Ce travail a été soutenu par le NIH subventions K08NS065157 (à TAF) En outre, le Centre de Penn pour les troubles musculo-squelettiques, P30AR050950 Nombre Prix de l&#39;Institut national de l&#39;arthrite, les maladies musculo-squelettiques et la peau ont soutenu ce travail (TAF et Steven S. Scherer). Enfin, les Shriners de financement pédiatrique semences Research Center (TAF) a soutenu ce travail. Nous tenons à remercier le Dr Young-Jin Fils pour démontrer d&#39;abord toute la montagne procédure et Amy A. Kim pour son aide dans la production des croquis de la figure 1.

<ol>
	<li>Pan, Y. A., Misgeld, T., Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+neurotoxic+and+neuroprotective+agents+on+peripheral+nerve+regeneration+assayed+by+time-lapse+imaging+in+vivo.">Effects of neurotoxic and neuroprotective agents on peripheral nerve regeneration assayed by time-lapse imaging in vivo.</a> J. Neurosci. 23, 11479-11488 (2003).</li><li>Magill, C., Tong, A., Kawamura, D., Hayashi, A., Hunter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinnervation+of+the+tibialis+anterior+following+sciatic+nerve+crush+injury:+A+confocal+microscopic+study+in+transgenic+mice.">Reinnervation of the tibialis anterior following sciatic nerve crush injury: A confocal microscopic study in transgenic mice.</a> Exp. Neurol. 207, 64-74 (2007).</li><li>Amado, S., Sim&#241;es, M. J., Armada-da-Silva, P. A. S., Lu&#237;s, A. L., Shirosaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hybrid+chitosan+membranes+and+N1E-115+cells+for+promoting+nerve+regeneration+in+an+axonotmesis+rat+model.">Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model.</a> Biomaterials. 29, 4409-4419 (2008).</li><li>Lu&#237;s, A. L., Rodrigues, J. M., Geuna, S., Amado, S., Sim&#240;es, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+cell+transplantation+effects+on+sciatic+nerve+regeneration+after+a+standardized+crush+injury+in+the+rat.">Neural cell transplantation effects on sciatic nerve regeneration after a standardized crush injury in the rat.</a> Microsurgery. 28, 458-470 (2008).</li><li>Lu&#237;s, A. L., Amado, S., Geuna, S., Rodrigues, J. M., Sim&#240;es, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+functional+and+morphological+assessment+of+a+standardized+rat+sciatic+nerve+crush+injury+with+a+non-serrated+clamp.">Long-term functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.</a> J. Neurosci. Methods. 163, 92-104 (2007).</li><li>Baptista, A. F., de Souza Gomes, J. R., Oliveira, J. T., Santos, S. M. G., Vannier-Santos, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+assess+function+after+sciatic+nerve+lesion+in+the+mouse+-+adaptation+of+the+sciatic+static+index.">A new approach to assess function after sciatic nerve lesion in the mouse - adaptation of the sciatic static index.</a> J. Neurosci. Methods. 161, 259-264 (2007).</li><li>Ronchi, G., Raimondo, S., Varej&#227;o, A. S. P., Tos, P., Perroteau, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+crush+injury+of+the+mouse+median+nerve.">Standardized crush injury of the mouse median nerve.</a> J. Neurosci. Methods. 188, 71-75 (2010).</li><li>Berg, A., Zelano, J., Cullheim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrin+G-2+ligand+mRNA+is+downregulated+in+spinal+motoneurons+after+sciatic+nerve+lesion.">Netrin G-2 ligand mRNA is downregulated in spinal motoneurons after sciatic nerve lesion.</a> Neuroreport. 21, 782-785 (2010).</li><li>Girolami, E. I., Bouhy, D., Haber, M., Johnson, H., David, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+potential+role+of+SOCS1+and+SOCS3+in+Wallerian+degeneration+in+injured+peripheral+nerve.">Differential expression and potential role of SOCS1 and SOCS3 in Wallerian degeneration in injured peripheral nerve.</a> Exp. Neurol. 223, 173-182 (2010).</li><li>Hossain-Ibrahim, M. K., Rezajooi, K., Stallcup, W. B., Lieberman, A. R., Anderson, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+axonal+regeneration+in+the+central+and+peripheral+nervous+systems+of+the+NG2-deficient+mouse.">Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse.</a> BMC Neurosci. 8, 80-80 (2007).</li><li>Thornton, M. R., Mantovani, C., Birchall, M. A., Terenghi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+N-CAM+and+N-cadherin+expression+in+axotomized+and+crushed+rat+sciatic+nerve.">Quantification of N-CAM and N-cadherin expression in axotomized and crushed rat sciatic nerve.</a> J. Anat. 206, 69-78 (2005).</li><li>Beer, G. M., Steurer, J., Meyer, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardizing+nerve+crushes+with+a+non-serrated+clamp.">Standardizing nerve crushes with a non-serrated clamp.</a> J. Reconstr. Microsurg. 17, 531-534 (2001).</li></ol>

Nous n&#39;avons rien à communiquer.

Nous avons présenté une méthode pour obtenir un écrasement du nerf sciatique avec fiabilité complète marquage précis du site d&#39;écrasement. Tel que mentionné précédemment, écrasement du nerf sciatique est un modèle commun de lésion du nerf périphérique chez la souris et les rats. Bien que chaque méthode d&#39;écrasement a ses avantages et ses inconvénients, nous avons trouvé cette méthode produit un écrasement complet qui a été facile de marquer avec un minimum d&#39;équipements spéciaux (p....

<table border="1"><tbody><tr><td> Nom (description / quantité) </td><td> Fournisseur </td><td> Numéro de catalogue </td></tr><tr><td> Clipper Mini n ° 0000 de la lame </td><td> Roboz </td><td> RC-5903 </td></tr><tr><td> Hair Remover Nair (9 onces) </td><td> Eglise et Dwight Co., Inc </td><td> N / A </td></tr><tr><td> Betadine scrub chirurgical (1 gallon) </td><td> Fisher Scientific </td><td> 19066452 </td></tr><tr><td> Pommade ophtalmique (1 oz) </td><td> Fisher Scientific </td><td> 19082795 </td></tr></tbody></table> Outils chirurgicaux: <table border="1"><tbody><tr><td> Nom (description / quantité) </td><td> Fournisseur </td><td> Numéro de catalogue </td></tr><tr><td> FST 250 Hot stérilisateur à billes </td><td> Outils belle science </td><td> 18000-45 </td> &lt;/ Tr&gt; <tr><td> Ciseaux Iris (11 cm de long) </td><td> Instruments de précision du monde </td><td> 500216 </td></tr><tr><td> Potts-Smith forceps (droite; 18cm; 1x2 dents) </td><td> Outils belle science </td><td> 11024-18 </td></tr><tr><td> McPherson Vannas Ciseaux (5 lames mm) </td><td> Instruments de précision du monde </td><td> 14124-G </td></tr><tr><td> Dumont # 5 - Pince Dumoxel standard Tip </td><td> Outils belle science </td><td> 11252-30 </td></tr><tr><td> Dumont # 5/45 - Pince Dumoxel standard Tip </td><td> Outils belle science </td><td> 11251-35 </td></tr><tr><td> Ultra fin (cylindrique hémostatiques; surfaces intérieures lisses) </td><td> Outils belle science </td><td> 13020-12 </td></tr><tr><td> Charbon actif en poudre (500 g) </td><td> Fisher Scientific </td><td> C272-500 </td></tr><tr><td> Taille 6-0 sutures avec C-1 aiguille (stérile, de la soie, noir, tressé,Non-absorbable; 18 &quot;Longueur; Boîte de 36) </td><td> Roboz </td><td> SUT-1073-1011 </td></tr><tr><td> Applicateur de clips Reflex (pour 9 clips mm) </td><td> Instruments de précision du monde </td><td> 500345 </td></tr><tr><td> 9mm en acier inoxydable Clips Reflex (100/boîte) </td><td> Instruments de précision du monde </td><td> 500346 </td></tr></tbody></table> Protection des animaux: <table border="1"><tbody><tr><td> Nom (description / quantité) </td><td> Fournisseur </td><td> Numéro de catalogue </td></tr><tr><td> Injectable à 0,9% de chlorure de sodium (agent de conservation; 20 ml) </td><td> Hospira </td><td> 0409-4888-20 </td></tr><tr><td> Système complet de couverture homéothermes avec sonde flexible (moyenne, 115 VAC, 60 Hz) </td><td> Harvard Apparatus </td><td> 507222F </td></tr></tbody></table> Platfor chirurgicalem et écarteurs: (Acheté chez votre quincaillerie locale) <table border="1"><tbody><tr><td> Nom (description /) </td><td> Quantité </td></tr><tr><td> Plate-forme en acier inoxydable (usiné; ~ 12 &quot;x12&quot; x1 / 8 &quot;) </td><td> 1 </td></tr><tr><td> Aimants bouton (Exemple: Eclipse E825) </td><td> 3 </td></tr><tr><td> Boulons en acier inoxydable (3 &quot;long; diamètre déterminé par aimants bouton) </td><td> 3 </td></tr><tr><td> Écrous en acier inoxydable (dimensionnée pour s&#39;adapter à Boulons) </td><td> 9 </td></tr><tr><td> Elastiques (légère tension) </td><td> 3 </td></tr><tr><td> Pins d&#39;insectes (les deux extrémités pour former des crochets Bent) </td><td> 3 </td></tr></tbody></table> Semi-Minces: <table border="1"><tbody><tr><td> Nom (description / quantité) </td><td> Fournisseur &lt;/ Strong&gt; </td><td> Numéro de catalogue </td></tr><tr><td> Paraformaldéhyde (1 kg) </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Phosphate de sodium dibasique anhydre (500 g; Utilisé pour Préparer un tampon phosphate) </td><td> Fisher Scientific </td><td> S375-500 </td></tr><tr><td> Phosphate de sodium monobasique anhydre (1 kg; Utilisé pour Préparer un tampon phosphate) </td><td> Fisher Scientific </td><td> AC38987-0010 </td></tr><tr><td> Glutaraldéhyde (50%; 10 x 10 ml) </td><td> Ted Pella, Inc </td><td> 18431 </td></tr><tr><td> Tétroxyde d&#39;osmium (aqueuse à 4%, 10 x 10 ml) </td><td> Ted Pella, Inc </td><td> 18465 </td></tr><tr><td> Oxyde de Propylène (450 ml) </td><td> Ted Pella, Inc </td><td> 18601 </td></tr><tr><td> Embed 812 (Kit, pour les blocs durs ou de contraste d&#39;image élevée) </td><td> Sciences Electron Microscopy </td><td> 14120 </td></tr><tr><td> Toluidine Bleu (25 g) </td><td> Ted Pella, Inc </td><td> 19451 </td></tr></tbody></table> Whole Préparation musculaire mont et immunohistochimie: <table border="1"><tbody><tr><td> Nom (description / quantité) </td><td> Fournisseur </td><td> Numéro de catalogue </td></tr><tr><td> Paraformaldéhyde (1 kg) </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Phosphate de sodium dibasique anhydre (500 g; Utilisé pour Préparer un tampon phosphate) </td><td> Fisher Scientific </td><td> S375-500 </td></tr><tr><td> Phosphate de sodium monobasique anhydre (1 kg; Utilisé pour Préparer un tampon phosphate) </td><td> Fisher Scientific </td><td> AC38987-0010 </td></tr><tr><td> 10% de diluant BSA / solution de blocage (200 ml) </td><td> Kirkegaard &amp; Perry Laboratories, Inc </td><td> 50-61-00 </td></tr><tr><td> Triton X-100 (100 mL) </td&gt; <td> Dot scientifique Incorporated </td><td> 9002-93-1 </td></tr><tr><td> Glycine, 98% (1 kg) </td><td> Fisher Scientific </td><td> AC12007-0010 </td></tr><tr><td> Tissue-Tek Cryo-OCT composé (Caisse de 12, 4 bouteilles oz) </td><td> Fisher Scientific </td><td> 14-373-65 </td></tr><tr><td> Sylgard DOW 170 (2 Kit lb) </td><td> Fisher Scientific </td><td> NC9492579 </td></tr><tr><td> Inoxydable Pins insectes en acier, taille 1 (100/PKG) </td><td> Outils belle science </td><td> 26001-40 </td></tr><tr><td> Tétraméthylrhodamine-A-bungarotoxine (0,5 mg) </td><td> Sigma-Aldrich </td><td> T0195-.5MG </td></tr><tr><td> Anticorps monoclonal de souris contre SMI-312 (0,1 mL) </td><td> Covance </td><td> SMI-312R </td></tr><tr><td> Anticorps monoclonal de souris contre SV2 (0,1 ml) </td><td> Des études de développement de la Banque d&#39;hybridome (DSHB) </td><td> SV2 </td></tr><tr><td> Lapin PolyAnticorps contre clonale GAP-43 </td><td> Novus Biologicals </td><td> NB300-143 </td></tr><tr><td> Flourescein conjugué de chèvre anti-IgG de souris, sous-classe 1 Fcy spécifique </td><td> Jackson ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=115-095-205&amp;Attributes=Yes&amp;Quantity=1">11 5-095-205</a> </td></tr><tr><td> DyLight 649 conjugué d&#39;âne anti-IgG de lapin </td><td> Jackson ImmunoResearch </td><td> <a href="http://www.jacksonimmuno.com/MERCHANT2/merchant.mv?Screen=BASK&amp;Store_Code=JI&amp;Action=ADPR&amp;Product_Code=711-495-152&amp;Attributes=Yes&amp;Quantity=1">711-495-152</a> </td></tr><tr><td> 4 &#39;,6-diamidino-2-phénylindole (DAPI, dilactate; 10 mg) </td><td> Invitrogen </td><td> D3571 </td&gt; </tr><tr><td> Vectashield Milieu de montage (10 ml) </td><td> Vector Laboratories </td><td> H-1000 </td></tr><tr><td> Superfrost Plus Lames de microscope (blanc; Taille: 75 x 25 mm, paquet de 144) </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Fisherfinest Lunettes de couverture Premium (Taille: 40 x 22 mm; Pack de 1 oz.) </td><td> Fisher Scientific </td><td> 12-548-5C </td></tr></tbody></table>

reproducible mouse sciatic nerve crush and subsequent assessment of regeneration by whole mount muscle analysis

La régénération dans le système nerveux périphérique (SNP) est largement étudié à la fois pour sa pertinence à la maladie humaine et de comprendre la réponse robuste régénérative montée par les neurones du SNP ce qui pourrait éclairant les échecs de la régénération du SNC 1. Écrasement du nerf sciatique (axonotmésis) est l&#39;un des modèles les plus courants de lésion du nerf périphérique chez les rongeurs 2. Broyage interrompt tous les axones, mais lames de cellules de Schwann basale sont conservés afin que la régénération est optimal 3,4. Cela permet à l&#39;enquêteur d&#39;étudier précisément la capacité d&#39;un axone en croissance pour interagir avec la cellule de Schwann et lames basales 4. Les rats ont généralement été les modèles animaux préférés pour écrasement du nerf expérimentale. Ils sont largement disponibles et leur sang-froid lésé sciatique fournit une approximation raisonnable de lésions nerveuses humaines 5,4. Bien que plus petit en taille que chez le rat nerf, le nerf de la souris a de nombreuses qualités similaires. Mais surtout, énormemodèles électroniques sont de plus en plus précieux en raison de la grande disponibilité de lignées transgéniques permet maintenant une dissection détaillée des molécules individuelles critiques pour la régénération du nerf 6, 7. Enquêteurs antérieures ont utilisé de multiples méthodes pour produire un écrasement du nerf ou d&#39;une blessure, y compris de simples pinces angulaires, pinces, pinces hémostatiques réfrigérés, pinces vasculaires, et les colliers enquêteur conçus 8,9,10,11,12. Les enquêteurs ont également utilisé diverses méthodes de marquage du site, y compris des blessures de suture, des particules de carbone et de billes fluorescentes 13,14,1. Nous décrivons notre méthode pour obtenir un écrasement du nerf sciatique reproductible complète des informations exactes et persistante de marquage de l&#39;écrasement site à l&#39;aide d&#39;une pince fine hémostatiques et de carbone suite à l&#39;écrasement du site de marquage. Dans le cadre de notre description de la procédure écrasement du nerf sciatique, nous avons également inclus une méthode relativement simple de l&#39;ensemble des muscles que nous utilisons pour monter ensuite de quantifier la régénération.

Watch this Scientific Journal Video about Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis at JoVE.com

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Dans ce rapport, nous décrivons une méthode pour écraser la souris nerf sciatique. Cette méthode utilise facilement disponibles pinces hémostatiques et facilement et de façon reproductible produit complet écrasement du nerf sciatique. En outre, nous décrivons une méthode pour préparer montures de muscles entiers appropriés pour l&#39;analyse de la régénération nerveuse après écrasement du nerf sciatique.

Reproductible écrasement du nerf sciatique de souris et l&#39;évaluation subséquente de la régénération par l&#39;analyse du Mont muscle entier

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

reproducible-mouse-sciatic-nerve-crush-subsequent-assessment

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JoVE Journal

Neuroscience

33.4K vues.  Temple University. Dans ce rapport, nous décrivons une méthode pour écraser la souris nerf sciatique. Cette méthode utilise facilement disponibles pinces hémostatiques et facilement et de façon reproductible produit complet écrasement du nerf sciatique. En outre, nous décrivons une méthode pour préparer montures de muscles entiers appropriés pour l'analyse de la régénération nerveuse après écrasement du nerf sciatique.

Vidéo: Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Isolement axoplasme du nerf sciatique du rat

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

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Neurosciences

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

Préparation Mont entiers de l&#39;Adulte Drosophile Chaîne nerveuse ventrale pour injection fibre Dye Géant

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Dissection du muscle Transversus Abdominis pour l’analyse de jonction neuromusculaire à montage entier

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Whole-monter imagerie de l&#39;embryon de souris projections sensorielles Axon

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Mont entiers Dissection et immunofluorescence de la cochlée de souris adultes

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

In Vivo Transfert de gènes à des cellules de Schwann dans le nerf sciatique Rongeur par électroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Analyse cinématique 3D pour l’évaluation fonctionnelle dans le modèle de rat de la blessure d’écrasement de nerf sciatique

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Coloration, visualisation et analyse en monture entière des papilles gustatives fongiformes, circonvallates et palatines

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

Évaluation multifactorielle du comportement moteur chez les rats après une blessure unilatérale d’écrasement du nerf sciatique

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

Dissection des fibres musculaires squelettiques simples pour les analyses immunofluorescentes et morphométriques des jonctions neuromusculaires de mont entier

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...