| Part1A Morphological Study of Atrophy of Denervated Intrinsic Muscle of Anterior Paw and Biceps MuscleObjective Clinically in obstetric brachial plexus palsy (OBPP), atrophy of denervated intrinsic musculature of the hand is much faster to irreversible than that of denervated muscles of the arm. We conjecture that different modulating modes of proteins and genes differ biological behaviors of denervated intrinsic musculature of the hand from those of big muscles in the arm:when the ability of denervated muscle fibers for self-repair and self-reconstruction is balanced with that for inflammation and apoptosis, the muscle atrophy is reversible; if the ability for self-repair and self-reconstruction is diminished to be out of balance, the denervated atrophy is irreversible. The purpose of the experiment is to compare the atrophy of denervated intrinsic muscle of anterior paw with that of biceps muscle, which will clarify the morphological basis of these two kinds of musculature on the differential atrophic process, and further for proteome research of differential protein expression.Methods32pup rats whose C5-T1nerve roots on the right side had been totally avulsed were divided into4groups of8each. The group-dividing was based on the time after nerve avulsion of one, five, ten and15weeks respectively. The myelinated axon analyzation was performed on the ulnar nerve at the wrist and the musculocutaneous nerve just before the entrance to the biceps to ensure whether there was any synkinetic innervation. Morphometric analysis of ulnar-nerve-innervated intrinsic muscle of the anterior paw and of the biceps was carried out. Hematoxylin-erosin staining was applied for observation of muscle fibers, Masson trichrome staining for collagen and TUNEL staining for apoptosis, the latter two of which were only performed for muscles at the right side. Images were observed under microscope with the image analysis system. The average cross-sectional area of the muscle fiber was measured on both side, and value on the experimental side was expressed as a percentage of that on the control side. The cross-sectional area percentage of collagen was calculated by dividing the area of collagen by that of collagen plus muscle fibers and multiplying by100percent. The apoptosis index of the muscle was defined as the percentage of apoptotic cell nuclei relative to the total nuclei, and that was calculated by dividing the number of TUNEL positive staining nuclei by1000nuclei which were counted in the random areas of3-5visual fields in one section, and multiplying by100percent. Data were expressed as median (p25-p75). The Wilcoxon signed-rank test was used to compare the difference between intrinsic muscle of anterior paw and biceps. All values of probability were two-tailed, and statistical significance was set at0.05.Results Results of cross-sectional area of the muscle fiber showed that, biceps were statistically superior to intrinsic muscle of the anterior paw when five and ten weeks after nerve avulsion, but the difference was not statistically significant when one week and15weeks after nerve avulsion. In intrinsic muscle of the forepaw, the cross-sectional area percentages of collagen was found statistically more than in biceps when one week, five and ten weeks after nerve avulsion, and there was no statistical difference between them when15weeks after nerve avulsion. Apoptosis indexes for intrinsic musculature of the forepaw were statistically higher than those for biceps when one week after nerve avulsion; but were statistically lower when ten weeks after nerve avulsion; and the difference was not statistically significant between intrinsic musculature and biceps when five and15weeks after nerve avulsion. Apoptosis index had a tendency of rising at the early stage and descending at the late in the denervated muscle.Conclusion Atrophy of intrinsic musculature of the forepaw was morpho-logically proved faster than that of muscles of the arm, which would contribute to further proteome research of differential protein expression between these two skeletal muscles. Part2Differential Protein Expression between the Atrophy Processes of Denervated Intrinsic Muscle of Anterior Paw and Biceps MuscleObjective The large number of membrane-associated proteins, the exceptionally high molecular mass of many muscle components, extensive posttranslational modifications in various muscle proteins and their organisation in highly complex supramolecular structures make it extremely difficult to carry out conventional biochemical studies of potential changes in protein clusters during physiological adaptations or pathological processes. Proteomics is an unbiased and technology-driven approach for the comprehensive cataloging of entire protein complements and represents an ideal analytical tool for the high-throughput discovery of protein alterations in health and disease. The purpose of the experiment is to screen the differential protein expression between denervated intrinsic muscle of anterior paw and biceps muscle by using iTRAQ-based quantitative proteomic technique. Organizing modes will be disclosed of different proteins which can maintain the atrophy of all kinds of denervated muscles reversible. Results of this study may provide theoretical basis for constructing clinical diagnosing criteria to indicate if the atrophy of denervated muscles has been in a state of irreversibility.Methods32pup rats whose C5-T1nerve roots on the right side had been totally avulsed were divided into4groups of8each. The group-dividing was based on the time after nerve avulsion of one, five, ten and15weeks respectively. The myelinated axon analyzation was performed on the ulnar nerve at the wrist and the musculocutaneous nerve just before the entrance to the biceps to ensure whether there was any synkinetic innervation. After extracted, quantified, purified and digested, proteins were labeled with the iTRAQ tags. iTRAQ-2DLC-ESI-MS/MS technique was used to screen the differential protein expression between denervated intrinsic muscle of anterior paw and biceps muscle. Protein identification and quantification for the iTRAQ experiment was performed with the ProteinPilot software. GO analysis and Pathway analysis were applied to determine the roles of these differentially expressed proteins played in the GO terms and biological pathways. Finally, we selected interested regulation pathway and functional proteins involved in denervated muscle atrophy.Results569proteins were quantified by iTRAQ proteome analysis, in which227,71,60and131proteins upregulated in intrinsic muscle of the forepaw, and58,188,163,34proteins upregulated in biceps muscle by the standards of expression fold change>1.5or<0.66. GO analysis showed that most upregulated proteins were found in ribosome, cytoskeleton and vesicle of cell component, involved in binding molecular function, and involved in translation, regulation of cell death and apoptosis, collagen organization of biological processes; most downregulated proteins were found in contractile fiber and mitochondrion of cell component, involved in binding and transporter activity of molecular function, and involved in biosynthetic and catabolic process, energy metabolism and muscle contraction of biological processes. Pathway analysis showed that up-regulated proteins were enriched in16pathways such as Proteasome and Leukocyte transendothelial migration, and down-regulated proteins were enriched in18pathways such as Glycolysis/Gluconeogenesis, Citrate cycle (TCA cycle) and Oxidative phosphorylation. Cluster analysis showed that the protein expression level was up-regulated both in intrinsic muscle of forepaw and biceps muscle from one week to five weeks after nerve avulsion, was up-then down-regulated in intrinsic muscle from five weeks to15weeks, and was down-regulated in biceps muscle frome ten weeks to15weeks.Conclusion Synthesis and degradation of protein and inflammation processes were showed more active in denervated intrinsic muscle of forepaw, but proteins involved in energy metabolism were down-regulated. Regulation of MAPK cascade such as PEBP1was up-regulated in denervated biceps muscle. |
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