| OBJECTIVE:To investigate the influence on histomorphology and myosin heavy chain(MHC) composition of gastrocnemius muscles after Intramuscular injection of Botulinum toxin type A (BoTx-A) in order to provide scientific base for clinical use.METHOD:Sixty adult male SD rats were divided equally into 10 groups at random: (1) 5 treatment groups,the rats was weighed per week and sacrificed at the 3rd day, 10th day, 4th week, 12th week and the 24th week after injecting Bo Tx-A5U(0.1ml) into gastrocnemius muscles. Muscular power was scored in the digit abduction score(DAS) before the rats were sacrificed. (2) 5 control groups, the rats was weighed per week and sacrificed at the 3rd day, 10th day, 4th week, 12th week and the 24th week after injecting 0.1ml saline into gastrocnemius muscles. Muscular power was scored in the digit abduction score(DAS) before the rats were sacrificed. The injected and contralateral gastrocnemius muscles was removed for weighing and sampling when the rats were sacrificed. The sample slices stained by HE and adenosine triphosphatase (ATPase) was investigated under light microscope and analyzed with image analysis system. MHC composition was separated by sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE).Spss 13.0 software was used for statistical analysis.Analysis of variance and independent sample t-test were used to establish statistically significant difference. RESULT:1. Muscular power of gastrocnemius muscles was scored in DAS, the score of injected gastrocnemius muscles of treatment groups was 2.50±0.54, 3.00±0.89, 3.17±0.98, 0.33±0.52 and 0 at the 3rd day, 10th day, 4th week, 12th week and the24th week after injecting BoTx-A into gastrocnemius muscles. The muscle weakness was obvious at the 3rd day, the peak of muscle weakness emerged at the 10th day and 4th week, the muscular power restored at the 12th week.2. The body weight between control groups and treatment groups had no significant difference in our study.3. The wet weight of injected gastrocnemius muscles in treatment groups was significantly lower than that of injected gastrocnemius muscles in control groups at the 3rd day (P<0.01) , the largest decrease was 58.7% contrast to the control value at the 4th week. The wet weight of injected gastrocnemius muscles in treatment groups recovered to 50.2% of control groups at the 12th week, to 81.3% of control groups at the 24th week. The wet weight of injected gastrocnemius muscles in treatment groups was significantly lower than that of injected gastrocnemius muscles in control groups at the 24th week (P<0.01) .The wet weight of uninjected gastrocnemius muscles between control groups and treatment groups had no significant difference at the 3rd day, 10th day, 4th week, 12th week and the 24th week (P>0.05) .4. Under the light microscope, the injected gastrocnemius muscles of treatment groups presented different degree scattering and grouping muscle fibers which were decreased in cross section area(CSA) at the 3rd day, 10th day, 4th week, 12th week and the 24th week. Most transverse section showed triangle or long strip shapes, and type grouping was found. It's consistent with neurogenic muscular atrophy.5. In the injected gastrocnemius muscles of treatment groups, the CSA of type II muscle fibers appeared significant decrease at the 3rd day after BoTx-A injection (P<0.05) , the most marked decrease was apeared at the 10th day and 4th week, and then the CSA came back slowly. The CSA of type II was significantly lower than that of control groups at thel2th and 24th week (P<0.01) .The CSA of type I showed decrease at the 10th day (P<0.05) and almost restored at the 4th week(P>0.05) . The CSA of uninjected gastrocnemius muscles between control groups and treatment groups had no significant difference at the 3rd day, 10th day, 4th week, 12th week and the 24th week (P>0.05) .6. In the injected gastrocnemius muscles of treatment groups, electrophoreticanalysis showed that the relative amount of MHC I isoform increased several fold and the share of MHCII b isofonn decreased heavily during the first 12 weeks. The percentage of MHC I isofonn displayed marked decrease but still higher than that of control groups at the 24th week (P<0.01) .The relative amount of MHC II b isoform was obviously increased but still lower than that of control groups at the 24th week after BoTx-A injection (P<0.01) . In sum, MHC composition was shifted toward slower isoforms.CONCLUSION:1. BoTx-A injection could induce rat gastrocnemius muscles atrophy. The atrophy of type II muscle fibers played a major role in gastrocnemius muscles atrophy. Rat gastrocnemius muscles atrophy was appeared at the 3rd day, reached the peak of atrophy at the 4th week, restored a little at the 12th week, was still obvious at the 24th week.2. Single injection of BoTx-A 5 U has no noticeable effect to induce remote skeletal muscle atrophy.3. MHC composition was shifted toward slower isoforms after BoTx-A injection., the MHC composition was still far from normal at the 24th week.
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