A fundamental study on postmortem submersion interval estimation by metabolomics analyzing of gastrocnemius muscle from submersed rat models in freshwater.

In forensic practice, determining the postmortem submersion interval (PMSI) and cause-of-death of cadavers in aquatic ecosystems has always been challenging task. Traditional approaches are not yet able to address these issues effectively and adequately. Our previous study proposed novel models to predict the PMSI and cause-of-death based on metabolites of blood from rats immersed in freshwater. However, with the advance of putrefaction, it is hardly to obtain blood samples beyond 3 days postmortem. To further assess the feasibility of PMSI estimation and drowning diagnosis in the later postmortem phase, gastrocnemius, the more degradation-resistant tissue, was collected from drowned rats and postmortem submersion model in freshwater immediately after death, and at 1 day, 3 days, 5 days, 7 days, and 10 days postmortem respectively. Then the samples were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the dynamic changes of the metabolites. A total of 924 metabolites were identified. Similar chronological changes of gastrocnemius metabolites were observed in the drowning and postmortem submersion groups. The difference in metabolic profiles between drowning and postmortem submersion groups was only evident in the initial 1 day postmortem, which was faded as the PMSI extension. Nineteen metabolites representing temporally-dynamic patterns were selected as biomarkers for PMSI estimation. A regression model was built based on these biomarkers with random forest algorithm, which yielded a mean absolute error (± SE) of 5.856 (± 1.296) h on validation samples from an independent experiment. These findings added to our knowledge of chronological changes in muscle metabolites from submerged vertebrate remains during decomposition, which provided a new perspective for PMSI estimation.

Detection of multiple biomarkers associated with satellite cell fate in the contused skeletal muscle of rats for wound age estimation.

From the perspective of forensic wound age estimation, experiments related to skeletal muscle regeneration after injury have rarely been reported. Here, we examined the time-dependent expression patterns of multiple biomarkers associated with satellite cell fate, including the transcription factor paired box 7 (Pax7), myoblast determination protein (MyoD), myogenin, and insulin-like growth factor (IGF-1), using immunohistochemistry, western blotting, and quantitative real-time PCR in contused skeletal muscle. An animal model of skeletal muscle contusion was established in 30 Sprague-Dawley male rats, and another five rats were employed as non-contused controls. Morphometrically, the data obtained from the numbers of Pax7 + , MyoD + , and myogenin + cells were highly correlated with the wound age. Pax7, MyoD, myogenin, and IGF-1 expression patterns were upregulated after injury at both the mRNA and protein levels. Pax7, MyoD, and myogenin protein expression levels confirmed the results of the morphometrical analysis. Additionally, the relative quantity of IGF-1 protein > 0.92 suggested a wound age of 3 to 7 days. The relative quantity of Pax7 mRNA > 2.44 also suggested a wound age of 3 to 7 days. Relative quantities of Myod1, Myog, and Igf1 mRNA expression > 2.78, > 7.80, or > 3.13, respectively, indicated a wound age of approximately 3 days. In conclusion, the expression levels of Pax7, MyoD, myogenin, and IGF-1 were upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting the potential for using them as candidate biomarkers for wound age estimation in skeletal muscle.

A preliminary study on early postmortem submersion interval (PMSI) estimation and cause-of-death discrimination based on nontargeted metabolomics and machine learning algorithms.

Postmortem submersion interval (PMSI) estimation and cause-of-death discrimination of corpses in water have long been challenges in forensic practice. Recently, many studies have linked postmortem metabolic changes with PMI extension, providing a potential strategy for estimating PMSI using the metabolome. Additionally, there is a lack of potential indicators with high sensitivity and specificity for drowning identification. In the present study, we profiled the untargeted metabolome of blood samples from drowning and postmortem submersion rats at different PMSIs within 24 h by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 601 metabolites were detected. Four different machine learning algorithms, including random forest (RF), partial least squares (PLS), support vector machine (SVM), and neural network (NN), were used to compare the efficiency of the machine learning methods. Nineteen metabolites with obvious temporal regularity were selected as candidate biomarkers according to "IncNodePurity." Robust models were built with these biomarkers, which yielded a mean absolute error of 1.067 h. Additionally, 36 other metabolites were identified to build the classifier model for discriminating drowning and postmortem submersion (AUC = 1, accuracy = 95%). Our results demonstrated the potential application of metabolomics combined with machine learning in PMSI estimation and cause-of-death discrimination.

Candidate biomarkers in brown adipose tissue for post-mortem diagnosis of fatal hypothermia.

Post-mortem diagnosis of fatal hypothermia (FHT) is challenging in forensic practice because traditional morphological and biochemical methods lack specificity. Recent studies have reported that brown adipose tissue (BAT) is activated during cold-induced non-shivering thermogenesis in mammals, but BAT has not been used to diagnose FHT. The aim of this study was to identify novel biomarkers in BAT for FHT based on morphological changes and differential protein expression. Two FHT animal models were created by exposing mice to 4 or -20 °C at 50% humidity. Morphologically, the unilocular lipid droplet content was significantly increased in BAT of FHT model mice compared with that of control mice. Proteomics analysis revealed a total of 283 and 266 differentially expressed proteins (DEPs) between the 4 or -20 °C FHT subgroups and control group, respectively. In addition, 140 proteins were shared between the FHT subgroups. GO and KEGG analyses revealed that the shared DEPs were mainly enriched in pathways associated with metabolism, oxidative phosphorylation, and thermogenesis. Further screening (|log2FC| > 1.6, q-value (FDR) < 0.05) identified GMFB, KDM1A, DDX6, RAB1B, SHMT-1, CLPTM1, and LMF1 as candidate biomarkers of FHT. Subsequent validation experiments were performed in FHT model mice using classic immunohistochemistry and western blotting. RAB1B and GMFB expression was further verified in BAT specimens from human cases of FHT. The results demonstrate that BAT can be used as a target organ for FHT diagnosis employing RAB1B and GMFB as biological markers, thus providing a new strategy for the post-mortem diagnosis of FHT in forensic practice.

Inferring Postmortem Submersion Interval in Rats Found in Water Based on Vitreous Humor Metabolites.

OBJECTIVES: The metabolomics technique of LC-MS/MS combined with data analysis was used to detect changes and differences in metabolic profiles in the vitreous humor of early rat carcasses found in water, and to explore the feasibility of its use for early postmortem submersion interval (PMSI) estimation and the cause of death determination. METHODS: The experimental model was established in natural lake water with 100 SD rats were randomly divided into a drowning group (n=50) and a postmortem (CO2 suffocation) immediately submersion group (n=50). Vitreous humor was extracted from 10 rats in each group at 0, 6, 12, 18 and 24 h postmortem for metabolomics analyses, of which 8 were used as the training set to build the model, and 2 were used as test set. PCA and PLS multivariate statistical analysis were performed to explore the differences in metabolic profiles among PMSI and causes of death in the training set samples. Then random forest (RF) algorithm was used to screen several biomarkers to establish a model. RESULTS: PCA and PLS analysis showed that the metabolic profiles had time regularity, but no differences were found among different causes of death. Thirteen small molecule biomarkers with good temporal correlation were selected by RF algorithm. A simple PMSI estimation model was constructed based on this indicator set, and the data of the test samples showed the mean absolute error (MAE) of the model was 0.847 h. CONCLUSIONS: The 13 metabolic markers screened in the vitreous humor of rat corpses in water had good correlations with the early PMSI. The simplified PMSI estimation model constructed by RF can be used to estimate the PMSI. Additionally, the metabolic profiles of vitreous humor cannot be used for early identification of cause of death in water carcasses.

Application Prospect of MicroRNA in Skin Wound Age Estimation.

Wound age estimation is one of the major tasks in forensic practice. However, relatively accurate estimation of the wound age is still a conundrum and research spotlight world-widely. Studies show that microRNAs (miRNAs) are involved in the whole process of the skin wound repair, and miRNAs, as biomarkers, might be used to estimate the time of skin injury owing to their characteristic advantage. This paper summarizes the miRNA fundamental function, properties, current research progress in the estimation of wound age, and its limitations, and put forward prospect of potential application and research based on miRNAs in estimation of wound age.

A novel approach for the forensic diagnosis of drowning by microbiological analysis with next-generation sequencing and unweighted UniFrac-based PCoA.

The diagnosis of drowning is one of the major challenges in forensic practice, especially when the corpse is in a state of decomposition. Novel indicators of drowning are desired in the field of forensic medicine. In the past decade, aquatic bacteria have attracted great attention from forensic experts because they can easily enter the blood circulation with drowning medium, and some of them can proliferate in the corpse. Recently, the advent of next-generation sequencing (NGS) has created new opportunities to efficiently analyze whole microbial communities and has catalyzed the development of forensic microbiology. We presumed that NGS could be a potential method for diagnosing drowning. In the present study, we verified this hypothesis by fundamental experiments in drowned and postmortem-submersed rat models. Our study revealed that detecting the bacterial communities with NGS and processing the data in a transparent way with unweighted UniFrac-based principal coordinates analysis (PCoA) could clearly discriminate the skin, lung, blood, and liver specimens of the drowning group and postmortem submersion group. Furthermore, the acquired information could be used to identify new cases. Taken together, these results suggest that we could build a microbial database of drowned and postmortem-submersed victims by NGS and subsequently use a bioinformatic method to diagnose drowning in future forensic practice.

Evaluation of specific neural marker GAP-43 and TH combined with Masson-trichrome staining for forensic autopsy cases with old myocardial infarction.

It has been a puzzling forensic task to determine the cause of death as a result of old myocardial infarction (OMI) in the absence of recognizable acute myocardial infarction. Recent studies indicated that the heterogeneous cardiac nerve sprouting and sympathetic hyperinnervation at border zones of the infarcted site played important roles in sudden cardiac death (SCD). So, the present study explored the value of growth associated protein-43 (GAP-43) and tyrosine hydroxylase (TH) as objective and specific neural biomarkers combined with Masson-trichrome staining for forensic autopsy cases. Myocardium of left ventricle of 58 medicolegal autopsy cases, 12 OMI cases, 12 acute/OMI cases, and 34 control cases, were immunostained with anti-GAP-43 and anti-TH antibodies. Immunoreactivity of GAP-43 and TH identified nerve fibers and vascular wall in OMI cases and acute/OMI cases. Specifically, TH-positive nerve fibers were abundant at border zones of the infarcted site. There were a few GAP-43 and TH expressions in the control cases. With Masson-trichrome staining, collagen fibers were blue and cardiac muscle fibers were pink in marked contrast with the surrounding tissue, which improved the location of nerve fibers. Thus, these findings suggest that immunohistochemical detection of GAP-43 and TH combined with Masson-trichrome staining can provide the evidence for the medicolegal expertise of SCD due to OMI, and further demonstrate a close relationship between sympathetic hyperinnervation and SCD.

Detection of satellite cells during skeletal muscle wound healing in rats: time-dependent expressions of Pax7 and MyoD in relation to wound age.

The study was focused on time-dependent expressions of paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) during skeletal muscle wound healing. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17, and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. By morphometric analysis, the data based on the number of Pax7(+)/MyoD(-), Pax7(+)/MyoD(+), and Pax7(-)/MyoD(+) cells were highly correlated with the wound age. Pax7 and MyoD expressions were upregulated after injury by Western blot and quantitative real-time PCR assays. The relative quantity of Pax7 protein peaked at 5 days after injury, which was >1.13, and decreased thereafter. Similarly, the relative quantity of MyoD mRNA expression peaked at 3 days after injury, which was >2.59. The relative quantity of Pax7 protein >0.73 or mRNA expression >2.38 or the relative quantity of MyoD protein >1.33 suggested a wound age of 3 to 7 days. The relative quantity of MyoD mRNA expression >2.02 suggested a wound age of 1 to 7 days post-injury. In conclusion, the expressions of Pax7 and MyoD are upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting that Pax7 and MyoD may be potential markers for wound age estimation in skeletal muscle.

[Effects of PI3K/Akt Pathway in Wound Healing Process of Mice Skin].

OBJECTIVE: To investigate the expressions and time-dependent changes of phosphatidylinositol-3-kinase (PI3K), phospho-PI3K (p-PI3K), protein kinase B (PKB/Akt) and phospho-Akt (p-Akt) during wound healing process of mice skin. METHODS: The changes of PI3K, p-PI3K, Akt and p-Akt expression in skin wound were detected by immunohistochemistry, Western blotting and real-time PCR. RESULTS: Immunohistochemistry showed the expression of PI3K and p-Akt were observed in mononuclear and fibroblast after skin wound, and reached peak in reconstruction. The positive bands of PI3K, p-PI3K, Akt and p-Akt were observed in all time points of the wound healing process by Western blotting. The expression peak of p-PI3K and p-Akt showed in inflammation and proliferation; the expression peak of PI3K and Akt in reconstruction. Real-time PCR showed the expression peak of PI3K mRNA in inflammation and reconstruction and the peak of Akt mRNA in reconstruction. CONCLUSION: During the wound healing process, the expressions of PI3K, Akt, p-PI3K and p-Akt show different changes with significant correlation to wound time. The expression of PI3K/Akt may be a valuable marker for wound time estimation.

Time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and its application for wound age determination.

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.

Molecular pathology of brain matrix metalloproteases, claudin5, and aquaporins in forensic autopsy cases with special regard to methamphetamine intoxication.

Methamphetamine (METH) is a highly addictive drug of abuse and toxic to the brain. Recent studies indicated that besides direct damage to dopamine and 5-HT terminals, neurotoxicity of METH may also result from its ability to modify the structure of blood-brain barrier (BBB). The present study investigated the postmortem brain mRNA and immunohistochemical expressions of matrix metalloproteases (MMPs), claudin5 (CLDN5), and aquaporins (AQPs) in forensic autopsy cases of carbon monoxide (n = 14), METH (n = 21), and phenobarbital (n = 17) intoxication, compared with mechanical asphyxia (n = 15), brain injury (n = 11), non-brain injury (n = 21), and sharp instrument injury (n = 15) cases. Relative mRNA quantification using Taqman real-time PCR assay demonstrated higher expression of AQP4 and MMP9, lower expression of CLDN5 in METH intoxication cases and lower expression of MMP2 in phenobarbital intoxication cases. Immunostaining results showed substantial interindividual variations in each group, showing no evident differences in distribution or intensity among all the causes of death. These findings suggest that METH may increase BBB permeability by altering CLDN5 and MMP9, and the self-protective system maybe activated to eliminate accumulating water from the extracellular space of the brain by up-regulating AQP4. Systematic analysis of gene expressions using real-time PCR may be a useful procedure in forensic death investigation.

[Correlation between percentages of PMN, MNC, FBC and wound age after skeletal muscle injury in rats].

OBJECTIVE: To study the percentages of polymorphonuclear leukocytes (PMN), mononuclear cells (MNC) and fibroblastic cells (FBC) in different post-traumatic intervals after skeletal muscle mechanical injury in rats. METHODS: The rat model of skeletal muscle mechanical injury was established. The rats were divided into injured groups (6 h, 12 h, 1 d, 3 d, 7 d, 10 d and 14 d after injury) and control group. The percentages of PMN, MNC and FBC in different post-traumatic intervals after skeletal muscle mechanical injury were assessed with HE staining and image analysis. RESULTS: At post-injury 6-12h, the percentages of PMN and MNC infiltration appeared in injured sites and that of PMN reached peak. At 1 d, the percentage of MNC infiltration appeared and reached peak, while that of PMN decreased. At 3-7 d, the percentage of FBC gradually increased, while that of PMN and MNC decreased. At 10-14d, the percentage of FBC reached peak. CONCLUSION: The percentages of PMN, MNC and FBC in injured zones showed time-dependent changes, which might be used as reference index for determination of age of skeletal muscle injury.

Molecular pathology of brain edema after severe burns in forensic autopsy cases with special regard to the importance of reference gene selection.

Brain edema is believed to be linked to high mortality incidence after severe burns. The present study investigated the molecular pathology of brain damage and responses involving brain edema in forensic autopsy cases of fire fatality (n = 55) compared with sudden cardiac death (n = 11), mechanical asphyxia (n = 13), and non-brain injury cases (n = 22). Postmortem mRNA and immunohistochemical expressions of aquaporins (AQPs), claudin5 (CLDN5), and matrix metalloproteinases (MMPs) were examined. Prolonged deaths due to severe burns showed an increase in brain water content, but relative mRNA quantification, using different normalization methods, showed inconsistent results: in prolonged deaths due to severe burns, higher expression levels were detected for all markers when three previously validated reference genes, PES1, POLR2A, and IPO8, were used for normalization, higher for AQP1 and MMP9 when GAPDH alone was used for normalization and higher for MMP9, but lower for MMP2 when B2M alone was used for normalization. Additionally, when B2M alone was used for normalization, higher expression of AQP4 was detected in acute fire deaths. Furthermore, the expression stability values of these five reference genes calculated by geNorm demonstrated that B2M was the least stable one, followed by GAPDH. In immunostaining, only AQP1 and MMP9 showed differences among the causes of death: they were evident in most prolonged deaths due to severe burns. These findings suggest that systematic analysis of gene expressions using real-time PCR might be a useful procedure in forensic death investigation, and validation of reference genes is crucial.

[Progress in myofibroblast and its application in forensic medicine].

The myofibroblasts have dual characteristics of smooth muscle cells and fibroblasts. In repairing tissular wound, myofibroblasts are involved in fibrogenesis and remodeling the extracellular matrix of the fibrotic cascades reaction. The review describes the morphological characteristics and biological behaviors of myofibroblasts and the application of skin wound age determination, which may provide reference for research in forensic medicine.

Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper.

In forensic molecular pathology, quantitative real-time polymerase chain reaction (RT-qPCR) provides a rapid and sensitive method to investigate functional changes in the death process. Accurate and reliable relative RT-qPCR requires ideal amplification efficiencies of target and reference genes. However, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements. The present study evaluated 32 potential reference genes in the human brain of 15 forensic autopsy cases using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On RT-qPCR data analyses using a completely objective and noise-resistant algorithm (Real-time PCR Miner), 24 genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested IPO8 and POLR2A as the most stable ones, and NormFinder indicated that IPO8 and POP4 had the highest expression stabilities, while BestKeeper highlighted ABL1 and ELF1 as reference genes with the least overall variation. Combining these three algorithms suggested the genes IPO8, POLR2A, and PES1 as stable endogenous references in RT-qPCR analysis of human brain samples, with YWHAZ, PPIA, HPRT1, and TBP being the least stable ones. These findings are inconsistent with those of previous studies. Moreover, the relative stability of target and reference genes remains unknown. These observations suggest that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of target genes in practical applications.

Molecular pathology of pulmonary edema after injury in forensic autopsy cases.

The lung is vulnerable to trauma; pulmonary edema starts quickly as part of the systemic responses involved in shock. The present study investigated the molecular pathology of posttraumatic alveolar damage and responses involving pulmonary edema in forensic autopsy cases of injury (n = 66) compared with acute cardiac death cases (n = 13). Intrapulmonary mRNA and immunohistochemical expressions of matrix metalloproteinases (MMPs; MMP-2 and MMP-9), intercellular adhesion molecule-1, claudin-5, and aquaporins (AQPs, AQP-1 and AQP-5) were examined. Subacute injury deaths showed an increase in lung weight similar to that in acute cardiac death, but relative mRNA quantification using the Taqman real-time PCR assay demonstrated different findings among the causes of death; higher expressions were detected for all markers, except for AQP-5 in sharp instrument injury, for MMP-2 in blunt brain injury, and for MMP-9 in non-brain blunt injury, but these expression levels were lower in acute cardiac death. In immunostaining, only MMPs showed differences among the causes of death: MMP-2 expression was evident in most subacute deaths due to blunt brain injury and sharp instrument injury, whereas MMP-9 was intensely positive in those of non-brain blunt injury and sharp instrument injury. These findings suggest significant differences in the mechanism of pulmonary edema among fatal injuries and acute cardiac death, especially between blunt and sharp instrument injury. Systematic analysis of gene expressions using real-time PCR in combination with immunohistochemistry may be useful in evaluating pulmonary damage and responses after injury in death investigations, especially in connection with posttraumatic shock.

Cannabinoid receptor type 2 is time-dependently expressed during skin wound healing in mice.

Dynamic localization of CB2R and quantitative analysis of CB2R mRNA during skin wound healing in mice were performed. Co-localization of CB2R with F4/80 or α-SMA was detected by double-color immunofluorescence microscopy. A total of 110 male mice were divided into control, injury, and postmortem groups. Sixty-five mice were sacrificed, followed by sampling at 0.5 h-21 days post-injury. Five mice without incision were used as control. The other 40 mice that received incised wound were sacrificed at 5 days after injury. The samples were collected at 0 h-3 days postmortem. In the uninjured controls, CB2R immunoreactivity was detected in the epidermis, hair follicles, sebaceous glands, dermomuscular layer, and vascular smooth muscle. In the incision groups, polymorphonulcear cells, macrophages, and myofibroblasts showed positive staining for CB2R. Morphometrically, the average ratios of CB2R-positive cells were more than 50 % at 5 days post-wounding, whereas it was <50 % at the other posttraumatic intervals. The average ratios of CB2R-positive macrophages maximized at 3 days post-wounding, and the average ratios of CB2R-positive myofibroblasts peaked at 5 days post-wounding. The relative quantity of CB2R mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >4.10, which was also confirmed by Western blotting. There was no significant change for CB2R protein within 6 h postmortem and for mRNA within 3 h postmortem as compared with the control group. In conclusion, dynamic distribution and expression of CB2R suggest that CB2R is involved in modulating macrophages and myofibroblasts in response to inflammatory event and repair process in mouse skin wound healing, and CB2R is available as a marker for wound age determination.

[Establishment of skin scald model in mice].

OBJECTIVE: To establish the model of skin scald in mice for the study of skin thermal injuries. METHODS: After anaesthetization mice were scalded in a 1 cm-diameter circle area on the central dorsum by boiling water at contact times: 10s or 25s. The mice were sacrificed at 1, 3, 5, 7 and 10 days after scald. The skin samples were collected and analyzed by gross and histopathological examinations. RESULTS: Deep II degree thermal injury involving full-thickness skin was observed in the 10s scald group. III degree thermal injury involving full-thickness skin and the dorsal skeletal muscle was observed in the 25 s scald group. CONCLUSION: A mouse skin scald model is established which is stable and can be used on the skin thermal injury in future research.

[The number of circulating fibrocytes of skeletal muscle in rats after contusion].

OBJECTION: To investigate the time-dependent appearance of circulating fibrocytes of skeletal muscle in rats after contusion. METHODS: The model of skeletal muscle wound was established in rat. The circulating fibrocytes in contused skeletal muscle were detected by CD45 and procollagen I double immunofluorescence staining method. RESULTS: In the control group, CD45- and procollagen I-positive cells were not detected in skeletal muscle. A few CD45 cells were observed aged from 6 h to 1 d after contusion. A few CD45- and procollagen I-positive cells (fibrocytes) initially gathered in injury area 3d after injury. The ratio of positive fibrocytes significantly increased 5 d after injury. The ratio of fibrocytes was highest at 7 d after contusion and then decreased. The volume of fibrocytes showed bigger with injury time increase compared with 3 d group. The expression of procollagen I and CD45 were weakened at 14d after injury. CONCLUSION: The circulating fibrocytes are detected in contused skeletal muscle in time-dependent pattern. Circulating fibrocytes may be a marker in the wound age determination for contused skeletal muscle.