Name: laminectomy and spinal cord window implantation in the mouse
Uploaded: 2019-10-23T12:30:00
Description: Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

S.E. Lutz, Grant KL2TR002002 ve University of Illinois Chicago College of Medicine başlangıç fonları altında Ulusal Çeviri Bilimleri, Ulusal Sağlık Enstitüleri, Geliştirme Merkezi tarafından desteklenir. Simon Alford RO1 MH084874 tarafından desteklenir. İçerik sadece yazarların sorumluluğundadır ve NIH'nin resmi görüşlerini temsil etmek zorunda değildir. Yazarlar Tg eGFP için Columbia Üniversitesi Tıp Merkezi Nöroloji Bölümü'nde Dritan Agalliu teşekkür:Claudin-5 fareler, bilimsel tartışmalar, ve cerrahi protokol ve görüntüleme uygulamalarının geliştirilmesi içine anlayışlar. Yazarlar California Üniversitesi Nörobiyoloji ve Davranış Bölümü Sunil P. Gandhi teşekkür, Irvine stereotaktik cihaz ve hayvan sıcaklık kontrolörü ilk prototip tasarımı için, cerrahi protokol tartışma, ve iki foton lu mikroskopi eğitimi. Yazarlar ayrıca Steve Pickens (W. Nuhsbaum, Inc) cerrahi stereomikroskop özelleştirme de yardım için teşekkür ve Ron Lipinski (Balina İmalat) stereotaktik parçaları işleme için.

Tüm deneyler Illinois Üniversitesi, Chicago Kurumsal Hayvan Bakım ve Kullanım Komitesi protokollerine uyar. Bu ölümcül bir prosedür.
1. Reaktif Hazırlama
<ol><li>DdH 2 O. Steril filtrede 125 mM NaCl, 5 mM KCl, 10 mM Glukoz, 10 mM HEPES, 2 mM MgCl2·6H2O, 2 mM CaCl2·2H2O ddH2O. Steril filtre içerecek şekilde suni serebral spinal sıvı (aCSF) hazırlayın ve bireysel kullanımda dondurulun. Kullanmadan önce 39 °C'ye kadar bi...

<ol>
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E., 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=Caveolin1+Is+Required+for+Th1+Cell+Infiltration,+but+Not+Tight+Junction+Remodeling,+at+the+Blood-Brain+Barrier+in+Autoimmune+Neuroinflammation.">Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.</a> Cell Reports. 21 (8), 2104-2117 (2017).</li><li>Harrison, M., 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=Vertebral+landmarks+for+the+identification+of+spinal+cord+segments+in+the+mouse.">Vertebral landmarks for the identification of spinal cord segments in the mouse.</a> Neuroimage. 68, 22-29 (2013).</li><li>Cupido, A., Catalin, B., Steffens, H., Kirchhoff, F., Bakota, L., Brandt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue. , 37-50 (2014).</li><li>Sekiguchi, K. 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Burada açıklanan yöntem, cam pencereden farelerdeki omuriliğin kararlı bir şekilde görüntülenmesine olanak sağlar. Bu yöntem transgenik eGFP BBB remodeling değerlendirmek için uygulanmıştır:Claudin5 +/- bir floresan BBB sıkı kavşak proteinifade fareler, ancak omurilik herhangi bir floresan protein veya hücrelerin çalışmaları için eşit derecede iyi uygulanabilir.
Laminektomi ve omurilik stabilizasyonu için birden fazla yöntem geliştirilmiştir. Tüm protokoller, ilg...

Floresan proteinleri ifade eden transgenik hayvan modelleri, intravital mikroskopi ile birleştirildiğinde, biyoloji ve patofizyolojiye yönelik güçlü bir platform sağlar. Omurilik bu teknikleri uygulamak için, görüntüleme için omurilik hazırlamak için özel protokoller gereklidir. Böyle bir strateji bir laminektomi ve omurilik penceresi implantasyonu yapmaktır. Mikroskopi için ideal bir laminektomi protokolünün temel özellikleri arasında doğal doku yapısı nın ve fonksiyonunun korunması, görüntüleme alanının stabilitesi, hızlı işlem süresi ve sonuçların tekrarlanabilirliği sayılabilir. Belirli bir sorun solunum ve kalp atışı tarafından indüklenen harekete karşı görüntüleme alanı stabilize etmektir. Birden fazla ex vivo ve in vivo stratejileri bu hedeflere ulaşmak için bildirilmiştir1,2,3,4,5. En in vivo yöntemleri spinal kolonun kenarlarına kenetleme içerir2,4 ve genellikle sert bir metalcihazimplante takip 3,4 ameliyat sırasında istikrar için ve downstream görüntüleme uygulamaları. Omurilik kıskaç potansiyel kan akışını tehlikeye ve kan-beyin bariyerini neden olabilir (BBB) protein remodeling.
Bu yöntemin amacı, protokolün invazivliğini en aza indirirken ve sonuçları iyileştirirken, omurganın canlı farede optik görüntüleme için kullanılabilir hale getirilmesidir. Biz hala sağlam mekanik kararlılık elde minimal invaziv oval plastik 3D baskılı arka plaka ile eşleştirilmiş tek bir laminektomi ve kapak cam implantasyon prosedürü açıklar. Arka plaka doğrudan diş çimentosu ile anterior ve posterior vertebral dikenler yapıştırılır. Arka plaka sert bir metal kol ile mikroskop aşamasına eklemek vida delikleri ile yanal uzatma kolları ile donatılmıştır. Bu etkili mikroskop aşamasına bozulmamış anterior ve posterior vertebra demirler, aksi takdirde solunum ve kalp atışı ile tanıtılacak hareket artifakı mekanik direnç sağlayan. Yöntem, torasik düzey 12'de tek bir vertebranın laminektomi için optimize edilmiş ve in vivo görüntüleme sırasında stabilite için alternatif stratejilerde kullanılan kıskaçlar atlanmıştır. İşlem hızlıdır ve fare başına yaklaşık 30 dakika süredilir.
Bu protokol BBB'nin hastalık mekanizmalarını incelemek için kullanılabilir. BBB, merkezi sinir sistemi (CNS) için son derece seçici bir ortam sağlayan endotel hücreleri, vasküler düz kas, perisitler ve astrosit ayak süreçlerinden oluşan dinamik bir mikrovasküler sistemdir. Temsili veriler, gelişmiş yeşil floresan proteini (eGFP): BBB sıkı kavşak proteini Claudin-5'i ifade etmek üzere tasarlanmış transgenik farelerde bu protokolün uygulanmasını betimlemelidir. Sağlanan arka plaka yazdırma dosyaları da alternatif uygulamalar için özelleştirilebilir.

<table>
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>Raise3D</td>
			<td>Pro2</td>
			<td>For printing backplates</td>
		</tr>
		<tr>
			<td>PLA 3D printing filament</td>
			<td>Inland</td>
			<td>PLA+-175-B</td>
			<td>Black plastic 3D printing material</td>
		</tr>
		<tr>
			<td>3D CAD software</td>
			<td>Dassault Systemes</td>
			<td>Solidworks software</td>
			<td>used to design 3D shapes</td>
		</tr>
		<tr>
			<td>3D printer software</td>
			<td>Raise3D</td>
			<td>Ideamaker software</td>
			<td>software used to interface with the 3D printer</td>
		</tr>
		<tr>
			<td>3D printed oval backplate</td>
			<td>custom</td>
			<td></td>
			<td>Stabilizing imaging field</td>
		</tr>
		<tr>
			<td>Surgical dissecting microscope</td>
			<td>Leica</td>
			<td>M205 C</td>
			<td>Equipped with Leica FusionOptics, Planapo 0.63x M-series objective, and gliding stage</td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Leica</td>
			<td>MC170</td>
			<td>HD color camera for visualizing surgical field</td>
		</tr>
		<tr>
			<td>Gliding stage</td>
			<td>Leica</td>
			<td>10446301</td>
			<td>The gliding stage is constructed of two metal plates. The base plate is fixed. The upper plate slides on greased interface to allow rotational and linear movement.</td>
		</tr>
		<tr>
			<td>Surgical station and stabilization fork</td>
			<td>Whale Manufactoring</td>
			<td>custom</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>SomnoSuite low-flow isoflurane delivery unit</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td>Surgical anesthesia administration with integrated digitial vaporizer</td>
		</tr>
		<tr>
			<td>Stainless steel 1.5 inch mounting post</td>
			<td>ThorLabs</td>
			<td>P50/M</td>
			<td>For mounting surgical station onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Counterbored Clamping Fork for 1.5&#34; mounting Post</td>
			<td>ThorLabs</td>
			<td>PF175</td>
			<td>For stabilizing surgical station mount onto optical table for two-photon imaging</td>
		</tr>
		<tr>
			<td>Ideal bone microdrill</td>
			<td>Harvard apparatus</td>
			<td>72-6065</td>
			<td>Thinning bone for laminectomy</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10</td>
			<td>Warming saline</td>
		</tr>
		<tr>
			<td>Cautery gun</td>
			<td>FST</td>
			<td>18010-00</td>
			<td>Cauterizing minor bleeds</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Benchmark</td>
			<td>BF11222</td>
			<td>1.9&#8221; x 4.5&#8221; silicone heater with 20&#8221; Teflon leads, 10W, 5V</td>
		</tr>
		<tr>
			<td>K type thermocoupled rectal probe</td>
			<td>Physitemp</td>
			<td>RET3</td>
			<td>Measuring mouse body temperature</td>
		</tr>
		<tr>
			<td>petroleum jelly</td>
			<td>Sigma</td>
			<td>8009-03-8</td>
			<td>Lubricating rectal probe</td>
		</tr>
		<tr>
			<td>Feedback-regulated thermal controller</td>
			<td>custom</td>
			<td>NA</td>
			<td>Commercially available alternatives include the Physitemp TCAT series</td>
		</tr>
		<tr>
			<td>PVA Surgical eye spears</td>
			<td>Beaver-visitec international</td>
			<td>40400-8</td>
			<td>Absorbing blood</td>
		</tr>
		<tr>
			<td>Electric trimmer</td>
			<td>Wahl</td>
			<td>41590-0438</td>
			<td>Trimming mouse fur</td>
		</tr>
		<tr>
			<td>Blade, #11</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #5</td>
			<td>FST</td>
			<td>11254-20</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Forceps, #4</td>
			<td>FST</td>
			<td>14002-14</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titatnium toothed forceps</td>
			<td>WPI</td>
			<td>555047FT</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Titanium Iris scissors</td>
			<td>WPI</td>
			<td>555562S</td>
			<td>Surgical tool</td>
		</tr>
		<tr>
			<td>Vetbond tissue adhesive</td>
			<td>3M</td>
			<td>084-1469SB</td>
			<td>Preparing tissue surface for dental acrylic</td>
		</tr>
		<tr>
			<td>Ceramic mixing tray</td>
			<td>Jack Richeson</td>
			<td>420716</td>
			<td>Mixing dental acrylic agent with accelerant</td>
		</tr>
		<tr>
			<td>Orthojet dental acrylic</td>
			<td>Lang Dental</td>
			<td>1520BLK, 1503BLK</td>
			<td>Permanently bonding backplate to tissue</td>
		</tr>
		<tr>
			<td>Small round cover glass, #1 thickness, 3 mm</td>
			<td>Harvard apparatus</td>
			<td>64-0720</td>
			<td>optical window</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>7447-40-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>50-99-7</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>7365-45-9</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td>For aCSF</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>QM01AE91</td>
			<td>Analgesia</td>
		</tr>
		<tr>
			<td>Bacteriostatic water</td>
			<td>Henry Schein</td>
			<td>2587428</td>
			<td>Diluent for carprofen</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Lactated ringer solution</td>
			<td>Baxter</td>
			<td>0338-0117-04</td>
			<td>Hydration for mouse</td>
		</tr>
		<tr>
			<td>Agarose High EEO</td>
			<td>Sigma</td>
			<td>A9793</td>
			<td>gel point 34-37 degrees C</td>
		</tr>
		<tr>
			<td>Opthalmic lubricating ointment</td>
			<td>Akwa Tears</td>
			<td>68788-0697</td>
			<td>Prevent corneal drying</td>
		</tr>
		<tr>
			<td>MOM Two-Photon Microscope</td>
			<td>Sutter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

laminectomy and spinal cord window implantation in the mouse

Bu protokol, fare omuriliğinin in vivo görüntülemesi için spinal kord laminektomi ve cam pencere implantasyonu için bir yöntem tanımlanmaktadır. Entegre bir dijital buharlaştırıcı isoflurane düşük akış hızında anestezi istikrarlı bir düzlem elde etmek için kullanılır. Tek bir vertebral omurga kaldırılır, ve ticari olarak kullanılabilir bir kapak-cam ince bir agarose yatak üzerine kaplanmış. 3D baskılı plastik bir arka plaka daha sonra doku yapıştırıcı ve diş çimentosu kullanılarak komşu vertebral dikenlere yapıştırılır. Bir sabitleme platformu solunum ve kalp atışı hareket artifakı azaltmak için kullanılır. Bu hızlı ve kelepçesiz yöntem akut multi-foton floresan mikroskopi için uygundur. Bu tekniğin transgenik farelerde spinal kord vaskülatürünün iki foton mikroskobuna uygulanması için temsili veriler eklenmiştir: Claudin-5 — sıkı bir kavşak proteini.

İmplante cam pencereler ve intravital iki foton mikroskopi CNS proteinlerinde dinamik değişiklikleri değerlendirmek için yararlı bir araç sağlar. BBB'nin fonksiyonel bütünlüğü, sıkı bağlantı proteinlerinin ekspresyonu, hücre altı lokalizasyonu ve ciro oranlarından etkilenir7. Önceki çalışmalar, sıkı kavşak proteinlerinin sabit durumda hızlı ve dinamik remodeling geçmesi olduğunu göstermiştir8. Şu anda açık...

Watch this Scientific Journal Video about Laminectomy and Spinal Cord Window Implantation in the Mouse at JoVE.com

Laminectomy and Spinal Cord Window Implantation in the Mouse

Bu protokol, intravital mikroskopi ile görselleştirmeyi kolaylaştırmak için bir farenin omuriliğine cam pencere nin yerleştirilmesini açıklar.

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 ...

Bu yöntem, bozulmamış merkezi sinir sistemi içinde in vivo protein dinamiğinin incelenmesini sağlayarak nörobilim ve immünoloji alanındaki soruları ele alır. Bu prosedürün başlıca avantajları görüntü edinimi için mükemmel bir hareket stabilitesi, kısa cerrahi süre, gaz anestezi azaltılmış operatör maruz kalma ve ucuz özelleştirilebilir backplates vardır. Bu yöntemin görsel gösterimi, prosedürün laminektomi adımları sırasında altta yatan sinir sistemi dokusuna zarar vermeden kemiğin çıkarılması zor olduğu için önemlidir.Yordamı başlamadan önce, belirtilen boyutlara bir model oluşturmak için 3B bilgisayar destekli tasarım yazılımı kullanın. Yani elimizde üç boyutlu bir nesne var, bununla başlayabiliriz. Daha sonra yapabileceğim şey, baskı sürümü için ayarları koyabiliyoruz.Bu yüzden bunu edit'i açarsam, Advanced'e gidin ve tüm bu ayarlara bakmamı sağlayan bir pencere açın. Meme sıcaklığını 205 dereceye, yatak sıcaklığını 45 dereceye, yazdırma hızını ise 3D yazıcıda saniyede 45 milimetreye ayarlayın. Arka plakaları yazdırmak için 0,4 milimetresıcak ent nozul ve 0,2 milimetrelik katman yüksekliği kullanın.Daha sonra yazdırılan arka plakaları yapısal bütünlükleri için görsel olarak değerlendirin. Brüt yapısal hatalar yazdırma hatalarını gösterir. Arka plakalar hazır olduğunda, sekiz ila 12 haftalık anestezili farenin başını cerrahi bir engelleyicinin kulak çubukları arasında bir ısı yastığına yerleştirin ve hayvanın gözlerine merhem uygulayın.Parmak ucurmasına yanıt eksikliğini doğruladıktan sonra, alt torasik üst lomber bölge üzerinde kaudal orta hat kesisine 1,5 santimetrelik rostral yapmak ve cildi peritondan ayırmak için 11 numaralı bıçağı kullanın. Yüzeysel kas ortaya çıkarmak için deri altında kalan şeffaf bağ dokusu geri soymak için forceps kullanın. Hedef vertebra kalan derin kas ile birlikte bir köpük cerrahi mızrak ile kasları yerinden.Arka plaka için bir koltuk oluşturmak için, torasik 11 ve torasik 13 ön yönü arka kısmından kas temizleyin. Gerekli tendondan kalan kas kaldırmak için forceps kullanarak. Tüm kas çıkarıldığında, dikkatle görselleştirme ve omurilik manipüle için yeterli alan elde edilene kadar forseps ile tendonu kesti.İntervertebral uzayın dura mater, yarı saydam laminar kemik, kemik altında merkezi yüzeysel kan damarı, ve anterior yayılan arter açıkça görülebilir. Bölgeyi sıcak yapay serebral spinal sıvı ile ısıtın ve omuriliğin uzun eksenine paralel düz vuruşlar kullanarak laminar kemiği inceltmek için mikro matkap kullanın. Hafifçe forseps ile yüzeysel spinous süreci kavrayarak, vertebra kaldırın.Kemik kolayca kaldırılmalıdır. Herhangi bir kemik kırıkları temizlemek için sayısı dört forceps kullanın, bir cerrahi mızrak ve gerektiği gibi nazik sabit basınç ile herhangi bir kanama kontrol. Sonra sıcak yapay beyin omurilik sıvısı ile doku durulayın.Kapak camı implantasyonu için, açıktaki kabloya üç milimetrelik borosilikat kapakca hafifçe uygulayın. 39 derece Santigrat uygulamak için küçük bir spatula kullanın cam kenarına% 2 agarose ısıtılır ve kapak cam yüzeyinin altında agarose çizmek için kılcal eylem sağlar. Torasik düzey 11 ve 13 vertebra iktisadi sinde bozulmamış komşu vertebranın maruz kalan kemikli eklem süreçlerine doku yapıştırıcısı uygulayın.Komşu tendon ve enine işlem üzerinde laminektomi siteetrafında bir halka ek doku yapıştırıcı uygulayın. Daha sonra, doku yapışkan tabakası üzerine hızlandırıcı ile karışık diş çimentosu uygulamak için yeni bir spatula kullanın ve kapak cam pencere üzerinde merkezli cerrahi alana bir arka plaka yerleştirin. Diş çimentosu birden fazla ince tabaka uygulanması daha güçlü bir arka plaka yapışmasonuçları, ancak diş çimentosu kurumaya başlamadan önce hızlı bir şekilde arka plaka yerleştirmek için emin olun.Diş çimentosu 10 dakika tedavi için izin verdikten sonra, ek yapıştırıcı ile arka yerin iç tabanı ve alt doldurun. Diş çimentosu ince tabakalar halinde uygulanması, tüm protokolü tehlikeye sokabilir kapak cam ve agarose, çimento yayılması riskini azaltmaya yardımcı olabilir. Diş çimentosu kuruduğunda, çatallı arka plaka tutucuyu pencerenin üzerinde uygun konuma uygulayın ve arka plakayı arka plaka tutucuya vidalayın.Daha sonra sızıntı yı test etmek için arka plakaya tuzlu uygulayın. Hayvan ve cerrahi platform daha sonra kapak cam pencereden görüntüleme için iki foton mikroskobun optik tabloya transfer edilebilir. Bu temsili deneyde, EGFP pozitif claudin-5 bir vasküler pleksus boyunca floresan etiketli sıkı kavşaklar içinde görselleştirilmiştir.Z projeksiyonundaki sıkı kavşak yapılarının net bir şekilde tanımlaması, başarılı bir laminektomi, pencere yerleşimi ve arka plaka implantasyonundan sonra minimal XY görüntü deplasmanının üretildiğini gösterir. Bu prosedürü denerken bu mikrocerrahinin zor olduğunu ve ustalaşmanın zaman alabileceğini unutmamak önemlidir. Ancak bir kez hakim, bu ameliyat yaklaşık 30 dakika içinde yapılabilir.Bu prosedürü takiben, diğer yöntemler, iki foton mikroskopi ve in vivo görüntüleme gibi, nörovasküler remodeling ve omurilik içeren diğer hastalık süreçleri hakkında ek sorulara cevap için yapılabilir.

laminectomy-and-spinal-cord-window-implantation-in-the-mouse

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Embriyonik Omurilik Commissural Nöronlar Diseksiyon ve Kültür

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Nörobilim

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Fare Omurilik İki foton Mikroskop kullanarak in vivo Görüntüleme

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Fare Omurilik Koşullu Gen Manipülasyon için bir Viral Vector Stereotaksik Enjeksiyon

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Larva Zebra balığı Omurilik Kesisi

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Boyuna için bir Spinal Odası implantasyon için bir Prosedürü<em&gt; İn Vivo</em&gt; Fare Omurilik Görüntüleme

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Fare Omurilik Hücresel Dynamics İki foton Görüntüleme

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Kullanımı Netlik Fare Omurilik içinde Serotonerjik Elyaf Görüntüleme / KÜBİK Tekniği

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

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 ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Nöral Kök Hücrelerin Sıçan ve Fare Beynine İntra-Arteriyel Teslimatı: Serebral İskemiye Uygulama

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Fare Omuriliğinden Mikroglia'nın Ekstraksiyonu ve Enzimatik Saflaştırılması

Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and function of the mammalian central nervous system rely significantly on the activity of resident macrophages known as microglia. Microglia display heterogeneity and multifunctionality, enabling distinct gene expression and behavior within the spinal cord and brain. Numerous studies have explored cerebral microglia function, detailing purification methods extensively. However, the purification of microglia from the spinal cord in mice lacks a comprehensive description. In contrast, the utilization of a highly purified collagenase, as opposed to an unrefined extract, lacks reporting within central nervous system tissues. In this study, the vertebral column and spinal cord were excised from 8-10 week-old C57BL/6 mice. Subsequent digestion employed a highly purified collagenase, and microglia purification utilized a density gradient. Cells underwent staining for flow cytometry, assessing viability and purity through CD11b and CD45 staining. Results yielded an average viability of 80% and a mean purity of 95%. In conclusion, manipulation of mouse microglia involved digestion with a highly purified collagenase, followed by a density gradient. This approach effectively produced substantial spinal cord microglia populations.

Yazar Spotlight: Omurilik Heterojenliği ve Saflaştırılması Üzerine Mikroglia Araştırması 

Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.

Fare omurilik mikrogliasının hızlı ve verimli bir şekilde zenginleştirilmesi

The vertebral column defines a vertebrate and shapes the spinal canal, a cavity that encloses and safeguards the spinal cord. Proper development and ...