| Background:Skeletal muscle impact injury is so common in sports,falls and road traffic accidents.Impact has a significant influence on the athletic function of the injured,and even some athletes have difficulty returning to the field.Currently,the injury mechanism and repair process of impact injuries have not been fully clarified.At present,the research on skeletal muscle injury is mostly carried out through animal models.After searching the literature,it is found that skeletal muscle impact injury is mostly made by animal models through heavy objects falling.However,this kind of models have some limitations.On the one hand,it is difficult to determine the mechanical parameters related to skeletal muscle injury,and it is difficult to ensure the uniformity of injury;On the other hand,most of the existing injury models are made with self-made equipment,which is difficult to copy.Therefore,we plan to develop a new rat skeletal muscle injury model to overcome those above shortcomings.Objects:A small pneumatic driven biological impactor was used to create a model of gastrocnemius muscle injury in rats.On the first,second,third,and fifth days after injury,the dynamic evolution of skeletal muscle after impact injury was observed at three levels:microstructure,medical imaging,and macroscopic function,and the possible relationship between the macroscopic functional changes and microscopic pathological changes was further explored,providing a new idea for clinical treatment of skeletal muscle injury.Methods:1.Establishment of experimental animal models:SD rats were selected as experimental objects,and experimental animal models were established using a small pneumatic driven biological impact machine to induce injury.The PVDF force sensor inherited from the impact head is used to collect and record the peak force generated by the impact and the compression time of the impact head on the lower limbs of rats.The acceleration and movement of the impact head are recorded using high-speed cameras,and the speed at the moment of impact and the details of the impact are estimated based on this.2.Observation indicators:(1)On the 1st,2nd,3rd,and 5th days after injury,changes in food intake and mental state of the rats were measured,as well as changes in the circumference of the affected limbs and legs;(2)On the 1st,2nd,3rd,and 5th days after impact injury,rats were killed with excessive anesthetic.The gastrocnemius muscles of the rats were taken for HE staining and IF staining to observe the microscopic pathological changes;(3)On the 1st,2nd,3rd,and 5th days after impact injury,the gastrocnemius muscle was taken for TEM(transmission electron microscopy)observation to detect the subcellular structural changes of the gastrocnemius muscle after impact injury.(4)On the 1st,2nd,3rd,and 5th days after impact injury,the affected limbs of anesthetized rats were imaged using 7T MR;(5)Before measurement,rats were trained to successfully pass the track of the CatWalk gait analysis system,and the baseline level before injury was recorded.On the 1st,2nd,3rd,and 5th days after impact injury,the gait changes of the rats were measured and recorded using the animal gait analysis system;3.Statistical method:Using repeated measurement analysis of variance for the above measured quantitative data,p<0.05 indicates a statistically significant difference.Results:(1)The gastrocnemius was injured at an impact speed of 6.63 m/s±0.25 m/s and a peak force of 1,556.80 N±110.79 N.(2)The calf circumference of rats was 6.11cm±0.15cm before injury,6.87cm±0.23 cm on1st day after injury,6.55cm±0.16 cm on 2nd day,6.36cm±0.20 cm on 3rd day,and 6.17 cm±0.11cm on 5th day.The circumference of the calf increased abruptly after injury and then decreased slowly.By day 5,the circumference of the calf had not recovered to the pre-injury level(p<0.05)(3)HE staining showed that compared with the normal control group,the HE staining of gastrocnemius muscle after injury showed significant changes.On the 1st day after the impact injury,there was interstitial edema and fibrous exudation in the injured area,local striated muscle cells were ruptured and degenerated,and a small amount of to moderate inflammatory cells were found around the injured area.A small number of macrophages can be seen in the injured muscle tissue.At the injured site,the cytoplasm of skeletal muscle showed homogeneous light staining.On the 3rd day,interstitial edema and inflammatory cell infiltration were further aggravated,and inflammatory cell infiltration reached a peak.On the 5th day,large area of interstitial edema,accompanied by moderate cellulosic exudation,a large number of inflammatory cell infiltration near the injury target,and accompanied by hemosiderin can be seen.(4)Immunofluorescence staining showed that on the 1st day after the impact injury,a small number of macrophages appeared near the impact target.By the 2nd day,the number of macrophages increased.By the 3rd day,the number of macrophages reached the peak.By the5th day,the number of macrophages did not increase or decrease significantly compared with the 3rd day.(5)TEM showed that obvious sarcoplasmic reticulum expansion was observed on the 1st day after injury;Obvious vacuolation of mitochondria can be seen on the 2nd day;On the 3rd day,the degree of mitochondrial vacuolation was lower than that on the 2nd day,but glycogen was deposited near sarcoplasmic reticulum;Glycogen deposition increased significantly on the5th day,significantly higher than that on the 3rd day.(6)MRI showed that the impact did not cause the lower limb fracture of rats,and a large number of long T2 signals were visible in the calf gastrocnemius muscle,and the muscle bundle morphology was visible,indicating muscle edema.Irregular clumps of low signal can be seen in it,and the muscle bundle morphology disappears,indicating hemorrhage.In addition,the long T2 signal distributed along the muscle space indicates intramuscular effusion.Long T2signal can be seen in the subcutaneous fat layer,indicating subcutaneous edema at the impact site.The above signs all suggest muscle injury with hematoma formation.On the 5th day,we can observe that the abnormal signal range is reduced to half of that on the 1st day,and the shape of muscle bundle is basically restored.(7)The baseline footprint of the right hind foot was 1.06 cm~2±0.14 cm~2 before injury.On the 1st day after injury,the footprint decreased to 0.64cm2±0.16cm2.On the 2nd day after injury,the footprint increased to 0.77 cm~2±0.18 cm~2 on 3rd day.The footprint area continued to increase,and was 0.90 cm~2±0.16 cm~2 on the 5th day,the footprint area recovered to 1.06cm~2±0.14 cm~2,but it was still smaller than the level before injury(p<0.05).Conclusion:(1)In this experiment,we developed a new,reliable and reproducible experimental animal model.It can be used as a reference model for the study of skeletal muscle impact injury and provide new ideas for the experimental animal research of skeletal muscle impact injury.(2)On the pathological level,after HE staining,the injured skeletal muscle showed"map-like"changes under the light microscope due to the infiltration of inflammatory cells,and there was a peak of inflammatory cell infiltration around the injury on the 3rd day after injury.(3)We found that in the subcellular structure,the injured skeletal muscle cells showed sarcoplasmic reticulum dilation and mitochondrial vacuolation.(4)In imaging,the gastrocnemius muscle after impact injury is mainly edema,with or without bleeding.The edema is the most serious on the 1st day after injury,and then gradually subsides.(5)The gait analysis of rats showed that the foot print area of the affected limb decreased the most on the first day after injury,and then gradually recovered. |
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