Electro-Acupuncture Promote Skeletal Muscle Regeneration After Contusion In Rabbits

In all types of unintentional injuries about 60% are injuries of musculoskeletal system, in which skeletal muscle injuries account for 60-67%, and the most common type of injury is blunt injury or contusion. It has been observed that injured muscles can regenerate promptly, however, this tendency, usually hindered by excessive fibrosis or the formation of scar tissue which may affect the recovery of skeletal muscles and increase economic burdens on society, is often inefficient in the healing process. Although methods of Therapeutic Ultrasound, Low-energy laser and Cell Transplantation can promote the regeneration of skeletal muscles, these don't significantly inhibit the excessive fibrosis in animal experiments and clinical researches. Growth factors, such as IGF-I,are able to promote regenerations of skeletal muscles and inhibit the excessive fibrosis, but they are restricted in some fields and special diseases. Hence it is very important and difficult to find a method to not only promote skeletal muscle regeneration but also reduce fibrosis.Preliminary study confirmed that Electro-Acupuncture (EA) can promote skeletal muscle regenerations and reduce fibrosis, however, the mechanism of EA in these fields were unclear. Therefore, it is necessary to carry out experimental researches to solve these problems.1 ObjectiveThrough performing EA on Living acupoint and Zu Sanli, we interposed the healing process of acute contusion in rabbits, observing effects of EA on the local micro-circulation perfusion, collagen deposition and ultrastructure of the injured gastrocnemius and expressions of myosin heavy chain(MHC),myostatin (GDF-8) and p-Smad2/3. EA was conducted on rabbits 24h after injury to detect the mechanism of EA on the regeneration and fibrosis process and the specificity of acupoints. Simultaneously, this study was also aimed at providing scientific guidances for EA on skeletal muscle regenerations in clinic.2 MethodsSeventy-five adult New Zealand rabbits (male or female,2.0±0.2kg)were randomly and equally divided into five groups:control group,Living acupoint,Zu Sanli,Living acupoint+Zu Sanli and IGF-I treatment groups. The acute skeletal muscle contusion model (1cm2,9.75J) was produced on the right hind limb gastrocnemius by mass-drop technique after anesthesia. EA or IGF-I treatments were beginning at the 24th hour after contusion. EA on the treatment group, Living acupoint group,.Zu Sanli group and Living acupoint+Zu Sanli group was respectively performed for 15 minutes every two days with 0.4mA (2Hz).0. lmg/ml IGF-I, 0.25ml per rabbit, was injected intramuscularly into gastrocnemius. Injections, once a week, were no more than 3 times. The rabbits in the control group performed a natural healing process, but got the same fixed position like the EA treatment group. Materials were picked up on the 7th d,14th d and 28th d (n= 5). Microcirculation changes of the right gastrocnemius muscle were observed and recorded by Laser-Doppler Perfusion Imaging (LDPI) on the 28th day after injury, and were compared with other groups. On the 7th, 14th, and 28th day after injury five rabbits were killed respectively in each group. When the rabbits were dead, the gastrocnemius muscle was harvested and subjected for histologic study. The ultrastructure of myofibrils were observed by Transmission Electron Microscopy. All specimens were observed or tintaged by the following research methods: Masson stain and immunohistochemistry (MHC, GDF-8, p-Smad2/3).3 Results3.1 Microcirculation changes of gastrocnemius on the 28th day after injury. (Chart 1)PU values of the control group was significantly lower than the other groups (P< 0.05) and PU values of the IGF-I group was significantly higher than the other groups(P< 0.05).There were significant differences between the Zusanli+Living acupoint group and the other groups (P< 0.05), and few differences between the Living acupoint group and the Zusanli group(P> 0.05). It could be concluded that relations of experimental data about five groups were:IGF-I group> Zusanli+Living acupoint group> Zusanli group, Living acupoint group > control group. In brief, the microcirculation of the gastrocnemius muscle in the treatment group was better than that in the control group, and the IGF-I group got the best results. As to the EA treatment, there was synergistic effect between Living acupoint and Zusanli.Chart 1 Comparisons of microcirculation changes of gastrocnemius on the 28th day after injury. (PU)Notes:compared with the other groups,▲▲P<0.01; compared with the Living acupoint group and the IGF-I group,P< 0.05; compared with the Zusanli group and the control group,※※P< 0.01.3.2 Ultrastructure changes of skeletal muscles in different groups.(1) 7d post-injury:After trauma to muscle, disruptions of the sarcomeres were found in different groups, including disorder of Z-Line streaming, vacuolization changes of mitochondria and expansion of sarcoplasm. Z-Line disappeared in the control group, but was obvious in the other groups. Swelling of the mitochondria and sarcoplasm was found in the Zusanli group, while less swelling changes occurred in the Living acupoint group and the Zusanli+Living acupoint group. Diameter of myofibrils was different among control group,Living acupoint group, Zusanli group and Zusanli+Living acupoint group.(2) 14d post-injury:The structure of myofibrils and sarcomeres was more clear and regular, compared with data of 7d post-injury, and the swelling of the mitochondria reduced. Varying degrees of disorder of the sarcomeres were found among control group, Living acupoint group, Zusanli group and Zusanli+Living acupoint group, especially in the control group. Myofibrils were better in the Living acupoint group and some mitochondria and sarcoplasm swelled. Myofibrils and myofilaments were more regular in the Zusanli+Living acupoint group with lightly Z-Line streaming and mitochondria swelling.(3) 28d post-injury: Z-Lines disappeared in some myofibrils in the control group, diameter of myofibrils was uneven and some were atrophic. Swelling of the mitochondria was also found in the control group. Structure of myofibrils, sarcomeres and Z-Line were rather regular in the Living acupoint group and the Zusanli+Living acupoint group. Less swelling mitochondria and sarcoplasm were found in the above groups. Structures of myofibrils and sarcomeres were almost integrate and regular in the Zusanli+Living acupoint group and the IGF-I group, so did the mitochondria and sarcomeres.3.3 Density of collagen fiber in different groups. (Chart 2)(1) The density of'collagen fiber of the control group was significantly higher than the other groups (P< 0.01) on the 7th,14th, and 28th day post-injury. There were significant differences between the Zusanli+Living acupoint group and the other groups (P< 0.01) on the 7th,14th and 28th day post-injury.(2) There were significant differences among control group, Zusanli group, Living acupoint group and Zusanli+Living acupoint group on 7th day post-injury (P< 0.01). It could be concluded that relations of experimental data about five groups were:control group, Living acupoint group> Zusanli group> Zusanli+Living acupoint group> IGF-I group.(3) There were significant differences among five groups on 14th and 28th day post-injury (P<0.01). It could be concluded that relations of experimental data about five groups were:control group> Living acupoint group>Zusanli group> Zusanli+Living acupoint group> IGF-I group. Chart 2 Comparisons of density of collagen fiber at different times among five groups. (%)Notes:compared with the other groups,▲▲P<0.01.3.4 The expression of MHC in different groups. (Chart 3) The MOD value of MHC in the Zusanli+Living acupoint group was significantly higher than control group, Living acupoint group and Zusanli group on 7th,14th and 28th day post-injury (P< 0.01)., but lower than that of IGF-I group (P< 0.01).(1) On the 7th day post-injury: The MOD value of MHC in the Living acupoint group was significantly higher than that of the control group (P< 0.01) and Zusanli group (P = 0.052). There were no significantly differences between the control group and Zusanli group (P> 0.05). It could be concluded that relations of experimental data about five groups were:control group, Zusanli group< Living acupoint group< Zusanl i+Living acupoint group<IGF-I group.(2) On the 14th day post-injury:There were no significantly differences among the control group, the Living acupoint group and the Zusanli+Living acupoint group (P> 0.05). However, there was an upward trend of the MOD value of MHC in the Living acupoint group. It could be concluded that relations of experimental data about five groups were:control group, Living acupoint group, Zusanli group< Zusanli+Living acupoint group<IGF-I group. (3) On the 28th day post-injury:The MOD value of MHC in the Zusanli group was significantly higher than that in the control group (P< 0.05), but was similar to the Living acupoint group (P> 0.05). There were no significant differences between the Living acupoint group and the control group (P> 0.05).Chart 3 Comparisons of expressions of MHC at different times among five groups.Notes:compared with the other groups,▲P< 0.05,▲▲P< 0.01; compared with the control group,P< 0.01.3.5 The expression of GDF-8 in different groups. (Chart 4)The MOD value of GDF-8 in the control group was significantly higher than the other groups on 7th,14th and 28th day post-injury (P< 0.01), but lower than that in the IGF-I group (P< 0.01).(1) On the 7th day post-injury:The MOD value of GDF-8 in the Living acupoint group was significantly higher than that in the Zusanli group (P< 0.05) and Zusanli+Living acupoint group (P< 0.01). There were no significant differences between the Zusanli group and the Living acupoint group (P> 0.05). It could be concluded that relations of experimental data about five groups were: control group>Living acupoint group> Zusanl i group, Zusanli+Living acupoint group>IGF-I group.(2) On the 14th day post-injury:The MOD value of GDF-8 in the Zusanli+Living acupoint group was significantly lower than that in the Living acupoint group (P< 0.01) and Zusanli group (F< 0.05). There were no significant differences between Living acupoint group and Zusanli group (P> 0.05). It could be concluded that relations of experimental data about five groups were: control group> Living acupoint group, Zusanli group> Zusanli+Living acupoint group>IGF-I group.(3) On the 28th day post-injury:There were no significant differences among Living acupoint group, Zusanli group and Zusanli+Living acupoint group (P> 0.05). It could be concluded that relations of experimental data about five groups were: control group>Living acupoint group, Zusanl i group, Zusanli+Living acupoint group>IGF-I group.Chart 4 Comparisons of expressions of GDF-8 at different times among five groups.Notes:compared with the other groups,(?)P< 0.05, (?)P<0.01.3.6 The expression of p-Smad2/3 in different groups. (Chart 5)(1) On the 7th day post-injury: There were significant differences about the MOD value of p-Smad2/3 among Zusanli group, Zusanli+Living acupoint group and IGF-I group (P < 0.01) on the 7th day post-injury. There were no significant differences between Living acupoint group and control group (P> 0.05). It could be concluded that relations of experimental data about five groups were: control group,Living acupoint group> Zusanli group> Zusanli+Living acupoint group>IGF-I group.(2) On the 14th day post-injury:The MOD value of p-Smad2/3 in the Zusanli+Living acupoint group was significantly lower than that in the Zusanli group (P< 0.01) and Living acupoint group (P< 0.01), and the MOD value of p-Smad2/3 in the Zusanli group was significantly lower than that in the Living acupoint group (P<0.01). It could be concluded that relations of experimental data about five groups were:control group> Living acupoint group> Zusanli group> Zusanli+Living acupoint group,IGF-I group.(3) On the 28th day post-injury:There were significant differences among five groups (P< 0.01 or P < 0.05). It could be concluded that relations of experimental data about five groups were:control group> Living acupoint group> Zusanli group > Zusanli+Living acupoint group>IGF-I group. As to all, There were significant differences between Zusanli+Living acupoint group and the other groups (P< 0.01 or P< 0.05) on the 7th and 28th day post-injury. And there were significant differences among Living acupoint group, Zusanli group and Zusanli+Living acupoint group(,P< 0.01 or P< 0.05) on the 7th,14th and 28th day post-injury. But there were significant differences between Zusanli+Living acupoint group and IGF-I group (P> 0.05).Chart 5 Comparisons of expressions of p-Smad2/3 at different times among five groups.Notes:compared with the other groups,(?)P<0.01; compared with the Zusanli group,(?)P< 0.05. 4 Conclusions4.1 In the healing process after contusion, Local microcirculation and the structure of myofibrils of skeletalmuscle were well recovered by EA treating on Zusanli and Living acupoint. Accompanied by the activation of the mitochondria and sarcoplasm.4.2 EA on Zusanli and Living acupoint were able to reduce the expression of GDF-8 and increase the expression of MHC to promote the regenetation of muscle fibres, while the effects of EA on Zusanli were in the later stage.4.3 EA on Zusanli and Living acupoint were able to restrain the signal paths of GDF-8/p-Smad2/3 to reduce the fibrosis during the healing process.However, EA on Zusanli or Living acupoint has different effect on the expression of GDF-8.As to all, EA on Zusanli and Living acupoint have dual effects on promoting the regenetation of muscle fibres and reducing fibrosis, but have different mechanisms.