New approach to treating spinal cord injury using PEG-TAT-modified,cyclosporine-A-loaded PLGA/polymeric liposomes

Cyclosporine-A (CsA) is an immunosuppressant agent that has shown effectiveness as a neuroprotective drug; however, it does not readily cross the blood-spinal cord barrier (BSCB), which constrains the clinical applications of CsA for the treatment of spinal cord injury (SCI). Our group recently tested the ability of novel polyethylene glycol (PEG)-transactivating-transduction protein (TAT)-modified CsA-loaded cationic multifunctional polymeric liposome-poly(lactic-co-glycolic acid) (PLGA) core/shell nanoparticles (PLGA/CsA NPs) to transport and deliver CsA across the BSCB to treat SCI. The PLGA/CsA NPs were successfully constructed. In vitro drug release studies have demonstrated that the sustained release of CsA from PLGA/CsA NPs occurs over ～25?h. The in vivo study presented here showed that injured animals that received PLGA/CsA NPs through the tail vein, exhibited a significant up-regulation of growth-associated protein-43 (GAP-43) expression and an increased number of GAP-43-stained neurons compared with animals that received CsA or the vehicle alone. The improvement in neurological function was also evaluated by the Basso–Beattie–Bresnahan (BBB) open-field test. Moreover, fluorescein isothiocyanate (FITC)-attached PLGA/CsA NPs were successfully aggregated in the intact spinal cord 4?h after injection. Our data suggest that PLGA/CsA NPs have the potential for use as a new treatment method for SCI.     

Pathophysiology of blood-spinal cord barrier in traumatic injury and repair

Blood-spinal cord barrier (BSCB) plays an important role in the regulation of the fluid microenvironment of the spinal cord. Trauma to the spinal cord impairs the BSCB permeability to proteins leading to vasogenic edema formation. Several endogenous neurochemical mediators and growth factors contribute to trauma induced BSCB disruption. Studies carried out in our laboratory suggest that those drugs and neurotrophic factors capable to attenuate the BSCB dysfunction following trauma are neuroprotective in nature. Whereas, agents that do not exert any influence on the BSCB disruption failed to reduce cell injury. These observations are in line with the idea that BSCB disruption plays an important role in the pathophysiology of spinal cord injuries. The probable mechanism(s) of trauma induced BSCB dysfunction and its contribution to cell injuries are discussed.     

Current options for drug delivery to the spinal cord

Introduction: Spinal cord disorders (SCDs) are among the most devastating neurological diseases, due to their acute and long-term health consequences, the reduced quality of life and the high economic impact on society. Here, drug administration is severely limited by the blood–spinal cord barrier (BSCB) that impedes to reach the cord from the bloodstream. So, developing a suitable delivery route is mandatory to increase medical chances.

Areas covered: This review provides an overview of drug delivery systems used to overcome the inaccessibility of the cord. On one side, intrathecal administration, either with catheters or with biomaterials, represents the main route to administer drugs to the spinal cord; on the other side, more recent strategies involve chemical or electromagnetic disruption of the barrier and synthesis of novel functionalized compounds as nanoparticles and liposomes able to cross BSCB.

Expert opinion: Both the multifactorial pathological progression and the restricted access of therapeutic drugs to the spine are probably the main reasons behind the absence of efficient therapeutic approaches for SCDs. Hence, very recent highlights suggest the use of original strategies to overcome the BSCB, and new multidrug delivery systems capable of local controlled release of therapeutic agents have been developed. These issues can be addressed by using nanoparticles technology and smart hydrogel drug delivery systems, providing an increased therapeutic compound delivery in the spinal cord environment and multiple administrations able to synergize treatment efficacy.     

大鼠脊髓损伤后NEK6表达的实验研究

目的 探讨大鼠急性脊髓损伤后脊髓组织中NEK6的表达变化及意义.方法 采用改良Allen's打击法,构建大鼠脊髓T8-T10撞击伤模型,利用蛋白免疫印迹、免疫荧光双标的方法,研究大鼠脊髓损伤后脊髓组织中NEK6表达的时间和空间分布特征.结果 脊髓损伤后NEK6表达于撞击后2h有轻微抬高趋势,损伤后8h达到高峰,随后逐渐...     

L-type Amino Acid Transporter 1 (SLC7A5)-Mediated Transport of Pregabalin at the Rat Blood-Spinal Cord Barrier and its Sensitivity to Plasma Branched-Chain Amino Acids

Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.     

REPAIR AND RECOVERY FOLLOWING SPINAL CORD INJURY IN A NEONATAL MARSUPIAL (MONODELPHIS DOMESTICA)

1. Repair and recovery following spinal cord injury (complete spinal cord crush) has been studied in vitro in neonatal opossum (Monodelphis domestica), fetal rat and in vivo in neonatal opossum. 2. Crush injury of the cultured spinal cord of isolated entire central nervous system (CNS) of neonatal opossum (P4–10) or fetal rats (E15–E16) was followed by profuse growth of fibres and recovery of conduction of impulses through the crush. Previous studies of injured immature mammalian spinal cord have described fibre growth occurring only around the lesion, unless implanted with fetal CNS. 3. The period during which successful growth occurred in response to a crush is developmentally regulated. No such growth was obtained after P12 in spinal cords crushed in vitro at the level of C7–8. 4. In vivo, in the neonatal (P4–8) marsupial opossum, growth of fibres through, and restoration of, impulse conduction across the crush was apparent 1–2 weeks after injury. With longer periods of time after crushing a considerable degree of normal locomotor function developed. 5. By the time the operated animals reached adulthood, the morphological structure of the spinal cord, both in the region of the crush and on either side of the site of the lesion, appeared grossly normal. 6. The results are discussed in relation to the eventual longterm possibility of devising effective treatments for patients with spinal cord injuries.     

Treatment with green tea extract attenuates secondary inflammatory response in an experimental model of spinal cord trauma

In this study, we evaluated the effect of green tea extract (that was administered 25 mg/kg intraperitoneal at 1 and 6 h after injury) in experimental animal model of spinal cord injury. The spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterised by oedema, neutrophilic infiltration and apoptosis. Also, immunohistochemical examination demonstrated a marked increase in immune reactivity for nitrotyrosine. All parameters of inflammation were attenuated by green tea extract. The degree of spinal cord inflammation, nitrotyrosine, poli (ADP-ribosio) synthetase (PARS) and neutrophilic infiltration was markedly reduced. Green tea extract significantly ameliorated the recovery of limb function. Values shown are mean ± SE mean of ten mice for each group. *p < 0.01 versus sham, °p < 0.01 versus spinal cord injury. Taken together, our results clearly demonstrate that green tea extract treatment ameliorates spinal cord injury oxidative stress. Irene Paterniti, Tiziana Genovese and Salvatore Cuzzocrea contributed equally to this work.     

周围神经移植联合神经营养因子治疗损伤脊髓的实验研究

目的 探讨周围神经移植联合神经生长因子(NGF)和脑源性神经生长因子(BDNF)对脊髓损伤的疗效。方法 用SD大鼠在胸8～胸9平面切除半侧4mm长脊髓制成半切模型，取自体肋间神经桥接脊髓缺损，并在局部持续灌注含NGF、BDNF生理盐水3天。3个月后由结构及功能二方面评价脊髓功能恢复情况。结果 术后3个月试验鼠后肢感觉、运动及运动功能改良Tarlov评分、斜板试验均优于对照组；肋间神经植入区见轴突再生，胶质细胞增生减少，雪旺氏细胞增殖；示踪剂通过植入区的近远端界面；体感诱发电位(SEP)磁刺激运动诱发电位(MEP)大部分恢复；MR示脊髓连续无中断。结论 肋间神经移植 NGF、BDNF能促进神经轴突的生长，抑制胶质细胞增生，在局部使雪旺氏细胞增殖，利于脊髓再生。     

多功能阳离子脂质体跨血脊髓屏障的实验研究

目的探讨异硫氰酸荧光素(FITC)标记阳离子脂质体通过尾静脉注入后,跨越大鼠血脊髓屏障并在局部聚集的情况。方法 30只成年Wistar大鼠随机等分为3组,每组10只,荧光显微镜动态观察脂质体在中枢神经系统(CNS)中靶向聚集与扩散情况,通过组织学观测进一步分析其与CNS细胞的关系。结果相同浓度下(75⁶00μg/m L),TAT-PEG-阳离子脂质体较未经TAT修饰者具更低细胞毒性,且更易被MCF-7细胞摄取;FITC标记TAT-PEG-阳离子脂质体可跨越BSCB并聚集,低倍镜下观察到许多荧光微粒聚集于CNS组织,高倍镜形象地显示出荧光微粒位于神经细胞内。结论经跨膜肽(TAT)、聚乙二醇(PEG)修饰的阳离子脂质体具有良好的生物相容性与靶向定位特性,可跨过BSCB,聚集于CNS细胞中,安全用作药物载体,为中枢神经系统疾病的治疗提供新的途径。     

人神经干细胞移植修复大鼠脊髓损伤的实验研究

目的研究人神经干细胞（hNSCs）移植对大鼠脊髓损伤的修复作用，并初步探讨其作用原理。方法分离、培养和鉴定hNSCs。24例成年SD大鼠分为移植组12例和对照组12例，均采用NYU-Ⅱ型脊髓打击器制作脊髓损伤模型，第9天移植组于损伤脊髓中心分别注入经CM-DiI标记的hNSCs混悬液，对照组注入人DMEM／F12培养液。术后第4、8周取损伤部位脊髓，免疫组织化学染色检测移植细胞的存活和分化；术后每7天行BBB评分，评定大鼠后肢运动功能恢复情况。数据进行成组设计资料的t检验。结果成功建立hNSCs的体外培养体系；移植的hNSCs在大鼠脊髓内存活超过8周，并向脊髓损伤头尾端迁移，免疫组织化学荧光染色示移植细胞可分化为神经元、少突胶质细胞和星形胶质细胞；移植组大鼠BBB评分高于对照组（P〈0．05）。结论移植到大鼠损伤脊髓中的人神经干细胞可分化为神经元和神经胶质细胞，并可促进脊髓损伤大鼠后肢运动功能恢复。     

Upregulation of S100A4 after spinal cord transection in adult rats

AIM: To investigate whether spinal cord transection induces changes of gene expression of S100A4 protein. METHODS: In a spinal cord transection model, S100A4 expression and cellular localization were examined using cDNA microarray, Northern blot, immunohistochemistry, and immunofluorescence double-labeling methods. RESULTS: There was very limited S100A4 mRNA expression in the control spinal cord. However, S100A4 mRNA expression was increased significantly in both the rostral and caudal spinal cord segments adjacent to the injury site. Specifically, S100A4 gene expression was substantially increased at d 2, peaked at d 7 and d 14, and remained high up to 28 d post-injury. During its peak expression, S100A4 protein was localized in astrocytes of the spinal cord within 5 mm from the site of spinal transection. CONCLUSION: Spinal cord transection induces prolonged S100A4 expression at both mRNA and protein levels in areas close to the injury site. Increased expression of S100A4 in astrocytes after spinal cor     

水下爆炸致急性脊髓损伤动物模型的构建

目的 构建水下爆炸致急性脊髓损伤（SCI）动物模型，为研究水下爆炸致急性SCI的治疗提供依据。方法 选取新西兰兔24只，随机分为A组（爆炸距离0.3 m）、B组（爆炸距离0.8 m）和C组（爆炸距离1.5 m），每组8只。将实验兔固定在中国科学技术大学研制的水下实验装置中，分别在3种冲击波压力强度下致伤，选取T9～T10段脊髓，观察脊髓组织病理形态学、动物存活情况以及运动功能。结果 同样爆炸当量，A组、B组、C组测得的平均冲击波压力峰值分别为18、5、2 MPa， B组实验兔成活率高，致伤后双下肢运动功能障碍，病理学显示脊髓内见大量散在出血坏死灶，满足实验需要。B组实验兔爆炸前后、爆炸前与致伤后2 h、爆炸前与致伤后24 h的改良Tarlov评分，差异有统计学意义（P<0.05），致伤后2 h与24 h的改良Tarlov评分差异无统计学意义（P>0.05）；C组实验兔爆炸前后、爆炸前与致伤后2 h、爆炸前与爆炸后24 h的改良Tarlov评分差异有统计学意义（P<0.05），致伤后2 h与24 h的改良Tarlov评分差异无统计学意义（P>0.05）。结论 水下爆炸装置冲击波压力峰值5 MPa时能够建立急性SCI模型。     

Motor control in the human spinal cord and the repair of cord function

In this review we describe clinical and neurophysiological features of motor control in human spinal cord injury based on two models. First, motor control is considered in subjects with injury-induced complete division of the spinal cord from brain and brainstem structures, and second, in those in which the division is partial. We describe motor control in terms of segmental and plurisegmental reflex activity that dominates motor unit output to the muscles following complete separation from the brain motor structures by accidental injury. With incomplete separation of the spinal cord from brain structures, motor control is defined as the voluntary manipulation of reflex and automatic activity integrated with internal and external feedback signals. We review here motor control found after complete spinal cord injury with paradigm of single and regular-repeating stimuli applied to elicit cutaneous and muscle stretch reflex responses. We argue, that isolated spinal cord neural circuitry is capable of organizing characteristic reflex events that depend on the characteristics of the stimulus. Also, the profile of residual brain and brainstem, modified by the reduction in descending long spinal tract fibers arriving at their targets in the spinal gray matter, produces characteristic changes in motor output to the muscles that leads to the development of new neural strategies for control of segmental and plurisegmental neural circuitry. In the second part of this review, we discuss available treatment modalities for impaired cord function and briefly outline neurobiological interventions under development for repair of spinal cord injury.     

骨髓间充质干细胞和脑源性神经营养因子悬液移植对大鼠脊髓损伤的治疗作用

目的探讨骨髓间充质干细胞(BMSCs)移植兼用脑源性神经营养因子(BDNF)促进大鼠脊髓半横断损伤修复的影响。方法建立成年大鼠脊髓T12半横断损伤模型,将体外培养纯化的BMSCs植入脊髓损伤处,同时局部应用BDNF,术后2个月用斜板试验及改良Tarlov评分评价动物后肢运动功能恢复情况;用光镜及电镜观察移植后脊髓的形态结构变化及移植物存活情况;用相对分子质量为200 000的神经微丝(NF)、相对分子质量为51 000的胶质原纤维酸性蛋白(GFAP)免疫组织化学活性的变化来评价移植对再生的影响。结果移植物能很好地与宿主融合和存活,移植细胞的大鼠后肢自主运动功能有显著的恢复,BMSCs和BDNF联合应用对脊髓损伤的修复作用最强。结论BMSCs移植可促进大鼠半横断脊髓结构和功能恢复,联合应用BMSCs和BD-NF在脊髓损伤修复治疗中具有协同作用。     

Administration of carnosine in the treatment of acute spinal cord injury

l-Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and l-histidine. It acts as a free radical scavenger and possesses antioxidant properties. l-Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha in different experimental settings. In the present study, we investigated the efficacy of l and d-carnosine on the animal model of spinal cord injury (SCI). The spinal cord was exposed via a four-level T5–T8 laminectomy and SCI was produced by extradural compression of the spinal cord at level T6–T7 using an aneurysm clip with a closing force of 24 g. Treatment with d-carnosine (150 mg/kg administered i.p., 1 h and 6 h, after SCI), but not l-carnosine significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation, inducible NO synthase (iNOS) and Hsp70 expression, (d) proinflammatory cytokines, and (e) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Furthermore, d-carnosine (150 mg/kg administered i.p., 1 h and 6 h, after SCI) significantly ameliorated the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate the strong difference between l-carnosine and d-carnosine. The result strongly suggests that d-carnosine treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

Therapeutic vaccination for spinal cord injury: helping the body to cure itself

Inflammation is thought to exacerbate the outcome of spinal cord injury. However, our findings have led us to view inflammation as a healing response that needs the help of a systemic immune response mediated by T helper 1 (Th1) cells that are specific to the abundant antigens residing in the lesion site. Strains differ in their ability to manifest, at the right time and intensity, a spontaneous T-cell response to antigens at the lesion site and therefore in their ability to generate a local inflammatory response whose outcome is beneficial (maintenance and repair). All strains, however, can benefit from immune intervention that boosts and regulates the inflammatory response. Because recovery comprises multi-step processes, pharmacological intervention will be less effective than well-synchronized, self-healing immune activity. Risk-free neuroprotective intervention might be achieved by post-traumatic vaccination with a weak, non-pathogenic, auto-antigen, causing autoimmune T cells to home to the lesion site where they become activated and therefore activate local phagocytic cells to remove hostile elements and provide growth factors.     

簇集蛋白在大鼠急性脊髓损伤组织中的表达及其意义

目的　探讨簇集蛋白 (clusterin)在急性脊髓损伤组织中的表达及其在继发性脊髓损伤中可能发挥的重要作用。方法　采用改良Allen重物打击法制成SD大鼠脊髓急性损伤模型 ,通过苏木素 伊红 (HE)染色及免疫组织化学染色观察损伤组和假手术组伤后 0. 5、1、3、7、14、2 1d脊髓损伤组织变性坏死及簇集蛋白的表达情况。结果　损伤组在伤后 1d簇集蛋白阳性表达开始增加 ,约在伤后 7d达到高峰 ,之后表达逐渐减少 ,伤后2 1d趋于稳定 ,随时间延长存在动态变化过程 ,并与脊髓损伤程度相一致。损伤组在伤后 1、3、7、14、2 1d簇集蛋白表达与假手术组及正常对照组差异有统计学意义 (P <0 . 0 1) ;假手术组及正常对照组伤后各时间点均有簇集蛋白阳性表达 ,但表达随时间延长无动态变化过程。结论　簇集蛋白在急性脊髓损伤组织中表达明显增加 ,并可能通过补体调节、抗凋亡及热休克蛋白活性等机制减轻继发性脊髓损伤。     

生物素葡聚糖胺示踪观察脊髓腹侧损伤大鼠模型下行传导通路的改变

目的观察大鼠脊髓腹侧损伤后下行传导通路的改变。方法清洁级雌性SD大鼠28只,随机分为实验对照组(6只)和脊髓腹侧损伤15、30、50g3个小组(7、7、8只),使用标准化脊髓损伤动物模型实验装置在T9～T10水平压迫脊髓腹侧制备动物模型,对照组大鼠仅咬除椎弓、横突,不损伤脊髓。术后12h,所有大鼠立体定向开颅,生物素葡聚糖胺(BDA)注射于大脑皮层,并采用改良的Tarlov分级评分、斜板实验及BBB评分评价实验动物的运动功能。BDA注射2周后取出损伤节段以下脊髓组织,行BDA染色成像。统计学方法采用双因素方差分析或秩和检验。结果脊髓损伤组大鼠改良的Tarlov分级评分、斜板实验及BBB评分均明显低于对照组(P<0.05);增加砝码质量,使损伤组大鼠受到的腹侧向上提拉力量增大,双下肢改良的Tarlov分级评分逐渐下降,鼠斜板实验临界角逐渐下降,BBB评分呈降低趋势;BDA神经示踪后免疫组织化学显色后阳性的神经元呈棕褐色深染,脊髓损伤组较对照组明显减少,且随着损伤外力的加大,阳性神经元数量明显减少。结论脊髓腹侧受损后大脑中枢下行传导通路神经传导异常程度越重,其造成的功能障碍程度也越重。     

Beneficial effects of nitric oxide synthase inhibition on the recovery of neurological function after spinal cord injury in rats

We have demonstrated recently that treatment with N(G)-monomethyl-L-arginine (L-NMMA) accelerates electrophysiological recovery after transient spinal cord ischaemia in anaesthetized cats. To determine whether nitric oxide synthase (NOS) inhibition in the acute phase of spinal cord injury results in better functional recovery in the chronic phase, we evaluated the influence of L-NMMA on the time course of changes of neurological function and the histopathological changes after spinal cord compression in rats. Experimental spinal cord injury was produced in anaesthetized rats by short-term (5 min) compression with a thread placed around the spinal cord at T13. The recovery of motor function was assessed by a treadmill test 10, 20 and 30 days after spinal cord compression. The latency of potentials evoked by hindlimb stimulation was measured at the funiculus posterior at C1 10 days after the spinal cord injury in anaesthetized rats. Histological examinations were also performed at the same time. The compression-induced spinal cord injury resulted in motor dysfunction of hindlimbs, an increase in the latency of the evoked potentials and neuronal degeneration in funiculus posterior at T13. Repeated administration of L-NMMA for 1 day significantly accelerated the recovery of the motor function, shortened the latency of the evoked potentials and attenuated the myelin vacuolization in the spinal cord. These beneficial effects of L-NMMA on neurological function and histopathological changes were abolished by coadministration of L- but not D-arginine. These results suggest that NOS inhibition during the early stage of spinal cord injury has beneficial effects on the recovery of neurological function and the histopathological changes in the chronic stage.     

大鼠急性脊髓损伤热休克蛋白70的表达

目的：观察大鼠急性脊髓损伤后脊髓组织中热休克蛋70的表达。方法：Wistar大鼠56只,随机分成7组,6组制作成急性脊髓损伤模型,另一组作为对照组。分别于伤后2h、6h、12h、24h、48h、72h。建立脊髓损伤动物模型。应用West-blot方法观察各个时段损伤脊髓组织HSP70的表达。结果：急性脊髓损伤后2h,脊髓组织内出现HSP70的表达,损伤后24h脊髓组织中HSP70染色达到高峰,并可维持至损伤后72h。结论：在遭遇损伤性刺激后,脊髓组织HSP70的表达明显增加,表明HSP70有可能在阻止脊髓的继发性损伤方面发挥一定的作用。