Delivery of neurotrophin-3 from fibrin enhances neuronal fiber sprouting after spinal cord injury.

Neurotrophins have been shown to promote axonal growth and regeneration after spinal cord injury. The therapeutic utility of neurotrophins may be enhanced by using a controlled delivery system to increase the duration of neurotrophin availability following injury. Such a delivery system can be incorporated into a bioactive scaffold to serve as a physical bridge for regeneration. This study assessed the effect of controlled delivery of neurotrophin-3 (NT-3) from fibrin scaffolds implanted in spinal cord lesions immediately following 2-mm ablation injury in adult rats. Nine days after injury, fibrin scaffolds containing the delivery system and NT-3 (1000 ng/mL) elicited more robust neuronal fiber growth into the lesion than did control scaffolds or saline (1.5- to 3-fold increase). Implantation of fibrin scaffolds resulted in a dramatic reduction of glial scar formation at the white matter border of the lesion. Hindlimb motor function of treated animals did not improve relative to controls at 12 weeks post-injury. Thus, controlled delivery of NT-3 from fibrin scaffolds enhanced the initial regenerative response by increasing neuronal fiber sprouting and cell migration into the lesion, while functional motor recovery was not observed in this model.     

Controlled release of neurotrophin-3 from fibrin gels for spinal cord injury.

The goal of this work was to assess the feasibility of using affinity-based delivery systems to release neurotrophin-3 (NT-3) in a controlled manner from fibrin gels as a therapy for spinal cord injury. A heparin-based delivery system (HBDS) was used to immobilize NT-3 within fibrin gels via non-covalent interactions to slow diffusion-based release of NT-3, thus allowing cell-activated degradation of fibrin to mediate release. The HBDS consists of three components: immobilized linker peptide, heparin and NT-3. The linker peptide contained a Factor XIIIa substrate and was covalently cross-linked to fibrin during polymerization. This immobilized linker peptide sequesters heparin within fibrin gels, and sequestered heparin binds NT-3, preventing its diffusion. Mathematical modeling was performed to examine the effect of heparin concentration on the fraction of NT-3 initially bound to fibrin. In vitro release studies confirmed that heparin concentration modulates diffusion-based release of NT-3. Fibrin gels containing the HBDS and NT-3 stimulated neural outgrowth from chick dorsal root ganglia by up to 54% versus unmodified fibrin, demonstrating that the NT-3 released is biologically active. In a preliminary in vivo study, fibrin gels containing the HBDS and NT-3 showed increased neural fiber density in spinal cord lesions versus unmodified fibrin at 9 days.     

神经营养素-3对大鼠脊髓损伤后caspase-3基因表达的影响

【目的】研究神经营养素-3(NT-3)对大鼠脊髓损伤后caspase-3基因表达的影响,探讨NT-3在脊髓损伤修复中的作用。【方法】SD大鼠80只,分为生理盐水对照组和NT-3组。用RT-PCR分析方法观察两组caspase-3基因的表达情况。【结果】①脊髓损伤后caspase-3基因mRNA转录水平升高;②NT-3组与生理盐水对照组相比caspase-3基因转录水平下降(P<0.05)。【结论】NT-3抑制caspase-3基因的表达,可能是促进神经纤维损伤后再生的一个重要因素。     

Preliminary study of a genetically engineered spinal cord implant on urinary bladder after experimental spinal cord injury in rats

The objective of this study was to determine the effect of neurotrophin-secreting Schwann cell implants on the urinary bladder after spinal cord contusion. One hour after severe spinal cord contusion at the T8 to T11 level, carbon filaments containing nonsecreting Schwann cells, brain-derived neurotrophic factor (BDNF)-secreting Schwann cells, neurotrophin-3 (NT-3)-secreting Schwann cells, or Schwann cells secreting both BDNF and NT-3 were implanted into the spinal cord. Untreated spinal cord injured (SCI) rats and noncontused rats (C) were also studied. Two months after spinal cord injury, cystometry was performed and the bladders were studied using light microscopy. SCI rats had significantly increased bladder mass, thickness, and smooth muscle mass compared to C rats. Bladder capacity of SCI rats and rats with spinal cord implants were both significantly greater than that of C rats. This preliminary study suggests that neurotrophin-secreting Schwann cell implants may lead to improved bladder structure after spinal cord injury.     

Local delivery of FTY720 and NSCs on electrospun PLGA scaffolds improves functional recovery after spinal cord injury

Spinal cord injury (SCI) is a common issue in the clinic that causes severe motor and sensory dysfunction below the lesion level. FTY720, also known as fingolimod, has recently been reported to exert a positive effect on the recovery from a spinal cord injury. Through local delivery to the lesion site, FTY720 effectively integrates with biomaterials, and the systemic adverse effects are alleviated. However, the effects of the proper mass ratio of FTY720 in biomaterials on neural stem cell (NSC) proliferation and differentiation, as well as functional recovery after SCI, have not been thoroughly investigated. In our study, we fabricated electrospun poly (lactide-co-glycolide) (PLGA)/FTY720 scaffolds at different mass ratios (0.1%, 1%, and 10%) and characterized these scaffolds. The effects of electrospun PLGA/FTY720 scaffolds on NSC proliferation and differentiation were measured. Then, a rat model of spinal transection was established to investigate the effects of PLGA/FTY720 scaffolds loaded with NSCs. Notably, 1% PLGA/FTY720 scaffolds exerted the best effects on the proliferation and differentiation of NSCs and 10% PLGA/FTY720 was cytotoxic to NSCs. Based on the Basso, Beattie, and Bresnahan (BBB) score, HE staining and immunofluorescence staining, the PLGA/FTY720 scaffold loaded with NSCs effectively promoted the recovery of spinal cord function. Thus, FTY720 properly integrated with electrospun PLGA scaffolds, and electrospun PLGA/FTY720 scaffolds loaded with NSCs may have potential applications for SCI as a nerve implant.

Spinal cord injury (SCI) is a common issue in the clinic that causes severe motor and sensory dysfunction below the lesion level.     

Multiple drug delivery hydrogel system for spinal cord injury repair strategies

The multifactorial pathological progress of spinal cord injury (SCI) is probably the main reason behind the absence of efficient therapeutic approaches. Hence, very recent highlights suggest the use of new multidrug delivery systems capable of local controlled release of therapeutic agents. In this work, a biocompatible hydrogel-based system was developed as multiple drug delivery tool, specifically designed for SCI repair strategies. Multiple release profiles were achieved by loading gel with a combination of low and high steric hindrance molecules. In vitro, in vivo and ex vivo release studies showed an independent combination of fast diffusion-controlled kinetics for smaller molecules together with slow diffusion-controlled kinetics for bigger ones. A preserved functionality of loaded substances was always achieved, confirming the absence of any chemical stable interactions between gel matrix and loaded molecules. Moreover, the relevant effect of the cerebrospinal fluid flux dynamics on the drug diffusion in the spinal cord tissue was here revealed for the first time: an oriented delivery of the released molecules in the spinal cord tract caudally to the gel site is demonstrated, thus suggesting a more efficient gel positioning rostrally to the lesion.     

人发角蛋白植入大鼠损伤脊髓部位的电镜观察

目的:脊髓外伤后的继发性病理改变,是影响脊髓神经组织再生修复的重要因素。采用人发角蛋白(humanhairkeratin,HHK)植入脊髓损伤(spinalcordinjury,SCI)部位,以期达到减轻继发性损害,诱导和促进损伤脊髓组织的再生。方法:采用改制Ⅱ型纽约大学(NewYorkUniversity,NYU)装置,在建立大鼠脊髓损伤模型基础上,将经过特殊处理后能在体内降解的HHK植入大鼠损伤脊髓部位,对植入后1,4,12,26周的损伤脊髓组织进行电镜观察。结果:第1周为急性炎症时期,HHK周围结构紊乱,集聚大量的炎症细胞,灰质出现坏死;第4周时,炎症细胞减少,巨噬细胞吞噬髓鞘,胶质细胞增生;第12周时,多核巨噬细胞出现在HHK周围,HHK开始崩解,崩解物被多核巨细胞所吞噬;第26周时,神经轴突沿HHK间隙排列生长,灰质中神经元数量增加,HHK周边细胞有序生长。结论:植入的人发角蛋白具有诱导神经胶质细胞增生,阻止脊髓空洞的形成,从而减轻了脊髓损伤组织的继发性伤害的程度,改善了神经元再生的外环境,并可以桥接诱导神经轴突定向再生的作用。     

神经营养因子对成年大鼠脊髓损伤后皮质脊髓束再生的影响

背景胶质细胞源性神经营养因子(GDNF)对脊髓运动神经元的存活有明显的保护作用,但GDNF对成年大鼠脊髓损伤后皮质脊髓束再生是否有促进作用仍不清楚.目的研究胶质细胞源性神经营养因子对脊髓损伤后皮质脊髓束再生的保护作用.设计随机对照实验.地点和材料本实验在第二军医大学神经生物实验室完成.实验动物采用雄性成年SD大鼠64只,体质量250～300 g.干预采用Nystrom法制备大鼠胸髓后路压迫损伤模型,压迫质量为50 g,时间为5 min,造成大鼠胸段脊髓急性重度损伤.脊髓损伤后24 h,对照组注射6μL阳离子脂质体DC-Chol和2μg pCDNA3的混合物,实验组将6μL DC-Chol和2μg重组质粒pEGFP-GDNF cDNA混合后注入大鼠损伤脊髓.主要观察指标应用RT-PCR技术和荧光显微镜检测GDNF体内转基因后在局部的表达,通过辣根过氧化物酶(HRP)顺行追踪技术和神经微丝(NF)免疫组化活性的变化来评价GDNF基因体内转染对大鼠皮质脊髓束再生的影响.结果GDNF基因体内转染1周后发现在基因注射局部有转录和蛋白水平表达,4周后在脊髓损伤周围仍检测到GDNF蛋白表达.脊髓损伤后4周,实验组脊髓损伤区皮质脊髓束HRP标记明显多于对照组,仅在实验组可见部分神经纤维穿过损伤区至远端5～9 mm.损伤区NF阳性轴突数(524.33±80.55/低倍视野)较对照组(309.84±56.65/低倍视野)明显增多(P＜0.01),GFAP阳性细胞数(186.68±16.25)明显少于对照组(239.78±21.44)(P＜0.01).结论阳离子脂质体介导GDNF体内转基因能促进近端皮质脊髓束再生和神经骨架蛋白的修复,提示神经营养因子基因治疗可用来治疗创伤性脊髓损伤.     

神经营养素-3修复脊髓损伤的研究进展

脊髓损伤的修复是神经科学研究领域的一大难题,原因之一是损伤脊髓的再生能力极其有限。近年来大量实验证明神经营养素－3（NT－3）对脊髓损伤的再生修复具有至关重要的作用,其中又以其基因移植效果为佳。本文就NT－3对脊髓损伤的修复研究进展作一综述。     

Potential of olfactory ensheathing cells for cell-based therapy in spinal cord injury

Contusive spinal cord injury (SCI) results in a complex lesion that includes cellular and axonal loss, microglia and macrophage activation, and demyelination. These changes result in permanent neurological deficits in people with SCI and in high financial costs to society. Unlike the peripheral nervous system (PNS), in which axonal regeneration can occur, axonal regeneration in the central nervous system (CNS) is extremely limited. This limited regeneration is thought to result from a lack of a permissive environment and from active inhibitory molecules that are present in the CNS but minimal in the PNS. Currently, cell transplantation approaches are among several experimental strategies being investigated for the treatment of SCI. In the olfactory system, a specialized glial cell called the olfactory ensheathing cell (OEC) has been shown to improve functional outcome when transplanted into rodents with SCI, and clinical studies transplanting OECs into patients with SCI are ongoing in China, Portugal, and other sites. Yet, a number of controversial issues related to OEC biology and transplantation must be addressed to understand the rationale and expectations for OEC cell therapy approaches in SCI. This review provides information on these issues for spinal cord medicine clinicians.     

神经营养因子3 壳聚糖支架诱导成年大鼠脑损伤后海马神经网络的形成

目的观察神经营养因子3(NT-3)壳聚糖支架诱导神经突触形成,修复成年大鼠创伤性脑损伤。方法60 只成年雄性Wistar 大鼠平均分为单纯损伤组、单纯壳聚糖支架组和NT-3 壳聚糖支架组,分别于术后3 d、7 d、14 d、28 d 和60 d,通过免疫组织化学方法检测损伤区神经再生。术后30 d 和60 d 应用神经示踪方法与免疫电镜技术观察损伤区内再生的神经突触。结果NT-3壳聚糖支架组海马损伤区内nestin+、微管蛋白β-tubulin-Ⅲ+、微管相关蛋白2 (MAP2)+神经细胞较单纯壳聚糖支架组和单纯损伤组明显增加(P<0.01)。NT-3 壳聚糖支架组在海马损伤区内观察到5-溴脱氧尿嘧啶(BrdU)/MAP2+双阳性新生神经元,并形成突触联系。结论NT-3 壳聚糖支架可激活脑损伤区神经前体细胞增殖,分化为成熟神经元并形成神经突触,参与脑神经网络的重建。     

Methacrylate‐endcapped caprolactone and FM19G11 provide a proper niche for spinal cord‐derived neural cells

Spinal cord injury (SCI) is a cause of paralysis. Although some strategies have been proposed to palliate the severity of this condition, so far no effective therapies have been found to reverse it. Recently, we have shown that acute transplantation of ependymal stem/progenitor cells (epSPCs), which are spinal cord‐derived neural precursors, rescue lost neurological function after SCI in rodents. However, in a chronic scenario with axon repulsive reactive scar, cell transplantation alone is not sufficient to bridge a spinal cord lesion, therefore a combinatorial approach is necessary to fill cavities in the damaged tissue with biomaterial that supports stem cells and ensures that better neural integration and survival occur. Caprolactone 2‐(methacryloyloxy) ethyl ester (CLMA) is a monomer [obtained as a result of ε‐caprolactone and 2‐hydroxyethyl methacrylate (HEMA) ring opening/esterification reaction], which can be processed to obtain a porous non‐toxic 3D scaffold that shows good biocompatibility with epSPC cultures. epSPCs adhere to the scaffolds and maintain the ability to expand the culture through the biomaterial. However, a significant reduction of cell viability of epSPCs after 6 days in vitro was detected. FM19G11, which has been shown to enhance self‐renewal properties, rescues cell viability at 6 days. Moreover, addition of FM19G11 enhances the survival rates of mature neurons from the dorsal root ganglia when cultured with epSPCs on 3D CLMA scaffolds. Overall, CLMA porous scaffolds constitute a good niche to support neural cells for cell transplantation approaches that, in combination with FM19G11, offer a new framework for further trials in spinal cord regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Acellularized spinal cord scaffolds incorporating bpV(pic)/PLGA microspheres promote axonal regeneration and functional recovery after spinal cord injury

Spinal cord injury (SCI) is a traumatic injury to the central nervous system (CNS) with a high rate of disability and a low capability of self-recovery. Phosphatase and tensin homolog (PTEN) inhibition by pharmacological blockade with bisperoxovanadium (pic) (bpV(pic)) has been reported to increase AKT/mTOR activity and induce robust axonal elongation and regeneration. However, the therapeutic effect of bpV(pic) in treating SCI is limited due to the lack of efficient delivery approaches. In this study, a composite scaffold consisting of an acellular spinal cord (ASC) scaffold and incorporated bpV(pic) loaded poly (lactic-co-glycolic acid) (PLGA) microspheres was developed, in order to improve the therapeutic effect of bpV(pic) on SCI. The inhibition of PTEN activity and activation of the mTORC1/AKT pathway, the axonal regeneration and the markers of apoptosis were analyzed via western blot and immunofluorescence in vitro. The bpV(pic)/PLGA/ASC scaffolds showed excellent biocompatibility and promoted the viability of neural stem cells and axonal growth in vitro. Implantation of the composite scaffold into rats with hemi-sectioned SCI resulted in increased axonal regeneration and functional recovery in vivo. Besides, bpV(pic) inhibited the phosphorylation of PTEN and activated the PI3K/mTOR signaling pathway. The successful construction of the composite scaffold improves the therapeutic effect of bpV(pic) on SCI.

Spinal cord injury (SCI) is a traumatic injury to the central nervous system (CNS) with a high rate of disability and a low capability of self-recovery.     

An overview of tissue engineering approaches for management of spinal cord injuries

Severe spinal cord injury (SCI) leads to devastating neurological deficits and disabilities, which necessitates spending a great deal of health budget for psychological and healthcare problems of these patients and their relatives. This justifies the cost of research into the new modalities for treatment of spinal cord injuries, even in developing countries. Apart from surgical management and nerve grafting, several other approaches have been adopted for management of this condition including pharmacologic and gene therapy, cell therapy, and use of different cell-free or cell-seeded bioscaffolds. In current paper, the recent developments for therapeutic delivery of stem and non-stem cells to the site of injury, and application of cell-free and cell-seeded natural and synthetic scaffolds have been reviewed.     

胸段完全性脊髓损伤患者的三维步态分析

目的通过对胸段完全性脊髓损伤(TCSCI)患者配戴交互式步行矫形器(RGO)的三维步态分析,比较分析脊髓损伤(SCI)平面与步行能力之间的关系,探讨重建SCI患者步行能力的量化指标。方法选择在本中心住院且配戴RGO进行步行训练3个月以上的TCSCI患者10例,采用Vicon三维步态分析系统进行步态检测与分析。应用Spearman秩和相关系数对TCSCI患者的不同SCI平面与其步态的运动学和时空参数等的相关性进行统计学检验。结果步频和跨步长分别为(37.4±2.15) 步/min和(91.6±9.09) cm;髋关节摆动角度及髋关节伸展和屈曲时相的角速度分别为(42.57 °±5.43 °)、(20.88 °±2.18 °)/s和(124.75 °±9.31 °)/s。步速(r=0.80,P<0.01)、跨步长(r=0.78,P<0.01)、助行架的压力峰值(r=0.82,P<0.01)、髋关节摆动角度(r=0.77,P<0.01)、助行架的压力均值(r=-0.67,P<0.05)和髋关节伸展的角速度(r=0.75,P<0.05)与SCI平面之间均有显著的相关性。结论TCSCI患者双上肢过度负载和髋关节摆动幅度受限是其步行能力受限的主要原因;降低过度负载的康复训练方法有助于改善重建的步行功能。     

电针治疗大鼠慢性脊髓损伤疗效观察及分子机制的实验研究

目的 探索电针治疗慢性脊髓损伤的作用机理.方法 采用大鼠后路渐进性脊髓压迫动物模型,然后手术减压,并进行电针治疗.通过体诱发电位和BBB评分观察后肢功能,采用免疫组化和蛋白印迹法观察神经营养因子-3 （NT-3）及其受体（TrkC）的变化.结果 脊髓损伤后NT-3和TrkC在神经元及胶质细胞表达增强,经过电针治疗后, NT-3和TrkC在神经元和胶质细胞的表达下降.诱发电位检测和BBB评分显示,电针组疗效优于减压组（P〈0.05）.结论 电针治疗可促进脊髓损伤大鼠的行为功能恢复,这可能是通过内源性神经营养因子及其受体介导的.     

Exogenous administration of glial cell line-derived neurotrophic factor improves recovery after spinal cord injury

The aim of present study was to examine whether systemically delivered glial cell-derived neurotrophic factor (GDNF) was beneficial in reversing the spinal cord injury (SCI) in a spinal cord compression model. Rats were divided into three major groups: (1) sham operation (laminectomy only); (2) laminectomy+SCI+normal saline (1ml/kg, i.v.); (3) laminectomy+SCI+GDNF (50ng/kg, i.v.). Spinal cord injury was induced by compressing the spinal cord for 1min with an aneurysm clip calibrated to a closing pressure of 55g. GDNF or saline was administered immediately after SCI via the tail vein. Behavioral tests of motor function measured by maximal angle an animal could hold to the inclined plane were conducted at days 1-7 after SCI. The triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis, respectively. Both GDNF and vascular endothelial growth factor (VEGF) in the injured spinal cord were assayed by immunofluorescence. It was found that systemically delivered GDNF, but not vehicle solution, significantly attenuated the SCI-induced hind limb dysfunction and spinal cord infarction and apoptosis. Both GDNF and VEGF could be detected in the injury spinal cord after GDNF, but not vehicle solution, therapy. The results indicate that GDNF treatment may be beneficial in reversing hind limb dysfunction by reducing spinal cord infarction and apoptosis in a spinal cord compression model.     

直流电场与甲基强的松龙联合应用促进脊髓再生的实验研究

背景直流电场能促进脊髓再生,但伤后6 h置入电刺激器的疗效比伤后立即置入电刺激器的疗效为差,可能于脊髓损伤后出现脊髓水肿有关.目的探讨直流电场与大剂量甲基强的松龙联合应用治疗完全性脊髓损伤的疗效.设计随机对照实验.地点和对象实验在海南省人民医院完成.对象为33只中国家犬,体质量10～12kg,犬龄1.5～2岁,由海南省动物中心提供.干预将33只家犬随机分成3组,用AllenWD法致脊髓完全损伤.A组为对照组;B组脊髓损伤6 h时置入电刺激器组;C组脊髓损伤2 h静滴大剂量甲基强的松龙,脊髓损伤6 h再置入电刺激器组.主要观察指标伤后各组1,2,3个月神经功能、皮层体感诱发电位、神经元数量、神经元截面积和内氏体密度恢复情况.结果神经功能评分A组伤后1～3个月均为1分;B组伤后1个月3分,伤后2,3个月均为4分;C组伤后1个月4分,伤后2,3个月均为5分.B,C组1,2,3个月神经功能、皮层体感诱发电位、神经元数量、神经元截面积和尼氏体密度恢复均优于同时期A组(P＜0.05或P＜0.01).此外,C组优于B组,差异有显著性意义.结论直流电场能有效地促进脊髓再生,直流电场与大剂量甲基强的松龙联合应用能有效地协同治疗脊髓损伤,特别是促进神经功能早期更好的恢复.     

Collagen scaffold combined with human umbilical cord‐derived mesenchymal stem cells promote functional recovery after scar resection in rats with chronic spinal cord injury

Effective therapeutic strategies for treating chronic spinal cord injury (SCI) are currently unavailable. Scar tissue in the lesion area is a main inhibitory factor for axonal regeneration and repair of chronic SCI. In this study, scar tissue was surgically resected from adult rats with 12 week chronic SCI and then collagen scaffold (NeuroRegen Scaffold; NRS) and human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) were implanted into the resected cavity to repair chronic SCI. The results demonstrated that the locomotor function of rats was not affected by surgical scar resection, indicating its safety in treating chronic SCI. Implanting NRS and hUC‐MSCs promoted locomotion in rats and improved cortical motor‐ and somatosensory‐evoked potentials. Furthermore, implanting NRS and hUC‐MSCs promoted neurofilament‐ and β‐tubulin‐III‐positive neural regeneration and remyelination, elicited β‐tubulin‐III‐positive neuron production in the lesion area and blocked astrocyte growth outside the lesion area. In conclusion, implanting NRS in combination with hUC‐MSCs provided a beneficial microenvironment for neural regeneration, showing significant therapeutic effects for chronic SCI.     

Strategies for neurotrophin‐3 and chondroitinase ABC release from freeze‐cast chitosan–alginate nerve‐guidance scaffolds

Freeze casting, or controlled unidirectional solidification, can be used to fabricate chitosan–alginate (C–A) scaffolds with highly aligned porosity that are suitable for use as nerve‐guidance channels. To augment the guidance of growth across a spinal cord injury lesion, these scaffolds are now evaluated in vitro to assess their ability to release neurotrophin‐3 (NT‐3) and chondroitinase ABC (chABC) in a controlled manner. Protein‐loaded microcapsules were incorporated into C–A scaffolds prior to freeze casting without affecting the original scaffold architecture. In vitro protein release was not significantly different when comparing protein loaded directly into the scaffolds with release from scaffolds containing incorporated microcapsules. NT‐3 was released from the C–A scaffolds for 8 weeks in vitro, while chABC was released for up to 7 weeks. Low total percentages of protein released from the scaffolds over this time period were attributed to limitation of diffusion by the interpenetrating polymer network matrix of the scaffold walls. NT‐3 and chABC released from the scaffolds retained bioactivity, as determined by a neurite outgrowth assay, and the promotion of neurite growth across an inhibitory barrier of chondroitin sulphate proteoglycans. This demonstrates the potential of these multifunctional scaffolds for enhancing axonal regeneration through growth‐inhibiting glial scars via the sustained release of chABC and NT‐3. Copyright © 2014 John Wiley & Sons, Ltd.