Ce protocole permet l&#39;isolement axoplasme avec minimisation de la contamination tissu glial et vasculaire. La méthode des rendements d&#39;environ 70 à 100 mg de protéines totales par axoplasme une semaine de 80 à 10 Wistar vieux rat. 1. Disséquer nerfs sciatiques <ol><li> Euthanasier deux rats par inhalation de CO 2 suivie par dislocation cervicale. Confirmer la mort par la palpation de l&#39;absence de battement avant la dissection début. </li><li> Nettoyer la zone de dissection avec 70% d&#39;éthanol. </li><li> Coupez la peau, les muscles séparer et isoler nerf sciatique en utilisant des ciseaux et des pinces attentivement, sans endommager les vaisseaux sanguins. Disséquer les deux nerfs sciatiques (environ 1,5 cm chacune) de chaque animal et de collecter toutes les quatre nerfs dans un eppendorf de 500 pl de PBS 0,2 X + inhibiteurs, conservés sur la glace. </li><li> Transférer le culot de segments plats en plastique contenant au moins 2 mL de PBS inhibiteurs de protéase 0,2 x +. </li><li> Retirez l&#39;épinèvre des nerfs à l&#39;aide de pinces fines dans le champ binoculaire. Séparez les fascicules très soigneusement jusqu&#39;à ce qu&#39;ils deviennent troubles et commencent à flotter sur la surface de solution. Cette étape devrait être pratiqué jusqu&#39;à ce qu&#39;il puisse être effectué rapidement et avec précision (ne pas prendre plus de 10 minutes par nerf). </li></ol> 2. L&#39;incubation, lavage et d&#39;élution <ol><li> Transfert séparés fascicules d&#39;un nouveau tube Eppendorf avec 500 ul de PBS contenant 0,2 x inhibiteurs de protéase pour une incubation de 2 h à température ambiante. </li><li> Laver au moins 3 fois avec 1 ml du même tampon en transférant les fascicules du tube Eppendorf à tube Eppendorf et agiter pendant 5 min après le transfert. </li><li> Pour enlever l&#39;excès de liquide mis les fascicules dans un tube eppendorf de nouveaux vides et puis de transférer les fascicules d&#39;un nouveau tube Eppendorf avec 300 ul de PBS 1X contenant inhibiteur de protéase pour une incubation pendant 20-30 min à température ambiante. </li><li> Centrifuger 10 min à 10000 xg à 4 ° C. </li><li> Prenez le surnageant et mesurer la concentration de protéines. Ceci est votre préparation axoplasme. </li></ol> 3. Les résultats représentatifs <img src="/files/ftp_upload/2087/2087fig1.jpg" alt="Figure 1" /> Figure 1. Axoplasme Western blot comparant isolé par la méthode manuelle serrer avec des échantillons isolés axoplasme comme indiqué dans ce protocole. Notez la réduction des niveaux d&#39;albumine et de la GFAP, protéine du sang qui représente et les contaminations de cellules gliales, respectivement. En revanche, la tubuline axonale b3 est enrichi dans cette préparation, indiquant un niveau élevé de protéines axonales.

<ol>
	<li>Hanz, S., Perlson, E., Willis, D., Zheng, J. Q., Massarwa, R., Huerta, J. J., Koltzenburg, M., Kohler, M., van-Minnen, J., Twiss, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axoplasmic+importins+enable+retrograde+injury+signaling+in+lesioned+nerve.">Axoplasmic importins enable retrograde injury signaling in lesioned nerve.</a> Neuron. 40, 1095-1104 (2003).</li><li>Michaelevski, I., Medzihradszky, K. F., Lynn, A., Burlingame, A. L., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+proteomics+reveals+mobilization+of+translation+machinery+to+the+lesion+site+in+injured+sciatic+nerve.">Axonal transport proteomics reveals mobilization of translation machinery to the lesion site in injured sciatic nerve.</a> Mol Cell Proteomics. 9 (5), 976-987 (2010).</li><li>Rishal, I., Michaelevski, I., Rozenbaum, M., Shinder, V., Medzihradszky, K. F., Burlingame, A. L., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axoplasm+isolation+from+peripheral+nerve.">Axoplasm isolation from peripheral nerve.</a> Dev Neurobiol. 70, 126-133 (2010).</li><li>Twiss, J. L., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomes+in+axons--scrounging+from+the+neighbors.">Ribosomes in axons--scrounging from the neighbors.</a> Trends Cell Biol. 19, 236-243 (2009).</li></ol>

Les analyses biochimiques et protéomiques ont confirmé que cette procédure réduit la contamination de cellules gliales sérum et 3 par rapport aux méthodes décrites précédemment pour l&#39;isolement axoplasme de Squeeze mécanique 1. Nous avons utilisé ce protocole d&#39;isolement axoplasme à explorer dynéine basé signalisation rétrograde après lésion du nerf sciatique 2, et s&#39;attendre à ce qu&#39;il ont une utilité dans l&#39;exploration de nombreux autres processus...

<ul><li> Des rats Wistar mâles (8-10 semaines) </li><li> PBS 0,2 X et PBS 1X contenant des solutions inhibiteurs de protéase (Roshe) 2 comprimés par 50 ml </li><li> Eppendorfs pour 1,5 ml </li><li> Stérilisé plats en plastique de 35 mm </li><li> Des outils de dissection, y compris: des ciseaux et des pinces fines </li><li> La lumière binoculaire et la table </li></ul> Anticorps Souris anti-GFAP clone GA-5 a été de Sigma (G6171). Lapin anti-albumine a été d&#39;Cedarlane (CLAG5140). Souris anti-tubuline β3 et de lapin anti-Gerk provenaient de chez Sigma (T2200 et M5670, respectivement).

axoplasm isolation from rat sciatic nerve

Isolement du cytoplasme axonal pur (axoplasme) de nerf périphérique est cruciale pour les études biochimiques de nombreux processus biologiques. Dans cet article, nous démontrons et décrire un protocole pour l&#39;isolement du nerf axoplasme sciatique de rat adulte sur la base des étapes suivantes: (1) la dissection des fascicules nerveux et la séparation des tissus conjonctifs; (2) incubation de courts segments de fascicules nerveux au milieu hypotonique pour libérer de la myéline et Lyse non axonale des structures, et (3) l&#39;extraction de l&#39;axone reste-matériau enrichi. Caractérisation protéomique et biochimique de cette préparation a confirmé un haut degré d&#39;enrichissement pour les composants axonale.

Watch this Scientific Journal Video about Axoplasm Isolation from Rat Sciatic Nerve at JoVE.com

Axoplasm Isolation from Rat Sciatic Nerve

Nous démontrons un protocole pour l&#39;isolement du nerf axoplasme sciatique de rat adulte repose sur la dissection des fascicules nerveux et l&#39;incubation dans le milieu hypotonique pour libérer de la myéline et Lyse non axonale structures, suivie de l&#39;extraction de l&#39;axone reste-matériau enrichi.

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 ...

axoplasm-isolation-from-rat-sciatic-nerve

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Neuroscience

13.5K vues.  Weizmann Institute of Science. Nous démontrons un protocole pour l'isolement du nerf axoplasme sciatique de rat adulte repose sur la dissection des fascicules nerveux et l'incubation dans le milieu hypotonique pour libérer de la myéline et Lyse non axonale structures, suivie de l'extraction de l'axone reste-matériau enrichi.

Vidéo: Axoplasm Isolation from Rat Sciatic Nerve

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

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

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 ...

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Neurosciences

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolement et culture de cellules embryonnaires de rat neurones: un protocole rapide

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

En Vivo mesures électrophysiologiques sur les nerfs sciatiques souris

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Technique mini-invasive pour injection dans Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

Enregistrement Potentiel évoqué visuel dans un modèle de rat of Experimental Optic Nerve démyélinisation

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

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 ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

In Vivo Mesures électrophysiologiques du nerf cubital Rat avec test d’excitabilité axonale

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Analyse des cellules immunitaires dans unique des nerfs sciatique et Ganglion de la racine dorsale d’une souris à l’aide de cytométrie en flux

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

Chirurgie du nerf facial dans le modèle de rat pour étudier l’inhibition et la régénération axonales

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Utilisation de l'enregistrement in vivo à fibre unique et du ganglion de racine dorsal intact avec le nerf sciatique attaché pour examiner le mécanisme de l'échec de conduction

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...