The authors thank members of the Valdez laboratory for intellectual input on experiments and comments on the manuscript.

Tüm deneyler Virginia Tech Kurumsal Hayvan Bakım ve Kullanım Komitesi tarafından onaylanmış NIH kurallar ve hayvan protokol çerçevesinde yürütülmüştür. Cerrahi 1. hazırlanması Hayvanlar <ol><li> steril 1 ml insülin şırınga ile ketamin (90 mg / kg) ve deri altı enjeksiyon yoluyla inguinal ksilazin (10 mg / kg) karışımı ile fareler anestezi. Taşıyıcı çözeltisi% 0.9 tuzlu su, 17.4 mg / ml ketamin ve 2.6 mg / ml ksilazin içeren bir karışım içerir. yürürlüğe girmesi için ilaç beklerken kafeslerde geri hayvanları yerleştirin. Not: yükleme dozu prosedür süresince yeterli anestezi sağlamıyorsa, yükleme dozunun% 25 ek enjekte edilebilir. </li><li> Monitör hayvanlar sürekli solunum oranları ve uyarılma seviyelerinin uygun depresyon kontrol etmek için enjeksiyon sonrası. yeterince anestezi zaman hiçbir tepki ortaya çıkarmak gerekir bir arka ayak tutam ile uyarılma seviyesini kontrol edin. NOT: Bu genellikle 3-5 alır25 ila 30 g ortalama bir genç yetişkin fare için min. Hayvan de 10 dakika sonrası enjeksiyonundan sonra yanıt ise, anestezi yükleme dozunun% 25 ek enjekte edilebilir. </li><li> kuruluğunu önlemek için hayvanın gözlerine vazelin ve hafif mineral yağ oftalmik merhem sürün. temiz, düz bir yüzey üzerine kafes ve yerden hayvanları kaldırın. ekstremitenin sadece yanal yönünü açığa bir elektrikli saç düzeltici kullanarak pelvise ayak istenen arka ekstremite tıraş. </li><li> 1 dakika için traş sitesine bir kimyasal saç çıkarıcı uygulanır. El ile laboratuvar mendil kullanarak tüylerden. laboratuvar ile temizleyin depilated alan etanole batırılmış mendil. </li></ol> 2. Cerrahi Prosedür <ol><li> otoklav veya başka uygun yöntem ile cerrahi aletleri sterilize edin. % 80 etanol / H 2 0 ile cerrahi sitesi ve cerrahi kurulu temizleyin. proviodine cerrahi siteyi dezenfekte edin. cerrahi gemide fare yerleştirin ve uzuv sınırlamalar uyum. tutmakDiz eklemi ile anatomik doğal bir pozisyonda hedef arka bacak hafifçe iç veya dış rotasyon olmadan uzatıldı. </li><li> Cerrahi mikroskop altında hayvan ve tahta yerleştirin. yüzeysel noktalarına özellikle kemik diz ekleminde ve tibialis anterior ve gastroknemius kaslar arasındaki sırtın palpasyon yoluyla uygun kesi sitesine yönlendirmek. </li><li> kavrama için genel forseps kullanırken bir neşter veya yaylı makas kullanılarak deri yoluyla yaklaşık 3 cm&#39;lik kesi olun. Ortak fibula sinir yatan tabii dik kesi yapmak. </li><li> Yüzeyel fasya ile kesi Devam biceps femoris açığa ve vastus lateralis kasları. bağlayan derin fasya boyunca keserek bu kasları ayırın. Bir 1-2 cm kesilmiş yeterli olmalıdır. </li><li> pazı geri çekin kaudale ortak fibula siniri açığa mekanik retraktörler kullanarak kas femoris. </li><li> onun Inter kadar proksimal sinir Tracegastroknemius kasının lateral başının tendonu ile bölüm bulunur. Not: Poz geri kas ve deri ek manipülasyonunu gerekebilir. Bu kesişme sinir hasarı için kararlı bir dönüm noktası olarak kullanılır. </li><li> gastroknemyus tendonun lateral sınırına paralel bir şekilde ipuçları hizalama ince bir forseps ile sinir kavrayın. 5 saniye boyunca sabit, sabit basınç uygulayarak ortak fibula sinir Crush. </li><li> Cerrahi kapsamı ile görsel muayene ile sinirin tam ezmek desteklemektedir. Bu yaralanma yerinde saydam görünür. Periferik aksonların floresan proteinleri ifade fareler kullanıyorsanız, floresan yaralanma yerinde kaybolur. </li><li> retraktörler çıkarın ve anatomik pozisyonlarında kasları realign. 6-0 ipek sütür ile kesi kapatın. 1-3 basit kesintiye dikişler yeterlidir. Temiz bir kafes içinde bir ısıtma pedi üzerinde fare recuperating yerleştirin. </li><li> 2 saat sonrası opera için tüm hayvanları izlemekyon nefes ve anestezi herhangi bir olumsuz reaksiyonlara kontrol etmek için. Ameliyattan hemen kurtarma aşağıdaki subkutan inguinal enjeksiyon yoluyla buprenorfin 0.05-0.10 mg / kg bir başlangıç ​​dozunu. 3 ek doz sonraki 48 saat boyunca her 12 saatte verin. tam iyileşme sonrasında, hayvan bakım tesisine fareler dönün. </li></ol> 3. İzolasyon ve boyanması ekstansör digitorum longus (EDL) Kaslar <ol><li> izofluran kullanarak hayvanların Kurban. Emici labwipes ile dolu bir 50 ml&#39;lik bir tüp içine 0.5 ml sıvı izofluran koyun. Mühürlü 2500 cm3 odasında hayvan kapaksız tüp yerleştirin. maruz en az 4 dakika yeterlidir. İkili üst kapağında, ayak-tutam, ve kuyruk-tutam reflekslerinin kaybı için test her hayvan perfüzyon başlamadan önce bilinçsiz olduğundan emin olmak için. </li><li> Transkardiyal ilk 10 mi 0.1 M PBS ile 16 hayvan serpmek, daha sonra 0.1 M PBS (pH 7.4) içinde% 4 paraformaldehit 25 mi. Heparin (30 ünite / 20g Hayvan ağırlık) / ml) 10 birim perfüzyon sonuçları geliştirmek küçük kapiler yatak kan pıhtılaşmasını önlemek için (PBS ile ilave edilebilir. </li><li> karın çevresi etrafında deri yoluyla enine kesmek için makas kullanarak arka bacaklarda kapsayan cilt kaldırmak. Arka bacaklarda ve ayaklarda forseps kullanarak geçmiş cildi aşağı soyun. </li><li> kavrayarak ve forseps ile soyulması ile arka bacaklarda yüzeysel kaplamasını çıkarın. Periferik aksonların floresan proteinleri ifade fareler kullanıyorsanız, 50 ml&#39;lik tüplerde% 4 PFA gecede bütün fareler sonrası düzeltmek. PBS ile üç kez yıkayın. Not: Sabit farenin 4 ° C&#39;de PBS içinde saklanabilir. Değilse, bu adımı atlayın ve post-sabitleme hayvanlar olmadan 3,6 adıma geçin. </li><li> Mümkün olduğunca sağlam proksimal ve distal tendon tutmak için emin olmak, fare arka bacaklarda EDL kasları 18 parçalara ayır. </li><li> en az 1 saat süre ile (1 x PBS,% 0.5 Triton X-100,% 3 BSA ve% 5 keçi serumu ihtiva eden) blokaj tamponunda EDS kasları inkübe edin. </li><live synaptotagmin-2 (1: 250), 3 gün boyunca blokaj tamponu içinde seyreltilmiş antikorlar:&gt; olarak sinir lifi (1.000 1) ihtiva eden tüpler içinde, motor aksonları ve sinir terminalleri, bir yer kasları görselleştirmek için. kasları 1x PBS ile üç kez 10 dakika her biri bir zaman yıkayın. Not: Periferik aksonların floresan proteinleri (XFP) ifade fareler kullanılarak bu adımı atlayın. </li><li> tam NMJ innervasyonundan bir pozitif kontrol olarak EDL yaralanmamış karşı taraf Leke. Negatif kontroller, 4 gün sonrası yaralanma, NMJ tamamen Denerve bir zaman noktasında elde edilen bir EDS, hem de yalnızca sekonder antikor ile boyanmış bir EDL içermelidir. </li><li> 1 gün sinir lifi ve synaptotagmin-2 algılamak için uygun floresan etiketli sekonder antikor ile kasları kuluçkaya yatmaktadır. kasları 1x PBS ile üç kez 10 dakika her biri bir zaman yıkayın. Not: Bu adım adım 3.10 ile birlikte yapılabilir. Periferik aksonların floresan proteinleri ifade fareler kullanılarak bu adımı atlayın. </li><li> postsy görselleştirmek içinNMJ naptic bölgesi, 5 ug / ml, en az 2 saat boyunca blokaj tamponu içinde seyreltilmiş Alexa-555 konjuge alfa bungarotoxin kasların inkübe edin. kasları 1x PBS ile üç kez 10 dakika her biri bir zaman yıkayın. </li><li> pozitif yüklü cam slaytlar üzerinde tüm kasları monte etmek için, slayt üzerinde orta montaj dayalı gliserol birkaç damla ekleyin ve lamel ile kaplayın, slayt doğrudan kas yerleştirin. kas düzleştirmek için slayt karşı lamel basın. slayt ve lamel kullanarak laboratuvar mendil çevresinden montaj medya kapalı ıslatın. lamel ve slayt arasındaki kenarları mühür oje sürün. </li></ol> 4. Görüntüleme ve Veri Analizi <ol><li> NMJs, görüntü 488, 555 ve 633 nm ışık heyecanlandırmak ve 20X ve 40X hedefleri ile yayılan ışığı yakalamak için donatılmış bir konfokal lazer tarama mikroskobu kullanılarak EDL kas yapısını analiz etmek. </li><li> Bütün NMJs görselleştirmek için, optik sn maksimum intensite projeksiyon görüntüler oluşturmakleri ayrı NMJ en yüksek görünen bölgeler düşük uzanan 1 mikron 2 aralıklı. piyasada mevcut görüntüleme yazılımı kullanarak maksimum yoğunluk projeksiyonlar oluşturun. </li><li> 1) Tamamen Denerve = postsinaptik sitesi akson ve AChRs arasında akson ile temas,% 5&#39;ten az ko tamamen yoksun: olarak reinnervation oranlarını belirlemek için, NMJs sınıflandırır. 2) Kısmen innerve = akson kısmen akson ve AChRs arasındaki postsynapse,% 5-95 ko çakışıyor. 3) Tüm innervasyonu = öncesi ve sonrası sinaps arasında neredeyse mükemmel appozisyon, akson ve AChRs arasında% 95&#39;ten fazla ko. görüntüleme düzlemine dik yalan veya tamamen görüntüde görselleştirilemediği NMJs hariç. Not: Tüm bu deneyler, en az 3 hayvan ve hayvan başına 50 NMJs incelenmiştir. Sonuçlar 0,05&#39;ten P değerine sahip bir öğrenci t-testi kullanılarak anlamlı kabul edildi. </li><li> operatörü kör, ayrı bireyler yapabilirCerrahi ve resim analizi. tedavi gruplarının bilgisi olmadan, analizörü NMJ puanlama ile objektif olabilir. Seçenek olarak ise, resim, randomize ve kaynak hayvan bilgisi olmadan analiz için operatöre sunulabilir. </li></ol> 5. Nicel PCR <ol><li> izofluran ve servikal dislokasyon kullanarak hayvanların Kurban. Cildi ve 3.3 adıma göre bacak kaslarını örten yüzeyel fasya çıkarın. tibialis anterior ve 3.4 adıma göre EDL kasları parçalara ayır. </li><li> Flaş sıvı nitrojen üzerinde 1.5 ml tüp bütün tibialis anterior ve EDS kasları donma. kısmen sıvı azot içine daldırıldığında, bir ön-soğutulmuş havan içinde tüp ve yerden doku çıkarın. bir havan ve havan tokmağı kullanılarak ince bir toz haline dondurulmuş kas öğütün. </li><li> Bir ticari olarak temin edilebilen RNA ekstraksiyon reaktifi içine donmuş kas tozu çözmek ve ticari olarak temin edilebilen bir kit üreticisinin I&#39;e göre olan RNA ekstraksiyonu ve genomik DNA giderme gerçekleştirmeknstructions. </li><li> üreticinin talimatlarına göre bir ticari olarak temin edilebilir ters transkriptaz karışımı ile ters transkripsiyon gerçekleştirin. </li><li> qPCR uygun temizlik genler kullanılarak ticari olarak mevcut bir kit kullanılarak gerçekleştirin (malzeme tabloya bakınız). (Malzeme tabloya bakınız) PCR yürütmek için piyasada mevcut olan kantitatif PCR termal döngü kullanın. </li><li> 58 ° C olarak ayarlandı tavlama sıcaklığı. Taq polimeraz / SYBR yeşil karışımı imalatçısının şartnamesine ek bisiklet parametrelerini ayarlamak. Primer özgüllük için test dimer oluşumunu astar 95 ° C ila 65 ° C ila 0.5 ° C artan artar oluşan termal döngü programına bir son erime eğrisi aşamasını içerir. </li><li> Kontrol geni olarak 18S RNA kullanılarak ΔΔCT yöntem 21-2 göreceli mRNA ekspresyon seviyelerini belirlemek. </li></ol>

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The authors have nothing to disclose.

Bu yazıda sunulan yöntem nöromüsküler kavşaklar (NMJ) tamir katılan mekanizmaları tanımlamak için benzersiz fırsatlar sunar. o diz yakın gastroknemius tendonu üzerinden geçerken Bu yöntem yaygın fibula siniri ezme içerir. Bir forseps ile sinir sıkışmasının sadece 5 saniye sonra, tam bir dejenerasyonu yaralanması 4 gün sonra belirtilen olduğunu göstermektedir. genç yetişkin farelerde, alfa-motor aksonlar 12 gün yaralanmamış farelere olanlardan ayırt edilemez presinaptik sitelerin Reformasy...

In young adult and healthy animals, the neuromuscular junction (NMJ) is a highly stable connection between the presynapse, the nerve ending of an &#945;-motor axon, and the postsynapse, the specialized region of an extrafusal muscle fiber where nicotinic acetylcholine receptors (AChRs) selectively aggregate1. The nearly perfect apposition of the pre- and post-synaptic apparatuses is necessary for proper neurotransmission, survival of &#945;-motor neurons and muscle fibers and motor function. Unfortunately, the function of the NMJ is adversely affected by aging, diseases such as amyotrophic lateral sclerosis (ALS), autoimmune diseases and injury to muscles and peripheral nerves2-5. These insults often result in degeneration of presynaptic nerve endings, leaving muscles denervated and significantly altering motor skills. For this reason, the identification of molecules that function to maintain and repair the NMJ has become a priority. Because peripheral nerves regenerate and reinnervate targets, peripheral nerve injury models have been used to identify molecular changes associated with regenerating NMJs. 
Peripheral nerve injury models often involve either completely cutting or crushing specific nerve branches6. Following a cut, the endoneurial tube has to be reformed, delaying axonal regeneration and reinnervation of target cells and tissues. The severity of this type of injury also causes axons to meander away from their original path, resulting in their failure to reach original targets. This is in contrast to nerves injured via crush where the endoneurium remains contiguous, providing a path for efficient and proper regrowth of regenerating axons. It also allows axons to find and reinnervate their original muscle fiber partners. Irrespective of injury model, there are a number of cellular and molecular changes that must occur for axons to regenerate and reinnervate targets. After an injury, the nerve segment proximal to the target is broken down and removed via a process termed Wallerian Degeneration7. This process involves reprogramming and de-differentiation of Schwann cells into non-myelinating cells that secrete regenerative factors, clear myelin, and recruit macrophages to the site of injury8. Macrophages in turn complete the clearance of myelin and axonal debris, which would otherwise impede growth of the regenerating axon9. In parallel, motor and sensory neurons activate mechanisms needed to promote regeneration of their severed axons. Once the regenerating axon reaches the target, it must transform from a growth cone to a nerve ending capable of properly transmitting (for motor axons) or receiving (for sensory axons) information10. In this regard, alpha-motor axons undergo a series of well-orchestrated changes that culminate in their growth cone differentiating into a fully functional presynaptic nerve ending that nearly perfectly opposes the post-synaptic site on the target muscle fiber11. 
The sciatic, tibial and accessory nerves have been the primary choices for studying axonal and NMJ regeneration12-14. However, there are a number of drawbacks when using these models to examine cellular and molecular changes associated with regenerating NMJs between animals and under different conditions. Firstly, the sciatic nerve supplies the majority of the muscles of the hind limb, with injury significantly limiting both movement and sensation. It is therefore not possible to use this method to study the impact of exercise alone or in combination with other factors. Additionally, the sciatic nerve is a rather thick structure and thus requires a large amount of compressive force to fully injure all axons. This in turn may result in complete transection of the more superficial axons while leaving the endoneurial tube of deeper lying axons intact, introducing significant variability in the rate and fidelity of regeneration among these axons. Complete transection of this nerve is even less desirable given that many axons will fail to reconnect with the same muscle fibers. Complicating matters, the sciatic nerve possesses intrinsic anatomic variability, both in the number and site of origin of its terminal nerve branches. It is therefore very difficult to lesion the same site. While the tibial nerve is smaller and more amenable to crush injuries, there is also no readily available landmark to serve as a lesion site for this nerve branch. 
The accessory nerve branch (part of cranial nerve XI) that supplies the sternocleidomastoid muscle has also been used to study regeneration of NMJs15. This nerve is particularly attractive because NMJs in the sternocleidomastoid muscle can be more readily imaged in live animals compared to NMJs in other muscles. But similar to the sciatic and tibial nerves, there is no specific landmark that can be used to injure this nerve in the same location, limiting it as a model for comparing regeneration of NMJs among individual animals of an experimental cohort. An inconsistent lesion site introduces variability in the rates of NMJ reinnervation. Due to these shortcomings, the procedure presented here utilizes the injury of a different peripheral nerve branch to examine regenerating NMJs. 
The common fibular nerve, also called the common peroneal nerve, contains many features that make it a reliable nerve to examine regeneration of NMJs between animals and across different treatments. The common fibular nerve has a predictable anatomic course as it runs over the tendon of the lateral head of the gastrocnemius muscle in the knee, the intersection serving as a stable landmark for lesions. The nerve is accessed through a small and minimally invasive incision near but anatomically segregated from the muscles of interest. The findings presented here demonstrate that regenerating motor axons begin to reform NMJs in the extensor digitorum longus (EDL) muscle 8 days after crushing the fibular nerve in 70 days old young adult female mice. Importantly, the pattern and rate of reinnervation is consistent among animals of the same age and sex and therefore provide a reliable injury model that will significantly hasten our understanding of the cellular and molecular changes required to maintain and repair NMJs.

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			<td>Spring Scissors</td>
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			<td>Braintree Scientific</td>
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			<td>Dumont</td>
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			<td>&#160;Suture Express</td>
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			<td>Braintree Scientific</td>
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			<td>Alexa 555 conjugated alpha-BTX </td>
			<td>Molecular Probes</td>
			<td>B35451</td>
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			<td>Vectashield</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
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the fibular nerve injury method: a reliable assay to identify and test factors that repair neuromuscular junctions

nöromüsküler bileşke (NMJ) yaşlanma, yaralanma ve hastalık sonucu zararlı yapısal ve fonksiyonel değişikliklere uğrar. Nedenle, bakım ve onarım NMJs katılan hücresel ve moleküler değişiklikleri anlamak için şarttır. Bu amaçla, güvenilir ve sürekli olarak farelerde NMJs rejenere incelemek için bir yöntem geliştirdik. o diz yakın gastroknemius kası tendonun lateral başının üzerinden geçerken Bu sinir yaralanması yöntemi yaygın fibula siniri ezme içerir. 70 günlük dişi fareler kullanarak, motor aksonlar 7 gün sonrası ezmek içinde önceki postsinaptik hedefleri reinnervate başlar göstermektedir. Bunlar tamamen 12 gün önceki sinaptik alanları yeniden işgal. Bu yaralanma yöntemin güvenilirliğini belirlemek için, bireysel 70 günlük dişi fareler arasında reinnervation oranları karşılaştırıldı. Biz tekrar innerve olmuş postsinaptik sitelerin sayısı 7, 9 fareler arasında benzer olduğunu buldu ve 12 gün sonrası crush. olmadığını belirlemek içinBu yaralanma deneyi, kas moleküler değişiklikleri karşılaştırmak için kullanılabilir biz kas nikotinik reseptör (y-AChR) ve kas özel kinaz (MuSK) gama alt-biriminin seviyeleri araştırılmıştır. gama-AChR alt birim ve MuSK yüksek aşağıdaki denervasyon regüle ve NMJs reinnervation aşağıdaki normal seviyelere geri oluştururlar. Biz bu genlerin ve kasların innervasyonu durumu transkript seviyeleri arasında yakın bir ilişki bulundu. Biz bu yöntem NMJ ve diğer sinaps tamir katılan hücresel ve moleküler değişiklikler anlayışımız hızlandırmak inanıyoruz.

Ortak fibula sinir, aynı zamanda, peroneal sinir olarak adlandırılan bacak (Şekil 1A) ön yönüne fibula başının etrafında sallanır popliteal fossada yukarıdaki siyatik sinirin, doğar. yüzeysel ve derin fibula sinir içine dalları birbirine ayak ve ayak dorsifleksör tedarik (anterior tibialis, extensor digitorum longus ve brevis ve ekstansör halluces longus kasları) ve ayağın everters (peroneus kaslar) Orada olmuyor. Bu sinir de ayak ve bacağın alt y...

Watch this Scientific Journal Video about The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions at JoVE.com

The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle.

Fibula Sinir Yaralanması Yöntem: Güvenilir Testi tanımak ve Test O Tamir Nöromüsküler Junctions Faktörleri

The neuromuscular junction (NMJ) undergoes deleterious structural and functional changes as a result of aging, injury and disease. Thus, it is imperative ...

the-fibular-nerve-injury-method-reliable-assay-to-identify-test

Research

JoVE Journal

Neuroscience

15.6K Görüntüleme.  Virginia Tech. We have developed a nerve injury method to reliably examine muscle reinnervation, and thus regeneration of neuromuscular junctions in mice. This technique involves injuring the common fibular nerve via a simple and highly reproducible surgery. Muscle reinnervation in then assessed by whole-mounting the extensor digitorum longus muscle. 

Video: The Fibular Nerve Injury Method: A Reliable Assay to Identify and Test Factors That Repair Neuromuscular Junctions

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Retina Ganglion Hücre Survival Eğitim Bir Optik Sinir Crush Yaralanma murin Modeli

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Nörobilim

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

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

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

Santral Sinir Sistemi Onarım İnceleyecek Bir Sakatlık Paradigma<em&gt; Drosophila</em

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

İnme sonrası Denge Rehabilitasyon bir Nöromüsküler Elektriksel Stimülasyon Sistemi ile düşük maliyetli Sensörler entegre bir İnsan-makine arayüzü

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Kök Ganglia Nöronlar ve Farklılaştırılmış Yağ-türetilmiş Kök Hücreler Dorsal: bir<em&gt; İn Vitro</em&gt; Co-kültür Modeli Periferik Sinir Rejenerasyon Eğitim

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Kapalı Başkanı Travmatik Beyin yaralayabilecek Bir Yöntem<em&gt; Drosophila</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Perkütan Elektriksel Sinir Stimülasyonu kullanma Nöromüsküler Fonksiyonu Değerlendirilmesi

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Kurbağa Sinir Uçlarına Sinir Kütüğü Vasıtasıyla Kalsiyum Boyası Yükleme: Kurbağa Nöromüsküler Kavşağında Kalsiyum Geçici Kayıt

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Sırt kök ganglion Enjeksiyon ve Dorsal Kök Duyusal Axon Rejenerasyon Modeli Olarak Ezilme Yaralanması

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...