The distribution and time-dependent expression of HIPK2 during the repair of contused skeletal muscle in mice.

HIPK2 is an evolutionarily conserved serine/threonine kinase and is considered a co-regulator of an increasing number of transcription factors modulating a variety of cellular processes, including inflammation, proliferation and fibrosis. Skeletal muscle injuries repair is an overlapping event between inflammation and tissue repair. There are no reports about HIPK2 expression in skeletal muscles after trauma. A foundational study on distribution and time-dependent expression of HIPK2 was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR, which is expected to obtain a preliminary insight into the functions of HIPK2 during the repair of contused skeletal muscle in mice. An animal model of skeletal muscle contusion was established in 50 C57B6/L male mice. Samples were taken at 1, 3, 5, 7, 9, 14, 17, 21 and 28 days after contusion, respectively (5 mice at each posttraumatic interval). 5 mice were employed as control. No HIPK2-positive staining was detected in uninjured skeletal muscle. Intensive immunoreactivties of HIPK2 were observed in polymorphonuclear cells, round-shaped mononuclear cells, regenerated multinucleated myotubes and spindle-shaped fibroblastic cells in the contused tissue. The HIPK2-positive cells were identified as neutrophils, macrophages and myofibroblasts by double immunofluorescent procedure. HIPK2 protein and mRNA expression were remarkably up-regulated after contusion by Western blotting and qPCR analysis. The results demonstrated that the expression of HIPK2 is distributed in certain cell types and is time-dependently expressed in skeletal muscle after contusion, which suggested that HIPK2 may participate in the whole process of skeletal muscle wound healing, including inflammatory response, muscle regeneration and fibrogenesis.

The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats.

Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and pro-inflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB1 and CB2 receptors.

Heme oxygenase-1 and heme oxygenase-2 expression in bruises.

The first step in catabolism of hemoglobin in a bruise is performed by the enzyme heme oxygenase, which produces biliverdin that is then reduced to bilirubin. The development of yellow coloration in bruises can be attributed to local accumulation of degradation products of hemoglobin, including bilirubin, but it is not clear why there is a delay before this color change is apparent. One explanation may be that time is required for the establishment of heme oxygenase activity at the bruise site. This study used immunohistochemistry to examine the time course of expression of heme oxygenase-1 and heme oxygenase-2 in a rat bruise model. Heme oxygenase-1 levels rose above background from 6 h to peak from days 1 to 3. There was strong expression by macrophages, but only occasional neutrophils expression of heme oxygenase-1. Heme oxygenase-2 did not change significantly from background levels. The results suggest that the delay in the development of yellow coloration of bruises may in part be attributed to the requirement for macrophages to be recruited to the site of injury.

Hyperbaric oxygen therapy treatment for the recovery of muscle injury induced in rats.

INTRODUCTION: We evaluated the effect of hyperbaric oxygen treatment (HBOT) in the recovery of muscle injury in rats. MATERIALS AND METHODS: Twelve female Wistar rats, weighing 200-250 g, were submitted to contusion of the right gastrocnemius muscle. Animals were then randomly assigned to an untreated control group or an HBOT-treated group. The HBOT group was given three, 60-minute sessions of HBOT at 253 kPa pressure at 24, 48 and 72 hours post injury. After the last session all animals were sacrificed and both gastrocnemius muscles removed, the left muscle as an internal control. Blood samples were taken for creatine phosphokinase (CPK). Using a standard traction technique, the muscles were analysed for their biomechanical properties: hardness, maximum elongation and maximum weight. RESULTS: Significant differences were found between uninjured and injured muscles and between untreated and HBOT groups in maximum weight and hardness: maximum weight in the non-treated group 18.27 ± 2.99 N versus 26.18 ± 2.84 N in the HBOT group (P = 0.007); hardness in the non-treated group 2.24 ± 0.38 103 N m⁻¹ versus 3.19 ± 0.32 10³ N m⁻¹ in the HBOT group (P = 0.001). The difference in maximum elongation was not significant (P = 0.793). CPK was significantly different between the two groups (non-treated 6,445 ± 387 i.u. L⁻¹; HBOT 4,551 ± 80 i.u. L⁻¹; P = 0.009). CONCLUSIONS: HBOT seems to play a positive role in the recovery of induced muscle injury in rats. However relevant, these results cannot be extrapolated to humans, for whom further clinical studies are warranted.

Acute molecular perturbation of inducible nitric oxide synthase with an antisense approach enhances neuronal preservation and functional recovery after contusive spinal cord injury.

Inducible nitric oxide synthase (iNOS) is a key mediator of inflammation and oxidative stress produced during pathological conditions, including neurodegenerative diseases and central nervous system (CNS) injury. iNOS is responsible for the formation of high levels of nitric oxide (NO). The production of highly reactive and cytotoxic NO species, such as peroxynitrite, plays an important role in secondary tissue damage. We have previously demonstrated that acute administration of iNOS antisense oligonucleotides (ASOs) 3 h after moderate contusive spinal cord injury (SCI) potently inhibits iNOS-mediated increases in NO levels, leading to reduced blood-spinal cord barrier permeability, decreased neutrophil accumulation, and less neuronal cell death. In the current study we investigated if iNOS ASOs could also provide long-term (10-week) histological and behavioral improvements after moderate thoracic T8 contusive SCI. Adult rats were randomly assigned to three groups (n=10/group): SCI alone, SCI and mixed base control oligonucleotides (MBOs), or SCI and iNOS ASOs (200 nM). Oligonucleotides were administered by spinal superfusion 3 h after injury. Behavioral analysis (Basso-Beattie-Bresnahan [BBB] score and subscore) was employed weekly for 10 weeks post-SCI. Although animals treated with iNOS ASOs demonstrated no significant differences in BBB scores compared to controls, subscore analysis revealed a significant improvement in foot positioning, trunk stability, and tail clearance. Histologically, while no gross improvement in preserved white and gray matter was observed, greater numbers of surviving neurons were present adjacent to the lesion site in iNOS ASO-treated animals than controls. These results support the effectiveness of targeting iNOS acutely as a therapeutic approach after SCI.

Distinct time courses of secondary brain damage in the hippocampus following brain concussion and contusion in rats.

Secondary brain damage (SBD) is caused by apoptosis after traumatic brain injury that is classified into concussion and contusion. Brain concussion is temporary unconsciousness or confusion caused by a blow on the head without pathological changes, and contusion is a brain injury with hemorrhage and broad extravasations. In this study, we investigated the time-dependent changes of apoptosis in hippocampus after brain concussion and contusion using rat models. We generated the concussion by dropping a plumb on the dura from a height of 3.5 cm and the contusion by cauterizing the cerebral cortex. SBD was evaluated in the hippocampus by histopathological analyses and measuring caspase-3 activity that induces apoptotic neuronal cell death. The frequency of abnormal neuronal cells with vacuolation or nuclear condensation, or those with DNA fragmentation was remarkably increased at 1 hr after concussion (about 30% for each abnormality) from the pre-injury level (0%) and reached the highest level (about 50% for each) by 48 hrs, whereas the frequency of abnormal neuronal cells was increased at 1 hr after contusion (about 10%) and reached the highest level (about 40%) by 48 hrs. In parallel, caspase-3 activity was increased sevenfold in the hippocampus at 1 hr after concussion and returned to the pre-injury level by 48 hrs, whereas after contusion, caspase-3 activity was continuously increased to the highest level at 48 hrs (fivefold). Thus, anti-apoptotic-cell-death treatment to prevent SBD must be performed by 1 hr after concussion and at latest by 48 hrs after contusion.

Detection and possible role of proteasomes in the bronchoalveolar space of the injured lung.

Recent observations suggest the presence of 20S proteasomes (20S) in the lung epithelial lining fluid. However, the physiological relevance of 20S in the alveolar space and possible contribution to disease processes are unknown. Thus, we evaluated whether extracellular proteasomes could have a pathophysiological role in the injured lung using a rat model of lung contusion (LC). Bronchoalveolar lavage fluids (BALF) were obtained at various time points for up to 168 h after LC or sham procedure. Enzyme activities, ELISA and Western blots indicated enzymatically active 20S, the 19S subunit Rpt5 and ubiquitin in BALF. 20S and ubiquitin increased significantly after LC, peaked at 24 h and normalized within 168 h. Mg(2+)/ATP-dependent peptidase activities were detectable 6-24 h after LC. BALF after LC also contained ubiquitin-protein-ligase activity. Addition of Mg(2+)/ATP to BALF after LC led to significant proteolysis and could be prevented with epoxomicin and EDTA. These data suggest for the first time that the Mg(2+)/ATP-dependent 26S proteasome complex exists outside the cell, is released into the lung epithelial lining fluid after LC and contributes to the proteolysis of the bulk of protein in the alveolar space of the injured lung. We infer that proteasome complexes may have a pathophysiological role during lung edema clearance.

Expression levels of mRNAs for catecholamine biosynthetic enzymes as markers of acute response to contusion stress during the early postmortem period.

Various stresses can be followed by sudden unexpected deaths, and autopsies sometimes fail to identify pathological findings that determine the cause of death. Pathologists occasionally explain such deaths as being due to overstimulation of sympathoadrenal systems, but postmortem assessment of antemortem sympathoadrenal activity has not been established. An animal model of weight injuries was used to quantify sympathoadrenal response to contusion stress, which is common in forensic fields. A weight was dropped from a given height onto the right dorsal limb of each anesthetized rat, with a control group and three stress groups (n = 4, each): 1000 g-80 cm, 1000 g-40 cm, and 500 g-40 cm. To explore the postmortem changes, we also included ten groups comprised of control and 1000 g-80 cm groups, whose tissues were harvested during 12 hours after euthanasia. Real-time quantitative polymerase-chain reaction was performed to quantify relative expression levels of mRNAs for catecholamine biosynthetic enzymes in the adrenals and the anterocervical ganglia: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). The expression levels of all target mRNAs in the adrenals increased with the intensity of impact (TH, p < 0.0005; DBH and PNMT, p < 0.005), and particularly, TH mRNA level exhibited near-stepwise elevation (p < 0.05). In contrast, no significant differences were detected in the anterocervical ganglia. Moreover, these mRNA levels in the adrenals decreased with increasing postmortem interval length. Thus, TH mRNA level may be a good marker of sympathoadrenal response to contusion stress during the early postmortem period.

[Time-dependent expression of caspase-3 during contused skeletal muscle wound healing in rats].

OBJECTIVE: To investigate the expression of caspase-3 during contused skeletal muscle wound healing in rats and to explore the applicability of caspase-3 in wound age estimation. METHODS: Male Sprague-Dawley rats undergone anesthesia with 2% pentobarbital sodium (45 mg/kg) were struck by a single strike at the skeletal muscle of the right leg, then were sacrificed by cervical dislocation at different post-traumatic intervals. The changes of caspase-3 activity were assessed by imunohistochemistry, Western blot and caspase-3 colorimetric assay. The skeletal muscle of non-contusion rat's were used as controls. RESULTS: No caspase-3 was detected in skeletal muscle in the control group. The expression level of caspase-3 in the peripheral area of contused rat skeletal muscle increased gradually and maximized on the fifth day after injury, and then decreased gradually. Caspase-3 was also detected in the regenerated skeletal muscle within 7-14 days after injury. CONCLUSION: The expression of capase-3 in the peripheral area of contused rat skeletal muscle indicated that it may play a pivotal role in apoptosis of the wounded skeletal muscle and may be used as a marker for wound age estimation. The expression of capase-3 in regenerating skeletal muscle suggests that caspase-3 activity may be used for skeletal muscle differentiation.

Altered beta-1,4-galactosyltransferase I expression during early inflammation after spinal cord contusion injury.

Post-traumatic inflammation has been implicated in secondary tissue damage after spinal cord injury (SCI). beta-1,4-Galactosyltransferase I (beta-1,4-GalT-I) is a key inflammatory mediator that plays a critical role in the initiation and maintenance of inflammatory reaction in diseases. The aim of the current study was to investigate whether beta-1,4-GalT-I is expressed in SCI. Spinal cord contusion model was established in adult rats. Real-time PCR and Western blot analysis were used to detect the spatio-temporal expression of beta-1,4-GalT-I after SCI. Lectin-fluorescent staining with RCA-I was used to detect the galactosylation of the membrane glycoproteins. The interaction and colocalization between beta-1,4-GalT-I and E-selectin in the injured spinal cords were also assessed by immunoprecipitation of E-selectin and double immunofluorescent staining, respectively. Real-time PCR revealed that beta-1,4-GalT-I mRNA reached the peak at 1d after spinal cord contusion. In situ hybridization indicated that beta-1,4-GalT-I mRNA was mainly distributed in the local inflammatory cells, adjacent to the center of injury. Double immunofluorescent staining showed that beta-1,4-GalT-I mostly overlapped with ED1-positive macrophages 1d after SCI, partly colocalized with microglia, neutrophils and a few with oligodendrocytes and astrocytes. The result of Lectin-fluorescent staining with RCA-I was similar to that of double immunofluorescent staining. Terminal galactosylation of E-selectin underwent obvious changes between sham and 3d after SCI by immunoprecipitation of E-selectin. Thus, the transient expression of high levels of beta-1,4-GalT-I may provide new insight into the early inflammation after SCI.

Time-dependent expression of caspase-3 during contused skeletal muscle wound healing in rats / 法医学杂志

OBJECTIVE@#To investigate the expression of caspase-3 during contused skeletal muscle wound healing in rats and to explore the applicability of caspase-3 in wound age estimation.@*METHODS@#Male Sprague-Dawley rats undergone anesthesia with 2% pentobarbital sodium (45 mg/kg) were struck by a single strike at the skeletal muscle of the right leg, then were sacrificed by cervical dislocation at different post-traumatic intervals. The changes of caspase-3 activity were assessed by imunohistochemistry, Western blot and caspase-3 colorimetric assay. The skeletal muscle of non-contusion rat's were used as controls.@*RESULTS@#No caspase-3 was detected in skeletal muscle in the control group. The expression level of caspase-3 in the peripheral area of contused rat skeletal muscle increased gradually and maximized on the fifth day after injury, and then decreased gradually. Caspase-3 was also detected in the regenerated skeletal muscle within 7-14 days after injury.@*CONCLUSION@#The expression of capase-3 in the peripheral area of contused rat skeletal muscle indicated that it may play a pivotal role in apoptosis of the wounded skeletal muscle and may be used as a marker for wound age estimation. The expression of capase-3 in regenerating skeletal muscle suggests that caspase-3 activity may be used for skeletal muscle differentiation.

Matrix metalloproteinase-2 facilitates wound healing events that promote functional recovery after spinal cord injury.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are involved in both injury and repair mechanisms in the CNS. Pharmacological blockade of MMPs, limited to the first several days after spinal cord injury, improves locomotor recovery. This beneficial response is, however, lost when treatment is extended beyond the acutely injured cord to include wound healing and tissue remodeling. This suggests that some MMPs play a beneficial role in wound healing. To test this hypothesis, we investigated the role of MMP-2, which is actively expressed during wound healing, in white matter sparing and axonal plasticity, the formation of a glial scar, and locomotor recovery after spinal cord injury. MMP-2 increased between 7 and 14 d after injury, where it was immunolocalized in reactive astrocytes bordering the lesion epicenter. There was reduced white matter sparing and fewer serotonergic fibers, caudal to the lesion in injured MMP-2 null animals. MMP-2 deficiency also resulted in increased immunoreactivity to chondroitin sulfate proteoglycans and a more extensive astrocytic scar. Most importantly, locomotion in an open field, performance on a rotarod, and grid walking were significantly impaired in injured MMP-2 null mice. Our findings suggest that MMP-2 promotes functional recovery after injury by regulating the formation of a glial scar and white matter sparing and/or axonal plasticity. Thus, strategies exploiting MMPs as therapeutic targets must balance these beneficial effects during wound healing with their adverse interactions in the acutely injured spinal cord.

[The effect of carnosine on inflammatory processes during contusion of the eyeball].

The effect of dipeptide carnosine on proteolytic processes accompanying inflammation was investigated in lacrimal fluid of patients with eyeball contusion. Eye drops containing 5% carnosine in combination with traditional treatment reduced elastase-like activity in lacrimal fluid and increased effectiveness of therapy.

Case reports. 1. An autopsy case of fatal arrhythmia induced by injuries of the atrioventricular conduction system: a case report.

A 65-year-old woman died three days after being involved in a traffic accident, following an episode of ventricular fibrillation. She was diagnosed as having suffered cardiac contusion, liver contusion, mediastinal hematoma and rib fracture on admission. Her electrocardiogram showed complete right bundle branch block, complete atrioventricular block, and right axis deviation. Aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and creatine kinase-MB were found to be elevated on biochemical blood analysis. These findings recovered and her condition appeared to improve daily. At autopsy, epicardial and intramyocardial haemorrhage were macroscopically seen in the posterior wall of the bilateral ventricles. On microscopic examination, there was evidence of fresh haemorrhage and coagulative necrosis with inflammatory reaction in the ordinary myocardium and adipose tissue around the atrioventricular node, which had spread to the proximal portion of the His' bundle. It is considered that these findings caused ventricular fibrillation to occur, and that the cause of death in this case was myocardial contusion due to blunt thoracic injury. This case would indicate that myocardium nearby atrioventricular junction is vulnerable to external force. Moreover, it would seem that fatal arrhythmia occasionally occurs during the follow-up stage, despite the lack of any significant clinical findings.

Constitutive and inducible nitric oxide synthase activities after spinal cord contusion in rats.

Nitric oxide (NO) plays a role in the secondary damage after spinal cord (SC) injury. NO is produced by the activity of two classes of enzymes: calcium-dependent constitutive nitric oxide synthase (NOS) and calcium-independent inducible NOS. To determine the time course of both NOS activities after SC injury, 50 Wistar rats were submitted to severe SC contusion. NOS activities were assayed at the site of SC injury at several times after lesion. Results showed a significant increase of 138 and 96% in the constitutive NOS activity at 4 and 8 h after the lesion, respectively, as compared to sham-operated rats. iNOS activity was increased 72 h after lesion by 103% (P<0.05). In conclusion, both isoforms of NOS increase their activity at different time periods after SC injury.

[Overall trypsin-like, elastase-like and antitryptic activity in patients with eye contusion]. / Obshchaia tripsinopodobnaia élastazopodobnaia i antitripticheskaia aktivnost' u patsientov s kontuziei glaza.

The investigations of tear fluid of eye after contusion injury revealed on increase of trypsin-like activity in 1-3 day and 10-19 day after trauma, the progressively elevation of elastase-like activity and lowering of inhibitory potential along 2-3 week. It was detected indirect correlation between the elastase-like activity and severity of the contusion injury of the eye, the grade of corneal edema, corneal erosion and conjunctival wound, hyphema. This results was shown the participation and the role of proteolytic and inflammatory processes in pathogenesis of eye blunt trauma. It was establish that the during of local inflammation is 2 week end more and is necessary antiinflammatory therapy with use the proteases inhibitors.

Release kinetics of cardiac troponin I and cardiac troponin T in effluents from isolated perfused rabbit hearts after graded experimental myocardial contusion.

BACKGROUND: Few experimental studies report effects of direct contusion on cardiac enzyme release. Cardiac troponins I (cTnI) and T (cTnT) have been shown to be highly sensitive and specific markers of myocardial cell injury. This investigation was designed to determine and compare the acute effects of quantified magnitudes of blunt cardiac trauma upon release of cTnI and cTnT in comparison with creatine kinase (CK) and lactate dehydrogenase (LD). METHODS: In 24 rabbit hearts prepared on a standard Langendorff apparatus, myocardial contusion (MC) was produced by a single blow with a ball falling from a predefined height, delivered directly to the surface of the heart. Hearts were divided into control (n = 6) and various quantified impacts: 75 mJoules (mJ) (n = 6), 100 mJ (n = 6), 200 mJ (n = 6). Coronary effluent samples for cTnI, cTnT, CK, and LD were collected at baseline, immediately after MC and 5, 15, 30, 45, and 60 minutes after MC. At the end of experiment, histologic condition was evaluated. RESULTS: The anti-cTnI and cTnT MAbs used in the cTnI (Access) and cTnT (Elecsys) assays cross-react with cTnI and cTnT of the rabbit. The time-courses of cTnI, cTnT, CK, and LD were monophasic in form. After MC, all parameters rose significantly compared with baseline and with control group. The maximal release occurred immediately after MC. The area under the cTnI curve and the maximal cTnI concentration were linked to the contusion energy when increased at 200 mJ. Maximal concentrations and areas under cTnT, CK, LD time activity curve were not linked to the contusion energy level and showed no between-energy group differences. The correlation found between maximal cTnI and maximal cTnT concentrations was 0.70 (p = 0.0001). Histologic examination showed cellular disruption and after the more severe impact, the extent of pathologic changes was more extensive. CONCLUSION: After graded experimental MC, maximal cTnI concentration and area under cTnI curve increase with the power of impact kinetic energy. Levels of cTnI allow a much higher accuracy in detecting the extent of myocardial injury postMC in comparison with cTnT, CK, and LD in this experimental study. These results should be consistent with the more extensive cTnI release with more severe impact in patients with blunt chest trauma. Furthermore, because specificity and time-course of release, both cTnI and cTnT should have a role in the diagnosis and evaluation of such patients.

Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat.

The induction of heme oxygenase-1 (HO-1) was studied in intact spinal cords and injured spinal cords after a moderate, thoracic contusion injury. HO-1 was immunolocalized in the normal cord and along the axis of the cord at 1, 2, 3 and 4 days after contusion. Induction of this enzyme in astrocytes and microglia/macrophages was evaluated using immunofluorescent double labeling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor. HO-1 was expressed in neurons in the normal spinal cord. After contusion, HO-1 was induced in both gray and white matter at the impact site. In segments of cord that were 1 cm proximal or distal to the injury, HO-1 was primarily induced in the dorsal columns and occasionally in the lateral white matter. This pattern of induction was noted at all time points. The HO-1 was induced primarily in microglia/macrophages. The distribution of the HO-1 positive cells closely correlated with the pattern of intraparenchymal hemorrhage. These findings demonstrate acute induction of HO-1 in non-neuronal cells in the injured spinal cord. Induction of HO-1 in glia may be a consequence of multiple factors including exposure to heme proteins, hypoxia and oxidative stress.