第806回 千葉医学会・第10回 歯科口腔外科例会 22.

Lack of CNS demyelination in LPS-injected Cx32 mutant mice. a-l: Images of brain sections at the level of the corpus callosum immunostained with axonal marker RT97 (green) in combination with myelin marker MBP (red) (a-f) or with myelin marker MOG (g-l) and DAPI nuclear staining (blue). There is no apparent alteration of myelin immunoreactivity in the brains of LPS-injected mice (b, d, f, h, I, l) compared to saline controls (a, c, e, g, j, k) from all three genotypes, as indicated. Scale bar: 50 Οm. m-o: Immunoblot analysis of MBP levels in brainstem tissue lysates show no significant change induced by LPS injection (LPS) compared to saline-injected mice (S) in WT (m), Cx32 KO (n) or KO T55I (o) groups as indicated. All blots were re-probed for tubulin to demonstrate the loading, and quantification of normalized MBP band intensity is shown next to each blot. (TIF 19222 kb

AAV9-mediated Schwann cell-targeted gene therapy rescues a model of demyelinating neuropathy

Funder: The Republic of Cyprus through the Research and Innovation Foundation Foundation (Project: CULTURE/BR-NE/0416/07)Funder: Wallenberg Scholar and the fluid biomarker measurements in the lab of HZ and AJH were supported by the UK Dementia Research Institute at UCLAbstract: Mutations in the GJB1 gene, encoding the gap junction (GJ) protein connexin32 (Cx32), cause X-linked Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We developed a gene therapy approach for CMT1X using an AAV9 vector to deliver the GJB1/Cx32 gene under the myelin protein zero (Mpz) promoter for targeted expression in Schwann cells. Lumbar intrathecal injection of the AAV9-Mpz.GJB1 resulted in widespread biodistribution in the peripheral nervous system including lumbar roots, sciatic and femoral nerves, as well as in Cx32 expression in the paranodal non-compact myelin areas of myelinated fibers. A pre-, as well as post-onset treatment trial in Gjb1-null mice, demonstrated improved motor performance and sciatic nerve conduction velocities along with improved myelination and reduced inflammation in peripheral nerve tissues. Blood biomarker levels were also significantly ameliorated in treated mice. This study provides evidence that a clinically translatable AAV9-mediated gene therapy approach targeting Schwann cells could potentially treat CMT1X

The effect of stress conditions PN the function of the sciatic nerve of vertebrates

The peripheral nervous system (PNS) is the part of the nervous system that can undergo various types of stresses. Contrast to the central nervous system (CNS), the PNS is vulnerable and easily accessible to the various stress factors. The effects of PNS can be either chemical or mechanical and can be made through direct or indirect contact with the agent that causes stress. The effects on the PNS can have as objectives structural and functional components of the nerve fibres. This work focused on the study of various chemical and mechanical stress factors on electrophysiological properties of sciatic nerve of vertebrates. The study of chemical factors included local anaesthetics, anticancer drugs, insecticides, naturally produced toxins and neurotransmitters while the study of the mechanical stresses included: the expansion of nerve and its the regeneration.The report of the sciatic nerve from chemically produced local anaesthetics showed that the local anaesthetics reversible influence the compound action potential (CAP) through their action on the voltage-gated sodium channels (VGNaCs). Similar action had and linalool, a naturally derived chemical, which has demonstrated to have local anaesthetic properties compared to lidocaine and acting through deactivation of VGNaCs. Effects on voltage gated potassium channels (VGKCs) had oxaliplatin, a widely used anticancer drug, which presented a significant action in the CAP waveform with the appearance of secondary depolarizations. In contrast, another anticancer product was studied versus the oxaliplatin. It had no significant effects and only the CAP amplitude has been affected without presenting any changes in the CAP waveform. In addition, the insecticide rotenone was found to causes a reduction in the CAP amplitude and this was not connected with the production of free radicals or change in the structure of the microtubule system observed after exposure of the sciatic nerve to rotenone. Palytoxin, a naturally produced toxin caused reduction of CAP amplitude in significant low concentrations (nM) through the action on the Νa+/K+-ATPase- and the increase in the resting membrane action potential of the nerve fibres. In comparison, naturally produced palytoxin analogs were studied which again resulted in a reduction of CAP amplitude. Quantification of their action was also made. Also the action of γ-aminobutiric acid (GABA), a neurotransmitter of CNS, in the PNS was studied. GABA caused a reduction in the CAP amplitude which was reversed after pre-incubation of sciatic nerve with picrotoxin, an antagonist of GABA receptors. Further study clarifying how the GABA causes the reduction of CAP amplitude using intracellular recordings showed that GABA causes increase of the membrane action potential and therefore causing a decrease of the action potential of nerve fibre. Finally, in the context of chemical stress the role of the perineurium on the entrance of various chemical compounds in nerve fibers was also studied. The results showed that the perineurium is a type of selective barrier for the various compounds entering the nerve fibers. As mentioned two cases mechanical stress were studied: nerve expansion and nerve regeneration. The expansion of nerve showed that there are elastic components in nerve fibres capable of absorbing the forces acting on it up to a point where a gradual disruption begins. An increase of the expansion force applied to the nerve fibres caused a change to the electrophysiological parameters that were restored after the end of the expansion. Finally, the regeneration of sciatic nerve was studied after section and suturing of the nerve. The results showed that the nerve is capable to be functional after 6 months while after 3 months of regeneration the function of the regenerated nerve is oxygenate dependent. There were differences in the innervation pattern of the sciatic nerve branches after the operation indicated by the differences observed at the values of conduction velocity and the percentage that the branches represent. Combination of the two mechanical stresses showed that regenerated nerve has the ability to maintain its flexibility and to behave in a similar manner to the control.The results of this work give information on how different factors affect the PNS leading to neuropathy.Το περιφερικό νευρικό σύστημα (ΠΝΣ) είναι το τμήμα του νευρικού συστήματος το οποίο μπορεί να υποστεί ευκολότερα διαφόρων τύπων καταπονήσεις. Σε αντίθεση με το κεντρικό νευρικό σύστημα το ΠΝΣ είναι πιο ευάλωτο και πιο εύκολα προσβάσιμο στους διάφορους παράγοντες καταπόνησης. Οι καταπονήσεις του ΠΝΣ μπορεί να είναι είτε χημικές είτε μηχανικές και μπορεί να προέλθουν διαμέσου άμεσης ή έμμεσης επαφής με τον παράγοντα που προκαλεί την καταπόνηση. Οι καταπονήσεις του ΠΝΣ μπορεί να έχουν ως στόχους δομικά και λειτουργικά στοιχεία των νευρικών ινών. Η παρούσα εργασία εστίασε στη μελέτη διαφόρων χημικών και μηχανικών παραγόντων καταπόνησης στις ηλεκτροφυσιολογικές ιδιότητες του ισχιακού νεύρου σπονδυλωτών.Η μελέτη των χημικών καταπονήσεων περιλάμβανε τοπικά αναισθητικά, αντικαρκινικά φάρμακα, εντομοκτόνα, φυσικά παραγόμενες τοξίνες και νευροδιαβιβαστές και η μελέτη των μηχανικών καταπονήσεων περιλάμβανε τη διάταση και την αναγέννηση του νεύρου. Η έκθεση του ισχιακού νεύρου σε χημικά παραγόμενα τοπικά αναισθητικά έδειξε ότι τα τοπικά αναισθητικά επιδρούν με αναστρέψιμο τρόπο στο σύνθετο δυναμικό ενέργειας (ΣΔΕ) μέσω της δράσης τους στα τασεοελεγχόμενα κανάλια νατρίου (ΤΕΚΝa). Παραπλήσια δράση είχε και η λιναλόλη, μια φυσικά παραγόμενη ένωση, η οποία όπως αποδείχθηκε έχει ιδιότητες τοπικού αναισθητικού συγκρινόμενη με τη λιδοκαΐνη δρώντας κι αυτή μέσω της απενεργοποίησης των ΤΕΚΝa. Δράση στα τασεοελεγχόμενα κανάλια καλίου (ΤΕΚΚ) βρέθηκε να έχει η οξαλιπλατίνα, ένα ευρέως χρησιμοποιούμενο αντικαρκινικό φάρμακο, η οποία παρουσίασε μια χαρακτηριστική δράση στη μορφή του ΣΔΕ με την εμφάνιση δευτερευουσών εκπολώσεων. Αντίθετα, ένα ακόμη αντικαρκινικό φάρμακο το οποίο μελετηθήκε σε αντιπαραβολή με την οξαλιπλατίνα είχε αμελητέα επίδραση στο ύψος μόνο του ΣΔΕ χωρίς να παρουσιάσει οποιεσδήποτε αλλαγές στη μορφή του ΣΔΕ. Επιπλέον, εξετάστηκε το εντομοκτόνο ροτενόνη, το οποίο στη παρούσα εργασία βρέθηκε ότι προκαλεί μείωση του ύψους του ΣΔΕ η οποία όμως δεν συνδέεται με την παραγωγή ελευθέρων ριζών ή την μεταβολή στη δομή των μικροσωληνίσκων η οποία παρατηρείται μετά από έκθεση του ισχιακού νεύρου σε ροτενόνη. Η παλιτοξίνη με τη σειρά της, μια φυσικά παραγόμενη τοξίνη, προκάλεσε μείωση του ύψους του ΣΔΕ σε πολύ μικρές συγκεντρώσεις (nM) μέσω της δράσης της στην Νa+/K+-ATPάση και αύξηση του μεμβρανικού δυναμικού ηρεμίας των νευρικών ινών. Εξετάστηκαν επίσης φυσικά παραγόμενα ανάλογα παλιτοξίνης τα οποία και πάλι προκάλεσαν μείωση του ύψους του ΣΔΕ και έγινε ποσοτικοποίηση της δράσης τους. Μελετήθηκε επίσης η δράση του γ-αμινοβουτυρικού οξέος (GABA), ενός νευροδιαβιβαστή του κεντρικού νευρικού συστήματος, στο ΠΝΣ. Το GABA παρουσίασε ανασταλτική δράση στις νευρικές ίνες του ΠΝΣ προκαλώντας μείωση του ύψους του ΣΔΕ η οποία ήταν αναστρέψιμη μετά από προεπώαση του ισχιακού νεύρου με την πικροτοξίνη, έναν ανταγωνιστή των GABA υποδοχέων. Περαιτέρω μελέτη για διασαφήνιση του τρόπου με τον οποίο το GABA προκαλεί μείωση του ύψους του ΣΔΕ με τη βοήθεια ενδοκυτταρικών καταγραφών έδειξε ότι το GABA προκαλεί αύξηση του μεμβρανικού δυναμικού ηρεμίας προκαλώντας κατά συνέπεια και μείωση του ύψους του δυναμικού ενέργειας της νευρικής ίνας. Τέλος, στο πλαίσιο της χημικής καταπόνησης μελετήθηκε και ο ρόλος του περινευρίου στην είσοδο διαφόρων χημικών ενώσεων στις νευρικές ίνες. Τα αποτελέσματα έδειξαν ότι το περινεύριο αποτελεί ένα τύπο επιλεκτικού φραγμού για τις διάφορες ενώσεις που εισέρχονται στις νευρικές ίνες.Όπως αναφέρθηκε μελετήθηκαν και δύο περιπτώσεις μηχανικής καταπόνησης, η διάταση και η αναγέννηση. Η διάταση του νεύρου έδειξε ότι στο νεύρο υπάρχουν ελαστικά στοιχεία τα οποία μπορούν να απορροφούν τις δυνάμεις που ασκούνται σε αυτό μέχρι ενός σημείου όπου ξεκινάει η σταδιακή διάρρηξη των ελαστικών του στοιχείων. Αύξηση της τάσης που ασκείται προκαλεί αλλαγή στις ηλεκτροφυσιολογικές παραμέτρους των νευρικών ινών οι οποίες όμως επανέρχονται μετά την διακοπή της ασκούμενης τάσης. Τέλος, μελετήθηκε η αναγέννηση του ισχιακού νεύρου του επίμυος μετά από τομή του και επινευριακή επανασυρραφή. Τα αποτελέσματα έδειξαν ότι το νεύρο έχει την ικανότητα να επανέρχεται λειτουργικά μετά το πέρας 6 μηνών ενώ η λειτουργία του μετά το πέρας 3 μηνών από την επέμβαση εξαρτάται από την οξυγόνωση. Βρέθηκαν διαφορές στην εννεύρωση των κλάδων του ισχιακού μετά την επέμβαση που φαίνονται τόσο από τις τιμές της ταχύτητας αγωγής όσο και από το ποσοστό του συνολικού ΣΔΕ που αντιπροσωπεύουν. Συνδυασμός των δύο μηχανικών καταπονήσεων έδειξε ότι το αναγεννημένο νεύρο έχει την ικανότητα να διατηρεί την ελαστικότητά του και να συμπεριφέρεται με παρόμοιο τρόπο με το μάρτυρα. Τα αποτελέσματα της παρούσας εργασίας δίνουν στοιχεία για τον τρόπο δράσης διαφόρων παραγόντων καταπόνησης στο ΠΝΣ που επηρεάζουν τη λειτουργία του οδηγώντας σε νευροπάθειες

Gene replacement therapy in two Golgi-retained CMT1X mutants before and after the onset of demyelinating neuropathy

X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants

The Bite of the Honeybee: 2-Heptanone Secreted from Honeybee Mandibles during a Bite Acts as a Local Anaesthetic in Insects and Mammals

The research was directed and funded by Vita (Europe) Limited, UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors have read the journal's policy and have the following conflicts: the research was funded through Vita (Europe) Limited, a small UK company specialising only in honeybee health. MW works for this company. The original studies examined honeybee behaviour and they discovered the anaesthetisation properties of 2-H in the mammalian peripheral nervous systems as an extension to the honeybee biology work. Vita has now filed a patent on the potential use of 2-heptanone as a local anaesthetic (International Patent Application number: PCT/GB2012/050157). This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. All other authors have declared that no competing interests exist.We thank the Cypriot Union of Beekeepers for providing the specimens for the trials. We thank Evagelos Gikas for analytical advice, Dimitris Fotaroudis for the statistical support, ChanTest Corporation (Cleveland OH, US) for the support on patch-clamp experiments and Sharilynn Wardrop for reviewing the paper. We thank Anais Carpentier for the support during SEM photography, which benefited from the facilities and expertise of the Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Conseil General de l' Essonne.Honeybees secrete 2-heptanone (2-H) from their mandibular glands when they bite. Researchers have identified several possible functions: 2-H could act as an alarm pheromone to recruit guards and soldiers, it could act as a chemical marker, or it could have some other function. The actual role of 2-H in honeybee behaviour remains unresolved. In this study, we show that 2-H acts as an anaesthetic in small arthropods, such as wax moth larva (WML) and Varroa mites, which are paralysed after a honeybee bite. We demonstrated that honeybee mandibles can penetrate the cuticle of WML, introducing less than one nanolitre of 2-H into the WML open circulatory system and causing instantaneous anaesthetization that lasts for a few minutes. The first indication that 2-H acts as a local anaesthetic was that its effect on larval response, inhibition and recovery is very similar to that of lidocaine. We compared the inhibitory effects of 2-H and lidocaine on voltage-gated sodium channels. Although both compounds blocked the hNav1.6 and hNav1.2 channels, lidocaine was slightly more effective, 2.82 times, on hNav.6. In contrast, when the two compounds were tested using an ex vivo preparation-the isolated rat sciatic nerve-the function of the two compounds was so similar that we were able to definitively classify 2-H as a local anaesthetic. Using the same method, we showed that 2-H has the fastest inhibitory effect of all alkyl-ketones tested, including the isomers 3- and 4-heptanone. This suggests that natural selection may have favoured 2-H over other, similar compounds because of the associated fitness advantages it confers. Our results reveal a previously unknown role of 2-H in honeybee defensive behaviour and due to its minor neurotoxicity show potential for developing a new local anaesthetic from a natural product, which could be used in human and veterinary medicine.Vita (Europe) Limited, UK, Vita (Europe) Limited, ChanTest Corporation (Cleveland OH, US), Conseil General de l' Essonn

Additional file 2: Figure S2. of Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease

LPS causes diffuse CNS inflammation in the cerebellum and cerebral cortex. Images of fixed coronal sections of cerebellum (a-f) and cerebral cortex (g-l), as indicated, immunostained for microglial marker Iba1 (red). Cell nuclei are counterstained with DAPI (blue). Microglia are diffusely activated in both CNS areas and in all genotypes in LPS (b, d, f, h, j, l) compared to saline (a, c, e, g, i, k) injected mice. Scale bars: 50 Οm. (TIF 7715 kb

Additional file 12: Figure S10. of Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease

Lack of oligodendrocyte apoptosis after LPS-induced neuroinflammation in Cx32 mutant mice. Images of fixed longitudinal sections of cerebellar white matter double stained with apoptosis marker caspase-3 (red) along with oligodendrocyte marker CC1 (green) and counter stained with DAPI (blue). Caspase-3 immunoreactivity does not increase in LPS-tissues (b, d, f) compared to saline controls (a, c, e) in any of the three genotypes, indicating lack of oligodendrocyte apoptosis up to 1 week after LPS-induced inflammation. Scale bar: 50 Οm. (TIF 8299 kb

The effects of 2-heptanone and lidocaine on the isolated sciatic nerve of the rat.

<p>The effects of 2-heptanone and lidocaine on the isolated sciatic nerve of the rat.</p

The effect of 2-heptanone on hNav1.2 (a) and hNav1.6 (b) sodium channels

<p>. Data represent tonic (a and c) and phasic (b and d) effects. Solid curves represent when data are fitted with a logistic equation with minimum and maximum effects fixed at 0 and 100% (n = 6).</p