A Study On The Time-dependent Expression Of Caspase-3 During Repair Of Contused Skeletal Muscle In Rats

A Study on the Time-Dependent Expression of Caspase-3 During Repair of Contused Skeletal Muscle in RatsIntroductionProgrammed cell death, or apoptosis, is a physiological process of cellular autodestruction, which is governed by series of genes. Lots of researches have been conducted to study the phenomena of apoptosis in different tissues and cells, including skeletal muscle. On one hand, the apoptosis of skeletal muscle is crucial for normal development and tissue homeostasis. On the other hand, untoward apoptosis is associated with some damages and diseases of skeletal muscle. Some studies indicated that apoptosis occurs and plays an important role in denervated skeletal muscle.The members of caspases family are crucial proteases inducing apoptosis. As the most powerful executioner, caspase-3 have been intensively investigated. However little is known about the expression of caspase-3 after skeletal muscle contusion.To investigate the mechanism of caspase-3 after skeletal muscle contusion and explore the applicability of caspase-3 to determination of wound age, the expression of caspase-3 was studied by immunohistochemical, Western blot techniques and colorimetric measurement of proteolytic activity in skeletal muscle contusion at different posttraumatic intervals in rat.Materials and Methods1. Establishment of animal modelA total of 30 male, healthy rats, weighting 220g-250g was anesthesized by intraperitoneal injection of 2% pentobarbital sodium (0.45mg/kg), and received a single impact trauma at the skeletal muscle of the right rear leg. After wounding, each rat was individually housed in a cage and given sterilized chow and redistilled water. Muscles of right lower limb were taken after the animals were anaesthetized and sacrificed by cervical dislocation at 3h, 6h, 12h, ld, 3d, 5d, 7d, 10d, 14d (3 rats in each group) postwounding. The remaining 3 rats were used as control.2. Immunohistochemical detection of caspase-3Immunohistochemistry SP method was applied to investigate the immunoreactivity of caspase-3 in the contused skeletal muscle. After serum incubated, sections were incubated with polyclonal antibody against caspase-3 at 4℃overnight at a dilution 1: 600 followed by incubition with biotinylated goat anti-rabbit IgG antibody at 25℃for 20 minuts. The slides were incubated with SP at 25℃for 20 minutes. DAB was used as chromogen for visualization of the specific reaction. Finally, the sections were counterstained with hematoxylin, dehydrated and mounted. Additionally, H.E staining of the sections was routinely conducted.3. Analysis of caspase-3 by Western blotProteins extracted from whole cells were loaded at 50μg per lane[w/v]on 12% SDS-PAGE. Protein bands were transferred to PVDF membrane, blocked in 5% milk powder and 0.1%Tween-20. Membrane was probed with polyclonal antibody against caspase-3, which was then incubated with secondary antibody. Immunoreactive bands were visualized by ECL for detecting the bands of the protein.4. Determination of caspase-3 activityThe enzymatic reaction for caspase-3 activity is carried out in a 96 well flat bottom microplate that can be read with a microplate reader. Each reaction requires 50μL of cell lysate, 50μL of 2×Reaction Buffer. Prior to using the 2×Reaction Buffer, add 10μL of fresh DTT stock per 1 ml of 2×Reaction Buffer. Then add 5μL of caspase-3 colorimetric substrate (DEVD-pNA). Incubate the plate at 37℃for 1.5 hrs. Read the plate on a microplate reader at 405nm wave length. The results were compared with that of the control group. Fold-increase of caspase-3 activity was calculated and expressed as mean±SD. Results1. Morphological changes of the contused skeletal muscle wound3 houres after wounding, hemorrhage, oedema, degeneration and tom muscle fiber could be detected in the injuried site with a few polymorphonuclear cells were detected. 6 houres after wounding, carcoplasm clarifixation appeared, caryon disappeared. From 12 houres to 1 day after wounding, a large number of inflammatory cells accumulated. 3 days after wounding, lots of cell nucli came out. 5 days after wound, myocyte nucli proliferate, nearly all the nucli located in the middle of the cell. From 7 days to 14 days after wound, skeletal muscle regeneration and collagen emerged.2. Expression of caspase-3 after skeletal muscle contusionIn the control group, caspase-3 could not be detected. In the peripheral zone of the contused skeletal muscle, caspase-3 was expressed at low level in the wound aged 3h-6h, and maximized in the group aged 5d. Thereafter, the expression decreased and minimized in traumatized muscle aged 14d, which was still kept at a certain level as detected by immunostaining and Western blot.Meanwhile, the regenerated skeletal muscle express caspase-3 too.3. Measurement of caspase-3 activityIn comparison with the control, fold-increase of caspase-3 activity was detected in its climax in the contused muscle aged 5d. Thereafter, the enzymatic activity reduced and minimized in the group aged 14d, although it might be detected at a certain level.DiscussionIn recent years, lots of studies have been conducted to investigate the phenomena of apoptosis in skeletal muscle. The apoptosis of skeletal muscle is crucial for maintaining normal development and tissue homeostasis. Fidzianska's work showed that in mammalian, including human, more than 40% of the myocytes were eliminated by apoptosis in the early period of muscle fiber development. In addition, apoptosis is associated with the mechanism of some injury and pathogenesis of skeletal muscle disorders. Liu's work showed that the progression of DMD was related to over-apoptosis. Jin's later work confirmed that the change of atrophy, necrosis in DMD were related to apoptosis. Strenuous exercise would increase the level of apoptosis, Arslam's work showed that apoptotic index increased after strenuous exercise. Apoptosis in skeletal muscle has also been observed in vivo in suspended hindlimb and after denervation injury. Hurme's research showed that skeletal muscle contusion would lead to denervation.In the present study, caspase-3 was detected by immunohistochemical, Western blot techniques and caspase-3 activity was determined by colorimetric assay method after skeletal muscle contusion. We found that caspase-3 was detected at the peripheral zone of contused skeletal muscle in rats, which was activated, while caspase-3 can not be detected at non-traumatized zone. We presumed that at the initial stage of contusion, there is certain amount ATP(Adenosine triphosphate) in skeletal muscle, although the level is low, which is enough for apoptotic invents and cells that engulf the apoptotic cells may release immunosuppressive cytokines. Apoptotic cells express specific cell surface molecules on the cell membrane, which are recognized by certain receptors on phagocytes (macrophages or DC) This recognition leads to a rapid and efficient phagocytosis in the early stages of apoptosis. Antigens from phagocytosed cells are processed and presented to the naive T-cells. Since apoptotic cells and phagocytes produce immunosuppressive cytokines, the antigens from apoptotic cells can not elicit an immune response, instead of leading to immunosuppression. Under such a circumstance, the phagocytic antigen-presenting cells do not express co-stimulating molecules. Thus, T-cells become tolerant of antigens from apoptotic cells.Caspase-3 is also expressed in the regenerated skeletal muscle, which may indicate that caspase-3 activity was required for skeletal muscle differentiation.The results suggest that:①The peripheral area of contused rat skeletal muscle express caspase-3, which is activated. It suggests that apoptosis take place in the peripheral area of contused rat skeletal muscle.②Caspase-3 maybe used as a maker for the wound age determination, since caspase-3 was expressed time-dependently after skeletal muscle contusion.③Caspase-3 is required for skeletal muscle differentiation.Conclusions1. Caspase-3 can not be detected at non-contused zone of skeletal muscle.2. The peripheral area of contused rat skeletal muscle expressed caspase-3, which was activated. It is suggested that apoptosis should take place in the peripheral zone of contused skeletal muscle in rats.3. Caspase 3 may be required for skeletal muscle differentiation in rats.4. Caspase-3 may be used as a maker for the skeletal muscle wound age determination.