Name: two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver
Uploaded: 2018-02-06T14:30:00
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

Denne undersøgelse blev støttet af tilskud fra National Research Foundation af Korea (NRF) finansieret af Korea regeringen (MSIP; grant no. 2016R1A2B4008199 til Y-M. H.), og sundhedsministerium & velfærd, Republikken Korea (giver nummer: HI14C1324).

Alle procedurer blev gennemført i overensstemmelse med retningslinjerne i den institutionelle Animal Care og brug Udvalget af Yonsei University College of Medicine, Korea. LysM-grøn fluorescerende proteiner (normal god landbrugspraksis; normal god landbrugspraksis / +) mus8 og CX3Cr1-normal god landbrugspraksis (NGL / +)9 mus på 8-12 ugens i alder blev brugt til intravital billeddannelse. Alle kirurgiske værktøjer blev autoklaveres og desinficeres med 70% alkohol....

<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>

Leveren har været aktivt studeret af mange grupper3,10, på grund af betydningen af dens funktion. For eksempel, foreslået Heymann et al. et delikat metode ved hjælp af agarosegelelektroforese. Selvom denne metode fandtes for at være meget præcis, tager indstillingen Agarosen tid. Dog med vores metode, alle procedurer kan gennemføres inden for 30 min, minimere andre forstyrrende faktorer. Andet, stabilisere leveren er ekstremt vigtigt, fordi hjerteslag kan ...

Leveren er den store metaboliske orgel i pattedyr; det fungerer som et kontrol tårn for energi, hormoner og afgiftning1. På grund af dens betydning, har forskerne udført mange i in vitro og i vivo eksperimenter for at belyse forandringer i leveren under betændelse. Intravital mikroskopi er blevet brugt til at fange levende billeder fra leveren2 . Faktisk, forskellige leveren Billeddannende metoder har været indført2,3,4,5,6. Men for at udvikle en mere forenklet kirurgisk tilgang og opnå stabilisering af målorgan og immunceller, vi har designet en enkel protokol, der kan guide forskere for at minimere deres indsats for intravital billeddannelse.
I denne undersøgelse indførte vi en stabil og roman billeddiagnostiske afdeling og kirurgisk teknik, der kunne bruges til at minimere blødning og mulige infektioner. Vi bekræftede, at leveren forblev intakt i mere end 2 timer. Med denne metode identificeret vi med held morfologier LCMs7 og neutrofile septisk betingelse. Da denne metode minimerer fysiske og biologiske konfunderende faktorer som bævende og uventede betændelse under kirurgisk procedure, forventer vi, at denne metode kan bruges til at observere akutte reaktioner af immunceller i leveren som reaktion på reagenser som LP'ER.

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			<td height="61" style="height:61px;width:260px;">Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td>
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two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

Sepsis er en type af svær infektion, som kan forårsage organsvigt og væv skader. Selvom dødelighed og sygelighed priser forbundet med sepsis er ekstremt høje, ingen direkte behandling eller Organrelaterede mekanisme er blevet undersøgt i detaljer i realtid. Leveren er det centrale organ, der administrerer toksiner og infektioner i den menneskelige krop. Heri, vi havde til formål at udføre intravital billeddannelse af mus lever efter induktion af endotoxemia spore motilitet af immunceller, såsom neutrofile og leveren kapsulær makrofager (LCMs). Derfor, vi har designet en roman kirurgisk metode til eksponering af leveren med minimalt invasiv kirurgi. Mus blev intraperitoneal injiceret med LPS (LPS), en fælles endotoxin. Ved hjælp af vores nye kirurgisk fremgangsmåde for eksponering og intravital billeddannelse af mus lever, fandt vi neutrofile rekruttering i LPS-behandlede LysM-grøn fluorescerende proteiner (NGL) var mus lever steget sammenlignet med det i fosfatbufferet saltvand-behandlede leveren. Efter LPS behandling, antallet af neutrofile steget betydeligt med tiden. Derudover visualiseret bruger CX3Cr1-NGL mus, vi med held lever fastboende makrofager kaldet LCMs. Derfor, for at undersøge effekten af nye reagenser til at styre immun mobilitet i vivo, fastlæggelsen af motilitet og morfologi af neutrofile og LCMs i leveren kan give os mulighed at identificere terapeutiske effekt i orgel bankerot og væv skader forårsaget af leukocytter aktivering i sepsis.

En enkel og meget stabil billeddiagnostiske afdeling blev udtænkt. Bunden af kammeret var dækket med silikone, som forhindrede glider musen kroppen og organer. Derudover fordi silicium har den rette mængde af elasticitet, kunne det forhindre forventede skader induceret af presset af diasset cover (figur 1A, C). Andet, væv-safe lim (Vet bond) blev anvendt for at fastsætte den udpakkede leveren på cirkel-formede silikone seng. Denne metod...

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

Vi etablerede en roman kirurgisk protokol for to-foton billeddannelse af levende mus lever med minimal invasion. Med denne teknik identificeret vi den detaljerede struktur i leveren under LPS-induceret endotoxemia. Vi forventer, at denne metode kan bruges til at bestemme effektiviteten af forskellige reagenser behandling af hepatisk leukocyt migration.

To-foton Intravital billeddannelse af leukocytter under immunrespons i LPS-behandlede mus lever

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 ...

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