The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

Todos los experimentos se llevaron a cabo bajo las directrices del NIH y los animales protocolos aprobados por el Cuidado y Uso de Animales Institucional de Virginia Tech. 1. Los animales se preparan para la cirugía <ol><li> Anestesiar los ratones con una mezcla de ketamina (90 mg / kg) y xilazina (10 mg / kg) mediante inyección subcutánea inguinal con un 1 ml jeringa de insulina estéril. solución portadora contiene una mezcla de 0,9% de solución salina, 17,4 mg / ml de ketamina, y 2,6 mg / ml de xilazina. Coloque de nuevo los animales en jaulas a la espera de la medicación surta efecto. NOTA: Si la dosis de carga no proporciona anestesia suficientes para la duración del procedimiento, un 25% adicional de la dosis de carga se puede inyectar. </li><li> Seguir los animales después de la inyección para comprobar la frecuencia respiratoria y la depresión constante adecuada de los niveles de excitación. Comprobar el nivel de excitación con una pizca pata trasera, lo que debería provocar ninguna respuesta cuando anestesiado suficientemente. NOTA: Esto suele tardar 3-5min para un ratón adulto joven con un promedio de 25 a 30 g. Si el animal es todavía sensible después de la inyección posterior 10 min, un 25% adicional de la dosis de carga anestésico se puede inyectar. </li><li> Aplicar vaselina y aceite mineral ligero pomada oftálmica a los ojos del animal para evitar la sequedad. Retirar los animales de la jaula y el lugar sobre una superficie limpia y plana. Afeitar el miembro posterior deseada del pie a la pelvis usando un cortador de pelo eléctrico, exponiendo sólo el aspecto lateral de la extremidad. </li><li> Aplique un removedor de pelo química al sitio afeitado durante 1 min. quitar manualmente el cabello utilizando toallitas de laboratorio. Limpiar la zona depilada con el laboratorio trapo empapado en etanol. </li></ol> 2. Procedimiento Quirúrgico <ol><li> Esterilizar instrumentos quirúrgicos a través de autoclave u otro método apropiado. Limpiar la zona quirúrgica y tabla quirúrgica con un 80% de etanol / H 2 0. Desinfectar el sitio quirúrgico con proviodine. Coloque el ratón a bordo quirúrgica y se alinean con las restricciones de las extremidades. Guardarla extremidad posterior de destino en una posición anatómicamente natural con la articulación de la rodilla ligeramente extendida sin rotación interna o externa. </li><li> Lugar de los animales y la tabla bajo el microscopio quirúrgico. Orientar al sitio de la incisión adecuada mediante palpación de puntos superficiales, específicamente la articulación de la rodilla y de la cresta ósea entre los músculos gemelos y tibial anterior. </li><li> Hacer una incisión de aproximadamente 3 cm a través de la piel con un bisturí o tijeras de primavera, mientras que el uso de fórceps generales para el agarre. Hacer la incisión perpendicular al supuesto subyacente del nervio peroneo común. </li><li> Continuar la incisión a través de la fascia superficial, exponiendo el bíceps femoral y los músculos vasto lateral. Separar estos músculos mediante el corte a través de la fascia profunda conexión. Un 1-2 cm de corte debe ser suficiente. </li><li> Repliegue del músculo bíceps femoral caudal utilizando retractores mecánicos, dejando al descubierto el nervio peroneo común. </li><li> Trace el nervio proximal hasta su intersección con el tendón de la cabeza lateral del músculo gastrocnemio se encuentra. Nota: La exposición puede requerir la manipulación adicional de la piel y el músculo retraído. Esta intersección se usa como punto de referencia estable para la lesión del nervio. </li><li> Agarre el nervio con unas pinzas finas, alineando la punta de un modo paralelo al borde lateral del tendón del gastrocnemio. Aplastar el nervio peroneo común mediante la aplicación de presión constante, dura durante 5 segundos. </li><li> Corroborar completo aplastamiento del nervio mediante inspección visual a través del ámbito quirúrgico. Se aparecerá translúcida en el sitio de la lesión. Si el uso de ratones que expresan proteínas de fluorescencia en los axones periféricos, la fluorescencia desaparece del lugar de la lesión. </li><li> Retire retractores y realinear los músculos en sus posiciones anatómicas. Cerrar el sitio de la incisión con suturas de seda 6-0. 1-3 suturas interrumpidas simples son suficientes. Coloque recuperación del ratón sobre una almohadilla térmica en una jaula limpia. </li><li> Supervisión de todos los animales de 2 horas después de la óperación para comprobar la respiración y cualquier reacción adversa a la anestesia. Administrar una dosis inicial de buprenorfina 0,05-0,10 mg / kg mediante inyección subcutánea inguinal inmediatamente después de la recuperación de la cirugía. Dar a 3 dosis adicionales cada 12 h durante las siguientes 48 horas. Después de la recuperación total, los ratones regresar a las instalaciones de cuidado de animales. </li></ol> 3. El aislamiento y la tinción de extensor largo de los dedos (EDL) Músculos <ol><li> Sacrificar animales utilizando isoflurano. Coloque 0.5 ml isoflurano líquido en un tubo de 50 ml lleno de labwipes absorbentes. Se coloca el tubo destapado con el animal en una cámara de 2.500 cm 3 sellada. Al menos 4 min de exposición es suficiente. Prueba para la pérdida de palpebral bilateral, dedo del pie para los dedos, y los reflejos de estricción de rabo para asegurar que cada animal está inconsciente antes de proceder con la perfusión. </li><li> Transcardially perfundir 16 animales primero con 10 ml 0,1 M PBS, a continuación, 25 ml de paraformaldehído al 4% en PBS 0,1 M (pH 7,4). Heparina (30 unidades / 20g de peso del animal) se puede añadir con PBS (10 unidades / ml) para prevenir la coagulación de la sangre en pequeños lechos capilares, mejorar los resultados de perfusión. </li><li> Retire la piel que cubre extremidades traseras mediante el uso de tijeras para cortar transversalmente a través de la piel alrededor de la circunferencia del abdomen. Pelar abajo la piel más allá de las extremidades traseras y los pies con unas pinzas. </li><li> Retire la fascia superficial de las extremidades traseras agarrando y se pela con un fórceps. Si el uso de ratones que expresan proteínas de fluorescencia en los axones periféricos, posterior a solucionar los ratones toda la noche en PFA al 4% en tubos de 50 ml. Enjuague tres veces con PBS. NOTA: Se ha corregido los ratones pueden ser almacenados en PBS a 4 ° C. Si no es así, omita este paso y vaya al paso 3.6 y sin animales de arreglo de correos. </li><li> Diseccionar los músculos EDL 18 de las patas traseras del ratón, asegurándose de mantener proximal y distal tendones lo más intacta posible. </li><li> Incubar músculos EDL en tampón de bloqueo (1x PBS que contenía 0,5% de Triton X-100, 3% de BSA y suero de cabra al 5%) durante al menos 1 hr. </li><li&gt; Para visualizar los axones motores y sus terminales nerviosas, lugar músculos en tubos que contienen neurofilamentos (1: 1000) y sinaptotagmina-2 (1: 250) anticuerpos diluidos en tampón de bloqueo durante 3 días. Lavar los músculos tres veces con PBS 1x y 10 minutos cada vez. Nota: omitir este paso si el uso de ratones que expresan proteínas de fluorescencia (XFP) en los axones periféricos. </li><li> Manchar el contralateral ileso EDL como un control positivo para la completa inervación NMJ. Los controles negativos deben incluir un EDL obtenida a los 4 días después de la lesión, un punto de tiempo en el que el NMJ es completamente denervado, así como un EDL se tiñeron con anticuerpo secundario solamente. </li><li> Incubar los músculos con anticuerpos secundarios marcados con fluorescencia apropiados para detectar los neurofilamentos y sinaptotagmina-2 por 1 día. Lavar los músculos tres veces con PBS 1x y 10 minutos cada vez. Nota: Este paso puede llevarse a cabo en conjunto con el paso 3,10. Omitir este paso si el uso de ratones que expresan proteínas de fluorescencia en los axones periféricos. </li><li> Para visualizar el postsyregión Naptic de la NMJ, incubar músculos con 5 mg / ml Alexa-555 conjugado de alfa-bungarotoxina diluido en tampón de bloqueo durante al menos 2 hr. Lavar los músculos tres veces con PBS 1x y 10 minutos cada vez. </li><li> Para montar músculos enteros en portaobjetos de vidrio cargados positivamente, colocar el músculo directamente en la diapositiva, añadir unas gotas de glicerina basado en la diapositiva medio de montaje y cubrir con un cubreobjetos. Pulse el cubreobjetos contra la corredera para aplanar el músculo. Empapa del medio de montaje desde el perímetro de la diapositiva y cubreobjetos utilizando toallitas de laboratorio. Aplicar esmalte de uñas para sellar los bordes entre el cubreobjetos y portaobjetos. </li></ol> 4. Análisis de los datos de imagen y <ol><li> Para analizar la estructura de NMJs, la imagen del músculo EDL utilizando un microscopio láser confocal de barrido equipado para excitar a 488, 555 y 633 nm de luz y capturar la luz emitida con 20X y 40X. </li><li> Para visualizar NMJs conjunto, crear imágenes de proyección de intensidad máxima de seg ópticaciones espaciadas 1 a 2 micras se extiende aparte de los más bajos a regiones más altas visibles de la UNM. Crear proyecciones de máxima intensidad utilizando software de imagen disponible en el mercado. </li><li> Para determinar las tasas de reinervación, clasificar NMJs como: 1) completamente denervado = sitio postsináptica está completamente desprovisto de contacto con axón, a menos de 5% colocalización entre el axón y AChR. 2) parcialmente inervado = el axón se solapa parcialmente el postsynapse, 5-95% colocalización entre el axón y AChR. 3) inervación completa = casi perfecta aposición entre el pre y post-sinapsis, más de un 95% colocalización entre el axón y AChR. Excluir NMJs que se encuentran perpendiculares al plano de la imagen o no se visualizan totalmente en la imagen. Nota: En todos estos experimentos, al menos 3 animales y 50 NMJs por animal fueron examinados. Los resultados se consideraron significativos utilizando un test t de Student con un valor de p menor de 0,05. </li><li> Para cegar el operador, los individuos pueden realizar por separadola cirugía y análisis de imágenes. Sin el conocimiento de los grupos de tratamiento, el analizador puede ser objetivo con NMJ de puntuación. Alternativamente, las imágenes pueden ser asignados al azar y se presentan al operador para el análisis sin el conocimiento del animal fuente. </li></ol> 5. PCR cuantitativa <ol><li> Sacrificar los animales, utilizando isoflurano y dislocación cervical. Retire la piel y fascia superficial que cubre músculos de la pierna según la etapa 3.3. Diseccionar músculos EDL según la etapa 3.4 tibial anterior y. </li><li> Flash congelar todo el tibial anterior y los músculos EDL en un tubo de 1,5 ml de nitrógeno líquido. Extraer tejido del tubo y colocar en un mortero previamente enfriado parcialmente sumergido en nitrógeno líquido. Moler músculo congelado en un polvo fino usando un mortero y mano de mortero. </li><li> Disuelva el polvo músculo congelado en un reactivo de extracción de ARN comercialmente disponible y realizar la extracción de ARN y la extracción de ADN genómico con un kit disponible comercialmente de acuerdo con i del fabricanterecauciones. </li><li> Realizar la transcripción inversa con una mezcla de transcriptasa inversa disponible en el mercado de acuerdo con las instrucciones del fabricante. </li><li> Realizar qPCR utilizando un kit disponible comercialmente usando genes de limpieza adecuados (véase el cuadro de materiales). Use un termociclador de PCR cuantitativa disponible en el mercado para llevar a cabo la PCR (véase la tabla de materiales). </li><li> Ajuste la temperatura de recocido a 58 ° C. Ajustar parámetros de los ciclos adicionales en las especificaciones del fabricante de la mezcla verde taq polimerasa / SYBR. Incluir un paso final curva de fusión en el programa de termociclador que consiste en 0,5 ° C aumentos incrementales de 65 ° C a 95 ° C para probar la especificidad del cebador y el cebador formación de dímeros. </li><li> Determinar los niveles de expresión del ARNm de la 2 - método ΔΔCT 21 usando ARN 18S como gen de control. </li></ol>

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The authors have nothing to disclose.

El método presentado en este manuscrito ofrece oportunidades únicas para identificar los mecanismos implicados en la reparación de las uniones neuromusculares (NMJ). Este método implica la trituración el nervio peroneo común a medida que pasa sobre el tendón gastrocnemio cerca de la rodilla. Se demuestra que después de sólo 5 segundos de compresión de los nervios con una pinza, una degeneración completa se observó por 4 días después de la lesión. En los ratones adultos jóvenes, los axones alfa-motoras co...

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an &#945;-motor axon, and the postsynapse, the specialized region of an extrafusal muscle fiber where nicotinic acetylcholine receptors (AChRs) selectively aggregate1. The nearly perfect apposition of the pre- and post-synaptic apparatuses is necessary for proper neurotransmission, survival of &#945;-motor neurons and muscle fibers and motor function. Unfortunately, the function of the NMJ is adversely affected by aging, diseases such as amyotrophic lateral sclerosis (ALS), autoimmune diseases and injury to muscles and peripheral nerves2-5. These insults often result in degeneration of presynaptic nerve endings, leaving muscles denervated and significantly altering motor skills. For this reason, the identification of molecules that function to maintain and repair the NMJ has become a priority. Because peripheral nerves regenerate and reinnervate targets, peripheral nerve injury models have been used to identify molecular changes associated with regenerating NMJs. 
Peripheral nerve injury models often involve either completely cutting or crushing specific nerve branches6. Following a cut, the endoneurial tube has to be reformed, delaying axonal regeneration and reinnervation of target cells and tissues. The severity of this type of injury also causes axons to meander away from their original path, resulting in their failure to reach original targets. This is in contrast to nerves injured via crush where the endoneurium remains contiguous, providing a path for efficient and proper regrowth of regenerating axons. It also allows axons to find and reinnervate their original muscle fiber partners. Irrespective of injury model, there are a number of cellular and molecular changes that must occur for axons to regenerate and reinnervate targets. After an injury, the nerve segment proximal to the target is broken down and removed via a process termed Wallerian Degeneration7. This process involves reprogramming and de-differentiation of Schwann cells into non-myelinating cells that secrete regenerative factors, clear myelin, and recruit macrophages to the site of injury8. Macrophages in turn complete the clearance of myelin and axonal debris, which would otherwise impede growth of the regenerating axon9. In parallel, motor and sensory neurons activate mechanisms needed to promote regeneration of their severed axons. Once the regenerating axon reaches the target, it must transform from a growth cone to a nerve ending capable of properly transmitting (for motor axons) or receiving (for sensory axons) information10. In this regard, alpha-motor axons undergo a series of well-orchestrated changes that culminate in their growth cone differentiating into a fully functional presynaptic nerve ending that nearly perfectly opposes the post-synaptic site on the target muscle fiber11. 
The sciatic, tibial and accessory nerves have been the primary choices for studying axonal and NMJ regeneration12-14. However, there are a number of drawbacks when using these models to examine cellular and molecular changes associated with regenerating NMJs between animals and under different conditions. Firstly, the sciatic nerve supplies the majority of the muscles of the hind limb, with injury significantly limiting both movement and sensation. It is therefore not possible to use this method to study the impact of exercise alone or in combination with other factors. Additionally, the sciatic nerve is a rather thick structure and thus requires a large amount of compressive force to fully injure all axons. This in turn may result in complete transection of the more superficial axons while leaving the endoneurial tube of deeper lying axons intact, introducing significant variability in the rate and fidelity of regeneration among these axons. Complete transection of this nerve is even less desirable given that many axons will fail to reconnect with the same muscle fibers. Complicating matters, the sciatic nerve possesses intrinsic anatomic variability, both in the number and site of origin of its terminal nerve branches. It is therefore very difficult to lesion the same site. While the tibial nerve is smaller and more amenable to crush injuries, there is also no readily available landmark to serve as a lesion site for this nerve branch. 
The accessory nerve branch (part of cranial nerve XI) that supplies the sternocleidomastoid muscle has also been used to study regeneration of NMJs15. This nerve is particularly attractive because NMJs in the sternocleidomastoid muscle can be more readily imaged in live animals compared to NMJs in other muscles. But similar to the sciatic and tibial nerves, there is no specific landmark that can be used to injure this nerve in the same location, limiting it as a model for comparing regeneration of NMJs among individual animals of an experimental cohort. An inconsistent lesion site introduces variability in the rates of NMJ reinnervation. Due to these shortcomings, the procedure presented here utilizes the injury of a different peripheral nerve branch to examine regenerating NMJs. 
The common fibular nerve, also called the common peroneal nerve, contains many features that make it a reliable nerve to examine regeneration of NMJs between animals and across different treatments. The common fibular nerve has a predictable anatomic course as it runs over the tendon of the lateral head of the gastrocnemius muscle in the knee, the intersection serving as a stable landmark for lesions. The nerve is accessed through a small and minimally invasive incision near but anatomically segregated from the muscles of interest. The findings presented here demonstrate that regenerating motor axons begin to reform NMJs in the extensor digitorum longus (EDL) muscle 8 days after crushing the fibular nerve in 70 days old young adult female mice. Importantly, the pattern and rate of reinnervation is consistent among animals of the same age and sex and therefore provide a reliable injury model that will significantly hasten our understanding of the cellular and molecular changes required to maintain and repair NMJs.

<table><tbody><tr><td>Ketamine</td><td>VetOne</td><td>501072</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Inc.&amp;nbsp;</td><td>003437&amp;nbsp;</td><td></td></tr><tr><td>Buprenorphine</td><td>&amp;nbsp;Zoopharm</td><td>1Z-73000-150910&amp;nbsp;</td><td></td></tr><tr><td>Nair</td><td>Nair</td><td></td><td></td></tr><tr><td>Kim-wipes</td><td>Kimtech</td><td>34155</td><td></td></tr><tr><td>Electric Razor</td><td>Braintree Scientific</td><td>CLP-64800</td><td></td></tr><tr><td>80% EtOH/H&lt;sub&gt;2&lt;/sub&gt;0</td><td></td><td></td><td></td></tr><tr><td>10% Proviodine</td><td></td><td></td><td></td></tr><tr><td>1 ml Insulin Syringe</td><td></td><td></td><td></td></tr><tr><td>Spring Scissors</td><td>Vannas</td><td>91500-09</td><td></td></tr><tr><td>No. 15 scalpel</td><td>Braintree Scientific</td><td>SSS 15</td><td></td></tr><tr><td>#5 Forceps</td><td>Dumont</td><td>11252-00</td><td></td></tr><tr><td>6-0 silk suture on reverse cutting needle</td><td>&amp;nbsp;Suture Express</td><td>752B&amp;nbsp;</td><td></td></tr><tr><td>Rodent Heating Pad</td><td>Braintree Scientific</td><td>AP-R-18.5</td><td></td></tr><tr><td>Alexa 555 conjugated alpha-BTX</td><td>Molecular Probes</td><td>B35451</td><td></td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>Olympus Stereo Zoom Microscope</td><td>Olympus</td><td>562037192</td><td></td></tr><tr><td>Zeiss 700 Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Variable-flow peristaltic perfusion pump</td><td>Fisher Scientific</td><td>13-876-3</td><td></td></tr><tr><td>Aurum Total RNA Mini Kit</td><td>Bio-Rad</td><td>7326820</td><td></td></tr><tr><td>Bio-Rad iScript RT Supermix</td><td>Bio-Rad</td><td>1708840</td><td></td></tr><tr><td>SsoFast Evagreen Supermix</td><td>Bio-Rad</td><td>1725200</td><td></td></tr><tr><td>Bio-Rad CFX96</td><td>Bio-Rad</td><td>1855196</td><td></td></tr><tr><td>Puralube Vet ointment</td><td>Puralube</td><td>1621</td><td></td></tr><tr><td>Synaptotagmin-2 antibody</td><td>Antibodies-Online</td><td>ABIN401605</td><td></td></tr><tr><td>Neurofilament antibody</td><td>Antibodies-Online</td><td>ABIN2475842</td><td></td></tr></tbody></table>

the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions

La unión neuromuscular (UNM) sufre cambios estructurales y funcionales deletéreos como resultado del envejecimiento, lesiones y enfermedades. Por lo tanto, es imprescindible para entender los cambios celulares y moleculares implicados en el mantenimiento y la reparación de NMJs. Para este propósito, hemos desarrollado un método para examinar fiable y consistente regeneración NMJs en ratones. Este método consiste en la lesión del nervio de aplastamiento del nervio peroneo común, ya que pasa por encima de la cabeza lateral del tendón del músculo gemelo cerca de la rodilla. Utilizando viejos ratones hembra de 70 días, se demuestra que los axones motores comienzan a reinervar objetivos postsinápticos anteriores dentro de los 7 días posteriores a aplastamiento. Que reingresen completamente sus áreas sinápticas anteriores por 12 días. Para determinar la fiabilidad de este método de la lesión, se compararon las tasas de reinervación entre 70 días de edad ratones hembras individuales. Se encontró que el número de sitios postsinápticos reinervados fue similar entre los ratones a los 7, 9, y 12 días post-aplastamiento. Para determinar sieste ensayo lesión también se puede utilizar para comparar los cambios moleculares en los músculos, se examinaron los niveles de la subunidad gamma del receptor nicotínico muscular (gamma-AChR) y la cinasa específica de músculo (MuSK). La subunidad gamma-AChR y almizcle que están altamente regulados por incremento tras la denervación y vuelven a los niveles normales después de la reinervación de NMJs. Hemos encontrado una estrecha relación entre los niveles de transcripción de estos genes y el estado de la inervación de los músculos. Creemos que este método se acelerará la comprensión de los cambios celulares y moleculares implicados en la reparación de la NMJ y otras sinapsis.

El nervio peroneo común, también llamado nervio peroneo común, surge del nervio ciático por encima de la fosa poplítea, donde se hace pivotar alrededor de la cabeza del peroné a la cara anterior de la pierna (Figura 1A). No se ramifica en los nervios peroneo superficial y profunda, junto suministro de los flexores dorsales del pie y los dedos (tibial anterior, extensor largo de los dedos y corto, y halluces músculos extensores largo del dedo gordo), y los everters...

Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

El nervio peroneo Método de lesiones: un ensayo fiable para identificar y Factores de prueba de que la reparación neuromusculares uniones

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

the-fibular-nerve-injury-method-reliable-assay-to-identify-test

Research

JoVE Journal

Neuroscience

15.6K vistas.  Virginia Tech. We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle. 

Video: The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

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.

Ratón reproducible aplastamiento del nervio ciático y posterior evaluación de la regeneración mediante el análisis de músculo entero Monte

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

Investigación

Neurociencias

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Evaluación de la función neuromuscular Usando percutánea estimulación nerviosa eléctrica

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Medir la funcionalidad de la placa neuromuscular

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those individuals with extremity weakness. However, their use remains largely confined to research institutions, frequently operating as a means of static extremity support as motor detection methods remain unreliable. Peripheral nerve interfaces have arisen as a potential solution to this shortcoming; however, due to their inherently small amplitudes, these signals can be difficult to differentiate from background noise, lowering their overall motor detection accuracy. As current interfaces rely on abiotic materials, inherent material breakdown can occur alongside foreign body tissue reaction over time, further impacting their accuracy. The Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI) was designed to overcome these noted complications. Consisting of a segment of free muscle graft secured circumferentially to an intact peripheral nerve, the construct regenerates and becomes reinnervated by the contained nerve over time. In rats, this construct has demonstrated the ability to amplify a peripheral nerve's motor efferent action potentials up to 100 times the normal value through the generation of compound muscle action potentials (CMAPs). This signal amplification facilitates high accuracy detection of motor intent, potentially enabling reliable utilization of exoskeleton devices.

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

La interfaz nerviosa periférica regenerativa del manguito muscular para la amplificación de señales nerviosas periféricas intactas

Robotic exoskeletons have gained recent acclaim within the field of rehabilitative medicine as a promising modality for functional restoration for those ...

Bioingeniería

Functional and Physiological Methods of Evaluating Median Nerve Regeneration in the Rat

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different methods of simulating postoperative physiotherapy are presented. Multiple standardized strategies are used to assess motor and sensory recovery using an MN model of peripheral nerve lesion and repair, thus permitting easy comparison of the results. Several options are included for providing a postoperative physiotherapy-like environment to rats that have undergone MN injuries. Finally, the paper provides a method to evaluate the recovery of the MN using several noninvasive tests (i.e., grasping test, pin prick test, ladder rung walking test, rope climbing test, and walking track analysis), and physiological measurements (infrared thermography, electroneuromyography, flexion strength evaluation, and flexor carpi radialis muscle weight determination). Hence, this model seems particularly appropriate to replicate a clinical scenario, facilitating extrapolation of results to the human species.
Although the sciatic nerve is the most studied nerve in peripheral nerve research, analysis of the rat MN presents various advantages. For example, there is a reduced incidence of joint contractures and automutilation of the affected limb in MN lesion studies. Furthermore, the MN is not covered by muscle masses, making its dissection easier than that of the sciatic nerve. In addition, MN recovery is observed sooner, because the MN is shorter than the sciatic nerve. Also, the MN has a parallel path to the ulnar nerve in the arm. Hence, the ulnar nerve can be easily used as the nerve graft for repairing MN injuries. Finally, the MN in rats is located in the forelimb, akin to the human upper limb; in humans, the upper limb is the site of most peripheral nerve lesions.

Presented is a protocol to produce different types of median nerve (MN) lesions and repair in the rat. Additionally, the protocol shows how to evaluate the functional recovery of the nerve using several noninvasive behavioral tests and physiological measurements.

Métodos funcionales y fisiológicos de evaluación de la regeneración media nerviosa en la rata

The main goal of this investigation is to show how to create and repair different types of median nerve (MN) lesions in the rat. Moreover, different ...

Medicina

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.

Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article.

Medición De La Fuerza Teánica Isométrica Máxima Del Músculo Tibial Anterior En La Rata

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve ...

Implantation and Control of Wireless, Battery-free Systems for Peripheral Nerve Interfacing

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces can be sensors, actuators, or both. Traditional methods of peripheral nerve interfacing must either tether to an external system or rely on battery power that limits the time frame for operation. With recent developments of wireless, battery-free, and fully implantable peripheral nerve interfaces, a new class of devices can offer capabilities that match or exceed those of their wired or battery-powered precursors. This paper describes methods to (i) surgically implant and (ii) wirelessly power and control this system in adult rats. The sciatic and phrenic nerve models were selected as examples to highlight the versatility of this approach. The paper shows how the peripheral nerve interface can evoke compound muscle action potentials (CMAPs), deliver a therapeutic electrical stimulation protocol, and incorporate a conduit for the repair of peripheral nerve injury. Such devices offer expanded treatment options for single-dose or repeated dose therapeutic stimulation and can be adapted to a variety of nerve locations.

This is a protocol for the surgical implantation and operation of a wirelessly powered interface for peripheral nerves. We demonstrate the utility of this approach with examples from nerve stimulators placed on either the rat sciatic or phrenic nerve.

Implantación y control de sistemas inalámbricos sin batería para la interfaz de nervios periféricos

Peripheral nerve interfaces are frequently used in experimental neuroscience and regenerative medicine for a wide variety of applications. Such interfaces ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>Modelo modificado de cirugía de lesión nerviosa preservada del dolor neuropático en ratones

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Modelo modificado de cirugía de lesión nerviosa preservada del dolor neuropático en ratones

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.

The present protocol describes traumatic peripheral nerve injuries (TPNIs), including precisely calibrated crush, strictly aligned and misaligned laceration, as well as grafted and non-grafted gaps of the sciatic nerve in mice. Custom-designed sensors are developed to gauge nerve trauma, induced with commonly available tools to ensure reproducible post-TPNI outcomes.

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve ...

El nervio peroneo Método de lesiones: un ensayo fiable para identificar y Factores de prueba de que la reparación neuromusculares uniones

Resumen

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