Post-traumatic Interval Research Articles

Post-Concussion Syndrome after a Mine Blast Injury: Neuropsychological Consequences and Changes of the Cognitive Evoked Potentials (P 300)

In 115 male patients with the post-concussion syndrome (PCS) after a mine blast injury (MBI) and 30 control healthy subjects, complex clinical and instrumental diagnostics were performed. The age of the patients varied from 18 to 45 years, the post-traumatic MBI interval was 0.5 to 3 years, and the MBIs were accompanied by an acoustic (AT) or combined acoustic/barotrauma in 92% of the cases. The standard examination procedure included MRI of the brain, recording of EEG and of auditory cognitive evoked potentials (CEP) (elicited within the oddball paradigm, with special attention to the cognitive component, wave P 300) using the Cicerone and Kalmar NSI questionnaire [7], and neurological and neuropsychological examination using the MoCA, HADS, and asthenic disorder (AD) scales. It was found that, despite a mild intensity of traumatic brain ingury (mTBI) and a rather long duration of post-traumatic period, neuropsychological consequences of the trauma are not fully compensated in a majority of the patients. Up to 90% of persons with PCS demonstrated abnormally high anxiety and depression estimates, psychasthenic disorders, and a more or less expressed decrease in the working ability (WA). These patients were characterized by noticeably longer latencies and much lower amplitudes of P 300 wave at application of the target stimuli than control subjects. Such differences in the parameters of P 300 were clearly associated with the presence of cognitive impairment, severe asthenia, depression, and drop in the WA. It was found that such associations were most intense (statistically significant) in the cases where increments in the P300 latency exceeded the upper limits of age normatives calculated for the population. As is concluded, it is expedient to use recording and analysis of CEP P 300 in the complex diagnostics of cognitive disorders in patients with PCS.

Chemical Biomarkers of Diffusse Axonal Injury

Cranio cerebral trauma is the most common cause of death and post-traumatic disability in people under 45 years of age. In Romania, the annual incidence shows, that for every 100,000 inhabitants, there are 300 cases of craniocerebral trauma that require specialized medical assistance. Craniocerebral traumas are the most common types of traumas encountered in current forensic practice. Research on the mechanisms of injury, the timing of head trauma and the establishment of causes of death remain relevant. Establishing the traumatic moment implies both the distinction between pre-mortem and post-mortem injuries but also considerations regarding the post-traumatic survival interval. Regarding the elucidation of the moment of occurrence of the craniocerebral trauma from the forensic point of view, a satisfactory result has not been reached so far. The classic hypothesis regarding the development of traumatic brain injuries shows that they are the result of primary traumatic injuries due to cell necrosis combined with the inflammatory brain response that causes secondary brain injuries. It was considered that post-traumatic neuronal losses are strictly due to necrosis and inflammation, and cellular apoptosis being a physiological process, does not play a role in this process. Due to recent experimental data, brain cell apoptosis has begun to be reevaluated. The pathophysiology of traumatic brain injury is far from being fully understood, with the idea that apoptosis would play an even more important role than originally thought. Specifically, damaged brain cells release neuromodulatory substances that can lead to late-onset neuronal damage long after necrotic and inflammatory brain phenomena have ceased to act. These neuronal cell losses are responsible for the development of various neurological deficits and post-traumatic sequelae.

Role of Interleukin 6 And Tumor Necrosis Factor Alpha In Determination Of Skin Wound Age In Human Cadavers : Histopathological And Immunohistochemical Study

Estimation of wound age is of at most importance in medicolegal death cases. Wound age can be evaluated using morphological, cytological, and molecular biological techniques. The purpose of this study was to evaluate the role of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the determination of wound age in autopsy cases. The present study included (20) autopsy cases; (16) with chronologically dated stab wounds (wound age varied from 30 to 300 minutes) and (4) cases without any injury to provide skin specimens serving as a negative control (Control group). Injured cases were examined, full-thickness skin specimens were collected and classified chronologically according to age of wound into 4 groups each group consisted of 4 specimens: Group I: (Time of injury ≤ 30 min), Group II: (Time of injury 31-60 min), Group III: (Time of injury 61-120 min), Group IV: (Time of injury >120min). Each group of injured cases was divided into two subgroups: Subgroup A: At uninjured contralateral site, Subgroup B: At the site of injury. All specimens were stained and studied and subjected to histological, immunohistochemical followed by a histomorphometric. The times passed since injury infliction (stab wound) caused a change in the area percentage of the released cytokines (TNF-α and IL6) whereas, early detection of (TNF-α and IL6) were at 30 min and a marked reaction became obvious at 31-60 min and from 30 min to 300 min, there was a significant and progressive time-dependent increase in the area percentage of the two markers, evaluated semi-quantitatively. We concluded that pro-inflammatory cytokines (IL-6) and (TNF-α) could serve as a useful tool for the estimation of wound age, in particular in the early post-traumatic interval prior to leukocyte reaction.

Histological dating of subarachnoid hemorrhage and retinal hemorrhage in infants

BackgroundDating the traumatic event is usually done on subdural hematoma (SDH). After infant deaths due to Abusive head trauma (AHT) without SDH available, the magistrates still ask experts to date the traumatic event. To do so, the expert only has tools based on adult series of AHT. We aimed to develop a subarachnoid hemorrhage (SAH) and retinal hemorrhage (RH) dating system applicable to infants aged under 3 years. Methods and resultsWe studied a retrospective multicenter collection of 235 infants who died between the ages of 0 and 36 months, diagnosed with SAH and/or RH by forensic pathological examination and with known posttraumatic interval (PTI). Two pathologists assessed blindly and independently 12histomorphological features in 83 infants (35 girls, 48 boys) whose median age was 3.8 months. For SAH, histopathological changes were significantly correlated with PTI for the appearance of red blood cells, of fibrino-plaquetted organization, the quantity of lymphocytes and macrophages and the presence or absence of siderophages, collagen and fibroblast formation and presence or absence of neovascularization. For RH, histopathological changes were significantly correlated with PTI for the appearance of red blood cells, the presence or absence of siderophages and sclerosis of the retina. ConclusionOur HAS dating system improves the precision and reliability of forensic pathological expert examination of AHT, when SDH are not available, for age estimation in infants. The study of RH histomorphological changes does not allow for reliable dating.

Histological dating of subdural hematoma in infants.

After infant deaths due to non-accidental head injury (NAHI) with subdural hematoma (SDH), the magistrates ask experts to date the traumatic event. To do so, the expert only has tools based on adult series of NAHI. We aimed to develop an SDH dating system applicable to infants aged under 3years. We studied a retrospective multicenter collection of 235 infants who died between the ages of 0 and 36months, diagnosed with SDH by forensic pathological examination and with known posttraumatic interval (PTI). Two pathologists assessed blindly and independently 12 histomorphological criteria relating to the clot and 14 relating to the dura mater in 73 victims (31 girls, 42 boys) whose median age was 3.8months. Histopathological changes were significantly correlated with PTI for the appearance of red blood cells (RBCs) and the presence or absence of siderophages, and regarding the dura mater, the quantity of lymphocytes, macrophages, and siderophages; presence or absence of hematoidin deposits; collagen and fibroblast formation; neomembrane thickness; and presence or absence of neovascularization. Dating systems for SDH in adults are not applicable to infants. Notably, neomembrane of organized connective tissue is formed earlier in infants than in adults. Our dating system improves the precision and reliability of forensic pathological expert examination of NAHI, particularly for age estimation of SDH in infants. However, the expert can only define a time interval. Histopathology is indispensable to detect repetitive trauma.

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.

Clinical significance of dynamic morphological changes of circle cistern observed by computed tomography scanning in patients with severe traumatic brain injury

Objective To investigate the clinical significance of observing the dynamic morphological changes of circle cistern on CT scanning in the patients with acute severe traumatic brain injury (TBI). Methods The CT imaging data of circle cistern of 135 patients with acute severe TBI were analyzed retrospectively. According to the morphological changes of circle cisterns on the initial CT images, 135 cases were divided into 5 groups, and 135 cases carried on prognostic evaluation with Glasgow outcome score (GOS) 6 months later. The relationship between the dynamic morphological changes of circle cistern and the prognosis of patients were analyzed statistically. Results There was a significant relationship between the state of circle cistern and the prognosis of patients. The more obvious the dynamic morphological changes of circle cistern were, the worse prognosis and the higher the fatality was, especially higher mortality in patients with exacerbation of the dynamic morphological changes of circle cistern (79.5%), and the difference was statistically significant among 5 groups (P<0.01). Conclusions There was a significant relationship between the state of circle cistern and the prognosis of the patients with acute severe TBI. The dynamic morphological changes of circle cistern on CT scanning might be used as a promising marker to evaluate the severity of severe TBI, especially in the patients at earlier posttraumatic interval. It is necessary to observe the dynamic change of circle cistern. Key words: Craniocerebral trauma/RA/PA; Tomography, X-ray computed

Detection of RAGE expression and its application to diabetic wound age estimation.

With the prevalence of diabetes, it is becoming important to analyze the diabetic wound age in forensic practice. The present study investigated the time-dependent expression of receptor for advanced glycation end products (RAGE) during diabetic wound healing in mice and its applicability to wound age determination by immunohistochemistry, double immunofluorescence, and Western blotting. After an incision was created in genetically diabetic db/db mice and control mice, mice were killed at posttraumatic intervals ranging from 6h to 14days, followed by the sampling of wound margin. Compared with control mice, diabetic mice showed the delayed wound healing. In control and diabetic wound specimens, RAGE immunoreactivity was observed in a small number of polymorphonuclear cells (PMNs), a number of macrophages, and fibroblasts. Morphometrically, the positive ratios of RAGE in macrophages or fibroblasts considerably increased in diabetic wounds during late repair, which exceeded 60% at 7 and 10days post-injury. There were no control wound specimens to show a ratio of >60% in macrophages or fibroblasts. By Western blotting analysis, the ratios of RAGE to GAPDH were >1.4 in all diabetic wound samples from 7 to 10days post-injury, which were >1.8 at 10days after injury. By comparison, no control wound specimens indicated a ratio of >1.4. In conclusion, the expression of RAGE is upregulated and temporally distributed in macrophages and fibroblasts during diabetic wound healing, which might be closely involved in prolonged inflammation and deficient healing. Moreover, RAGE is promising as a useful marker for diabetic wound age determination.

Below the Callus Surface: Applying Paleohistological Techniques to Understand the Biology of Bone Healing in Skeletonized Human Remains

Objectives: Bone trauma is a common occurrence in human skeletal remains. Macroscopic and imaging scrutiny is the approach most currently used to analyze and describe trauma. Nevertheless, this line of inquiry may not be sufficient to accurately identify the type of traumatic lesion and the associated degree of bone healing. To test the usefulness of histology in the examination of bone healing biology, we used an integrative approach that combines gross inspection and microscopy. Materials and Methods: Six bone samples belonging to 5 adult individuals with signs of bone trauma were collected from the Human Identified Skeletal Collection from the Museu Bocage (Lisbon, Portugal). Previous to sampling, the lesions were described according to their location, morphology, and healing status. After sampling, the bone specimens were prepared for plane light and polarized light analysis. Results: The histological analysis was pivotal: (1) to differentiate between types of traumatic lesions; (2) to ascertain the posttraumatic interval, and (3) to diagnose other associated pathological conditions. Conclusion: The outer surface of a bone lesion may not give a complete picture of the biology of the tissue's response. Accordingly, microscopic analysis is essential to differentiate, characterize, and classify trauma signs.

Dating of Early Subdural Haematoma: A Correlative Clinico-Radiological Study.

Determination of post-traumatic interval remains one of the foremost important goals of any forensic investigation related to human crimes. The estimation of time since injury in cases of subdural haemorrhage has been studied only by a few investigators on the histological and radiological front. The purpose of this study was to determine the post-traumatic interval of Subdural Haemorrhage (SDH) based on Hounsfield Unit measurements (HU) on Computed Tomography (CT) in surviving victims of head injury. The study included a total of 100 cases of closed head injury with subdural haemorrhage. The Post-traumatic Time Interval (PTI) varied from 0.5 hours to a maximum of 249 hours, with a mean of 54.2 hours. Statistically significant results were obtained between the HU measurements of the SDH and the post-traumatic intervals and were found to be statistically significant. A rough attempt was made to determine the effect of haematoma volume on attenuation and was found out to be statistically insignificant. The density of the subdural haematoma decreases with increase in the post-traumatic interval that concurs with the limited number of studies being conducted in the past. We concluded that further sorting of cases could be done according to its age with additional research and uniformity in the methodology.

Dating of Acute and Subacute Subdural Haemorrhage: A Histo-Pathological Study.

Microscopic study of the organization of the Subdural Haemorrhage (SDH) verified against the time period can help us in the determination of its age which has serious medico-legal implications. Very few studies concerning the dating of SDH are present in the literature. This study was conducted for dating the early subdural haemorrhage by routine histopathological stains. A prospective analytical study was conducted during July 2009 to December 2010. A total of 100 cases (50 males and 50 females) fulfilling the inclusion and exclusion criteria were included in this study. Routine histopathological staining of the subdural haematoma was done. Correlation between the frequency of a given histomorphological phenomenon and the length of the Post-Traumatic Interval (PTI) was evidential. All the histomorphological features, when correlated with PTI groups, were found to be statistically significant, except for Polymorphonuclear Leukocytes (PMN). We concluded that routine histopathology was reliable in the dating of early subdural haemorrhages.

α7nAChR is expressed in satellite cells at different myogenic status during skeletal muscle wound healing in rats

Recent study has reported that α7 nicotine acetylcholine receptor (α7nAChR) is expressed in regenerated multinucleated myotubes. But the distribution of α7nAChR in satellite cells in different myogenic status is unknown. A preliminary study on the dynamic distribution of α7nAChR in satellite cells was performed by double indirect immunofluorescent procedures during skeletal muscle wound healing in rats. 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. In normal muscle specimens, weak immunoreactivity for α7nAChR was detected in a few satellite cells (considered as quiescent). α7nAChR-positive signals were observed in proliferated and differentiated satellite cells and regenerated multinucleated myotubes in the wounded areas. By morphometric analysis, the average number of α7nAChR+/Pax7+ and α7nAChR+/MyoD+ cells climaxed at 5 days post-injury. The average number of α7nAChR+/myogenin+ cells was significantly increased from 3 to 9 days post-injury as compared with other posttraumatic intervals. The protein level of α7nAChR maximized at 9 days post-injury, which implies that α7nAChR was associated with the satellite cells status. Our observations on expression of α7nAChR in satellite cells from quiescence to myotube formation suggest that α7nAChR may be involved in muscle regeneration by regulating satellite cell status.

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.

The distribution and time-dependent expression of MAGL during skeletal muscle wound healing in rats.

Monoacylglycerol lipase (MAGL) is widely distributed in mammals and largely responsible for metabolizing 2-arachidonoylglycerol (2-AG). Little is known about its expression in skeletal muscles after trauma. A preliminary study on time-dependent expression and distribution of MAGL was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR (qPCR) during skeletal muscle wound healing in rats. 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 contusion, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. Weak immunoreactivity of MAGL was observed in the sarcoplasm of myofibers in control rats. Intensive immunoreactivities of MAGL were observed in polymorphonuclear cells (PMNs), round-shaped mononuclear cells (MNCs), spindle-shaped fibroblastic cells (FBCs) and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as MAGL-positive cells by double immunofluorescent procedure. MAGL expression was remarkably up-regulated after contusion by qPCR and Western blot analysis. The results demonstrate that the expression of MAGL is distributed in certain cell types and time-dependently expressed in skeletal muscles after trauma, suggesting that MAGL may be involved in inflammatory response, fibrogenesis and muscle regeneration during skeletal muscle wound healing.

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.

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

Nrf1 is time-dependently expressed and distributed in the distinct cell types after trauma to skeletal muscles in rats.

Our goal was to elucidate the dynamic expression and distribution of the nuclear factor erythroid-derived factor 2-related factor 1 (Nrf1) by immunohistochemistry, Western blotting, and real-time PCR during wound healing of contused skeletal muscle in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male healthy rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. A weak immunoreactivity of Nrf1 was observed in the sarcoplasm and nuclei of normal myofibers in control rats. Prominent immunostaining for Nrf1 was seen in a large number of polymorphonuclear cells, round-shaped mononuclear cells and spindle-shaped fibroblastic cells, and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as expressing Nrf1 by double immunofluorescent procedures. By real-time PCR analysis, Nrf1 expression was up-regulated and peaked at inflammatory phase. The expression tendency was also confirmed by Western blot. In conclusion, Nrf1 is time-dependently expressed in certain cell types, such as neutrophils, macrophages, myofibroblasts and regenerated multinucleated myotubes, suggesting that Nrf1 may modulate oxidative stress response and regeneration after trauma to skeletal muscles.

Time-dependent expression and distribution of Egr-1 during skeletal muscle wound healing in rats

Recent studies have shown that early growth response factor-1 (Egr-1) plays an important role in regulation of inflammation and tissue repair, but little is known about its expression after trauma to skeletal muscles. A preliminary study on time-dependent expression and distribution of Egr-1 was performed by immunohistochemistry, immunofluorescence and Western blotting during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 45 Sprague-Dawley male rats. Samples were taken at 6h, 12h, 1day, 3days, 5days, 7days, 10days, 14days and 21days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. In the uninjured controls, Egr-1 positive staining was observed in the sarcoplasm and nuclei of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells (PMNs), a number of mononuclear cells (MNCs), fibroblastic cells (FBCs) and regenerated multinucleated myotubes showed positive reaction for Egr-1 in contused zones. By morphometric analysis, an increase in Egr-1 expression was verified at inflammatory phase after contusion, which reached a peak in the regenerated phase overlapping with the fibrotic phase during skeletal muscle wound healing. The expression tendency was further confirmed by Western blotting assay. By immunofluorescent staining for co-localization, the Egr-1-positive MNCs and FBCs in wounds were identified as macrophages and myofibroblasts. The results demonstrate that the expression of Egr-1 is up-regulated and temporally distributed in certain cell types after trauma to skeletal muscles, which may be closely involved in inflammatory response, fibrotic repair and muscle regeneration during skeletal muscle wound healing.

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.

Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice

The study investigated the expression of monoacylglycerol lipase (MGL) during the skin-incised wound healing in mice and applicability of the time-dependent expression of MGL to wound age determination by immunofluorescent staining, Western blotting, and real-time PCR. Furthermore, cell types were identified by double immunofluorescence. A total of 45 BALB/c male mice were used in this study. After a 1.5-cm-long incision in the central dorsum skin, mice were killed at intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. In the control, there was a low-level expression of MGL in the epidermis, hair follicles, and glandulae sebaceae. In the injured skin, MGL immunoreactivity was mainly detected in the neutrophils, macrophages, and myofibroblasts. Morphometrically, the average ratios of MGL-positive cells were more than 50% at 5 and 7 days post-wounding, whereas it was <50% at the other posttraumatic intervals. By Western blotting analysis, the average ratio of MGL protein expression was highest at 5 days after injury, which had a ratio of >2.30. Similarly, the relative quantity of MGL mRNA expression maximized at posttraumatic 5 days in comparison with control as detected by real-time PCR, with an average ratio of >2.54. In conclusion, MGL expression is detected in neutrophils, macrophages, and myofibroblasts and significantly up-regulated, suggesting that it may play roles in response to inflammation during skin-incised wound healing. From the viewpoint of forensic pathology, MGL detection is applicable to skin wound age determination.