| Part1 The Differential Expression Profile of microRNAs between the Biceps and Intrinsic Forepaw Muscles of Rat Models of Brachial Plexus Injuries Simulating OBPPObiective:The purpose of this study was to perform a comprehensive analysis of the differential expression profile of microRNAs between the biceps and intrinsic forepaw muscles of rat models of brachial plexus injuries simulating OBPPMethods:24 pup rats underwent total root avulsion of the brachial plexus on the right side.1,5,10, or 15 weeks after operation (n=6 each group), the intrinsic forepaw muscles and biceps on both sides were excised, Masson trichrome staining was applied for observation of the level of muscle atrophy and collagen proliferation in different time points. The 5 weeks denervated the intrinsic forepaw muscles and biceps in 9 pup rats whose C5-T1 root on the right side had been avulsed were analyzed with Affymetrix Rat microRNA array to detect the expressed microRNAs against those from the contralateral normal controls. To test the result of microRNA array, we used real-time PCR to measure the expression of differential expressed microRNAs (miR-322-5p, miR-495, miR-221-3p, miR-466b-1-3p, miR-466b-2-3p, miR-30a-5p).Results:After brachial plexus avulsion simulating OBPP, the retention rate of cross-sectional area of denervated the intrinsic forepaw muscles and biceps dramatically decline during the first 5 weeks. Ten weeks later, muscular tissue was almost replaced by collagenous fiber in denervated the intrinsic forepaw muscles and biceps. Thirty and 11 microRNAs of rat microRNA tested were significantly up-regulated and down-regulated between the denervated intrinsic forepaw muscles and the contralateral normal controls, respectively.36 and 26 microRNAs of rat microRNAs tested were significantly up-regulated and down-regulated between the denervated biceps and the contralateral normal controls, respectively. The gene expression levels of miR-322-5p, miR-495, miR-221-3p, miR-466b-1-3p, miR-466b-2-3p and miR-30a-5p which were measured by real-time PCR were similar to those of microRNA array.Conclusion:MicroRNA widely involve in the denervated atrophy of the intrinsic forepaw muscles and biceps in 5 weeks after brachial plexus avulsion simulating OBPP. There are differences in differential expressed microRNAs.Part 2 Bioinformatic Analysis of Target Genes of Differential Expressed microRNAs from the Biceps and Intrinsic Forepaw Muscles of Rat Models of Brachial Plexus Injuries Simulating OBPPObjective:The purpose of this study was to perform a bioinformatic analysis of the differential expression profile of microRNAs between the biceps and intrinsic forepaw muscles in rat models of brachial plexus injuries simulating OBPPMethods:A combined approach using computational prediction by the miRanda algorithm and Mirdb algorithm was performed to indentify the potential target genes of these differential expressed microRNAs. To be able to extract more biological information from the microarray data, gene ontology (GO) analysis and Pathway Analysis was performed on the potential target genes of these differential expressed microRNAs. Furthermore, microRNA-Gene-Networks were builded according to the relationship between differential expressed microRNAs and the potential target genes. The 5 weeks denervated the intrinsic forepaw muscles and biceps were used to analyze the morphology of end-plate by immunohistochemistryResults:The target genes of differential expressed microRNAs in the denervated intrinsic forepaw muscles were 1143. The target genes of differential expressed microRNAs in the denervated biceps were 2204. The target genes of up-regulated and down-regulated microRNAs in denervated intrinsic forepaw muscles were found involved in 36 and 42 GO functions with high enrichment(≥10), respectively. The target genes of up-regulated and down-regulated microRNAs in denervated biceps were found involved in 33 and 18 GO functions with high enrichment(≥10), respectively. The target genes of up-regulated and down-regulated microRNAs in denervated intrinsic forepaw muscles were found involved in 17 and 21 pathways, respectively. The target genes of up-regulated and down-regulated microRNAs in denervated biceps were found involved in 48 and 42, respectively. Comparing to the 5 weeks denervated biceps, total endplate perimeter, stained endplate perimeter, total endplate area, stained endplate area, receptor dispersion, the normalized persynaptic NMJ area in the denervated intrinsic forepaw muscles more significantly decline during the first 5 weeks.Conclusion:MicroRNA widely involve in the denervated atrophy of the intrinsic forepaw muscles and biceps in 5 weeks after brachial plexus avulsion simulating OBPP. There are differences in target genes, GO functions and pathways between the denervated atrophy of the intrinsic forepaw muscles and the denervated biceps. The end-plate in the denervated intrinsic forepaw muscles had more serious injury than that of the biceps during the first 5 weeks.Part 3 Differences in Postnatal Neuromuscular Junctions Development of the Biceps and Intrinsic Forepaw Muscles in Rat ForelimbObjective:The purpose of the study was to verify hypothesis that the postnatal development of NMJs of the intrinsic hand muscles is slower than that of the biceps.Methods The intrinsic forepaw muscles and biceps from twelve normal SD rats at the age of seven days and twelve normal adult SD rats at the age of ten months were used to analyze the morphology of NMJ by immunohistochemistry and electromicroscopy. Total RNA from the intrinsic forepaw muscles and biceps isolated at days 3,7,14,21,28 and 35 of postnatal age and from those of adult rats (n=6 each time point) to analysis of gene expression of AChR y subunit and AChR ε subunit.Results In term of biceps NMJ morphology, vesicle total perimeter, vesicle stained perimeter, vesicle total area, vesicle stained area, total branch length, average branch length, branching complexity, total endplate perimeter, stained endplate perimeter, total endplate area, stained endplate area, diameter of muscle fiber and the number of terminal Schwann cell had been found statistically significant differences between the rats at the age of seven days and the adult rats at the age of ten months. Vesicle dispersion, branch number, vesicle area/branch length, receptor dispersion, the normalized persynaptic NMJ area had not been found statistically significant differences between the rats at the age of seven days and the adult rats at the age of ten months. In term of the intrinsic forepaw muscles NMJ morphology, all of the above presynaptic and postsynaptic variables exhibited statistically significant differences between the rats at the age of seven days and the adult rats at the age of ten months. Using electromicroscopy we found NMJs in the intrinsic forepaw muscles of 7-day-old rat had numerous unmyelinated axons which contained fewer synaptic vesicle and been covered by terminal Schwann cell, the primary synaptic cleft was already appearing and the secondary synaptic cleft in the postsynaptic membrane was not yet formed. In contrast, NMJs in biceps of 7-day-old rat contained many axon terminals with larger diameters which been covered by larger terminal Schwann cell and had more synaptic vesicle, the primary synaptic cleft and the secondary synaptic cleft in the postsynaptic membrane could be observed. In the intrinsic forepaw muscles, ε subunit genes could been found by 7 days of age and peaked at 14 days of age before reaching to adult levels at 35 days of age (P<0.05). Y subunits tended to decrease from 3 days of age to 35 days of age and could not be test at 35 days of age. In the biceps,ε subunit genes could been found by 3 days of age and peaked at 21 days of age before reaching to adult levels at 35 days of age(P<0.05). γ subunits tended to decrease from 3 days of age to 21 days of age and could not be test at 21 days of age.Conclusions In the postnatal development, the morphology and subunits of the nicotinic acetylcholine receptor of NMJ in the intrinsic hand muscles more slowly develop to maturity than that of the biceps. |
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