Effect of combined treatment with methylprednisolone and soluble Nogo-66 receptor after rat spinal cord injury

Methylprednisolone (MP) is a synthetic glucocorticoid used for the treatment of spinal cord injury (SCI). Soluble Nogo-66 receptor (NgR) ectodomain is a novel experimental therapy for SCI that promotes axonal regeneration by blocking the growth inhibitory effects of myelin constituents in the adult central nervous system. To evaluate the potential complementarity of these mechanistically distinct pharmacological reagents we compared their effects alone and in combination after thoracic (T7) dorsal hemisection in the rat. Treatment with an ecto-domain of the rat NgR (27-310) fused to a rat IgG [NgR(310)ecto-Fc] (50 microm intrathecal, 0.25 microL/h for 28 days) or MP alone (30 mg/kg i.v., 0, 4 and 8 h postinjury) improved the rate and extent of functional recovery measured using Basso, Beattie, Bresnahan (BBB) scoring and footprint analysis. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NgR(310)ecto-Fc was not apparent until 2 weeks after SCI. NgR(310)ecto-Fc or MP treatment resulted in increased axonal sprouting and/or regeneration, quantified by counting biotin dextran amine-labeled corticospinal tract axons, and increased the number of axons contacting motor neurons in the ventral horn gray matter caudal to the lesion. Combined treatment with NgR(310)ecto-Fc and MP had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone. The data demonstrate that NgR(310)ecto-Fc and MP act in a temporally and mechanistically distinct manner and suggest that they may have complementary effects.     

以NgR为靶点治疗脊髓损伤的研究进展

NgR是最近发现并克隆的一种糖基磷脂酰肌醇（GPI）锚定膜蛋白，因其在髓磷脂抑制轴突再生信号转导过程中特殊的靶分子效应，日益受到重视。以NgR为靶点促进脊髓轴突再生的尝试为中枢神经系统（CNS）损伤的治疗提供了有益的启示。     

Expression profile of receptors for myelin-associated inhibitors of axonal regeneration in the intact and injured mouse central nervous system

Although CNS neurons have the potential to regenerate their axons after injury, myelin debris carrying axon growth inhibitors rapidly induce growth cone collapse. Receptors (NgR1, NgR2) and coreceptors (LINGO-1, p75(NTR), TROY) for these inhibitors have been characterized and transduction pathways partially identified. However, little is known about the expression of these receptors in intact and lesioned supraspinal projection neurons. Using in situ hybridization, immunohistochemistry and neuronal tract-tracing, we found that NgR1, NgR2 and LINGO-1 are strongly expressed in several neuronal populations of the adult mouse brain projecting to the spinal cord, including neurons projecting through the corticospinal, rubrospinal, caerulospinal, reticulospinal, raphespinal and vestibulospinal tracts. As expected, p75(NTR) expression was restricted to neuronal descending pathways from the brainstem. TROY was absent from most brain regions and from all neuronal projection systems, suggesting that additional signal-transducing coreceptors exist. Qualitative and quantitative analyses revealed that brain expression for these receptors was not affected by a severe T10 spinal cord contusion.     

Nogo, a star protein in reticulon family

Nogo is widely expressed in higher vertebrate animals. Nogo gene gives rise to multiple isoforms. All the subtypes of Nogo proteins are characterized by a 200-amino-acid C-terminal domain, including two long hydrophobic sequences. Biological functions of Nogo include inhibition of neurite growth from the cell surface via specific receptors, intracellular trafficking, cell division and apoptosis. Here, we briefly review the elementary structure, taxonomic distribution and tissue expression of Nogo, summarize recent discoveries about localization of Nogo and mechanism of action, and discuss the possible functions of Nogo.     

Nogo,a star protein in reticulon family

1 Introduction In higher vertebrate animals, from amphibian to mammalian, when the central nervous system (CNS) is injured, the neural tissue itself is difficult for most animals to regenerate. Earlier functional studies indicated that in- hibitory proteins, such as myelin-associated glycoprotein (MAG)[1], oligodendrocyte-myelin glycoprotein (OMgp)[2 ] and Nogo[3], are enriched in myelin and oligodendrocytes. Among these proteins, Nogo has attracted more and more interest, although the fun…     

Selective temporal and regional alterations of Nogo-A and small proline-rich repeat protein 1A (SPRR1A) but not Nogo-66 receptor (NgR) occur following traumatic brain injury in the rat

Axons show a poor regenerative capacity following traumatic central nervous system (CNS) injury, partly due to the expression of inhibitors of axonal outgrowth, of which Nogo-A is considered the most important. We evaluated the acute expression of Nogo-A, the Nogo-66 receptor (NgR) and the novel small proline-rich repeat protein 1A (SPRR1A, previously undetected in brain), following experimental lateral fluid percussion (FP) brain injury in rats. Immunofluorescence with antibodies against Nogo-A, NgR and SPRR1A was combined with antibodies against the neuronal markers NeuN and microtubule-associated protein (MAP)-2 and the oligodendrocyte marker RIP, while Western blot analysis was performed for Nogo-A and NgR. Brain injury produced a significant increase in Nogo-A expression in injured cortex, ipsilateral external capsule and reticular thalamus from days 1-7 post-injury (P < 0.05) compared to controls. Increased expression of Nogo-A was observed in both RIP- and NeuN positive (+) cells in the ipsilateral cortex, in NeuN (+) cells in the CA3 region of the hippocampus and reticular thalamus and in RIP (+) cells in white matter tracts. Alterations in NgR expression were not observed following traumatic brain injury (TBI). Brain injury increased the extent of SPRR1A expression in the ipsilateral cortex and the CA3 at all post-injury time-points in NeuN (+) cells. The marked increases in Nogo-A and SPRR1A in several important brain regions suggest that although inhibitors of axonal growth may be upregulated, the injured brain is also capable of expressing proteins promoting axonal outgrowth following TBI.     

Combined NgR vaccination and neural stem cell transplantation promote functional recovery after spinal cord injury in adult rats

C‐J. Xu, L. Xu, L‐D. Huang, Y. Li, P‐P. Yu, Q. Hang, X‐M. Xu and P‐H. Lu (2011) Neuropathology and Applied Neurobiology 37, 135–155
Combined NgR vaccination and neural stem cell transplantation promote functional recovery after spinal cord injury in adult rats Aims: After spinal cord injury (SCI), there are many adverse factors at the lesion site such as glial scar, myelin‐derived inhibitors, cell loss and deficiency of neurotrophins that impair axonal regeneration. Therefore, combination therapeutic strategies might be more effective than a single strategy for promoting functional recovery after SCI. In the present study, we investigated whether a Nogo66 receptor (NgR) vaccine, combined with neural stem cell (NSC) transplantation, could promote better functional recovery than when NgR vaccine or NSCs were used alone. Methods: Adult rats were immunized with NgR vaccine at 1 week after a contusive SCI at the thoracic level, and the NSCs, obtained from green fluorescent protein transgenic rats, were transplanted into the injury site at 8 weeks post injury. The functional recovery of the animals under various treatments was evaluated by three independent behavioural tests, that is, Basso, Beattie and Bresnahan locomotor rating scale, footprint analysis and grid walking. Results: The combined therapy with NgR vaccination and NSC transplantation protected more ventral horn motor neurones in the injured spinal cord and greater functional recovery than when they were used alone. Furthermore, NgR vaccination promoted migration of engrafted NSCs along the rostral‐caudal axis of the injured spinal cords, and induced their differentiation into neurones and oligodendrocytes in vivo. Conclusions: The combination therapy of NgR vaccine and NSC transplantation exhibited significant advantages over any single therapy alone in this study. It may represent a potential new therapy for SCI.     

Introduction of the MASH1 gene into mouse embryonic stem cells leads to differentiation of motoneuron precursors lacking Nogo receptor expression that can be applicable for transplantation to spinal cord injury

ES cells transfected with the MASH1 gene yielded purified spinal motoneuron precursors expressing HB9 and Islet1. The cells lacked the expression of Nogo receptor that was of great advantage for axon growth after transplantation to an injured spinal cord. After transplantation, mice with the complete transection of spinal cord exhibited excellent improvement of the motor functions. Electrophysiological assessment confirmed the quantitative recovery of motor-evoked potential in the transplanted spinal cord. In the grafted spinal cord, gliosis was inhibited and Nogo receptor expression was scarcely detected. The transplanted cells labeled with GFP showed extensive outgrowth of axons positive for neurofilament middle chain, connected to each other and expressed Synaptophysin, Lim1/2 and Islet1. Thus, the in vivo differentiation into mature spinal motoneurons and the reconstitution of neuronal pathways were suggested. The grafted cell population was purified for neurons and was free from teratoma development. These therapeutic strategies may contribute to a potent treatment for spinal cord injury in future.     

实验性恒河猴脊髓半横切损伤模型的建立

目的通过脊髓半侧横切的方法建立灵长类恒河猴脊髓半横切损伤动物模型,用于神经再生的研究。方法 5只成年恒河猴称重麻醉后,颈部后正中切口显露C3-5脊髓,于C4用虹膜刀切开脊髓左半侧0．5cm,通过体感诱发电位(somatosensory evoked potential SSEP)监测,确保波形改变并观察恒河猴术后24h、7d肢体运动功能和SSEP变化。结果术后24h、7d同侧肢体瘫痪,肢体运动评分由术前的5分降到1分,SSEP显示术前潜伏期(15．88±2．53)毫秒,术后24h(22．26±4．12)毫秒;7d(23．12±3．92)毫秒延长明显(P<0．01),波幅术前(11．92±5．48)μV,术后24h(5．34±3．04) μV,7d(5．78±2．98)μV,降低明显(P<0．01)。结论恒河猴麻醉后用虹膜刀脊髓半横切能损伤皮质脊髓束,引起肢体瘫痪,结果稳定可靠,操作较为简便,可建立脊髓半侧横切损伤动物模型,重复性较好,为脊髓损伤的神经修复实验研究提供了模型基础。     

Design and testing of a controlled electromagnetic spinal cord impactor for use in large animal models of acute traumatic spinal cord injury

BackgroundSpinal cord injury (SCI) causes debilitating neurological dysfunction and has been observed in warfighters injured in IED blasts. Clinical benefit of SCI treatment remains elusive and better large animal models are needed to assess treatment options. Here, we describe a controlled electromagnetic spinal cord impactor for use in large animal models of SCI.MethodsA custom spinal cord impactor and platform were fabricated for large animals (e.g., pig, sheep, dog, etc.). Impacts were generated by a voice coil actuator; force and displacement were measured with a load cell and potentiometer respectively. Labview (National Instruments, Austin, TX) software was used to control the impact cycle and import force and displacement data. Software finite impulse response (FIR) filtering was employed for all input data. Silicon tubing was used a surrogate for spinal cord in order to test the device; repeated impacts were performed at 15, 25, and 40 Newtons.ResultsRepeated impacts demonstrated predictable results at each target force. The average duration of impact was 71.2 ± 6.1 ms. At a target force of 40 N, the output force was 41.5 ± 0.7 N. With a target of 25 N, the output force was 23.5 ± 0.6 N; a target of 15 Newtons revealed an output force of 15.2 ± 1.4 N. The calculated acceleration range was 12.5–21.2 m/s².ConclusionsThis custom spinal cord impactor reliably delivers precise impacts to the spinal cord and will be utilized in future research to study acute traumatic SCI in a large animal.     

构建脊髓慢性压迫损伤模型大鼠巢蛋白的表达规律*

背景：既往应用的脊髓损伤动物模型难以达到一种慢性渐进性的压迫效果,与人体慢性脊髓压迫损伤机制有很大的不同。 目的：构建一种新的脊髓慢性压迫性损伤模型大鼠,探究慢性压迫损伤后脊髓损伤区域巢蛋白的表达规律及其意义。 方法：Wistar大鼠40只随机分为实验组30只和对照组10只。实验组大鼠取下胸7、8椎板,植入压迫材料,形成慢性压迫脊髓损伤模型。植入后第1,3,7,14,28天,取压迫处脊髓组织,行病理学检查及巢蛋白免疫组织化学染色,半定量反转录PCR反应测定巢蛋白mRNA的表达,同时测量压迫段椎管直径及缓膨胀材料侵占厚度。 结果与结论：随压迫时间的延长,实验组大鼠椎管侵占率逐渐增加,脊髓组织出现坏死等情况,大鼠BBB评分降低,压迫处脊髓组织中Nestin mRNA及蛋白表达至伤后7 d时达到高峰,而后表达逐渐下降,说明实验成功建立慢性脊髓压迫损伤动物模型,且慢性脊髓压迫损伤大鼠脊髓组织Nestin mRNA及蛋白呈动态变化。     

兔正中神经F波对脊髓压迫损伤的评估

目的探讨正中神经F波对兔急性椎管狭窄导致脊髓压迫损伤的评估作用及对运动功能的预测价值。方法20只新西兰大白兔用随机分组法分实验组(n=15)和对照组(n=5),实验组分为A、B和C三个亚组(n=5),分别在C6-C7椎板间隙向尾端方向植入直径为1.0 mm、1.5 mm和2.0 mm的实心硅胶柱,分别记录术前和术后波形平稳后的F波,麻醉前和术后2 d进行运动功能评分。对照组用于排除麻醉和手术对F波的影响。结果A、B和C组F波波幅和潜伏期均对椎管狭窄脊髓压迫损伤敏感,分别经过(0.36±0.17)min、(0.36±0.22)min和(0.32±0.13)min波变平稳;A、B和C组术后波形平稳后波幅与基线波幅百分比(71.93%±6.90%)、(34.74%±6.05%)和(8.32%±4.25%)经配对t检验,与本组术前基线波幅与基线波幅百分比(100.00%±0.00%)、(100.00%±0.00%)和(100.00%±0.00%)比较,差异有统计学意义(均P<0.05);A、B和C组手术后波形平稳后潜伏期(9.48±1.31)ms、(12.04±0.48)ms和(10.55±0.21)ms经配对t检验,与术前(8.48±0.76)ms、(9.08±0.91)ms和(8.45±0.71)ms比较,差异有统计学意义(均P<0.05);各组F波术后早期的波幅变化和术后2 d前肢运动功能评分经Spearman相关性分析呈显著性正相关(r=0.960,P<0.05)。结论兔正中神经F波对发现椎管狭窄引起的脊髓损伤非常敏感;早期F波的波幅变化可以预测脊髓损伤后的肢体运动功能。     

Nogo-66受体分子在体外培养的星形胶质细胞中的表达

目的检测Nogo-66受体（NgR）在体外培养的大鼠星形胶质细胞中的表达。方法利用RT—PCR、WesteiTl Blot和间接免疫荧光染色技术，检测原代培养并纯化的星形胶质细胞中NgR mRNA和蛋白分子的表达。结果利用RT—PCR技术，从星形胶质细胞总RNA中扩增出特异的NgR条带；利用Western Blot技术，从星形胶质细胞抽提物中检测到64kD特异性的NgR反应条带；间接免疫荧光染色和Cotffocal显微镜进一步显示，星形胶质细胞中的NgR免疫反应物主要位于细胞内。同时，来源于大鼠的C6胶质瘤细胞也证明表达NgR蛋白。结论体外培养的星形胶质细胞中表达NgR分子，这为证明NgR在体内胶质细胞中的表达提供了更直接的证据。     

不完全性脊髓缺血损伤动物模型的建立及价值

目的 探讨不完全性脊髓缺血损伤动物模型的建立方法,为不完全性脊髓缺血损伤机制研究提供理想的载体. 方法 24只新西兰大白兔按照随机数字表法分为对照组及3根组、4根组,每组8只.对照组用于排除麻醉和手术对运动诱发电位的影响;3根组、4根组分别结扎3根、4根腰动脉.各组麻醉后记录基线诱发电位,手术/结扎后30 min、2d、7d记录诱发电位;麻醉清醒后、手术/结扎后2d、7d进行运动功能评分;手术/结扎后7d后取缺血中心区标本进行HE染色,镜下观察. 结果 3根组动物结扎后30 min诱发电位波幅与对照组比较差异有统计学意义(P＜0.05),结扎后2d、7d与对照组比较差异无统计学意义(P＞0.05);4根组动物结扎后30 min、2d、7d3个时间点诱发电位波幅与对照组比较差异均有统计学意义(P＜0.05).3组动物手术/结扎后30min、2d、7d3个时间点的潜伏期与对照组比较差异均无统计学意义(P＞0.05).各组动物运动功能评分结果与诱发电位波幅变化一致. 结论 结扎3根腰动脉可以造成可逆性不完全性脊髓缺血损伤,结扎4根腰动脉可以造成不可逆性不完全性脊髓缺血损伤.     

Pharmacologic and cellular therapies in the treatment of traumatic spinal cord injuries: A systematic review

ObjectiveThe objective of this review is to synthesize and consolidate the existing literature on the treatment of SCI, focusing on drugs in development and cellular therapeutics, including stem-cell treatments.MethodsStudies were identified through a systemic search of PubMed, Ovid MEDLINE, Embase and the Cochrane database from their respective inceptions through January 1, 2020. We used the keywords “spinal cord injuries”, “therapeutics”, “stem cells”, and “pharmacology.”Study selectionStudies that assessed treatment strategies for SCI were included.Data extraction and synthesisData on SCIs were processed according to the Preferred Reporting Items for Systematic Reviews and meta-Analyses (PRISMA) guidelines.FindingsIn total, 62 articles were found in the literature search and 13 clinical trials were identified and included in this study. This review article discusses the management and treatment of SCI with an emphasis on the pharmacology, molecular approaches, and the use of stem cells. Presently, none of the treatments examined has shown to be clearly effective.ConclusionsPresent management strategies of SCI are focused on improving spinal cord perfusion and decreasing secondary injuries such as hypoxia, inflammation, edema, excitotoxicity and disturbances of ion homeostasis. This review hopes to demonstrate the significant advances made in the field of SCI and the new methodologies and practices being employed by researchers to improve our knowledge of the pathology. Our hope is that by consolidating the past and current research, improvements can be made in the management, treatment, and outcomes for these patients and other who suffer from spinal pathologies.     

促甲状腺释放激素类似物YM-14673对大鼠脊髓损伤后水肿的影响

目的研究促甲状腺释放激素（ＴＲＨ）类似物,ＹＭ－１４６７３大鼠脊髓损伤后水肿的影响。方法用改良Ａｌｌｅｎ氏法建立大鼠脊髓损伤模型,分设正常组、对照组和治疗组,治疗组在损伤后１５分钟注射ＹＭ－１４６７３,用称重法测量脊髓的水含量,公式：（湿重－干重）÷湿重×１００％。结果对照组示伤后２４小时脊髓水肿,治疗组显示在２４小时脊髓水肿减轻。结论早期应用ＴＲＨ类似物,ＹＭ-１４６７３可减轻脊髓损伤后的脊随水肿。     

Delayed myelopathy following lightning strike: a demyelinating process

Summary Although the clinical findings in over 30 cases of delayed myelopathy after electrical injury have been described, there are scantly reports of the pathology of the spinal cord in such cases. We report a case in which progressive myelopathy presented 1 month after electrical injury by lightning strike and death occurred 4 months after injury. The spinal cord showed widespread demyelination of white matter from the medulla to just above the conus medullaris. This consisted of selective degeneration of myelin sheaths without inflammation or injury to axons, other cord components, or nerve roots. Infarction of more circumscribed segments of the thoracic cord where demyelination was greatest was probably due to edema associated with myelin degeneration, causing cord swelling limited by the pia mater. Raised pressure within the cord then resulted in secondary infarction.     

Unstable upper pediatric cervical spine injuries: report of 28 cases and review of the literature

Objective Traumatic lesions of upper cervical spine are rare in children. To evaluate their experience with this lesions and factors affecting outcome, authors conducted a retrospective study of 28 cases of upper pediatric cervical spine injuries treated in the last 25 years. Materials and methods To help in treatment of these lesions, we studied our series and reviewed the literature. Patients were divided into three groups: 0 to 2, 3 to 8, and 9 to 16 years, and managed according to status at presentation and type of injury. Results Seven patients were managed surgically and 21 nonsurgically (3 halo braces, 18 hard collars or molded braces). Patients in the younger age group sustained more neurological injuries than the others. In group 1, 33% present a fracture/luxation of C0/C1 or odontoid. In group 2, 80% had sustained fracture/luxations of C2. In group 3, 60% present odontoid fractures. At late follow-up review, solid fusions were demonstrated in all patients. Neurological deterioration occur in six patients. The mortality rate was 12%. Compared with other authors’ report, incidence of this lesions increased but not the number of those managed surgically. Conclusions Management must be tailored to the patient’s age, neurological status, type, and level of injury. Compared with other author’s experience, fusion and instrumentation procedures were used less frequently. Signs of medullary compression, significant spine deformation, dynamic instability, and age higher than 8 years are the criteria for surgery. The criteria for instability in children are different from those used in adults because the residual spinal growth is a major concern. The best treatment is therefore preventive.     

The Effectiveness of Early Tracheostomy (within at least 10 Days) in Cervical Spinal Cord Injury Patients

Objective

This study aimed to determine the optimal time for tracheostomy by evaluating the benefits and safety of early versus late tracheostomy in spinal cord injury (SCI) patients.

Methods

We retrospectively reviewed a total of 254 patients with spinal cord injury. Of them, we selected 21 spinal cord injury patients who required tracheostomy due to long-term mechanical ventilation and analyzed their medical records. The patients were categorized into two groups. Early tracheostomy was performed day 1-10 from intubation in 10 patients and the late tracheostomy was performed after day 10 in 11 cases. We also evaluated the duration of mechanical ventilation, stay in the ICU and complications related to tracheostomy, the injury level of and clinical severity. All data was analyzed using SPSS 18.0/WIN.

Results

The early tracheostomy offered clear advantages for shortening the total ICU stay (20.8 day vs. 38.0 day, p=0.010). There was also statistically significant reduction in the total length of time on mechanical ventilation (5.2 day vs. 29.2 day, p=0.009). However, the reductions in the incidence of pneumonia (40% vs. 82%) and the length of ICU stay post to tracheostomy (6 day vs. 15 day) were found to be statistically not significant. There were also no statistically significant differences in the injury level and clinical severity between the groups.

Conclusion

We concluded that the early tracheostomy (at least 10 days) is beneficial for SCI patients who are likely to require prolonged mechanical ventilation.