| Objective:The purpose of this study is to investigate the changes of cytoskeleton vimentin and nebulin and ultrastructure in skeletal muscle during recovery period after exhaustive swimming exercise in rats.Method:36 SD adult rats that only 6 weeks old were randomly divided in to 6 groups, and each group had six rats. Control group(C),Immediately after exhaustive exercise group(E0), 12 hours after exhaustive exercise group(E12),24 hours after exhaustive exercise group(E24),48 hours after exhaustive exercise group(E48),72 hours after exhaustive exercise group(E72). All exhaustive exercise groups were carried on tail-loaded swimming with the weight of 3% of body weight for seven days. After the last exhaustive exercise at different time points, each rat was anesthetized intraperitoneally to Slaughter with Urethane, and the gastrocnemius,soleus,vastus lateralis,rectus femoris muscle were taken. Some were used for cryopreservation in Liquid nitrogen and the others were used to fix with 4% paraformaldehyde or 2.5% glutaraldehyde. The Protein expression of the cytoskeleton vimentin and nebulin were detected by immunohistochemistry SABC in Rat soleus and vastus lateralis. The contents of them were measured by the automated image analysis system. A side of the rectus femoris muscle sample was fixed for producing ultrathin sections by transmission electron microscope observation of the ultrastructural changes.Result:(1) The results of immunohistochemistry showed that the stain and positive areas of vimentin and nebulin enlarged,not only at cytoplasm,but also at the surrounding of connective tissue and peripheral blood vessels after exhaustive exercise in vastus lateralis muscle of E0.The expression of vimentin and nebulin were higher in E12 than in the control,but lower than in E0(P<0.05).E24 and E48 were similar with a large staining area and the weak colour.The stain in E72 was weakest. The results of soleus muscle was similar to vastus lateralis.(2)The automated image analysis results of vimentin and nebulin by immunohistochemistry were similar. The mean optical density of vimentin and nebulin was lowest in control group, but the valus were highest in E0 group after exhaustive exercise. Then the one of vimentin and nebulin were higher in E12 than in the control, but lower than in E0(P<0.05).E24 and E48 were similar with a large staining area and the weak colour.The stain in E72 was weakest.On the other hand, the percentages of positive area (PSA) of vimentin and nebulin were all higher in exhaustive exercise groups than in control with a peak at E24.As a whole ,the peak of mean optical density was at EO,but the one of PSA was at E24.The results of immunohistochemistry showed that the expressions of vimentin and nebulin increased at different times after exhaustive exercise.(3) By observing the electron micrographs of the rectus femoris ,we found a normal ultrastructure of skeletal muscle in the control group. The endoplasmic reticulum and mitochondria were clearly visible.The nuclei showed normal structure.The myofibril arranged regular, and the H band,I band,M line and Z line of the boundaries were clear.There are only few vacuole matrix. On the other hand the skeletal muscle structure in exhaustive exercise groups (E0,E24) were significantly damaged with an interstitial edema, and muscle gap widened. The endoplasmic reticulum and mitochondria were slightly deformed.The nucleus and nuclear membrane preserved also;The myofibril become thinner and more incompact.H band and I band took ill -defined.M lines and Z lines blurred.Z lines were distorted and disappeared.Some matrix become shallow,and mitochondrials were deformed,distorted and swollen.Conclusion:(1) The results of immunohistochemistry in rat skeletal muscles showed that the stain and positive area of vimentin and nebulin enlarged after exhaustive exercise with a peak at E0. It indicated that the cytoskeleton vimentin and nebulin can be sensitive morphological indicators of exercise-induced micro-injury.(2)The automated image analysis results of vimentin and nebulin by immunohistochemistry were similar. The mean optical density and the percentages of positive area (PSA) of vimentin and nebulin were all higher in exhaustive exercise groups than in control.As a whole ,the peak of mean optical density was at EO,but the one of PSA was at E24.The results of immunohistochemistry showed that the expressions of vimentin and nebulin increased at different times after exhaustive exercise.(3) Under an electron microscope, a week of hevay load swimming exercise can result a significant effect on ultrastructure in skeletal muscle such as A band stretched, myofibril disordered, Z line fractured, Z line flow and so on. This showed that the heavy load swimming exercise can be one of important models to observe the changes of skeletal muscle.
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