Biz Sağlık Araştırması (CIHR) Kanada Enstitüleri bu iş için finansman desteği kabul etmek istiyorum, katkı için Krembil Aile Vakfı, Nöral Onarım Halbert Başkanı ve Yenilenme, Phillip ve Peggy DeZwirek ve Gordon Yao Şekil 2&#39;ye . Klaus Zweckberger &quot;Deutsche Forschungsgesellschaft&quot; (DFG) bir hibe ile finanse edildi.

NOT: Aşağıdaki deneysel protokol Üniversitesi Sağlık Ağı (Toronto, Kanada) hayvan bakımı komitesi tarafından onaylanmış ve bakım ve hayvan bakım Kanadalı konseyi tarafından hazırlanan deney hayvanlarının kullanımı kılavuzda oluşturulan politikalar doğrultusunda dir . 1. Servikal Anevrizma Klip Kontüzyon / Sıkıştırma Modeli <ol><li> Cerrahi otoklav cihazları ve% 70 alkol banyosunda aletleri koyarak tüm surgcial prosedür sırasında steril koşullar tutun önce. </li><li> (1: 1) bir oksijen kombinasyonu (O 2), azot oksit (N2 O), Wistar sıçanları (250-270 g), uyuşturan ve 1.8-2.2% İzofluran ve bir gaz anestezi ekranı üzerinden spontan solunum desteği. Anestezinin indüksiyonu için 1 dakika için,% 5 İzofluran ile başlar ve daha sonra azaltır. Bir acı uyaran (örneğin. Pençeleri at) vererek cerrahi, anestezi derinliği kontrolü başlamadan önce. Yağlı merhemler Uygulagözlerde kuruluk önlemek için ve sonraki enfeksiyonları önlemek için. </li><li> Bir ısıtma yastık (37 ° C) üzerine sıçan koyun ve bir stereotaktik çerçevenin içine kafasını düzeltin. </li><li> Servikal omurga etrafında cerrahi bölgeyi Tıraş ve povidon iyot ve% 70 alkol ile dezenfekte edin. </li><li> Torasik vertebra gövde 2 (T2) belirgin processus ulaşan servikal vertebra (C2) den omurga üzerinde bir orta hat kesi yapmak. </li><li> Doğrudan orta hat üzerinde vertebra kasların dış tabakası kesti bir kranyokaudal yönde (kanamayı önlemek için) ve daha ileri processus spinosus ve laminalar ulaşana kadar dobra dobra derin kas katmanları incelemek. Ekartörleri yerleştirin. </li><li> Laminektomi için hedeflenen seviyelere gözlemlemek için vertebra gövdesi T2 belirgin prosesus spinosus yönlendirmek. Kimlik ve seçilen lamina mikro-cerrahi hazırlıktan sonra, laminalar ve processus spinosus gevşetmek için ligamenti flavae kesti. Son olarak, Comuriliğe bir kemik kesme yanal ile lamina ile ut ve omurilik kendisi herhangi bir sıkıştırma kaçınarak, yavaşça o çıkarın. NOT: En yaygın seviyeleri C5 / 6, C6 / 7 veya C7 / T1 vardır. perioperatif mortalite oranı yaralanma seviyesi rostral daha artar. Paravertebral venöz sinüs kanama yaygındır ve sünger ile dikkatli sıkıştırma ile ele alınabilir. </li><li> Kablosunu travmatize klibi takmadan önce, (özellikle seviye C5 / 6) kırpma onları yedek sinir köklerini ortaya çıkan tespit. </li><li> Pürüzsüz klip indüksiyon sağlamak amacıyla, bir kanca ile vertebralarda dorsal tarafına ventral dura gevşetin ve klip için bir koridor hazırlamak. </li><li> Son olarak, açık klibi eklemek ve bir kafa travması ulaşmak için kapattı (hızlı kapatma) ek verelim. Klip kapanma gücü ve klip kapatma süresi travmanın şiddetini ve sıkıştırma derecesini belirler. Yaygın olarak kullanılan 15-35 g ve clippi arasında klip kuvvetleri vardırörneğin 1 dakika ng süresi. (Şekil 1). </li><li> Klibin uzaklaştırılmasından sonra, 2 kat kasları adapte yarayı kapatmak. </li><li> Anestezi durdurun ve sternum yatma için yeterli bilince kavuşur kadar sürekli gözlem altında hayvan uyandırma izin. Son olarak, bir tek kafese koymak sıçan ve post-operatif tedavi yönergeleri izleyin. </li><li> Hayvanlar yaralanma bu tip şiddeti ile mücadele olduğundan, ameliyat sonrası tedavilere özel dikkat gerekir: <ol><li> (3 gün ve 5 gün, sırasıyla ve klinik semptomlara göre için Buprenorphine ve meloksikamın) ağrı kesiciler yönetme. </li><li> 3 gün için ek tuz çözeltisi deri altına vermek (günde 2 kez 5-10 mililitre (mi)). </li><li> 2 gün önce içme suyu antibiyotik sağlayın ve 7 gün sonrası ameliyat kadar (örn moksifloksasin) </li><li> Mesane fonksiyon kurtarma sabit oluncaya kadar Mesaneyi günde 2-3 kez sıkınly belirgin. </li><li> Bir günde en az bir kez nörolojik açıkları ve işletilen hayvanların fizyolojik durumunu gözlemlemek. </li></ol></li></ol> 2. (14 gün yaralanma sonrası) SAP&#39;larını ve NPC enjekte <ol><li> 1.1 açıklandığı gibi anestezi neden olur. 1.3, bir stereotaktik çerçeve içinde sıçan başını düzeltmek dikiş veya yara klipleri kaldırmak ve yara ve povidon iyot ve% 70 alkol ile cerrahi alan dezenfekte edin. </li><li> Dikkatle, Ekartörleri eklemek, paravertebral kaslar teşrih dura gelen mikroskobik skar dokusu kaldırmak ve lezyon sitesini yeniden maruz. </li><li> % 1 bir konsantrasyonda SAP&#39;larını hazırlanması (a / h) bir hücre dışı matris jel yapmak. QL6 SAP&#39;ler fizyolojik olarak uyumlu bir pH değerine sahip ve enjeksiyondan önce tamponlanmalıdır gerek yoktur. Omurilikte SAP&#39;lerin görselleştirme için, QL6 (QL6-FITC) bir floresan türevini kullanın. </li><li> 2 porsiyon, her dağıtılan lezyonun merkezine içine SAP (5 mikrolitre (ul) enjekteOrta hat 2.5 ul ikili. Bir mikro cam kapiller (100 mikrometre (mikron) dış çapı (OD)) ile stereotaktik çerçeve bağlı bir Hamilton şırınga kullanın. Keskin bir iğne ucu ile dikkatlice dura açın ve travmatize omuriliğe cam kılcal stereotaktik 2 milimetre (mm) takın. </li><li> Hacim 1/3 enjekte ettikten sonra, 1,5 mm derinliğe kadar iğne kaldırmak ve sonra daha 1/3 1 mm. Tüm hacminin enjekte edildikten sonra ve şırınga uzaklaştırılmasından önce, jel oluşumu stabilize etmek için 5 dakika bekleyin. </li><li> NPC oluşturmak için, yetişkin farelerin DsRed (ya da, yeşil YFP pozitif fareler) kullanın ve izole ve paraventrical bölge 4,18,21 onları yetiştirmek. </li><li> Hücre süspansiyonu 90% canlı hücrelerin ~ varlığını gösteren Tripan Mavi boyama ile NPC canlılığını değerlendirmek. Büyüme ortamı (50 x 103 canlı hücre / ul) hücreleri seyreltin ve daha sonra hücre nakli için bunları kullanın. </li><li> Dört 2 ul olun (8 ul toplam volume, 2 mm rostral ve yaralanma yerinde kaudal de bilateral 4 x 10 5 NPC) İntraspinal enjeksiyonları içeren. Dura açılışından sonra, Hamilton mikro cam kapiller omurilik dorsal yüzeyinin altında 1.5 mm eklemek ve hücre süspansiyonu 2 ul enjekte. Yaklaşık 0.5 ul / dk (dk) bir enjeksiyon hızı seçin. </li><li> Her bir enjeksiyonun ve kordon üzerinden kılcal uzaklaştırılmasından önce sonunda, yeni hücre hacmine uygun olarak uzanan en az 1 dakika sağlayan doku bekleyin. (Şekil 2) </li></ol> Subdural 3. implantasyonu Büyüme Faktörü Uygulama için pompalar <ol><li> Hücre yaşamını destekleyen bir büyüme faktörü olan beyin omurilik sıvısı (CSF) zenginleştirmek için, 0.5 ul / saat arasında bir seyreltme oranı, 0.04 kateter çapa sahip olan, 7-14 gün boyunca, alt-durally büyüme faktörleri seyreltilmesi mikro-ozmotik pompalar kullanımı cm OD, 100 ul bir hazne hacmi. </li><li> Tercih büyüme faktörleri seçin(Örneğin, beyin türevi büyüme faktörü (BDGF), epidermal büyüme faktörü (EGF), fibroblast büyüme faktörü (FGF)), pompa 6-10 saat önce implantasyonu dolgu ve 37 ° C sıcaklıkta bir su banyosu içinde pompalar dengeye getirin. </li><li> Hemen NPC enjeksiyondan sonra, pompayı yerleştirmek için bir deri altı girinti hazırlamak. Tercih yerleri pompa kendisinin neden büyük yerel rahatsızlık kaçınarak torakoskopik karın bölgesi lateral kenarlar vardır. </li><li> Büyüme faktörlerinin en yüksek konsantrasyonu elde etmek için, kateterin açık ucu lezyon yerinde yakın biter emin olun. Bu nedenle, komşu üst veya alt düzey bir konteyner-laminektomi gerçekleştirmek. SCI C7 / T1 ise, örneğin, C5 küçük bir laminektomi gerçekleştirmek. </li><li> , Deri altı oyuk içine pompayı koymak gerekli uzunlukta kateter kısaltmak, ve herhangi bir hareket-ilişkili çıkık kaçınarak paravertebral kaslar birkaç dikişlerle (6.0) ile sabitleyin. </li><li> Sivri uçlu (C5, örneğin) dura açtıktan sonraBir iğne, subdural uzaya kateter tanıtmak ve herhangi bir direnişle karşılaşmadan ve kablosunu zarar vermeden bir kuyruk yönde kayma sağlar. Örneğin C6 Atlanan lamina kateter tespit ve istikrar gibi ek noktasını vermektedir. </li><li> Kateter sorunsuz çalışır ve pas olmadığından emin olun. </li><li> Dikişler ya da klips ile yakın bir tabaka ile kas ve deri. (Şekil 3) </li><li> Anestezi durdurun ve sternum yatma için yeterli bilince kavuşur kadar sürekli gözlem altında hayvan uyandırma izin. Son olarak, bir tek kafese onları geri koydu ve post-operatif tedavi yönergeleri izleyin. </li><li> Olsa bile, hayvanlar bu zaman noktasında kısmen kurtarıldı olabilir, özel ameliyat sonrası tedavi sağlayın: <ol><li> (3 gün ve 5 gün, sırasıyla ve klinik semptomlara göre için Buprenorthine ve meloksikamın) ağrı kesiciler yönetme. </li><li> 2 gün önce kadar su şişeleri antibiyotik sağlayın7 gün ameliyat sonrası (örn moksifloksasin). </li><li> Gerekli eğer hala, mesane sıkma devam edin. </li><li> Sıçanlar susuz görünürse, ek deri altı sıvıları verin. </li><li> Bir günde en az bir kez çalıştırılan hayvanların nörolojik fonksiyonu ve fizyolojik durumunu gözlemleyerek tutun. </li><li> 2 gün önce NPC enjeksiyon immünosupresyon tedavisi yönetme ve 7 gün sonrası transplanation (minosiklin) ve kurban kadar (Sandimmune) kadar sırasıyla. </li></ol></li></ol> 4. Doku Değerlendirmesi <ol><li> Gözlem süresi sonunda (örn., Omurilik yaralanması sonrasında 4 hafta) derin anestezi (% 5 İzofluran 2-3 dakika için) olarak hayvanların kurban ve 4, soğuk ve 150 ml, 50 ml soğuk (4 ° C), tuzlu su ile transkardiyal bunları serpmek 0.1 M fosfat tamponlu salin (PBS) içinde% paraformaldehid. </li><li> Omurilik çıkarın ve 24 saat süre ile, 0.1 M fosfat tamponlu salin (PBS) içinde% 4 paraformaldehid içinde koydu. </li><li&gt; 48 saat boyunca donmaya karşı koruma sağlamak için,% 25 sükroz solüsyonunda omurilik koyun. </li><li> &lt;30 um bir kalınlığa sahip uzunlamasına cryosections sağlar. </li><li> Bir arka plan kullanım DAPI (: 1,000 1) sağlayan tüm hücre çekirdekleri immünohistokimyasal boyama için. DsRed pozitif NPC kırmızı görünür, QL-6 FITC yeşil görünür, hem de böylece özel lekeli olması gerekmez. (Şekil 4). </li><li> Tarama elektron mikroskopi (SEM) için örnekler, 2 saat süre ile 4 ° C &#39;de, glutaraldehid emmek 5 dakika boyunca etanol,% 10 artış adımda yavaş yavaş bunları kurutmak ve 1 saat boyunca basınçlı bir sıvı CO2 sifon koyun sağlar. Bir püskürtmeli kaplayıcı kullanılarak altın ile kaplayın iskelelerinin. Hitachi S-3400N taramalı elektron mikroskobu görüntüleri çekin. (Şekil 5) </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+trasplantation+of+adult+neural+presursor+cells+promotes+remyelination+and+functional+neurological+recovery+after+spinal+cord+injury.">Delayed trasplantation of adult neural presursor cells promotes remyelination and functional neurological recovery after spinal cord injury.</a> J Neurosci. 26 (13), 3377-3389 (2006).</li><li>Burdick, J. A., Ward, M., Liang, E., Young, M. J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+neurite+outgrowth+by+neurotrophins+delivered+from+degradable+hydrogels.">Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels.</a> Biomaterials. 27, 452-459 (2006).</li></ol>

Covidien supplied the QL6 SAP for this study.

Bu protokol sıçanlarda bir servikal yaralanma modeli gerçekleştirmek için okuyucu sağlamak ve servikal SKY sonrası daha iyi iyileşme teşvik SAP&#39;lerin ve NPC ile kombine tedavi yaklaşımı kullanmak için geliştirilmiştir. Özellikle böyle (hemi) -transection modeli veya kilo düşüşü ve sarsıntı modelleri, klip kontüzyonu / sıkıştırma modeli büyük patofizyolojik travma mekanizmaları hem temsil gibi diğer servikal travma modelleri, göre - ezilme ve basınç ve ...

Spinal kord yaralanmalarının% 50&#39;den fazlası servikal omurga ile ilişkilidir. Klinik olarak iki ana patofizyolojik mekanizma tanımlanmıştır ayarı: initial omurilik ezilme ve kemik kırıkları, kanamalar veya doku şişmesi nedeniyle sürekli sıkıştırma. anevrizma klibi kontüzyonu / sıkıştırma modeli taklit hem patofizyolojik mekanizmalar: klibi yakalamaya Bir ezilme üretir ve kırpma süresi, kemik kırıkları, kanamalar veya doku neden olduğu klinik ortamlarda sıkıştırma son önemli uzun şişme olduğunu yemeden, sıkıştırma bileşeni temsil eder. kullanılan anevrizma klibi kesin ve tekrarlanabilir kırpma kuvveti garanti altına alan bir halka yay ile değiştirilir. Özellikle yarı-kesisi veya kontüzyon modeli, bu anevrizma klibi modeli taklit iyi klinik ortamlarda göre. Toraks yaralanması olan hastalar servikal inj ile parapleji, hastaların çoğunda muzdarip ikenuries tetraplejik ve tamamen bağımlıdır. servikal kord anatomik yapısı, ancak, göğüs veya lomber kıyasla önemli farklılıklar gösterir ve bu nedenle, bu protokolde özellikle ele almaktadır. intramedüller boşlukların ve doku skar gelişimi kurtarma ve rejenerasyon için engellerdir. Bu engelleri iskele malzemesinin kullanımını aşmak için umut verici bir yaklaşımdır. Kendi toplanan peptidler lezyon üssü doğrudan enjekte edilebilir. Orada boşluğu köprüleme nano-lif iskeleleri içine monte ve inflamasyon ve doku korkutuyorsun azaltarak inhibitör ortamını iyileştirmek. Katı maddeler, implantasyon esnasında omurilik önemli hasara neden olsa da, akışkan peptidler ve güvenli bir ek ağır zarar vermeden enjekte edilebilir. Kök hücre nakli öncesinde kendinden montaj peptitler ile inhibitör çevrenin iyileştirilmesi, dolayısıyla, hücre destek integration, servikal spinal kord yaralanması sonrası farklılaşma ve nihayet, fonksiyonel iyileşme.

<table border="0" cellpadding="0" cellspacing="0" width="541">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:268px;">Company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aneurysmal clip</td>
			<td>SharpTech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical microscope</td>
			<td>Leica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro injection system</td>
			<td>World Precision Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small animal stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe</td>
			<td>Hamilton company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subdural pumps</td>
			<td>Alzet osmotic micro pump 1007D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instrument</td>
			<td>Fine Science tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane USP</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% Sodium Chloride injection USP</td>
			<td>Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Povidone iodine</td>
			<td>Purdue Pharma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropyl alcohol USP</td>
			<td>GreenField Ethanol Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QL6 SAP</td>
			<td>Covidien</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan blue</td>
			<td>Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platelet-Derived Growth Factor (PDGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor (FGF)</td>
			<td>Sigma</td>
		</tr>
	</tbody>
</table>

synergetic use of neural precursor cells and self-assembling peptides in experimental cervical spinal cord injury

Omurilik yaralanmaları (SKY) hastalar ve aileleri için ciddi nörolojik bozukluk ve, psikolojik, ekonomik, ve sosyal sonuçlara neden. Klinik, SCI fazla% 50 servikal omurga 1 etkiler. Birincil yaralanma sonucu olarak, enflamasyon, apoptoz, ve demiyelinasyon dahil olmak üzere ikincil mekanizmaların bir kademeli sonunda intramedüler kavite 2,3 doku yara ve gelişmesine yol meydana gelir. Her iki hücre nakli, entegrasyon ve yenilenme fiziksel ve kimyasal engelleri temsil eder. Bu nedenle, inhibitör ortamı şekillendirme ve hücre transplantasyonu ve yenilenme için bir destekleyici ortam yaratmak için boşluklar köprü umut verici bir tedavi hedefi 4. Burada, bir anevrizma klibi ile servikal SKY bir ezilme / sıkıştırma modeli tarif edilmektedir. Tam transeksiyonu veya kordonun yırtılmalar nadirdir çünkü bu model, daha klinik açıdan diğer deneysel modellere daha. Ayrıca compariso içindeÖzellikle hasar Sırt taraf sütunlar, omurilik çevresel sıkıştırma avantajlı görünen ağırlık damla modeli, n. Klip kapanma gücü ve süresi farklı yaralanma şiddetini elde etmek için ayarlanabilir. Bir halka yay hassas kalibrasyon ve klip kuvvet süreklilik kolaylaştırır. Fizyolojik koşullar altında, sentetik kendinden montaj peptitler (SAP) nanoliflerden halinde kendi kendine birleşebilen ve böylece, SCI 5 uygulama için hitap etmektedir. Onlar kord hasarı minimize lezyon içine doğrudan enjekte edilebilir. SAP intramedüller boşlukları köprü ve böylece, rejeneratif tedaviler için hasarlı kablosunu donatmak için iskele kurulması biyouyumlu yapılardır. K2 (QL) (QL6), diğer peptidler ile karşılaştırıldığında Dong et l. 6 tarafından tanıtılan yeni SAP olduğunu, QL6, 6 .14 gün SKY sonrası nötr pH β-tabakalar halinde 6K2 kendine araya akut evre, SAP&#39;lerin sonra lezyon ve nöral öncü hücreler (NPC) merkezine enjekte edilir Inje vardırbitişik sırt sütunlara cted. Hücre yaşamını desteklemek amacıyla, transplantasyon, 7 gün boyunca ozmotik mikro pompa, büyüme faktörlerinin sürekli subdural tatbikat ile bir araya getirilmektedir.

<html> Yukarıda tarif edilen prosedür gerçekleştirirken,. SAP iskele boşluğu köprü ve önleyici bir ortamının iyileştirilmesi, daha az doku korkuturuz NPC sağkalımda bir artış sağlar elde Şekil 4 olacak elde edilen bir sıçan omurilik bir uzunlamasına kesitini göstermektedir yaralanma sitesi SKY sonrası 6 hafta ve QL6 SAP enjeksiyonu ve NPC transplantasyon sonrası 4 hafta. QL6 peptidler başarıyla merkez üssü toplu, kordon içine enjekte ve yarı gölge postero-kaudal dağınık ...

Watch this Scientific Journal Video about Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury at JoVE.com

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Deneysel Servikal Spinal Kord Yaralanmalarında Sinir Öncü Hücreler ve Öz-montaj Peptitler sinerjik Kullanımı

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

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9.1K Görüntüleme.  University Health Network, Krembil Neuroscience Centre. Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established. 

Video: Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Amyotrophic Lateral Sclerosis ve Travmatik Spinal Kord Yaralanması Servikal Ventral Horn Hedefleme intraspinal Hücre Transplantasyonu

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Tıp

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Sıçanlarda Spinal Kord Yaralanma sonrası Akut ve Kronik Dokunsal Duyu Testi

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Sonsuz Horizon Darbeli kullanarak tek taraflı Servikal Spinal Kord Yaralanması bir Contusive Modeli

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Farelerde Kontrollü Servikal yırtığı Sakatlık

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

Kronik Spinal Kord Yaralanması Bireylerde Solunum Motor Kontrol Değerlendirmesi (RMCA) kullanarak Solunum Kas Aktivasyon Değerlendirilmesi

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

Post-lezyonlu Solunum nöroplastisite Eğitim için Servikal Spinal Kord Yaralanması bir fare modeli

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Travmatik Beyin Hasarı için Kontrollü Kortikal Etki Modeli

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

A Radyo-telemetrik Sistemi Omurilik transeksiyonu ve Embriyonik Nöral Hücre Greftler Kök Sıçanlarda Kardiyovasküler Fonksiyonu Monitör

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Omurilik Yaralanması Deneysel Kontuzif Modelinde Sinir Kök Hücre Nakli

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Omurilik Sıkıştırma Yaralanma Kalibre Forseps Modeli

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...