Yazarlar, yardım için Verlene Henry bu model geliştirme ve Lindsay Holmes teşekkür etmek istiyorum. Bu çalışma kısmen James S. McDonnell Vakfı (220.020.173) tarafından finanse edildi.

1. Hücre dizileri <ol><li> U87MG glioma hücrelerinin% 5 fetal sığır serumu (FBS) ile desteklenmiş DMEM-F12 ile büyük doku kültürü şişeleri kültür. </li><li> Hücreler şişeler sıcak Fosfat ile iki kez yıkanarak hasat Tamponlu Salin (PBS) ve 37 ° kuluçka ° C,% 0.25 &#39;lik tripsin ve% 0.05 EDTA içeren 10 ml PBS ile 5 dakika. Bir kez hücreleri içeren bir kültür ortamı ve santrifüj (4 dakika için 300 X g) 10 ml 50 ml tüp yerleştirilir kaldırılır. </li><li> Yıkama sonrasında, hücrelerin 10 konsantrasyonda yeniden süspanse x 10 6 / ml ul 50.000 hücre / 5 bir aşılama için izin verilen kültür ortamı,. </li><li> Hücreler intrakranial enjeksiyon kadar buz üzerinde tutulur. </li></ol> 2. Intrakranial Rehberi vida civatalama Bu prosedür, hücre enjeksiyonu öncesinde birkaç gün yapılabilir. Burada açıklanan tüm prosedürler katı steril koşullar altında yapılmaktadır. <ol><li> Fare (nu BALB / c / nu kadın, 5-6 hafta yaklaşık 18g), tanıtım amaçlı numaralı tartılır ve ketamin (100 mg / kg) ve ksilizin (5 mg karışımı intraperitoneal (IP) enjeksiyonu ile anesthetised / kg). </li><li> Cilt iyot çözeltisi ile silinir ve küçük bir kesi (2-3 mm), orta hat ve interaural çizgi anterior sağ tarafı boyunca yapılır. Bu kafatasının koronal ve sagital sütür ortaya koyar. Bregma, bu iki sütür kavşakta konumlandırılmış. </li><li> 2,5 mm lateral ve bregma anterior 1 mm bir noktadan sonra kılavuz vida giriş noktası işaretlenir. Bu nokta, doğrudan kaudat nükleus 1 yukarıda yer almaktadır . </li><li> 1 x 1 mm derin delik dura kafatası yoluyla twist drill düzenlenen steril bir eli ile delinmiş. </li><li> Dura içine kafatasları yüzeyinin altında 1.6 mm uzatacaktır sterilize kılavuz vida sonra bir tornavida ile delik içine cıvatalı kafatası (Şekil 1A) ile aynı hizada kadar. </li><li> Steril bir stile veya vida kukla sonra açılması (Şekil 1B) kapatmak için kılavuz vida merkezi deliğe yerleştirilir. </li><li> Yara Vetbond Doku Yapıştırıcı (n-butil siyanoakrilat) ve fareler ile kapatılır reversine bir intraperitoneal enjeksiyon (küçük hayvanlar) (0.1 ml / kg) ve analjezi carprofen (5 mg/kg/100 ul) verilmiştir. Fare sonra 20 dakika kadar sürebilir bir ısınma mat (36 ° C) kurtarmak için izin verilir. Tamamen bilinçli kadar Fare sık sık izlenir ve bu kurtarma süre içinde gözlenir. </li></ol> 3. İntrakranial hücresel engraftman <ol><li> Steril kelepçeli Hamilton şırınga, iğne ucu kılavuz vida çıkışı (Şekil 1B) altında genişletmek için sadece 2 mm iğne sağlayan küçük plastik bir halka uygulanarak hazırlanmıştır. Bu nedenle, son aşılama noktası kafatası yüzeyinden 3.5 mm altında. </li><li> Dört gün kılavuz vida cerrahisi sonrası, farelerin yukarıdaki gibi tekrar anestezi (2.1) ve stile kaldırmak için kılavuz vida üzerinde küçük bir kesi yapılır. </li><li> Kelepçeli Hamilton şırınga sonra 5 ul herhangi bir hava kabarcığı oluşturabilir veya yukarı çekmek için önlem alarak iyice karıştırılır hücrelerinin ile doludur. </li><li> Şırınga perfüzyon pompa güvenli ve iğne kılavuz vidasını (Şekil 1C) eklenir. Hücreler daha sonra saatte 30 ul hızında infüze. </li><li> Otomatik cihazları (Şekil 1D), sabit bir akış hızında bir kerede bize 10 hayvanlara enjekte etmek için izin verdi. Hücreler aynı zamanda kelepçeli hamilton şırınga kullanılarak elle enjekte edilebilir ya da alternatif şırınga, iğne ucu 2 mm kılavuz vida ötesine izin 3 mm koparılmış 200 ul pipet ucu tarafından kelepçeli olabilir. Enjeksiyonu sonra sürekli bir sabit hızda yapılmalıdır. </li><li> Infüzyon tamamlandığında, şırınga dikkatlice çıkarılır ve stile kılavuz vida değiştirilir. Yara kapalı yapıştırılmış ve fareler (2.7) daha önce olduğu gibi kurtarma ilaçlar verilir ve bir ısınma mat kurtarmak için izin verilir. </li></ol> 4. Terapötik meydan okuma <ol><li> Dört gün U87MG hücre aşılama sonra, fare tartılır ve rastgele kontrol ve tedavi grupları ayrılmıştır. </li><li> Tedavi grubunda AMG102 (PBS 100 ul 100 mikrogram) bir IP enjeksiyonu ise kontrol grubu, PBS (100 ul) bir-periton içi (IP) enjeksiyonu. </li><li> Enjeksiyonlar, 14 gün boyunca her iki günde toplam 6 enjeksiyonları (Şekil 2) bir tekrarlanır. </li><li> Son enjeksiyondan sonra, farelerin günlük olarak izlenmekte ve her ikinci gün tartılır. </li><li> Farelerde ciddi nörolojik işlev bozuklukları (denge bozukluğu, felç), dehidratasyon, ya da% 10&#39;dan fazla kilo kaybı ya da can çekişen gösterilecek başladığında, onlar insanca ötenazi uygulandı. Bu insani bir uç nokta gün sonra, Kaplan-Meier sağkalım eğrisi kaydedildi. </li></ol> 5. Tedavi verimliliğinin değerlendirilmesi </p&gt; <ol><li> GraphPad Yazılım, Windows için GraphPad Prism 4.03 San Diego Kaliforniya ABD, Kaplan-Meier sağkalım eğrisi kullanılarak oluşturulan <a href="http://www.graphpad.com">www.graphpad.com</a> 3. Ölümler veya euthanizations son nokta olayları olarak işaretlenir ve 1 olarak puanlandı. Deneyin sonunda kurtuldum Hayvanlar olmayan olaylar olarak kaydedilir ve 0 olarak skorlandı. </li></ol> 6. Temsilcisi sonuçları: Kontrol hayvanların hücrelerinin ilk aşılama sonrası nörolojik bozukluğu ve kilo kaybı 23 gün belirtileri göstermeye başladı. Bu önemli ölçüde, 35 gün AMG102 tedavi grubunda ertelendi. Gerçekten de 35 günde,% 77 (n = 7 / 9), kontrol grubunda ötenazi olmuştu. Kaplan-Meier sağkalım eğrisi açıkça AMG102 tedavi (Şekil 3) yanıt sağkalım önemli bir artış göstermektedir. 70 gün, kontrol farelerin% 88,% 37 ile karşılaştırıldığında (n = 8 / 9) ötenazi olmuştu AMG102 tedavi edilen grupta (n = 3 / 8). Beyinleri histolojik analiz, kontrol grubunda ise tüm 9 hayvanlar gelişmiş tümörler sadece 3 8 AMG102 tedavi edilen farelerde (Şekil 4) ile karşılaştırıldığında doğruladı. <img alt="Şekil 1" src="/files/ftp_upload/3157/3157fig1.jpg" /> Şekil 1: Görüntü (A) doğrudan kaudat nükleus üstünde yer alan bir alanda konumlandırılmış intrakranial kılavuz vida gösterir. 2 mm iğne kılavuzu ötesine uzatır ve böylece hücreler kaudat nükleus içinde 3,5 mm enjekte edilir kelepçeli hamilton şırınga (hücreleri ile önceden yüklenmiş) sonra kılavuz vida içine yerleştirilir. Şırınga sonra bir stile (B) tarafından kaldırılır ve değiştirilir. Otomatik infüzyon cihazı (C) bir defada 10 hayvanlara enjekte edebilir. Simültane beş enjeksiyonları (D) burada gösterilir. <img alt="Şekil 2" src="/files/ftp_upload/3157/3157fig2.jpg" /> Şekil 2: Şematik Çalışma protokolü. Hücreler, tedavi günde 4 başlamadan gün 0 enjekte edildi. Kontrol hayvanlar PBS (100 ul) IP enjeksiyon tedavi grubunda AMG102 (100 μg/100 ul) IP enjeksiyon alırken aldı. Bu enjeksiyonlar, 10 gün boyunca her ikinci gün verildi. Toplam 6 enjeksiyonları verildi. Hayvanlar daha sonra nörolojik ve fiziksel bozuklukları belirtileri açısından 70 gün süreyle takip edildi. <img alt="Şekil 3" src="/files/ftp_upload/3157/3157fig3.jpg" /> Şekil 3: Kaplan-Meier sağkalım eğrisi kontrolü ve AMG102 tedavi edilen farelerde karşılaştırarak. Anlamlı sağkalım AMG102 (p = 0.005) tedavi sonrasında elde edildi. <img alt="Şekil 4" src="/files/ftp_upload/3157/3157fig4.jpg" /> Şekil 4: koronal beyin bölümlerinin kontrol hayvanlarda tümör gelişimi (A) ve AMG102 tedavi hayvanlarda hiçbir beyin tümörü geliştirme (B) gösteren Microphotographs. Ölçek çubuğu 1 mm.

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. 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+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

Bu çalışma Amgen Inc Araştırma Fonu tarafından desteklenen

Burada sunulan intrakranial kılavuz vida yöntemi intrakranial xenografts hızlı ve tekrarlanabilir kurulmasını sağlar ve eğitimli bir hayvan teknisyen elinde, bu işlem çok kolay bir stereotaktik cihaz için gerek kalmadan yapılır. Bizim yöntemi doğru in vivo tümör gelişimi için en uygun beyin bölgesi ve bir seferde 10 hayvanların aynı anda aşılama sağlayan otomatik bir infüzyon pompası bulmak için bir kılavuz vida kullanımı birleştiriyor. Kılavuz vida büyütme yordamı gerçekleştirmek için en az 5 dakika sürer. 30 ul / saat hücre otomatik infüzyon 5 ul hacmi için sadece 10 dakika sürer. Bu sabit bir oranda doğru dozaj sağlar ve artmış intrakranial basınç nedeniyle ekstra kranial alana hücre çıkarılma riskini azaltır. Ayrıca 10 ul hacimli bu yordamı var ve hücresel reflü nedeniyle herhangi bir ekstra kranial tümör büyüme görülmez. Otomatik infüzyon pompası aynı zamanda çeşitli hayvanların, saatte yaklaşık 15 hayvan sınırlı bir stereotaktik prosedüre göre saatte 60 hayvanlara enjekte edebilir aynı anda inoküle olması elverdiğince. Konumunu tümör toplu nihai konumunu belirleyen en önemli adım olarak, bu yordamı kafatası kılavuz vida konumudur. Kılavuz vida çok merkezi ise, hücreler kılavuzu ileriye doğru yerleştirilir ise iki beyin hemisfer arasında bir bölgeye enjekte edilecektir, tüm hücrelerin beyin içine enjekte edilir olmaz. Bu kılavuz yanal 2.5 mm ve 1 mm bregma 1 anterior bulmak mümkün olduğunca doğru olması çok önemlidir. Plastik bir kılavuz vida gibi MR veya PET 6 gibi beyin görüntüleme gerektiren çalışmalar için de kullanılabilir olmasına rağmen, bu çalışmada biz, bir çelik kılavuz vida kullanılır. Otomatik infüzyon pompası, bazı gruplar için engelleyici maliyet olsa bile, hücreler hâlâ elle daha hızlı stereotaktik yaklaşımlar daha enjekte edilebilir. Genel olarak, bu hızlı, doğru ve tekrarlanabilir bir yöntem glioma, medulloblastom ve beyin sapı tümörleri 7 incelemek için kullanılabilir . Bu prosedür iki laboratuarlar arasında beklenmedik anestezi reaksiyonları (&lt;% 1) bağlı ölümlerin 500 fareler üzerinde yapılmıştır. Fare kılavuz vida ile sabitledim ve hücreler, herhangi bir nörolojik komplikasyonlara prosedürün bir sonucu olarak ölmek ya da zarar vermedi ile sonradan infüze edildi. Hayvanlar herhangi bir komplikasyon olmadan 100 gün geçmişte kalmıştır. SCID gibi saçlı tüysüz bir fare, immün yetmezlikli fare kullanmayı tercih rağmen uygun kafatası saç sitesi temiz tutmak ve kılavuz vida ile müdahale saç önlemek için ameliyat öncesi kaldırıldı gibi olurdu. Glioma 8-10 tedavisi için geliştirilmiş olan birçok romanı terapötikler vardır. Yakın zamanda subkutan U87MG glioma xenografts AMG102 3 ile tedavisi ile inhibe edilebilir olduğunu gösterdi; humanize antikor glioma 5 klinik değerlendirme yapılan HGF yönetti. Biz çıplak farelerin beyinlerinde, intrakranial U87MG tümörlerin kurmak yöntemi tanımlamak için kullanılan. Bizim çalışmamız açıkça yöntemin yararını gösterir ve AMG102 ortotopik U87MG xenografts büyüme önemli ölçüde inhibe ettiğini göstermektedir. Biz daha etkili tümör büyüme inhibisyonu elde etmek için diğer terapötikler AMG102 ile birlikte kullanıldığında ne belirlemek için bu modeli kullanmaya devam edecektir.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 12634010 </td><td></td></tr><tr><td> FCS </td><td> Bovogen </td><td> SFB&#39;ler </td><td></td></tr><tr><td> Tripsin </td><td> Invitrogen </td><td> 15050065 </td><td></td></tr><tr><td> EDTA </td><td> Sigma </td><td> E6758 </td><td></td></tr><tr><td> Balb / c nu / nu fareler </td><td> ARC Perth </td><td></td><td></td></tr><tr><td> Vidalı Tutucu </td><td> Plastik Bir </td><td> SD-1 </td><td></td></tr><tr><td> Matkap Tutucu </td><td> Plastik Bir </td><td> DH-1 </td><td></td></tr><tr><td> Matkap ucu </td><td> Plastik Bir </td><td> D # 60 </td><td></td></tr><tr><td> Vida kılavuzu (metal) </td><td> Plastik Bir </td><td> C212SG </td><td></td></tr><tr><td> Vida Dummy (stylus) </td><td> Plastik Bir </td><td> C212SD </td><td></td></tr><tr><td> Hamilton Şırınga (10 ul 26g çimentolu iğne) </td><td> Grace Bölümü Discovery Science </td><td> 80075 </td><td></td></tr><tr><td> PHD 22/2000 infüzyon pompası </td><td> Harvard Apparatus </td><td> 70-2003 </td><td> Isteğe bağlı </td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> Thermo pedi </td><td> Harvard Apparatus </td><td> 340390 </td><td></td></tr></tbody></table>

a simple guide screw method for intracranial xenograft studies in mice

Insan tümör hücreleri bağışıklık farelerin beyin içine aşılama çalışması için, glioblastoma (glioma) ve medulloblastom beyin kanserleri dahil olmak üzere kurulmuş bir yöntemdir. Yaygın olarak kullanılan stereotaktik yaklaşım, sadece tek bir seferde hayvan enjeksiyon için, emek yoğun ve son derece özel ekipman gerektirir. Başlangıçta, Lal ve arkadaşları, 1 tarafından geliştirilen kılavuz vida yöntemi, hantal stereotaktik prosedürleri ortadan kaldırmak için geliştirilmiştir . Şimdi hızlı ve son derece güvenli, modifiye edilmiş bir kılavuz vida yaklaşım tarif, her ikisi de kritik etik kaygılar. Özellikle, bizim prosedür şimdi tümör hücreleri ile aynı anda enjekte edilmek üzere 10 hayvan sağlayan bir infüzyon pompası içermektedir. HGF / dağılım faktörü şu anda klinik değerlendirme 2-5 geçiren bir insan antikor; bu prosedürün yardımcı programı göstermek için, biz sonra AMG102 ile tedavi edilen farelerde intrakranial xenografts olarak insan U87MG glioma hücreleri kurdu . AMG102, Sistemik enjeksiyon intrakranial U87MG xenografts tüm farelerin hayatta kalma önemli ölçüde uzun süreli ve tam kürleri bir dizi sonuçlandı. Bu çalışmada, kılavuz vida yöntemi intrakranial xenografts kurmak için ucuz, yüksek tekrarlanabilir bir yaklaşım olduğunu göstermektedir. Ayrıca, beyin kanserlerinin tedavisinde yeni tedavi stratejilerinin doğrulamak için ilgili fizyolojik bir model sunar.

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Glioma tedavisi için yeni tedavi paradigmalar değerlendirmek için, fizyolojik ilgili modeller gereklidir. Biz stereotaktik yaklaşımlar daha hızlı ve daha güvenli bir intrakranial xenograft modelleri kurulması için implante edilebilir bir kılavuz vida prosedürü kullanır.

Basit Fare İntrakranial Xenograftlarında Çalışmaları Kılavuzu Vida Yöntemi

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

a-simple-guide-screw-method-for-intracranial-xenograft-studies-in-mice

Research

JoVE Journal

Medicine

23.0K Görüntüleme.  Monash Institute of Medical Research. Glioma tedavisi için yeni tedavi paradigmalar değerlendirmek için, fizyolojik ilgili modeller gereklidir. Biz stereotaktik yaklaşımlar daha hızlı ve daha güvenli bir intrakranial xenograft modelleri kurulması için implante edilebilir bir kılavuz vida prosedürü kullanır.

Video: A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Monokoryonik ikiz plasenta Damar Haritalama Kılavuz Tel Destekli Kateterizasyon ve Renkli Boya Enjeksiyon

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

Tıp

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Bir Fiber optik Basıç kullanarak Sıçanlarda Epidural İntrakraniyal Basınç Ölçümü

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Fare Akciğer Entübasyonun Basit Bir Yöntem

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Yeni Bir Yüksek çözünürlüklü<em&gt; In vivo</emTümör Büyüme ve Tedavi için İntrakranial Yapıların Dinamik Tepki Eğitim&gt; Görüntüleme Tekniği

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; Ovo</emSarkoma için bir Xenograftmodel olarak&gt; CAM-testi

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Farelerde Taksanlar Farmakokinetik ve Farmakodinamik Analiz Karotis Arter İnfüzyonlar

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Farelerde Basit Kritik ölçekli Femoral Hata Modeli

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Farmakolojik ve Toksikoloji Araştırmaları bir Oral Çözme Strip kullanmak: Hayvanlar için ağızdan sonda ile Basit ve İnsanca Alternatif

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Farelerde Trakea hızlı intübasyon için geliştirilmiş bir yöntem

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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. ...