Repurposing of FDA-Approved Drugs for Treating Iatrogenic Botulism: A Paired 3D-QSAR/Docking Approach†

Botulinum neurotoxin (BoNT) is widely used for the treatment of spasticity, focal dystonia, chronic migraine, facial hemispasm, and facial aesthetic treatments. Generally, treatment with botulinum toxin is a safe procedure when conducted by clinicians with expertise, and local side effects are rare and transient. However, occasionally adverse effects can occur due to the spread of the drug to nontargeted muscles and organs, producing dry mouth, fatigue, and flu-like symptoms, up to signs of systemic botulism, which appears to be more frequent in children treated for spasticity than in adults. In silico 3D-QSAR and molecular docking studies were performed to build a structure-based model on selected potent known botulinum neurotoxin type A inhibitors; this was used to screen the US Food and Drug Administration (FDA) database. Thirty molecules were identified as possible light-chain BoNT/A inhibitors. In this study, we applied a well-established ligand- and structure-based methodology for the identification of hit compounds among a database of FDA-approved drugs. The identification of budesonide, protirelin, and ciclesonide followed by other compounds can be considered a starting point for investigations of selected compounds that could bypass much of the time and costs involved in the drug approval process.     

Generation of Magnetized Olfactory Ensheathing Cells for Regenerative Studies in the Central and Peripheral Nervous Tissue

As olfactory receptor axons grow from the peripheral to the central nervous system (CNS) aided by olfactory ensheathing cells (OECs), the transplantation of OECs has been suggested as a plausible therapy for spinal cord lesions. The problem with this hypothesis is that OECs do not represent a single homogeneous entity, but, instead, a functionally heterogeneous population that exhibits a variety of responses, including adhesion and repulsion during cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. In this paper, we report a system based on modified OECs carrying magnetic nanoparticles as a proof of concept experiment enabling specific studies aimed at exploring the potential of OECs in the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) in vitro, their migration can be modulated by magnetic fields; and (iii) their transplantation in organotypic slices of spinal cord and peripheral nerve showed positive integration in the model. Altogether, these findings indicate the therapeutic potential of magnetized OECs for CNS injuries.     

Engineering electrospun nanofibrillar surfaces for spinal cord repair: a discussion

BACKGROUND: The design of implants comprised of biodegradable electrospun nanofibers for the purpose of bridging injuries in damaged spinal cord is discussed. Electrospun nanofibers structurally mimic the extracellular matrix on which neurons and other cell types grow in vivo. This property has created great interest for their use in tissue engineering applications. However, their employment as biomimetic surfaces for such in vivo applications is still in its infancy. RESULTS: A nonwoven fabric comprised of electrospun polyamide nanofibers supported modest axonal regeneration in injured adult rat spinal cord. Covalent modification of the nanofibers with a bioactive peptide derived from the neuroregulatory extracellular matrix molecule tenascin‐C enhanced the ability of the nanofibers to facilitate axonal regrowth. However, the random orientation of the nanofibrillar fabric folds was an impediment to the forward movement of axons. CONCLUSIONS: Polyamide nanofibers covalently modified with neuroactive molecules provide a promising material for grafts to promote spinal cord regeneration. However, for the proper guidance of regrowing axons, attention must be paid to the engineering of ordered nanofibrillar structures. Copyright © 2007 Society of Chemical Industry     

The Temporal Mechanisms Guiding Interneuron Differentiation in the Spinal Cord

Neurogenesis timing is an essential developmental mechanism for neuronal diversity and organization throughout the central nervous system. In the mouse spinal cord, growing evidence is beginning to reveal that neurogenesis timing acts in tandem with spatial molecular controls to diversify molecularly and functionally distinct post-mitotic interneuron subpopulations. Particularly, in some cases, this temporal ordering of interneuron differentiation has been shown to instruct specific sensorimotor circuit wirings. In zebrafish, in vivo preparations have revealed that sequential neurogenesis waves of interneurons and motor neurons form speed-dependent locomotor circuits throughout the spinal cord and brainstem. In the present review, we discuss temporal principals of interneuron diversity taken from both mouse and zebrafish systems highlighting how each can lend illuminating insights to the other. Moving forward, it is important to combine the collective knowledge from different systems to eventually understand how temporally regulated subpopulation function differentially across speed- and/or state-dependent sensorimotor movement tasks.     

肉毒毒素的应用现状

肉毒毒素是肉毒杆菌在生长繁殖中产生的一种神经毒素。注入肌肉后，能迅速与突触前胆碱能神经终板结合，减少周围运动神经末梢和神经肌肉接头处乙酰胆碱的释放，抑制细胞外乙酰胆碱，造成肌肉松弛和麻痹。20世纪70年代末肉毒毒素被开发并逐步应用于临床，以治疗某些神经肌肉疾思。目前，肉毒毒素已经用于眼科、神经科、康复科、消化科及皮肤科(多汗症、美容)等领域50余种病症的治疗，取得了诸多成果。本文就肉毒毒素在临床医疗、医学美容、生物农药等方面的应用情况进行了概述。     

Evaluation of Injured Axons Using Two-Photon Excited Fluorescence Microscopy after Spinal Cord Contusion Injury in YFP-H Line Mice

Elucidation of the process of degeneration of injured axons is important for the development of therapeutic modules for the treatment of spinal cord injuries. The aim of this study was to establish a method for time-lapse observation of injured axons in living animals after spinal cord contusion injury. YFP (yellow fluorescent protein)-H transgenic mice, which we used in this study, express fluorescence in their nerve fibers. Contusion damage to the spinal cord at the 11th vertebra was performed by IH (Infinite Horizon) impactor, which applied a pressure of 50 kdyn. The damaged spinal cords were re-exposed during the observation period under anesthesia, and then observed by two-photon excited fluorescence microscopy, which can observe deep regions of tissues including spinal cord axons. No significant morphological change of injured axons was observed immediately after injury. Three days after injury, the number of axons decreased, and residual axons were fragmented. Seven days after injury, only fragments were present in the damaged tissue. No hind-limb movement was observed during the observation period after injury. Despite the immediate paresis of hind-limbs following the contusion injury, the morphological degeneration of injured axons was delayed. This method may help clarification of pathophysiology of axon degeneration and development of therapeutic modules for the treatment of spinal cord injury.     

Differential Role of p53 in Oligodendrocyte Survival in Response to Various Stresses: Experimental Autoimmune Encephalomyelitis,Cuprizone Intoxication or White Matter Stroke

Promoting oligodendrocyte viability has been proposed as a therapeutic strategy for alleviating many neuronal diseases, such as multiple sclerosis and stroke. However, molecular pathways critical for oligodendrocyte survival under various stresses are still not well known. p53 is a strong tumor suppressor and regulates cell cycle, DNA repair and cell death. Our previous studies have shown that p53 plays an important role in promoting neuronal survival after insults, but its specific role in oligodendrocyte survival is not known. Here, we constructed the mice with oligodendrocyte-specific p53 loss by crossing TRP53^flox/flox mice and CNP-cre mice, and found that p53 was dispensable for oligodendrocyte differentiation and myelin formation under physiological condition. In the experimental autoimmune encephalomyelitis (EAE) model, p53 loss of function, specifically in oligodendrocytes, did not affect the EAE disease severity and had no effect on demyelination in the spinal cord of the mice. Interestingly, p53 deficiency in oligodendrocytes significantly attenuated the demyelination of corpus callosum and alleviated the functional impairment of motor coordination and spatial memory in the cuprizone demyelination model. Moreover, the oligodendrocyte-specific loss of p53 provided protection against subcortical white matter damage and mitigated recognition memory impairment in mice in the white matter stroke model. These results suggest that p53 plays different roles in the brain and spinal cord or in response to various stresses. Thus, p53 may be a therapeutic target for oligodendrocyte prevention in specific brain injuries, such as white matter stroke and multiple sclerosis.     

Epidermal Stem Cells in Orthopaedic Regenerative Medicine

In the last decade, great advances have been made in epidermal stem cell studies at the cellular and molecular level. These studies reported various subpopulations and differentiations existing in the epidermal stem cell. Although controversies and unknown issues remain, epidermal stem cells possess an immune-privileged property in transplantation together with easy accessibility, which is favorable for future clinical application. In this review, we will summarize the biological characteristics of epidermal stem cells, and their potential in orthopedic regenerative medicine. Epidermal stem cells play a critical role via cell replacement, and demonstrate significant translational potential in the treatment of orthopedic injuries and diseases, including treatment for wound healing, peripheral nerve and spinal cord injury, and even muscle and bone remodeling.     

Ischemic Preconditioning Protects against Spinal Cord Ischemia-Reperfusion Injury in Rabbits by Attenuating Blood Spinal Cord Barrier Disruption

Ischemic preconditioning has been reported to protect against spinal cord ischemia-reperfusion (I-R) injury, but the underlying mechanisms are not fully understood. To investigate this, Japanese white rabbits underwent I-R (30 min aortic occlusion followed by reperfusion), ischemic preconditioning (three cycles of 5 min aortic occlusion plus 5 min reperfusion) followed by I-R, or sham surgery. At 4 and 24 h following reperfusion, neurological function was assessed using Tarlov scores, blood spinal cord barrier permeability was measured by Evan’s Blue extravasation, spinal cord edema was evaluated using the wet-dry method, and spinal cord expression of zonula occluden-1 (ZO-1), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-α (TNF-α) were measured by Western blot and a real-time polymerase chain reaction. ZO-1 was also assessed using immunofluorescence. Spinal cord I-R injury reduced neurologic scores, and ischemic preconditioning treatment ameliorated this effect. Ischemic preconditioning inhibited I-R-induced increases in blood spinal cord barrier permeability and water content, increased ZO-1 mRNA and protein expression, and reduced MMP-9 and TNF-α mRNA and protein expression. These findings suggest that ischemic preconditioning attenuates the increase in blood spinal cord barrier permeability due to spinal cord I-R injury by preservation of tight junction protein ZO-1 and reducing MMP-9 and TNF-α expression.     

Human umbilical cord blood stem cells: rational for use as a neuroprotectant in ischemic brain disease

The use of stem cells for reparative medicine was first proposed more than three decades ago. Hematopoietic stem cells from bone marrow, peripheral blood and human umbilical cord blood (CB) have gained major use for treatment of hematological indications. CB, however, is also a source of cells capable of differentiating into various non-hematopoietic cell types, including neural cells. Several animal model reports have shown that CB cells may be used for treatment of neurological injuries. This review summarizes the information available on the origin of CB-derived neuronal cells and the mechanisms proposed to explain their action. The potential use of stem/progenitor cells for treatment of ischemic brain injuries is discussed. Issues that remain to be resolved at the present stage of preclinical trials are addressed.     

Therapeutic Hypothermia in Spinal Cord Injury: The Status of Its Use and Open Questions

Spinal cord injury (SCI) is a major health problem and is associated with a diversity of neurological symptoms. Pathophysiologically, dysfunction after SCI results from the culmination of tissue damage produced both by the primary insult and a range of secondary injury mechanisms. The application of hypothermia has been demonstrated to be neuroprotective after SCI in both experimental and human studies. The myriad of protective mechanisms of hypothermia include the slowing down of metabolism, decreasing free radical generation, inhibiting excitotoxicity and apoptosis, ameliorating inflammation, preserving the blood spinal cord barrier, inhibiting astrogliosis, promoting angiogenesis, as well as decreasing axonal damage and encouraging neurogenesis. Hypothermia has also been combined with other interventions, such as antioxidants, anesthetics, alkalinization and cell transplantation for additional benefit. Although a large body of work has reported on the effectiveness of hypothermia as a neuroprotective approach after SCI and its application has been translated to the clinic, a number of questions still remain regarding its use, including the identification of hypothermia’s therapeutic window, optimal duration and the most appropriate rewarming rate. In addition, it is necessary to investigate the neuroprotective effect of combining therapeutic hypothermia with other treatment strategies for putative synergies, particularly those involving neurorepair.     

Exerting the Appropriate Application of Methylprednisolone in Acute Spinal Cord Injury Based on Time Course Transcriptomics Analysis

Methylprednisolone (MP) is an anti-inflammatory drug approved for the treatment of acute spinal cord injuries (SCIs). However, MP administration for SCIs has become a controversial issue while the molecular effects of MP remain unexplored to date. Therefore, delineating the benefits and side effects of MP and determining what MP cannot cure in SCIs at the molecular level are urgent issues. Here, genomic profiles of the spinal cord in rats with and without injury insults, and those with and without MP treatment, were generated at 0, 2, 4, 6, 8, 12, 24, and 48 h post-injury. A comprehensive analysis was applied to obtain three distinct classes: side effect of MP (SEMP), competence of MP (CPMP), and incapability of MP (ICMP). Functional analysis using these genes suggested that MP exerts its greatest effect at 8~12 h, and the CPMP was reflected in the immune response, while SEMP suggested aspects of metabolism, such as glycolysis, and ICMP was on neurological system processes in acute SCIs. For the first time, we are able to precisely reveal responsive functions of MP in SCIs at the molecular level and provide useful solutions to avoid complications of MP in SCIs before better therapeutic drugs are available.     

治疗用A型肉毒梭菌神经毒素全基因克隆及序列分析

目的对我国治疗用A型肉毒毒素生产用菌株Hall株神经毒素(BoNT)全基因进行克隆及序列分析,了解其遗传特性,为该制剂的质量控制提供依据。方法取生产用主代种子、工作种子,通过PCR扩增BoNT全基因片段,将其克隆至载体pGEM-T中,构建重组克隆质粒pGEM-T-BoNT,对克隆的BoNT基因进行序列测定与分析。结果测得的Hall株BoNT全基因序列3891bp,共编码1297个氨基酸。4种核苷酸的比例分别为:A:40.58%,G:16.17%,T:33.13%,C:10.13%;GC含量为26.29%,AT含量为73.71%。主代种子、工作种子核苷酸序列3591位的G变成A,为无义突变,未导致其推导的氨基酸的改变。序列测定结果与GenBank中登录的Hall标准株进行比较,主代种子、工作种子核苷酸和氨基酸序列的同源性分别为99.99%和100%。结论 A型肉毒梭菌Hall株在实验室长期的保存和生产传代过程中,BoNT基因遗传特性非常稳定。     

Inhibition of Spinal TRPV1 Reduces NMDA Receptor 2B Phosphorylation and Produces Anti-Nociceptive Effects in Mice with Inflammatory Pain

Transient receptor potential vanilloid 1 (TRPV1) has been implicated in peripheral inflammation and is a mediator of the inflammatory response to various noxious stimuli. However, the interaction between TRPV1 and N-methyl-D-aspartate (NMDA) receptors in the regulation of inflammatory pain remains poorly understood. This study aimed to investigate the analgesic effects of intrathecal administration of capsazepine, a TRPV1 antagonist, on carrageenan-induced inflammatory pain in mice and to identify its interactions with NMDA receptors. Inflammatory pain was induced by intraplantar injection of 2% carrageenan in male ICR mice. To investigate the analgesic effects of capsazepine, pain-related behaviors were evaluated using von Frey filaments and a thermal stimulator placed on the hind paw. TRPV1 expression and NMDA receptor phosphorylation in the spinal cord and glutamate concentration in the spinal cord and serum were measured. Intrathecal treatment with capsazepine significantly attenuated carrageenan-induced mechanical allodynia and thermal hyperalgesia. Moreover, carrageenan-enhanced glutamate and phosphorylation of NMDA receptor subunit 2B in the spinal cord were suppressed by capsazepine administration. These results indicate that TRPV1 and NMDA receptors in the spinal cord are associated with inflammatory pain transmission, and inhibition of TRPV1 may reduce inflammatory pain via NMDA receptors.     

Conjugated fluorescent polymer sensor for proteolytic activity detection with designed specificity

A portable fluorescence assay for direct endopeptidase activity detection has been developed with the use of a cyclophane‐based conductive conjugated fluorescent polymer and peptide substrates. The substrates, carrying internal quenching amino acid, were designed to be cleaved in a sequence‐specific manner by a protease of interest. Intact substrates were incapable of quenching the fluorescence of the polymer due to steric constraints. Upon specific cleavage, the quencher became exposed and could interact with the ring structure of the fluorescent polymer, disrupting the conjugation and quenching the fluorescence along the polymer chain. The approach was developed using a model Glu‐C endopeptidase from Staphylococcus aureus strain V8, detected in the picomolar to micromolar concentration range. The developed assay was tested for the detection of endopeptidase activity of botulinum toxin. The feasibility study showed that botulinum neurotoxin (BoNT)‐A could be detected down to picomolar concentration, with limit of detection of 5 pg, or 33 amol in 5 µL of sample, and a total assay time under 2 h. The assay exhibited high specificity and no cross‐reactivity with BoNT‐B was detected. Following this proof‐of‐concept work, the assay can be further optimized and expanded to differentiate between various botulinum toxin serotypes in their active proteolytic form, or modified for detection of proteases with other specificities. © 2015 Society of Chemical Industry     

Neuromyelitis Optica Spectrum Disorder Treatment—Current and Future Prospects

Neuromyelitis optica (NMO) is an immune-mediated demyelinative disorder of the central nervous system affecting mainly the optical nerves and the spinal cord. The recurrent course of the disease, with exacerbations and incomplete remissions, causes accumulating disability, which has a profound impact upon patients’ quality of life. The discovery of antibodies against aquaporin 4 (AQP4) and their leading role in NMO etiology and the formulation of diagnostic criteria have improved appropriate recognition of the disease. In recent years, there has been rapid progress in understanding the background of NMO, leading to an increasing range of treatment options. On the basis of a review of the relevant literature, the authors present currently available therapeutic strategies for NMO as well as ongoing research in this field, with reference to key points of immune-mediated processes involved in the background of the disease.     

Effect of Acupuncture on Diabetic Neuropathy: A Narrative Review

Diabetic neuropathy, a major complication of diabetes mellitus, refers to a collection of clinically diverse disorders affecting the nervous system that may present with pain. Although the number of patients suffering from severe neuropathy is increasing, no optimal treatment method has been developed yet. Acupuncture is well known for its ability to reduce various kinds of pain, and a number of studies have also reported its effect on diabetes mellitus; however, its effect and underlying mechanism against diabetic neuropathy are not yet clearly understood. In this review, ten and five studies performed in humans and animals, respectively, were analyzed. All studies reported that acupuncture significantly relieved diabetic neuropathy. ST36, BL13, BL20, SP6, and SP9 were the most widely used acupoints. Five studies used electro-acupuncture, whereas other studies used manual acupuncture. Furthermore, the effect of acupuncture was shown to be mediated through the various molecules present in the peripheral nerves and spinal cord, such as P65, GPR78, and TRPV1. Five studies reported side effects, such as swelling, numbness, and nausea, but none were reported to be serious. Based on these results, we suggest that acupuncture should be considered as a treatment option for diabetic neuropathy.     

肉毒毒素生物学活性及肉毒中毒病原检测方法的研究进展

肉毒毒素生物学活性的检测和肉毒中毒诊断方法的研究进展迅速,对食品检验、肉毒中毒实验室诊断、生物反恐和肉毒毒素制品的开发及应用具有十分重要的意义。本文就肉毒毒素生物学活性及肉毒中毒病原检测方法的研究进展作一综述。     

Neuroprotective Effects of Sigma 1 Receptor Ligands on Motoneuron Death after Spinal Root Injury in Mice

Loss of motor neurons (MNs) after spinal root injury is a drawback limiting the recovery after palliative surgery by nerve or muscle transfers. Research based on preventing MN death is a hallmark to improve the perspectives of recovery following severe nerve injuries. Sigma-1 receptor (Sig-1R) is a protein highly expressed in MNs, proposed as neuroprotective target for ameliorating MN degenerative conditions. Here, we used a model of L4–L5 rhizotomy in adult mice to induce MN degeneration and to evaluate the neuroprotective role of Sig-1R ligands (PRE-084, SA4503 and BD1063). Lumbar spinal cord was collected at 7, 14, 28 and 42 days post-injury (dpi) for immunohistochemistry, immunofluorescence and Western blot analyses. This proximal axotomy at the immediate postganglionic level resulted in significant death, up to 40% of spinal MNs at 42 days after injury and showed markedly increased glial reactivity. Sig-1R ligands PRE-084, SA4503 and BD1063 reduced MN loss by about 20%, associated to modulation of endoplasmic reticulum stress markers IRE1α and XBP1. These pathways are Sig-1R specific since they were not produced in Sig-1R knockout mice. These findings suggest that Sig-1R is a promising target for the treatment of MN cell death after neural injuries.