This work is supported by the SFB 824 subprojects C6 and C7 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany.

Not: Aşağıdaki adımlar kurumumuzun kuralları ve Bavyera, Almanya&#39;nın yerel yönetimler çerçevesinde yapılmıştır. Yerel hayvan bakımı ve kullanımı komitesi tarafından dikte edildiği gibi bu protokolü üretmek için tüm girişimler, hayvanların insanca tedavi için yerel kurallarına bağlı kalınarak yapılmalıdır. Örneğin, birçok enstitü steril eldivenler kemirgen ameliyat sırasında giyilen gerektirir. Ayrıca, virüsler ile çalışma, kişisel ve çevre güvenliği için dikkat çekici, yerel yönetmeliklere göre yapılmalıdır. Bakım düzgün kontamine atık imha etmek ve (otoklav ve üreticinin talimatlarına göre uygun bir antiviral dezenfektanla temizleyerek, yani) aletleri ve çalışma alanı temizlemek için alınmalıdır. Başlangıç ​​Cerrahi öncesi 1. Hazırlıklar <ol><li> (Otoklav yoluyla, yani) cerrahi aletler sterilize. </li><li> appropr virüs seyreltinkullanılan spesifik virüs toksisitesi ve sıçan soyunun duyarlılığına bağlı olarak değişecektir iate konsantrasyonu. Not: bir erkek Buffalo sıçanlarda veziküler stomatit virüsü kullanılarak, örneğin,, genellikle bu modelde maksimum tolere edilen doz, 1 ml hacim içinde 10 7 PFU enjekte edilir. <ol><li> 1 mi şırıngalarda (aseptik teknik kullanılarak) virüs önceden doldur ve şırınga ve iğne hava ortadan kaldırmak için dikkat edilerek, 30 G iğne ekleyin. </li></ol></li><li> gerekli tüm malzeme ve ekipman ile cerrahi alan hazırlayın ve dezenfekte etmek için etanol ile bölgeyi püskürtün. gazlı çubukları ve pamuk uçlu aplikatörler nemlendirilmesi steril tuzlu su çözeltisi (% 0.9 NaCl) ile küçük bir kap doldurun. </li></ol> Sıçan 2. Hazırlık <ol><li> (Yerel hayvan bakımı ve kullanımı komitesi tarafından belirtildiği gibi) uygun doz 30 dakikada önce cerrahi prosedür başlamadan, bir analjezik uygulayın. Örneğin,metamizol yüksek bir doz (100-200 mg / kg) tatbik. </li><li> Inhalasyon (izofluran) ya da enjekte edilebilir (örneğin, MMF şekilde bilinmektedir medetomidin bir karışımı (0.15 mg / kg), midazolam (2 mg / kg) ve fentanil (0.005 mg / kg),) anestezi kullanılarak sıçan anestezisi. % 1-3 bir buharlaştırıcı vasıtasıyla isolflurane yönetmek ve normal solunumu ve kalp hızını devam ettirebilmek için gerektiği gibi ayarlayın. <ol><li> Devam etmeden önce, sıçan tamamen uyuşturulmuş ve ayak pedi kısma ve bir reaksiyon için gözlemleyerek ağrı hissediyorum mümkün olduğundan emin olun. </li><li> anestezi sırasında kurumasını önlemek için bir veteriner göz merhemi sürün. </li></ol></li><li> sadece küçük bir veteriner saç kesme makinesi kullanarak genital üzerinde aşağı sternum karın alan tıraş. Cildi zarar vermeden yakın bir tıraş elde etmek için özen gösterin. </li><li> inhalasyon anestezi ile, uygun bir büyüklükte burun konisi içine burun yerleştirirseniz, ön ve arka ayakların üzerinde bant kullanarak cerrahi yatağa sıçan düzeltmek ve. bir ısıtma yastığı yerleştirinSıçan altında vücut ısısını korumak için. antiseptik boya bir alkol çalkalama ve uygulama, ardından cerrahi sınıfa ait bir dezenfektan ile karın bölgesi, dezenfekte. </li></ol> 3. Laparotomi <ol><li> Böyle 15. olarak, küçük bir eğri neşter kullanılarak, doğrudan orta hat boyunca deri tabakası içinde küçük bir dikey bir kesi yapmak. xiphoid sürecine kadar kesi uzatın. Sonra, yavaşça bir neşter kesi yapmak için Adson forseps bir çift ile kaldırarak kas tabakası kesilirken, ve daha sonra cerrahi makas ile xiphoid sürecine kadar kesi uzatmak. karın boşluğu organları delmek veya xiphoid sürecin her iki tarafında küçük damarları zarar vermemeye özen gösterin. </li><li> kas ve cilt yaymak ve tüm karın bölgesi ortaya çıkarmak için karın karşısında retractor yerleştirin. </li><li> Steril serum fizyolojik ile iki 7.5 x 7.5 cm 2 gazlı bezlerden ıslatın ve cerrahi üst kısmında birini yayıldıŞekil 2A&#39;da gösterildiği gibi, açma ve alt kısmında bir. </li></ol> Karaciğer Arter 4. İzolasyonu <ol><li> yavaşça karın boşluğundan dışarı bağırsak ve çekum kaldırın ve alt gazlı bez üzerine koyun, steril serum fizyolojik ile nemlendirilmiş iki nemli pamuk aplikatör bezlerden kullanarak. nemli organları tutmak için ve yolumdan onları tutmak için üzerine gazlı bez katlayın. </li><li> İki nemlendirilmiş pamuk aplikatör bezlerden kullanarak, yavaşça bu aşamada görünümünde en baskın lob olacak sol lateral karaciğer lobu, yukarı kaldırın. lob tamamen yukarı doğru çevrilmiş ve daha önce yerleştirilen gazlı bez üstünde dinlenmeye sağlayan, lobun alt lifli zarından kesilmiş yaylı makas küçük bir çifti kullanılarak, yukarı lob çevirin ve. yerinde tutmak için lob üzerinde gazlı bez bez katlayın. </li><li> Şimdi görünümünde en ön lob sonraki lobun (anterior kaudat) ile devam edin. thbir mikroskop ve ince forseps e yardım, bırakın ve serbestçe yukarı çevrilmiş izin için lobu çevreleyen ince bir zar teşrih. sol lateral lob ile birlikte gazlı bez bez altına lob yerleştirin. </li><li> artık görünür olacak arka kaudat lobu, gözlemleyin. tamamen arka kaudat lobun incelemek ve yukarı ve gazlı bez içinde birlikte önceden yerleştirilen karaciğer lobları ile çevirmek için adımı yineleyin 4.3. </li><li> Sadece kaudat lobun orijinal konumuna altında, şimdi görünür ve kolayca güçlü nabız tarafından tanınan olacaktır hepatik arter, bulmak. anatomik sağ arter ardından, görünümünden gastroduodenal ve uygun karaciğer arterleri engellemeyecek kalın zar malzeme gözlemlemek. Ince forseps kullanarak, bu malzemeyi yukarı kaldırın ve yavaşça bu alanda herhangi bir küçük damarları önlemek için özen, onu gözyaşı. meydana gelebilecek herhangi bir kanamayı durdurmak için pamuklu aplikatör bezlerden kullanın. <ol><li> a bulunmembranlar düzgün disseke zaman bir mikroskop yardımıyla açıkça görünür olacak rterial dalları. </li></ol></li><li> Iki güzel, atravmatik forseps kullanarak, dikkatli bir hepatik arter, gastroduodenal arter ve onlar çevreleyen zarların (Şekil 2B) kurtulmuş şekilde uygun hepatik arter teşrih. </li></ol> Enjeksiyon için Arter 5. Hazırlık <ol><li> Gastroduodenal arter altında bir 7-0 Prolen sütür geçirin ve sadece çatallanma (uygun yerleştirme için bakınız Şekil 1) Yukarıdaki, uzak ucunda kalıcı bir bitişik harfleri bağlamak için mikro iğne tutucu kullanın. Ucunda kalan dikiş materyali yaklaşık 2 inç izin ve Şekil 2C olarak, öğretilen arteri çekmek için bir iğne tutucu ile kelepçe. </li><li> Geçici olarak kan akımını (Şekil 2C) engellemek için ortak hepatik arter üzerinde küçük bir kelepçe yerleştirin. Not: Bu impİğne arterden çıkarıldıktan sonra ortant büyük kanamanın önlenmesi için. Bundan başka, kan akışı bloke yavaş virüs yönetim ve daha transdüksiyon verimliliği sağlar. <ol><li> Önemli: Kan akışı durur o zaman miktarını sınırlamak için hızlı bir şekilde çalışın - hepatik arter sıkma 5 dakika içinde arterin enjeksiyon ve müteakip ligasyonu gerçekleştirin. </li></ol></li><li> Mümkünse, gastroduodenal arter keskin odak ve doğru 30 G iğne eklemek için yeterince büyük görünen, öyle ki diseksiyon mikroskobu büyütme artırır. gastroduodenal arterin lümenine enjeksiyon şırınga ucunu yerleştirin ve pistonu itmek yavaş yavaş. Not: İğnenin Uygun yerleşim doğru hepatik arter yoluyla ve karaciğere doğru doğrudan akan enjekte çözüm neden olacaktır. <ol><li> transdüksiyon verimliliği maksimize etmek çok yavaş enjeksiyon yapın. </li></ol></li> &lt;li&gt; tüm enjeksiyon hacmi tatbik edildikten sonra, yavaş yavaş arterden iğne geri. Not: enjeksiyon yerinde kanama olmamalıdır. meydana gelir kanama durumunda, bu kelepçe düzgün yerleştirilmiş değildir ve hızlı bir şekilde ayarlanması gerektiğini belirtir. </li></ol> 6. Kapanış <ol><li> gastroduodenal arter altında ikinci bir 7-0 Prolen sütür geçirin ve enjeksiyon yerinde üzerinde ikinci bir kalıcı bitişik harfleri kravat. </li><li> hepatik arterden kelepçeyi çıkarın. karaciğere doğru düzgün hepatik arter yoluyla kan akışını onaylamak restore edilmiştir. <ol><li> bitişik harfler gevşek uçları kesilir. </li></ol></li><li> karın boşluğuna bağırsak ve karaciğer dönmeden önce hiçbir kanama olmadığından emin olun. ek orijinal yönü döndürülür dikkat edin. </li><li> kavisli bir iğne ile 4-0 vikril dikiş materyali kullanarak ayrı katmanlar halinde kas ve cilt dikin. Yeri continuous sütür yaklaşık 1 mm aralıklı ve sıkı çekin. <ol><li> dikiş aşamasında, bir analjesik örneğin buprenorfin gibi (0.05 mg / kg) tatbik. </li></ol></li><li> anestezi sıçan çıkarın. izofluran kullanılmış ise, sıçan birkaç dakika içinde bilincini kazanmak gerekir. MMF enjekte edilebilir bir anestezi gibi kullanılmış ise, anestezinin ters olan, atipamezol (0.75 mg / kg), flumazenil (0.2 mg / kg), ve nalokson (9.12 mg / kg) deri altından enjeksiyonu ile antagonize ve hayvanlar 20 dakika içinde bilincini kazanmak için izin verir. İlk wakening aşamasında kızılötesi ısınma lambası ile sıcak sıçan tutun. <ol><li> sternal yatma korumak için yeterli bilinci yerine kadar gözetimsiz bir hayvan bırakmayın. </li><li> Tamamen iyileşene kadar diğer hayvanların şirkete ameliyat geçirmiş bir hayvan iade etmeyin. </li></ol></li><li> sıkıntı patolojik belirtileri postoperatif sıçan Monitör ve devamıanaljezikler ile yerel hayvan bakımı ve kullanımı komitesi tarafından gerektiği gibi. Örneğin, buprenorfin / 0.05 mg uygulanan her 12 saat kg olabilir. </li></ol>

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	<li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+health+policy+-+lessons+from+the+Global+Burden+of+Disease+Study.">Evidence-based health policy - lessons from the Global Burden of Disease Study.</a> Science. 274, 740-743 (1996).</li><li>Parkin, D. M., Bray, F., Ferlay, J., Pisani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+world+cancer+burden:+Globocan+2000.">Estimating the world cancer burden: Globocan 2000.</a> Int J Cancer. 94, 153-156 (2001).</li><li>Bruix, J., Sala, M., Llovet, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoembolization+for+hepatocellular+carcinoma.">Chemoembolization for hepatocellular carcinoma.</a> Gastroenterology. 127, 179-188 (2004).</li><li>Miraglia, R., Pietrosi, G., Maruzzelli, L., Petridis, I., Caruso, S., Marrone, G., Mamone, G., Vizzini, G., Luca, A., Gridelli, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+transcatheter+embolization/chemoembolization+(TAE/TACE)+for+the+treatment+of+single+hepatocellular+carcinoma.">Efficacy of transcatheter embolization/chemoembolization (TAE/TACE) for the treatment of single hepatocellular carcinoma.</a> World J Gastroenterol. 13, 2952-2955 (2007).</li><li>Breedis, C., Young, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+supply+of+neoplasms+in+the+liver.">The blood supply of neoplasms in the liver.</a> Am J Pathol. 30, 969-977 (1954).</li><li>Nakashima, T., Kojiro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathologic+characteristics+of+hepatocellular+carcinoma.">Pathologic characteristics of hepatocellular carcinoma.</a> Semin Liver Dis. 6, 259-266 (1986).</li><li>Heo, J., 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=Randomized+dose-finding+clinical+trial+of+oncolytic+immunotherapeutic+vaccinia+JX-594+in+liver+cancer.">Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer.</a> Nat Med. 19, 329-336 (2013).</li><li>Altomonte, J., Marozin, S., Schmid, R. M., Ebert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+newcastle+disease+virus+as+an+improved+oncolytic+agent+against+hepatocellular+carcinoma.">Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma.</a> Mol Ther. 18, 275-284 (2010).</li><li>Shinozaki, K., Ebert, O., Kournioti, C., Tai, Y. S., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolysis+of+multifocal+hepatocellular+carcinoma+in+the+rat+liver+by+hepatic+artery+infusion+of+vesicular+stomatitis+virus.">Oncolysis of multifocal hepatocellular carcinoma in the rat liver by hepatic artery infusion of vesicular stomatitis virus.</a> Mol Ther. 9, 368-376 (2004).</li><li>Shinozaki, K., Ebert, O., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+advanced+hepatocellular+carcinoma+in+rats+via+repeated+hepatic+arterial+infusions+of+recombinant+VSV.">Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV.</a> Hepatology. 41, 196-203 (2005).</li><li>Altomonte, J., 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=Synergistic+antitumor+effects+of+transarterial+viroembolization+for+multifocal+hepatocellular+carcinoma+in+rats.">Synergistic antitumor effects of transarterial viroembolization for multifocal hepatocellular carcinoma in rats.</a> Hepatology. 48, 1864-1873 (2008).</li></ol>

The authors have nothing to disclose.

Although direct intratumoral injection is undoubtedly the simplest method to result in efficient tumor transduction of a single tumor nodule, hepatic arterial infusion represents an ideal administration route to target multifocal, orthotopic HCC. This method has proven to be both safe and effective for treating HCC in immune competent rats with oncolytic viruses. Furthermore, since HCC patients are routinely treated by transarterial application of chemoembolization, the method described here is readily translatable to ...

Hepatosellüler karsinom (HCC) dünya çapında beşinci en yaygın kanser ve önemli bir sağlık sorunu 1,2 yapma kansere bağlı ölümlerin üçüncü lider nedenidir. Transarteryal embolizasyon (TAE) ya da transarteryal kemoembolizasyon (TACE) içeren tümör rezeksiyonu veya bu bekleyen karaciğer nakli, lokal tedavi için uygun olmayan hastalar için standart palyatif bakım 3,4 olarak uygulanır. Çevreleyen karaciğer portal ven 5,6 onun kan kaynağının çoğunluğunu alırken bu tedaviler, tümörler hepatik arter kan akışının neredeyse sadece beslenen sayede karaciğerde ikili kan kaynağının benzersiz bir özellik istifade eder. HCC için mevcut tedavilerin son derece sınırlı etkinliklerinin nedeniyle, onkolitik virüs umut verici bir alternatif terapötik maddeler olarak ortaya çıkmıştır. JX-594, son zamanlarda PEXa-VEC adını, bir timidin kinaz silinmiş aşı vektörü, granülosit-m ile silahlı olduğunuHCC 7 Faz II klinik deneme tamamladı acrophage koloni uyarıcı faktör (GM-CSF). Daha yakın bir zamanda, insan interferon-beta ifade eden bir yeniden birleştirici veziküler stomatit virüs vektörü (VSV), sorafenib dirençli HCC (NCT01628640) bir safha I klinik deneme girmiştir. onkolitik virüsler yakın hastalarda HCC için klinik uygulama için onay alma taşımak gibi, etkili bir uygulama yolu için ihtiyaç multifokal hastalık açıktır hedef. Sistemik teslim verimsiz tümör iletimi için büyük ölçüde etkisiz olmakla birlikte, tümör içi uygulamalar hastalığın ilerlemesine karşı duyarlı uninjectable mikroskobik lezyonlar bırakarak, enjekte edilen tümör tedavisinin etkinliğini sınırlayabilir. Biz ortotopik HCC hedeflemek için lokoregional şekilde onkolitik virüs tedavisi yönetmek için sıçanlarda hepatik arter izole bir yöntem kurduk. Biz göstermiştir ki güvenli ve verimli olarak bu yönetim rota sonuçlarıBağışıklık yetkili sıçanlarda 8-10 anlamlı sağkalım uzaması sonucu, multifokal HCC nodüllerin transdüksiyonunu ettik. Burada, biz, erişme diseksiyon ve sıçanlarda hepatik artere enjekte yöntemi tarif etmektedir. Prosedürün bir şema, Şekil 1 &#39;de gösterilmiştir (daha önce 9 yayınlanmıştır)

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">Veterinary clippers</td>
			<td style="width:157px;">Aesculap</td>
			<td style="width:88px;">GT415</td>
			<td style="width:208px;">Small, cordless trimmer ideal for removing fur from surgical area</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Stereomicroscope</td>
			<td style="width:157px;">Zeiss</td>
			<td style="width:88px;">Stemi SV6</td>
			<td style="width:208px;"></td>
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			<td height="22" style="height:22px;width:144px;">30G Needles</td>
			<td style="width:157px;">Braun</td>
			<td align="right" style="width:88px;">4656300</td>
			<td style="width:208px;">30G x &#189;&#8221;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">1ml syringes</td>
			<td style="width:157px;">Braun</td>
			<td style="width:88px;">9161406V</td>
			<td style="width:208px;">Tuberculin syringe</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Disposable scalpel</td>
			<td style="width:157px;">Feather</td>
			<td style="width:88px;">2975#15</td>
			<td style="width:208px;">#15 blade&#160;</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Standard surgical scissors</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">14001-13&#160;</td>
			<td style="width:208px;">Sharp/blunt, for opening skin and muscle</td>
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			<td height="43" style="height:43px;width:144px;">Adson forcep</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">1101-12</td>
			<td style="width:208px;">With teeth, for grasping skin and muscle</td>
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			<td height="64" style="height:64px;width:144px;">Alm retractor</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">17008-07</td>
			<td style="width:208px;">With blunt teeth, for spreading abdominal cavity open during surgery</td>
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			<td height="43" style="height:43px;width:144px;">Gauze swabs</td>
			<td style="width:157px;">Lohmann &#38; Rauscher</td>
			<td align="right" style="width:88px;">18504</td>
			<td style="width:208px;">7.5 x 7.5 cm, should be autoclaved prior to use</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Cotton-tipped applicator swabs</td>
			<td style="width:157px;">Lohmann &#38; Rauscher</td>
			<td align="right" style="width:88px;">11970</td>
			<td style="width:208px;">Sterile</td>
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		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Fine-tipped foreceps</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">11063-07</td>
			<td style="width:208px;">0.4mm, angled tip, for dissecting hepatic artery</td>
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			<td height="43" style="height:43px;width:144px;">Vannas spring scissors</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">91500-09</td>
			<td style="width:208px;">For delicate cutting</td>
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			<td height="43" style="height:43px;width:144px;">Micro-needle holder</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">12076-12</td>
			<td style="width:208px;">For ligating gastroduodenal artery</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Needle holder</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">12005-15</td>
			<td style="width:208px;">Tungsten carbide jaws</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">7-0 Prolene sutures</td>
			<td style="width:157px;">Ethicon</td>
			<td style="width:88px;">8648H</td>
			<td style="width:208px;">Polypropylene suture with curved needle, for ligating gastroduodenal artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">4-0 Vicryl sutures</td>
			<td style="width:157px;">Ethicon</td>
			<td style="width:88px;">V3040H</td>
			<td style="width:208px;">With curved needle attached</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Infrared warming lamp</td>
			<td style="width:157px;">Beurer</td>
			<td style="width:88px;">IL11</td>
			<td style="width:208px;">For maintaining body temperature post-operatively</td>
		</tr>
	</tbody>
</table>

transarterial administration of oncolytic viruses for locoregional therapy of orthotopic hcc in rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

tecrübesi ile, (arter başarıyla disseke ve enjekte ve hayvan ameliyatı hayatta olduğu anlamına gelir) neredeyse% 100 başarı oranı elde edilebilir. Ancak, (vb tümör yükü derecesi, altta yatan karaciğer fonksiyon) ameliyat öncesinde sıçan sağlık durumu açıkça cerrahi müdahale sonucu bir rol oynayacaktır. Cerrahi müdahalenin ardından, fareler, tek başına tampon bile virüs olmadan enjekte edilmiştir, Geçici kilo kaybı ve karaciğer enzimlerinin hafif,...

Watch this Scientific Journal Video about Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats at JoVE.com

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Sıçanlarda Ortotopik HCC Lokorejyonel Tedavisi için oncolytic virüs transarteryel İdaresi

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

transarterial-administration-oncolytic-viruses-for-locoregional

Research

JoVE Journal

Medicine

9.4K Görüntüleme.  Klinikum rechts der Isar der TU München. Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Video: Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Vivo Enfeksiyon Oncolytic Virüsler Canlı Doku

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

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Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Kanserler Üretim ve reaktif oksijen türlerinin (ROS Algılama)

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Sıçanlarda Ortotopik Küçük Bağırsak Nakli

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Sıçanlarda Ortotopik Karaciğer Nakli

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Bir Fetal Fare Modelinde Biyoaktif Ajan intratrakeal Yönetimi İçin Yeni Bir Cerrahi Yaklaşım

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

Osteosarkom Hücrelerde Fotodinamik Tedavi sitotoksik etkinliği<em&gt; In Vitro</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Oncolytic virüs klinik öncesi Değerlendirme Çalışmaları Pamuk Rat Taşıma

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of outcome measures of upper extremity motor performance has grown. However, clinicians and researchers lack practical, quantitative measures of the hand&#8217;s natural role as a receptor of the environment. The Hand Active Sensation Test (HASTe), developed by Williams and colleagues in 2006, is a valid and reliable measure of haptic performance. Though not available commercially, the HASTe can be fabricated from inexpensive materials, and it has been used successfully to identify impairments in haptic touch in individuals with stroke. (Williams, 2006). This paper presents the methods of design and fabrication of the HASTe testing kit, as well as a visual screen to be used during administration, and instructions for the tests administration and scoring.

Design, Fabrication, and Administration of the Hand Active Sensation Test HASTe

The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe.

Tasarım, ve Yönetim El Fabrikasyon Aktif Sensation Testi (Haste)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

Ivmeölçer kullanan Günlük Yaşamda Üst Ekstremite Performans miktarının Bir Yöntem

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...