Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that are driven by impaired growth of denervated muscles. A rare form of NBPI, which maintains afferent muscle innervation despite motor denervation, does not cause contractures. As afferent innervation regulates various aspects of skeletal muscle homeostasis through NRG/ErbB signaling, our current study investigated the role of this pathway in modulating contracture development. Through pharmacologic modification with an ErbB antagonist and NRG1 isoforms, we discovered that NRG/ErbB signaling does not modulate the development of contractures in neonatal mice. Instead, ErbB inhibition impeded growth in nondenervated skeletal muscles, whereas increased ErbB activation exacerbated denervation‐induced skeletal muscle atrophy. This potential regulatory effect of NRG/ErbB signaling on neonatal muscle growth warrants deeper investigation.

Muscle contractures are a prominent and disabling feature of many neuromuscular disorders, including the 2 most common forms of childhood neurologic dysfunction: neonatal brachial plexus injury (NBPI) and cerebral palsy. There are currently no treatment strategies to directly alter the contracture pathology, as the pathogenesis of these contractures is unknown. We previously showed in a mouse model of NBPI that contractures result from impaired longitudinal muscle growth. Current presumed explanations for growth impairment in contractures focus on the dysregulation of muscle stem cells, which differentiate and fuse to existing myofibers during growth, as this process has classically been thought to control muscle growth during the neonatal period. Here, we demonstrate in a mouse model of NBPI that denervation does not prevent myonuclear accretion and that reduction in myonuclear number has no effect on functional muscle length or contracture development, providing definitive evidence that altered myonuclear accretion is not a driver of neuromuscular contractures. In contrast, we observed elevated levels of protein degradation in NBPI muscle, and we demonstrate that contractures can be pharmacologically prevented with the proteasome inhibitor bortezomib. These studies provide what we believe is the first strategy to prevent neuromuscular contractures by correcting the underlying deficit in longitudinal muscle growth.

Purpose: Neonatal brachial plexus injury (NBPI) causes disabling contractures that cannot be fully prevented or corrected, largely because their pathophysiology is incompletely understood. Research in a mouse model has shown that contractures result at least in part from impaired postnatal muscle growth, but the mechanism of this impaired muscle growth is unknown. The current work uses a mouse model to experimentally assess and correct muscle protein imbalance (synthesis versus degradation) as a mechanism of impaired muscle growth and contractures following NBPI. Methods: Unilateral global (C5-T1) NBPIs were surgically created in 5-day-old mice, which permanently denervates the forelimb and reliably causes shoulder and elbow contractures 4 weeks post-NBPI. Protein synthesis was measured in denervated and contralateral control elbow flexor muscles within 4 weeks post-NBPI by incorporation of puromycin, a nucleoside analog, and by expression of major structural and contractile proteins by RNA-sequencing and Western blot. Protein degradation was similarly measured by K48-linkage specific polyubiquitin, an indicator of protein degradation by the proteasome pathway, and by expression of protein degradation markers by RNA-sequencing. Subsequently, to test the ability of proteasome inhibition to prevent contractures, bortezomib, a 20S proteasome inhibitor, was co-administrated with [Gly14]-Humanin G ([Gly14]-HN, to limit bortezomib toxicity) systemically for 4 weeks post-NBPI. Saline and [Gly14]-HN alone were administered separately as controls. Shoulder and elbow contractures were measured 4 weeks post-NBPI, and denervated and control elbow flexor muscles were assayed for 20S proteasome activity, volume and cross-sectional area (CSA) by microCT, and sarcomere length by DIC microscopy. Results: NBPI did not reduce muscle protein synthesis, measured either by puromycin incorporation or by RNA and protein levels of all major structural and contractile proteins. However, NBPI increased protein degradation, as indicated by a doubling of K48-linkage specific polyubiquitin and increased expression of Trim63/MurF1, a driver of proteasome-mediated protein degradation. Bortezomib + [Gly14]-HN effectively prevented contractures following NBPI compared to saline and [Gly14]-HN alone (p<0.001). Bortezomib blunted the denervation-induced increase in proteasome activity (p<0.001), and rescued muscle growth in volume (p<0.0001), CSA (p<0.001), and sarcomere length (p=0.03). Conclusion: Contractures following NBPI are associated with increased muscle protein degradation counteracting normal protein synthesis. Inhibition of the proteasome pathway of protein degradation improves growth of denervated muscle and prevents contractures following NBPI. Significance: This study identifies a pathophysiologic mechanism of impaired muscle growth and contracture formation following neonatal brachial plexus injury, and demonstrates the first ever successful pharmacologic strategy to prevent these contractures by targeting a causative molecular mechanism.

Satellite cells remain abundant in vivo in neonatally denervated muscle. Following neonatal denervation, satellite cells proliferate normally, but a disproportionate number of daughter cells return to quiescence instead of differentiating into the myoblasts necessary for neonatal muscle growth. Correcting this imbalance between SC self-renewal and differentiation, potentially by manipulating Notch signaling, could allow us to harness this population of SCs to maintain muscle growth and prevent contractures following neonatal brachial plexus injury.

Purpose We used an established mouse model of elbow flexion contracture after neonatal brachial plexus injury (NBPI) to test the hypothesis that preservation of afferent innervation protects against contractures and is associated with preservation of muscle spindles and ErbB signaling.Methods A model of preganglionic C5 through C7 NBPI was first tested in mice with fluorescent axons using confocal imaging to confirm preserved afferent innervation of spindles despite motor end plate denervation. Preganglionic and postganglionic injuries were then created in wild-type mice. Four weeks later, we assessed total and afferent denervation of the elbow flexors by musculocutaneous nerve immunohistochemistry. Biceps muscle volume and cross-sectional area were measured by micro computed tomography. An observer who was blinded to the study protocol measured elbow flexion contractures. Biceps spindle and muscle fiber morphology and ErbB signaling pathway activity were assessed histologically and immunohistochemically.Results Preganglionic and postganglionic injuries caused similar total denervation and biceps muscle atrophy. However, after preganglionic injuries, afferent innervation was partially preserved and elbow flexion contractures were significantly less severe. Spindles degenerated after postganglionic injury but were preserved after preganglionic injury. ErbB signaling was inactivated in denervated spindles after postganglionic injury but ErbB signaling activity was preserved in spindles after preganglionic injury with retained afferent innervation. Preganglionic and postganglionic injuries were associated with upregulation of ErbB signaling in extrafusal muscle fibers.Conclusions Contractures after NBPI are associated with muscle spindle degeneration and loss of spindle ErbB signaling activity. Preservation of afferent innervation maintained spindle development and ErbB signaling activity, and protected against contractures.Clinical relevance Pharmacologic modulation of ErbB signaling, which is being investigated as a therapy for congestive heart failure, may be able to recapitulate the protective effects of afferent innervation in spindle development and contracture prevention. Muscle spindle preservation may also have implications in proprioception and motor learning, both of which are impaired in NBPI. (J Hand Surg Am. 2015;40(10):2007-2016. Copyright (C) 2015 by the American Society for Surgery of the Hand. All rights reserved.)

Introduction: We investigated the contribution of muscle fibrosis to elbow flexion contractures in a murine model of neonatal brachial plexus injury (NBPI). Methods: Four weeks after NBPI, biceps and brachialis fibrosis were assessed histologically and compared with the timing of contracture development and the relative contribution of each muscle to contractures. Modulus of elasticity and hydroxyproline (collagen) content were measured and correlated with contracture severity. The effect of halofuginone antifibrotic therapy on fibrosis and contractures was investigated. Results: Elbow contractures preceded muscle fibrosis development. The brachialis was less fibrotic than the biceps, yet contributed more to contractures. Modulus and hydroxyproline content increased in both elbow flexors, but neither correlated with contracture severity. Halofuginone reduced biceps fibrosis but did not reduce contracture severity. Conclusions: Contractures after NBPI cannot be explained solely by muscle fibrosis, arguing for investigation of alternate pathophysiologic targets for contracture prevention and treatment. Muscle Nerve49:398-404, 2014

The pathophysiology of paradoxical elbow flexion contractures following neonatal brachial plexus injury (NBPI) is incompletely understood. The current study tests the hypothesis that this contracture occurs by denervation-induced impairment of elbow flexor muscle growth. Unilateral forelimb paralysis was created in mice in four neonatal (5-day-old) BPI groups (C5-6 excision, C5-6 neurotomy, C5-6 neurotomy/repair, and C5-T1 global excision), one non-neonatal BPI group (28-day-old C5-6 excision), and two neonatal muscle imbalance groups (triceps tenotomy ± C5-6 excision). Four weeks post-operatively, motor function, elbow range of motion, and biceps/brachialis functional lengths were assessed. Musculocutaneous nerve (MCN) denervation and reinnervation were assessed immunohistochemically. Elbow flexion motor recovery and elbow flexion contractures varied inversely among the neonatal BPI groups. Contracture severity correlated with biceps/brachialis shortening and MCN denervation (relative axon loss), with no contractures occurring in mice with MCN reinnervation (presence of growth cones). No contractures or biceps/brachialis shortening occurred following non-neonatal BPI, regardless of denervation or reinnervation. Neonatal triceps tenotomy did not cause contractures or biceps/brachialis shortening, nor did it worsen those following neonatal C5-6 excision. Denervation-induced functional shortening of elbow flexor muscles leads to variable elbow flexion contractures depending on the degree, permanence, and timing of denervation, independent of muscle imbalance.

目的 探讨共聚物P85与黏附增强型绿色荧光蛋白(enhanced green fluoresent protein,EGFP)质粒的微泡造影剂(microbubble,MB)结合后联合一定强度的超声(ultrasound,US)辐照,是否增强人肝癌细胞(HepG2)的质粒转染及表达.方法 以人肝癌细胞(HepG2)为研究对象,质粒DNA是可表达绿色荧光蛋白的pEGFP,添加微泡造影剂或共聚物P85后进行脉冲多普勒超声辐照(频率1 MHz,声强1 W/cm2,工作周期20%,时间20 s).24 h后台盼蓝染色评价细胞存活率,荧光显微镜和流式细胞仪评价细胞的基因转染率.结果 有超声辐照组的pEGFP转染率明显高于无超声辐照组(P＜0.01),有超声辐照组中pEGFP+30%MB+P85+US组转染效率(22.14±3.06)%优于单独使用微泡组(11.34±2.2)%或P85组(9.72±1.21)%(P＜0.01).结论 微泡造影剂与共聚物P85结合同时给予超声辐照可增强基因转染效率,在基因治疗上有待于进一步关注和研究.

Objective To investigate the appropriate microbubble(MB) concentration with pluronic P85 that can enhance the gene transfection and expression to HepG2 cell under ultrasound(US) irradiation.Methods Plasmid encoding enhanced green fluorescent protein(pEGFP) was used as a report gene.HepG2 cell and plasmids DNA with P85 and different microbubble concentration were exposed to US,and after 24 h the transfection rates and cell viability were assessed with fluorescence microscopy,FACS and trypan blue exclusion.Results The approving transfection efficiency was got at 30% microbubble concentration,with ideal gene transfection efficiency(22.14±3.06)% and without obvious decreasing of survival rate(55.73±3.32)%.Higher transfection rate than pEGFP+US+P85 could be obtained through adding different concentration of microbubble.Conclusion Microbubble combined with pluronic P85 can enhance the gene transfection under ultrasound irradiation,and the approving transfection rate can be obtained at 30% microbubble concentration.

This study examined the effect of P85（a pluronic block copolymer）and microbubble（MB）ultrasound contrast agents under ultrasound irradiation on gene transfection and expression.The pEGFP plasmids that can encode enhanced green fluorescent protein（pEGFP）served as a report gene and were mixed with different concentrations of MB/0.05%（w/v）P85.Then the plasmids were transfected into human hepatoma G2（HepG2）cells.The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound（US parameters:1 MHz,1.0 W/cm2,20 s,20% duty cycle）.Twenty-four hours later,the transfection efficiency was assessed by fluorescence microscopy and fluorescence activated cell sorting（FACS）analysis.The cell viability was evaluated by Trypan blue exclusion test.The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation.HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein.The expression efficiency reached（22.14±3.06）% and the survival rate was as high as（55.73±3.32）% in the 30% MB plus P85 group.It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.

Objective To investigate the relationship of gene transfection efficiency with different ultrasound exposure time and different dose of microhuhble,and to find the appropriate ultrasound parameters for gene transfection. Methods Plasmid encoding enhanced green fluorescent protein(pEGFP) was chosen as a report gene and HepG2 cells were chosen as research object. The HepG2 cells plus pEGFP and different dose of microbubble were exposed to ultrasound(1 MHz,0.5 W/cm2) with varying time. Twenty-four hours later, the expression of EGFP in the cells was observed by fluorescence microscope,the transfection efficiency was assessed by FACS and the cell viability was observed by trypan blue exclusion. Results The expression of EGFP in all experimental groups was different,and the approving transfection efficiency was got by ultrasound exposed for 20 s when the dose of microbubble was 60 μl. Conclusions With fixed ultrasound frequency and power, different transfection efficiency was got when the exposure time and dose of microbubble were different. The appropriate parameter was 20 s,60 μl, which can supply information for further study.

Objective To explore the correlation between contrast-enhanced ultrasound(CEUS)and the histopathological grading of hepatocellular carcinoma(HCC).Methods Forty-seven patients with HCC underwent examination of CEUS performed with SonoVue.The results of the CEUS were compared with histopathological grand and tumor differentiation.Results Nine(100%)well differentiated HCC's TIC showed quick-up and slow-down;15 of 24(20.80%)moderately differentiated HCC's TIC showed quick-up and slow-down;19 of 24(79.20%)moderately differentiated HCC's TIC showed quick-up and quick-down;8 of 14(57.10%)poorly differentiated HCC's TIC showed quick-up and slow-down,while 6 of 14(42.9%)poorly differentiated HCC's TIC showed quick-up and quick-down.Pathologic grading of HCC was correlated to washing-out time(P0.05),but was not correlated to the arrival time,enhancement time or peak intensity(P0.05).Conclusion The type of TIC and acoustic quantified parameters of CEUS are useful for the diagnosis and histological differentiation of HCC.

<正>近年,超声医学不仅在诊断领域取得了突飞猛进的发展,而且在治疗领域也呈现出潜在的应用前景,尤其在基因治疗方面愈来愈受到超声医学和分子生

Ultrasonography is a simple and reliable non-invasive method in tumor vascularity detection of breast neoplasms. Tumor vascularity morphometric and topological characteristic in breast neoplasms provides an important basis for differentiation between benign and malignant lesions. Studies indicate that visual blood vessel shape and direction, velocity, properties of blood flow can be satisfactorily shown by color Doppler flow imaging(CDFI). But CDFI is confined by the angle between acoustic beam and blood flow. Power Doppler ultrasound (PDU) can overcome the difficulties mentioned above, but can not offer the information of blood velocities and motion error. The intratumoral flow features can clearly and visually shown by three-dimensional ultrasonography (3DUS) and contrast- enhanced ultrasound (CEUS). 3 DUS and CEUS will be new potential diagnosis techniques in vascularity estimation of breast neoplasms.

对106例各种肝脏疾病合并腹泻的住院病人资料进行总结和分析。结果:(1)各种肝脏疾病中,肝源性腹泻发生率由高到低依次为:各型肝硬化(64.15%)、慢性乙型病毒性肝炎(18.87%)、原发性肝癌(11.32%)、脂肪肝(3.77%)和药物性肝病(1.89%),其临床表现无特征性;(2)腹泻发生在肝病早期的较少,大多出现在肝病的中后期;(3)腹泻的严重程度与肝功能密切相关。早期诊断和改善肝功能是治疗肝源性腹泻的关键。