Bu çalışma CIHR Nöromusküler Araştırma Ortaklığı (-; JAEB için 90959 JNM) hibe tarafından desteklenmiştir.

Bundan önce modelini kullanarak, araştırmacılar vücut yönetim kendi kurumun hayvan kullanımından cerrahi protokolü için onay almış olmalıdır. Model Araştırma Etik Kurulu, Hamilton Sağlık Bilimleri Kurumu, McMaster Üniversitesi (KKS # 10-04-24) tarafından onaylanmıştır ve Hayvan Bakım Kanada Konseyi önerileri ile tam uyum içinde gerçekleştirilir. 1. Fare Hazırlık <ol><li> Fare tartılır. 5 izofluran% veya% 2 halotan ile anestezi neden. Cerrah korumak için yeterli bir anestezik atma kullanılan devre sağlamalıdır. 2-3 dakika sonra hayvanın solunum yavaş olacaktır. Göz kırpma refleksi yoktur emin olun ve cerrahi anestezi (fare ile cevap yani) onaylamak için pençe en arasi bosluklari çimdik. Ameliyat sırasında, korneanın kurumasını önlemek için, göz oftalmik yağ uygulanır. </li><li> Siyatik gelen lateral uyluk ve kalça tıraşdiz çentik ve proviodine ile dezenfekte edin. Tıraş cerrahi alanın yeterli görselleştirme sağlamak ve sinir diseksiyonu ve transection müdahale en aza indirmek için kesi saç site ücretsiz devam edecektir. Üstün ve femur posterior olan siyatik çentik, palpasyonla tespit edilebilir. </li></ol> 2. Operatif Prosedürü <ol><li> 2% (halotan% 1) inhalasyon izofluran azaltın ve bir işletim ya da mikroskop altında yan (yukarı bakacak şekilde ameliyat için tasarlanmıştır tarafı), üzerinde fare yerleştirin. 3,5 kat büyütme, bir yetişkin için (20-25 g), fare için yeterli olduğu Alternatif olarak cerrahi cerrahi loupes ile yapılabilir. </li><li> Steril eldiven Don. Palpasyonla siyatik çentik belirleyin. Siyatik çentik gelen diz (yaklaşık 1 cm) için lateral uyluk bir neşter, insizyon deri kullanma. </li><li> Nazikçe cilt yayıldı. Biceps femoris kas belirlemek, hangi lateral uyluk düz yüzeysel kashemen derinin altında. Ince makas kullanarak, kas lifleri boyunca biceps femoris kas bölünmüş ve siyatik sinir ve dalları ortaya çıkarmak için bir yay retraktörü ile açık tutun. </li><li> Biceps femoris kas hemen derin siyatik sinir belirleyin. Bu karakteristik parlak beyaz renk ile tanımlanan ve çapı yaklaşık 0.8 mm olabilir. Bu popliteal fossa düzeyinde, tibial peroneal ve sural sinirler içine dallanma, siyatik çentik gelen diz çalışır. </li><li> Yavaşça ultra ince forseps ve yaylı Microdissecting makas ile peroneal ve sural sinir dallarından tibia ayırın. Tibial sinir en büyük dalıdır ve genellikle merkezi. Bu dalları ayıran sinir ezmek için değil önemlidir. Ultra ince forseps ile sadece dış adventicial katman üzerinde sinir Holding ve sinir gevşek (öğretti değil) tutarak, sinir ezilme ve çekiş yaralanmaları önlemek olacaktır. </li><li> Tam ve kalıcı bir denervati içinüzerinde, dikkatle popliteal damarlar kaçınarak, gibi distalde mümkün olduğunca makas Microdissecting ile tibial sinir kesti. Alternatif olarak, 2-4 hafta içinde beklenen tam reinnervation ile geçici denervasyon için, tibial sinir sadece kesilen 15 sn yerine için ultra ince forseps ile ezilebilir. (Periferik sinirler aşağıdaki yaralanma regrow ve hedef kas reinnervate olacaktır.) </li><li> Tam denervasyon gerekiyorsa, gastroknemius yeniden innervasyonu anormal önlemek için 5-0 Vicryl ile 10-0 Naylon ve yeniden yaklaşık biceps femoris ile biseps femoris kasının ön yüzeyine kesilen tibial sinir sonunda dikiş ve soleus kasları. </li><li> Bir çalışan 5-0 Vicryl dikiş ile cilt kapatın. </li></ol> 3. Operatif Bakım Mesaj <ol><li> Inhalasyon anestezi kapatın ama oksijen akışını sağlamak. Subkutan analjezik buprinorphine (veya yerine) yönetmek. </li><li> Olmayan temiz bir kafes için fareyi transferyatak anestezi uyanma sırasında. Ambulasyon kadar kafes içinde ve doğrudan gözlem altında bir ısınma battaniye tutun. </li><li> Bol yumuşak yatak yumuşak alt kafeste transferi ve ev (değil tel). </li><li> Dekübit topuk ülser veya çiğneme kanıtı gelişimi için cerrahi yara ve ayak durumu için günlük ameliyat bacak kontrol edin. Ufak sorunlar topikal antibiyotik ya da proviodine gibi antiseptikler ile tedavi edilebilir. Hayvan ötenazi gerektiren Endpoint göstergeleri kilo kaybı, kendine bakım kanıtı (karıştırdı kürk), ve kambur duruş vardır. Buna ek olarak, büyük yara bozulma veya topikal antibiyotiklerle 1-2 hafta içinde iyileşmek ya da ağrı görünmüyor ülseri ile hayvanların kurban edilmelidir. </li></ol> 4. Denerve Gastrocnemius ve Soleus Kas Hasat <ol><li> İstenilen sonrası ameliyat süresi noktada, CO 2 doz aşımı ile fare ve kurban tartın. </li><li> Hem de medial Tıraşoperatif ve karşı kontrol bacakları ve alkol ile temiz. Bir işletim ya da mikroskop altında fare yerleştirin, ya da alternatif büyütme için cerrahi görüntüleme loupes kullanın. </li><li> Operatif bacak üzerinde, bir diz ayak bileği neşter ve çevresel olarak ayak bileği etrafında ile medial buzağı cilt insizyon. Yavaşça kas kapalı ve proksimal uyluk doğru forseps ile cilt çekin. Bu, bacak bölgesinin tüm kas ortaya koyar. Diz bacak arka yönü üzerinde ayak bileği için çalışır ve cilt altında hemen yatıyor baldır kas gastroknemius kas, tanımlayın. Diz medial yönü üzerinde gastroknemius kas proksimal biseps femoris kas distal ekleme, tanımlayın. Distal ekleme de biseps femoris ince ve şeffaf görünür ve gastroknemius en proksimal kısmını örter. Makas ve künt sonunda diseksiyon kullanarak, yavaşça gazdan biceps femoris distal ekleme ayırmaktrocnemius kas. </li><li> Calcaneous içine distal ekleme de, gastroknemius kas Aşil tendonu içine tapers. Beyaz ve zinde görünen Aşil tendonu, tanımlayın. Tutun veya gastroknemius kas ezmek için özen, forseps ile Aşil tendonu tutun ve makas kullanarak kalkaneus ekleme gelen Aşil tendonu bölün. </li><li> Hala tendon tutarak, yavaşça diz aslına doğru distal ekleme (soleus ayrı ayrı hasat edilebilir) gelen, derin soleus (derin kırmızı) kapalı gastroknemius kas (soluk kırmızı) kaldırın. </li><li> Makas kullanarak medial ve lateral femur chondyles gelen gastroknemius kökeni bölerek bacak kapalı gastrocnemius parçalara ayır. Kas çok nazik çekiş bu süreci kolaylaştırır. Kas ezmek için dikkatli olun. </li><li> Soleus şimdi hemen gastroknemius sitenin altında, açıkça görülebilir. Için Aşil tendonu üzerindeki ekleme gelen soleus kaldırın onun veyaarka buzağı üzerinde igin. Soleus yanlışlıkla gastroknemius ile kaldırılırsa, yavaşça hasat örnekten ayırmak. Gastrokinemius (açık kırmızı) ve soleus (koyu kırmızı), renk farklılıkları nedeniyle hasat örnekte kolayca tanımlanabilir kalır. </li><li> , Bir hassas terazi üzerinde ayrı ayrı kas tartılır. </li><li> (Sonraki protein ve / veya RNA ekstraksiyonu için) sıvı azot içinde ek dondurma için dikey yarım kaslar ayrıldı ve histoloji (yani morfolojik değerlendirmesi, immünohistoloji) için yarım izopentan içinde% 10 formalin ile, ya da dondurulmuş sabitleme ya da sabitleme sıvı azot ile soğutulmuş istendiği gibi. </li><li> Kontrolü gastroknemius ve soleus kasları hasat için kontrol yukarıdaki adım 4.3, un-çalışan, taraftan tekrarlayın. </li></ol>

<ol>
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Çıkar çatışması ilan edilir.

Denervasyon bağlı iskelet kas atrofi tibial sinir kesisi modeli sıçanlarda yaygın olarak kullanılan ve iyi onaylanmış modelidir. Biz araştırmacı genetik farenin varlığını yararlanmak ve kas kütlesi 7,8 düzenlenmesinde çok önemli proteinlerin yokluğunda in vivo olarak kas atrofisi süreci çalışma sağlar farenin olarak kullanımı Bu modeli adapte edilmiştir. Gastroknemius ve soleus kas, bu modelde denerve hem de böylece daha sonraki moleküler analiz için mükemmel kalitede ...

Travmatik periferik sinir yaralanma, hastalık veya farmakolojik müdahale nedeniyle İskelet kası denervasyon, kas gönüllü kasılma fonksiyonu hemen kaybına neden olur. Kas eş zamanlı atrofi başlar ve zamanında, kaliteli reinnervation 1,2 oluşursa bu atrofi tersine çevrilebilir. Reinervasyon yokluğunda, myofiber atrofi ilerler ve geri dönüşü olmayan bir biyolojik kas değişiklikleri kas fibroz ve myofiber ölüm meydana gelir. Burada farelerde tibial sinir transection modeli, denervasyon-bağlı iskelet kas atrofisi ve fibrozis bir model, göstermektedir. Bu model gastroknemius ve soleus kaslarında in vivo kas atrofi altında yatan, fizyolojik hücresel ve moleküler biyolojik mekanizmaları incelemek için bilim adamları sağlar. Tarihsel sıçanlarda ağırlıklı olarak kullanılır iken, nakavt ve özellikle transgenik fare hatları, bu modelin daha yeni uygulama, araştırmacılar kendi rolünü değerlendirmenizi sağlarin vivo indüksiyon, geliştirme ve bakım veya alternatif çözümü, kas atrofi ve fibrozis ilgi özellikle protein (ler). Tibial sinir kemirgen bacak bir karma motor duyusal periferik sinir ve siyatik sinirin üç terminali dallarından biridir. Tibial sinir kesisi gastroknemius, soleus ve plantaris kas (ve posterior tibial, fleksör digitorum longus ve fleksör hallicus longus dahil olmak üzere ayağın üç küçük derin fleksör kaslar) denervates ve sıçanlarda 3,4 bir iyi standart ve onaylanmış model . Gastroknemius ve soleus kasları kolayca tibial sinir kesisi, sabit ve eğitim amacıyla kas RNA ve protein çıkarılması için dondurulmuş kas histoloji ve kas lifi morfometrik, veya flash değerlendirilmesi için işlenmiş, örneğin, sonrası seri zaman noktalarında disseke edilebilir kas atrofi düzenleyen hücresel sinyal ağları. Gastrocnemius kas karışık elyaf tipi kas (tip I ve tip II, tip II, ancak ağırlıklı olarak) ve soleus kasının ve böylece 5,6 değerlendirilmesi için hızlı ve yavaş hem kasılan kas sağlayarak, tip I lifleri büyük bir kısmı oluşmaktadır. Tibial sinir kesisi modeli (ay hafta) kısa vadeli (gün) 7 ve uzun vadeli hem de 4,8 de denervasyon bağlı kas atrofi süreci çalışmak için uygundur. Siyatik sinir transeksiyonu modeli (genellikle kemirgenlerde kullanılan denervasyon bağlı kas atrofi bir ikinci model) aksine, tibial sinir kesisi bir daha çekici model yapımı, hayvan daha az morbidite neden olur. Tibial sinir kesisi siyatik sinirin peroneal ve sural sinir dalları olduğu gibi bırakır ise siyatik sinir kesisi böylece koruyarak, 2 ambulasyon için hayvanın yeteneğini bozarak, bacak (diz altında) ve ayak tüm kasları denervateshedef kas ve duyusal toprakları. Fare plantar esnek yapamaz veya ayak invert, ancak kolayca ambulasyon yapabiliyor ve ağırlığı böylece önemli ölçüde modelin morbidite azalan, her iki arka ayakları üzerinde eşit taşımaktadır. Yürüyüş desen değerlendiren yürüme analizi çalışmaları tibial ve siyatik sinir yaralanmaları sonrasında sıçanlarda yapılan ve ayak izi ve yük daha tibia yaralanma 9,10 ile korunmuş olduğunu göstermektedir. Edilmiştir Çalışma tasarımı 3 gerektiriyorsa, ek olarak, tibial sinir transection modelinde, peroneal sinir daha sonra bir noktada seferber edilebilir ve gecikmiş reinnervation kaynağı olarak aktarılır. Buna karşılık, siyatik sinir kesisi modelinde gecikmeli reinnervation çok önemli modelin teknik zorluk artan ve yetenekli cerrahlar onun kullanımını sınırlayan, siyatik sinir açığı bir sinir greft kullanılması gerekmektedir. Ise tibial sinir transeksiyonu model rbu innervates tibial sinir ve baldır kasları hem hayvan cerrahi steril ameliyat tekniği ile operatörün aşinalık, equires cerrahlar değil, ya da çok hayvan cerrahi deneyimli kişiler, kolayca bu model ana böylece, manipülasyon için kolay erişilebilir ve tanımlanabilir .

<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

Tibial sinir kesisi modeli kemirgenlerde denervasyon bağlı iskelet kas atrofisi bir, iyi tolere doğrulanmış ve tekrarlanabilir bir model. Geliştirildiği ve daha büyük boyutu nedeniyle sıçan yaygın olarak kullanılmasına rağmen, farelerde tibial sinir siyatik sinirin peroneal ve sural sinir dalları sağlam ve böylece muhafaza bırakarak, kolayca ezmeyin veya kesisi ya ile manipüle edilebilir yeterince büyük hedef kasları. Bu nedenle, bu model siyatik sinir transeksiyonu modeli daha az morbidite ve ambulasyon engel uyaran avantajları sunuyor ve aynı zamanda araştırmacılar genetik olarak farelerde kas atrofi süreci düzenleyen, fizyolojik hücresel ve moleküler biyolojik mekanizmalar çalışma sağlar. Tibial sinir gastroknemius, soleus ve plantaris kasları sağlar, bu yüzden onun transection hızlı kasılan tip II lifleri ve / veya yavaş kasılan tip oluşan Denerve iskelet kası çalışma izin Ilifler. Burada C57Black6 fare tibial sinir transeksiyonu modeli göstermektedir. Biz parafine gömülü histolojik bölümlerde kas ağırlık ve lif tipi özel kesit alanı ölçerek 1, 2, bir temsilci kas olarak, gastroknemius kas atrofi değerlendirmek ve 4 hafta sonrası denervasyon hızlı kasılan miyozin için immunohistokimyasal.

<html> Tibial sinir kesisi gastroknemius, soleus ve buzağı plantaris kasları denervates. Burada bir temsilci kas olarak, gastroknemius kas atrofi gelişimini değerlendirmek. Gastrokinemius kası, 1, 2 ya da 4 hafta boyunca denerve 2-3 aylık C57Black 6 fareleri (Jackson Laboratuarları) hasat edilmiştir. Kas ağırlıkları kademeli olarak zaman içinde tip II hızlı kasılan kas lifleri (Şekil 2), enine kesit alanı yapar (Şekil 1) azalır. Gastrokinemius karışık lif tipi kas...

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

Tibial sinir kesisi modeli iskelet kas atrofi bir, iyi tolere doğrulanmış ve tekrarlanabilir bir model. Model cerrahi protokol C57Black6 farelerde açıklanan ve gösterilmiştir.

Tibial Sinir transeksiyonu - Farelerde denervasyon bağlı İskelet Kas Atrofi için bir Standart Model

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 Görüntüleme.  St Michaels Hospital.  Tibial sinir kesisi modeli iskelet kas atrofi bir, iyi tolere doğrulanmış ve tekrarlanabilir bir model. Model cerrahi protokol C57Black6 farelerde açıklanan ve gösterilmiştir. 

Video: 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.

İskelet Kası Cinsiyet Proteomik Dimorfizm

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

Tıp

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.

Amyotrofik Lateral Skleroz Tanısında Disosiye ıntrinsic El kas Atrofi Utility

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.

Farelerde Nöropatik Ağrı Siyatik Sinir cuffing Modeli

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.

Farelerde Yüz Sinir Aksotomi: Sakatlık Motonöron Yanıtı Eğitim için Bir Model

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.

Farelerde Basınç Aşırı Modeli Koroner Arter Akış ve Koroner Akım Rezervi Ultrason Dayalı Değerlendirme

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.

İnsan<em&gt; Vastus lateralis</em&gt; Weil-Blakesley Conchotome kullanılması İskelet Kası Biyopsi

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.

Domuz Tibial Allotransplantation cerrahi angiogenez: bileşik Allotransplantation Model yeni bir büyük hayvan kemik skarların

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.

Fare kas iskelet morfoloji ve Fiber türü kompozisyon yarı otomatik analizi

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.

Endochondral standart biyomekanik koşullar altında farelerde şifa kırık çözümlemek için minimal invaziv bir Model

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.

Non-invaziv Dorsiflexor kas fonksiyonu farelerde değerlendirilmesi

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