Questo lavoro è stato sostenuto da sovvenzioni dal partenariato Cihr neuromuscolare della ricerca (JNM - 90.959; al Jaeb).

Prima di utilizzare questo modello, i ricercatori devono aver ricevuto l&#39;approvazione per il protocollo chirurgico dall&#39;uso degli animali della loro istituzione organo di governo. Il modello è approvato dal Consiglio di etica della ricerca, Hamilton Health Sciences Corporation, McMaster University (AUP # 10-04-24) e viene effettuata in stretta conformità con le raccomandazioni del Consiglio canadese sulla cura degli animali. 1. Topo Preparazione <ol><li> Pesare il mouse. Indurre l&#39;anestesia con il 5% isoflurano o 2% alotano. Il circuito utilizzato dovrebbe garantire un&#39;adeguata scavenging dell&#39;anestetico per proteggere il chirurgo. Dopo 2-3 minuti di respirazione dell&#39;animale rallenterà. Assicurarsi che il riflesso corneale è assente e pizzicare gli spazi interdigitali della zampa per confermare l&#39;anestesia chirurgica (cioè nessuna risposta dal mouse). Applicare oftalmica lubrificante per gli occhi, per evitare l&#39;essiccazione della cornea durante l&#39;intervento chirurgico. </li><li> Shave laterale della coscia e gluteo dalla sciaticotacca per il ginocchio e disinfettare con proviodine. Rasatura manterrà il sito del pelo libero incisione per garantire un&#39;adeguata visualizzazione del campo chirurgico e per minimizzare l&#39;interferenza con dissezione e transezione del nervo. La tacca sciatico, che è superiore e posteriore al femore, può essere identificato con la palpazione. </li></ol> 2. Procedura Operativa <ol><li> Ridurre inalatoria isoflurano al 2% (alotano 1%) e posizionare il mouse su un fianco (lato destinata all&#39;intervento rivolto verso l&#39;alto), sotto un microscopio operatorio o dissezione. In alternativa, la chirurgia può essere eseguita con gli occhialini chirurgico poiché ingrandimento 3,5 x è soddisfacente per un topo adulto (20-25 g). </li><li> Indossare guanti sterili. Identificare la tacca sciatico con la palpazione. Usando un bisturi, incidere la pelle della coscia laterale dalla tacca sciatico al ginocchio (circa 1 cm). </li><li> Diffondere delicatamente la pelle. Identificare il muscolo bicipite femorale, che è il muscolo superficiale piatto della coscia lateraleimmediatamente sotto la pelle. Utilizzando le forbici sottili, dividere il muscolo bicipite femorale lungo le fibre muscolari e tenere aperto con un divaricatore molla per esporre il nervo sciatico e suoi rami. </li><li> Identificare il nervo sciatico immediatamente profondo al muscolo bicipite femorale. Esso può essere identificato dal suo caratteristico colore bianco lucida ed è circa 0,8 mm di diametro. Si corre dalla tacca sciatico al ginocchio, ramificazione nei nervi tibiale, peroneale e surale a livello della fossa poplitea. </li><li> Separare delicatamente il tibiale dai rami del nervo peroneo e surale con pinze ultrafine e la primavera forbici microdissecting. Il nervo tibiale è il più grande ramo ed è solitamente centrale. E &#39;importante non schiacciare il nervo mentre separando i rami. Tenendo il nervo solo sullo strato esterno adventicial con le pinze ultrafini, e mantenendo il gioco nervo (non insegnato), eviterà schiacciamento del nervo e lesioni trazione. </li><li> Per una completa e duratura denervatisu, tagliare il nervo tibiale con microdissecting forbici come distalmente possibile, evitando accuratamente i vasi poplitea. In alternativa, per la denervazione temporaneo previsto reinnervazione completa in 2-4 settimane, il nervo tibiale può essere semplicemente schiacciato con una pinza ultrafini per 15 sec invece di transected. (Nervi periferici ricrescere seguenti lesioni e saranno Reinnervate muscolo bersaglio.) </li><li> Se è richiesta la completa denervazione, suturare l&#39;estremità del nervo tibiale transected alla superficie anteriore del muscolo bicipite femorale con 10-0 Nylon e ri-approssimare il bicipite femorale con 5-0 Vicryl per impedire aberrante reinnervazione dei gastrocnemio e soleo. </li><li> Chiudere la pelle con una corsa 5-0 sutura Vicryl. </li></ol> 3. Posta operatorio <ol><li> Spegnere l&#39;anestetico inalatorio, ma mantenere il flusso di ossigeno. Somministrare buprinorphine (o sostituto) analgesico per via sottocutanea. </li><li> Passa il mouse in una gabbia pulita senzabiancheria da letto, mentre risveglio dall&#39;anestesia. Tenere su una coperta di riscaldamento all&#39;interno della gabbia e sotto diretta osservazione fino deambulazione. </li><li> Il trasferimento e la casa in morbido movimento centrale (non filo) con ampio letto morbido. </li><li> Ispezionare l&#39;arto operativa quotidiana per la condizione della ferita chirurgica e il piede per lo sviluppo di ulcere da decubito del tallone o prova di masticazione. I problemi minori possono essere gestiti con antibiotici topici o antisettici come proviodine. Indicatori Endpoint richiedono l&#39;eutanasia degli animali sono la perdita di peso, la prova di scarsa cura di sé (pelo arruffato), e postura ricurva. Inoltre, gli animali con gravi disagi ferite o ulcere che non guariscono in 1-2 settimane con antibiotici topici o sembrano avere dolore dovrebbero essere sacrificati. </li></ol> 4. Gastrocnemio e soleo Denervato Harvest muscolare <ol><li> Al punto di tempo post-operatorio desiderato, pesare il mouse e il sacrificio con il sovradosaggio di CO 2. </li><li> Shave faccia mediale sia deloperative e le gambe di controllo controlaterale e pulito con l&#39;alcol. Posizionare il mouse sotto un microscopio operatorio o dissezione, o in alternativa utilizzare lenti di ingrandimento di visualizzazione chirurgico per l&#39;ingrandimento. </li><li> Sull&#39;arto operativa, incidere la pelle mediale vitello con un bisturi dalla caviglia al ginocchio e circonferenzialmente intorno alla caviglia. Estrarre delicatamente la pelle con una pinza fuori il muscolo e prossimalmente verso la coscia. Ciò espone tutti i muscoli della gamba. Identificare il muscolo gastrocnemio, che è il muscolo del polpaccio che va dal ginocchio alla caviglia sulla faccia posteriore della gamba e si trova immediatamente sotto la pelle. Identificare l&#39;inserzione distale del muscolo bicipite femorale, prossimale al muscolo gastrocnemio sulla faccia mediale del ginocchio. Alla sua inserzione distale del bicipite femorale appare sottile e vaporosa e sovrappone la parte più prossimale del gastrocnemio. Utilizzando le forbici e la dissezione estremità smussata, separare delicatamente l&#39;inserzione distale del bicipite femorale dal gasmuscolo trocnemius. </li><li> Alla sua inserzione distale nel calcagno, il muscolo gastrocnemio rastrema nel tendine di Achille. Identificare il tendine di Achille, che appare bianco e nervosa. Tenere il tendine di Achille con una pinza, facendo attenzione a non tenere o schiacciare il muscolo gastrocnemio, e dividere il tendine di Achille dall&#39;inserimento calcagno con le forbici. </li><li> Sempre tenendo il tendine, sollevare delicatamente il muscolo gastrocnemio (rosso chiaro) dai soleo profonde (più profonde rosso), da l&#39;inserzione distale verso la sua origine al ginocchio (il soleo può essere raccolto separatamente). </li><li> Sezionare il gastrocnemio largo della gamba dividendo l&#39;origine delle gastrocnemio dai chondyles femore mediale e laterale con le forbici. Trazione molto delicato sul muscolo facilita questo processo. Fare attenzione a non schiacciare il muscolo. </li><li> Il soleo saranno ora chiaramente visibile, immediatamente sotto il sito del gastrocnemio. Sollevare il soleo dalla sua inserzione sul tendine di Achille alla sua oigin sul polpaccio posteriore. Se il soleo viene inavvertitamente sollevato con il gastrocnemio, separare delicatamente dal campione raccolto. Il gastrocnemio (luce rossa) e soleo (rosso scuro) rimangono facilmente identificabile nel campione raccolto a causa delle loro differenze di colore. </li><li> Pesare i muscoli separatamente su una bilancia di precisione. </li><li> Dividere i muscoli verticalmente, metà per scatto congelamento in azoto liquido (per successiva proteine ​​e / o estrazione di RNA), e metà per istologia (cioè la valutazione morfometrica, immunoistologia) fissa sia in formalina al 10%, o fissazione congelati in isopentano raffreddato con azoto liquido , se lo desideri. </li><li> Ripetere dal punto 4.3 sopra il controllo, non operato, fianco a raccogliere gastrocnemio di controllo e muscoli soleo. </li></ol>

<ol>
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Il nervo modello di resezione tibiale di atrofia del muscolo scheletrico denervazione-indotta è un modello comunemente impiegato e ben convalidato nei ratti. Abbiamo adattato questo modello per l&#39;uso in topi, che consente al ricercatore di sfruttare la presenza di topi geneticamente ingegnerizzati e studiare il processo di atrofia muscolare in vivo, in assenza di proteine ​​fondamentali per la regolazione della massa muscolare 7,8. I muscoli gastrocnemio e soleo, sia denervato in questo mode...

Denervazione del muscolo scheletrico, a causa di una lesione traumatica dei nervi periferici, malattie o intervento farmacologico, comporta l&#39;immediata perdita della funzione contrattile del muscolo volontario. Muscle inizia in concomitanza ad atrofizzarsi e questa atrofia è reversibile se tempestivo, reinnervazione di buona qualità si verifica 1,2. In assenza di reinnervazione, myofiber atrofia progredisce, e cambiamenti biologici irreversibili nel muscolo si verifica con fibrosi muscolare e morte miofibre. Qui mostriamo il modello nervo tibiale resezione, un modello di denervazione indotta-atrofia del muscolo scheletrico e la fibrosi, nei topi. Questo modello consente agli scienziati di studiare le fisiologiche, cellulari e molecolari meccanismi biologici che sono alla base atrofia muscolare in vivo nei muscoli gastrocnemio e soleo. Mentre storicamente utilizzato prevalentemente nei ratti, di più recente applicazione di questo modello ad eliminazione diretta e le linee di topi transgenici in particolare, consente agli investigatori di valutare il ruolo dei loroparticolare proteina (s) di interesse nella induzione, sviluppo e manutenzione, o in alternativa la risoluzione, atrofia muscolare e fibrosi in vivo. Il nervo tibiale è un nervo periferico motore-sensoriale mista nel hindlimb roditore, ed è uno dei tre rami terminali del nervo sciatico. Recisione del nervo tibiale denervates gastrocnemio, soleo e muscoli plantare (e le tre piccoli muscoli flessori profondi del piede compreso tibiale posteriore, flessore lungo delle dita e flessore hallicus longus), ed è un modello ben standardizzati e validati nei ratti 3,4 . I muscoli gastrocnemio e soleo può essere facilmente sezionato in momenti seriali postare recisione del nervo tibiale, fissati e trattati per la valutazione di istologia dei muscoli e fibre muscolari morfometria, o Flash congelati per l&#39;estrazione di RNA e proteine ​​muscolari a scopo di studio, per esempio, le reti cellulari di segnalazione che regolano l&#39;atrofia muscolare. La gastrocnemius muscolare è un tipo di fibra muscolare misto (tipo I e tipo II, anche se prevalentemente di tipo II) e il muscolo soleo è composto da una grande percentuale di fibre di tipo I, fornendo in tal modo sia veloce e lenta contrazione muscolare per la valutazione 5,6. Il modello di resezione del nervo tibiale è adatto per studiare il processo di denervazione indotta da atrofia muscolare, sia a breve termine (giorni) 7 e lungo termine (settimane o mesi) 4,8. In contrasto con il modello transezione del nervo sciatico (un secondo modello di denervazione indotta atrofia muscolare comunemente usato nei roditori), transezione del nervo tibiale induce meno morbilità nell&#39;animale, rendendolo un modello più attraente. Transezione del nervo sciatico denervates tutti i muscoli della gamba (sotto il ginocchio) e piede, riducendo la capacità del animale di deambulare 2, mentre transezione del nervo tibiale lascia i rami del nervo peroneo e surale del nervo sciatico intatto, preservando cosìi loro muscoli bersaglio e territori sensoriali. Il mouse è in grado di flessione plantare oppure invertire il piede, ma è in grado di deambulare facilmente e sopporta il peso equamente sui due arti posteriori, diminuendo così significativamente la morbidità del modello. Andatura studi di analisi che valutano i modelli a piedi sono stati condotti in ratti dopo lesioni del nervo tibiale e sciatico e dimostrare che ingombro e peso del cuscinetto è meglio conservato con lesione tibiale 9,10. Inoltre, nel modello transezione del nervo tibiale, il nervo peroneo può essere mobilizzato in un punto temporale successivo e trasferita come fonte di ritardata reinnervazione, se il disegno studio richiede 3. Al contrario, ritardato reinnervazione nel modello transezione del nervo sciatico richiede l&#39;uso di un innesto nervoso al deficit nervo sciatico, molto aumentando notevolmente la difficoltà tecnica del modello e limitazione all&#39;uso chirurghi qualificati. Mentre il nervo tibiale modello transection requires familiarità dell&#39;operatore con tecnica operatoria sterile nella chirurgia degli animali, sia il nervo tibiale e muscoli del polpaccio si innerva sono facilmente accessibili e identificabili per la manipolazione, in modo che gli individui che non sono chirurghi, o di grande esperienza con la chirurgia degli animali, possono facilmente padroneggiare questo modello .

<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

Il modello di resezione del nervo tibiale è un modello ben tollerato, validato e riproducibile di denervazione indotta atrofia del muscolo scheletrico nei roditori. Benché originariamente sviluppato e ampiamente utilizzato nel ratto causa delle sue grandi dimensioni, il nervo tibiale nei topi è abbastanza grande da poter essere facilmente manipolato con uno schiacciamento o transezione, lasciando i rami peroneale e surale del nervo sciatico intatto e preservando così i loro muscoli bersaglio. Così, questo modello offre i vantaggi di indurre minore morbilità e impedimento della deambulazione rispetto al modello resezione del nervo sciatico e permette anche ai ricercatori di studiare i fisiologici, cellulare e molecolare dei meccanismi biologici che regolano il processo di atrofia muscolare nei topi geneticamente modificati. Il nervo tibiale fornisce il gastrocnemio, soleo e muscoli plantare, quindi la sua recisione permette lo studio del muscolo scheletrico denervato composto da fibre a contrazione rapida di tipo II e / o tipo di contrazione lenta iofibre. Qui mostriamo il modello recisione del nervo tibiale nel topo C57Black6. Valutiamo l&#39;atrofia del muscolo gastrocnemio, come un muscolo rappresentante, a 1, 2 e 4 settimane dopo la denervazione misurando pesi muscolari e l&#39;area specifica il tipo di fibra trasversale su paraffina sezioni istologiche immunocolorate per una rapida contrazione della miosina.

<html> Recisione del nervo tibiale denervates gastrocnemio, soleo e muscoli plantare del polpaccio. Qui valutiamo lo sviluppo di atrofia nel muscolo gastrocnemio, come un muscolo rappresentante. Muscolo gastrocnemio è stato raccolto da 2-3 mesi vecchi C57Black 6 topi (Jackson Laboratories) denervati per 1, 2, o 4 settimane. Pesi muscolari diminuiscono progressivamente (figura 1), così come l&#39;area di sezione trasversale di tipo II fibre muscolari a contrazione rapida (Figura 2), nel...

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

Il modello di resezione del nervo tibiale è un modello ben tollerato, validato e riproducibile di atrofia del muscolo scheletrico. Il protocollo chirurgico modello è descritto e dimostrato in C57Black6 topi.

Resezione del nervo tibiale - Un modello standardizzato per Denervation indotta scheletrico atrofia muscolare nei topi

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

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JoVE Journal

Medicine

24.3K Views.  St Michaels Hospital.  Il modello di resezione del nervo tibiale è un modello ben tollerato, validato e riproducibile di atrofia del muscolo scheletrico. Il protocollo chirurgico modello è descritto e dimostrato in C57Black6 topi. 

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-meccanico Analisi dei isolata del muscolo scheletrico

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

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Medicina

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Risparmiato il Nerve pregiudizio (SNI) Modello di Allodinia meccanica indotta nei topi

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

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.

Sesso Dimorfismo muscolo scheletrico da Proteomica

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

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.

Il nervo sciatico ammanettare modello di dolore neuropatico nei topi

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

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Scheletrici Procedure biopsia muscolare dell&#39;uomo con la tecnica Bergström Modificato

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Un semplice Critical dimensioni femorale Difetto Modello in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

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.

Facial assotomia Nerve in topi: un modello per studiare motoneuroni risposta al danno

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.

Valutazione Ecografia base di coronaropatia Flow e flusso coronarico Reserve Utilizzando la pressione Sovraccarico modello nei topi

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.

Umano<em&gt; vasto laterale</em&gt; Biopsia muscolare scheletrico Utilizzando la 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.

Chirurgica dell'angiogenesi in porcina Allotransplantation Tibial: un nuovo grande osso animale vascolarizzato Allotransplantation composito modello

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

Resezione del nervo tibiale - Un modello standardizzato per Denervation indotta scheletrico atrofia muscolare nei topi

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