This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

Not: sıçan kullanımını kapsayan tüm deney prosedürleri, Duke Üniversitesi Hayvan Bakımı ve Kullanımı Komitesi tarafından onaylanmıştır protokollere uygun olarak yapıldı. Trakeal Entübasyon Kanül 1. Hazırlık <ol><li> Pense kesme ile 2 inç 14 G angiocathether iğnenin iğne ucunu kes. </li><li> İğnenin lümen iğne burunlu pense ile kapalı sıkıştırın. </li><li> Kesme pense ile açık-lümen bölümünün kenarına aşağı sıkışmak bölümü kesin, ve sonra 30 kalan sonu her iki tarafında kenarları kesilmiş - İğne kenarına 45 ° açı. </li><li> Düz kayma-eklem pense açık alana kanül 4 mm ve yukarı doğru hafif bir eğri ucunda oluşmuş kadar yukarı çekin - alt 3 koyun. NOT: Bu trakea (Şekil 1) içine kateter yönetmenlik kolaylaştıracaktır. </li><li> Kanül sonunda kenarlarını aşağı pürüzsüz ince zımpara kağıdı kullanın. </li><li&gt; Anjiyokateter tüp içine küntleşmiş enstrüman dönün. Boru küntleşmiş iğne biraz daha kısa olduğundan emin olun. </li></ol> 2. Ön-Cerrahi İşlemleri <ol><li> Çalışma alanı üzerinde 10X büyütme ayarlanmış bir işletim mikroskop yerleştirerek prosedür için site kurmak. </li><li> Çalışma alanında bir ısınma yastık yerleştirin ve temiz bir emilim ped ile kaplayın. </li><li> Cerrahi alan yakın bir kalp hızı ve kan oksijenlenme monitörü ayarlayın. </li><li> Ağırlık esaslı ilaç doz (örn, postoperatif analjezi, antibiyotikler veya lenfosit tüketen antikor) için sıçan tartılır. NOT: - 450 gr Tipik erişkin sıçan 350 arasında tartın. </li><li> Bir indüksiyon odacığı kullanılarak buharlaştırılmış% 3 izofluran-O 2 (3 L / dak) ile oturaklı sıçan aktif kömür ihtiva eden bir atık anestezi gazı tahliye sisteminden tahliye edilebilir. Anestezi indüksiyonu için 5 dakika entübasyon için gerekli derin anestezi sağlamaktayız. </li><li> Subkutan sıçan önce entübasyon ve cerrahi ile devam etmek Carprofen veya Meloxicane (4.4 mg / kg) uygulayınız. </li><li> Entübasyon öncesi anestezi sıçan boyun ve göğüs tıraş elektrik makasları kullanın. </li></ol> 3. Entübasyon <ol><li> Entübasyon kanül ucunda yağlama küçük bir miktar (örneğin, KY jel) sürterek entübasyon kurulumu hazırlayın. Konnektör tüp yoluyla izofluran gaz akışını başlatmak için ventilatör açın. </li><li> Entübasyon aparatı sıçan aktarın ve üst kesici dişlerin (Şekil 2A) tarafından metal çubuğun üzerine sıçan askıya. NOT: Bazı entübasyon aygıtları çevrimiçi satın alınabilir. Burada gösterilen model bütan meşale kullanarak 2 x 4 inç lomber parçasının bölümler etrafında kalıp bir akrilik sayfasını kullanarak ev yapımı olduğu ve takviyeler kloroform ile bağlıdırlar. bar (ya da metal tel) s yerleştirilen maskeleme bandı üzerinde sondaj delikleri ile bağlıakrilik ide parçalanmayı veya çatlamasını önlemek için. </li><li> Boyun (Şekil 2B) ventral yüzeyinden 2 cm - esnek, yüksek yoğunluklu ışık kaynağını 1 konumlandırarak sıçan boynunu Trans-aydınlatmak. </li><li> Yavaşça yukarı ve alt dişlerin tarafına dilini çekin öğrenci standart desen forseps bir çift kullanın. Kavrama baş ve bir elin işaret parmağı dilin alt ucuna karşı forseps bir tarafında düz iç yüzeyi yerleştirerek arasında dilidir. Basın ventral gırtlak (Şekil 2C) epiglottis ve diyafram ortaya çıkarmak. </li><li> Ses telleri görselleştirmek ve angiocatheter göbek kesiciler dokunana kadar trakea içine açık glottisin ile öne entübasyon kanül kalkık, körelmiş ucunu kılavuz. </li><li> Metal stile çıkarın ve izofluran ile havalandırma başlamak için anjiyokateter açılması için anestezi boru takın. NOT: Tipik oksijen floventilatöre w oranı% 3 izofluran ile 3 L / dk. <ol><li> 14 mmHg - ~ 12 bir basınç elde hacim kontrol ventilatör modunda 60 respirations / dakikalık bir oranda ventilatörü ayarlayın. 3 cm H 2 O bir pozitif ekspirasyon sonu basıncı (PEEP) kullanın </li></ol></li><li> Endotrakeal tüpün doğru yerleşimi sağlamak için ventilatör ile senkronize ikili göğüs duvarı genişleme gözlemleyin. </li><li> Anestezi altında iken kuruluğu önlemek için sıçan gözlerine veteriner tarafından önerilen göz merhemi sürün. </li><li> Anestezi altında iken kuruluğu önlemek için sıçan gözleri veteriner merhem sürün. </li><li> Bez bant şerit ile sıçan başkanı endotrakeal tüp sabitleyin. Endotrakeal tüp ve anestezik giriş tüp arasında sıkı bir bağlantı sağlayın. </li><li> Sıçan ayak kan oksijenasyonu ve kalp hızı monitörü takın ve izleme başlar. Hiçbir tepki için orada onaylayarak uygun anesthetization onaylayınE-tutam. </li></ol> 4. Torakotomi ve Timektomi <ol><li> Prosedürün bütünlüğü için standart aseptik teknik izleyin. <ol><li> Çalışma alanı ve çalışma masası temizleyin ve% 70 etanol çözeltisi ile dezenfekte edin. </li><li> İşlem sırasında steril cerrahi eldiven kullanın ve işlem sırasında kullanılan tüm alet ve malzemeleri otoklav. </li><li> Tüm göğüs bölgesine providon-iyot uygulayın ve kurumaya bırakın. Daha sonra tül üzerinde% 70 etanol ile cildin temizlenmesi gerekir. Steril çalışma alanını ortaya çıkarmak için bir delik kesim şeffaf plastik wrap ile sıçan örtün. </li></ol></li><li> Üst torakal bölgede Suprasternal çentik belirleyin. Suprasternal çentik üstünde 3 mm ve künt uçlu Shea makas kullanarak sternum boyunca distale orta hat uzanan - 2 başlangıç ​​deri yoluyla 2 cm orta hat kesi olun. </li><li> Yine künt uçlu Shea makas kullanarak Suprasternal çentik bir 1,5 cm medyan sternotomi gerçekleştirin. KSadece yavaş yavaş sternum ve peşin altında makas alt kenarını EEP. </li><li> Ön-trakeal kayış kasları (sternohyoid ve sternothyroid kasları) ortaya çıkarmak için sadece ayrılmış sternum ve açık altında küçük bir Alm retraktör yerleştirin. Künt Graefe forseps kullanarak ön-trakeal kayış kasları ayırın. NOT: Bu noktada, nefes borusu görülebilir ve entübasyon tüp nefes borusuna içinde görsel edilmelidir. </li><li> Ayrılmış kayış kasları ve sternum altında küçük Alm retraktörün çatallar yerleştirin. Timus üstün yönünü ortaya çıkarmak için retraktör açın. </li><li> Alt timik lobları timus dokusunun yan kenarlarını serbest ve ortaya çıkarmak için ince Dumont forseps kullanın. </li><li> Vena kava, subklavian ve karotid gemileri ile temasını engellemek için özen, açık kesi içine yavaşça superiorda timus çekin ve timus ve akciğerler arasındaki hassas plevral astar kesintileri en aza indirmek için. </li><li> T AdıO, gemiler disseke ve ortaya çıkar timik onları bölmek için mikro-makas kullanın. Gerekirse hemostaz için gemilerin üzerindeki baskıyı tutmak için pamuklu bir bez kullanın. </li><li> Kesi içine alt timik lobları sunun ve sonra keskin arka ekleri parçalanmayabilir. Sağlam timus çıkarın ve dikkatlice eksik bölümler için kaldırılmıştır timus inceleyin. NOT: Küçük göğüs lenf düğümleri timus çevreleyen görsel ve genellikle timik doku ayırt etmek zordur olabilir. Bu timik dokuya benzer bir görünüme sahip ayrık yuvarlak düğümleri olacak ama timus ile süreklilik içinde olmayacaktır. </li><li> Alm retraktörü çıkarın ve iki 5-0 Maxon sütür kesintiye ile sternohyoid ve sternothyoid kasları kapatın. </li><li> Pozitif basınçlı ventilasyon altında mediasteni mühür dikişlerle arasında cerrahi sınıf siyanoakrilat doku yapıştırıcısı 2 damla uygulayın. NOT: Bu pnömotoraks ve hematom insidansını azaltacaktır. </li><li> St kapatıniki ile ernum bir kesim iğne 4-0 ipek sütür kesildi. Altta yatan kas tabakası önlemek için hemen sternum altında iğneyi rehberlik özen, kaburga arasındaki boşluklar yoluyla iğne takın. </li><li> Bir çalışan 4-0 Naylon sütür ile cilt katmanı kapatın. Post-operatif anestezi iyileşme süresini kısaltmak için bu noktada izofluran durdurun. </li><li> Tuzlu-nemlendirilmiş gazlı bez ile kesi yeri ve çevresindeki cildi temizleyin. </li><li> Kesi mühür kesi üzerinde siyanoakrilat tutkal 2 damla - 1 ardından lokal anestezi için bupivakain (% 0.25) birkaç damla uygulayın. </li><li> Sıçan bağımsız solunum çaba belirtileri gösterir ve ekstremitelerde hareket etmeye başlayana kadar havalandırmak için devam edin. Sıçan ekstübe ve bir ısınma pad üzerinde yerleştirilen bir kafes yakın gözlem altında kurtarmak için izin verir. </li><li> Buprenorphine yönetme (0,01-0,05 mg / kg) deri sıçan hemen ameliyat sonrası ve 48 saat po için her 8-12 saat tekrarst-operatif (normalde 5 toplam doz). 24 ve 48 saat post-operatif, karprofen veya Meloxicane (4.4 mg / kg) deri (pre-operatif doz olmak üzere 3 doz toplam) yönetmek. </li><li> Sıçan kafes etrafında hareket kadar izlemeye devam edin. Solunum sıkıntısı herhangi bir işaret anda, yeniden entübasyon ve göğüs yeniden keşif yapmak. <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> Cilt tabakası dikişler 1 hafta operasyon sonrası çıkarın. </li></ol>

<ol>
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The authors have nothing to disclose.

tam Timektomiye mevcut protokol komplikasyonları en aza indirmek için tasarlanmış cerrahi tutkal uygulaması ile üç katmanlı kesi kapatılması ile minimal invaziv bir yaklaşım sağlar. Timus tamamen çıkarılması saf T hücrelerinin kaybı ve sitokeratin mediastinal lenf dokusu IHC boyamasıyla gösterilmiştir. 20% nedeniyle plevra, kalp ve timus ile alt lob samimi derneğe ilişkilendirilebilen çoğu peri-operatif komplikasyonlar için - yetişkin sıçan Timektomiye prosedür...

1960&#39;ların başından beri, timus merkezi immünolojik tolerans gelişiminde kritik rol için kabul edilmiştir. Kemirgen timektomi organ transplantasyonu ve tümör metastazının bir ortamda lenfosit farklılaşması, kendi kendine tolerans ve bağışıklık toleransı, timus rolünü tanımlamak için gerekli bir yöntem olduğu kanıtlanmıştır. Fare timus çıkarılması, T-hücresi kaybı ya da doğal saf T hücrelerinin yeniden ortaya çıkması tarif edilmiş T hücre popülasyonlarının adoptif transfer edilmesini içeren çalışmalar için izin verir. Yenidoğan kemirgenlerde timektomi güvenilir sonuçlar 1 bir emme tekniği kullanılarak gerçekleştirilebilir. Yetişkin farelerde, bu yöntem yaklaşık% 20 ölüm oranı ile ilişkilidir ve sıklıkla yetersiz timektomi 2 ile sonuçlanır. Sürekli yetişkin sıçanlarda tam timektomi ulaşmak için, medyan sternotomi ile mediasten açık poz gereklidir. Bununla birlikte, bu% 6 2- 4 - prosedürü 1.5 arasında değişen bir genel mortalite oranı açan trakea yaralanması, kanama ve pnömotoraks dahil komplikasyonlar ile ilişkilidir. Son iki yıldır timektomi teknikleri gelişmeler perioperatif komplikasyonlar azalmış ve sağkalım oranları düzeldi. Pozitif basınçlı ventilasyon sağlayan Endotrakeal entübasyon pnömotoraks oranları 5 azalmıştır. Entübasyon Yöntemleri önceden doğrudan vokal kord görselleştirme kullanarak daha az invaziv yöntemler trakea açık maruz aralığı nitelendirdi. Entübasyon prosedürü ile ilişkili komplikasyonlar kardiyak delme veya vena kava kesiden kaynaklanan trakeal yaralanma, vokal kord rüptürü, istenmeyen özofagus entübasyon ve kanama sayılabilir. Buna ek olarak, plevral astar alt timik lob yakınlığı pnömotoraks neden olabilir. Burada bir teknik tarifBir körelmiş sonu angiocatheter ve deri trakea aydınlatma kullanarak entübasyon basit bir yöntemi izlenerek minimal invaziv 2 cm&#39;lik cilt kesisi ile timektomi. timektomi 1.5 cm sternotomi ve mediasteni mühür ve kanama ve solunum komplikasyonları en aza indirmek için cerrahi tutkal uygulaması ile üç katmanlı kapatma içerir. Timektomi ve IHC boyamada timik doku aradan sonra CD4 + ve CD8 + naiv T hücrelerinin yok olması ile kanıtlandığı gibi bu yöntem, güvenilir bir tam bir timektomi sonuçlanır. Operatif kez ve peri-prosedürel ölüm minimumda tutulur.

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete thymectomy in adult rats with non-invasive endotracheal intubation

Yenidoğan kemirgenlerde Timektomi immünolojik çalışmalar için bir köklü ve güvenilir bir yöntemdir. Ancak, yetişkin sıçanlarda, plevra bozulma gelen kanama ve pnömotoraks komplikasyonları önemli bir mortalite oranı neden olabilir. Bu protokol, mini-sternotomi ve endotrakeal entübasyon kullanan sıçan Timektomiye basit bir yöntemdir. Entübasyon bir non-invaziv ve kolay tekrarlanabilir yöntemle gerçekleştirilir ve pnömotoraks önlemek için pozitif basınçlı ventilasyon ve plevra kesintileri en aza indirmek için dikkatli timus diseksiyon için yeterli zaman sağlar kontrollü hava yolu için izin verir. Hala timus tam görselleştirme sağlarken bir 1,5 cm sternum insizyon, mediastinal damarlar ve plevra ile temas azalır. Mediasten ortaya çıkmasının ardından, timus büyütme altında künt diseksiyon ile kaldırılır. plevral boşluk daha sonra cerrahi yapıştırıcı uygulanması ve ardından ön-trakeal kas dikiş kapatma ile sızdırmaz kılınmıştır.Toraks sonra cilt sütür kapanması ardından sternum sütür kapanması ile kapatılır. Akış sitometrisi ile saf T-hücrelerinin immünohistokimyasal (IHK), mediastinal doku boyama ve olmaması ile kanıtlandığı gibi tüm timektomi tamamlandı ve prosedür,% 96 hayatta kalma oranlarına sahipti. En az komplikasyon ile tam timektomi atimik yetişkin farelerde de immünolojik çalışmalar için arzu edilir, bu yöntem uygundur.

Bu prosedür, yetişkin Lewis sıçanlarına (n = 26) uygulandı. Ortalama operasyon süresi 15 ± 3 dk oldu. İntraoperatif mortalite yoktu. Ortalama entübasyon süresi - Havalandırma kurulmasına entübasyon cihazında sıçan yerleştirme - 45 ± 5 sn. 24 sıçan post-operatif gün (POD) POD 4 14. Bir sıçan geliştirilen solunum güçlüğü ile solunum güçlükleri ya da kanama bulgusu ile operasyon tam bir iyileşme vardı ve mediasteni keşfetmek için yeniden ameliyata alındı. Sıçan çökmüş akciğer l...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Non-invaziv Endotrakeal Entübasyon Erişkin Sıçanlarda Komple Timektomi

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K Görüntüleme.  Duke University Medical Center. Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described. 

Video: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Lökosit Homing Göç ve non-invaziv Görüntüleme In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

İmmünoloji ve Enfeksiyon

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Zebra balığı Larva yılında Dissemine Kandidiyazis non-invaziv Görüntüleme

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

Bir Mouse Lenfatik damarları Non-invaziv Optik Görüntüleme

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Yenidoğan ve Erişkin Merkezi sinir sisteminin Kültüre Mikroglia

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic olmayan İnvaziv Modeli<em&gt; Escherichia coli</em&gt; Yenidoğan Sıçan Enfeksiyon

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Farelerde Deneysel Bakteriyel Pnömoni İndüksiyonunun ve fenotipleme bir Non-invaziv ve Teknik Olmayan yoğun Yöntem

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

Non-invaziv<emIn vivo</em&gt; Aktive Edilebilir Floresan Görüntüleme Ajanını Kullanan İnflamatuvar MMP Aktivitesinin Flüoresan Optik Görüntülemesi

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Bir kullanma Lenfosit Ekstravazasyonunun analizi<em&gt; İn Vitro</em&gt; İnsan Kan-beyin bariyeri Model

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Conjunktiva&#39;dan Hücreleri İzole Etmek ve Fenotip Hedeflemek İçin İnvaziv Olmayan Bir Yöntem

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...