Realtid Digital Imaging af leukocyt-endotel Interaktion med iskæmi-reperfusionsskade (IRI) af rotte Cremaster Muscle
Summary
Digital intravital epifluorescens-mikroskopi af postkapillære venuler i den cremasteric mikrocirkulation er en bekvem metode til at få indblik i leukocyt-endotel-interaktion<em> In vivo</em> I iskæmi-reperfusionsskade (IRI) af tværstribet muskelvæv. Vi her give en detaljeret protokol til sikkert at udføre teknikken og diskutere dens anvendelsesmuligheder og begrænsninger.
Abstract
Iskæmi-reperfusionsskade (IRI) har været impliceret i en lang række patologiske tilstande, såsom cerebralt slagtilfælde, myocardieinfarkt, intestinal iskæmi såvel som efter transplantationen og kardiovaskulær kirurgi. En Reperfusion af tidligere iskæmisk væv, mens afgørende for forebyggelse af irreversibel vævsskade, fremkalder overdreven inflammation af det påvirkede væv. Tilgrænsende til produktion af reaktive oxygenarter, aktivering af komplementsystemet og forøget mikrovaskulær permeabilitet, aktivering af leukocytter er en af de vigtigste aktører i patologiske kaskade af inflammatorisk vævsbeskadigelse under reperfusion. 2, 3 leukocytaktivering er en flertrinsproces, der består rullende, faste adhæsion og transmigration og er medieret af et komplekst samspil mellem adhæsionsmolekyler som reaktion på kemoattraktanter såsom komplementfaktorer, kemokiner eller blodplade-aktiverende faktor. 4
<p class = "jove_content"> Mens leukocyt rulning i postkapillare venuler hovedsageligt medieres ved interaktion af selectiner 5 med deres modparter ligander, faste adhæsion af leukocytter til endotelet, er selectin-styret via binding til intercellulære adhæsionsmolekyler (ICAM) og vaskulær cellulære adhæsionsmolekyler (VCAM). 6, 7Guldstandarden til in vivo observation af leukocyt-endotel-interaktion er en teknik intravital mikroskopi, først beskrevet i 1968. 8
Selvom forskellige modeller af IRI (iskæmi-reperfusionsbeskadigelse), er blevet beskrevet til forskellige organer, 9-12 kun få er egnede til direkte visualisering af leukocytrekruttering i mikrovaskulære leje på et højt niveau af billedkvaliteten. 8
Vi her fremme den digitale intravital epifluorescens-mikroskopi af postkapillære venulen i cremasteric mikrocirkulationaf rotte 13 som en bekvem metode til kvalitativt og kvantitativt at analysere leukocytrekruttering for IRI-forskning i tværstribet muskelvæv og giver en detaljeret manual for udførelse af teknikken. Vi yderligere at belyse almindelige faldgruber og give nyttige tips, der skal gøre det muligt for læseren at virkelig værdsætte, og sikkert udføre metoden.
I en trinvis protokol, som vi skildrer, hvordan du kommer i gang med åndedræt kontrolleret anæstesi under tilstrækkelig overvågning til at holde dyret fast bedøvet i længere perioder. Vi så beskriver cremasteric præparatet som en tynd flad plade for fremragende optisk opløsning og giver en protokol for leukocyt billeddannelse i IRI, der er blevet veletableret i vores laboratorier.
Protocol
Discussion
Leukocyt-endothelial interaktion, produktion af reaktive ilt arter og aktivering af komplementsystemet er de vigtigste funktioner i IRI-induceret væv dysfunktion. 26 mikrocirkulationen af det angrebne væv betragtes som integralet stedet for den inflammatoriske debut. Bortset fra ex vivo eksperimenter såsom flow kammer analyser 27, 28 er det obligatorisk at give veletablerede modeller for intravital billedbehandling til yderligere at evaluere in vivo relevans. Selvom IRI er blev…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af en bevilling af "Deutsche Forschungsgemeinschaft" til SU Eisenhardt (EI 866/1-1).
Materials
| Name of the equipment: | Company: | Catalogue No.: | Comments: |
| Forene 100% (V/V) | Abbot | B506 | API isoflurane |
| Terylene Suture | Serag Weissner | OC108000 | |
| Portex Fine Bore Polythene Tubing | Smiths Medical | 800/100/100 | 0.28 mm inner Diameter |
| 0,9% saline solution | Fresinus Kabi | 808771 | |
| Change-A-tip deluxe cautery kit | Bovie Medical | DEL1 | |
| Abbocath -T 14G | Venisystems | G713 – A01 | used as lens tube |
| Servo Ventilator 900C | Maquet | used as animal ventialtor | |
| Logical pressure transducer | Smiths Medical | MX1960 | |
| Sirecust 404 Monitor | Siemens | ||
| ABL 700 Benchtop Analyzer | Radiometer | for blood gas measurement | |
| Heating pad | Effenberger | 8319 | |
| Aluminum stage | Alfun | AW7022 | |
| Surgical microscope OPMI 6-SDFC | Carl Zeiss | ||
| Microsurgical instruments lab set | S&T | 767 | |
| Biemer vessel clip | Diener | 64.562 | |
| Applying forceps | Diener | 64.568 | for Biemer vessel clip |
| Rhodamine 6G | Sigma-Aldrich | R4127 | |
| Vaseline white DAB | Winthrop | 2726853 | |
| Cover glasses | 32×32 mm | ||
| Intravital setup | |||
| Zeis Axio Scope A-1 MAT | Carl Zeis | 490036 | epifluorescence microscope |
| 470 nm LED | Carl Zeis | 423052 | fluorescence light source |
| Colibri 2 System | Carl Zeis | 423052 | |
| W Plan-Apochromat 20x/1,0 DIC | Carl Zeis | 421452 | water immersion objective |
| AxioCam MRm Rev. 3 FireWire | Carl Zeis | 426509 | high resolution digital camera |
| Axio vision LE software | Carl Zeis | 410130 | use for offline analysis |
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