Biz mükemmel teknik destek için Daniel Bukala Natalie Altmeyer ve Funda Cay teşekkür ederim. Biz taslağın düzenlenmesi için Jonathan Cotton Greg Bowden ve Paul Soubiran teşekkür ederim. Bu çalışma CRC 156 (proje C3) üzerinden Werner Siemens-Vakfı ve Eberhard Karls Üniversitesi Tübingen ( &#39;&#39; Promotionskolleg &#39;&#39;) Tıp Fakültesi tarafından ve DFG tarafından desteklendi.

Bu makalede açıklanan tüm prosedürler, laboratuar hayvanlarının bakımı ve kullanımı ile ilgili uluslararası standartlara ve kurallara uymuş ve Almanya&#39;nın Tuebingen Ülke Komisyonu&#39;nun Hayvan Refah ve Etik Komitesi tarafından onaylanmıştır. 8 - 12 haftalık BALB / c ve C57BL / 6 fareleri, 12 saatlik bir süreyle 12 saat süreyle tutuldu: 12 saatlik ışık: karanlık döngüde, 2-5 gruplar halinde 22 ± 1 ° C&#39;de IVC&#39;ler ve standart çevre koşulları altında su ve Gıda erişimi ad libitum . 1. Malzeme Hazırlama <ol><li> Enjeksiyondan önce doğrudan ilgili veri sayfasına göre OI boyasını yakın kızılötesi floresans görüntüsü için inceltin. MMP&#39;leri in vivo ölçmek için aktive olabilen OI boyası (680 nm&#39;de uyarımlı ticari prob), 1.5 mL 1x PBS&#39;de 20 nmol konsantrasyonda kullanıma hazırdır. Çözümü kullanmadan önce hafifçe çalkalayın veya vorteksleyin. OI boyası ışığa karşı korunduğunda 2-8 ° C&#39;de 6 aya kadar depolanabilir. </li&gt; <li> İntravenöz ( İV ) enjeksiyonlar için bir venöz kateteri hazırlayın. % 0.9 salin solüsyonu (50 mL&#39;de 10 enjeksiyon ünitesi içeren) ve 30 gauge iğne ile polietilen katetere bağlanmış 20 U (0.5 mL) insülin şırınga kullanın. OI boya fare başına önerilen 2 nmol dozunu enjekte edin. </li></ol> 2. Romatoid Artritin İndüksiyonu ve Kronik Temas Hipersensitivitesi Reaksiyonu <ol><li> Romatoid artrit: <ol><li> RA uyarmak için 1x PBS (200 μL) ile glikoz-6-fosfat izomeraz (GPI) 1: 1&#39;e karşı antikorlar (AB) içeren serum 100 μL&#39;yi (K / BxN farelerinden elde edilen 10 ) seyreltin. Seyreltilmiş serumu -80 ° C&#39;de saklayın. RA indüksiyonu için ayrıntılı prosedürler Monach ve ark. Tarafından anlatılmıştır . 8 . </li><li> RA indüklemek için, her kuyruğunu kuyruğuyla hafifçe kaldırın ve t 0 gününde 200 μL seyreltilmiş serum intraperitoneal ( ip ) enjekte edinDeniyor. Enjeksiyondan sonra fareyi doğrudan kafesin içine geri koyun. </li><li> İsteğe bağlı olarak, AB enjeksiyonlarından önce ayak bileği çaplarını ölçün ve bir &quot;artritik skor&quot; ( Şekil 1A ) tanımlayın. Mekanik bir ölçüm cihazı (mikrometre) kullanarak GPI serumundan 6. güne kadar günlük ayak bileği şişlik ölçümüne devam edin. </li><li> Aktifleştirilebilir OI boyasını enjekte edin (adım 1.1 - 1.2), in vivo MMP aktivitesi iv&#39;yi RA farelerinin kuyruk damarına ölçün GPI-serum enjeksiyonundan sonra 5. günde ve kontrol farelerinin kuyruk venine uygulandı. Kuyruk damarı enjeksiyonundan 24 saat sonra optik görüntüleme deneyleri uygulayın. </li></ol></li><li> Aşırı duyarlılık reaksiyonuyla temas: <ol><li> C57BL / 6 farelerinin sensitizasyonu için, 4: 1 aseton / yağ karışımı içinde çözülmüş% 5 TNCB çözeltisi hazırlayın. </li><li> % 100 oksijen (1.5 L / dk) buharlaştırılmış% 1.5 izofluran kullanarak C57BL / 6 farelerde anestezi uygulayın. Fareler uyuşturulduktan sonra, kendiliğinden çılgınca yerleştirin<html> Burun konisi (hacimce% 1.5 izofluran tüpüne bağlı kesilmiş bir 5 mL polietilen şırınga) ve anestezi korumalıdır. Anestezi uygulanırken göz kuruluğunu önlemek için anestezi uygulanmış hayvanlara veteriner merhemi uygulayın. </li><li> Küçük bir hayvan tüy düzeltici kullanarak karnınızı dikkatle tıraş edin (2 x 2 cm). Cildin yaralanmasından kaçının; zira bu, hayvandaki spesifik olmayan flüoresan sinyallerine neden olabilir ve çalışmanın sonuçlarını etkileyebilir. </li><li> Hayvanı hassaslaştırmak için 100 uL&#39;lik bir pipet kullanarak traşlı karın bölgesine yavaş yavaş 80 μL% 5 TNCB solüsyonu uygulayın. Fare, korneanın hasar görmesini önlemek ve iyileşme safhasında düşük vücut ısısından kaçınmak için fare gözlerinin yataklarla temas etmemesine özen göstererek nazikçe kafesin içine geri koyun. </li><li> Duyarlılığın altıncı gününde% 1 TNCB solüsyonu (9: 1 aseton / yağ karışımı içinde çözülmüş) hazırlayın ve akut ve kronik CHSR&#39;yi elde etmek için bir pipet yardımıyla sağ kulakta 20 mcL uygulayın.&quot;&gt; 1 <ol><li> 100 μL&#39;lik bir pipet kullanarak 20 μL% 1 TNCB çözeltisiyle sağ kulağın her iki tarafındaki 1. meydan okumayla başlayın ve CHR&#39;yi ortaya çıkarmak için her saniye ikinci günde beş kez tekrarlayın (hassasiyetten sonra 15 gün sonra) . Kulak kalınlığını her gün bir mikrometre ölçüm cihazı kullanarak ölçün. </li></ol></li><li> Meydan okumadan 12 saat sonra CHR farelerinde in vivo olarak MMP aktivitesini ölçmek için aktive olan OI boyasını (680 nm&#39;de uyarımlı ticari bir prob) enjekte edin (adım 1.1-1.2). 24 saat post kuyruk damarı enjeksiyonu ( iv ) ile optik görüntüleme deneyleri yapın. </li></ol></li></ol> 3. Optik Görüntüleme İçin Hayvan Hazırlığı <ol><li> Kullanılmış OI ajanlarının flüoresan sinyaliyle otomatik flüoresansın (yaklaşık 700 nm) parazitlenmesini önlemek için fare yemini (görüntülemeden en az 3 gün önce) düşük veya floresan olmayan çaylara ( örn. , Manganez içermez) çevirin. </li><li> Hayvanı yerleştirinBir anestezi kutusuna yerleştirin ve oksijen ile buharlaştırılmış hacimce% 1.5 izofluran kullanarak (oksijen hava ile değiştirilebilir, ancak bir denemede standart haline getirilmesi gerekir) anestezi altına alın. </li><li> Fareye görünür şekilde anestezi uygulandığında, kendiliğinden üretilen burun konisine yerleştirin (hacimce% 1.5 izofluran tüpüne bağlı, kesilmiş bir 5 mL&#39;lik polietilen şırınga ile oluşturun) ve anestezi uygulayın. </li><li> Hayvanı, küçük bir hayvan tüy düzeltici kullanarak hedef bölgede (2 cm x 2 cm) dikkatle tıraş edin. Cildin yaralanmasından kaçının; zira bu, hayvandaki spesifik olmayan flüoresan sinyallerine neden olabilir ve çalışmanın sonuçlarını etkileyebilir. NOT: Saçlar, ilgilenilen bölgeye (ROI) bağlı olarak, OI boyunca flüoresan sinyalini absorbe edebilir (fare suşuna bağlıdır, daha fazla veya daha az emilim gözlenir). </li><li> İV enjeksiyonu için, vazodilatasyon başlatmak için fare kuyrukunu ılık suya yerleştirin, alkolle enjeksiyon yerinde deriyi nazikçe temizleyin ve kateterin üzerine yerleştirilmesine başlayınkuyruğunun uzak bir site. kuyruk damarına 20 ° &#39;lik bir açı ile, iğnenin kenar &quot;kesme&quot; ve şırınga yeniden süspansiyon haline getirerek kateterin doğru yerleştirilmesi test etmek yerleştirin. </li><li> Kateter doğru yerleştirilir, şırınga yerine ve Ol probu (2 nmol) enjekte edilir. Enjeksiyondan sonra, şırınga yerine ve% 0.9 salin çözeltisi polietilen borunun tam olarak net ölü hacmi 25 uL enjekte edilir. Not: kullanılan OI boyanın dokusu yarılanma ömrü süresi, in vivo olarak, MMP aktivitesinin ölçülmesi için (680 nm&#39;de eksıtasyonla ticari bir prob) 72 saattir. Tam bir açıklık sağlamak için, boya yeniden enjeksiyon önceki enjeksiyondan sonra daha önceki 7 günden önerilmez. </li></ol> 4. Optik Görüntüleme <ol><li> görüş alanı (FOV) merkezinde OI-tarayıcının siyah kutu içinde bir siyah plastik ya da kağıt tabaka yerleştirin. </li><li> Bir ölçüm protokolünü kurmak ve sağ dalga boyu tercih (eksitasyon: 680 ± 10 nm, eGörev: 700 ± 10 nm) ve görüntüleme parametreleri. NOT: Bazı OI sistemlerinde, çeşitli görüntüleme boyalarının kurulumu önceden tanımlanmıştır. </li><li> Floresan görüntüleme için doğru protokolü seçmek için görüntüleme yazılımını açın (üretici tarafından sağlanır) ve sistemi başlatın. Çoğu CCD kameranın çalışma sıcaklığına soğuması gerekir ve bu işlem 10 dakika sürebilir. Güvenilir sonuçlar için, sistem hazır oluncaya kadar bekleyin. </li><li> İn vivo görüntüleme sistemi edinme kontrol panelinin açılmasını göz önünde bulundurun ve seçilen her filtre çifti dizideki bir görüntüyü temsil edecektir. Bu durumda, sırasıyla 680 ± 10 nm ve 700 ± 10 nm&#39;lik bir uyarılma ve emisyon dalga boyuna sahip olan ticari boya için filtre çiftleri ile bir görüntü elde edin ve ölçümü başlatın (&quot;Alınan sıralama&quot; tuşuna basın). Daha ayrıntılı talimatlar için üreticinin el kitabına bakın. </li><li> Görüntüleri uygun şekilde etiketleyin ve bilgileri &quot;Görüntü Düzenle&quot; ye kaydedin.Labels &quot;penceresi açılır ve&quot; Acquire sequence &quot;i izleyecektir. </li><li> OI boya enjeksiyonundan önce her bir hayvanın temel taramasını yapın veya arka plan sinyalini ayırt etmek için naif kontrol hayvanları kullanın. </li><li> Saat sonra OI boya kuyruk damar enjeksiyonu, hayvanlar OO sisteminde en yüksek sinyali ölçmek için bir pozisyonda, FOV ortasına yerleştirin ve ölçümleri başlatmak. NOT: Önemli: Hipotermi, görüntüleme maddelerinin dağılımını önemli ölçüde etkileyebilir. Hayvanların hipotermiden kaçınmak için sahnenin 37 ° C&#39;ye kadar ısıtıldığından emin olun. Görüntüleme, 1-5 fareden oluşan gruplar halinde eşzamanlı olarak gerçekleştirilebilir. Aynı anda ölçülecek hayvan sayısına bağlı olarak FOV boyutunu seçin. Her fare görünür olup olmadığını kontrol etmek için parlak bir alan resmi alabilirsin. </li></ol> 5. Veri Analizi NOT: Aşağıdaki görüntü yazılımını kullanarak veri analizi yapınÜreticinin protokolü. <ol><li> RA ölçümleri için, Şekil 2&#39;de gösterildiği gibi foton emisyonunun kalibre edilmiş birimi kullanılırken, kronik CHSR yüzde olarak (etkinlik) sinyal yoğunluğu olarak resmedilmiştir. </li><li> ROI&#39;leri el ile çizmek için alet plakasını kullanın. RA görüntülerinin analizi için, her hayvanın tüm ayak bilekleri ve pençeleri arasında en yüksek sinyalin etrafına yerleştirilmiş standart bir daire kullanın. CHSR&#39;yi analiz etmek için ROI&#39;leri parlak alan görüntüsüne göre sağ ve sol kulak çevresine yerleştirin. </li><li> Belirli çizilen ROI&#39;de foton emisyonunu veya sinyal yoğunluğu değerlerini ölçmek için &quot;ölçme&quot; ye basın. Sistem betimsel istatistiksel analiz için çizilen ROI&#39;de değerler sağlayacaktır. </li></ol>

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S., Nicolay, K., Grull, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+targeting+of+MMP-2/9+activatable+cell-penetrating+imaging+probes+is+caused+by+tumor-independent+activation).">Tumor targeting of MMP-2/9 activatable cell-penetrating imaging probes is caused by tumor-independent activation).</a> J Nucl Med. 52 (2), 279-286 (2011).</li><li>Schafers, M., Schober, O., Hermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-metalloproteinases+as+imaging+targets+for+inflammatory+activity+in+atherosclerotic+plaques.">Matrix-metalloproteinases as imaging targets for inflammatory activity in atherosclerotic plaques.</a> J Nucl Med. 51 (5), 663-666 (2010).</li><li>Wagner, S., 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=A+new+18F-labelled+derivative+of+the+MMP+inhibitor+CGS+27023A+for+PET:+radiosynthesis+and+initial+small-animal+PET+studies.">A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: radiosynthesis and initial small-animal PET studies.</a> Appl Radiat Isot. 67 (4), 606-610 (2009).</li><li>Waschkau, B., Faust, A., Schafers, M., Bremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+new+fluorescence-labeled+MMP+inhibitor+to+image+tumor+MMP+activity+in+vivo+in+comparison+to+an+MMP-activatable+probe.">Performance of a new fluorescence-labeled MMP inhibitor to image tumor MMP activity in vivo in comparison to an MMP-activatable probe.</a> Contrast Media Mol Imaging. 8 (1), 1-11 (2013).</li><li>Vogl, T., 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=Alarmin+S100A8/S100A9+as+a+biomarker+for+molecular+imaging+of+local+inflammatory+activity.">Alarmin S100A8/S100A9 as a biomarker for molecular imaging of local inflammatory activity.</a> Nat Commun. 5, 4593 (2014).</li></ol>

Yazarların açıklayacak bir şeyi yok.

OI klinik öncesi araştırma, in vivo, moleküler görüntüleme yayılmayan için çok yararlı hızlı ve pahalı olmayan bir araçtır. OI özel bir mukavemeti iltihabik cevaplar gibi yüksek dinamik işlemleri izlemek için özelliğidir. Dahası, OI bir hafta gün arasında değişen geniş bir zaman süresi için bir hastalığın seyrini takip etmesini sağlar. Çok zamana ve maliyet açısından verimli olarak OI, pozitron emisyonu tomografisi (PET) ya da manyetik ...

Romatoid artrit (RA) veya psoriyaz vulgaris gibi otoimmün hastalıklar, gecikmiş tip hipersensitivite reaksiyonları (DTHR) olarak derecelendirilmektedir. 1 RA yaygın erozif sinovit ve eklem yıkımı ile karakterize otoimmün bir hastalıktır. 2 İnflamasyonlu artritik eklemler inflamatuar hücrelerin infiltrasyonu ve proliferasyonunu, proinflamatuar hücrelerin artmış ekspresyonunu, pannus oluşumuna, kıkırdağa ve kemik yıkımına yol açtığını göstermektedir. Matriks metalloproteinazlar (MMP&#39;ler) ile kollajen gibi ekstraselüler matriks moleküllerinin bölünmesi, doku dönüştürme ve anjiyogenez için gereklidir ve doku parçalanmasına neden olur. 5 , 6 Kontak aşırı duyarlılık reaksiyonları (CHR), nötrofillerin bir araya toplanması ile karakterizedir ve oksidatif patlamaya neden olur. 7 Benzer şekilde RA, CHR&#39;deki MMP&#39;ler invol&#39;durkronik enflamasyonu oluşturmak amacıyla doku dönüşümü, hücre göçü ve anjiyogenez ved. RA araştırmak için, glukoz-6-fosfat izomeraz (GPI) Serumda enjeksiyon fare modeli kullanılmıştır. GPI karşı antikorlar ihtiva eden transgenik K / BxN farelerden 8 serum, romatizmal iltihabı, GPI-serumu enjekte edildikten sonra 6. günde şişme ayak bileği, maksimum 24 saat (1.1) içinde gelişmeye başlayan sonra saf BALB / c farelerine enjekte edildi. Kronik CHR analiz etmek için, C57BL / 6 fareleri karın trinitrochlorobenzene (TNCB) ile duyarlı hale getirildi. sağ kulak hassasiyeti 1 hafta sonra başlayarak 5 kata kadar meydan (aynı zamanda 1.1 ve 1.2). Invaziv olmayan küçük hayvan OI esas olarak klinik öncesi araştırmalarda kullanılır fluorescent- chemiluminescent- ve biyolojik olarak ışık veren sinyallerin, in vivo araştırma dayanan bir tekniktir. alınan yarı nicel veriler Molec yönelik anlayışıSağlıklı ve deneysel hayvan modellerinin organ ve dokularındaki ular mekanizmalarını ve uzunlamasına takip ölçümlerini ( örn . , In vivo terapötik yanıt profillerini değerlendirmek için) sağlar. Uzunlamasına çalışmaların büyük bir avantajı, hayvan sayılarının azaltılmasıdır; çünkü aynı hayvanlar, zaman noktası başına farklı fareler kullanmak yerine, birkaç zaman noktasında yapılan takip çalışmalarında ölçülebilir. OI&#39;nın çözümü, deney hayvanlarında organların ve hatta daha küçük doku yapılarının ayrıntılı fonksiyonel görüntülemesine olanak tanır. Dar transmisyon spektrumu, ışık geçirmez bir &quot;karanlık kutu&quot; ile dağınık ışığa karşı koruma ve -70 ° C&#39;ye kadar birçok cihazda soğutulan hassas bir şarj kuplajlı cihaz (CCD) kamera ile özel uyarma ve emisyon filtreleri kullanımı , Floresans sinyallerinin son derece spesifik ve hassas ölçümlerini sağlar. Uyarıcı ile floresan ajanlar kullanarak - vekızıl ötesine yakın fluoresans penceresinde emisyon spektrumu (650-950 nm), bir sinyal-gürültü oranı önemli ölçüde geliştirilebilir. yakın kızılötesi fluoresans pencere hemoglobin ve su ile sinyalin nispeten düşük bir emme yanı sıra düşük bir arkaplan otomatik floresans ile karakterize edilir. 9 Bu küçük hayvan dokusu içinde yukarı 2 cm bir penetrasyon derinliğine sağlar. OI-sondalar ile doğrudan (bir floresan etiketli antikor ile) bir hedef adres ya da (proteazlar ile), hedef doku içinde aktif hale getirilebilir. Aktive OI problar bağlı başka bir etki, molekül içindeki uyarım enerji aktaran bir söndürme kısım, Förster rezonans enerji transferi (FRET) kendi inaktive edilmiş biçimde optik olarak sessizdir. boya (örneğin, bir proteaz tarafından) ile bölünmesi durumunda enerji artık molekül içinde aktarılır ve bir flüoresan sinyal OI ile tespit edilebilir. Bu yüksek specificit ile OI sondaları tasarlanmasını sağlayanFarklı biyolojik süreçler ve mükemmel sinyal / gürültü oranları için. Aşağıdaki protokol, in vivo MMP-2, -3, -9 ve -13 aktivitesini ve inflamasyonun iki deneysel modelini (RA, CHR) görüntülemeye yönelik bir Aktive Edilebilir OI probu kullanarak hayvanların hazırlanmasını, OI ölçümlerini detaylı olarak açıklamaktadır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cornergel</td>
			<td>Gerhard Mann GmbH</td>
			<td>1224635</td>
			<td>ophthalmic ointment&#160;</td>
		</tr>
		<tr>
			<td>Forene</td>
			<td>Abbott GmbH</td>
			<td>4831850</td>
			<td>isoflurane</td>
		</tr>
		<tr>
			<td>U40 insulin syringe</td>
			<td>Becton Dickinson and Company</td>
			<td>324876</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sintetica</td>
			<td>6093089</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Med-PE 0.28x0.61mm</td>
			<td>Reichelt Chemietechnik GmbH+Co</td>
			<td>28460</td>
			<td>polyethylene tubing, inner diameter 0.28 mm, outer diameter 0.61&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 30 G</td>
			<td>Becton Dickinson &#38; Co. Ltd.</td>
			<td>305106</td>
			<td>30 G injection cannula</td>
		</tr>
		<tr>
			<td>RTA-0011 isoflurane vaporizer</td>
			<td>Vetland Medical Sales and Services LLC</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Artagain drawing paper</td>
			<td>Strathmore Artist Paper</td>
			<td>446-8</td>
			<td>coal black</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td>Optical imaging system</td>
		</tr>
		<tr>
			<td>BD Regular Bevel Needles, 25 G</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Chloro-1,3,5-trinitrobenzene</td>
			<td>Sigma Aldrich GmbH</td>
			<td>7987456F</td>
			<td>TNCB</td>
		</tr>
		<tr>
			<td>MMPSense 680</td>
			<td>Perkin Elmer&#160;</td>
			<td>NEV10126</td>
			<td>fluorescent imaging dye</td>
		</tr>
		<tr>
			<td>Oditest&#160;</td>
			<td>Koreplin GmbH</td>
			<td>C1X018</td>
			<td>mechanical measurment</td>
		</tr>
		<tr>
			<td>Miglyol 812</td>
			<td>SASOL</td>
			<td>-</td>
			<td>Oil</td>
		</tr>
		<tr>
			<td>&#160;BALB/C, C57BL/6</td>
			<td>Charles River Laboratories&#160;</td>
			<td>-</td>
			<td>Mice used for experiements</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich GmbH</td>
			<td></td>
			<td>For dilution of the RA serum&#160;</td>
		</tr>
		<tr>
			<td>Pipette (100&#181;l)</td>
			<td>Eppendorf&#160;</td>
			<td></td>
			<td>Used for TNCB application&#160;</td>
		</tr>
		<tr>
			<td>shaver&#160;</td>
			<td>Wahl&#160;</td>
			<td>9962</td>
			<td>Animal hair trimmer</td>
		</tr>
		<tr>
			<td>Living Image&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td></td>
			<td>Imaging software to measure OI</td>
		</tr>
	</tbody>
</table>

non-invasive in vivo fluorescence optical imaging of inflammatory mmp activity using an activatable fluorescent imaging agent

Bu çalışma, enflamasyon, iki farklı fare modellerinde, görüntüleme matriks metalloproteinaz (MMP) bir aktive flüoresan sonda ile -Aktiflik yoluyla in vivo olarak floresans optik görüntüleme (Ol) için invazif olmayan bir yöntem açıklanmaktadır: romatoid artrit (RA), ve bir kayıt hipersensitivite reaksiyonu (CHR) modeli. yakın kızılötesi (NIR) penceresinde bir dalga boyuna sahip ışık (650-950 nm) 650 nm&#39;nin altında kıyasla daha derin bir doku penetrasyonu ve en az bir sinyal emilimini sağlar. floresan OI kullanılarak en önemli avantajlardan biri, ucuz, hızlı ve farklı hayvan modellerinde uygulanması kolay olmasıdır. Aktive floresan probları inaktive eyalette optik sessiz, ancak bir proteaz tarafından aktive edildiği zaman, son derece flüoresan hale gelir. Aktif MMP&#39;ler doku tahribatına yol açan ve RA ve CHR olarak gecikmeli tipte aşırı duyarlılık reaksiyonları (DTHRs) hastalık ilerlemesinde önemli bir rol oynar. Ayrıca, MMPler olanKıkırdak ve kemik bozulması için anahtar proteazlar ve pro-inflamatuar sitokinlere yanıt olarak makrofajlar, fibroblastlar ve kondrositler tarafından indüklenir. Burada, MMP-2, -3, -9 ve -13 gibi anahtar MMP&#39;ler tarafından aktive edilen bir prob kullanıyor ve RA ve kontrol farelerinde, hastalık indüksiyondan 6 gün sonra da MMP aktivitesinin yakın kızılötesi floresan OI için bir görüntüleme protokolü tanımlıyoruz Sağlıklı kulaklara kıyasla sağ kulakta akut (1x meydan okuma) ve kronik (5x meydan okuma) CHR bulunan farelerde olduğu gibi.

Sığır BALB / c farelerinde romatizmal artrit (RA) indüklemek için hayvanlar 0 günde GPI&#39;ye karşı oto-antikorlar (1x PBS ile 1: 1 seyreltme) ile ip enjekte edildi. Bu GPI serumunda indüklenen maksimum iltihaplanma (ayak bileği şişmesi) RA modeli enjeksiyon sonrası 6. günde 11 . Bu nedenle, aktive edilebilir OI boya 2 nmol hazırlandı ve 5. günde artritik farelerin ve sağlıklı kontrol hayvanlarının kuyruk damarına iv enjek...

Watch this Scientific Journal Video about Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent at JoVE.com

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

Bu yazıda, iltihaplanmanın iki farklı deneysel modelindeki temel matris metalloproteinazların in vivo aktivitesini görselleştirmek için aktive edilebilir bir optik görüntüleme probu kullanılarak floresan görüntülemenin uygulanmasını açıklanmaktadır.

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 ...

non-invasive-vivo-fluorescence-optical-imaging-inflammatory-mmp

Research

JoVE Journal

Immunology and Infection

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

8.7K Görüntüleme.  Eberhard Karls University of Tübingen.  Bu yazıda, iltihaplanmanın iki farklı deneysel modelindeki temel matris metalloproteinazların in vivo aktivitesini görselleştirmek için aktive edilebilir bir optik görüntüleme probu kullanılarak floresan görüntülemenin uygulanmasını açıklanmaktadır. 

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

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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dinamik Canlı Hücre Görüntüleme Konukçu-Patojen Etkileşimleri Çalışma Optik Tuzak

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

Farklı Hayvan Modelleri NADPH Oksidaz Aktivitesinin Biyoparlaklık Görüntüleme

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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 ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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 ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo Görüntüleme Yöntemi Akut ve Kronik İltihap ayırt

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

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 ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

tarafından Kemirgen Lenfosit Etiketleme<sup&gt; 64</sup&gt; Cu-Antikor Reseptör için hedefleme<em&gt; In Vivo</em&gt; PET / CT ile hücre Trafik

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Non-invaziv Vivo içinde Bioluminescence sistemi tarafından fare konak Enterohemorrhagic Escherichia Coli kolonizasyon algılama

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...