Creation of a contusion injury method for skeletal muscle in rats with differing impacts

Abstract Purpose: To realize a morphological examination of the musculoskeletal tissue, assessing the effect of a contusion method for the production in rat gastrocnemius, comparing the inflammatory responses generated by different impacts. Methods: For the analysis of a contusion method, twelve female Wistar rats were distributed into four groups. The lesion was generated by 324 g of mass that was dropped from different predetermined heights for each group (30, 45, 60 and 70 cm). Results: In the analysis of musculoskeletal tissue, the response to injury varied according to the mass of the height drop onto the muscle. Only the group that was injured from 70 cm responded with uniform and severe inflammation, whereas the groups 30, 45 and 60 cm showed inflammation in some regions of the tissue with mild and moderate infiltrates. Conclusion: The method with the 324-gram mass dropped from a 70-cm height onto the gastrocnemius muscle of rats seems to be the most suitable for the production of muscle injury in these animals after 72 hours, showing an important inflammatory infiltrate.

Time-dependent Expression of MT1A mRNA and MT2A mRNA in the Contused Skeletal Muscle of Rats / 法医学杂志

OBJECTIVES@#To investigate the time-dependent expression of metallothionein （MT） 1A mRNA and MT2A mRNA in contused skeletal muscle of rats.@*METHODS@#A total of 54 Sprague-Dawley rats were used in this study. The rats were divided into two parts: control group （n=6） and contusion groups （0.5, 1, 6, 12, 18, 24, 30, and 36 h after contusion, n=6）. Total RNA was extracted from skeletal muscle. The expression levels of MT1A mRNA and MT2A mRNA were detected by SYBR Green I real-time PCR.@*RESULTS@#The expression trends of the two potential marker genes were related to wound age. In addition to 0.5 h, there were significant contrasts between the control group and contused group （P<0.05）, about the expression levels of MT1A mRNA and MT2A mRNA in different phases. As the extension of wound age, the relative expression of MT1A mRNA and MT2A mRNA at 1 h, 6 h, 12 h and 18 h after contusion demonstrated upgrade tendency until its expression levels in 18 h peak with 239.41±15.20 and 717.42±50.76, respectively. When time extends to 24 h after injury, the expression of above two marks decreased, respectively. The MT1A mRNA and MT2A mRNA expression levels increased at 30 h and then decreased.@*CONCLUSIONS@#Determination of MT1A mRNA and MT2A mRNA levels by real-time PCR may be useful for the estimation of wound age.

Comparison of the Skin and Skeletal Muscle Contusion in Rats Induced by Blunt Force with Different Heights / 法医学杂志

OBJECTIVES@#To explore the differences in the repair process of skin and skeletal muscle after contusion caused by blunt force attack with different heights.@*METHODS@#Three degrees of contusion were performed on SD rats' right hind limbs by a designed free-dropping device falling from 15, 30 and 50 cm heights, which as a main consideration factor for degree of injury. The repair process of skin and skeletal muscle at 6 h, 24 h, 3 d, 7 d and 13 d after contusion were observed using routine histological methods.@*RESULTS@#Hematoma within skin and/or muscle was found in the rats' hind limbs after contusion with three different heights. The repair processes were similar at 24 h after contusion. However, with the increase of height, the display degree was more obvious. At 3 d after contusion, the RBC of the hemorrhagic region would be decomposed and elapsed in 15 cm contusion group, but for 30 cm contusion group, it delayed to 7 d. At 13 d after contusion, the similar result was found in 15 cm and 30 cm contusion groups, in contrast, the 50 cm contusion group was still in the proliferative phase.@*CONCLUSIONS@#With the increase of height, the occurring rate of hematoma within skin and muscle at the same time increases, and the more serious histological appearance after contusion, including inflammation and proliferation, the longer healing process are observed. According to the results of present study and considering forensic application, the contusion model with 50 cm height （2.58 J/cm²） is recommended as the experimental animal model for the future study of wound age estimation on contusion.

Time-related effects of general functional training in spinal cord-injured rats

OBJECTIVES: This prospective, randomized, experimental study with rats aimed to investigate the influence of general treatment strategies on the motor recovery of Wistar rats with moderate contusive spinal cord injury. METHODS: A total of 51 Wistar rats were randomized into five groups: control, maze, ramp, runway, and sham (laminectomy only). The rats underwent spinal cord injury at the T9-T10 levels using the NYU-Impactor. Each group was trained for 12 minutes twice a week for two weeks before and five weeks after the spinal cord injury, except for the control group. Functional motor recovery was assessed with the Basso, Beattie, and Bresnahan Scale on the first postoperative day and then once a week for five weeks. The animals were euthanized, and the spinal cords were collected for histological analysis. RESULTS: Ramp and maze groups showed an earlier and greater functional improvement effect than the control and runway groups. However, over time, unexpectedly, all of the groups showed similar effects as the control group, with spontaneous recovery. There were no histological differences in the injured area between the trained and control groups. CONCLUSION: Short-term benefits can be associated with a specific training regime; however, the same training was ineffective at maintaining superior long-term recovery. These results might support new considerations before hospital discharge of patients with spinal cord injuries.

Analysis of 28 death cases involved with tumbling injury / 法医学杂志

OBJECTIVE@#To explore the general features and the keypoints of forensic medical examination in tumbling injury cases.@*METHODS@#Twenty-eight cases dying of tumbling injury were collected and the locations and features of injury were analyzed.@*RESULTS@#The occiput of head was the common position for the tumbling injury cases. Force, disease and alcohol were the main reasons for tumbling injury.@*CONCLUSION@#The injury is mild outside and severe inside from tumbling injury cases. The craniocerebral contrecoup is the significant feature in tumbling injury cases.

Different expression of TNF-alpha in brain and peripheral organs after cerebral contusion of rats / 法医学杂志

OBJECTIVE@#To compare the expression of tumor necrosis factor-alpha (TNF-alpha) between brain and peripheral organs after cerebral contusion in order to provide the scientific theoretical basis for forensic pathological diagnosis and wound age estimation.@*METHODS@#Brain and peripheral organs including heart, liver, spleen, lung, kidney tissues of 45 SD rats after the cerebral contusion were obtained and TNF-alpha of these tissues were analyzed with immunohistochemistry methods.@*RESULTS@#TNF-alpha was detected at 1 h in brain, reaching maximum at 6 h and 3 d after the cerebral contusion, and then decreased but still kept at high expression level at 7 d. TNF-alpha was detected at 1 h after the cerebral contusion in heart, liver, spleen, lung and kidney tissues. The number of cells expressing TNF-alpha increased gradually, reaching maximum at 3 d after the contusion of brain, and then decreased but still kept at high expression level at 7 d.@*CONCLUSION@#Besides the change of cerebral contusion, this study considered both the brain and peripheral organs. It is helpful for forensic pathological diagnosis and wound age estimation after contusion of brain.

The number of circulating fibrocytes of skeletal muscle in rats after contusion / 法医学杂志

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

Homer protein expression after focal brain contusion in rats / 法医学杂志

OBJECTIVE@#To investigate Homer protein expression after focal brain contusion and explore the relationship between expression and injury time.@*METHODS@#Focal brain contusion in rats was established and Homer protein expression in brain at different injury intervals after contusion was detected by immunohistochemistry and Western blotting.@*RESULTS@#A small amount of Homer positive expression cells were detected in control group, sham operated group and experimental group (0.5 h after contusion). The amount of Homer positive expression cells increased after 3 h and reached peak 12 h after contusion. The amount of positive cells continued to decrease 1 d after contusion and to the base level 7 d after contusion. Homer protein expression based on immunohistochemistry and Western blotting had statistical difference among adjacent groups.@*CONCLUSION@#Expression of Homer protein near the focal contusion area shows time dependence after brain contusion in rats.

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

OBJECTIVE@#To investigate the expression of intercellular adhesion molecular-1 (ICAM-1) mRNA in contused skeletal muscle of rats using fluorescence in situ hybridization (FISH) and its relationship with the contusion interval.@*METHODS@#To make the contusion models with rats skeletal muscle. The samples were taken to extract mRNA at 0.5 h, 1 h, 6 h, 12 h, 18 h, 24 h, 30 h and from the control group after contusion respectively. FISH was performed on frozen section samples and the sections were observed using LSCM.@*RESULTS@#The expression of ICAM-1 mRNA peaked at 6 h in skeletal muscle after contusion. Its level fell to 3.46 times the level of control group at 18 h and then increased again.@*CONCLUSION@#The time-order expression of ICAM-1 mRNA in 30 hours after contusion is potentially useful for estimation of early wound age.

Bcl-2 expression following the brain concussion in rats / 法医学杂志

OBJECTIVE@#To evaluate the expression of Bcl-2 protein after brain concussion.@*METHODS@#Expression levels of Bel-2 protein in cortex, pontine and cerebellum of rats were investigated using immunohistochemistry.@*RESULTS@#There was no expression of Bcl-2 protein in control group seen. The expression of Bcl-2 protein in brain concussion groups was detected at l hour, and the expression level reached its peak 4 days after the concussion and then declined gradually.@*CONCLUSION@#Our findings suggest that the detection of Bel-2 protein could be an indicator for diagnosis of brain concussion and for estimation of the post injury time interval.

Expression of beclin1 and LC3 after rat's skin contusion / 法医学杂志

OBJECTIVE@#This study aimed at examining the expression of Beclin1 and LC3 in the skin of rat after contusive injury and at observing the process of autophagy in response to injury or trauma. Their significance in diagnosis of trauma and application in estimation of the post injury time interval were also investigated.@*METHODS@#Immunohistochemistry and imaging analysis were used to examine the expression level of Beclinl and LC3 at the injury site of the rat skin at different post-traumatic times.@*RESULTS@#Expression of Beclin1 and LC3 in normal rat skin or immediately after injury maintained at a very low level, but their level started to elevate 6 hours after injury and showed increasing strong expression at post injury time of day 3, day 5 and day 7.@*CONCLUSION@#Over-expression of Beclin1 and LC3 as well as increased autophagy, a response to injury, can be observed in the rat skin at the injury site. The expression level of Beclin1 and LC3 starts to increase 6 hours and shows a linear increase up to 7 days after injury. Beclin1 and LC3 may serve as a marker for estimation of the post injury time interval.

A study on the time-dependent expression of caspase-6 during repair of skin contusion in rats / 法医学杂志

OBJECTIVE@#To investigate the expression of caspase-6 in rat skin contusion and its surrounding areas during repairment.@*METHODS@#Immunohistochemical SP method and Western blot technique were used to study the expression and activation of caspase-6 in rat skin contusion and its surrounding areas.@*RESULTS@#Weak expression of caspase-6 was detected in cytoplasms of polymorphonuchear cells (PMNs) infiltrated in the injured area at 3 hours post-contusion. The ratio of the caspase-6 positive cells was low (25.78 +/- 1.38)%. The expression of caspase-6 was increased prominently (47.70 +/- 5.14)% at 12 hours post-contusion. Almost all of the PMNs, mononuclear cells (MNCs) and fibroblastic cells (FBCs) were caspase-6 positive with both cytoplasm and nucleus staining (54.58 +/- 5.64)% on post-contusion day 3. The expression of caspase-6 decreased gradually thereafter. The expression of the 34-kDa pro-caspase-6 was detected by Western blot in both control and the post-contusion groups with time dependent dynamics.@*CONCLUSION@#These results suggest that caspase-6 may play a major role in trauma-induced inflammatory response. Since caspase-6 shows a timely dependent expression in PMNs, MNCs and FBCs during skin injury repair in rat, it may be used as a marker for the contusion age determination,

Expression of c-fos protein after muscle contusion in experimental rats / 法医学杂志

OBJECTIVE@#To study the relationship between the expression of c-Fos protooncogene and skeleton muscle contusion of rats, and to search for a sensitive marker of timing for skeleton muscle contusion.@*METHODS@#Fifty Sprague-Dawley rats were randomly distributed into control group and experimental groups. The expression of c-fos was microscopically observed by immunohistochemical method.@*RESULTS@#With the time prolonged the c-fos positive intensity and area were increased. Positive expression of c-fos protein appeared at 15 min after skeleton muscle contusion, and reached to the peak at 1h after skeleton muscle contusion, then decreased gradually and returned to the normal level on 1d after skeleton muscle contusion.@*CONCLUSION@#The detection of the expression of c-fos protein could be a sensitive marker for timing skeleton muscle contusion.

Regenerated rat skeletal muscle after periodic contusions

In the present study we evaluated the morphological aspect and changes in the area and incidence of muscle fiber types of long-term regenerated rat tibialis anterior (TA) muscle previously submitted to periodic contusions. Animals received eight consecutive traumas: one trauma per week, for eight weeks, and were evaluated one (N = 8) and four (N = 9) months after the last contusion. Serial cross-sections were evaluated by toluidine blue staining, acid phosphatase and myosin ATPase reactions. The weight of injured muscles was decreased compared to the contralateral intact one (one month: 0.77 + or - 0.15 vs 0.91 + or - 0.09 g, P = 0.03; four months: 0.79 + or - 0.14 vs 1.02 + or - 0.07 g, P = 0.0007, respectively) and showed abundant presence of split fibers and fibers with centralized nuclei, mainly in the deep portion. Damaged muscles presented a higher incidence of undifferentiated fibers when compared to the intact one (one month: 3.4 + or - 2.1 vs 0.5 + or - 0.3 percent, P = 0.006; four months: 2.3 + or - 1.6 vs 0.3 + or - 0.3 percent, P = 0.007, respectively). Injured TA evaluated one month later showed a decreased area of muscle fibers when compared to the intact one (P = 0.003). Thus, we conclude that: a) muscle fibers were damaged mainly in the deep portion, probably because they were compressed against the tibia; b) periodic contusions in the TA muscle did not change the percentage of type I and II muscle fibers; c) periodically injured TA muscles took four months to reach a muscle fiber area similar to that of the intact muscle