Name: the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions
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Description: 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 authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

Todos os experimentos foram realizados sob as diretrizes do NIH e protocolos de animais aprovados pela Comissão de Virginia Tech Institucional animal Cuidado e Uso. 1. Os animais que se preparam para Cirurgia <ol><li> Anestesiar ratos com uma mistura de cetamina (90 mg / kg) e xilazina (10 mg / kg) via injecção subcutânea inguinal com uma seringa de 1 ml estéril insulina. solução portadora contém uma mistura de solução salina a 0,9%, 17,4 mg / ml de cetamina e 2,6 mg / ml de xilazina. Coloque animais de volta em gaiolas enquanto espera para a medicação fazer efeito. NOTA: Se a dose de carga não proporcionar anestesia suficientes para a duração do processo, um adicional de 25% da dose de carga pode ser injectado. </li><li> animais do monitor após a injecção para verificar se há taxas respiratórias estáveis ​​e depressão adequada dos níveis de excitação. Verifique o nível de excitação com uma pitada pata traseira, o que deve provocar nenhuma resposta quando suficientemente anestesiados. NOTA: Isso normalmente leva 3-5min para um rato adulto jovem, com média de 25 a 30 g. Se o animal ainda é responsivo após a injecção de pós de 10 min, um adicional de 25% da dose de carga de anestésico pode ser injectado. </li><li> Aplicar vaselina e pomada oftálmica óleo mineral leve para os olhos do animal para evitar a secura. Remover animais de gaiola e coloque sobre uma superfície plana e limpa. Raspar o membro posterior desejado de pé para pelve, por um aparador de cabelo elétrico, expondo apenas o aspecto lateral do membro. </li><li> Aplicar um removedor de cabelo química para o local raspada durante 1 min. remover manualmente o cabelo usando lenços de laboratório. Limpe a área depilada com o laboratório toalhete embebido em álcool. </li></ol> 2. Procedimento Cirúrgico <ol><li> Esterilizar instrumentos cirúrgicos através de autoclave ou outro método adequado. Limpar o local da cirurgia e tábua cirúrgico com 80% de etanol / H 2 0. Desinfectar o local cirúrgico com proviodine. Coloque o mouse na placa cirúrgica e alinhar-se com as restrições dos membros. Guardao membro alvo traseira em uma posição anatomicamente natural com a articulação do joelho ligeiramente estendido sem rotação interna ou externa. </li><li> Coloque o animal e tábua sob o microscópio cirúrgico. Orientar para o local da incisão adequada através da palpação dos marcos superficiais, especificamente a articulação do joelho ossudo eo cume entre o tibial anterior e gastrocnêmio. </li><li> Fazer uma cerca de 3 cm da incisão através da pele utilizando um bisturi ou uma tesoura de mola enquanto estiver usando uma pinça gerais para emocionante. Fazer a incisão perpendicular ao curso subjacente do nervo fibular comum. </li><li> Continue a incisão através da fáscia superficial, expondo o bíceps femoral e vasto lateral. Separar estes músculos cortando a fáscia profunda ligação. A 1-2 cm corte deve ser suficiente. </li><li> Retrair o músculo bíceps femoral caudal com afastadores mecânicas, revelando o nervo fibular comum. </li><li> Trace o nervo proximal até suas interseção com o tendão da cabeça lateral do músculo gastrocnêmio é encontrado. Nota: A exposição pode requerer manipulação adicional da pele retraída e músculo. Esta intersecção é utilizado como o ponto de referência estável para a lesão do nervo. </li><li> Segure o nervo com uma pinça fina, alinhando as pontas de uma forma paralela à borda lateral do tendão gastrocnêmio. Esmagar o nervo fibular comum, aplicando pressão constante, dura por 5 s. </li><li> Corroboram queda completa do nervo por inspeção visual com o espaço cirúrgico. Ele aparece translúcido no local da lesão. Se utilizando camundongos que expressam proteínas fluorescentes em axônios periféricos, a fluorescência irá desaparecer do local da lesão. </li><li> Remover afastadores e realinhar os músculos em suas posições anatômicas. Feche o local da incisão com 6-0 seda. 1-3 suturas interrompidas simples é suficiente. Coloque recuperando do mouse sobre uma almofada de aquecimento em uma gaiola limpa. </li><li> Monitorar todos os animais para 2 horas pós-óperação para verificar se há respiração e quaisquer reacções adversas à anestesia. Administrar uma dose inicial de buprenorfina 0,05-0,10 mg / kg por via de injecção subcutânea inguinal imediatamente a seguir à recuperação da cirurgia. Dê 3 doses adicionais a cada 12 horas durante o próximo 48 horas. Após a recuperação completa, voltar ratos para a instalação de cuidados com os animais. </li></ol> 3. Isolamento e Coloração de extensor longo dos dedos músculos (EDL) <ol><li> Sacrificar animais com isoflurano. Pipetar 0,5 ml de isoflurano líquido para um tubo de 50 mL embalada com labwipes absorventes. Colocar o tubo destapado com o animal numa câmara selada 2,500 centímetros 3. Pelo menos 4 min de exposição é suficiente. Teste para perda de palpebral bilateral, toe-beliscão, e reflexos rabo-de aperto para garantir que cada animal está inconsciente antes de prosseguir com a perfusão. </li><li> Transcardialmente perfundir 16 animais em primeiro lugar com 10 ml de 0,1 M de PBS, em seguida, 25 ml de 4% de paraformaldeído em 0,1 M de PBS (pH 7,4). Heparina (30 unidades / 20g de peso do animal), pode ser adicionado com o PBS (10 unidades / ml) para evitar a coagulação do sangue em pequenos capilares, melhorando os resultados de perfusão. </li><li> Remover a pele que cobre os membros posteriores, utilizando uma tesoura para cortar transversalmente através da pele em torno da circunferência do abdómen. Descascar para baixo a pele após os membros e patas traseiras com a pinça. </li><li> Remover fáscia superficial de membros posteriores, segurando e peeling com fórceps. Se utilizando camundongos que expressam proteínas fluorescentes em axônios periféricos, pós-fix ratos toda a noite em 4% PFA em tubos de 50 ml. Lavar três vezes com PBS. NOTA: Fixa ratinhos podem ser armazenadas em PBS a 4 ° C. Se não, pule esta etapa e vá para a etapa 3.6, sem animais pós-fixação. </li><li> Dissecar músculos EDL 18 de rato membros traseiros, tendo a certeza de manter proximal e distal tendões tão intacto quanto possível. </li><li> Incubar músculos EDL em tampão de bloqueio (PBS 1x contendo 0,5% de Triton X-100, BSA a 3% e soro de cabra a 5%) durante pelo menos 1 h. </li><li&gt; Para visualizar os axônios a motor e seus terminais nervosas, músculos lugar em tubos contendo neurofilamentos (1: 1000) e sinaptotagmina-2 (1: 250) anticorpos diluídos em tampão de bloqueio durante 3 dias. Lave músculos três vezes com 1x PBS e 10 min de cada vez. Nota: Ignore esta etapa se utilizando camundongos que expressam proteínas fluorescentes (XFP) em axônios periféricos. </li><li> Manchar o contralateral ileso EDL como um controlo positivo para a inervação completa MNJ. Os controlos negativos devem incluir uma EDL obtido em 4 dias pós-ferimento, um ponto de tempo onde o JNM é completamente desnervado, bem como uma EDL coradas com anticorpo secundário única. </li><li> Incubar músculos com anticorpos secundários marcados fluorescentemente apropriadas para detectar neurofilamento e sinaptotagmina-2 durante 1 dia. Lave músculos três vezes com 1x PBS e 10 min de cada vez. Nota: Este passo pode ser realizado juntamente com o passo 3.10. Pule esta etapa se utilizando camundongos que expressam proteínas fluorescentes em axônios periféricos. </li><li> Para visualizar a postsynaptic região do MNJ, músculos incubar com 5 ug / mL de Alexa-555 conjugado alfa-bungarotoxina diluído em tampão de bloqueio durante, pelo menos, 2 h. Lave músculos três vezes com 1x PBS e 10 min de cada vez. </li><li> Para montar músculos inteiros em lâminas de vidro com carga positiva, coloque o músculo diretamente sobre o slide, adicionar algumas gotas de glicerol com base médio no slide de montagem e cobrir com uma lamela. Pressione a lamela contra o slide para achatar o músculo. Mergulhe off meios de montagem a partir do perímetro da lâmina e lamela usando lenços de laboratório. Aplique unha polonês para selar as arestas entre as lamelas e slide. </li></ol> 4. Imagem e Análise de Dados <ol><li> Para analisar a estrutura da NMJs, imagem do músculo EDL usando um microscópio confocal de varrimento laser equipado para excitar 488, 555 e 633 nm de luz e capturar a luz emitida com 20X e 40X objectivos. </li><li> Para visualizar toda NMJs, criar imagens projeção de intensidade máxima de sec ópticações espaçadas de 1 a 2 mm que se estende para além do menor para o maior regiões visíveis do MNJ. Crie projeções de intensidade máxima, utilizando software de imagens disponível no mercado. </li><li> Para determinar as taxas de reinervação, categorizar NMJs como: 1) completamente desnervado = sítio pós-sináptico é completamente desprovida de contacto com axônio, menos de 5% co-localização entre o axônio e AChRs. 2) parcialmente inervado = o axónio sobrepõe parcialmente o postsynapse, 5-95% de co-localização entre o axónio e AChRs. 3) inervação completa = cerca de aposição perfeita entre o pré e pós-sinapse, superior a 95% co-localização entre o axônio e AChRs. Excluir NMJs que se encontram perpendiculares ao plano de imagem, ou não são totalmente visualizadas na imagem. Nota: Em todas estas experiências, pelo menos, 3 animais e 50 NMJs por animal foram examinados. Os resultados foram considerados significativos usando um teste t de Student, com um valor P inferior a 0,05. </li><li> Para cegar o operador, indivíduos separados pode executara análise de cirurgia e imagem. Sem o conhecimento dos grupos de tratamento, o analisador pode ser objectivo com JNM de pontuação. Alternativamente, as imagens podem ser aleatorizados e apresentados ao operador para análise sem o conhecimento do animal de origem. </li></ol> 5. Quantitative PCR <ol><li> Sacrificar animais utilizando isoflurano e deslocamento cervical. Retire a pele e fáscia superficial que cobre os músculos da perna de acordo com o passo 3.3. Dissecar tibial anterior e músculos EDL acordo com o passo 3.4. </li><li> Flash congelar todo o tibial anterior e músculos EDL em um tubo de 1,5 ml sobre azoto líquido. Remover o tecido do tubo e colocar num almofariz pré-arrefecido parcialmente submerso em azoto líquido. Moer músculo congelado em um pó fino usando um almofariz e pilão. </li><li> Dissolve-se pó de músculo congelado em um reagente de extracção de ARN comercialmente disponível e efectuar a extracção de ARN e a remoção de DNA genómico com um kit disponível comercialmente de acordo com I do fabricantenstructions. </li><li> Realizar a transcrição reversa com uma transcriptase reversa mistura disponível comercialmente de acordo com as instruções do fabricante. </li><li> Execute qPCR utilizando um kit disponível comercialmente usando genes de limpeza adequados (ver tabela de materiais). Use um quantitativo termociclador PCR comercialmente disponível para realizar PCR (ver tabela de materiais). </li><li> Ajuste temperatura de recozimento de 58 ° C. Ajustar os parâmetros de ciclismo adicionais às especificações do fabricante de Taq polimerase / SYBR mix verde. Incluir um passo final da curva de fusão do programa de termociclador consistindo de 0,5 ° C aumentos incrementais de 65 ° C a 95 ° C para testar a especificidade do iniciador e o iniciador a formação de dímeros. </li><li> Determinar os níveis de expressão de ARNm relativa pelos 2 - Método ΔΔCT 21 utilizando 18S ARN como o gene de controlo. </li></ol>

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The authors have nothing to disclose.

O método apresentado neste manuscrito oferece oportunidades únicas para identificar mecanismos envolvidos na reparação de junções neuromusculares (JNM). Este método envolve o esmagamento do nervo fibular comum que passa sobre o tendão gastrocnêmio, perto do joelho. Mostramos que após apenas 5 segundos de compressão do nervo com um fórceps, degeneração completa é conhecida por 4 dias após a lesão. Em ratos adultos jovens, os axônios alfa-motoras começam a reinnervate sítios sinápticos anteriores no m...

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

A junção neuromuscular (MNJ) sofre alterações estruturais e funcionais deletérios, como resultado do envelhecimento, ferimentos e doenças. Assim, é imperativo compreender as alterações celulares e moleculares envolvidos na manutenção e reparação NMJs. Para esta finalidade, desenvolvemos um método fiável e consistente para examinar regenerar NMJs em ratinhos. Este método envolve a lesão do nervo esmagamento do nervo fibular comum, uma vez que passa por cima da cabeça lateral do tendão do músculo gastrocnêmio, perto do joelho. Usando ratos fêmeas mais velhas 70 dias, demonstramos que axônios motores começam a reinnervate metas pós-sinápticos anteriores no prazo de 7 dias após o esmagamento. Eles reocupar completamente suas áreas sinápticas anteriores por 12 dias. Para determinar a confiabilidade desse método de lesão, foram comparadas as taxas de reinervação entre camundongos fêmeas velhas 70 dias individuais. Verificou-se que o número de sítios pós-sinápticos reinervados foi semelhante entre os ratinhos em 7, 9, e 12 dias pós-esmagamento. Para determinar seeste ensaio de lesão também pode ser utilizado para comparar as alterações moleculares em músculos, que examinaram os níveis de gama-a subunidade do receptor nicotínico muscular (gama-AChR) e a quinase específica do músculo (almíscares). A subunidade gama-AChR e almíscar que são altamente regulada seguinte desnervação e retornar aos níveis normais após a reinervação dos NMJs. Encontramos uma relação estreita entre os níveis de transcrição para estes genes e status inervação dos músculos. Acreditamos que este método irá acelerar nossa compreensão das mudanças celulares e moleculares envolvidos na reparação do MNJ e outras sinapses.

O nervo fibular comum, também chamado o nervo fibular comum, surge a partir do nervo ciático acima da fossa poplítea, onde ele oscila ao redor da cabeça da fíbula ao aspecto anterior da perna (Figura 1A). Lá ramificações nos nervos fibulares superficial e profunda, em conjunto fornecendo os dorsiflexores do pé e dos dedos (tibial anterior, extensor longo dos dedos e brevis, e hálux músculos extensores longus), e os everters do pé (músculos fibulares). Este n...

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.

O nervo fibular método de lesão: um ensaio confiável para identificar e testar fatores que o reparo Junções Neuromusculares

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 visualizações.  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. 

Vídeo: 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.

Reprodutível mouse esmagamento do nervo ciático e subsequente avaliação de Regeneração por Análise Whole Mount muscular

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

Neurociência

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

aplicações de<em&gt; In Vivo</em&gt; Teste Funcional do Rato tibial anterior para a Avaliação da engenharia de tecidos Repair Músculo Esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Bioengenharia

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).

Avaliação da função neuromuscular Usando percutânea Estimulação Elétrica Nervosa

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 a funcionalidade da junção Neuromuscular

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

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Murino do tendão flexor lesão ea cirurgia de reparação

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Medicina

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 funcionais e fisiológicos de avaliação da regeneração do nervo mediano no rato

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 ...

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.

Medição máxima da Força Tetanica Isométrica do Músculo Tibialis Anterior no Rato

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.

Implantação e Controle de Sistemas Sem Fio e Sem Bateria para Interface de Nervos 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 de cirurgia de lesão nervosa poupada modificada de dor neuropática em camundongos

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.

O nervo fibular método de lesão: um ensaio confiável para identificar e testar fatores que o reparo Junções Neuromusculares

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