Name: two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver
Uploaded: 2018-02-06T14:30:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

Bu çalışmada bir hibe--dan Ulusal Araştırma Vakfı, Kore (Kore hükümeti (MSIP; grant No 2016R1A2B4008199 Y-m tarafından finanse edilen NMG) tarafından desteklenmiştir H.), Sağlık Bakanlığı ve refah, Güney Kore (sayı vermek: HI14C1324).

Tüm yordamları kurumsal hayvan bakım ve kullanım Komitesi Yonsei Üniversitesi Tıp Fakültesi, Kore kılavuzlarınıza uygun olarak yapılmıştır. LysM-yeşil flüoresan protein (GFP; gfp / +) fareler8 ve CX3Cr1-GFP (gfp / +)9 fare 8-12 hafta-in yaş, intravital görüntüleme için kullanılmıştır. Tüm cerrahi alet autoclaved ve % 70 alkol ile dezenfekte edildi.
1. özel tasarlanmış fare araçları destekleyici odası Imaging karaciğer
<ol><li>Fare karaciğer odası tasarım görüntüleme<ol>
<li>Fare karaciğer (şekil 1) görüntüleme için aşağıdaki boyut TMMOB olun: iç Odası (120 mm Uzunluk × 160 mm genişlik × 30 mm yükseklik), dış Kamara (100 mm Uzunluk × 100 mm genişlik × 24 mm yükseklik), cıvata (Ф 3 mm × 30 mm yükseklik), fındık (Ф 4 mm) ve kanat şeklindeki fındık (Ф 4 mm).</li>
		<li>Kür Ajan ve elastomer 1:9 oranı temel silikon 120 g toplam yapmak için ölçmek.</li>
		<li>Sıvı silikon karışımı bir 250 mL Erlenmeyer şişeye koyun ve iyice karıştırın. Sonra bir desiccator şişeye koy ve hava kabarcıkları vakum altında yaklaşık 1 h için pompa ile kaldırın.</li>
		<li>10 mm yükseklik gibi yüksek bir özel tasarlanmış fare odasına sıvı silikon karışımı dökün ve izin için bir kaç gün (şekil 1A) tedavi.</li>
	</ol>
</li>
	<li>Kapak slaytlar ve yatak sabitleme karaciğer hazırlanması<ol>
<li>Metal çerçeve üzerinde kapak cam silikon yapıştırıcı kullanarak ekleyin. 1 gündür (şekil 1B) kurumasını bekleyin.</li>
		<li>Etkili bir şekilde su daldırma lens için distile su tutmak için semipermanent su engelleme yapısı çok önemlidir. 1 mm silikon yapıştırıcı yuvarlama yapmak ve gecede sertleşmesine.</li>
		<li>Mix kür Ajan ve elastomer temel ve sonra içine 6-şey plaka bir yükseklikte, yaklaşık 8-10 mm. gerçekleştir sertleştirme işlemi 1.1.2-1.1.4 içinde açıklandığı gibi dökün.</li>
	</ol>
</li>
</ol>2. LPS hazırlanması
<ol><li>Steril 1 × fosfat tamponlu tuz (PBS) üzerinden Salmonella enterica serotip enteritidis LPS toz sulandırmak için hazırlayın. (1 × PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4).</li>
	<li>PBS su banyosu 37 ° C'de ön ısı ve LPS toz şişe (25 mg) dikkatlice açın.</li>
	<li>Damlalıklı yardımların kullanılması, steril PBS (5 mL) LPS toz şişe içine dökün ve hafifçe karıştırın veya hisse senedi çözüm hazırlamak için çözüm askıya alabiliriz. Not: LPS toz sıvılaşmış sonra sert vortexing kabarcıklar neden olabilir. Aliquots 1.5 mL tüplerde yaparken kaybı en aza indirgemek için baloncuklar yapmaktan kaçının.</li>
	<li>−20 ° C de hisse senedi LPS çözüm depolamak ve enjeksiyon için 1 mg/mL LPS çözüm içine oranında seyreltin.</li>
	<li>1 mg/mL LPS çözüm (20 mg/kg) 1-mL şırınga kullanarak bir sigara anestezi fare Periton enjekte. Denetim hayvan için PBS aynı hacmi enjekte.</li>
	<li>2s için yeterli inflamatuar yanıt bekleyin.</li>
</ol>3. kan damarları leke hazırlanması Texas-kırmızı Dextran
<ol><li>İki 15 mL konik boru ve 10 mL steril PBS hazırlayın.</li>
	<li>Texas-kırmızı Dextran toz (25 mg) PBS 3 ml seyreltik ve iyice karıştırın. Karışımı bir boş 15 mL konik tüp aktarın.</li>
	<li>Kalan PBS (7 mL) dextran karışımı (3 mL) içeren 15 mL konik tüp aktarın.</li>
	<li>0,45 µm şırınga filtre bir 10 mL şırınga ucunu bağlayın. Şırınga filtre iğne değiştirmeniz gerekir.</li>
	<li>Dextran karışımı (10 mL) filtre. Şırınga eşit dökülmesini önlemek için tuşuna basın.</li>
	<li>2.5 mg/mL dextran çözüm ışık bloke 1.5 mL tüp içinde 500 µL dağıtmak ve −20 ° C. saklayın Bir kez çözdürülen, 4 ° C'de depolayın</li>
</ol>4. cerrahi işlem
<ol><li>Cerrahi tablo ve tüm aletleri ameliyat öncesi % 70 alkol kullanarak dezenfekte edin. Sonra karaciğer ameliyatı için araçları seti ve ısıtma yüzeyi 37 ° C (şekil 2A, B) için ayarlayın.</li>
	<li>Anestezi için 30 mg/kg tiletamine/zolazepam ve 10 mg/kg xylazine ile fareler intraperitoneally enjekte et. Bu çözümler karışık olabilir ve aynı anda enjeksiyon için PBS içinde seyreltilmiş. Anesthetization yavaşça fare ayak basarak onaylayın.  Hiçbir refleks olduğunda sonraki adıma geçin.</li>
	<li>Fare'nın retina kuruluğu önlemek için göz merhem küçük miktarda uygulayın.</li>
	<li>Vücut saç saç dökücü krem (şekil 2C) kullanarak karın çevresinde kaldırın.</li>
	<li>Texas-kırmızı Dextran eriyik (2.5 mg/mL konsantrasyonu) kuyruk ven veya bir insülin şırınga (şekil 2B) kullanarak retro-orbital enjeksiyon yolu ile 200 µL enjekte.</li>
	<li>Şu subcostal alan bir marker ile işaretleyin. Çizgi xiphoid işleminden kaburga sonuna kadar çizilmiş olması. Forseps ve makas (şekil 2E, F) ile epidermis kesti.</li>
	<li>Karın boşluğu mikro-makas ve forseps kullanarak açın ve karaciğer dikkatle bulaşmasına neden. Sol yan lob pamuk temizleme bezi (Şekil 2 g, H) kullanılarak alınması.</li>
	<li>Steril PBS 500 µL kuruluk karaciğer önlemek için karın boşluğuna dökün.</li>
	<li>Özel olarak tasarlanmış odası sağ-decubitus konumda fareyi getirin ve doku yapıştırıcı az miktarda silikon yatağa uygulayın. Pamuk temizleme bezi (şekil 2I) kullanarak karaciğer yapıştırmanız dikkatle. Not: karaciğer dokusu son derece kırılgan olduğundan, mekanik karaciğerinden çekmeyin. Yavaşça pamuk temizleme bezi ile tomar dışarı.</li>
	<li>Birkaç saniye sonra Karaciğer karaciğer dışarı kurutma önlemek için steril PBS 500 µL ile tekrar ıslak. Metal çerçeve üzerinde karaciğer yerleştirin ve sıkıca fındık (şekil 2J) ile düzeltmek.</li>
</ol>5. İki fotonlu mikroskobu ile karaciğer intravital görüntüleme
<ol><li>ZEN yazılım (Carl Zeiss) üzerine lazer açmak, dalga boyunu ayarlamak için 880 nm ve set yeşil, çalıştırın ve kırmızı kanalları. Lazer güç floresan yoğunluğu (şekil 3A) göre ayarlayın. Emisyon filtre bilgi ise şöyle: yeşil/Alexa488, 500-550 nm ve kırmızı/Alexa 555, 575-610 nm.</li>
	<li>Odası görüntüleme sahnesinde yerini ve 20 X su-daldırma objektif lens kapağı cam yakın yerleştirin.</li>
	<li>Silikon kauçuk ısıtıcı (şekil 3B), vücut ısısını korumak için fare cesedi altında düzeltmek.</li>
	<li>500 µL distile su üzerinde metal çerçeve bırakın ve sonra karaciğer (şekil 3 c) yakın objektif lens çekin.</li>
	<li>Derinlik ve saati görüntüleme alanını belirlemek ve odağı ayarlayın.</li>
	<li>Kuralları görüntüleme.</li>
	<li>Uzun vadeli görüntüleme için her saat görüntüleme sırasında yarım doz tiletamine/zolazepam ve xylazine karışımı enjekte et. Vücut ısısı 37 ° C'de farelerin Bakımı sırasında bu sahne çok önemlidir. Her zaman anesthetization zaman her fare farklılık fare için göz kulak ol. Not: Bu yöntem cerrahi ve görüntüleme sonra kurtarma için uygun değildir. Etik fare hemen ameliyat sonrası kurban ediyoruz.</li>
	<li>Kararlı anesthetization için her 30 dakika pedal refleksleri kontrol edin.</li>
</ol>6. veri analizi
<ol><li>Veri analiz yazılımı Volocity açın ve sonra kütüphanede ham veri yazılım alın.</li>
	<li>Gürültü durumu uygun ve bir anlık görüntü almak için görüntüden kaldırın. Not: düzgün görüntü elde etmek, 'Para' veya 'Orta' filtresini kullanın. Ayrıca, karanlık ya da bulanık görüntülerin daha net yapmak için 'Kontrast geliştirme' özelliğini kullanın.</li>
	<li>Anlık görüntüler için JPEG veya TIFF dosyalarını ve AVI dosyalarını filmler için % 100 ve ihracat için düzenlenmiş öğe sığar.</li>
</ol>

<ol>
	<li>Girard, J. R., 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=Fuels,+hormones,+and+liver+metabolism+at+term+and+during+the+early+postnatal+period+in+the+rat.">Fuels, hormones, and liver metabolism at term and during the early postnatal period in the rat.</a> J Clin Invest. 52 (12), 3190-3200 (1973).</li><li>Marques, P. E., 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=Imaging+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat Protoc. 10 (2), 258-268 (2015).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy+Imaging+of+the+Liver+following+Leishmania+Infection:+An+Assessment+of+Hepatic+Hemodynamics.">Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.</a> J Vis Exp. (101), e52303 (2015).</li><li>Honda, M., 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=Intravital+imaging+of+neutrophil+recruitment+in+hepatic+ischemia-reperfusion+injury+in+mice.">Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.</a> Transplantation. 95 (4), 551-558 (2013).</li><li>Jenne, C. N., Wong, C. H., Petri, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PLoS One. 6 (9), e25109 (2011).</li><li>Ritsma, L., 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=Intravital+microscopy+through+an+abdominal+imaging+window+reveals+a+pre-micrometastasis+stage+during+liver+metastasis.">Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.</a> Sci Transl Med. 4 (158), 158ra145 (2012).</li><li>Sierro, F., 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+Liver+Capsular+Network+of+Monocyte-Derived+Macrophages+Restricts+Hepatic+Dissemination+of+Intraperitoneal+Bacteria+by+Neutrophil+Recruitment.">A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.</a> Immunity. 47 (2), 374-388 (2017).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 719-726 (2000).</li><li>Jung, 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Heymann, F., 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=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> J Vis Exp. (97), (2015).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının bildirin.

Karaciğer aktif olarak birçok gruplar3,10, onun işlevini önemi nedeniyle tarafından incelenmiştir. Örneğin, Heymann vd. özel kullanarak hassas bir yöntem önerdi. Bu yöntem son derece kesin olarak bulundu rağmen özel ayar sürüyor. Ancak, bizim metodoloji ile tüm yordamları karıştırıcı diğer faktörler minimize 30 dk içinde yürütülen. Kalp atışı video rahatsız edemez çünkü İkincisi, karaciğer sabitleme son derece önemlidir. Bu ...

Karaciğer önemli metabolik organ memelilerde olduğunu; enerji, hormonlar ve detoksifikasyon1için bir kontrol kulesi olarak görür. Onun önemi nedeniyle, araştırmacılar değişiklikleri karaciğerde iltihap sırasında aydınlatmak için birçok vitro ve in vivo deneyler yaptık. İntravital mikroskobu karaciğer2 canlı görüntüleri yakalamak için kullanılır. Gerçekten de,2,3,4,5,6çeşitli karaciğer görüntüleme yöntemleri olmuştur tanıttı. Ancak, daha basitleştirilmiş bir cerrahi yaklaşım geliştirmek ve sabitleme hedef organ ve bağışıklık hücreleri elde etmek için biz kendi çaba intravital görüntüleme için en aza indirmek araştırmacılar rehberlik eder basit bir protokol tasarlanmıştır.
Bu çalışmada, istikrarlı ve yeni görüntüleme odası ve kanama ve olası enfeksiyon en aza indirmek için kullanılabilir cerrahi tekniği tanıttı. Karaciğer daha 2 h için sağlam kaldı doğruladı. Bu yöntemle başarıyla türleri Morfoloji LCMs7 ve nötrofil septik koşul altında tanımlanan. Bu yöntem titreyen ve beklenmedik iltihabı gibi fiziksel ve biyolojik karıştırıcı faktörlerin cerrahi işlem sırasında en aza indirir bu yana, biz bu yöntemi yanıt olarak karaciğerde bağışıklık hücrelerinin akut reaksiyonlar gözlemlemek için kullanılan olabilir tahmin reaktifler LPS severim.

<table><tbody><tr><td>Custom designed chamber</td><td>Live Cell Instruments</td><td></td><td>Custom-designed size</td></tr><tr><td>Metal cover slide</td><td>Live Cell Instruments</td><td></td><td>Custom-designed size</td></tr><tr><td>Cover glass</td><td>Electron Microscopy Sciences</td><td>#72204-03</td><td></td></tr><tr><td>Sylgard 184 Silicone elastomer kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Erlenmeyer flask</td><td>DURAN</td><td>21 216 36</td><td></td></tr><tr><td>Cell culture plate (Flat type)</td><td>HYUNDAI Micro Co.</td><td>H31006</td><td></td></tr><tr><td>Desiccator</td><td>ADARSH</td><td>55204</td><td></td></tr><tr><td>High Q BOND SE 5 mL</td><td>BJM LAB</td><td></td><td>To make semi-permanent dam</td></tr><tr><td>Flexible Silicone Rubber Heaters</td><td>Omega</td><td>SRFR/SRFG SERIES, M1250/0800</td><td></td></tr><tr><td>DC power supply</td><td>Toyotech</td><td>DP30-03A</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td></td><td></td><td>Home-made</td></tr><tr><td>Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td><td>Sigma-Aldrich</td><td>L6011</td><td></td></tr><tr><td>Texas Red Dextran</td><td>Thermo Fisher Scientific</td><td>D1830</td><td></td></tr><tr><td>15 mL High-clarity polypropylene&lt;br/&gt; conical tube</td><td>FALCON</td><td>14-959-49B</td><td></td></tr><tr><td>0.45 &amp;mu;m filter (Minisart Syringe Filter)</td><td>Sartorius</td><td>16555k</td><td></td></tr><tr><td>1.5ml Micro Tube, Amber</td><td>Axygen</td><td>MCT-150-X</td><td>For light-blocking</td></tr><tr><td>1 mL syringe</td><td>Korea Vaccine</td><td>KV-S01</td><td></td></tr><tr><td>3 mL syringe</td><td>Korea Vaccine</td><td>KV-S03</td><td></td></tr><tr><td>10 mL syringe</td><td>Korea Vaccine</td><td>KV-S10</td><td></td></tr><tr><td>0.3 mL insulin syringe</td><td>Becton Dickinson</td><td>324900</td><td></td></tr><tr><td>Hair removal cream 100 mL</td><td>Body natur</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>ABDI</td><td></td><td></td></tr><tr><td>Zoletil 50 5 mL</td><td>Virbac</td><td></td><td></td></tr><tr><td>Rompun 10 mL</td><td>Bayer</td><td></td><td></td></tr><tr><td>Heating plate</td><td>Live Cell Instruments</td><td>PH-S-10</td><td>Custom-designed size</td></tr><tr><td>DC controller</td><td>Live Cell Instruments</td><td>CU-301</td><td></td></tr><tr><td>Microdessection Scissors</td><td>Harvard Apparatus</td><td>72-5418</td><td></td></tr><tr><td>Scissors</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-5137</td><td></td></tr><tr><td>Vet bond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>ZEN software (Black Edition)</td><td>Carl Zeiss</td><td></td><td>Program for LSM 7 MP</td></tr><tr><td>LSM 7 MP</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Volocity</td><td>PerkinElmer</td><td></td><td>Analysis program</td></tr></tbody></table>

two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

Sepsis organ yetmezliği ve doku neden olabilir şiddetli enfeksiyon türüdür hasar. Mortalite ve morbidite oranları sepsis ile ilişkili, ancak son derece yüksek, hayır doğrudan tedavi vardır veya mekanizma organ ile ilgili ayrıntılı gerçek zamanlı olarak inceledi. Karaciğer toksinleri ve enfeksiyonlar insan vücudunda yönetir anahtar organdır. Burada, nötrofil ve karaciğer kapsüler makrofajlar (LCMs) gibi bağışıklık hücreleri hareket izlemek için fare karaciğer intravital görüntüleme sonra endotoksemi indüksiyon gerçekleştirmek amaçladık. Buna göre bir roman cerrahi yöntemi pozlama minimal invaziv cerrahi ile karaciğer için tasarlanmış. Fareler intraperitoneally ile lipopolysaccharide (LPS), bir ortak endotoksin enjekte edildi. Pozlama ve LPS tedavi LysM-yeşil flüoresan protein (GFP) fare karaciğer, o nötrofil bulduk alımı intravital görüntüleme için bizim yeni cerrahi yaklaşım kullanarak fare karaciğer bununla fosfat tamponlu göre artış serum fizyolojik tedavi karaciğer. LPS tedaviden sonra nötrofil sayısı önemli ölçüde zamanla arttı. Ayrıca, CX3Cr1-GFP fare kullanarak, biz başarıyla karaciğer ikamet makrofajlar LCMs adı görüntülenir. Bu nedenle, bağışıklık hareketlilik vivo içindekontrol etmek için yeni reaktifler etkinliğini araştırmak için hareketliliği ve nötrofil ve LCMs morfolojisi karaciğerde belirleme organ yetmezliği ve doku hasarı nedeniyle tedavi edici etkisi tanımamıza izin verebilir Sepsis lökosit etkinleştirme.

Basit ve çok kararlı görüntüleme odası geliştirildi. Odası alt fare vücudu ve organları kaymasını engelleyen silikon ile kaplıydı. Ayrıca, silikon elastikiyet doğru miktarda olduğundan, beklenen hasar kapak slayt (şekil 1A, C) basınç tarafından indüklenen engelleyebilir. İkinci, doku-kasa tutkal (veteriner bond)'i ayıklanan karaciğer daire şeklinde silikon yatağa düzeltmek için uygulandı. Bu yöntem kalp atışı...

Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Bir roman cerrahi protokol İki fotonlu görüntüleme en az invaze olan canlı fare karaciğer için kurduk. Bu teknik ile karaciğer detaylı yapısını lipopolysaccharide kaynaklı endotoksemi sırasında tespit. Biz bu yöntemi hepatik lökosit göç için çeşitli reaktifler tedavinin etkinliğini belirlemek için kullanılması gereken tahmin.

İki fotonlu Lipopolysaccharide tedavi fare karaciğer bağışıklık yanıtında sırasında lökositlerin Intravital görüntüleme

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

two-photon-intravital-imaging-leukocytes-during-immune-response

Research

JoVE Journal

Immunology and Infection

10.1K Görüntüleme.  Yonsei University College of Medicine. Bir roman cerrahi protokol İki fotonlu görüntüleme en az invaze olan canlı fare karaciğer için kurduk. Bu teknik ile karaciğer detaylı yapısını lipopolysaccharide kaynaklı endotoksemi sırasında tespit. Biz bu yöntemi hepatik lökosit göç için çeşitli reaktifler tedavinin etkinliğini belirlemek için kullanılması gereken tahmin.

Video: Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

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

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Intravital Görüntüleme Fare Popliteal Lenf Nodu

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Aşağıdaki Karaciğer intravital Mikroskopi Görüntüleme<em&gt; Leishmania</em&gt; Enfeksiyon: Karaciğer Hemodinami Üzerine Bir Değerlendirme

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfp Muhabir Fare kullanma Sağlıklı ve hastalıklı Karaciğer Bağışıklık Hücreleri Uzun Vadeli intravital multiphoton Mikroskopi Görüntüleme

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Farelerde mesenteriumda Damarlardaki intravital Mikroskopi Lökosit-endotelyal ve Trombosit-lökosit Etkileşimleri

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Real Time Anestetize Farelerde Intravital Mikroskopi Mezenterik Damarlardaki nötrofiller ve monositler Görüntüleme

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

İn vivo Iki renkli 2-foton görüntüleme genetiği etiketlenmiş Reporter hücrelerinin cilt içinde

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Biyomühendislik

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Bir Fulminan Miyokardit Modelinde Işık Yeri Mikroskopisi İle Lökosit İnfiltrasyonunun Nicel Görselleştirilmesi

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Tıp

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Bakteriyel Pathogen sonda için hücresel ve Organismic düzeyi ana bilgisayar yanıt için kullanma

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

<meta charset="utf8"></head><body>Fare akciğer naklinde hücresel dinamiklerin gerçek zamanlı izlenmesi

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>Yazar Spotlight: İntravital Görüntüleme Yoluyla Akciğer Nakli İmmünolojisini İlerletmek

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Nakledilen Fare Akciğerinin İntravital İki Foton Mikroskobu

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

İki fotonlu Lipopolysaccharide tedavi fare karaciğer bağışıklık yanıtında sırasında lökositlerin Intravital görüntüleme

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Lökosit Homing Göç ve non-invaziv Görüntüleme In vivo