Name: transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time
Uploaded: 2013-03-10T14:00:00
Description: Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

Vi anerkender Drs. Camillo Ricordi, Antonello Pileggi, R. Damaris Molano, Stephan Speier og Daniel Nyqvist for frugtbare diskussioner. Vi takker også Eleut Hernandez og Diego Espinosa-Heidmann til teknisk bistand, og Mike Valdes og Margaret Formoso for at få hjælp med videooptagelse. Byron Maldonado optaget, redigeret og produceret den endelige video. Forskningsstøtte blev leveret af Diabetes Research Institute Foundation ( <a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a> ), NIH / NIDDK / NIAID (F32DK083226 til MHA; NIH RO3DK075487 til AC; U01DK089538 til PO.B.). Yderligere forskning støtte til PO.B blev tilvejebragt gennem midler fra Karolinska Institutet, det svenske forskningsråd, det svenske Diabetes Foundation, Family Erling-Persson Foundation, Familie Knut og Alice Wallenberg Foundation, Skandia Insurance Company Ltd, levende ( FP7-228.933-2), strategisk forskningsprogram i Diabetes på Karolinska Institutet, Novo Nordisk Fonden, og Berth von Kantzow Fond.

Den følgende procedure udføres under stereoskop i 2 trin, det første trin indebærer at indlæse holme i kanylen og det andet trin er den faktiske transplantation ind i ACE. Alle procedurer udført på dyr blev godkendt af den institutionelle dyrepleje og brug komité (IACUC) fra University of Miami. 1. Ilægning Islets i tungeholder til transplantation <ol><li> Center holme i dyrkningsskål ved at dreje fadet i indsnævrende cirkler. </li><li> Tag kanylen fra &quot;reservoir&quot; og p...

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Murine pancreasøer blev isoleret ved hjælp af collagenasefordøjelse efterfulgt af oprensning på densitetsgradienter som beskrevet tidligere 33. Isolerede øer blev dyrket natten over før transplantation. Selv om dette ikke kræves, anbefales det at lade øerne at komme sig efter isoleringsproceduren. Dette er kritisk, når transplantation udføres i diabetiske recipienter, da det vil sikre transplantation for at overleve / robuste øer. Transplantation er udført under genera...

Fremskridt i intravital mikroskopi har afsløret fysiologiske fænomener ikke forudsagt af in vitro-undersøgelser 1. Dette understreger udfordringen i at oversætte resultater opnået ved konventionel in vitro-metoder i den levende dyr. I det seneste årti var visualisering af væv i levende dyr væsentligt forbedret af de teknologiske fremskridt i billeddiagnostiske metoder 2, 3, 4, 5, 6. Dette har givet anledning et behov for in vivo imaging tilgange med mulig anvendelse i eksperimentelle dyremodeller for at muliggøre langsgående visualisering af målvæv ikke-invasivt. Billeddannelsesteknikker, såsom magnetisk resonans og positronemissionstomografi eller bioluminescens har muliggjort ikke-invasiv billeddannelse af organer / væv dybt inde i legemet 7-8, 9. Men disse teknikker kan ikke opnå enkelt celle opløsning som følge af høje baggrundssignaler og lav rumlig opløsning, på trods af anvendelsen of høj kontrast materialer eller vævsspecifik luminescens 4. Dette blev behandlet med fremkomsten af to-foton fluorescens konfokal mikroskopi 10. To-foton mikroskopi aktiveret intravital billeddiagnostiske undersøgelser for at visualisere og kvantificere cellulære begivenheder med hidtil usete detaljer 11, 12. Dette har ført til karakteriseringen af de vigtigste biologiske processer i sundhed og sygdom 13, 14, 15, 16. Mens banebrydende intravital billeddiagnostiske undersøgelser primært har &quot;efterlignet&quot; in vivo forhold i udskårne væv (f.eks lymfeknuder), har andre undersøgelser brugt invasive metoder til billede udsatte målvæv in situ 17, 18, ​​19, 20, 21. Andre undersøgelser har også brugt &quot;window kammer modeller&quot; at omgå begrænsninger i forbindelse med invasive metoder og begrænset imaging opløsning in vivo 22, 23, 24, 25. I vinduet kammeret model, er et kammer med et transparent vindue kirurgisk implanteret ind i huden i forskelleje steder (ryg eller ørehud, bryst-fedtpuden, lever, osv.) af dyret (fx mus, rotte, kanin). Selv om denne fremgangsmåde tydeligt giver høj opløsning in vivo billeddannelse, kræver det en invasiv kirurgi at implantere afdeling og kan ikke være i stand til at rumme langsgående billeddiagnostiske undersøgelser over flere uger eller måneder 22. Det blev for nylig vist, at en kombination af høj opløsning konfokal mikroskopi med en minimalt invasiv procedure, nemlig transplantation ind i det forreste kammer i øjet (ACE) tilvejebringer en &quot;naturlig organ vindue&quot; som en kraftfuld og alsidig in vivo-billeddannelse platform 26, 27. Transplantation ind i ACE er blevet anvendt i de sidste årtier til at studere biologiske aspekter af en række væv 28, 29, 30, og den seneste kombination med høj opløsning billeddannelse aktiveret undersøge fysiologien af pancreasøer med enkeltcelle-opløsning ikke- invasivt og i længderetningen &lt;sup&gt; 26, 27. Denne fremgangsmåde blev anvendt til at studere autoimmune responser under udviklingen af ​​type 1 diabetes i dyremodeller (upublicerede data). Det blev også anvendt til at undersøge pancreasudviklingen, såvel som, i studier af nyrefunktionen ved at transplantere ind i ACE pancreas knopper eller individuelle nyrernes glomeruli, henholdsvis (upublicerede data). En nylig rapport ved hjælp af denne tilgang yderligere demonstreret sin ansøgning om at studere immunresponser efter pancreasø transplantation 31. Vigtigere er det, denne undersøgelse viste, at transplantation ind i det forreste kammer i øjet tilvejebringer en naturlig organ vindue til at udføre: (1) langsgående, ikke-invasiv billeddannelse af transplanterede væv in vivo, (2) in vivo cytolabeling at vurdere cellulær fænotype og levedygtigheden i situ, (3) realtidssporing af infiltrerende immunceller i målvævet, og (4) lokal indgriben ved topisk påføring eller intraokulær injektion. Her vi demonstrate hvordan man udfører transplantation ind i det forreste kammer i øjet med Langerhanske øer.

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Beskrivelse / Bemærkninger </td></tr><tr><td> IsoTHESIA (Isofluran) </td><td> Buttler Animal Health Supply </td><td> 11695-6775-2 </td><td> 99,9% Isofluran / ml </td></tr><tr><td> Ketaset (Ketamin HCl) </td><td> Fort Dodge Animal Health </td><td> 0856-2013-01 </td><td> Alternativ injicerbar anæstesi </td></tr><tr><td> Beprenex (Buprenorphin HCL) </td><td> Reckitt Benckiser Health Care (UK) Ltd </td><td> 12496-075-7-1 </td><td> 0,3 mg / ml </td></tr><tr><td> Erythromycin ophthalmisk salve USP, 0,5% </td><td> Akron </td><td> 17478-070-35 </td><td> Anvendes profylaktisk til transplanterede øje </td></tr><tr><td> 0,9% natriumchlorid (saltvand) </td><td> Hospira Inc. </td><td> 0409-7983-03 </td><td> Til iv injektion. Sterile </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td> 1X. Sterile </td></tr><tr><td> CMRL medium 1066 </td><td> Cellgro </td><td> 98-304-CV </td><td> Suppleret, CIT modifikation. Foretrukne medier til øer </td></tr></tbody></table>

transplantation into the anterior chamber of the eye for longitudinal, non-invasive in vivo imaging with single-cell resolution in real-time

Intravital billedbehandling har vist sig som et uundværligt værktøj i biologisk forskning. Ved fremgangsmåden har mange billeddannelsesteknikker blevet udviklet til at studere forskellige biologiske processer hos dyr ikke-invasivt. Men en stor teknisk begrænsning i eksisterende intravital billeddiagnostiske metoder er den manglende evne til at kombinere non-invasiv, langsgående billeddannelse med encellede opløsning kapaciteter. Vi viser her, hvordan transplantation ind i det forreste kammer i øjet omgår så væsentlig begrænsning giver en alsidig eksperimentel platform, der muliggør ikke-invasiv, langsgående billeddannelse med cellulær opløsning in vivo. Vi demonstrerer transplantation procedure i mus og giver repræsentative resultater ved anvendelse af en model med klinisk relevans, nemlig pancreasø transplantation. Ud over at muliggøre direkte visualisering i en række væv transplanteret ind i det forreste kammer i øjet, giver denne fremgangsmåde en platform til urenn lægemidler ved at udføre langsigtet opfølgning og kontrol i målvæv. Grund af dens alsidighed, vævs / celle transplantation ind i forkammeret i øjet ikke blot fordele transplantation terapier, udvider den til andre in vivo anvendelser for at studere fysiologiske og patofysiologiske processer, såsom signaltransduktion og cancer eller autoimmune sygdomme udvikling.

Der er et par parametre, der definerer et &quot;godt&quot; transplantation. En god transplantation er en, der forløber uden blødning, når de foretager indsnittet som det kan ses i videoen. Blødning forhindres / minimeres ved at trænge kun toppen af skalpellen (p) i ACE (figur 3a). Dette vil også hjælpe med at forhindre kontakt og punktering af iris. Det vil også sikre et lille snit, som heler godt uden at bevirke uklarhed af hornhinden med tiden (figur 3c, d). Et andet vigtigt a...

Watch this Scientific Journal Video about Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time at JoVE.com

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

En ny metode, der kombinerer intraokulær transplantation og konfokal mikroskopi muliggør langsgående, non-invasiv real-time scanning med encellede løsning inden podede væv<em&gt; In vivo</em&gt;. Vi viser, hvordan transplantere pancreasøer i forkammeret af muse øjet.

Transplantation ind i det forreste kammer i øjet til langsgående, ikke-invasiv<em&gt; In vivo</em&gt; Imaging med Single-celle Opløsning i realtid

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

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JoVE Journal

Medicine

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

Non-invasiv Imaging af akut afstødelse efter Rat Renal Transplantation Brug<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Udvikling af en Audio-based Virtual Gaming Miljø til at hjælpe med Navigation Skills i Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Kemoterapi-induceret vaskulær toksicitet - Real-time<em&gt; In vivo</em&gt; Imaging af Vessel Nedskrivninger

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Ikke-invasiv vurdering af effekten af ​​nye lægemidler til behandling af Intestinal patologier Brug Serial Endoskopisk Imaging af levende mus

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Indkapsling Thermogenic præadipocytter for transplantation i fedtvæv Depoter

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Udvidet Time-lapse Intravital Imaging af Real-time flercellede Dynamics i Tumor mikromiljø

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Longitudinal<em&gt; In vivo</em&gt; Imaging af Cerebrovasculature: Relevans til CNS-sygdomme

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re Arterialized rotte delvis lever Transplantation med en i vivo fartøj-orienterede 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...