The lumbar motor activity in isolated spinal cords of 72 postnatal Balb/C mice aged 2, 5, 10 and 21 days (PN2-21) was electroneurographically recorded (ENG) via bilateral ventral roots following treatment with three different concentrations (25, 100 and 200 pM) of the neurotransmitter, 5-hydroxytryptamine (5-HT), i.e., serotonin, to determine its effects on spinal pattern generation.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Traumatic spinal cord injury (SCI) is a physically debilitating, emotionally devastating, financially costly, and life-changing condition that afflicts more than 1,000,000 people in the United States alone. Owing to the characteristic neuropathology and low regenerative capacity of the central nervous system, many victims of SCI are left permanently paralyzed. Though the tissue damage caused by the initial insult almost certainly cannot be reversed, intensive research in recent years to elucidate the cellular and molecular events that follows has provided new grounds for optimism. Accordingly, in this dissertation, we present a number of potential treatment strategies aimed to address some of these pathological sequelae seen post-SCI so as to facilitate the regeneration of axons and the recovery of physiological functions. After the initial traumatic insult, a prominent and lasting injury-induced proliferative response occur and results in the development of a gliotic scar that isolates the lesion from the surrounding viable tissue. Although this process aids to prevent the spread of uncontrolled tissue damage, the scar nevertheless acts as a physical barrier to axonal regeneration. Furthermore, cells within the scar are a major source of axon growth-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPG) and thus the scar acts concomitantly as a biochemical barrier. Concurrent to all this, inflammatory cells infiltrate the lesion and promote cell death through immunologic activation. Neuronal survival is also threatened from the lack of neurotrophic support caused by axonal severance. Finally, the pathology culminates in the formation of a fluid-filled cyst, which represents a gap that further hinders axonal regrowth. Since regeneration cannot physically occur in the presence of a cavity, we, by employing electrospinning techniques, generated a biocompatible matrix implant that can bridge and direct axonal elongation across the fluid-filled cyst. Given the complex array and scope of pathological sequelae post-SCI, it is generally recognized that a multifaceted approach is required to successfully treat SCI. In view of this, we presented novel approaches by which successful tried and true therapeutic strategies are combined to generate an enhanced matrix. An enzyme as well as a growth factor was incorporated into our matrix implants in order to respectively neutralize CSPGs and provide neurotrophic support. Using in vitro assays, we were able to demonstrate excellent protein bioactivity after incorporation. In vivo experimentation of these enhanced matrices is now ongoing. To address the injury-induced proliferative response, which represents an on-ramp off-ramp obstacle that prevents axonal regeneration onto our matrix implant, we showed how X-irradiation can be utilized to moderate this response by killing dividing cells so as to facilitate a more efficient penetration of regrowing axons into and beyond the gliotic scar. Finally, we demonstrate how a novel pharmacologic agent FTY720 can be used to attenuate the inflammatory response by preventing lymphocytic egress from lymphoid tissues. Collectively, these ideas and experimental results represent novel therapeutic strategies that can be combined in order to bring about meaningful functional recovery after SCI.

Spinal cord injury (SCI) has presented itself as a multifaceted pathology that is largely inhibitory to regeneration, and therefore to functional recovery, even though spinal cord neurons have been found to be innately regenerative. Thus, having identified the key players in the inhibition of this innate regeneration, SCI researchers have focused on two major types of approaches: (1) blocking inhibitory cues and (2) promoting innate regeneration. Schwann cells (SCs) have long been shown to promote and enhance functional recovery after SCI through providing supplemental myelination and trophic and tropic factors to regenerating axons, though singular approaches rarely address the complex SCI pathology. Guidance channels and scaffolds have been shown to provide physical support and directional cues to regeneration axons. Therefore, a combinatorial approach in which SCs migrate into and throughout a guidance scaffold would be an ideal research focus for treating SCI. However, cell migration into guidance scaffolds has been shown to be problematic. This study attempts to assess and improve two- and three-dimensional SC migration on electrospun scaffolds. Additionally, we evaluate the ability of SCs, seeded on Matrigel-coated electrospun scaffolds, to improve functional recovery in rats with completely transected spinal cords.

This study examines the potential of Schwann cells (SCs) to be manipulated by electric fields (EFs) in order to improve recovery from spinal cord injury (SCI). It had long been believed that the central nervous system (CNS) is incapable of regeneration, but recent studies have proven otherwise. SC transplants are known to be useful in promoting axon regeneration after SCI, but is not sufficient for functional recovery. EFs are known to exist in vivo, and have been known to drastically affect the morphology and behavior of cells in various tissues. It was the hypothesis of this study that the conditioning and observed alignment of SCs was a reproducible phenomenon that could promote the growth of axons. It was found that SCs could be aligned at various field strengths and preliminary data suggest that aligned SCs increased the length and directionality of axons extending from DRG explants.

Les douleurs neuropathiques ayant une origine à la suite de blessures traumatiques du SNC ou du SNP sont particulièrement difficiles à traiter en utilisant les moyens thérapeutiques actuellement disponibles. Il est donc nécessaire d'identifier de nouvelles stratégies thérapeutiques. Notre objectif était donc de définir les mécanismes impliqués dans ces douleurs neuropathiques. LIMK1 est l'un des acteurs possibles de la réorganisation épinière qui caractérise les lésions nerveuses. Une fonction très caractérisé de cette protéine, est la phosphorylation d'une famille de protéines appelées « cofilines ». Sa phosphorylation, ce qui induit la réorganisation du cytosquelette d'actine. Récemment, il a été montré qu’un microARN (miARNs) nomé miR-134 régule l'expression de LIMK1 en se liant au messager de LIMK1 (ARNm), inhibant sa traduction en protéine physiologiquement active. Notre hypothèse était que la régulation de LIMK1 par miR-134 pourrait jouer un rôle essentiel dans la sensibilisation à la douleur. Cette régulation pourrait ainsi être liée non seulement à la modulation neurochimique neuronale mais aussi à la plasticité fonctionnelle associée. Au cours de cette thèse, l’HIS a montré une diminution de miR-134 chez des rats SNL (neuropathique), cette sous-expression était concomitante à une augmentation de LIMK1 illustrée par l’IHC. Il est important de noter ici que l'ISH est une méthode de détection connue récemment et qui a été identifiée pour visualiser les miARNs. Des différents protocoles de l’HIS ont également été discutés dans le cadre de cette thèse. Ce résultat a été confirmé par Le qRT-PCR . Par la suite, afin de vérifier les changements comportementaux douloureux induits par miR-134 et LIMK1. Nous avons effectués des injections intrathécales de siRNA anti-LIMK1 pour inhiber l'expression endogène de LIMK1 chez les SNL. C’était intéressant de ne pas avoir trouvé aucun changement comportemenal chez les SNL après ce type d’injection. Une surexpression artificielle de miR-134 en utilisant un précurseur de miR-134 (premiR-134) chez les SNL a montré le même effet. Ensuite, nous avons essayé d'effectuer les mêmes injections chez les Sham (control), et c’était plus intéressant de trouver que ces injections (siRNA LIMK1 et premiR-134) ont provoqué une hypersensibilité douleureuse chez les sham. Cela a été illustré au moyen de deux tests de comportement; le Von Frey (VF) et la distribution pondérale dynamique (DWB). Pour etudier l'effet inverse, nous avons inhibé miR-134 en utilisant une sonde spécifique KD (Knock-Down); une diminution significative inattendue dans le seuil de retrait a été observée avec VF et DWB. qRT-PCR dans la plupart de ces cas, a confirmé la corrélation in vivo entre miR-134 et LIMK1. Enfin, nous avons cherché un mécanisme d'action possible qui pourrait réguler cette modulation. Des données récentes publiées ont montré une implication de l'ADF/cofiline sur le trafic des récepteurs AMPA (AMPAR). En accord avec les résultats mentionnés ci-dessus, la transfection du KD de miR-134 a montré une diminution dans AMPAR adressés à la membrane plasmique. Tout ensemble ces données suggèrent que l'effet antinociceptif de KD de miR-134 et la surexpression de LIMK1 sont indirectement régulé par l'insertion des AMPAR à la membrane plasmique.Il semble que miR-134 exerce un effet différent sur la douleur neuropathique que miR-103, discuté aussi dans le cadre de cette thèse. Il était demontré comme un régulateur de plusieurs cibles, les trois sous-unités formant les canaux calciques de type-L « Cav1.2 LTC ». MiR-103 a été trouvé également réprimés chez les SNL. La surexpression de miR-103 soulage la douleur neuropathique. Contrairement au miR-134, miR-103 exerce un rôle pronociceptive pendant la douleur neuropathique. / Pains having a neuropathic origin following CNS or PNS traumatic injury are particularly difficult to treat using the actually available therapeutic means. It is thus necessary to identify new therapeutic strategies. Hence, our aim was to define the mechanisms implicated in these neuropathic pains. Nervous lesions are characterised by an anatomical reorganization of the neuronal network of the dorsal horn. Neurochemical alterations are also involved. Some of the molecular mechanisms underlying the neuronal plasticity (a main feature of neuropathic pain) have been emphasized here by a variety of complementary technical approaches. LIMK1 is one of the possible actors of this reorganization. Among this protein’s known functions, and the most characterized is the phosphorylation of a family of proteins known as cofilins. Their phosphorylation induces the reorganization of actin cytoskeleton. Recently, it has been shown that a miR-134 miRNA regulates LIMK1 expression by binding to the LIMK1 messenger, inhibiting its translation into physiologically active protein. Our hypothesis is that LIMK1 regulation by miR-134 might play an essential role in pain sensitization by modulating neuron neurochemical reorganization and the associated functional neuronal plasticity. Firstly, by means of IHC and ISH, we studied miR-134/LIMK1 distribution within the dorsal horn of the spinal cord in sham animal (control group) and in neuropathic pain model (SNL model). Important to note here that ISH is a known detection method recently identified to visualize miRNA. Different protocols of ISH were discussed in a part of this thesis. ISH showed a decrease in miR-134 expression in SNL rats concomitantly with an increase in LIMK1 illustrated by IHC. This finding has been confirmed by qRT-PCR techniques. Afterward, in order to check for the possible behavioural-induced changes of miR-134 and LIMK1. We intrathecally injected an anti-LIMK1 siRNA to inhibit endogenous LIMK1 expression in SNL rats. Interestingly no significant changes in pain behaviour have been observed. Artificial overexpression of miR-134 using a PremiR-134, showed the same effect. Then we tried to perform the same injections on sham rats, and more interestingly, siRNA LIMK1 and premiR-134 evoked pain hypersensitivity in shams rats. This was illustrated by means of two behaviour tests; Von Frey (VF) and the Dynamic Weight bearing (DWB). To explore the reverse effect, we inhibited miR-134 using a specific KD probe in SNL rats; unexpectedly a significant decrease in pain withdrawal threshold was observed with VF and DWB. qRT-PCR in most cases confirmed the in vivo correlation between miR-134 and LIMK1. Finally, we searched for the possible mechanism of action that could regulate this modulation. Recent published data showed an involvement of ADF/cofilin on AMPAR trafficking. In line with the above mentioned findings, miR-134 KD transfection showed a decrease in AMPAR addressed to the plasma membrane. Altogether suggest that the antinociceptive effect of miR-134 KD and LIMK1 overexpression are mediated by AMPAR insertion at the plasma membrane. It seems that miR-134 exerts a different effect on neuropathic pain than miR-103 another miRNA discussed within the frame of this thesis. MiR-103 has been proved to regulate multiple targets, the three subunits forming Cav1.2 LTC. Pain sensitization involves Cav1.2 activation which consequently alters gene expression during this form of plasticity. MiR-103 was found downregulated also in the SNL model. Conversely to miR-134, overexpression of miR-103 partially alleviates pain. It decreases pain withdrawal threshold of the Von Frey test. Unlike miR-134, miR-103 exerts a pronociceptive role during neuropathic pain.

La mise en place des projections axonales est une étape clé du développement des circuits de la moelle épinière qui contrôle les fonctions motrices de l’individu. Il s’agit d’un processus complexe impliquant des mécanismes de spécification des neurones moteurs et des signalisations multiples assurant le guidage spécifique de chaque axone jusqu’à sa cible d’innervation. Les Sémaphorines de classe 3 (Sema3) sont des molécules secrétées dans l’environnement qui participent notamment au guidage des axones moteurs de la moelle épinière. De manière surprenante, les motoneurones expriment également eux-mêmes des Sema3. Mon équipe a déjà montré que l’expression intrinsèque de Sema3A par les motoneurones module leur sensibilité au Sema3A de l’environnement. Ce processus s’effectue par une régulation du niveau de son récepteur à la surface du cône de croissance, la structure terminale de l’axone responsable de la perception des signaux de guidage extracellulaires (Moret et al., 2007). Quelle est la portée de ce nouveau mécanisme modulatoire et peut-il être généralisé à d’autres membres de la famille des Sema3 exprimés dans les motoneurones ? Mon travail de thèse a permis de mettre en évidence ce rôle modulatoire d’un autre membre de la famille Sema3 : Sema3C, dons l’expression est restreinte à une sous-population de motoneurones, en plus d’une expression environnementale. Par des expériences de gain et de perte de fonction dans l’embryon de poulet, nous avons montré que l’expression motoneuronale de Sema3C module de manière différentielle ses deux récepteurs du niveau de récepteur entraînent une modulation de réponse à Sema3C mais aussi à Sema3A et Sema3F, qui se fixent respectivement sur Nrp1 et Nrp2. De plus, nous avons étudié la portée de ce mécanisme de modulation dans l’innervation des membres de l’embryon de poulet, ce modèle permettant l’étude des projections motoneuronales dans un environnement riche en Sema3. Ainsi, l’analyse in vivo des embryons de poulet pour lesquels l’expression intrinsèque de Sema3C dans les motoneurones a été manipulée a permis de montrer que la modulation de réponse aux Sema 3 de l’environnement contribue au positionnement stéréotypé des projection motrices dans le membre antérieur. Ce travail permet donc de proposer que l’expression d’un code de Sema 3 par les motoneurones confère aux différentes sous-populations une sensibilité spécifique aux gradients de Sema 3 présents dans l’environnement et assure ainsi une trajectoire stéréotypée des axones moteurs jusque dans leur territoire cible. / During spinal circuit’s development, the implementation of axonal projections is a key step as it ensures the integrity of motor functions. This complex process implicates several mechanisms such as motoneuton specification and various signaling cascades in order to guide specifically each axon toward its innervation target. Class 3 Semaphorins (Sema3s) are chemotactic cues, secreted in the environment, and contributing to the guidance of spinal motor axons. Surprisingly, these Sema3s are also expressed by motoneurons themselves. Previous work in our team showed that intrinsic expression of Sema 3A. This process arises from the regulation of its receptor availability at the growth cone surface, the terminal end of the axon that enables sensing of extracellular signals (Moret et al., 2007). But in what extent can we generalize this mechanism to other members of the Sema3 family, and what impact could it have, in vivo, during development? My PhD work revealed the modulatory role of another Sema3, Sema3C, expressed in a restricted motoneuronal subpopulation as well as in the environment. By again and loss of function experiments in the chick embryo, we showed that motoneuronal expression of Sema3C modulates differentially its two Neuropilin receptor level, Nrp1 and, Nrp2, at the growth cone surface. These variations of receptors’ availability lead to a response modulation to extrinsic Sema3, but also to Sema3A and Sema3F, that bind to Nrp1 and Nrp2, respectively. Moreover, we analyzed the role of such modulation mechanisms during limb innervation in the chick embryo. Indeed, this model enables the study of motor axon growth in a complex Semaphorin environment. In vivo analysis of chick embryos after intrinsic Sema3C manipulations revealed that response modulation to environmental Sema3s takes part in the stereotyped positioning motor axon projections in the forelimb. Hence, this Study suggest a model in which the expression of a Semaphorin code by motoneurons enables specific subpopulations to modulate their sensitivity Sema3 gradients expressed in their environment, thus contributing to the stereotyped trajectories of motor axon projections towards their final target.

Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn is a significant mechanism in the development and maintenance of different pathological pain states. Accumulating evidence indicates that the TRPV1 (transient receptor potential vanilloid 1) receptor and chemokine CCL2 (C-C motif ligand 2) may play a critical role in this process. The aim of this diploma thesis was to investigate the CCL2 induced modulation of nociceptive synaptic transmission in the dorsal horn of spinal cord and the role of the TRPV1 receptors. To investigate this aim patch-clamp recordings of spontaneous and miniature excitatory postsynaptic currents (sEPSC, mEPSC) from superficial dorsal horn neurons in acute rat lumbar spinal cord slices were used. After acute application of CCL2 on the slice preparation from naïve animals, a frequency increase of both sEPSC and mEPSC was present. This CCL2 induced increase in both sEPSC and mEPSC frequency was prevented by the TRPV1 receptor antagonist SB366791 application. No changes were observed in the amplitudes of sEPSC or mEPSC after application of the CCL2, SB366791, or co-application of CCL2 and SB366791. This suggests that the observed changes were mediated predominantly by presynaptic mechanisms. The preliminary results indicate that after chronic constriction...

Introduction - This thesis focuses on the reaction of cardiovascular system to Lokomat therapy in patients with lesion of the spinal cord. It observes reaction of the heart rate and systolic blood pressure and compares with the cardiovascular response to the exercise on an upper body ergometer. Methods - We recruited 25 patients with spinal cord lesion under Th 5 and 4 patients with lesion above Th 5. The blood pressure was measured. The heart rate was measured by by ambulatory blood pressure monitor and an auscultatory method using a mercury manometer and a stethoscope a sporttester. Patients were measured during the Locomat therapy and the exercise on an upper body ergometer during the 6-minute arm test. Results - The parameters of systolic blood pressure and heart rate had a tendency to increase with the peak in 25th minute of the therapy. There was no significant difference between the patients with lesion under or above Th5. The parameters of heart rate were significantly higher on the upper body ergometer than on Lokomat. Conclusion - The Lokomat therapy does have a positive influence to cardiovascular system of patients with complete or incomplete spinal cord injury. When using this approach, the most important benefit is the possibility to activate all parts of body. Keywords: spinal cord injury,...

La locomotion implique l'activité coordonnée de réseaux d'interneurones spinaux inhibiteurs et excitateurs qui génèrent le rythme et le pattern de la décharge des motoneurones et de la contraction musculaire. La maturation de la transmission inhibitrice au niveau des motoneurones chez le rat se produit durant la période périnatale. Dans cette fenêtre temporelle, les projections provenant du tronc cérébral commencent à envahir l'élargissement lombaire de la moelle épinière. Nous avons étudié les effets du blocage de la mise en place des afférences supraspinales dans la moelle lombaire par transection complète de la moelle épinière (SCT) à la naissance (P0), sur l'expression des sous-unités des récepteurs GABAA et glycine (RcGABAA et RcGly) au niveau des motoneurones lombaires. Nous concluons en une plasticité différentielle des récepteurs de l'inhibition en réponse à une section spinale néonatale. La première étape de notre seconde étude était d'évaluer les modifications de la transmission synaptique inhibitrice sur des motoneurones lombaires innervant des muscles fléchisseurs et extenseurs après SCT complète chez des rats adultes. Une étude longitudinale a montré une évolution différentielle de l'expression des RcGly et des RcGABAA au cours des mois qui suivent la lésion mais qui est a peu près identique sur les Mns fléchisseurs et extenseurs. Nos résultats montrent que chez les rats avec SCT, l'entrainement permet d'acquérir une « locomotion spinale » qui résulte d'interactions dynamiques entre un programme moteur dans la moelle sous lésionnelle, et des feedback proprioceptifs. / Maturation of inhibitory postsynaptic transmission onto motoneurons in the rat occurs during the perinatal period, a time window during which pathways arising from the brainstem reach the lumbar enlargement of the spinal cord. There is a developmental switch in miniature IPSCs (mIPSCs) from predominantly long-duration GABAergic to short-duration glycinergic events. We investigated the effects of a complete neonatal [postnatal day 0 (P0)] spinal cord transection (SCT) on the expression of Glycine and GABAA receptor subunits (GlyR and GABAAR subunits) in lumbar motoneurons. In P7 animals with neonatal SCT (SCT-P7), the GlyR densities were unchanged compared with controls of the same age, while the developmental down regulation of GABAAR was prevented. After spinal cord injury, the disruption of flexion/extension and left-right alternations is largely attributed to a deterioration of the inhibitory circuitry below the lesion, but most of the cellular mechanisms are still unknown. Our aim of this was to measure the alteration of the GABA and glycinergic synaptic transmission on lumbar motoneurons (Mns) after spinal cord transection (SCT) in the adult rat, and evaluate the benefit of manual training and stepping recovery on the inhibitory networks. All in all our results show that, the presynaptic and postsynaptic components of the glycinergic synapses are relatively preserved on lumbar Mns. We developed a manual training procedure, based on daily alternate phases of imposed stepping and free walking in enriched environment. Pharmacological treatment with 5-HT2 receptor agonists allowed a standing recovery and alternate stepping.