We wish to thank the study participants for making this work possible and the staff at the NIHR Southampton Biomedical Research Centre and the Wellcome Trust Clinical Research Facility, University Hospital Southampton for assistance with study measurements.
This work was supported by the NIHR Southampton Biomedical Research Centre, Nutrition, the MRC Lifecourse Epidemiology Unit and the University of Southampton. This report is independent research by the NIHR BRC Funding Scheme. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. AMB is supported by a NIHR Clinical Research Fellowship, HPP is supported by the NIHR Southampton Biomedical Research Centre.

Etik bildirimi: bizim kurumda izleyin kas biyopsisi işleminin bir açıklaması şimdi izler. kas biyopsisi Hertfordshire Araştırma Komitesi numarası 07 / Q0204 / 68 tarafından onaylandı Hertfordshire Sarkopeni Çalışmasında kullanılır. Tüm katılımcılar bilgilendirilmiş onam 20 yazılı verdi. NOT: Tek bir operatör kaygıyı azaltmak ve onlar her zaman rahat olmasını sağlamak amacıyla konuşma katılımcıyı meşgul olabilir bir asistan yardımıyla prosedürü gerçekleştirebilir. Prosedür odada lavabo, yüksekliği ayarlanabilir kanepe, temiz yüzeyler ve bir prosedür arabası ile donatılmış olmalıdır. 1. Katılımcı hazırlayın (Şekil 2) <ol><li> Bir kanepede yatar, rahat bırakmaya katılımcıdan. kasık kırışık tercih edilen uyluk Açığa. maruz uyluk altında atılabilir emici tabakanın yayıldı. bacak prosedürü boyunca düz ve rahat kalmasını sağlamak amauyluk vastus lateralis anahat an gerildi olduğunu. </li><li> Patellanın anterior superior iliyak hayali bir satır aşağı Mark prosedürü sitesi yaklaşık üçte ikisi. </li></ol> 2. Cilt hazırlayın (Şekil 2) <ol><li> Steril bir tıraş bıçağı ile önerilen biyopsi bölgesini çevreleyen çapı yaklaşık 4 cm cilt Tıraş ve alkollü bir bezle cildi temizleyin. NOT: Bu iyileşme sürecinde yara sonraki saç sıkışmasını önlemek içindir. Bu adım operatörün tercihine göre isteğe bağlıdır. </li><li> lokal anestezik ile deri ve örten fasya Infiltrate. Daha sonra 23 G iğne ile deri altı doku içine derin sızmak% 2 lidokain subkutan bleb 25 G iğne kullanın. Biyopsi yerinde deri altı doku içeriğine bağlı olarak bir inç en az yarısı nüfuz hedefliyoruz. Bu derinlik üzerinde uzanan Fas sağlamak için yeterli olmalıdırCIA anestezi. eylem anestezi için 5 dakika - 2 izin verin. İğne veya neşter bıçağı ucu ile cildi nazikçe sondalama ile anestezi onaylayın. </li></ol> 3. Steril Alan hazırlayın (Şekil 2) <ol><li> sabunla ellerinizi yıkayın ve steril bir cüppe giyilir kadar temiz eller korumak. Prosedür arabası üzerinde steril bir alan oluşturmadan önce steril eldiven ile el örtün. </li><li> % 2 klorheksidin / izopropil alkol solüsyonu veya iyot bazlı cilt dezenfektan ile biyopsi sitesini sterilize edin. biyopsi sitesi açığa çıkarmak için bir yapıştırıcı açıklığı ile bir steril örtüyü uygulayın ama aynı zamanda steril bir alan sağlamak için. </li></ol> 4. Prosedürü uygulayın (Şekil 2) <ol><li> ve aşağı ve örten fasya yoluyla deri üzerinde 10 mm kesi - bir boyut 11 neşter ile, bir 5 yapmak. bir inç bir derinlik ortalama yarıya, femur uzun eksenine dik açıyla kesikten conchotome kapandı ısırma ucu takın. Gerekli kullanım durumundaserbest el tutun ve conchotome ucu kası içinde açılan ve kapanan iken biyopsi bölgesini çevreleyen uyluk desteklemek. </li><li> kas kesmek için 180 ° - 90 ile conchotome döndürün. conchotome çekiniz ve steril normal salin ile nemlendirilmiş steril gazlı bez üzerine ucu açın. Gerekirse yeterli kas dokusu elde etmek, tek yara site içinde aynı işlemi tekrarlayın. buz üzerine yerleştirilir, bir kap içine gazlı bez aktarın. yerel kurumsal protokole göre kas işlemek için bir hazırlık laboratuara kabı aktarın. </li></ol> 5. Mesaj Prosedürü (Şekil 2) <ol><li> En fazla 5 dakika süreyle yaraya doğrudan basınç uygulayın. potansiyel skar dokusunun çıkartılması ve steri-şeritler yanlışlıkla kaldırılır eğer yara açılmasının önlemek için dik açılarda aksine onları yaraya paralel koyarak steri-şeritler ile yarayı kapatmak. steri-şeritler üzerinde steril bir emici pansuman yerleştirin ve iki katman kravatsıkıştırma için bandaj ve bant ile sabitleyin. </li><li> İşlemden sonra 4 gün - onlar bir giyinme bir değişiklik 3 gerçekleştirmek zorunda kalacak gibi katılımcıya yara pansuman yöntemi açıklamak. katılımcı daha sonra o akşam yatmadan önce sıkıştırma bandaj kaldırır emin olun. </li></ol> 6. Mesaj Biyopsi Önerileri <ol><li> O hafif egzersiz ile giderilebilir (örneğin., Yürüme) bazı uyluk sertlik yaşamaya yaygındır katılımcıyı bilgilendirin. asetaminofen gibi basit analjezi kullanın. iki haftaya kadar yanı sıra yara enfeksiyonu potansiyel komplikasyonu devam edebilir biyopsi site çevresinde geçici uyuşma ile ilgili katılımcıyı uyar. </li></ol> Konu Gönder Usulü 7. Talimatları <ol><li> (Örneğin, çalışan, ağır kaldırma tırmanma gibi) 72 saat boyunca şiddetli aktiviteyi engellemek için katılımcı isteyin. 72 saat - 48 su içinde daldırma önlemek için katılımcı isteyin. Bununla birlikte, ve katılımcı talimatsoyunma kuru tutmak için zaman duş sahip t sarılmak film veya biyopsi site çevresinde benzer sarmak için. </li><li> 4 gün ve bir hafta sonra steri-şeritleri kaldırmak - 3 sonra soyunma değiştirmek için katılımcı isteyin. NOT: katılımcı sonrası biyopsi bakımı, yedek sosları ve prosedürü uygulamadan sorumlu ekibin acil iletişim bilgileri için yazılı talimatlar verilmelidir. </li></ol>

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None declared.

katılımcı güvenliğini sağlamak ve verimli bir kas biyopsisi yapmak için, protokol dahilinde kritik adımlar dikkat etmek çok önemlidir. Katılımcılar dikkatle kanama ve kötü yara iyileşmesi olarak komplikasyonları önlemek için dışlama kriterleri dikkate sonra seçilmelidir. prosedür boyunca sıkı asepsi esastır. Doğru tekniğin uygulama, katılımcı konfor sağlamak katılımcının dokularına istenmeyen travmayı en aza indirmek ve analiz için yeterli bir örnek ve...

Bir miyopati ve diğer nöromüsküler dejeneratif hastalıkların tanısı için iskelet kası elde edilmesi kabul edilebilir ve hastalar 1 ile incapacitating olmayan güvenli ve etkin yöntemlere dayanır. Tarihsel olarak, analiz için kas dokusu elde etme yöntemleri, genel anestezi altında veya otopsi gerçekleştirilen açık biyopsi alındı. Bu teknikler kas doğrudan görselleştirme izin ve kas daha büyük bir parça biyopsi için izin verdi. bu teknikler hastanede kalış ve iyileşme açısından hastaya sahip potansiyel dezavantajları göz önüne alındığında, perkütan &#39;yarı açık&#39; kas biyopsi yöntemleri alternatif olarak tanıtıldı. Bu teknik ilk trokar ile kendinden inşa iğne kullanılan ve genel anestezi 2 olmadan yaşayan bir denekten kas örnek elde etmek mümkün oldu Duchenne (1806-1875) tarafından yapılmıştır. O zamandan beri, biyopsi iğne çeşitli biçimleri 3,4,5 kullanılmıştır. Bununla birlikte, biyopsi n1962 yılında Bergstrom tarafından geliştirilen eedle klinik pratikte en yaygın kullanılan olmuştur; Gerek çocuklarda gerekse yetişkinlerde de, yanı sıra araştırma 6,7,8,9,10 yılında. Bu keskin trokar, bir kesim kanül ve doku sonrası biyopsi çıkarmak için bir itme çubuğu sahiptir. Örnekleme 11,9 tekrarlanan sonra bu prosedür elde edilen kas verimleri 25 -293 mg arasında değiştiği rapor edilmiştir. Henriksson (Şekil 1) 12 alternatif yarı açık kas biyopsisi yöntemi olarak 1979 yılında Weil-Blakesley conchotome tanıttı. enstrüman Bergstrom yapısının aksine tek yapıdır ve keskin bir ısırma uçlu bir forsepsi gibi tasarlanmıştır. uç boyutu 4 mm genişliğinde 6 mm arasında olabilir. ısırma ucunun iki kenarı karşı zaman, içi boş bir kas yakalama sağlar olacağı şekilde oluşturulmuştur. conchotome 5 arasına sokulur - 10 mm cilt insizyonu ve bir scalpe olarak kas ve örten fasya nüfuz bir trokar ihtiyacını ortadan kaldırırl kas aşağı bir parça yapmak için kullanılabilir. Bu yöntem, manevra 13,14 yüksek derecede doku penetrasyonu kontrol sağlar. Bu yersiz basınç potansiyel örneğin nörovasküler veya altta yatan kemik yapısına zarar verebilir siteler için özellikle yararlıdır., Tibialis anterior 15,14 yerinde. conchotome kullanarak kas verim değişken olabilir ve pratikte 20 - kas 200 mg tekrar numune alındıktan sonra elde edilmiştir. Bergstrom iğne üzerinde conchotome potansiyel bir avantajı, sunduğu manevra kabiliyeti yüksek derecede ek olarak, ısırma ucu bilenmiş ya Bergstrom trokar 16 oluşturmak sıklıkta değiştirilmesi gerekmez olmasıdır. Weil Blakesley conchotome kullanarak kas biyopsisi genel amacı, hem teşhis ve resear için histokimya ve moleküler analiz sağlamak için kas dokusunun yeterli miktarda elde etmektirch çalışma. Kolayca öğrenilebilir basit ve güvenli bir işlemdir. Bu teknik sayesinde, biyopsi için uygun kas pazı, triseps, deltoid, gastroknemius, tibialis anterior, soleus ve sacrospinals 17,15,7,18,14 içerir. O uyluk içinde ana nörovasküler yapıları önler olarak vastus lateralis en dış kısmı biyopsisi için kullanılan en yaygın sitedir. Anterior ve superior iliak omurga ve patellayı birleştiren hayali bir çizgi aşağı yaklaşık üçte ikisini tanımlanır. Kas morfoloji verileri, vastus türetilmiş örnek myofibre alanı, myofibre oranlarda, kılcal yoğunluğu için örnekleme çalışmaları 11,19 arasında karşılaştırma sağlayan literatürde yaygın olarak kullanılabilir lateralis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Weil-Blakesley conchotome</td>
			<td>Gebr&#252;der Zepf Medizintechnik, D&#252;rbheim, Germany</td>
			<td>None</td>
			<td>6 mm biting tip</td>
		</tr>
		<tr>
			<td>5 ml 2% lidocaine</td>
			<td>Generic use</td>
			<td>None</td>
			<td>One 5ml vial should suffice for adequate anaesthesia</td>
		</tr>
		<tr>
			<td>Alcohol swab</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mls syringe</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>25G and 23G sterile needles</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel size 11</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze squares</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% 10 ml</td>
			<td>Fannin</td>
			<td>1122/002/001</td>
			<td></td>
		</tr>
		<tr>
			<td>Steri-Drape&#8482; Small Drape with Adhesive Aperture</td>
			<td>3M</td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>ChloraPrep 2% w/v /70% v/v cutaneous solution&#160;</td>
			<td>CareFusion</td>
			<td>270400</td>
			<td>Two</td>
		</tr>
		<tr>
			<td>Steri-Strip&#8482; 1/4 inch x 3 in Reinforced Adhesive Skin Closures</td>
			<td>3M</td>
			<td>R1541</td>
			<td></td>
		</tr>
		<tr>
			<td>PRIMAPORE sterile dressing 15cm x 8cm</td>
			<td>Smith &#38;&#160; 
			Nephew</td>
			<td>66000318</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton crepe bandage Hospicrepe 233 7,5 cm x 4,5 m</td>
			<td>Hartmann</td>
			<td>915562</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible cohesive bandage Co-Plus LF</td>
			<td>BSN medical</td>
			<td>2904565</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable absorbent sheet</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore or transpore tape</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gown with sterile paper towels</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gloves</td>
			<td>Generic use</td>
			<td>None</td>
			<td></td>
		</tr>
	</tbody>
</table>

human vastus lateralis skeletal muscle biopsy using the weil-blakesley conchotome

Weil-Blakesley conchotome kullanarak Perkütan kas biyopsisi de hem klinik hem de araştırma uygulamada kurulmuştur. Bu, güvenli, etkili ve iyi tolere tekniktir. 6 mm genişliğinde oyuk - Weil-Blakesley conchotome 4 ile keskin bir ısırma uca sahiptir. Bu 5 arasına sokulur - 10 mm cilt insizyonu ve kontrollü doku penetrasyonu için manevra olabilir. ucu açılır ve doku içinde kapatıldı ve sonra kas kesmek için 90 -180 ° döndürülür. Elde edilen kas miktarı numune histolojisi ve moleküler çalışmalar hem de işlenebilir 290 mg 20 mg arasında değişebilir tekrar et. Yara aktivitesi normal düzeyleri prosedür takip hemen yeniden rağmen, kuru ve şiddetli bir fiziksel aktivite, yaklaşık 72 saat için en aza tutulması gerekir. yakın ilgi konular, tam asepsi ve sonrası prosedürü bakım seçimi ödenir Bu prosedür, güvenli ve etkilidir. Sağ ve sol v Hemastus lateralis katılımcı tercihine göre biyopsi için uygundur.

Yukarıda açıklanan prosedür hem klinik ve araştırma ortamlarında güvenli ve kabul edilebilir. biyopsi tanı sürecinin bir parçası olarak gerçekleştirildiğinde, kas hastanın semptomları ve kas zayıflığı işaretleri göre seçilmelidir. Biyopsi sitesi önceki yaralanmalar, kontraktür veya enstrümantasyon 21,1 arındırılmış olmalıdır. Araştırmada, diyabet hastalarının oruç veya dışlanma olarak standartlaştırılmış şartlar 19 gere...

Watch this Scientific Journal Video about Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome at JoVE.com

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 ...

human-vastus-lateralis-skeletal-muscle-biopsy-using-weil-blakesley

Research

JoVE Journal

Medicine

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

16.6K Görüntüleme.  University of Southampton, University Hospital Southampton. This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

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

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.

İzole İskelet Kası Myo-mekanik Analizi

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 ...

Tıp

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

Vivo Köpek Kas Fonksiyon Testi

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

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 ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

İlköğretim İnsan Fibroblastlar türetilmesi için Cilt Punch Biyopsi Eksplantın Kültür

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

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

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

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. ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

İnsan Serum içinde N-metil-D-aspartat reseptör ya da dopamin-2 reseptör antikorları algılamak üzere yüksek verimli Flow Cytometry hücre bazlı bir deneyde

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

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.

Modifiye Bergström Tekniği İnsan İskelet Kası Biyopsi Prosedürleri

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

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Perkütan İğne Biyopsisi ile Elde Edilen İskelet Kası Doku İzole Mitokondri hazırlanması ve Respirometrik Değerlendirmesi

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

İskelet Kası Hastalığının kantitatif Manyetik Rezonans Görüntüleme

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

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 ...