Este trabalho foi financiado por doações da Parceria de Pesquisa Neuromuscular CIHR (JNM - 90959, para Jaeb).

Antes de usar este modelo, os investigadores devem ter recebido aprovação para o protocolo cirúrgico de uso de animais de sua instituição de órgão. O modelo é aprovado pelo Conselho de Ética de Pesquisa, Hamilton Health Sciences Corporation, da Universidade McMaster (AUP # 10-04-24) e é realizado em estrita conformidade com as recomendações do Canadian Council on Animal Care. 1. Rato Preparação <ol><li> Pesar o mouse. Induzir a anestesia com isoflurano 5% ou 2% de halotano. O circuito utilizado deve assegurar a eliminação adequada do anestésico para proteger o cirurgião. Após 2-3 min respiração do animal vai abrandar. Verifique se o piscar reflexo está ausente e prender os espaços interdigitais na pata para confirmar a anestesia cirúrgica (ou seja, sem resposta do mouse). Aplicar lubrificante oftálmica para o olho, para evitar a secagem da córnea, durante a cirurgia. </li><li> Raspar a lateral da coxa e nádega do ciáticoponto para o joelho e desinfectar com proviodine. Barbear vai manter o local do cabelo incisão livre para garantir a adequada visualização do campo cirúrgico e minimizar a interferência com dissecção do nervo e transecção. O entalhe ciático, que é superior e posterior do fêmur, pode ser identificado por palpação. </li></ol> 2. Procedimento operatório <ol><li> Reduzir inalatória isoflurano a 2% (1% de halotano) e colocar o rato sobre o seu lado (destinado a cirurgia virada para cima), sob um microscópio de operação ou dissecação. Alternativamente, a cirurgia pode ser executada com lupas cirúrgicas desde ampliação 3.5X é satisfatória para um adulto (20-25 g) do rato. </li><li> Vestir luvas estéreis. Identificar o entalhe ciático por palpação. Utilizando um bisturi, inciso na pele da coxa lateral da incisura ciática para o joelho (aproximadamente 1 cm). </li><li> Delicadamente espalhar a pele. Identificar o músculo bíceps femoris, o qual é o músculo superficial plana da face lateral da coxaimediatamente por baixo da pele. Usando uma tesoura fina, dividir o músculo bíceps femoral ao longo das fibras musculares e mantenha aberto com um afastador de primavera para expor o nervo ciático e seus ramos. </li><li> Identificar o nervo ciático imediatamente profundo ao músculo bíceps femoral. Ele pode ser identificado pela sua cor branca brilhante característica e é aproximadamente 0,8 mm de diâmetro. Ele roda a partir do entalhe ciático até o joelho, ramificando-se nos nervos tibial, fibular e sural ao nível da fossa poplítea. </li><li> Delicadamente separar o tibial dos ramos do nervo fibular e sural com uma pinça ultrafinas e primavera tesouras microdissecting. O nervo tibial é o maior ramo e é geralmente central. É importante para não esmagar o nervo enquanto separando os ramos. Segurando o nervo apenas na camada adventicial exterior com a pinça ultrafinas, e mantendo a folga nervo (não ensinado), vai evitar esmagamento do nervo e lesão de tração. </li><li> Para completa e duradoura denervatiem, cortar o nervo tibial com microdissecting tesoura como distal possível, evitando cuidadosamente os vasos poplíteos. Alternativamente, para a desnervação temporária com Reinervação completa esperado em 2-4 semanas, o nervo tibial podem ser simplesmente esmagados com uma pinça ultrafinas de 15 seg, em vez de seccionado. (Nervos periféricos regredir após lesão e reinervar músculo alvo.) </li><li> Se desnervação completo é necessária, a sutura da extremidade do nervo tibial seccionado na superfície anterior do músculo bíceps femoris com nylon 10-0 e re-aproximar a bíceps femoris com 5-0 Vicryl aberrante para evitar a re-inervação dos gastrocnémio e sóleo. </li><li> Feche a pele com uma corrida de sutura Vicryl 5-0. </li></ol> 3. Cuidados pós-operatória <ol><li> Desligue o anestésico inalatório, mas manter o fluxo de oxigênio. Administrar buprinorphine (ou substituto) analgésico por via subcutânea. </li><li> Transferir o mouse para a gaiola limpa, semcama ao acordar da anestesia. Mantenha em um cobertor aquecimento dentro da gaiola e sob observação direta até deambulação. </li><li> Transferência e casa na gaiola inferior macia (não condutores) com ampla cama macia. </li><li> Inspecione a parte operativa diária para a condição da ferida cirúrgica e do pé para o desenvolvimento de úlceras de decúbito ou provas de salto de mastigar. Pequenos problemas podem ser tratados com antibióticos ou anti-sépticos como proviodine. Indicadores de terminais que requerem a eutanásia de animais são perda de peso, a evidência de auto-cuidado pobres (pele enrugada) e postura arqueada. Além disso, animais com grande ruptura feridas ou úlceras que não cicatrizam em 1-2 semanas com antibióticos tópicos ou parecem ter dor deve ser sacrificado. </li></ol> 4. Gastrocnêmio desnervado Colheita músculo sóleo <ol><li> No ponto em tempo de pós-operatório desejado, pesar o mouse e sacrifício com dois overdose CO. </li><li> Raspar o aspecto medial de ambos osoperatório e pernas controle contralateral e limpo com álcool. Posicione o mouse sob um microscópio cirúrgico ou de dissecação, ou, alternativamente, utilizar lupas visualização cirúrgicos para ampliação. </li><li> No membro operativo, incisar a pele de vitelo medial com um bisturi a partir do tornozelo ao joelho e perifericamente em torno do tornozelo. Puxe delicadamente a pele com uma pinça fora do músculo e proximal em direção à coxa. Isto expõe todos os músculos da perna. Identificar o músculo gastrocnémio, a qual é o músculo bezerro que se estende desde o joelho para o tornozelo na parte posterior da perna e encontra-se imediatamente abaixo da pele. Identificar a inserção distal do bíceps femoral proximal do músculo gastrocnêmio sobre o aspecto medial do joelho. Na sua inserção distal do bíceps femoral parece fina e transparente e recobre a porção mais proximal do gastrocnêmio. Usando uma tesoura e dissecção fim brusco, separe delicadamente a inserção distal do bíceps femoral a partir do gásmúsculo gastrocnêmio. </li><li> Na sua inserção distal no calcâneo, o músculo gastrocnêmio afunila no tendão de Aquiles. Identificar o tendão de Aquiles, que aparece em branco e musculoso. Segure o tendão de Aquiles com uma pinça, tomando cuidado para não prender ou esmagar o músculo gastrocnêmio, e dividir o tendão de Aquiles da inserção do calcâneo com uma tesoura. </li><li> Ainda segurando o tendão, levante suavemente o músculo gastrocnêmio (vermelho pálido) fora o sóleo profundas (mais fundo vermelho), a partir da inserção distal em direção a sua origem na altura do joelho (o sóleo pode ser colhida separadamente). </li><li> Dissecar as gastrocnémio fora da perna dividindo a origem do gastrocnémio dos chondyles medial e lateral do fémur, utilizando uma tesoura. Tração muito suave no músculo facilita esse processo. Tenha cuidado para não esmagar o músculo. </li><li> Os sóleo será agora claramente visível, imediatamente abaixo do local do gastrocnêmio. Levante o sóleo de sua inserção no tendão de Aquiles para a sua ouigin na panturrilha posterior. Se o sóleo é, inadvertidamente, levantado com o gastrocnêmio, separe-o suavemente a amostra colhida. O gastrocnêmio (luz vermelha) e sóleo (vermelho escuro) permanecem facilmente identificáveis ​​na amostra colhida, devido às suas diferenças de cor. </li><li> Pesar os músculos separadamente em uma balança de precisão. </li><li> Dividir os músculos na vertical, para o congelamento de metade pressão em azoto líquido (por proteína posterior e / ou de extracção de ARN), e metade para a histologia (isto é, a avaliação morfométrica, imunohistologia) fixar ou em formalina a 10%, ou fixação congelados em isopentano arrefecido com azoto líquido , conforme o desejado. </li><li> Repita a partir do passo 4.3 acima no controle, não-operado, lado a colheita de controle gastrocnêmio e sóleo. </li></ol>

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O modelo de corte transversal do nervo tibial induzida por desnervação atrofia do músculo esquelético é um modelo vulgarmente utilizados e bem validado em ratos. Temos este modelo adaptado para utilização em ratinhos, o que permite ao investigador para tirar vantagem da existência de ratos geneticamente modificados e estudar o processo de atrofia muscular in vivo na ausência de proteínas essenciais para a regulação da massa muscular 7,8. Os músculos gastrocnemius e sóleo, ambos desnerva...

Desnervação do músculo esquelético, devido a uma lesão traumática do nervo periférico, doença ou intervenção farmacológica, resulta na perda imediata da função contrátil do músculo voluntário. Muscle concomitantemente começa a atrofiar e esta atrofia é reversível se oportuna, reinervação de boa qualidade ocorre 1,2. Na ausência de Reinervação, myofiber atrofia progride, e alterações biológicas irreversíveis no músculo ocorrer com fibrose muscular e da morte de células musculares. Aqui demonstramos o modelo transecção do nervo tibial, um modelo de atrofia induzida por desinervação do músculo esquelético e da fibrose, em ratinhos. Este modelo permite aos cientistas estudar os mecanismos biológicos fisiológicos, celulares e moleculares subjacentes atrofia muscular in vivo nos músculos gastrocnêmio e sóleo. Enquanto historicamente utilizados predominantemente em ratos, uma aplicação mais recente deste modelo KO e linhas de ratinhos transgénicos especificamente, permite aos investigadores para avaliar o papel da suaproteína específica (s) de interesse na indução, desenvolvimento e manutenção, ou em alternativa, a resolução de, atrofia muscular e fibrose in vivo. O nervo tibial é um nervo periférico sensório-motora mista no membro posterior do roedor, e é um dos três ramos terminais do nervo ciático. Transecção do nervo tibial denervates o gastrocnêmio, sóleo e plantar (e os três músculos flexores profundos pequenos do pé incluindo tibial posterior, flexor longo dos dedos e flexor hallicus longus), e é um modelo bem padronizados e validados em ratos 3,4 . Os músculos gastrocnêmio e sóleo pode ser facilmente dissecados em momentos de série postar secção do nervo tibial, fixados e processados ​​para avaliação da histologia muscular e morfometria das fibras musculares, ou flash congelado para a extração de RNA e proteínas musculares para a finalidade de estudar, por exemplo, as redes de sinalização celular que regulam a atrofia muscular. O gastrocnemius muscular é um tipo de fibra muscular mista (tipo I e tipo II, embora seja predominantemente do tipo II) e do músculo sóleo é composta por uma grande proporção de fibras do tipo I, proporcionando deste modo tanto rápido e lento contração muscular para avaliação 5,6. O modelo de corte transversal do nervo tibial é adequado para o estudo do processo de atrofia induzida por desnervação muscular quer a curto prazo (dias) 7 e de longo prazo (semanas a meses) 4,8. Em contraste com o modelo de corte transversal do nervo ciático (um segundo modelo de atrofia induzida por desinervação do músculo utilizada em roedores), transecção do nervo tibial induz menor morbidade no animal, tornando-a um modelo mais atraente. A transecção do nervo ciático denervates todos os músculos da perna (abaixo do joelho) e no pé, prejudicar a capacidade do animal para deambulação 2, enquanto que a transecção do nervo tibial folhas dos ramos do nervo sural e fibular do nervo ciático intacta, preservando assimseus músculos alvo e territórios sensoriais. O mouse é incapaz de flexão plantar ou inverter a pé, mas é capaz de deambular facilmente eo peso tem igualmente em ambos os membros posteriores, assim diminuindo significativamente a morbidade do modelo. Estudos de análise de marcha avaliando padrões de caminhada foram realizados em ratos após lesão do nervo ciático e tibial e demonstrar que a pegada eo peso do rolamento é melhor preservada com lesão tibial 9,10. Além disso, no modelo de corte transversal do nervo tibial, o nervo peroneal pode ser mobilizada num ponto de tempo posterior e transferido como uma fonte de Reinervação retardada, se o desenho do estudo requer 3. Em contraste, Reinervação retardada no modelo de corte transversal do nervo ciático que requer a utilização de um enxerto de nervo ao défice do nervo ciático, o aumento muito significativamente a dificuldade técnica do modelo e limitando a sua utilização para os cirurgiões especializados. Enquanto o modelo de secção do nervo tibial requires familiaridade do operador com técnica cirúrgica estéril em cirurgia animal, tanto o nervo tibial e que inerva os músculos da panturrilha são facilmente acessíveis e identificáveis ​​para a manipulação, de modo que os indivíduos que não são cirurgiões, ou altamente experiente com a cirurgia animal, podem facilmente dominar este modelo .

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>10-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>2850G</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Vicryl suture</td>
			<td>Ethicon</td>
			<td>J553G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Spring microdissecting scissors</td>
			<td>Fine Surgical Tools</td>
			<td>15021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Fine Surgical Tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Non locking micro needle holder (driver)</td>
			<td>Fine Surgical Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring retractor</td>
			<td>Fine Surgical Tools</td>
			<td>17000-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice

O modelo de corte transversal do nervo tibial é um modelo bem-tolerado, validado, e reprodutível de atrofia do músculo esquelético desnervação induzida em roedores. Embora originalmente desenvolvido e amplamente utilizado no rato, devido ao seu grande tamanho, o nervo tibial em ratinhos é grande o suficiente para que ele possa ser facilmente manipulada com qualquer esmagamento ou corte transversal, deixando os ramos do nervo sural e fibular do nervo ciático intacta e preservando assim seus músculos alvo. Assim, este modelo oferece as vantagens de induzir menor morbidade e impedimento de locomoção do que o modelo de secção do nervo ciático e também permite aos investigadores estudar os mecanismos biológicos fisiológicos, celulares e moleculares que regulam o processo de atrofia muscular em camundongos geneticamente modificados. O nervo tibial fornece os gastrocnêmio, sóleo e plantar, por isso seu corte transversal permite o estudo do músculo esquelético desnervado composto de fibras de contração rápida do tipo II e / ou tipo de contração lenta Ifibras. Aqui demonstramos o modelo de secção do nervo tibial no C57Black6 mouse. Avaliamos a atrofia do músculo gastrocnêmio, como um músculo representante, em 1, 2 e 4 semanas de pós-denervação medindo pesos musculares e tipo de fibra área transversal específica em parafina cortes histológicos histoquímica para miosina de contração rápida.

<html> Secção do nervo tibial denervates o gastrocnêmio, sóleo e plantar do bezerro. Aqui nós avaliar o desenvolvimento de atrofia do músculo gastrocnémio, como um representante do músculo. Músculo do gastrocnémio foi colhido 2-3 meses de idade C57Black seis ratinhos (Jackson Laboratories) desnervados por 1, 2 ou 4 semanas. Pesos do músculo diminuir progressivamente (Figura 1), assim como a área de secção transversal de fibras do tipo II rápidas contração muscular (Figura 2),</st...

Watch this Scientific Journal Video about Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice at JoVE.com

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

O modelo de corte transversal do nervo tibial é um modelo bem-tolerado, validado, e reprodutível de atrofia do músculo esquelético. O protocolo cirúrgico modelo é descrito e demonstrado em ratinhos C57Black6.

Secção do nervo tibial - um modelo padronizado para denervação induzida por atrofia muscular em ratos

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

tibial-nerve-transection-standardized-model-for-denervation-induced

Research

JoVE Journal

Medicine

24.2K visualizações.  St Michaels Hospital.  O modelo de corte transversal do nervo tibial é um modelo bem-tolerado, validado, e reprodutível de atrofia do músculo esquelético. O protocolo cirúrgico modelo é descrito e demonstrado em ratinhos C57Black6. 

Vídeo: Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Dimorfismo Sexo músculo esquelético de Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Medicina

Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P&#60;0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split&#160;hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split&#160;hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.

Dissociated atrophy of intrinsic hand muscles, termed the split hand, appears to be a specific feature of amyotrophic lateral sclerosis (ALS).&#160;Consequently, a novel neurodiagnostic test, termed the split hand&#160;index, was developed to quantify the clinical phenomenon of the split hand, which differentiated ALS from mimic disorders.

Utilitário de Dissociated Intrínseca Mão atrofia muscular no Diagnóstico de Esclerose Lateral Amiotrófica

The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

O ciático Nervo cuffing Modelo de Dor Neuropática em Ratos

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Axotomia do nervo facial em ratos: um modelo para estudo de resposta motoneuron para Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Avaliação ultra-som base de Artéria Coronária Flow e Fluxo Coronariano Reserve Usando o modelo de sobrecarga de pressão no Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Humano<em&gt; vasto lateral</em&gt; Biópsia de músculo esquelético Usando o Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Angiogênese cirúrgica em suínos Allotransplantation Tibial: um novo grande osso Animal vascularizado composto Allotransplantation modelo

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

Semi automatizados de análise da morfologia do músculo esquelético de rato e composição de fibra-tipo

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Um modelo minimamente invasivo para analisar a fratura endocondral cura em ratos sob condições biomecânicas padronizadas

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Avaliação não-invasiva da função de músculos levantadores em ratos

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...