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.

Expressions of TGF-β1 and EⅢA-FN after Rat Skeletal Muscle Contusion and Wound Age Estimation / 法医学杂志

Objective To study the expressions of transforming growth factor-β1 （TGF-β1） and EⅢA-fibronectin （EⅢA-FN） at different time points of antemortem injury, antemortem injury postmortem expression and postmortem injury and to explore their application value in wound age estimation. Methods A model of rat skeletal muscle contusion was established. The rats were randomly divided into normal control group （n=5）, antemortem contusion group （n=40）, antemortem contusion postmortem expression group （n=110） and postmortem injury group （n=25）. The expressions of TGF-β1 and EⅢA-FN after rat skeletal muscles antemortem contusion were detected with immunohistochemical staining. Expression changes of TGF-β1 and EⅢA-FN mRNA in each group were analyzed with real-time fluorescence quantitative PCR. Results Immunohistochemical staining results showed that a large number of polymorphonuclear leukocyte, mononuclear cells and fibroblastic cells showed a strong expression of TGF-β1 in wounded zones 12 h-14 d after antemortem contusion. EⅢA-FN was mainly distributed in the extracellular matrix, 3 to 7 d post-traumatic. Real-time fluorescence quantitative PCR results showed that TGF-β1 and EⅢA-FN mRNA in antemortem injury group reached the peak at 3 and 5 d post-traumatic respectively. The expressions of TGF-β1 and EⅢA-FN mRNA in antemortem contusion postmortem expression group peaked at 6 h and 12 h postmortem. The expression of TGF-β1 and EⅢA-FN mRNA in postmortem injury group 0.5-12 h postmortem was significantly lower than those of the normal control group and the antemortem contusion group. Conclusion TGF-β1 and EⅢA-FN might become a reference index for skeletal muscle wound age estimation.

Expression of SFRP5 mRNA in Rat Skeletal Muscle after Contusion / 法医学杂志

OBJECTIVE@#To investigate the relationship between the expression of secreted frizzled-related protein 5 (SFRP5) mRNA and the time interval after skeletal muscle injury in rats by real-time PCR.@*METHODS@#A total of ninety SD rats were randomly divided into the contusion groups at different times including 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h after contusion, incision groups at different times including 4h and 8h after incision and the control group. The samples were taken from the contused zone at different time points. The total RNA was isolated from the samples and reversely transcribed to analyze the expression levels of SFRP5 mRNA.@*RESULTS@#Compared to the control group, the expression of SFRP5 mRNA in contusion groups were down-regulated within 48 h after contusion and reached the lowest level at 20 h, and the expression of SFRP5 mRNA gradually increased from 20 h to 48 h after contusion. The expression of SFRP5 mRNA in the incised groups were significantly lower than that of the contusion groups at 4 h after injury. At the time of 8 h, the expression levels between the contusion and incision groups showed no statistically significant difference.@*CONCLUSION@#It is suggested that SFRP5 mRNA analysis may show regular expression and can be a marker for estimation of skeletal muscle injury age.

Relation between Injury Time and the Expression of COX6C mRNA in Skeletal Muscle of Rats after Contusion / 法医学杂志

OBJECTIVE@#To investigate the relation between injury time and the expression of cytochrome c oxidase subunit VIc (COX6C) mRNA in skeletal muscle of rat after contusion.@*METHODS@#A total of fifty-four SD rats were divided into the control group and the contusion groups (0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion), randomly. The contusion model was established by free fall drop of gravity hammer. At corresponding time point after contusion, the regular histology was examined and expression level of COX6C mRNA was tested by real-time PCR after extraction of total RNA from the tissues.@*RESULTS@#The main pathological features of 6 h after injury included edema and hemorrhage in myocytes with no inflammatory cells found. After 6 hours, the findings included myocyte degeneration and necrosis, inflammatory cells infiltration, and fibrous connective tissue proliferation in the contused zone. The expression level of COX6C mRNA was higher than that of the control group within 6 h after contusion. The expression level was lower than that of the control group from 6-36 h after contusion.@*CONCLUSION@#The level of COX6C mRNA expresses in a regular way after contusion. It may be useful for estimating wound age in combination with the results of pathological features.

Expression of p35 and p25 after Focal Cerebral Contusion in Rat / 法医学杂志

OBJECTIVE@#To study the expression of p35 and p25 in rat after focal cerebral contusion and to provide experimental data for estimating brain injury time.@*METHODS@#Fifty adult male SD rats were randomly divided into 0 h, 6 h, 12 h, 24 h, 3 d, 5 d, 7 d, 10 d after focal cerebral contusion, control and sham-operated groups (5 rats each group). The focal cerebral contusion rat model was established. The expression of p35 and p25 protein of the damage peripheral zone in brain were detected by HE staining, immunohistochemistry and Western blotting at different injury time.@*RESULTS@#A large number of p35 protein and a small amount of p25 protein were expressed in control group and sham-operated group. After focal cerebral contusion, p35 presented unimodal change with time and p25 presented bimodal changes with time.@*CONCLUSION@#Expression of p35 and p25 showed different regularity with good time correlation, which could help to estimate the brain injury time.

Time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and its application for wound age determination / 法医学杂志

OBJECTIVE@#To research the relation between the time-dependent appearances of myotibroblasts during the repair of contused skeletal muscle in rat and wound age determination.@*METHODS@#A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas.@*RESULTS@#Immunohistochemical staining showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval.@*CONCLUSION@#The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.

Expression of microtubule associated protein-2 mRNA after cerebral contusion / 法医学杂志

OBJECTIVE@#To explore the mRNA expression of microtubule associated protein-2 (MAP-2) after cerebral contusion, and to investigate its relationship with post-injury interval in a rat model of cerebral contusion.@*METHODS@#Based on Feeney's model of cerebral contusion, the time-dependent changes of MAP-2 mRNA were detected by real-time PCR method in different groups.@*RESULTS@#MAP-2 mRNA showed a lower expression in the brain of the control group. The expression of MAP-2 mRNA decreased 1 h post-injury, and reached the lowest level at 6 h, and the expression of MAP-2 mRNA gradually increased in the focus of the cerebral contusion from 12h to 14d after contusion, which kept a high level up to 14 d post-injury.@*CONCLUSION@#The time-dependent changes of MAP-2 mRNA expression may be a new method for estimating the wound age of cerebral contusion in forensic practice.

Changes of aquaporins 1 expression in the contused lung of rats / 法医学杂志

OBJECTIVE@#To investigate the expression changes of aquaporins 1 (AQP1) in contused lung tissue of rats and its relationship with pulmonary edema.@*METHODS@#SD rats were randomly divided into experimental and control groups. The pulmonary contusion models were then prepared. The expression and distribution of AQP1 in lung tissue of the rats were detected by immunohistochemistry.@*RESULTS@#The lung tissue showed edema, hemorrhage, inflammatory cell infiltration 1 h, 3 h after pulmonary contusion, and the inflammatory response aggravated after 5 h. AQP1 expression at 1 h, 3 h and 5 h in the contusion group were significantly higher than that of the control group (P < 0.01). The expression of AQP1 continued to increase with time and aggravation of edema compared to the control group. AQP1 was mainly distributed in the capillary endothelial cells and interstitial cells of the bronchial and alveolar walls. Although there were no observed changes in AQP1 expression location in contused lung tissue, the intergrated optical density(IOD) showed significant statistical difference (P < 0.01).@*CONCLUSION@#There might exist an dysregulation of AQPs gene expression in contused lung tissue, leading to a large number of abnormal transmembrane water transportation and abnormal water accumulation, which may be one of the reasons for pulmonary edema in contused lung tissue.

Changes of ICAM-1 mRNA expression in rats' skin and muscle after contusion / 法医学杂志

OBJECTIVE@#To investigate the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in contused skin and muscle of rats and the relationship between the ICAM-1 expression and the wound age.@*METHODS@#The samples were taken at 0.5, 1, 6, 12, 18, 24 and 30 h after contusion of rats, respectively. Total RNA was extracted both from the skin and muscle samples of each group and reverse transcription polymerase chain reaction (RT-PCR) was conducted to synthesize the 1st strand cDNA. The amount of ICAM-1 mRNA in each sample was quantified using rp132 intrinsic fluorescent assay and compared by the 2 (-Delta Delta Ct) method with that from control samples.@*RESULTS@#After contusion the expression of ICAM-1 mRNA in skin increased rapidly and peaked at 0.5 h, at 24 h degraded to the amount that was seven times as much as the control group, then rised again. The expression of ICAM-1 mRNA in muscle increased significantly within 0.5 h and peaked at 6 h, reached the minimum at 18 h, then increased again.@*CONCLUSION@#It is suggested that ICAM-1 mRNA analysis may be useful for estimation of early wound age because of its time-related expression after contusion in skin and muscle.