Biohybrids for spinal cord injury repair

Spinal cord injuries (SCIs) result in the loss of sensory and motor function with massive cell death and axon degeneration. We have previously shown that transplantation of spinal cord‐derived ependymal progenitor cells (epSPC) significantly improves functional recovery after acute and chronic SCI in experimental models, via neuronal differentiation and trophic glial cell support. Here, we propose an improved procedure based on transplantation of epSPC in a tubular conduit of hyaluronic acid containing poly (lactic acid) fibres creating a biohybrid scaffold. In vitro analysis showed that the poly (lactic acid) fibres included in the conduit induce a preferential neuronal fate of the epSPC rather than glial differentiation, favouring elongation of cellular processes. The safety and efficacy of the biohybrid implantation was evaluated in a complete SCI rat model. The conduits allowed efficient epSPC transfer into the spinal cord, improving the preservation of the neuronal tissue by increasing the presence of neuronal fibres at the injury site and by reducing cavities and cyst formation. The biohybrid‐implanted animals presented diminished astrocytic reactivity surrounding the scar area, an increased number of preserved neuronal fibres with a horizontal directional pattern, and enhanced coexpression of the growth cone marker GAP43. The biohybrids offer an improved method for cell transplantation with potential capabilities for neuronal tissue regeneration, opening a promising avenue for cell therapies and SCI treatment.     

Transplantation of Glial Progenitors That Overexpress Glutamate Transporter GLT1 Preserves Diaphragm Function Following Cervical SCI

Approximately half of traumatic spinal cord injury (SCI) cases affect cervical regions, resulting in chronic respiratory compromise. The majority of these injuries affect midcervical levels, the location of phrenic motor neurons (PMNs) that innervate the diaphragm. A valuable opportunity exists following SCI for preventing PMN loss that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxicity due to dysregulation of extracellular glutamate homeostasis. Astrocytes express glutamate transporter 1 (GLT1), which is responsible for the majority of CNS glutamate clearance. Given our observations of GLT1 dysfunction post-SCI, we evaluated intraspinal transplantation of Glial-Restricted Precursors (GRPs)—a class of lineage-restricted astrocyte progenitors—into ventral horn following cervical hemicontusion as a novel strategy for reconstituting GLT1 function, preventing excitotoxicity and protecting PMNs in the acutely injured spinal cord. We find that unmodified transplants express low levels of GLT1 in the injured spinal cord. To enhance their therapeutic properties, we engineered GRPs with AAV8 to overexpress GLT1 only in astrocytes using the GFA2 promoter, resulting in significantly increased GLT1 protein expression and functional glutamate uptake following astrocyte differentiation in vitro and after transplantation into C4 hemicontusion. Compared to medium-only control and unmodified GRPs, GLT1-overexpressing transplants reduced lesion size, diaphragm denervation and diaphragm dysfunction. Our findings demonstrate transplantation-based replacement of astrocyte GLT1 is a promising approach for SCI.     

Release of tetracycline hydrochloride from electrospun poly(ethylene-co-vinylacetate), poly(lactic acid), and a blend.

Electrospun fiber mats are explored as drug delivery vehicles using tetracycline hydrochloride as a model drug. The mats were made either from poly(lactic acid) (PLA), poly(ethylene-co-vinyl acetate) (PEVA), or from a 50:50 blend of the two. The fibers were electrospun from chloroform solutions containing a small amount of methanol to solubilize the drug. The release of the tetracycline hydrochloride from these new drug delivery systems was followed by UV-VIS spectroscopy. Release profiles from the electrospun mats were compared to a commercially available drug delivery system, Actisite (Alza Corporation, Palo Alto, CA), as well as to cast films of the various formulations.     

Neuronal Hyperexcitability: A Substrate for Central Neuropathic Pain After Spinal Cord Injury

Neuronal hyperexcitability produces enhanced pain transmission in the spinal dorsal horn after spinal cord injury (SCI). Spontaneous and evoked neuronal excitability normally are well controlled by neural circuits. However, SCI produces maladaptive synaptic circuits in the spinal dorsal horn that result in neuronal hyperexcitability. After SCI, activated primary afferent neurons produce enhanced release of glutamate, neuropeptides, adenosine triphosphate, and proinflammatory cytokines, which are known to be major components for pain transmission in the spinal dorsal horn. Enhanced neurochemical events contribute to neuronal hyperexcitability, and neuroanatomical changes also contribute to maladaptive synaptic circuits and neuronal hyperexcitability. These neurochemical and neuroanatomical changes produce enhanced cellular signaling cascades that ensure persistently enhanced pain transmission. This review describes altered neurochemical and neuroanatomical contributions on neuronal hyperexcitability in the spinal dorsal horn, which serve as substrates for central neuropathic pain after SCI.     

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.     

Effects of Etanercept and Minocycline in a rat model of spinal cord injury

Loss of function is usually considered the major consequence of spinal cord injury (SCI). However, pain severely compromises the quality of life in nearly 70% of SCI patients. The principal aim of this study was to assess the contribution of Tumor necrosis factor α (TNF‐α) to SCI pain. TNF‐α blockers have already been successfully used to treat inflammatory disorders but there are few studies on its effect on neuropathic pain, especially following SCI. Following T13 spinal cord hemisection, we examined the effects on mechanical allodynia and microglial activation of immediate and delayed chronic intrathecal treatment with etanercept, a fusion protein blocker of TNF‐α. Immediate treatment (starting at the time of injury) with etanercept resulted in markedly reduced mechanical allodynia 1, 2, 3 and 4 weeks after SCI. Delayed treatment had no effect. Immediate etanercept treatment also reduced spinal microglial activation assessed by OX‐42 immunostaining, a putative marker of activated microglia. To assess whether the effects of etanercept were mediated via decreased microglial activation, we examined the effects of the microglial inhibitor, minocycline which significantly reduced the development of pain behaviours at 1 and 2 weeks after SCI compared to saline treatment. Minocycline also significantly reduced microglial OX‐42 expression. Furthermore, minocycline decreased the expression of noxious‐stimulation‐induced c‐Fos, suggesting an effect on evoked neuronal activity. This study demonstrates that TNF‐α plays an important role in the establishment of neuropathic pain following SCI, seemingly dependent on microglial activation. Pharmacological targeting of TNF‐α may offer therapeutic opportunities for treating SCI pain.     

Hydrogel-electrospun fiber composite materials for hydrophilic protein release

Although hydrogels are widely used in controlled-release systems, obtaining extended, uniform drug release with little initial burst has been challenging. However, recently researchers have shown that combining hydrogels with another drug delivery material can dramatically improve release kinetics. Here we describe a novel hydrogel-based composite material that exhibits stable, near-linear, sustained release of a model hydrophilic protein (e.g., bovine albumin serum, BSA) for over two months with a significant reduction in initial burst release (7% vs. 20%). The composite is comprised of poly(ε-caprolactone) (PCL) electrospun fiber mats coupled with poly(ethylene glycol)-poly(ε-caprolactone) diacrylate (PEGPCL) hydrogels through photo-polymerization. It is believed that the additional diffusion barrier provided by hydrophobic electrospun fiber mats reduces hydrogel swelling and water penetration rates and increases the diffusion path length, resulting in delayed, more uniform drug release. Further, released proteins remain bioactive as demonstrated by PC12 cell neurite extension in response to released nerve growth factor (NGF). The use of electrospun fiber mats to modulate hydrogel drug release provides a new method to control release kinetics of hydrophilic proteins, reducing burst release and extending the release duration.     

Alpha‐Adrenoceptor Modulation in Central Nervous System Trauma: Pain,Spasms, and Paralysis – An Unlucky Triad

Many researchers have attempted to pharmacologically modulate the adrenergic system to control locomotion, pain, and spasms after central nervous system (CNS) trauma, although such efforts have led to conflicting results. Despite this, multiple studies highlight that α‐adrenoceptors (α‐ARs) are promising therapeutic targets because in the CNS, they are involved in reactivity to stressors and regulation of locomotion, pain, and spasms. These functions can be activated by direct modulation of these receptors on neuronal networks in the brain and the spinal cord. In addition, these multifunctional receptors are also broadly expressed on immune cells. This suggests that they might play a key role in modulating immunological responses, which may be crucial in treating spinal cord injury and traumatic brain injury as both diseases are characterized by a strong inflammatory component. Reducing the proinflammatory response will create a more permissive environment for axon regeneration and may support neuromodulation in combination therapies. However, pharmacological interventions are hindered by adrenergic system complexity and the even more complicated anatomical and physiological changes in the CNS after trauma. This review is the first concise overview of the pros and cons of α‐AR modulation in the context of CNS trauma.     

A‐Fibers Mediate Cold Hyperalgesia in Patients with Oxaliplatin‐Induced Neuropathy

Cold hyperalgesia is a common side effect of oxaliplatin treatment; still, the pathophysiological and molecular mechanisms as well as the contribution of different primary afferent fiber systems are unclear. Therefore, patients with oxaliplatin‐induced acute neuropathy with (n = 6) and without (n = 7) cold hyperalgesia were tested by applying a preferential blockade of peripheral myelinated A‐fiber afferents in combination with quantitative sensory testing. Additionally, an interview‐based questionnaire assessed the severity of symptoms and the impact on daily activities. Results indicate a deficit of cold perception in patients without cold hyperalgesia compared to patients with cold hyperalgesia prior to A‐fiber blockade. In patients with cold hyperalgesia, a preferential blockade of A‐fibers abolished cold hyperalgesia. This suggests that oxaliplatin‐induced cold hyperalgesia is mediated by A‐fibers and that a deficit in A‐fiber function might prevent the development of cold hyperalgesia. The work supports findings in rodents and in human sural nerve biopsies indicating that oxaliplatin interferes with axonal ion conductance in intact A‐fibers by sensitizing potassium and/or sodium channels. Drugs that act on these molecular targets might be of potential value to treat oxaliplatin‐induced cold hyperalgesia.     

Nogo及其受体在脊髓损伤修复中的作用机制

成体哺乳动物中枢神经系统(CNS)髓磷脂可影响神经的可塑性并抑制神经纤维的再生。Nogo-A被认为是中枢神经系统中抑制轴突生长最关键的一种髓磷脂抑制分子。在脊髓损伤(SCI)动物模型中,抑制Nogo-A的活性可明显促进轴突再生及功能改善。Nogo-A及其信号转导机制的研究日益成为SCI修复过程中的研究热点;Nogo-A及其信号转导分子特别是Nogo-66受体(NgR)、p75神经营养素受体(p75NTR)和LINGO-1成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制Nogo-A及其受体NgR/p75NTR/LINGO-1可能有助于SCI的修复,促进患者功能的恢复。     

BCNU-loaded PEG-PLLA ultrafine fibers and their in vitro antitumor activity against Glioma C6 cells.

The purpose of the present study was to develop implantable BCNU-loaded poly(ethylene glycol)-poly(L-lactic acid) (PEG-PLLA) diblock copolymer fibers for the controlled release of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was well incorporated and dispersed uniformly in biodegradable PEG-PLLA fibers by using electrospinning method. Environmental Scanning Electron Microscope (ESEM) images indicated that the BCNU-loaded PEG-PLLA fibers looked uniform and their surfaces were reasonably smooth. Their average diameters were below 1500 nm. The release rate of BCNU from the fiber mats increased with the increase of BCNU loading amount. In vitro cytotoxicity assay showed that the PEG-PLLA fibers themselves did not affect the growth of rat Glioma C6 cells. Antitumor activity of the BCNU-loaded fibers against the cells was kept over the whole experiment process, while that of pristine BCNU disappeared within 48 h. These results strongly suggest that the BCNU/PEG-PLLA fibers have an effect of controlled release of BCNU and are suitable for postoperative chemotherapy of cancers.     

Human gingival mesenchymal stem cells pretreated with vesicular moringin nanostructures as a new therapeutic approach in a mouse model of spinal cord injury

Spinal cord injury (SCI) is a neurological disorder that arises from a primary acute mechanical lesion, followed by a pathophysiological cascade of events that leads to further spinal cord tissue damage. Several preclinical and clinical studies have highlighted the ability of stem cell therapy to improve long‐term functional recovery in SCI. Previously, we demonstrated that moringin (MOR) treatment accelerates the differentiation process in mesenchymal stem cells inducing an early up‐regulation of neural development associated genes. In the present study, we investigated the anti‐inflammatory, anti‐apoptotic, and regenerative effects of gingival mesenchymal stem cells (GMSCs) pretreated with nanostructured liposomes enriched with MOR in an animal model of SCI. SCI was produced by extradural compression of the spinal cord at levels T6–T7 in ICR (CD‐1) mice. Animals were randomly assigned to the following groups: Sham, SCI, SCI + GMSCs (1 × 10⁶ cell/i.v.), SCI + MOR‐GMSCs (1 × 10⁶ cell/i.v.). Our data show that MOR‐treated GMSCs exert anti‐inflammatory and anti‐apoptotic activities. In particular, MOR‐treated GMSCs are able to reduce the spinal cord levels of COX‐2, GFAP, and inflammatory cytokines IL‐1β and IL‐6 and to restore spinal cord normal morphology. Also, MOR‐treated GMSCs influenced the apoptotic pathway, by reducing Bax, caspase 3, and caspase 9 expressions.     

Current and future therapeutic strategies for functional repair of spinal cord injury

Spinal cord injury (SCI) is one of the major disabilities dealt with in clinical rehabilitation settings and is multifactorial in that the patients suffer from motor and sensory impairments as well as many other complications throughout their lifetimes. Many clinical trials have been documented during the last two decades to restore damaged spinal cords. However, only a few pharmacological therapies used in clinical settings which still have only limited effects on the regeneration and functional recovery. This review presents recent clinical trials and recent advances in the development of strategies to restore locomotion after SCI. Several approaches toward functional recovery in SCI succeeded in acute and subacute phases in animal models.However, effective strategies against chronic phase of SCI have not been established yet. The strategy aiming to inhibit single molecule sometimes shows controversial results. In SCI, a lot of players participate in motor and sensory dysfunctions. Therefore, sufficient functional recovery may be achieved by regulating multiple targets. Regrowth of tracts connecting the brain and spinal cord, and axonal sprouting of propriospinal interneurons are fundamentally important for neuronal network working. In addition, remyelination, protection of neuronal death, inhibition of inflammation, and upregulation of beneficial influence of astrocytes are also quite crucial to supporting the axonal refining. Combination of several strategies might be useful as practical therapy. Several compounds such as a Sema3A inhibitor, estrogen, withanoside IV and their relating compounds or other neurotrophic factor-mimicking agents may be candidates for useful SCI therapeutic drugs since those have multi-effects on damaged spinal cord.     

Additive manufacturing of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] scaffolds for engineered bone development

A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue‐engineering scaffolds. In this study, the manufacture of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer‐controlled wet‐spinning system, was investigated. By optimizing the processing parameters, three‐dimensional scaffolds with different internal architectures were fabricated, based on a layer‐by‐layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79–88%, fibre diameter 47–76 µm and pore size 123–789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase‐inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell‐culture experiments employing the MC3T3‐E1 murine pre‐osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright © 2014 John Wiley & Sons, Ltd.     

Recent advances in the multitarget‐directed ligands approach for the treatment of Alzheimer's disease

With 27 million cases worldwide documented in 2006, Alzheimer's disease (AD) constitutes an overwhelming health, social, economic, and political problem to nations. Unless a new medicine capable to delay disease progression is found, the number of cases will reach 107 million in 2050. So far, the therapeutic paradigm one‐compound‐one‐target has failed. This could be due to the multiple pathogenic mechanisms involved in AD including amyloid β (Aβ) aggregation to form plaques, τ hyperphosphorylation to disrupt microtubule to form neurofibrillary tangles, calcium imbalance, enhanced oxidative stress, impaired mitochondrial function, apoptotic neuronal death, and deterioration of synaptic transmission, particularly at cholinergic neurons. Approximately 100 compounds are presently been investigated directed to single targets, namely inhibitors of β and γ secretase, vaccines or antibodies that clear Aβ, metal chelators to inhibit Aβ aggregation, blockers of glycogen synthase kinase 3β, enhancers of mitochondrial function, antioxidants, modulators of calcium‐permeable channels such as voltage‐dependent calcium channels, N‐methyl‐D ‐aspartate receptors for glutamate, or enhancers of cholinergic neurotransmission such as inhibitors of acetylcholinesterase or butyrylcholinesterase. In view of this complex pathogenic mechanisms, and the successful treatment of chronic diseases such as HIV or cancer, with multiple drugs having complementary mechanisms of action, the concern is growing that AD could better be treated with a single compound targeting two or more of the pathogenic mechanisms leading to neuronal death. This review summarizes the current therapeutic strategies based on the paradigm one‐compound‐various targets to treat AD. A treatment that delays disease onset and/or progression by 5 years could halve the number of people requiring institutionalization and/or dying from AD. © 2011 Wiley Periodicals, Inc. Med Res Rev     

Extrasynaptic glutamate release through cystine/glutamate antiporter contributes to ischemic damage

During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.     

Influence of the drug compatibility with polymer solution on the release kinetics of electrospun fiber formulation.

The electrospun fiber mat for drug delivery is a novel formulation with promising clinical applications in the future. The influence of the solubility and compatibility of drugs in the drug/polymer/solvent system on the encapsulation of the drug inside the poly(L-lactide) (PLLA) electrospun fibers and the release behavior of this formulation were examined by using paclitaxel, doxorubicin hydrochloride and doxorubicin base as model drugs. The burst release of the drugs can be avoided by using compatible drugs with polymers, and the drug release can follow nearly zero-order kinetics due to the degradation of the PLLA fibers in the presence of proteinase K.     

Transplantation of human bone marrow‐derived clonal mesenchymal stem cells reduces fibrotic scar formation in a rat spinal cord injury model

This study aimed to evaluate the therapeutic effect on tissue repair and scar formation of human bone marrow‐derived clonal mesenchymal stem cells (hcMSCs) homogeneously isolated by using a subfractionation culturing method, in comparison with the non‐clonal MSCs (hMSCs), in a rat spinal cord injury (SCI) model. The SCI was made using a vascular clip at the T9 level. Cells were transplanted into the lesion site 3 days after injury. A functional test was performed over 4 weeks employing a BBB score. Rats were killed for histological analysis at 3 days, 1 week and 4 weeks after injury. The transplantation of hMSCs and hcMSCs significantly reduced lesion size and the fluid‐filled cavity at 4 weeks in comparison with the control group injected with phosphate buffered saline (PBS) (p < 0.01). Transplantation of hcMSCs showed more axons reserved than that of hMSCs in the lesion epicentre filled with non‐neuronal tissues. In addition, hMSCs and hcMSCs clearly reduced the inflammatory reaction and intraparenchymal hemorrhaging, compared with the PBS group. Interestingly, hcMSCs largely decreased Col IV expression, one of the markers of fibrotic scars. hcMSCs yielded therapeutic effects more than equal to those of hMSCs on the SCI. Both hMSCs and hcMSCs created an increase in axon regeneration and reduced scar formation around the SCI lesion. Copyright © 2017 John Wiley & Sons, Ltd.     

Spinal cord injury

Purpose : This article is an overview of the newer therapeutic interventions employed in the care of the spinal cord injured individual and the theoretical rationale supporting them. Issue : Spinal Cord Injury (SCI) care was, until recently, a maintenance type treatment, addressing systems mostly affected by complications of the original injury (e.g. bladder, skin, spasiticity). Conclusion : With the recent advances in the neuroscience field, more aggressive interventions geared at secondary injury prevention, neuronal regeneration and functional restoration are emerging.     

Essential role of the synaptic vesicle protein synapsin II in formalin-induced hyperalgesia and glutamate release in the spinal cord

The synaptic vesicle protein synapsin II plays an important role in the regulation of neurotransmitter release and synaptic plasticity. Here, we investigated its involvement in the synaptic transmission of nociceptive signals in the spinal cord and the development of pain hypersensitivity. We show that synapsin II is predominantly expressed in terminals and neuronal fibers in superficial laminae of the dorsal horn (laminae I-II). Formalin injection into a mouse hindpaw normally causes an immediate and strong release of glutamate in the dorsal horn. In synapsin II deficient mice this glutamate release is almost completely missing. This is associated with reduced nociceptive behavior in the formalin test and in the zymosan-induced paw inflammation model. In addition, the formalin evoked increase in the number of c-Fos IR neurons is significantly reduced in synapsin II knockout mice. Touch perception and motor coordination, however, are normal indicating that synapsin II deficiency does not generally disrupt sensory and/or motor functions. Antisense-mediated transient knockdown of synapsin II in the spinal cord of adult animals also reduced the nociceptive behavior. As the antisense effect is independent of a potential role of synapsin II during development we suggest that the hypoalgesia in synapsin II deficient mice does involve a direct 'pain-facilitating' effect of synapsin II and is not essentially dependent on potentially occurring developmental alterations. The distinctive role of synapsin II for pain signaling probably results from its specific localization and possibly from a specific control of glutamate release.