Generation af Hook Ischemia-Reperfusion Model ved hjælp af en tre-dages udvikling Chick Embryo
Summary
Dette papir beskriver iskæmi-reperfusion (I / R) modellering i en 3-dages chick embryo ved hjælp af en spinal nål tilpasset krog til bedre at forstå I / R udvikling og behandling. Denne model er enkel, hurtig og billig.
Abstract
Iskæmi og reperfusion (I / R) lidelser, såsom myokardieinfarkt, slagtilfælde, og perifer vaskulær sygdom, er nogle af de førende årsager til sygdom og død. Mange in vitro – og in vivo-modeller er i øjeblikket tilgængelige til undersøgelse af I/R-mekanismen i sygdom eller beskadiget væv. Til dato er der imidlertid ikke rapporteret nogen in ovo I/R-model, hvilket ville give mulighed for en bedre forståelse af I/R-mekanismer og hurtigere lægemiddelscreening. Dette papir beskriver I / R modellering ved hjælp af en spinal nål tilpasset krog i en 3-dages chick embryo til at forstå I / R udvikling og behandlingsmekanismer. Vores model kan bruges til at undersøge anomalier på DNA, RNA, og protein niveauer. Denne metode er enkel, hurtig og billig. Den nuværende model kan bruges uafhængigt eller sammen med eksisterende in vitro – og in vivo I/R-modeller.
Introduction
Iskæmi-reperfusion vævsskade er blevet knyttet til en række patologier, herunder hjerteanfald, iskæmisk slagtilfælde, traumer, og perifer vaskulær sygdom1,2,3,4,5. Dette skyldes primært en mangel på en omfattende forståelse af sygdommens progression og manglen på en effektiv forskningsmodel. Iskæmisk skade opstår, når blodforsyningen til et bestemt område af vævet er afskåret. Som et resultat, iskæmisk væv i sidste ende nekrotizes, selv om satsen varierer afhængigt af vævet. Hvis blodforsyningen genoprettes, kan det derfor være med til at mindske skaderne. Det er imidlertid blevet observeret, i nogle tilfælde, at reperfusion forårsager mere vævsskade end iskæmi alene gør6,7,8. Derfor er det nødvendigt at forstå de molekylære og cellulære mekanismer i iskæmi-reperfusion for at udvikle en effektiv terapeutisk intervention. I øjeblikket er ingen effektiv behandling for I / R-skader kendt. Denne forskel har foranlediget oprettelsen af nye eksperimentelle modeller, lige fra in vitro til in vivo modeller, til at løse det eksisterende problem9,10,11,12,13.
Chick embryoner (Gallus gallus domesticus) er meget udbredt i forskning på grund af deres nem adgang, etisk accept, relativt stor størrelse (sammenlignet med andre embryoner), lave omkostninger, og hurtig vækst14. Vi brugte en chick embryo på 72 h af udvikling til at skabe en in ovo I / R ved okkludering og frigivelse af den rigtige vitelline arterie ved hjælp af en spinal nål. Vi kaldte det Hook-I/R iskæmi-reperfusion model (Figur 1). Den model, der anvendes i denne undersøgelse er i stand til præcist at simulere alle downstream processer, herunder oxidative og inflammatoriske veje, som ofte er forbundet med I / R skader15,16,17.
Protocol
Representative Results
Discussion
Målet med iskæmi-reperfusion forskning er at skabe terapeutiske strategier, der forhindrer celledød og fremme inddrivelse29,30. For at overvinde de nuværende begrænsninger i I / R forskning, vi designet en Hook I / R chick embryo model til at producere en pålidelig og reproducerbar I / R model. Så vidt vi ved, er vores den første I /R-model, der nogensinde er skabt i et 3-dages chick embryo til rutinemæssige I / R-eksperimenter, udover at studere stresss…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne udtrykke vores taknemmelighed over for Hari Shankar for hans kritiske input under videografi og redigering, Mr. Baqer Hussain for voice-over, Mr. Asghar Rizvi for videoredigering, Mr. Mohammad Haider for videooptagelser, Mr. Mohammad danske Siddiqui for hjælp under eksperimenterne.
Materials
| (-80°C) freezer | Haier, China | – | |
| 1.5mL Centrifuge tube | TARSONS, India | 500010X | |
| 100mm Petri dish (sterile) | Tarsons, India | 460050 | |
| 18G Needle (18G×1.5 (1.25×38mm) | Ramsons, India | 13990 | |
| 1mL Syringe | DISPO VAN | – | |
| 26G Needle (26G×1/2 (10.45x13mm) | DISPO VAN, india | 30722D | |
| 37°C egg incubator with adjustable percentage humidity | Gentek, India | GL-100 | |
| 37°C laboratory incubator | SCIENCE TECH, India | CB 101-14 | |
| 3-Methyladenine (3-MA) | Sigma Aldrich, USA | M9281 | |
| 3mL Pasture Pipette | TARSONS, India | 940050 | |
| 50mL Beaker | TARSONS, India | – | |
| 5mL Syringe | DISPO VAN, India | IP53 | |
| 70% ethanol | Merck Millipore, United States | 64-17-5 | |
| Adhesive tape/Cello tape | Sunrise, India | – | |
| Ambra1 primers | Applied Biosystems, Foster city, USA | Hs00387943_m1 | |
| Anti-mouse IgG | Cell Signaling Technology, USA | 7076S | |
| Anti-Rabbit IgG | Jackson Immuno Research Laboratories, USA | 711-035-152 | |
| Atg7 | R&D Systems, USA | MAB6608 | |
| Atg7 primers | Applied Biosystems, Foster city, USA | Hs00893766_m1 | |
| Autoclave Bag | Tarsons, India | 550022 | |
| Autoclave Machine | Local made, Lucknow, India | – | |
| Beclin-1 | Proteintech, USA | 66665-1-Ig | |
| Beta Actin | ImmunoTag, USA | ITT07018 | |
| Bovine Serum Albumin | Himedia, Mumbai, India | TC194 | |
| Calcium Chloride | Himedia, Mumbai, India | GRM534 | |
| Catalase | ImmunoTag, USA | ITT5155 | |
| Cleaning wipes | Kimberly-Clark, India | 370080 | |
| Cleaved Caspase3 | ImmunoTag, USA | ITT07022 | |
| di-Sodium hydrogen phosphate heptahydrate | Himedia, Mumbai, India | GRM39611 | |
| Doppler blood flowmeter | Moors instrument, United Kingdom | moorVMS-LDF1 | |
| Egg rack | – | – | |
| Egg rack | – | – | |
| GAPDH | ImmunoTag, USA | M1000110 | |
| GAPDH primers | Applied Biosystems, Foster city, USA | Hs02758991_g1 | |
| Glycine | Himedia, Mumbai, India | MB013 | |
| Kidney tray | HOSPITO | – | |
| LC3A/B | Cell Signaling Technology, USA | 4108S | |
| Methanol | Rankem laboratories, Mumbai, India | M0252 | |
| Micromanipulator | Narishige, Japan | M-152 | |
| N-acetyl-L-cysteine (NAC) | Sigma Aldrich, USA | A7250 | |
| Naringenin | Sigma Aldrich, USA | 67604-48-2 | |
| NF-kβ | Thermo Fisher Scientific, USA | 51-0500 | |
| NLRP3 | ImmunoTag, USA | ITT07438 | |
| Nose plier | Local made, Lucknow, India | – | |
| Ocular forceps | Stoelting, Germany | 52106-40 | |
| Ocular iris | Tufft Surgical Instruments, Jaipur, India | Hard Age Vannas Micro Scissors Angled 8CM / 3 1/8" | |
| OHP marker pen | Camlin, India | – | |
| ORP-150 | ImmunoTag, USA | ITT08329 | |
| Pointed sharp edge scissor | Stoelting, Germany | 52132-11 | |
| Potassium Chloride | Himedia, Mumbai, India | MB043 | |
| Potassium phosphate monobasic anhydrous | Himedia, Mumbai, India | MB050 | |
| Protease Inhibitor | Abcam, United States | Ab65621 | |
| SOD-1 | ImmunoTag, USA | ITT4364 | |
| Sodium Chloride | Fisher Scientific, Mumbai, India | 27605 | |
| Sodium dodecyl sulphate | Himedia, Mumbai, India | GRM886 | |
| Spinal needle 25GA; 3.50 IN (90.51 X 90mm) | Ramson, India | GS-2029 | |
| Stereo Zoom surgical microscope | Olympus, Japan | SZ2-STU3 | |
| Syringe discarder | BIOHAZARD | 882210 | |
| Toothed forceps | Stoelting, Germany | 52102-30 | |
| Tris Base | G Biosciences, United States | RC1217 | |
| Tris Hydrochloric Acid | Himedia, Mumbai, India | MB030 | |
| Tween 20 | G Biosciences, United States | RC1227 | |
| White Leghorn Chicken 0-day eggs | – | – | |
| Z-Val-Ala-Asp(OMe)-FMK | MP Biomedicals, LLC, USA | FK009 |
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