groups were set. The law of response of neutrophils that participated in the inflammation after injury was detected by immunohistochemical staining, and the relationship between neutrophil migration distance and injury time was detected by TissueFAXS PLUS software. Results The skeletal muscle was obviously infiltrated with neutrophils 2-6 h after injury. The positive rate of neutrophil was （28.75±0.94）% at 2 h post-traumatic, and reached the peak （45.50±3.63）% at 4 h post-traumatic, then decreased to （31.92±1.56）% at 6 h post-traumatic. The neutrophil migration distances increased with the progress of inflammation, and reached （124.80±12.32） μm, （229.03±21.45） μm and （335.04±16.75） μm at 2 h, 4 h and 6 h, respectively. Conclusion There is a relationship of neutrophil infiltrated number and migration distance and wound age within the 2-6 h after skeletal muscle injury, which could be used for the inference of skeletal muscle wound age.

Combination of mRNA of Repair-related Genes in Rat Skeletal Muscles for Wound Age Estimation / 法医学杂志

Objective To investigate the estimation of early and mid-term wound age by a combination of four mRNAs, the DNA polymerase delta-interacting protein 3 （POLDIP3） mRNA, regulator of chromosome condensation 1 like （RCC1L） mRNA, proline-rich 5 （PRR5） mRNA, and ribonucleic acid export 1 （RAE1） mRNA in rats skeletal muscles. Methods The model of rat skeletal muscle contusion was established, and then contusion area muscle tissue was extracted 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 h after injury. Histomorphological changes during the repair process after rat skeletal muscle contusion were observed. The relative expressions of Poldip3, Rcc1l, Prr5 and Rae1 mRNAs were detected by reverse transcription real-time quantitative polymerase chain reaction （RT-qPCR）. Different stages of wound age were classified by using the expression patterns of four genes at various time points after injury. The accuracy of the results was verified by Fisher discriminant analysis. Results Histomorphological results showed that the repair process after skeletal muscle contusion occurred with the prolonging of time. Through combination of the expression trends of the four kinds of mRNAs, the 48 h after injury could be divided into three periods, 4-12 h, 16-28 h and 32-48 h. The Fisher discrimination method showed that the classification accuracy rates of the three periods were 83.3%, 75.0% and 73.3%, respectively. Conclusion The classification discrimination based on the relative expression of every gene has a higher accuracy, and the accuracy of wound age estimation with combination of mRNA relative expressions is higher than that with a single indicator. By combining with Fisher discrimination method, this method can be used for early and mid-term wound age estimation.