Bu çalışma Ulusal Sağlık Enstitüleri [Grant no tarafından desteklenmiştir. R01NS069726] ve Yardımlaşma Grant Amerikan Kalp Derneği Grant [Hibe no. 13GRNT17020004] SD.

Not: Fareler (C57BL / 6J, erkek) yaşlı 10-12 haftalık, bu çalışmada kullanılmıştır. Tüm işlemler Laboratuvar Hayvanları Bakım ve Kullanımı için NIH Kılavuzu&#39;na uygun olarak yapılmış ve Missouri Kurumsal Hayvan Bakım ve Kullanım Kurulu Üniversitesi (IACUC) tarafından onaylanmıştır. 1. Ön Cerrahi <ol><li> ve cerrahi otoklav gün önce tüm cerrahi aletler sterilize edin. (121 o C, 15 psi, 30/30 çevrimi) kurutma 30 dakika, ardından 30 dakika boyunca 15 psi 121 o C&#39;de aletleri ve otoklav sarın. Daha sonra kullanmak kadar temiz ve steril bir ortamda aletleri yerleştirin. </li><li> Taze Rose Bengal (RB) solüsyonu (steril tuzlu su içinde 20 mg / ml) ameliyat öncesi her hazırlayın. Daha sur kullanılan tamamen kaybolana kadar RB, girdap daha sonra 19 W. Wrap alüminyum folyo ile boru ve ışıktan korumak için çıkış gücü ile 50/60 Hz, 5 dakika süreyle sese tabi tutularak ve ardından tüp çözünmesi içingery. </li><li> Steril tuzlu su içinde, ketamin / ksilazin bir karışım hazırlayın. Anestezik karışımın 1 ml&#39;lik bir son hacim sağlamak ve steril tuzlu su içinde 550 ul; ve ketmaine 325 ul (100 mg / ml stok konsantrasyonu): ksilazin 125 ul (20 mg / ml stok konsantrasyonu) ekleyin. </li><li> Homeothermic ısıtma yastığı ön ısıtmak. </li><li> Lamba gücünü dengelemek için 30 dakika süreyle metal halide lamba (FN1 epi-floresan mikroskop ışık kaynağını) Pre-sıcak. </li><li> 10X objektif ve dik FN1 epi-floresan mikroskop alan diyafram ayarlayarak bir retikülü kullanılarak 1 mm&#39;lik bir çapa ışıklı bölgenin boyutunu ayarlayın. </li></ol> 2. Cerrahi Prosedür <ol><li> Ketamin (130 mg / kg B. ağ.) Ve xylazine bir dozu ile fare anestezi (10 mg / kg B. ağ.). 1.3 kokteyl dayanarak, fare B. ağırlıkça gramı başına 4 ul. ihtiyaç vardır. Alkollü bir bezle enjeksiyon siteyi sterilize ve içi ile anestezi yönetmek-peritoneal (IP) rota. Hayvan indüksiyon ve kurtarma gecikme olacağını bu kadar kan damarları veya kas içine anestezik enjekte özen gösterin. </li><li> Kurumasını önlemek ve hipotermi önlemek için ısıtma pedi üzerinde hayvan yerleştirmek için fare hem gözlere suni gözyaşı merhemi sürün. </li><li> Hayvanın Hazırlanması <ol><li> Ayak tutam yanıtı kullanarak uygun cerrahi seviye anestezi hayvan kontrol edin. </li><li> Hayvan anestezi cerrahi düzeyine ulaştığında, bir elektrikli saç düzeltici kullanarak hayvanın orta hat çevresinde dorsal yüzeyi saç klibi. Betadin çözeltisi üç kez, ardından% 70 etanol ile cerrahi alan fýrçalayýn. Bir sonraki adıma kadar steril cerrahi örtü ile site örtün. </li></ol></li><li> İnce kemik cerrahi prosedür omurilik maruz <ol><li> Cerrahi platformu (Şekil 1A) üzerine homeothermic ısıtma yastığı üzerinde yüzükoyun pozisyonda fare yerleştirin. Düzgün fareyi sabitleyinBir burun kelepçe kullanarak duruş uzatılmış boyun bölgesini (Şekil 1A, B) korumak için. </li><li> T12 için göğüs omurları T9 uzanan dorsal orta hat boyunca cerrahi makas kullanarak (yaklaşık 1 cm uzunluğunda) bir kesi yapmak. Cerrahi alan maruz cilt ayırınız. </li><li> Bir neşter kullanılarak, dikkatli bir şekilde T9 dorsal dikenleri ortaya çıkarmak için kas temizlemek - T12 vertebra. Steril bir pamuklu çubukla hafif basınç uygulayarak her adımda kanamayı durdurmak. Ayrı T10 - Çevre kas T12 vertebra ve stabilize ve herhangi bir hareketi önlemek için omurga kelepçe kullanarak sabitleyin (Şekil 1A, B). </li><li> Dikkatlice ve yavaşça ince, T10 veya T11 vertebra dorsal yüzeyini kemik parlatma matkap ile yüksek devirli kullanma SC (Şekil 1C) dorsal yüzeyinde posterior spinal ven ve diğer küçük gemiler görselleştirmek için. </li><li> Nedeniyle inceltme işlemi sırasında oluşan ısı termal hasar görmesini önlemek için,artığın ayrılması için sürekli emme ile birlikte, normal tuzlu su, bir yumuşak ve sürekli bir akım uygulanır. </li><li> Ana damar açıkça görünür oluncaya kadar bir neşter kullanılarak dikkatle kemik yüzeyini pürüzsüz. Bu süreçte omurilik zarar vermemeye özen gösterin. </li><li> Kan damarı görüntülenmiştir sonra, bir insülin şırıngası kullanılarak retro-orbital sinüs yoldan 30 mg / kg (vücut ağırlığı) arasında bir dozda tatbik RB. </li><li> RB enjeksiyonu takiben 3 dakika sonra bir lazer Doppler debimetre kullanılarak kan akışını ölçmek gerekirse (Şekil 2A, B). Bütün işlem sırasında asepsi koruyun. </li></ol></li></ol> PT 3. İndüksiyon <ol><li> Yüksekliğini ayarlayabileceğiniz bir Lab-Jack üzerinde XY pozisyonu ayarlanabilir sahnede hayvan yerleştirin. T11 omuriliğin açıkta kalan bölge FN1 epi-floresan mikroskop (Şekil 3A) 10x objektif altında doğrudan böylece fare konumunu ayarlayın. </li><li> Li gücünü ayarlama% 12 GHT, kaynak ve inceltilmiş omurilik ortasında 0.75 mm bir çapa sahip bir bölge ışınlanmış T11: - 580 nm, yeşil ışık (dalga boyu 540 (Not: Bu bölge, posterior spinal damar ve kılcal damarları içeren) 2 dakika süreyle 10X objektif ile mikroskop filtre küp) tarafından elde etti. Başında ve ışınlama sonunda (Şekil 3B, C) ​​görüntüleri almak ve bu noktada deney zamanı kaydedin. </li><li> 2.4.8 gibi omurilik üzerinde aynı pozisyona lazer Doppler probu yerleştirilerek 10 dakika gerektiğinde tekrar kan akışını ölçmek (Şekil 2A, B). </li><li> Herhangi bir kanama ve eğer ışınlama kontrolünden sonra hiçbiri bulundu hayvan sütür devam edin. Emilebilen sütür veya 4-0 boyut ipek sütür kullanılarak omurilik iki tarafında kasların birlikte yüzeysel fasya dikin. Maruz SC zarar vermemeye özen gösterin. 4-0 ipek sütür ile cilt dikin. Betadine ya da iyot uygulanırsütür sonrası cildin kenarlarına. </li></ol> 4. Post-cerrahi Bakımı <ol><li> Sütür sonra, kurtarma için ısıtma pedi üzerinde hayvan yerleştirin. Kurtarma sonra her iki arka-bacaklarda hareket gözlemleyerek nörolojik defisit belirtileri hayvanları kontrol ediniz. Bu sternal yatma korumak için yeterli bilinci yerine kadar sahipsiz hayvan bırakmayın. </li><li> Evde kafes hayvanları aktarın. Tamamen iyileşene kadar diğer hayvanların şirkete ameliyat geçirmiş hayvan iade etmeyin. </li><li> Düzenli aralıklarla hayvanların kontrol edin. Ciddi nörolojik defisit durumunda, mesane boşaltılması, analjezik idaresi (- 0.1 mg / kg buprenorfin, 0.05) gibi uygun bakım sağlamak. Dehidrasyon için kontrol edin ve ağır durumda normal tuzlu su subkutan yönetmek. Genellikle, buprenorfın (0.1 mg / kg), cerrahi sitedeki ağrı gidermek için dikiş sonra tatbik edilecektir. </li><li> Hayvanlar hemen değilsenizhayvanların kolayca yiyecek ulaşabilir, böylece ameliyat sonrası ötenazi, biz kafes katta yüksek su içeriği diyet koyacağız. </li></ol> 5. Transcardial Perfüzyonu, Nissl boyama ve immün <ol><li> Daha önce tarif edildiği gibi 17-20 transkardiyal hayvan serpmek. <ol><li> Protokol daha önce tarif edilen ve transkardiyal buz soğukluğunda% 4 paraformaldehid, PBS içinde (PFA), ardından fosfat tamponlu salin (PBS, pH 7.4), serpmek hayvan anestezisi. </li><li> Perfüzyon sonra, omurilik (SC) çıkarın ve 4 ° CO / N PBS içinde% 4 PFA bunu post-düzeltin. % 30 sakaroz ile PBS içine sabit SC aktarın ve 2 için saklayın - bu tüpün dibine batar kadar 3 gün. </li><li> Bir kriyostat kullanarak 30 mikron kalınlığında bölümler halinde omurilik kesilmiş ve jelatin kaplı cam lam üzerine ya da 0.01 M PBS ile bir 48-çukurlu plaka içinde seri yerleştirin. </li></ol></li><li> Nissl boyama: Bir Nissl staini gerçekleştirmek PT kaynaklanan zararı incelemek içinspinal kord kesitlerinde ng daha önce tarif edildiği 17-20. <ol><li> Kısaca,% 0.25 cresyl menekşe ile her beşinci omurilik cam slaytlar dilim ve leke toplamak. Boyanan kesitler (Şekil 4) görüntülerini çekin. </li></ol></li><li> Immün: Daha önce bir yüzer bölüm yöntemi 17,18,20 kullanılarak açıklandığı gibi. <ol><li> , Tavşan anti-NeuN antikoru (1: 300), ve tavşan anti-: Kısaca, tavşan anti-glia fibriler asidik protein (GFAP) poliklonal antikor (300 1) ile 4 ° C &#39;de, O / N inkübe edilerek omurilik bölümleri leke Iba1 antikoru (1: 500) eşek anti tavşan Alexa 568-konjuge IgG (1: 400), ardından oda sıcaklığında 4 saat süre ile sekonder antikor. Floresan mikroskop (Şekil 5) ile görüntüleri çekin. </li></ol></li></ol>

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D., 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=The+evolution+of+chemokine+release+supports+a+bimodal+mechanism+of+spinal+cord+ischemia+and+reperfusion+injury.">The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion injury.</a> Circulation. 126 (11 Suppl 1), S110-S117 (2012).</li><li>Jia, Z., 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=Oxidative+stress+in+spinal+cord+injury+and+antioxidant-based+intervention.">Oxidative stress in spinal cord injury and antioxidant-based intervention.</a> Spinal Cord. 50 (4), 264-274 (2012).</li></ol>

The authors have nothing to disclose.

Bu çalışmada, biz SC iskemi bir Fototrombotik modelini tarif. Nedeniyle gelişmelere genetik mühendisliğinde mümkün SC iskemik patofizyolojisinde rol oynayan spesifik genlerin etkilerini incelemek için yapmıştır piyasada mevcut transgenik farelerin bir dalgalanma olmuştur. Bu çalışmanın amacı, omurilik iskemisi tekrarlanabilir bir fare modeli geliştirmek olmuştur. Burada farelerde SCI ikna etmek için bir kortikal PT modeli uyarladı. Cerrahisi T11 torakal vertebra seviyesinde fareler...

Travmatik omurilik yaralanması (SKY) SC sensörimotor ve otonomik fonksiyonları etkileyen yıkıcı bir klinik durumdur. SCI kalan hastalar sıklıkla önemli ölçüde günlük aktivitelerini ve yaşam kalitesini etkiler 1 parapleji zayıflatıcı ile bırakılır. Deneysel SCI modelleri SCI patofizyoloji ve ilgili nöral tamir süreçlerini anlamak için bilimsel araştırmaların vazgeçilmez bir araç olmuştur. Bu modeller aynı zamanda fonksiyonel iyileşme amaçlayan çeşitli deneysel nöroprotektif müdahalelerin klinik öncesi etkinliğini test etmek için kullanılmıştır. Şu anda uygulamada SCI modellerinin çoğunluğu mekanik bozabilir ve SC zarar fiziksel künt kuvvet kullanımını istihdam. Bu yöntemler SC 2 kontüzyon, sıkıştırma, çıkık ve transekte bulunmaktadır. Bu önerilmiştir birincil mekanik hasara yaralı SC iskemi setleri formunda ikinci bir yaralanma sonrası<html> 3,4. İkincil iskemi etyolojisi doku ödemi 5-7 kan damarlarının tıkanması bazen geniş doku dejenerasyonu, parankimal kanama ve kapsar. SC bütünlüğü daha da etkilenir sekonder yaralanmanın bir sonucu olarak, nöron ve glial hücreler ciddi fonksiyonu ve canlılığı bozulmaktadır ve iskemik penumbra, aşağıdaki inme büyüme benzer yaralanma, kronik evrede büyüme infarkt yol açar, apoptozi altına 8,9. Eksitotoksisite, serbest kök üretimi ve enflamasyon gibi çeşitli mekanizmalar SCI 10,11, aşağıdaki iskemik hücre ölümünden sorumlu olduğu bildirilmiştir. Buna ek olarak, SC iskemi sıklıkla hastalar 12,13 parapleji yol torako-abdominal aort anevrizma tamiri ameliyatları ciddi bir komplikasyonudur. Böyle yüksek klinik etkilerine rağmen yüksek tekrarlanabilirlik ile spinal kord iskemi çok az sayıda modelleri mevcut. nt &quot;&gt; Fototromboz (PT) tekniği yüksek oranda çoğaltılabilir oldukça non-invazif. beyinde 14-20 fokal iskemi indüksiyonu için yaygın olarak kullanılan bir yöntem olup, beyin 17 açıktaki alanına kesin bir fokal iskemi lezyon üretir -21. Bu Rose Bengal (RB) 16-20,22 ya eritrosin B gibi fotoaktif boyaların sistemli uygulanması ile elde edilir, uygun bir ışık kaynağı ile birlikte, kan damarlarının lokal ışınlama ve ardından 23. boyanın Foto-aktifleştirme serbest radikallerin üretilmesini neden olur pürüzsüz vasküler endotel bütünlüğünü bozabilir ve trombositler daha sonra geminin 24 tarafından sağlanan bölgede infarkt trombüs sonuçları. kan akışının tıkanıklık trombüs oluşturan, birikmesine neden olur. ötürü kontrolden kolaylığı yoğunluğu ve ışınlama süresi bu işlem oldukça düzgün ve çoğaltılabilir bir enfarktüs verir. Bundan başka, bu yöntem, bir infarktüsü uyarılması için kullanılabilecektiriskemi etkisi uzaysal (örneğin, vs beyaz cevher gri madde) aydınlatılmasını sağlayan çeşitli anatomik yerlerinde t. Bu çalışmanın amacı, farelerde SC iskemi kolay ve yüksek tekrarlanabilir bir model geliştirmektir. Biz farelerde SC iskemi PT modeli prosedürü tarif. Histoloji ve immün sonuçları PT etkili bir SC enfarktüs, nöron kaybı ve reaktif gliozis uyarabilir gösterdi.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Rose Bengal</td>
			<td style="width:233px;">Sigma-Aldrich</td>
			<td style="width:180px;">330000</td>
			<td style="width:421px;">20 mg/ml in sterile saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57Bl6/J</td>
			<td>Jackson lab</td>
			<td>664</td>
			<td>22-25g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine&#160;</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine solution</td>
			<td>Purdue</td>
			<td>NDC-67618-150-01</td>
			<td>10% povidone iodine topical solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal saline</td>
			<td>Abott Laboratories</td>
			<td>04930-04-10</td>
			<td>For diluting RB, anaesthesia and for preventing tissue from drying</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tears ointment&#160;</td>
			<td>Rugby</td>
			<td>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Decon labs.Inc</td>
			<td>2716</td>
			<td>70% ethanol for disinfection</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Metal halide lamp</td>
			<td>EXFO, Canada</td>
			<td>X-Cite 120 PC&#160;</td>
			<td>Set power at 12%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors&#160;</td>
			<td>Fine Science Tool</td>
			<td>15000-10</td>
			<td>for minor dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors (angled to side)</td>
			<td>Fine Science Tool</td>
			<td>14063-011</td>
			<td>No. 3 handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard scalpel</td>
			<td>Fine Science Tool</td>
			<td>10003-12</td>
			<td>for removing muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blade</td>
			<td>Feather</td>
			<td>2976</td>
			<td>No. 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (curved)</td>
			<td>Fine Science Tool</td>
			<td>11150-10</td>
			<td>for holding tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (straight)</td>
			<td>Fine Science Tool</td>
			<td>11151-10</td>
			<td>for holding tissue</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Needle holder&#160;</td>
			<td>Fine Science Tool</td>
			<td>12002-12</td>
			<td>for suturing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue adhesive glue</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>to adhere to edges of the cut skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monofilament polypropylene&#160;</td>
			<td>USSC Sutures</td>
			<td>VP-521</td>
			<td>Size = 4-0 (for fascia)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perma-hand silk</td>
			<td>Ethicon</td>
			<td>683G</td>
			<td>Size = 4-0 (for skin)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro drill</td>
			<td>Roboz Surgical Instrument Co. Inc.</td>
			<td>RS-6300</td>
			<td>with bone polishing drill bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser doppler flowmeter</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF1</td>
			<td>for monitoring change in blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating pad</td>
			<td>Fine Science Tool</td>
			<td>21052-00</td>
			<td>to prevent hypothermia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Jack</td>
			<td>Fisher scientific&#160;</td>
			<td>14-673-50</td>
			<td>4x4 in plate to adjust the height of the animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-Y gliding stage&#160;</td>
			<td>Amscope</td>
			<td>GT100</td>
			<td>for positioning the animal under microscope&#160;&#160;</td>
		</tr>
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photothrombosis-induced focal ischemia as a model of spinal cord injury in mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Bu çalışmanın amacı, PT modeli kullanılarak farelerde omurilik iskemisi üretmekti. Spinal kord (T10 - T12) Yukarıdaki kemik arzu edilen bölgeye sonra inceltilmiş, Rose Bengal, retro-orbital sinüs yoldan enjekte edildi ve iskemi PT indüklenen Şekil 1A, B yer alan fare göstermektedir ısmarlama bir cerrahi. Ameliyat sırasında platformu. Fare burun kelepçe ile yerinde düzenlenen ve iki ayarlanabilir omurgalı omuriliği stabilize kelepçeleri Şekil 1C T10 omurilik üzeri...

Watch this Scientific Journal Video about Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice at JoVE.com

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Farelerde Omurilik Yaralanma Modeli Olarak Odak iskemisi Fototromboz kaynaklı

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

photothrombosis-induced-focal-ischemia-as-model-spinal-cord-injury

Research

JoVE Journal

Medicine

13.1K Görüntüleme.  University of Missouri. Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice. 

Video: Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

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 ...

Tıp

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 ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

Sıçanlarda Şiddetli Spinal Kord Yaralanması bir bere modeli

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Farelerde Akut Böbrek Hasarı ve Post Yaralanma Fibrozis iskemi-reperfüzyon Model

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Reproducable Paraplegia by Thoracic Aortic Occlusion in a Murine Model of Spinal Cord Ischemia-reperfusion

Background 
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered advancements to decrease the occurrence this injury.&#160;The current model demonstrates reproducible lower&#160;extremity paralysis following thoracic aortic occlusion.
Methods 
Adult male C57BL6 mice were anesthetized with isoflurane. Through a cervicosternal incision the aorta was exposed. The descending thoracic aorta and left subclavian arteries were identified without entrance into pleural space. Skeletonization of these arteries was followed by immediate closure (Sham) or occlusion for 4 min (moderate ischemia) or 8 min (prolonged ischemia). The sternotomy and skin were closed and the mouse was transferred to warming bed for recovery.&#160; Following recovery, functional analysis was obtained at 12 hr intervals until 48 hr.
Results 
Mice that underwent sham surgery showed no observable hind&#160;limb deficit. Mice subjected to moderate ischemia for 4 min had minimal functional deficit at 12 hr followed by progression to complete paralysis at 48 hr. Mice subjected to prolonged ischemia had an immediate paralysis with no observable hind-limb movement at any point in the postoperative period. There was no observed intraoperative or post&#160;operative mortality.
Conclusion 
Reproducible lower extremity paralysis whether immediate or delayed can be achieved in a murine model. Additionally, by using a median sternotomy and careful dissection, high survival rates, and reproducibility can be achieved.

The lack of mechanistic understanding of spinal cord ischemia-reperfusion injury has hindered further adjuncts to prevent paraplegia following high risk aortic operations. Thus, the development of animal models is imperative. This manuscript demonstrates reproducible lower extremity paralysis following thoracic aortic occlusion in a murine model.

Spinal Kord İskemi-reperfüzyon bir Mürin Modelinde Torasik Aort Tıkanıklığı tarafından tekrarlanabilir Parapleji

Background
Lower extremity paralysis continues to complicate aortic interventions. The lack of understanding of the underlying pathology has hindered ...

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 ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Bir Yenidoğan Fare Omurilik Sıkıştırma Yaralanması Modeli

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...