Lentiviral-mediated silencing of glial fibrillary acidic protein and vimentin promotes anatomical plasticity and functional recovery after spinal cord injury.

In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)-mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral-mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral-mediated RNAi in SCI.

Potential adverse effects of cyclosporin A on kidneys after spinal cord injury.

STUDY DESIGN: Cell transplantation strategies are gaining increasing interest for spinal cord injury (SCI) with the objective of promoting spinal cord repair. To avoid allogenic graft rejection, an adequate immune suppression is required, and one of the most potent and commonly used immunosuppressives is cyclosporin A (CsA). In SCI, permanent sensory motor loss is combined with modifications of drug absorption, distribution and elimination. OBJECTIVES: The objectives of this study were to thoroughly explore histological and functional outcomes of CsA treatment in a rat model of spinal cord compression. SETTING: Experiments were carried out at the Institute for Neurosciences of Montpellier (France), the Integrative Biology of Neurodegeneration Laboratory (Spain) and in the Novartis Institutes for BioMedical Research (Switzerland) for CsA blood concentration determination. METHODS: We first evaluated histological outcomes of CsA treatment on kidneys and spinal cord after SCI. We then investigated whether SCI modified CsA blood concentration. Finally, using behavioral analysis, we assessed the potential CsA impact on functional recovery. RESULTS: When spinal-cord-injured rats were treated with a CsA dose of 10 mg kg(-1) per day, we observed deleterious effects on kidneys, associated with modifications of CsA blood concentration. Adding an antibiotic treatment reduced kidney alteration without modifying CsA blood concentration. Finally, we showed that CsA treatment per se modified neither functional recovery nor lesion extension. CONCLUSION: This study pinpoints the absolute requirement of careful CsA monitoring in the clinical setting for patients with SCI to minimize potential unexpected effects and avoid therapeutic failure.

Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.

3D Imaging of Nanoparticle Distribution in Biological Tissue by Laser-Induced Breakdown Spectroscopy.

Nanomaterials represent a rapidly expanding area of research with huge potential for future medical applications. Nanotechnology indeed promises to revolutionize diagnostics, drug delivery, gene therapy, and many other areas of research. For any biological investigation involving nanomaterials, it is crucial to study the behavior of such nano-objects within tissues to evaluate both their efficacy and their toxicity. Here, we provide the first account of 3D label-free nanoparticle imaging at the entire-organ scale. The technology used is known as laser-induced breakdown spectroscopy (LIBS) and possesses several advantages such as speed of operation, ease of use and full compatibility with optical microscopy. We then used two different but complementary approaches to achieve 3D elemental imaging with LIBS: a volume reconstruction of a sliced organ and in-depth analysis. This proof-of-concept study demonstrates the quantitative imaging of both endogenous and exogenous elements within entire organs and paves the way for innumerable applications.

Rehabilitación respiratoria para pacientes con distrofia muscular de Duchenne en etapas de pérdida de la marcha / Respiratory rehabilitation for patients with Duchenne muscular dystrophy in stages of loss of gait

Duchenne muscular dystrophy (DMD) is one of the most common neuromuscular diseases. Its evolution with well-defined stages related to motor and functional alterations, allows easily establishing relationships with respiratory function through a simple laboratory assessment including vital capacity (VC) measurements as well as peak cough flows. Without any treatment with respiratory rehabilitation, the main cause of morbidity and mortality is ventilatory failure, secondary to respiratory pump muscles weakness and inefficient cough. The VC plateau is reached during the non-ambulatory stages, generally after 13 years old. Respiratory rehabilitation protocols, including air stacking techniques, manual and mechanical assisted coughing and non-invasive ventilatory support, can effectively addressed the VC decline as well as the decrease in peak cough flows, despite advancing to stages with practically non-existent lung capacity. Non-invasive ventilatory support may be applied after 19 years old, initially at night and then extending it during the day. In this way, survival is prolonged, with good quality of life, avoiding ventilatory failure, endotracheal intubation and tracheostomy. This article proposes staggered interventions for respiratory rehabilitation based on the functional stages expected in the patient with DMD who has lost ambulation.

La distrofia muscular de Duchenne (DMD) es una de las enfermedades neuromusculares más frecuentes. Su curso evolutivo con etapas de declinación en la funcionalidad motora bien definidas, permite fácilmente establecer relaciones con la función respiratoria a través de un laboratorio de evaluación sencilla, básicamente de la capacidad vital (CV) y la capacidad tusígena. Sin intervenciones en rehabilitación respiratoria, la principal causa de morbimortalidad es la insuficiencia ventilatoria secundaria a debilidad de músculos de la bomba respiratoria e ineficiencia de la tos. En las etapas no ambulantes, se alcanza la meseta de la CV, generalmente después de los 13 años, su declinación junto con la disminución de la capacidad tusígena puede ser enfrentada efectivamente con la utilización de protocolos de rehabilitación respiratoria. Estos deben considerar la restitución de la CV con técnicas de insuflación activa o apilamiento de aire, tos asistida manual y mecánica, más soporte ventilatorio no invasivo, inicialmente nocturno después de los 19 años y luego diurno, pese a avanzar a etapas con capacidad pulmonar prácticamente inexistente. De esta manera, se prolonga la sobrevida, con buena calidad de vida, evitando el fallo ventilatorio, eventos de intubación endotraqueal y traqueostomía. Este artículo, hace propuestas escalonadas de intervención en rehabilitación respiratoria basadas en las etapas funcionales esperables en el paciente con DMD que ha perdido la capacidad de marcha.

Cuidados respiratorios de los pacientes con atrofia muscular espinal / Respiratory care approach for patients with spinal muscular atrophy

Spinal Muscular Atrophy (SMA) is a disease of the anterior horn of the spinal cord, which causes muscle weakness that leads to a progressive decrease in vital capacity and diminished cough flows. Respiratory morbidity and mortality are a function of the degree of respiratory and bulbar-innervated muscle. The former can be quantitated by the sequential evaluation of vital capacity to determine the lifetime maximum (plateau) and its subsequent rate of decline, progressing to ventilatory failure. SMA types 1 and 2 benefit from non-invasive respiratory care in early childhood and school age, improving quality and life expectancy. This document synthesizes these recommendations with special reference to interventions guided by stages that include air stacking, assisted cough protocols, preparation for spinal arthrodesis and non-invasive ventilatory support, even in those patients with loss of respiratory autonomy, minimizing the risk tracheostomy. Failure to consider these recommendations in the regular assessment of patients reduces the offer of timely treatments.

La Atrofia Muscular Espinal (AME) es una enfermedad genética del asta anterior de la medula espinal, que cursa con debilidad muscular progresiva. La intensidad y precocidad de la debilidad muscular presenta diferentes grados de afectación de los grupos musculares respiratorios, determinando la meseta en la capacidad vital y progresión a la insuficiencia ventilatoria, como también el compromiso de los músculos inervados bulbares. Los AME tipo 1 y 2, se benefician con cuidados respiratorios no invasivos en la infancia temprana y edad escolar, mejorando la calidad y esperanza de vida. Este documento sintetiza dichas recomendaciones, con especial referencia a intervenciones guiadas por etapas, que incluyan apilamiento de aire, protocolos de tos asistida, preparación para la artrodesis de columna y soporte ventilatorio no invasivo, incluso en aquellos pacientes con pérdida de la autonomía respiratoria, minimizando el riesgo de traqueostomía. La no consideración de estas recomendaciones en la valoración regular de los pacientes resta la oferta de tratamientos oportunos.

Inactivation of the glial fibrillary acidic protein gene, but not that of vimentin, improves neuronal survival and neurite growth by modifying adhesion molecule expression.

Intermediate filaments (IFs) are a major component of the cytoskeleton in astrocytes. Their role is far from being completely understood. Immature astrocytes play a major role in neuronal migration and neuritogenesis, and their IFs are mainly composed of vimentin. In mature differentiated astrocytes, vimentin is replaced by the IF protein glial fibrillary acidic protein (GFAP). In response to injury of the CNS in the adult, astrocytes become reactive, upregulate the expression of GFAP, and reexpress vimentin. These modifications contribute to the formation of a glial scar that is obstructive to axonal regeneration. Nevertheless, astrocytes in vitro are considered to be the ideal substratum for the growth of embryonic CNS axons. In the present study, we have examined the potential role of these two major IF proteins in both neuronal survival and neurite growth. For this purpose, we cocultured wild-type neurons on astrocytes from three types of knock-out (KO) mice for GFAP or/and vimentin in a neuron-astrocyte coculture model. We show that the double KO astrocytes present many features of immaturity and greatly improve survival and neurite growth of cocultured neurons by increasing cell-cell contact and secreting diffusible factors. Moreover, our data suggest that the absence of vimentin is not a key element in the permissivity of the mutant astrocytes. Finally, we show that only the absence of GFAP is associated with an increased expression of some extracellular matrix and adhesion molecules. To conclude, our results suggest that GFAP expression is able to modulate key biochemical properties of astrocytes that are implicated in their permissivity.

Transplantation of embryonic Raphe cells regulates the modifications of the gabaergic phenotype occurring in the injured spinal cord.

Transection of the spinal cord yields a permanent deficit due to the interruption of descending and ascending tracts which subserve the supraspinal control of spinal cord functions. We have shown previously that transplantation below the level of the section of embryonic monoaminergic neurons can promote the recovery of some segmental functions via a local serotonergic and noradrenergic reinnervation. Moreover, the up-regulation of the corresponding receptors resulting from the section was corrected by the transplants. The aim of the present work was to determine whether such a graft could also influence non-monoaminergic local neurons, the GABAergic interneurons of the spinal cord. Following spinal cord transection, the number of cells which express glutamate decarboxylase (mol. wt 67,000) messenger RNA--a marker of GABA synthesis--increased significantly below the lesion compared with the intact animal. In contrast, in lesioned animals which had been grafted one week later with raphe neuroblasts, this number was close to control level. These post-grafting modifications were further associated with increased GABA immunoreactivity in the host tissue. These data suggest that the graft of embryonic raphe cells which compensates the deficit of serotonin in the distal segment also regulates the expression of the GABAergic phenotype in the host spinal cord. This regulation could be mediated by the re-establishment of a local functional innervation by both serotonin and GABAergic transplanted neurons and/or by trophic factors released from the embryonic cells. It appears then that grafted cells influence the host tissue in a complex manner, through the release and/or regulation of several neurotransmitter systems.

Strategies for regeneration and repair in spinal cord traumatic injury.

Spinal cord injury is frequently followed by the loss of supraspinal control of sensory, autonomic and motor functions at the sublesional level. In order to enhance recovery in spinal cord-injured patients, we have developed three fundamental strategies in experimental models. These strategies define in turn three chronological levels of postlesional intervention in the spinal cord. Neuroprotection soon after injury using pharmacological tools to reduce the progressive secondary injury processes that follow during the first week after the initial lesion. This strategy was conducted up to clinical trials, showing that a pharmacological therapy can reduce the permanent neurological deficit that usually follows an acute injury of the central nervous system (CNS). A second strategy, which is initiated not long after the lesion, aims at promoting axonal regeneration by acting on the main barrier to regeneration of lesioned axons: the glial scar. Finally a mid-term substitutive strategy is the management of the sublesional spinal cord by sensorimotor stimulation and/or supply of missing key afferents, such as monoaminergic systems. These three strategies are reviewed. Only a combination of these different approaches will be able to provide an optimal basis for potential therapeutic interventions directed to functional recovery after spinal cord injury.

Gene therapy strategies in neurodegenerative diseases.

Treatment of neurodegenerative diseases by classical pharmacotherapy is restricted by blood-brain barrier which prevents access to the brain of potentially therapeutic molecules. Recent progress in the knowledge of pathophysiological molecular processes, and in the development of molecular biotechnology have opened the way to new therapeutic interventions for these disorders. This chapter reviews the most recent gene therapy strategies using experimental models for neurodegenerative diseases.

Neural stem cells and the quest for restorative neurology.

A great deal of interest has attracted the attention of researchers on the potential use of (neural) stem cells in cell replacement or restorative therapies for heretofore incurable CNS pathologies such as brain stroke, spinal cord injury, Parkinson's disease or multiple sclerosis. This short perspective illustrates our view of neural stem cell research with a focus on the stem cell concept, on the in situ identity of neural stem cells and on selected aspects of embryonic and adult neurogenesis. A brief survey of current stem cell-based experimental literature tries to provide a realistic picture of how far we have gone in the quest to establish a restorative neurology.

Recovery of locomotion following transplantation of monoaminergic neurons in the spinal cord of paraplegic rats.

Severe traumatic lesions of the spinal cord yield a permanent deficit of motricity in adult mammals and specifically a loss of locomotor activity of hindlimbs when the lesion is located at the lower thoracic level. To restore this function, we have developed a paradigm of transplantation in rats based on a transection model of the spinal cord and the subsequent injection at the sublesional level of a suspension of embryonic brainstem monoaminergic neurons which play a key role in the modulation of locomotion. A genuine locomotion was characterized in transplanted animals by electromyographic and electroneurographic recordings. This correlated with a specific reinnervation pattern of targets, where typical synapses were found, and with the normalization of biochemical parameters.

Specific and efficient gene transfer strategy offers new potentialities for the treatment of motor neurone diseases.

Several growth factors are candidates for the therapy of motor neurone diseases. However, there is no efficient, safe, and practicable administration route which hampers the clinical use of these potentially therapeutic agents. We show that specific and high yield gene transfer into motor neurones can be obtained by peripheral intramuscular injections of recombinant adenoviruses. These vectors are retrogradely transported from muscular motor units to motor neurone cell bodies. Gene transfer can thus be specifically targeted to particular regions of the spinal cord by appropriate choice of the injected muscle. The efficiency of gene transfer is high, with 58-100% of the motor neurones afferent to the injected muscle expressing the transgene. This new therapeutic protocol allows specific targeting of motor neurones without lesioning the spinal cord, and should avoid undesirable side effects associated with systemic administration of therapeutic factors.

Specific and efficient gene transfer strategy offers new potentialities for the treatment of motor neurone diseases.

Several growth factors are candidates for the therapy of motor neurone diseases. However, there is no efficient, safe, and practicable administration route which hampers the clinical use of these potentially therapeutic agents. We show that specific and high yield gene transfer into motor neurones can be obtained by peripheral intramuscular injections of recombinant adenoviruses. These vectors are retrogradely transported from muscular motor units to motor neurone cell bodies. Gene transfer can thus be specifically targeted to particular regions of the spinal cord by appropriate choice of the injected muscle. The efficiency of gene transfer is high, with 58-100% of the motor neurones afferent to the injected muscle expressing the transgene. This new therapeutic protocol allows specific targeting of motor neurones without lesioning the spinal cord, and should avoid undesirable side effects associated with systemic administration of therapeutic factors.

Transplantation of embryonic noradrenergic neurons in two models of adult rat spinal cord injury: ultrastructural immunocytochemical study.

The synaptic connections established by grafted noradrenergic (NA) neurons into the lesioned adult rat spinal cord were analysed using immunocytochemistry at the electron microscopic level. An embryonic cell suspension of the locus coeruleus region from E-13 rat embryos was transplanted into the spinal cord following either: (1) spinal cord transection or (2), partial selective denervation by 6-hydroxy dopamine (6-OH DA). One month after grafting, the NA-neurons established, in the two models, an innervation pattern similar to that found in the intact spinal cord. In both models, the transplanted NA-immunoreactive neurons formed extensive synaptic contacts with dendrites, spines and perikarya. The proportion of axodendritic and axospinous contacts was inverse in the two models. The first model thus reproduced more closely the normal synaptic pattern prefering dendritic targets, which could correspond to a better integration of the graft. In the second model, a partially NA-denervated spinal cord, there existed a competition between residual intrinsic and grafted neuron-derived fibres, which presumably affects synaptogenesis. In conclusion, the present study illustrate the complexity of cell interations conducting to the formation of a specific circuitry. Recognition phenomenon are likely modulated by space constraints, which ultimately shape-up the geometry of synaptic contacts.

Intraspinal noradrenergic-rich implants reverse the increase of alpha 1 adrenoceptors densities caused by complete spinal cord transection or selective chemical denervation: a quantitative autoradiographic study.

This study examined, in the adult rat, whether the intraspinal transplantation of a cell suspension of embryonic day (ED)13 rat locus coeruleus primordia was able to normalize the lesion-induced increase of spinal alpha 1-adrenoceptors. Two experimental models of spinal denervation were studied. The first model consisted of a complete spinal cord transection (thoracic vertebrae level T8-T9) and 1 week later, the cell suspension was transplanted below the section; the second one was obtained by a selective chemical lesion of the noradrenergic (NA) system and one month later, the cell suspension was implanted at the same level as in transected rats. Five weeks after grafting, all animals were sacrificed and spinal cord tissue sections were processed for immunohistochemical detection of noradrenaline or for quantification of alpha 1-adrenoceptors binding sites densities using [3H]prazosin as a ligand. After 6-OHDA lesion, as well as caudally to the transection, a significant increase by 21% (P < 0.01) to 68% (P < 0.001) of alpha 1-adrenoceptors densities was detected. The implantation of embryonic NA neurons into the denervated spinal cord led to a reversal of the lesion-induced increase of spinal alpha 1-adrenoceptors, five weeks later. Moreover, this reversal seems to be more effective after mechanical than after chemical denervation.

Influence of hypergravity on the development of monoaminergic systems in the rat spinal cord.

We have investigated in this study the influence of a moderate hypergravity (1.8 G) on the development of monoaminergic projections to the spinal cord in the rat. Pregnant dams and their offspring were submitted to hypergravity from day 11 of gestation to postnatal day 15. Some animals were sacrificed at birth, other at postnatal day 15 and other after 8 months of normal gravity. In newborn animals, a substantial delay of the development of monoaminergic projections to the spinal cord was evidenced. In 15 days and 8 months animals, the pattern of innervation appeared anarchic, with numerous dystrophic profiles, mainly of serotonergic system. Ultrastructural examination of serotonergic projections revealed a paucity of synapses, and the frequent enveloping of serotonergic boutons by thin astrocytic profiles. We conclude that rats submitted to hypergravity during the critical period of onset of monoaminergic projections to the spinal cord are affected durably in the organization and the ultrastructure of these projections. Future studies are directed to the functional analysis of hypergravity animals, and to the influence of microgravity on the same system.

[Neuronal regeneration and the glial barrier]. / Respousse axonale et obstacle glial.

The dogma of abortive axonal regrowth set by Cajal (1914) is now broken since the demonstration by Aguayo (1982) that severed axons can regrow in an appropriate environment. Over the last decade, the impediments to such a regrowth in the central nervous system of higher vertebrates have been identified, or, at least, some of them. On the one hand, the inhibitory molecules synthesized and secreted by oligodendrocytes have been counteracted by appropriate antibodies (Schnell & Schwab, 1990), which have permitted some regrowth of severed cortico-spinal axons in the rat spinal cord. On the other hand, the reduction by a pharmacological treatment of hypertrophy and hyperplasia of astrocytes has permitted some regrowth of monoaminergic axons in an hemisected cord (Gimenez y Ribotta et al. 1995). Finally, the identification of a subcategory of astrocytes, the tanycytes of the basal hypothalamus, as a permissive substrate for axonal regeneration opens a new avenue for future research.

Neurodegenerative changes are prevented by Erythropoietin in the pmn model of motoneuron degeneration.

Motoneuron diseases are fatal neurodegenerative disorders characterized by a progressive loss of motoneurons, muscle weakness and premature death. The progressive motor neuronopathy (pmn) mutant mouse has been considered a good model for the autosomal recessive childhood form of spinal muscular atrophy (SMA). Here, we investigated the therapeutic potential of Erythropoietin (Epo) on this mutant mouse. Symptomatic or pre-symptomatic treatment with Epo significantly prolongs lifespan by 84.6% or 87.2% respectively. Epo preserves muscle strength and significantly attenuates behavioural motor deficits of mutant pmn mice. Histological and metabolic changes in the spinal cord evaluated by immunohistochemistry, western blot, and high-resolution (1)H-NMR spectroscopy were also greatly prevented by Epo-treatment. Our results illustrate the efficacy of Epo in improving quality of life of mutant pmn mice and open novel therapeutic pathways for motoneuron diseases.