Novel ratio-expressions of genes enables estimation of wound age in contused skeletal muscle.

Given that combination with multiple biomarkers may well raise the predictive value of wound age, it appears critically essential to identify new features under the limited cost. For this purpose, the present study explored whether the gene expression ratios provide unique time information as an additional indicator for wound age estimation not requiring the detection of new biomarkers and allowing full use of the available data. The expression levels of four wound-healing genes (Arid5a, Ier3, Stom, and Lcp1) were detected by real-time polymerase chain reaction, and a total of six expression ratios were calculated among these four genes. The results showed that the expression levels of four genes and six ratios of expression changed time-dependent during wound repair. The six expression ratios provided additional temporal information, distinct from the four genes analyzed separately by principal component analysis. The overall performance metrics for cross-validation and external validation of four typical prediction models were improved when six ratios of expression were added as additional input variables. Overall, expression ratios among genes provide temporal information and have excellent potential as predictive markers for wound age estimation. Combining the expression levels of genes with ratio-expression of genes may allow for more accurate estimates of the time of injury.

MicroRNA-133b Dysregulation in a Mouse Model of Cervical Contusion Injury.

Our previous research studies have demonstrated the role of microRNA133b (miR133b) in healing the contused spinal cord when administered either intranasally or intravenously 24 h following an injury. While our data showed beneficial effects of exogenous miR133b delivered within hours of a spinal cord injury (SCI), the kinetics of endogenous miR133b levels in the contused spinal cord and rostral/caudal segments of the injury were not fully investigated. In this study, we examined the miR133b dysregulation in a mouse model of moderate unilateral contusion injury at the fifth cervical (C5) level. Between 30 min and 7 days post-injury, mice were euthanized and tissues were collected from different areas of the spinal cord, ipsilateral and contralateral prefrontal motor cortices, and off-targets such as lung and spleen. The endogenous level of miR133b was determined by RT-qPCR. We found that after SCI, (a) most changes in miR133b level were restricted to the injured area with very limited alterations in the rostral and caudal parts relative to the injury site, (b) acute changes in the endogenous levels were predominantly specific to the lesion site with delayed miR133b changes in the motor cortex, and (c) ipsilateral and contralateral hemispheres responded differently to unilateral SCI. Our results suggest that the therapeutic window for exogenous miR133b therapy begins earlier than 24 h post-injury and potentially lasts longer than 7 days.

Cortical stimulation leads to shortened myelin sheaths and increased axonal branching in spared axons after cervical spinal cord injury.

Neural activity and learning lead to myelin sheath plasticity in the intact central nervous system (CNS), but this plasticity has not been well-studied after CNS injury. In the context of spinal cord injury (SCI), demyelination occurs at the lesion site and natural remyelination of surviving axons can take months. To determine if neural activity modulates myelin and axon plasticity in the injured, adult CNS, we electrically stimulated the contralesional motor cortex at 10 Hz to drive neural activity in the corticospinal tract of rats with sub-chronic spinal contusion injuries. We quantified myelin and axonal characteristics by tracing corticospinal axons rostral to and at the lesion epicenter and identifying nodes of Ranvier by immunohistochemistry. Three weeks of daily stimulation induced very short myelin sheaths, axon branching, and thinner axons outside of the lesion zone, where remodeling has not previously been reported. Surprisingly, remodeling was particularly robust rostral to the injury which suggests that electrical stimulation can promote white matter plasticity even in areas not directly demyelinated by the contusion. Stimulation did not alter myelin or axons at the lesion site, which suggests that neuronal activity does not contribute to myelin remodeling near the injury in the sub-chronic period. These data are the first to demonstrate wide-scale remodeling of nodal and myelin structures of a mature, long-tract motor pathway in response to electrical stimulation. This finding suggests that neuromodulation promotes white matter plasticity in intact regions of pathways after injury and raises intriguing questions regarding the interplay between axonal and myelin plasticity.

Pathological hemodynamic changes and leukocyte transmigration disrupt the blood-spinal cord barrier after spinal cord injury.

BACKGROUND: Blood-spinal cord barrier (BSCB) disruption is a key event after spinal cord injury (SCI), which permits unfavorable blood-derived substances to enter the neural tissue and exacerbates secondary injury. However, limited mechanical impact is usually followed by a large-scale BSCB disruption in SCI. How the BSCB disruption is propagated along the spinal cord in the acute period of SCI remains unclear. Thus, strategies for appropriate clinical treatment are lacking. METHODS: A SCI contusion mouse model was established in wild-type and LysM-YFP transgenic mice. In vivo two-photon imaging and complementary studies, including immunostaining, capillary western blotting, and whole-tissue clearing, were performed to monitor BSCB disruption and verify relevant injury mechanisms. Clinically applied target temperature management (TTM) to reduce the core body temperature was tested for the efficacy of attenuating BSCB disruption. RESULTS: Barrier leakage was detected in the contusion epicenter within several minutes and then gradually spread to more distant regions. Membrane expression of the main tight junction proteins remained unaltered at four hours post-injury. Many junctional gaps emerged in paracellular tight junctions at the small vessels from multiple spinal cord segments at 15 min post-injury. A previously unnoticed pathological hemodynamic change was observed in the venous system, which likely facilitated gap formation and barrier leakage by exerting abnormal physical force on the BSCB. Leukocytes were quickly initiated to transverse through the BSCB within 30 min post-SCI, actively facilitating gap formation and barrier leakage. Inducing leukocyte transmigration generated gap formation and barrier leakage. Furthermore, pharmacological alleviation of pathological hemodynamic changes or leukocyte transmigration reduced gap formation and barrier leakage. TTM had very little protective effects on the BSCB in the early period of SCI other than partially alleviating leukocyte infiltration. CONCLUSIONS: Our data show that BSCB disruption in the early period of SCI is a secondary change, which is indicated by widespread gap formation in tight junctions. Pathological hemodynamic changes and leukocyte transmigration contribute to gap formation, which could advance our understanding of BSCB disruption and provide new clues for potential treatment strategies. Ultimately, TTM is inadequate to protect the BSCB in early SCI.

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.

Proteomics and bioinformatics reveal insights into neuroinflammation in the acute to subacute phases in rat models of spinal cord contusion injury.

Neuroinflammation is recognized as a hallmark of spinal cord injury (SCI). Although neuroinflammation is an important pathogenic factor that leads to secondary injuries after SCI, neuroprotective anti-inflammatory treatments remain ineffective in the management of SCI. Moreover, the molecular signatures involved in the pathophysiological changes that occur during the course of SCI remain ambiguous. The current study investigated the proteins and pathways involved in C5 spinal cord hemi-contusion injury using a rat model by means of 4-D label-free proteomic analysis. Furthermore, two Gene Expression Omnibus (GEO) transcriptomic datasets, Western blot assays, and immunofluorescent staining were used to validate the expression levels and localization of dysregulated proteins. The present study observed that the rat models of SCI were associated with the enrichment of proteins related to the complement and coagulation cascades, cholesterol metabolism, and lysosome pathway throughout the acute and subacute phases of injury. Intriguingly, the current study also observed that 75 genes were significantly altered in both the GEO datasets, including ANXA1, C1QC, CTSZ, GM2A, GPNMB, and PYCARD. Further temporal clustering analysis revealed that the continuously upregulated protein cluster was associated with immune response, lipid regulation, lysosome pathway, and myeloid cells. Additionally, five proteins were further validated by means of Western blot assays and the immunofluorescent staining showed that these proteins coexisted with the F4/80+ reactive microglia and infiltrating macrophages. In conclusion, the proteomic data pertaining to the current study indicate the notable proteins and pathways that may be novel therapeutic targets for the treatment of SCI.

Prolonged Chronic Stress and Persistent Iron Dysregulation Prevent Anemia Recovery Following Trauma.

Introduction Following major trauma, persistent injury-associated anemia is associated with organ failure, increased length of stay and mortality. We hypothesize that prolonged adrenergic stimulation following trauma is directly responsible for persistent iron dysfunction that impairs anemia recovery. Materials and Methods Naïve rodents, lung contusion and hemorrhagic shock followed by daily handling for 13 d (LCHS), LCHS followed by 6 d of restraint stress and 7 d of daily handling (LCHS/CS-7) and LCHS/CS followed by 13 d of restraint stress with day and/or night disruption (LCHS/CS-14) were sacrificed on day 14. Hemoglobin, plasma, urine, bone marrow/liver inflammatory and erythropoietic markers were analyzed. Results LCHS/CS-14 led to a significant decline in weight gain and persistently elevated plasma and urine inflammatory markers. Liver IL-6, IL-1ß and hepcidin expression were significantly increased following LCHS/CS-14. LCHS/CS-14 also had impaired anemia recovery with reduced plasma transferrin and erythropoietin receptor expression. Conclusion Prolonged chronic stress following trauma/hemorrhagic shock led to sustained inflammation with increased expression of IL-1ß, IL-6 and hepcidin with decreased iron availability for uptake into erythroid progenitor cells and a lack of anemia recovery.

Mediators of Prolonged Hematopoietic Progenitor Cell Mobilization After Severe Trauma.

BACKGROUND: This study investigated the molecular mediators of prolonged hematopoietic progenitor cell mobilization a trauma and chronic stress and the role of propranolol in modifying this response. METHODS: Sprague-Dawley rats were randomized to lung contusion (LC), LC plus hemorrhagic shock (LCHS), or LCHS with daily restraint stress (LCHS/CS). Propranolol was administered daily. Bone marrow (BM) and lung expression of high mobility group box 1 (HMGB1), granulocyte colony-stimulating factor (G-CSF), neutrophil elastase, stromal cell-derived factor 1 (SDF-1)/CXR4, and vascular cell adhesion protein 1 (VCAM-1)/very late antigen-4 were measured by real-time polymerase chain reaction. RESULTS: Bone marrow HMGB1, G-CSF, and neutrophil elastase expression were significantly elevated two- to four-fold after LCHS/CS, and all were decreased with the use of propranolol. SDF-1 and VCAM-1 were both significantly decreased after LCHS/CS. CONCLUSIONS: The increased expression of HMGB1 and G-CSF and decreased expression of BM anchoring molecules, SDF-1 and VCAM-1, after LCHS/CS, likely mediates prolonged hematopoietic progenitor cell mobilization. Propranolol's ability to reduce HMGB1, G-CSF, and neutrophil elastase expression suggests that the mobilization of hematopoietic progenitor cells was driven by persistent hypercatecholaminemia.

Noninvasive Monitoring of Dynamical Processes in Bruised Human Skin Using Diffuse Reflectance Spectroscopy and Pulsed Photothermal Radiometry.

We have augmented a recently introduced method for noninvasive analysis of skin structure and composition and applied it to monitoring of dynamical processes in traumatic bruises. The approach combines diffuse reflectance spectroscopy in visible spectral range and pulsed photothermal radiometry. Data from both techniques are analyzed simultaneously using a numerical model of light and heat transport in a four-layer model of human skin. Compared to the earlier presented approach, the newly introduced elements include two additional chromophores (ß-carotene and bilirubin), individually adjusted thickness of the papillary dermal layer, and analysis of the bruised site using baseline values assessed from intact skin in its vicinity. Analyses of traumatic bruises in three volunteers over a period of 16 days clearly indicate a gradual, yet substantial increase of the dermal blood content and reduction of its oxygenation level in the first days after injury. This is followed by the emergence of bilirubin and relaxation of all model parameters towards the values characteristic for healthy skin approximately two weeks after the injury. The assessed parameter values and time dependences are consistent with existing literature. Thus, the presented methodology offers a viable approach for objective characterization of the bruise healing process.

Novel insights into wound age estimation: combined with "up, no change, or down" system and cosine similarity in python environment.

Wound age estimation is a complex, multifactorial issue. It is considered to have great practical significance that combining multi-biomarkers and multi-methods for injury time estimation. We optimized our earlier "up, no change, or down" model by adding data on the expression levels of mRNAs encoding ABHD2, MAD2L2, and ARID5A, and we converted the relative quantitative expression levels of seven genes into a vector rather than a color model. We used Python to derive the cosine similarity (CS) between a test set and the vector matrix; the highest similarity most accurately reflected the injury time. For the optimized model, the internal and external verifications were approximately 0.71 and 0.66, respectively. The good double-blinded results indicated that the model was stable and reliable. In summary, we used a vector matrix and cosine similarities derived by Python to mine the levels of genes expressed in contused skeletal muscle. We are the first to combine several biomarkers and methods for wound age estimation.

Melittin - A bee venom component - Enhances muscle regeneration factors expression in a mouse model of skeletal muscle contusion.

Melittin is a major peptide component of sweet bee venom that possesses anti-allergic, anti-inflammatory, anti-arthritis, anti-cancer, and neuroprotective properties. However, the therapeutic effects of melittin on muscle injury have not been elucidated. We investigated the therapeutic effects of melittin on muscle injury in a mouse model of muscle contusion. The biceps femoris muscle of the mice was injured using drop mass method, and the animals were treated with melittin (4, 20, or 100 µg/kg) for 7 days. Melittin significantly increased: locomotor activity in open field test, and treadmill running activity in a dose-dependent manner to level comparable to the positive control, diclofenac (30 mg/kg). Melittin treatment attenuated the pro-inflammatory cytokine MCP-1, TNF-α and IL-6. The expression of muscle regeneration biomarkers, including MyoD (muscle differentiation marker), myogenin, smooth muscle actin, and myosin heavy chain was markedly increased in the injured muscle tissue of melittin-treated mice, as determined by western blotting and quantitative real-time polymerase chain reaction. These results demonstrate that melittin inhibits inflammatory response and improves muscle damage by regenerating muscles in a mouse model of muscle contusion. Taken together, the results of present study suggest that melittin is a promising candidate for the muscle injury treatment.

Combination of mRNA of Repair-related Genes in Rat Skeletal Muscles for Wound Age Estimation.

ABSTRACT: 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.

Wound Age Estimation by Neutrophil Migration Distance.

ABSTRACT: 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.

Expressions of TGF-ß1 and Eâ ¢A-FN after Rat Skeletal Muscle Contusion and Wound Age Estimation.

ABSTRACT: Objective To study the expressions of transforming growth factor-ß1 （TGF-ß1） 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-ß1 and EⅢA-FN after rat skeletal muscles antemortem contusion were detected with immunohistochemical staining. Expression changes of TGF-ß1 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-ß1 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-ß1 and EⅢA-FN mRNA in antemortem injury group reached the peak at 3 and 5 d post-traumatic respectively. The expressions of TGF-ß1 and EⅢA-FN mRNA in antemortem contusion postmortem expression group peaked at 6 h and 12 h postmortem. The expression of TGF-ß1 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-ß1 and EⅢA-FN might become a reference index for skeletal muscle wound age estimation.

Molecular Characterization of Hypoxic Alveolar Epithelial Cells After Lung Contusion Indicates an Important Role for HIF-1α.

OBJECTIVE: To understand the fate and regulation of hypoxic type II alveolar epithelial cells (AECs) after lung contusion (LC). BACKGROUND: LC due to thoracic trauma is a major risk factor for the development of acute respiratory distress syndrome. AECs have recently been implicated as a primary driver of inflammation in LC. The main pathological consequence of LC is hypoxia, and a key mediator of adaptation to hypoxia is hypoxia-inducible factor (HIF)-1. We have recently published that HIF-1α is a major driver of acute inflammation after LC through type II AEC. METHODS: LC was induced in wild-type mice (C57BL/6), luciferase-based hypoxia reporter mice (ODD-Luc), and HIF-1α conditional knockout mice. The degree of hypoxia was assessed using hypoxyprobe and in vivo imaging system. The fate of hypoxic AEC was evaluated by luciferase dual staining with caspases-3 and Ki-67, terminal deoxynucleotidyl transferase dUTP nick end labeling, and flow cytometry with ApoStat. NLRP-3 expression was determined by western blot. Laser capture microdissection was used to isolate AECs in vivo, and collected RNA was analyzed by Q-PCR for HIF-related pathways. RESULTS: Global hypoxia was present after LC, but hypoxic foci were not uniform. Hypoxic AECs preferentially undergo apoptosis. There were significant reductions in NLRP-3 in HIF-1α conditional knockout mice. The expression of proteins involved in HIF-related pathways and inflammasome activation were significantly increased in hypoxic AECs. CONCLUSIONS: These are the first in vivo data to identify, isolate, and characterize hypoxic AECs. HIF-1α regulation through hypoxic AECs is critical to the initiation of acute inflammation after LC.

The monocyte locomotion inhibitory factor inhibits the expression of inflammation-induced cytokines following experimental contusion in rat tibia.

Entamoeba histolityca produces the monocyte locomotion inhibitory factor (MLIF), a pentapeptide with powerful anti-inflammatory properties. MLIF may regulate trauma-induced inflammation through the effects it exerts directly or indirectly on immune cells, modulating the production and/or expression of the cytokines involved in the inflammatory processes that occur after damage. The aim of the present study was to evaluate the effect of MLIF on production of pro/anti-inflammatory cytokines after contusion in the rat tibia. Fifty-four Wistar rats were subjected to controlled contusion with a special guillotine-type device, and 36 rats were injected with MLIF or tenoxicam into the tibia. Eighteen animals received saline; the animals were sacrificed 24 or 48 hours after injection. Cytokine mRNA and protein production were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), immunofluorescence, and hematoxylin-eosin staining was performed to visualize cellular infiltration in the rats' injured tissue. Expression levels of the cytokines interferon gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-beta (TGF-ß) mRNA were inhibited significantly by MLIF at 24 hours post-contusion. MLIF significantly increased the expression levels of IL-10 at 24 hours compared with tenoxicam or the control group. These changes were associated with a significant decrease in protein production levels of TNF-α, IFN-γ, IL-6 and TGF-ß at 24 hours. Histological evaluation showed the presence of infiltration by neutrophils, monocytes and leucocytes in control tissues. This infiltration was decreased after MLIF administration, and intense infiltration was observed in tenoxicam-treated group. MLIF inhibited the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokine IL-10.

The influence of hyperbaric environment on the skeletal muscle mitochondrial energetic of rats after induced muscle contusion.

OBJECTIVE: Analyze the influence of the hyperbaric environment on skeletal muscle mitochondrial bioenergetic end-points of rats submitted to muscle contusion. METHODS: Twelve female Wistar rats were randomly assigned to three groups. All rats were submitted to muscle contusion in the right gastrocnemius through a standard protocol. The control group (C) remained under normobaric conditions without any treatment. The hyperbaric air (HB) and the hyperbaric oxygen (HBO2) groups had four sessions of HBO2 therapy 60 minutes, six, 12, 24 and 48 hours after the injury at 253.25 kPa (2.5 atmospheres absolute/ATA) with air or 100% oxygen, respectively. The animals were sacrificed 48 hours after muscle injury, and both muscles (injured and non-injured) were analyzed. Muscle mitochondrial bioenergetics and mitochondrial permeability transition pore (MPTP) susceptibility were evaluated. RESULTS: Significant differences were found in all parameters between the injured and the non-injured gastrocnemius in the C group. In the HB group, significantly better results concerning bioenergetics-related end points with complex I and II substrates where found in the right gastrocnemius, whereas in the HBO2 group the time to Vmax (time that elapsed until the faster swelling kinetics starts) was significantly higher and the swelling amplitude was significantly smaller than in other groups, which suggest a lower susceptibility to MPTP opening. CONCLUSION: The present data suggest that hyperbaric exposure, particularly with oxygen, positively modulates the efficiency of skeletal muscle mitochondria after muscle contusion.

CXCL1 and CXCR2 as potential markers for vital reactions in skin contusions.

Detection of the vitality of wounds is one of the most important issues in forensic practice. This study investigated mRNA and protein levels of CXCL1 and CXCR2 in skin wounds in mice and humans. Western blot analysis of CXCL1 and CXCR2 protein levels showed no difference between wounded and intact skin. However, mRNA levels demonstrated higher expression of CXCL1 and CXCR2 in contused mouse and human skin, compared with intact skin. At postmortem there were no remarkable changes in CXCL1 and CXCR2 mRNA levels in contused mouse skin. Increased mRNA expression was observed in contused mouse skin up to 96 h and 72 h after death for CXCL1 and CXCR2 respectively. In human samples of wounded skin, increased CXCL1 mRNA levels were detected up to 48 h after autopsy in all 5 cases, while increased CXCR2 mRNA levels were observed 48 h after autopsy in 4 of 5 cases. These findings suggest that the levels of CXCL1 and CXCR2 mRNA present in contused skin can be used as potential markers for a vital reaction in forensic practice.

Persistent injury-associated anemia: the role of the bone marrow microenvironment.

BACKGROUND: The regulation of erythropoiesis involves hematopoietic progenitor cells, bone marrow stroma, and the microenvironment. Following severe injury, a hypercatecholamine state develops that is associated with increased mobilization of hematopoietic progenitor cells to peripheral blood and decreased growth of bone marrow erythroid progenitor cells that manifests clinically as a persistent injury-associated anemia. Changes within the bone marrow microenvironment influence the development of erythroid progenitor cells. Therefore, we sought to determine the effects of lung contusion, hemorrhagic shock, and chronic stress on the hematopoietic cytokine response. MATERIALS AND METHODS: Bone marrow was obtained from male Sprague-Dawley rats (n = 6/group) killed 7 d after lung contusion followed by hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1ß, interleukin-10, stem cell factor, transforming growth factor-ß, high-mobility group box-1 (HMGB-1), and B-cell lymphoma-extra large. RESULTS: Seven days following LCHS and LCHS/CS, bone marrow expression of prohematopoietic cytokines (interleukin-1ß, interleukin-10, stem cell factor, and transforming growth factor-ß) was significantly decreased, and bone marrow expression of HMGB-1 was significantly increased. B-cell lymphoma-extra large bone marrow expression was not affected by LCHS or LCHS/CS (naïve: 44 ± 12, LCHS: 44 ± 12, LCHS/CS: 37 ± 1, all P > 0.05). CONCLUSIONS: The bone marrow microenvironment was significantly altered following severe trauma in a rodent model. Prohematopoietic cytokines were downregulated, and the proinflammatory cytokine HMGB-1 had increased bone marrow expression. Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia.

Time Course of Inflammatory Gene Expression Following Crush Injury in Murine Skeletal Muscle.

BACKGROUND: Early inflammation and secretion of proinflammatory cytokines such as IL-1ß, IL-6, and TNF-α act as the key drivers to regulate inflammation after muscle injury. However, the effects of these key proinflammatory drivers in a noninvasive crush injury model are not well known. Understanding these effects is important for treating crush injuries that occur during natural disasters and military conflicts. PURPOSE: We studied the timed mRNA expression of IL-1ß, IL-6, and TNF-α in a noninvasive murine crush injury model to further understand their impact on proinflammatory cytokine pathways that are activated within the first 48 hours after a crush muscle injury. METHODS: A total of 25 mice were anesthetized and placed on a crush injury apparatus platform with the apparatus piston situated in direct contact with intact skin overlying the right gastrocnemius muscle. Pressure at 45 psi was applied to the piston for 30 seconds for two applications. The mice recovered for either 4, 8, 24, or 48 hours postinjury, after which we harvested the gastrocnemius muscle of both legs. Microarray, confirmatory real-time polymerase chain reaction, and immunolabeling experiments were followed by a microarray time-course analysis. RESULTS: Muscle IL-1ß mRNA rose 270-fold within 4 hours and declined rapidly at 8 hours to 196-fold, 24 hours to 96-fold, and 48 hours to 10-fold. Muscle IL-6 followed the same pattern, with a 34-fold increase at 4 hours, 29-fold increase at 8 hours, 10-fold increase at 24 hours, and 5-fold increase at 48 hours. Ingenuity Pathway Analysis of IL-6 identified activation of two major downstream signaling pathways (IL-6/Stat3 and IL-1ß/Egr1) as key activators of inflammation, regeneration, and fibrosis. DISCUSSION: Closed crush muscle injury produced robust muscle cytokine expression levels, and the microarray findings allowed us to generate our most novel hypothesis: that high expression of IL-1ß, IL-6, and TNF-α may be related to the downregulation of mitochondrial genes early after injury and triggers activation of genes in the repair and fibrosis machinery. The significance of these findings and the identified expression pathways of IL1-ß, IL-6, and TNF-α and their downstream targets in skeletal muscle will allow us to further investigate targets for improved muscle recovery and limb-saving interventions.