Neuronal expression of the ubiquitin ligase Nedd4-2 in rat dorsal root ganglia: Modulation in the spared nerve injury model of neuropathic pain.

Neuronal hyperexcitability following peripheral nerve lesions may stem from altered activity of voltage-gated sodium channels (VGSCs), which gives rise to allodynia or hyperalgesia. In vitro, the ubiquitin ligase Nedd4-2 is a negative regulator of VGSC α-subunits (Na(v)), in particular Na(v)1.7, a key actor in nociceptor excitability. We therefore studied Nedd4-2 in rat nociceptors, its co-expression with Na(v)1.7 and Na(v)1.8, and its regulation in pathology. Adult rats were submitted to the spared nerve injury (SNI) model of neuropathic pain or injected with complete Freund's adjuvant (CFA), a model of inflammatory pain. L4 dorsal root ganglia (DRG) were analyzed in sham-operated animals, seven days after SNI and 48h after CFA with immunofluorescence and Western blot. We observed Nedd4-2 expression in almost 50% of DRG neurons, mostly small and medium-sized. A preponderant localization is found in the non-peptidergic sub-population. Additionally, 55.7±2.7% and 55.0±3.6% of Nedd4-2-positive cells are co-labeled with Na(v)1.7 and Na(v)1.8 respectively. SNI significantly decreases the proportion of Nedd4-2-positive neurons from 45.9±1.9% to 33.5±0.7% (p<0.01) and the total Nedd4-2 protein to 44%±0.13% of its basal level (p<0.01, n=4 animals in each group, mean±SEM). In contrast, no change in Nedd4-2 was found after peripheral inflammation induced by CFA. These results indicate that Nedd4-2 is present in nociceptive neurons, is downregulated after peripheral nerve injury, and might therefore contribute to the dysregulation of Na(v)s involved in the hyperexcitability associated with peripheral nerve injuries.

Neuronal potential of cells in early postnatal rat sciatic nerve.

A population of undifferentiated cells with neuronal potentialities were revealed in rat sciatic nerve. Explant cultures of sciatic nerve were prepared from newborn or early postnatal rat. Cultures were growth in F14 medium supplemented with 10% of fetal calf serum, incubated in a humidified 3% CO2, 97% air atmosphere. Within 2 weeks, refractile cells exhibiting the morphology of neurons were observed in all examined cultures. These cells had ovoid or multipolar refractile cells bodies with extended cytoplasmic processes. The neuronal nature of these cells was confirmed by their immunostaining with specific neuronal markers: neurofilament triplets, neuron-specific enolase, peripherin, microtubule-associated proteins, and brain spectrin. This neuronal population displayed various phenotypes. The CO2 concentration in the incubator plays an important role, since the number of differentiated neurons was lower in cultures incubated in 5% CO2. Since the sciatic nerve is devoid of nerve cell bodies in vivo, we concluded that early postnatal sciatic nerve contains crest cells with neuronal potentialities differentiating into neurons in response to the culture's environmental cues.

Patterns of extraocular muscle weakness in vasculopathic pupil-sparing, incomplete third nerve palsy.

OBJECTIVE: To determine the pattern of extraocular muscle (EOM) paresis in incomplete vasculopathic third nerve palsies (3NP) that have normal pupillary function. METHODS: A retrospective study in a private practice and academic neuro-ophthalmic practice. Patients diagnosed with vasculopathic 3NP within 4 weeks of symptom onset were identified. The chart of each patient was reviewed to determine pupillary function and the pattern and degree of EOM and levator palpebrae paresis at the time of presentation. RESULTS: Of 55 patients with vasculopathic 3NP, 42 (76%) had normal pupillary function. Of these 42, 23 (55%) demonstrated an incomplete EOM palsy, defined as partially reduced ductions affecting all third nerve-innervated EOMs and levator (diffuse pattern) or partially reduced ductions that involved only some third nerve-innervated EOMs and levator (focal pattern). Twenty (87%) of these 23 patients showed a diffuse pattern of paresis; only three (13%) showed a focal pattern of paresis, one that affected only the superior rectus and levator muscles (superior division weakness). CONCLUSIONS: Based on our series, most patients with EOM/levator involvement in pupil-sparing, incomplete 3NP of vasculopathic origin have a diffuse pattern of paresis. In contrast, our review of the literature suggests that pupil-sparing 3NP of aneurysmal origin usually have a focal pattern of paresis. We propose that distinguishing these two patterns of EOM paresis may be helpful in differentiating between vasculopathic and aneurysmal 3NP. Future studies will be needed to confirm the clinical utility of this hypothesis.

The spared nerve injury model of neuropathic pain.

The spared nerve injury (SNI) model mimics human neuropathic pain related to peripheral nerve injury and is based upon an invasive but simple surgical procedure. Since its first description in 2000, it has displayed a remarkable development. It produces a robust, reliable and long-lasting neuropathic pain-like behaviour (allodynia and hyperalgesia) as well as the possibility of studying both injured and non-injured neuronal populations in the same spinal ganglion. Besides, variants of the SNI model have been developed in rats, mice and neonatal/young rodents, resulting in several possible angles of analysis. Therefore, the purpose of this chapter is to provide a detailed guidance regarding the SNI model and its variants, highlighting its surgical and behavioural testing specificities.

Regulation of Nav1.7 and Nav1.8 voltage-gated sodium channels by Nedd4-2 and β-subunits and its implication in neuropathic pain

In mammals, the presence of excitable cells in muscles, heart and nervous system is crucial and allows fast conduction of numerous biological information over long distances through the generation of action potentials (AP). Voltage-gated sodium channels (Navs) are key players in the generation and propagation of AP as they are responsible for the rising phase of the AP. Navs are heteromeric proteins composed of a large pore-forming a-subunit (Nav) and smaller ß-auxiliary subunits. There are ten genes encoding for Navl.l to Nav1.9 and NaX channels, each possessing its own specific biophysical properties. The excitable cells express differential combinations of Navs isoforms, generating a distinct electrophysiological signature. Noteworthy, only when anchored at the membrane are Navs functional and are participating in sodium conductance. In addition to the intrinsic properties of Navs, numerous regulatory proteins influence the sodium current. Some proteins will enhance stabilization of membrane Navs while others will favour internalization. Maintaining equilibrium between the two is of crucial importance for controlling cellular excitability. The E3 ubiquitin ligase Nedd4-2 is a well-characterized enzyme that negatively regulates the turnover of many membrane proteins including Navs. On the other hand, ß-subunits are known since long to stabilize Navs membrane anchoring. Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of Navs expressed in dorsal root ganglion (DRG) sensory neurons as highlighted in different animal models of neuropathic pain. Among Navs, Nav1.7 and Nav1.8 are abundantly and specifically expressed in DRG sensory neurons and have been recurrently incriminated in nociception and neuropathic pain development. Using the spared nerve injury (SNI) experimental model of neuropathic pain in mice, I observed a specific reduction of Nedd4-2 in DRG sensory neurons. This decrease subsequently led to an upregulation of Nav1.7 and Nav1.8 protein and current, in the axon and the DRG neurons, respectively, and was sufficient to generate neuropathic pain-associated hyperexcitability. Knocking out Nedd4-2 specifically in nociceptive neurons led to the same increase of Nav1.7 and Nav1.8 concomitantly with an increased thermal sensitivity in mice. Conversely, rescuing Nedd4-2 downregulation using viral vector transfer attenuated neuropathic pain mechanical hypersensitivity. This study demonstrates the significant role of Nedd4-2 in regulating cellular excitability in vivo and its involvement in neuropathic pain development. The role of ß-subunits in neuropathic pain was already demonstrated in our research group. Because of their stabilization role, the increase of ßl, ß2 and ß3 subunits in DRGs after SNI led to increased Navs anchored at the membrane. Here, I report a novel mechanism of regulation of a-subunits by ß- subunits in vitro; ßl and ß3-subunits modulate the glycosylation pattern of Nav1.7, which might account for stabilization of its membrane expression. This opens new perspectives for investigation Navs state of glycosylation in ß-subunits dependent diseases, such as in neuropathic pain. - Chez les mammifères, la présence de cellules excitables dans les muscles, le coeur et le système nerveux est cruciale; elle permet la conduction rapide de nombreuses informations sur de longues distances grâce à la génération de potentiels d'action (PA). Les canaux sodiques voltage-dépendants (Navs) sont des participants importants dans la génération et la propagation des PA car ils sont responsables de la phase initiale de dépolarisation du PA. Les Navs sont des protéines hétéromériques composées d'une grande sous-unité a (formant le pore du canal) et de petites sous-unités ß accompagnatrices. Il existe dix gènes qui codent pour les canaux sodiques, du Nav 1.1 au Nav 1.9 ainsi que NaX, chacun possédant des propriétés biophysiques spécifiques. Les cellules excitables expriment différentes combinaisons des différents isoformes de Navs, qui engendrent une signature électrophysiologique distincte. Les Navs ne sont fonctionnels et ne participent à la conductibilité du Na+, que s'ils sont ancrés à la membrane plasmique. En plus des propriétés intrinsèques des Navs, de nombreuses protéines régulatrices influencent également le courant sodique. Certaines protéines vont favoriser l'ancrage et la stabilisation des Navs exprimés à la membrane, alors que d'autres vont plutôt favoriser leur internalisation. Maintenir l'équilibre des deux processus est crucial pour contrôler l'excitabilité cellulaire. Dans ce contexte, Nedd4-2, de la famille des E3 ubiquitin ligase, est une enzyme bien caractérisée qui régule l'internalisation de nombreuses protéines, notamment celle des Navs. Inversement, les sous-unités ß sont connues depuis longtemps pour stabiliser l'ancrage des Navs à la membrane. La douleur neuropathique périphérique est une condition débilitante résultant d'une atteinte à un nerf. Elle est caractérisée par la dérégulation des Navs exprimés dans les neurones sensoriels du ganglion spinal (DRG). Ceci a été démontré à de multiples occasions dans divers modèles animaux de douleur neuropathique. Parmi les Navs, Nav1.7 et Nav1.8 sont abondamment et spécifiquement exprimés dans les neurones sensoriels des DRG et ont été impliqués de façon récurrente dans le développement de la douleur neuropathique. En utilisant le modèle animal de douleur neuropathique d'épargne du nerf sural (spared nerve injury, SNI) chez la souris, j'ai observé une réduction spécifique des Nedd4-2 dans les neurones sensoriels du DRG. Cette diminution avait pour conséquence l'augmentation de l'expression des protéines et des courants de Nav 1.7 et Nav 1.8, respectivement dans l'axone et les neurones du DRG, et était donc suffisante pour créer l'hyperexcitabilité associée à la douleur neuropathique. L'invalidation pour le gène codant pour Nedd4-2 dans une lignée de souris génétiquement modifiées a conduit à de similaires augmentations de Nav1.7 et Nav1.8, parallèlement à une augmentation à la sensibilité thermique. A l'opposé, rétablir une expression normale de Nedd4-2 en utilisant un vecteur viral a eu pour effet de contrecarrer le développement de l'hypersensibilité mécanique lié à ce modèle de douleur neuropathique. Cette étude démontre le rôle important de Nedd4-2 dans la régulation de l'excitabilité cellulaire in vivo et son implication dans le développement des douleurs neuropathiques. Le rôle des sous-unités ß dans les douleurs neuropathiques a déjà été démontré dans notre groupe de recherche. A cause de leur rôle stabilisateur, l'augmentation des sous-unités ßl, ß2 et ß3 dans les DRG après SNI, conduit à une augmentation des Navs ancrés à la membrane. Dans mon travail de thèse, j'ai observé un nouveau mécanisme de régulation des sous-unités a par les sous-unités ß in vitro. Les sous-unités ßl et ß3 régulent l'état de glycosylation du canal Nav1.7, et stabilisent son expression membranaire. Ceci ouvre de nouvelles perspectives dans l'investigation de l'état de glycosylation des Navs dans des maladies impliquant les sous-unités ß, notamment les douleurs neuropathiques.

Effect of Vagus Nerve Stimulation in an Adult Patient with Dravet Syndrome: Contribution to Sudden Unexpected Death in Epilepsy Risk Reduction?.

We report on a patient who developed, from 5 months of age, multiple seizure types, including myoclonic, associated with severe psychomotor delay, leading to the diagnosis of Dravet syndrome. Over the years, he developed refractory epilepsy and was implanted with a vagus nerve stimulator at the age of 19. After 3 months, he experienced a progressive improvement of partial and generalized seizures, with a >90% reduction, and better alertness. This meaningful clinical improvement is discussed in the light of the sudden unexpected death in epilepsy risk, which is high in this setting, and seems remarkably diminished in our patient in view of the reduction of generalized convulsions.

Left recurrent laryngeal nerve palsy associated with silicosis.

Left recurrent laryngeal nerve palsy usually results from invasion or compression of the nerve caused by diseases localized within the aortopulmonary window. This study reports the case of a 76-yr-old male with vocal cord paralysis due to lymph node involvement by silicosis. This rare entity was identified by video-mediastinoscopy, which revealed a granulomatous and fibrosed recurrent lymph node encasing the nerve. The nerve was dissected and released from scar tissues. Progressive clinical improvement was observed followed by total and durable recovery of the voice after 15 weeks follow-up.

Fourth cranial nerve palsy and brown syndrome: two interrelated congenital cranial dysinnervation disorders?

Based on neuroimaging data showing absence of the trochlear nerve, congenital superior oblique palsy is now classified as a congenital cranial dysinnervation disorder. A similar absence of the abducens nerve is accompanied by misinnervation to the lateral rectus muscle from a branch of oculomotor nerve in the Duane retraction syndrome. This similarity raises the question of whether some cases of Brown syndrome could arise from a similar synkinesis between the inferior and superior oblique muscles in the setting of congenital superior oblique palsy. This hypothesis has gained support from the confluence of evidence from a number of independent studies. Using Duane syndrome as a model, we critically review the accumulating evidence that some cases of Brown syndrome are ultimately attributable to dysgenesis of the trochlear nerve.

Upregulation of the GABA transporter GAT-1 in the gracile nucleus in the spared nerve injury model of neuropathic pain.

Neuropathic pain is a major health issue and is frequently accompanied by allodynia (painful sensations in response to normally non-painful stimulations), and unpleasant paresthesia/dysesthesia, pointing to alterations in sensory pathways normally dedicated to the processing of non-nociceptive information. Interestingly, mounting evidence indicate that central glial cells are key players in allodynia, partly due to changes in the astrocytic capacity to scavenge extracellular glutamate and gamma-aminobutyric acid (GABA), through changes in their respective transporters (EAAT and GAT). In the present study, we investigated the glial changes occurring in the dorsal column nuclei, the major target of normally innocuous sensory information, in the rat spared nerve injury (SNI) model of neuropathic pain. We report that together with a robust microglial and astrocytic reaction in the ipsilateral gracile nucleus, the GABA transporter GAT-1 is upregulated with no change in GAT-3 or glutamate transporters. Furthermore, [(3)H] GABA reuptake on crude synaptosome preparation shows that transporter activity is functionally increased ipsilaterally in SNI rats. This GAT-1 upregulation appears evenly distributed in the gracile nucleus and colocalizes with astrocytic activation. Neither glial activation nor GAT-1 modulation was detected in the cuneate nucleus. Together, the present results point to GABA transport in the gracile nucleus as a putative therapeutic target against abnormal sensory perceptions related to neuropathic pain.

Intérêt de l'échographie du diamètre de l'enveloppe du nerf optique pour la détection non invasive de l'hypertension intracrânienne [Interest of optic nerve sheath diameter ultrasonography in dectecting non-invasively raised intracranial pressure].

Intracranial hypertension is an emergency suspected from clinical symptoms, imaging data and ophthalomologic signs. Intracranial hypertension is confirmed by invasive intracranial monitoring, which is the gold standard technique to measure intracranial pressure (ICP). Because of complications, hemorrhage or infection, non-invasive methods have been developed such as neuroimaging, transcranial Doppler sonography and optic nerve sheath diameter (ONSD) ultrasonography. We have reviewed ONSD technique that detects intracranial hypertension related volume variations of subarachnoid space along the retro bulbar segment of the optic nerve. Technique, indications and prospects are discussed.

Ulnar nerve transposition at the elbow : local anesthesia should be preferred to general anesthesia

Après la compression du nerf médian au niveau du tunnel carpien, la compression du nerf ulnaire au niveau du coude est le deuxième syndrome compressif le plus fréquent des nerfs périphériques. La chirurgie des nerfs périphériques consiste dans une décompression nerveuse et est caractérisée par un suivi post¬opératoire parfois très douloureux avec des douleurs qui peuvent chroniciser si insuffisamment traitées. Le traitement chirurgical de décompression nerveuse se fait traditionnellement sous anesthésie générale ou régionale. Une analgésie post-opératoire plus efficace et durable avec moindre risque de chronicisation avait justifié ce choix jusqu'à ce jour. Grâce au développement de la chirurgie ambulatoire ces dernières années, un grand nombre d'interventions chirurgicales au niveau de la main sont effectués sous anesthésie locale. Au vu d'une meilleure connaissance de cette technique d'anesthésie, son rôle dans la chirurgie des nerfs périphériques a été remis en question. Alors que plusieurs études ont démontré que l'anesthésie locale est aussi efficace que l'anesthésie générale et régionale au sujet de la chirurgie du tunnel carpien, son utilisation pour la chirurgie du nerf ulnaire reste peu connue. La raison de l'hésitation à l'utilisation de l'anesthésie locale pour le traitement du tunnel ulnaire est due au fait que dans plus de la moitié des cas, une simple décompression n'est pas suffisante et qu'il est souvent nécessaire de transposer le nerf ulnaire devant l'épicondyle ulnaire. La seule publication disponible au sujet de l'utilisation de l'anesthésie locale dans le traitement du tunnel ulnaire considère comme irréalisable d'utiliser cette méthode dans le cas d'une transposition. Malgré cette mise en garde, nous avons, depuis plusieurs années, des excellents résultats avec la transposition du nerf ulnaire sous anesthésie locale. Avec le but d'objectiver notre expérience dans ce domaine nous avons souhaité analyser nos résultats de façon rétrospective avec particulière attention aux douleurs post-opératoires et à la satisfaction des patients. Les dossiers de cinquante patients Consécutifs (26F, 24M) opérés par le même chirurgien dans notre service de 2002 à 2012 ont été analysés rétrospectivement. Les critères suivants ont été récoltés: l'âge du patient, la profession, la main dominante, les détails des techniques opératoires utilisées, le type d'anesthésie, l'intensité du suivi ainsi que les complications, le niveau de douleur dans l'immédiat post-opératorie ainsi que à une année de l'intervention. Les patients ont été divisés en 4 groupes: les opérés sous anesthésie générale avec transposition du nerf (n=17) ou sans transposition (n=10) et les opérés sous anesthésie locale avec transposition (n=12) ou sans transposition (n=11). Au premier jour la douleur était comparable dans tous les groupes. A une semaine, elle était deux fois plus importante lorsque la transposition avait été réalisée sous anesthésie générale par rapport à si une anesthésie locale avait été effectuée (p=0.03). La satisfaction s'est révélée plus élevée mais non significative chez les patients opérés sous anesthésie locale. Ces derniers étaient significativement plus enclins à répéter la chirurgie comparé a ceux opérés sous anesthésie générale (p=0.04). En conclusion, les résultats de cette étude suggèrent que l'anesthésie locale est au moins autant efficace que l'anesthésie générale en termes de complications et de douleurs post-opératoires indépendamment du fait qu'une transposition nerveuse soit effectuée ou pas. Un meilleur contrôle des douleurs à une semaine post-opératoire a contribué à une haute satisfaction des patients de notre étude.

Vagus nerve stimulator treatment in adult-onset Rasmussen's encephalitis.

We describe a patient with adult-onset Rasmussen's encephalitis (RE) responsive to vagus nerve stimulation. This previously healthy woman developed RE in the right hemisphere at the age of 27. Despite antiepileptic drug polytherapy, she continued to experience subcontinuous, simple-partial left-sided motor seizures and slowly progressive cognitive impairment. Resective surgery was not considered owing to the preservation of left motor skills. She was implanted with a vagus nerve stimulator at the age of 41; after 6 months she experienced a greater than 50% reduction in seizure frequency, which persisted over 2 years together with improvement of her neurological and cognitive status.

Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures.

The addition of nerve growth factor (2.5S NGF) to serum-free aggregating cell cultures of fetal rat telencephalon greatly stimulated the developmental increase in choline acetyltransferase activity. Two other neuronal enzymes, acetylcholinesterase and glutamic acid decarboxylase, showed only slightly increased activities after NGF treatment whereas the total protein content of the cultures and the activity of 2',3'- cyclic nucleotide phosphodiesterase remained unchanged. The stimulation of choline acetyltransferase was dependent on the NGF media concentrations, showing a 50% maximum effect (120% increase) at approximately 3 ng/ml (10-10 M 2.5S NGF). NGF treatments during different culture periods showed that the cholinergic neurons remained responsive for at least 19 days. The continued treatment was the most effective; however, an initial treatment for only 5 days still caused a significant stimulation of choline acetyltransferase on day 19. The observed stimulation appeared to be specific to NGF. Univalent antibody fragments (Fab) against 2.5S NGF completely abolished the NGF-dependent increase in choline acetyltransferase activity, whereas Fab fragments of control IgG were ineffective. Furthermore, angiotensin II, added in high amounts to the cultures, showed no stimulatory effect. The present results suggest that certain populations of rat brain neurons are responsive to nerve growth factor.