This work is supported by an R21 grant from the National Institutes of Health and by a grant from Hearing Research, Incorporated.

NOT: NIH etik kurallar ve Kaliforniya Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu, San Francisco onaylanan protokol şartlarına uyulması Tüm prosedürler ve hayvan taşıma. 1. Cerrahi Hayvan Hazırlama <ol><li> Temiz, özel alanda cerrahi işlemleri yürütmek. Ameliyat öncesinde bir cam boncuk sterilizatör ile sterilize, tüm cerrahi aletler otoklav. NOT: Bu protokolde, doğum sonrası geçen gün 10-12 (P10-12) FVB fareler kullanın. Farklı yaş ve farelerin suşları belirli bir projenin ihtiyaçlarını karşılamak için kullanılabilir. Bül kemik zorlaşıyor çünkü P40 daha yaşlı Fareler zor olabilir. </li><li> Ketamin hidroklorür (100 mg / kg), ksilazin hidroklorür (10 mg / kg) ve asepromazin (2 mg / kg) karışımı intraperitoneal enjeksiyonu ile farelere anestezi. Hayvan artık böyle ayak tutam gibi ağrılı uyaranlara, cevap sonra cerrahi hazırlık başlayın. Eğer Mutlakay orijinal anestezi uçağı geri anestezik kokteyl bir rapel doz (beşte biri orijinal doz) yönetmek. NOT: Bu yaşta görülen mortalitenin en anestezik doz aşımı nedeniyle çünkü bu aşamada dikkat edin. </li><li> Hayvanın göz kırpma refleksi baskılayarak, anestezi sırasında gözleri nemli tutmak için koruyucu bir oftalmik merhem ile hayvanın gözlerini örtün. </li><li> Fare tamamen uyanık olduğu kadar anestezi sonrası hipotermi önlemek için, işlem boyunca bir ısıtma yastığı genişletilmiş boyun ile fare yerleştirin. </li><li> Bir kesme makinesi ile sol sonrası kulaktan bölgeyi Tıraş ve cerrahi manipülasyon öncesi% 70 etanol ve povidon-iyot ile dezenfekte edin. </li></ol> 2. Cerrahi Prosedür ve Vektör Enjeksiyon <ol><li> Timpanik bülü maruz sonrası aurikular yaklaşımı kullanın. Post-aurikular kas maruz küçük makas ile deri altı doku İnsizyon. Adipoz dokusu geri çekilmeden inci sonraKesi e arka tarafında, zamansal kemik açığa kesi dik sağ ve sol tarafına kasları ayrı. Bu kesi yeterli görselleştirme ile rahat çalışmak için yeterince uzun olduğundan emin olun. </li><li> 25 G iğne ile kulak bülü perfore ve koklea bazal dönüş erişime izin vermek için geri kemik soyulması ile forseps ile gerekli delik genişletin ve sonra da stapes arter ve yuvarlak pencere membranı görselleştirmek için yeterince genişletmek (RWM) . </li><li> Bir borosilikat kılcal pipet ile merkezde hafifçe RWM delinme. Bu noktada RWM yoluyla sıvı akışını gözlemlemek; Bu normaldir. Akış stabilize kadar bekleyin (koklea gelen effluxed sıvı steril bir filtre kağıdı ile kurutulur). NOT: tutma ve RWM perforasyon mümkün olduğunca küçük olması, böylece cam iğne ilerleyen dikkatli olun. Kullanılan mikroskop bağlı olarak, pipetler, bir pipet kullanarak bir micromanipulator kullanarak veya elle tutuntutucu. </li><li> Aynı şekilde yama kelepçe pipetler hazırlanır çektirmenin bir pipet kullanarak enjeksiyon için pipetler hazırlayın. Uç çapı büyük (yaklaşık 15 mikron çapında), dışarı virüs pipetle ve kolayca yapılır böylece olduğundan emin olun. </li><li> Bir enjeksiyon pipet içine AAV1-VGLUT3, AAV1-GFP veya AAV2-GFP 1 ila 2 ul kadar çizin. Akış (5 dakika 10) stabilize microinject aynı delikten skala timpani&#39;ye içine bu sabit bir hacim, daha önce RWM yapılır sonra. </li><li> Kas küçük bir fiş ile pipet çekerek sonra hızla RW niş Seal ve yuvarlak penceresinden sızıntıyı önlemek için kas üzerine yerleştirilen doku yapıştırıcı küçük bir damla ile sabitleyin. Not: çok iyi iğne koklea-Bu aşama bir perilenf sızıntıyı önlemek için kas küçük bir tıpa ile kaldırıldıktan sonra, bu delik Seal işitme korumak için önemlidir. Sızdırmaz takdirde tamamen RWM zaman içinde işitme kaybı ile sonuçlanacaktır. </li><li> Enjeksiyon sonrası, kapakadipoz dokusu ile işitsel bül delik, normal pozisyona sonrası aurikular kasları ve adipoz dokusu dönmek ve 6-0 ya da daha küçük emilebilir kromik sütür ile katmanlar halinde yara sütür. </li><li> Povidon-iyot ile yarayı dezenfekte edin. </li></ol> 3. Ameliyat Sonrası Bakım <ol><li> Sıcak kafeste fareler yerleştirin ve tam geri anne ile taşıyabilirsiniz kurtarıldı kadar onları yalnız bırakmayın. Bu anne ile geri yavrular koymadan önce kafesinden erkek kaldırmak için tavsiye edilir. </li><li> Inflamasyon ve ağrı yönetmek için deri altına karprofen postoperatif analjezi ve 3 gün boyunca her 24 saat için (2 mg / kg), yönetme. Sıkıntı, anormal kilo kaybı, ağrı, ya da enfeksiyon belirtileri günlük hayvanları izleyin. Genellikle tüm fareler normal hareket edilmelidir ameliyat sonrası 3. gün. Sıkıntı veya hastalık belirtileri 3. günden sonra bir fare görünüyorsa, t euthanizing düşününkurumsal kurallarına göre o fare. </li></ol> İşitsel Beyin Sapı Yanıtı (ABR) Kayıtlar kullanma Viral Teslimat ardından Koklear Fonksiyon 4. Değerlendirme NOT: işitsel beyin sapı cevabı (ABR) işitsel bir kafa derisi altına yerleştirilen elektrotlar aracılığıyla beynin devam eden elektriksel aktivite çıkarılan ve kaydedilen potansiyeli uyarılmış olduğunu. Hayvan sesi ile uyarılır. Elde edilen kayıt işitsel uyarının başlamasından sonra ilk 10 msn işitsel yolda ardışık noktaları elektriksel aktivitesini yansıtan beş dalgalar oluşur. <ol><li> Asepromazin olmaması haricinde, bu protokolün Bölüm 1.2&#39;de tarif edildiği gibi ketamin hidroklorür ve ksilazin hidroklorür intraperitoneal enjeksiyonu ile farelere anestezi. </li><li> Fare tamamen uyanık olduğu kadar anestezi sonrası hipotermi önlemek için, işitme testi boyunca bir ısıtma yastığı üzerinde fare yerleştirin. NOT: kullanBu deneyde aşağıdaki ses uyaranlar: (; 31 Hz sunum hızı 5 ms süresi) tıklayın. </li><li> Sol kulakta (referans) kepçesi altında, tepe de deri altı iğne elektrotları yerleştirin ve kontralateral kulakta altında (toprak) ve daha önce bir ses geçirmez odasına 27,28 açıklandığı gibi kayıt ABRS başlar. Aşağı maksimum genlik 5 dB ses basınç seviyesi aralıklarla Tutanak ABR dalga. </li><li> ABR eşik Ameliyat sonrası erken 4 gün olarak viral doğumdan sonra belirleyin NOT: Eşik dalgalar için zirveleri tepki hangi düşük uyarıcı seviyesi olarak tanımlanan I-V açıkça ve görsel inceleme üzerine tekrar tekrar mevcut. </li><li> Ben koklear sinir aktivitesini analiz Dalga ölçün. saptanabilir ABR dalga verir düşük uyaran eşik seviyesi olarak tanımlanır. </li></ol> Immunofluorescence Kullanılması 5. Koklear Transgen Protein Ekspresyonu <ol><li> Intrap aşırı dozda fareler uyutmakketamin hidroklorür ve ksilizin hidroklorür karışımı eritoneal enjeksiyon asepromazin olmadan hariç, bu protokolün Bölüm 1.2&#39;de açıklandığı gibi. </li><li> Hayvan artık böyle ayak tutam gibi ağrılı uyaranlara, cevap sonra başını başını kesmek. </li><li> 26 anlatıldığı gibi tüm montaj Immünofloresan için dışarı ve süreç cochleae incelemek. Anti-VGLUT3 ve anti-miyozin 7a antikorları kullanılarak koklear tüm montaj imünofloresans ayrıntılı bir protokol Akil ve diğ., 2013, 29 tarif edilmiştir. </li><li> 250 seyreltme GFP etiketleme için, 1 ile bir tavşan anti-GFP antikor ile 4 ° C&#39;de bir gece boyunca koklear tam bağlar inkübe edin. </li><li> PBS içinde 4000: PBS ile 10 dakika boyunca iki kez cochleae yıkayın ve daha sonra 2&#39;ye ait konjuge keçi anti-tavşan IgG 2 saat süreyle inkübe 1 seyreltilmiştir. </li><li> 10 dakika boyunca PBS ile iki kez cochleae yıkayın ve 15 dakika boyunca DAPI ile inkübe edilir. </li><li> Cam slaytlar cochleae monte ve und gözlemlemekkonfokal immünflörosans bir mikroskop er. </li></ol>

<ol>
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H., Kanzaki, S., Brown, N., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Functional+and+Structural+Outcome+of+Inner+Ear+Gene+Transfer+via+the+Vestibular+and+Cochlear+Fluids+in+Mice.">The Functional and Structural Outcome of Inner Ear Gene Transfer via the Vestibular and Cochlear Fluids in Mice.</a> Mol. Ther. 4 (6), 575-585 (2001).</li><li>Lalwani, A. K., Han, J. J., Walsh, B. J., Zolotukhin, S., Muzyczka, N., Mhatre, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+reporter+for+gene+transfer+studies+in+the+cochlea.">Green fluorescent protein as a reporter for gene transfer studies in the cochlea.</a> Hear Res. 114 (1-2), 139-147 (1997).</li><li>Lalwani, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+cochlear+transgene+expression+mediated+by+recombinant+adeno-associated+virus.">Long-term in vivo cochlear transgene expression mediated by recombinant adeno-associated virus.</a> Gene Ther. 5 (2), 277-281 (1998).</li><li>Raphael, Y., Frisancho, J. C., Roessler, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+into+guinea+pig+cochlear+cells+in+vivo.">Adenoviral-mediated gene transfer into guinea pig cochlear cells in vivo.</a> Neurosci. Lett. 207 (2), 137-141 (1996).</li><li>Weiss, M. A., Frisancho, J. C., Roessler, B. J., Raphael, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+mediated+gene+transfer+in+the+cochlea.">Viral mediated gene transfer in the cochlea.</a> Int. J. Dev. Neurosci. 15 (4=5), 577-583 (1997).</li><li>Pettis, R. M., Han, J. J., Mhatre, A. N., Lalwani, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracochlear+infusion+of+recombinant+adeno+associated+virus:+Analysis+of+its+dissemination+to+near+and+distant+tissues.">Intracochlear infusion of recombinant adeno associated virus: Analysis of its dissemination to near and distant tissues.</a> Assoc. Res. Otolaryngol. Abstr. 21, 673 (1998).</li><li>Konish, i. M., Kawamoto, K., Izumikawa, M., Kuriyama, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+guinea+pig+cochlea+using+adeno-associated+virus+vectors.">Gene transfer into guinea pig cochlea using adeno-associated virus vectors.</a> J. Gene Med. 10 (6), 610-618 (2008).</li><li>Kaplitt, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+gene+expression+and+phenotypic+correction+using+adeno-associated+virus+vectors+in+the+mammalian+brain.">Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.</a> Nature Genetics. 8 (2), 148-154 (1994).</li></ol>

No conflicts of interest declared.

Bu çalışmada, ayrıntılı olarak yeniden veya bir genetik kusur tarafından tehlikeye normal işitsel fonksiyon kurtarmak amacı ile, koklear gen terapisi için kullanılan bir tekniği açıklar. Genellikle atravmatik olduğu için, bu yaklaşım koklear gen transferi ya da diğer olası moleküler tedaviler 30 için güvenlidir. Koklear tedavisi için başka bir yaklaşım, ventral yaklaşımla 24, fare ve gine domuzunda kokleostomisi 31,32 ve endolenfatik kese teslimat 33</su...

Gen tedavisi uzun genetik işitme kaybı için potansiyel bir tedavi olarak öne sürülmüştür, ancak bu alanda başarı 1 zor kalmıştır. Bugüne kadar, viral aracılı metodolojiler nispeten ulaşılmaz koklea içinde belirli hücre türlerini hedef teorik yeteneği nedeniyle hakim olması. Her iki adenovirüs (AV), adeno-bağlantılı virüs (AAV), koklear gen aktarımı için kullanılmıştır. AAVler bir takım nedenlerden için kokleadaki avantajlıdır. Onlar çoğaltma eksikliği virüsler ve verimli nöronlar, işitme kaybının nedenleri bir dizi için önemli bir hedef gibi farklı hücre tiplerine transgenik molekülleri aktarabilirsiniz. Hücreye AAV giriş spesifik reseptörler 2 aracılık eder; hücre tipleri transduse edilmesi ile, böylece, belirli bir serotip tercih uyumlu olmalıdır. AAVler etkili saç hücreleri 3 transfekte Transformatörün istikrarlı, uzun vadeli ifade Elde edilen konakçı genomu içine dahil edilebilirnsgenic proteini ve hücre 4 fenotipik bir değişiklik. Bu tür saç hücre yenilenmesi gibi kısa süreli uygulamalar için gerekli olmayan, avantajlı olmasına rağmen, uzun vadeli ifade, genetik kusurların sabit kurtarma için çok önemlidir. AAVler herhangi bir insan hastalık ya da enfeksiyon ile bağlantılı hiçbir ototoksisiteye 5,6,7 göstermeyen, çünkü bunlar kaybı 8 işitme kalıtsal formları için gen terapisinde kullanım için ideal bir aday olup. Viral vektörleri kullanılarak memeli iç kulak içine egzojen genetik madde aktarımı son on yıl içinde incelenmiştir ve zarar 9 işitme genetik ve elde edilen her iki formu da tedavisi için ümit vaadeden bir teknik olarak ortaya çıkmaktadır. koklea potansiyel çeşitli nedenlerle, gen terapisi için ideal bir hedeftir: küçük hacimli gerekli virüs sınırlı miktarda gerektiren 1); Diğer organ sistemleri limitleri yan etkilerinden 2) göreceli izolasyon; ve 3) kendi sıvı dolu odaları viral kolaylaştırmakLabirent 10, 11,12,13,14, 15 boyunca teslim. Konjenital sağırlık Fare modelleri sistematik, yinelenebilir bir şekilde iç kulak gelişimini izlemek için çalışmanın birçok yöntem kullanımına izin verir. Fare cochleae küçük boyutu, bazı cerrahi zorluk sunmak yok iken, fare diğer türlerin üzerinde 16 çeşitli deneysel avantajları ile genetik işitme kaybı çalışmada son derece önemli bir model olarak hizmet vermektedir. Fare modelleri genetik bağlantı analizi, detaylı morfolojik gözlemler toplanması ve patojenik senaryoları simüle edilerek özellikleri bir dizi değerlendirilmesine olanak; gibi, bu viral olarak aracılık edilen, gen terapisi için iyi adaylardır. Teknolojik gelişmeler ile birlikte farelerde Kapsamlı genetik çalışmalar mümkün laboratuvarlarda 17,18, 19, 20,21 arasında bir tekrarlanabilir şekilde genetiği değiştirilmiş fareler üretmek için yaptık. Furthermorhem edinilmiş ve bu hayvan modelinde 22, 23,24 titiz test sağlayan, farelerde işitme kaybı fenotipleri kalıtsal e, sayısız modelleri de bulunmaktadır. Bu durumda, bir fare modelinde, viral aracılı gen terapisi kullanılarak işitme düzeltmek insan hastalığı için bir tedavi arayışında uygun bir ilk adımdır. Biz daha önce veziküler glutamat taşıyıcı 3 (VGLUT3) eksik transgenik fareler nedeniyle İHK şerit sinaps 25 glutamat salınımı eksikliği sağır doğarlar olduğunu göstermiştir. Bu mutasyon duyusal saç hücrelerinin birincil dejenerasyonuna yol olmadığından, bu mutant fareler potansiyel doğumsal işitme kaybı için koklear gen tedavisi sınamak için mükemmel bir modeldir. Bugüne kadar, koklear gen terapisi için viral uygulama teknikleri sayıda kokleostomisi ile yuvarlak cam membran yayılma, yuvarlak cam membran enjeksiyonu ve verme dahil olmak üzere tarif edilmiştir. Güçlü vardırial avantajları ve bu yaklaşımların 9 her dezavantajları. Burada yuvarlak pencere membranı (RWM) üzerinden VGLUT3 KO fare iç kulağa viral aracılı gen aktarımı için cerrahi bir yöntem sunulmaktadır. sonrası auricular RWM enjeksiyon yöntemi mükemmel işitme koruma ile minimal invaziv ve nispeten hızlı. Daha önce yayınlanmış gibi, bu fare modelinde işitme geri bir çaba, VGLUT3 genini (AAV1-VGLUT3) taşıyan bir AAV1 vektör doğum sonrası 12. günde de bu sağır farelerin koklea içine tanıtıldı (P! @), Sonuçlanan 26 işitme restorasyonu. Transgen protein ifadesi immünoflüoresans (IF) ile doğrulandı iken VGLUT3 KO farelerde İşitme, işitsel beyin sapı cevabı (ABR) tarafından doğrulandı. Bu metodoloji, böylece viral-aracılı gen terapisi olur, aksi takdirde sağırlık neden genetik kusur düzeltmek olduğunu göstermektedir.

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surgical method for virally mediated gene delivery to the mouse inner ear through the round window membrane

Sensörinöral sağırlık fonksiyonel iyileşme elde etmek için kullanılan gen terapisi, işitme kaybı katkıda yatan moleküler ve genetik mekanizmaları daha iyi anlaşılmasını vermek için vaat ediyor. Iç kulak içine vektörlerin giriş mevcut yapılara yaralanmasına en aza yaygın koklea boyunca maddesi dağıtan bir şekilde yapılmalıdır. Bu yazının moleküler, farmakolojik ve fareler doğum sonrası gün 10 ve yuvarlak pencere membranı (RWM) üzerinden eski viral teslim kullanarak fare koklear tedavisi için kullanılabilecek bir post-aurikular cerrahi yaklaşım anlatılmaktadır. Kan kaybını en aza indirmek ve hayvan ölümleri kaçınarak bu cerrahi yaklaşım skala timpani&#39;ye içine hızlı ve doğrudan teslim sağlar. Bu teknik, temel iç ve orta kulak yapıları yanı sıra boyun kasları tamamen işitme koruyarak ihmal edilebilir veya hiç hasar içerir. Bu cerrahi teknik, veziküler glutam etkinliğini göstermek için3 nakavt (KO VGLUT3) fareler bir adeno-bağlantılı virüs (AAV-1) iç kulağa VGLUT3 teslim aldıktan sonra işitme kurtarır konjenital sağırlık bir fare modelinde bir örnek olarak kullanılacaktır taşıyıcı yedi.

Teknik özellikleri ve koklear moleküler tedavi için post-aurikular yaklaşımın faydasını doğrulamak için, AAV1-VGLUT3, AAV1-GFP ve AAV2-GFP RWM üzerinden P10-12 fareler iç kulak içine teslim edildi. Bu yaklaşım, iç saç hücreleri içinde başarılı bir şekilde, transgen ekspresyonunu gösterir (IHC); (VGLUT3 Şekil 1 ve GFP Şekil 2 ve GFP Şekil 3A), dış saç hücreleri (OHC) (GFP Şekil 2) ve destek hücreleri (GFP Şekil 2...

Watch this Scientific Journal Video about Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane at JoVE.com

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Yuvarlak Pencere Membran aracılığıyla Fare İç Kulak için virally ve Gen Teslimat Cerrahi Yöntemi

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Research

JoVE Journal

Neuroscience

15.1K Görüntüleme.  University of California, San Francisco. The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane. 

Video: Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Kapalı Kranial Pencere kullanarak Fare Beyin Mikrosirkülasyon intravital Mikroskopi

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Nörobilim

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Omurilik Elektrofizyoloji II: Ekstrasellüler Emiş Elektrot İmalatı

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Yalıtımlı LacZ ifade Sitometrisi kullanarak Fare İç Kulak Dokular Hücreler

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

Tarafından geliştirilmesi Fare İç Kulak için Gen Transferi<em&gt; In Vivo</em&gt; Elektroporasyon

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

Fare Beyin Uzun vadeli Görüntüleme için Parlak ve Takviyeli inceltilmiş-kafatası Pencere

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

İç Kulak Saç Hücreleri Patch Clamp Kayıtlar İzole Zebrafish gelen

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Görüntüleme İlköğretim Kültürlü Fare Nöronlar içinde Plazma Membran Reseptör Yerleştirme pHluorin etiketli

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Kombine Metodolojileri kullanan Akson Dallanman ve Nöronal Morfogenez sırasında Plazma Membran Teslimat Rolü tanımlayın

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

CNS içine Hedeflenen Madde Teslimat için Fare Mikrocerrahi İnfüzyon Tekniği<i&gt; yoluyla</i&gt; İç Karotis Arter

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Farede Laminektomi ve Omurilik Penceresi İmplantasyonu

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...