| Part1A Rat Model Study of Atrophy of Denervated Musculature of the Hand Being Faster than That of Denervated Muscles of the ArmObjective Clinical evidence in obstetric brachial plexus palsy (OBPP) has showed that atrophy of denervated intrinsic musculature of the hand is much faster to irreversible than that of muscles of the arm. It is difficult to restore the morphology and contractile function of skeletal musle even if the nerve regeneration is very successful when denervated muscle is under irreversible atrophy. OBPP lies obviously in the fact that, the atrophy of denervated intrinsic musculature of the hand is much faster to irreversible than that of denervated biceps. The clinical appearances imply that the self-regulating mechanism for denervated intrinsic musculature of the hand is different from that for denervated muscles of the arm. The purpose of the experiment is to build the rat model by simulating the clinical fact that the atrophy speed of intrinsic muscular of hand is faster than the muscle in the upper limb. It will provide an ideal animal model to clarify the morphological basis of these two kinds of muscle on the the sensitivity of the surgery timing and for further genetic research.Methods64pup rats whose C5C6had been divided and C7C8T1avulsed, one of the most common pathological types of OBPP, were divided into the reconstruction and the control group of32each. The former had subgroups R1, R5, R10and R15where the ulnar and musculocutaneous nerves were reconstructed one, five, ten and15weeks, respectively, after nerve injury and efficacy was evaluated12weeks after reconstruction. The proximal stump of the C6spinal nerve was transferred to the ulnar nerve by two strands of the free sural nerve and that of C5to the musculocutaneous nerve. The control group had C1, C5, C10and C15subgroups where denervated target muscles of those two nerves were assessed one, five, ten and15weeks after nerve injury. The next step of the study was performed by1) Nerve conduction study: it was performed for the32rats in the four subgroups of the experimental group. Biceps with musculocutaneous nerves and intrinsic musculature of the anterior paw with ulnar nerves were exposed on both sides. The values of latency and amplitude of CMAPs of the musculocutaneous and ulnar nerves at the right (experimental) side were expressed, respectively, as a percentage of those at the left one.2) Myelinated axon counts:histological examination of the ulnar nerve at the wrist and of the musculocutaneous nerve just before its entrance to the biceps was performed for the32rats in the reconstruction group. Images were analyzed quantitatively by the image analysis system.The number of myelinated axons in either nerve at the right was compared with that in its counterpart at the left.3) Muscle histomorphometry: morphometric analysis of ulnar-nerve-innervated intrinsic musculature of the anterior paw and of the biceps was carried out for the reconstruction and the control group. 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 a light microscope with the image analysis system. The average cross-sectional area of the muscle fiber measured the average value at the right was expressed as a percentage of the one at the left. 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. Values were expressed as median (p25-p75) or mean (±SD), according to the distribution of variables. The Mann Whitney U test was used to compare the difference between the reconstruction and control groups. All values of probability were two-tailed, and statistical significance was set at0.05.Results The compound muscle action potential in the ulnar nerve and the musculocutaneous nerve can be measured in both R1and R5subgroup. In the R10subgroup, the CMAPs can still be stimulated in musculocutaneous nerve while it was failed in the ulnar nerve on the experimental side. The nerve conduction could not be detected of both the musculocutaneous and ulnar nerves in the R15subgroup. The least percentage of regenerating myelinated axons for ulnar nerves at the right side was85in the R1,70in the R5,73in the R10and53in the R15subgroup; that for musculocutaneous nerves was79in the R1subgroup,66in the R5,62in the R10and67in the R15.Results of cross-sectional area of the muscle fiber for intrinsic musculature of the anterior paw showed that the R5, R10and R15subgroups were not statistically superior to C5, C10and C15ones, respectively, though the Rl was; those for biceps indicated, however, that the R1, R5and R10subgroups were better than C1, C5and C10ones, respectively, though the R15was not. Apoptosis index for intrinsic musculature of the anterior paw was statistically lower in the Rl and R5subgroups than in C1and C5, respectively; but the difference was not statistically significant between R10and C10, R15and C15, respectively. As to the biceps, apoptosis index was statistically lower in R1, R5and R10than in C1, C5and C10, respectively; but that was not statistically different between R15and C15. Apoptosis index had a tendency of rising at the early stage and descending at the late in the denervated muscle.Conclusions The rat model of OBPP is suitable for simulating clinical appearance of atrophy of denervated intrinsic musculature of the hand being faster than that of muscles of the arm. Part2Study of Different Genetic Expression between the Atrophy Processes of Denervated Intrinsic Musculature of Anterior Paw and Biceps by Gene ChipObjective Despite the current clinical repair of peripheral nerve injury in the form has reached almost perfect degree, but the recovery of neural function effect is still not ideal:recognized one of the main causes of poor recovery of denervated muscles has been irreversible atrophy before the regenerated nerve arrived. But the two distinct processes atrophy between intrinsic muscular of hand and upper arm muscle after denervation may imply that there are contraction factors in the muscle which may also affect atrophy speed.The iterative process of the presence of inflammation, apoptosis, and reconstruction, regeneration is working during the whole process of skeletal muscle atrophy from the timeline or space axis, and is under one or more genes in synergy results. The gene chip could help to detect various physiological and pathological conditions in expression levels and changes in large-scale, and also provide global view of gene expression and variation with comprehensive understanding of gene function and relations. The purpose of this study is to use gene chip technology to screening the course of the differentially expressed genes and related network control mode between intrinsic musculature of the anterior paw and biceps of rats. It will lay the foundation to find the influence of muscle atrophy in the process of key genes and pathways, so as to provide theoretical basis to establish the molecular state indicators in clinical irreversible muscle atrophy.Methods64pup rats whose C5-T1on the right side had been totally avulsed were divided into4sgroup of16each. The group-dividing was based on the time after nerve injury operation 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 its entrance to the biceps to make sure whether there was any synkinetic innervation. The intrinsic musculature of the anterior paw and of the biceps in both sides of each rat was carried out for expression microarray analysis. Total RNA from each sample was quantified using the NanoDrop ND-1000and the RNA integrity was assessed using standard denaturing agarose gel electrophoresis. For microarray analysis, Agilent Array platform was employed. The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols. Agilent Feature Extraction software (version10.7.3.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed using the GeneSpring GX v11.5.1software package (Agilent Technologies). Differentially expressed genes with statistical significance were identified through Volcano Plot and Scatter Plot filtering. Hierarchical Clustering was performed to show the distinguishable gene expression profiling among samples. Pathway analysis and GO Analysis were applied to determine the roles of these differentially expressed genes played in these biological pathways or GO terms. Finally, we selected interested regulation pathway and functional genes involved in denervated muscle atrophy.Results Gene expression profiles of32samples in16experimental muscle groups were analyzed by gene chip technology. The different gene expression between denervated atrophy of intrinsic musculature of the anterior paw and biceps in rats was screened utilizing bioinformatics and biostatistics. Furthermore, the bio-softwares are used to explore the signal pathway which may play a role in the denervated muscle atrophy. Some crucial signal pathways, for example, MLC, MHC in vascular smooth muscle contraction pathway and Smads in TGF-beta signaling pathway expressed higher in the intrinsic musculature of the anterior paw than that in the biceps. Els, E2s, E3s in Ubiquitin mediated proteolysis and NIK, SRF gene in MAPK pathways expressed higher in biceps.Conclusion The genes and signaling pathways are less in the atrophy process of self regulation after denervation in the intrinsic musculature of the anterior paw, which to promote injury, apoptosis and inhibit the repair, regeneration mainly. The genes and signaling pathways are more than intrinsic musculature of the anterior paw in the atrophy process of self regulation after denervation in biceps, which to promote the repair, regeneration and inhibit the damage and apoptosis mainly. |
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