IJzer-Chloride geïnduceerde Murine Trombose Models
Summary
Wij rapporteren een verfijnde procedure van de ijzerchloride (FeCl3) geïnduceerde trombose modellen op de halsslagader en mesenterica evenals ader, efficiënt gekarakteriseerd met behulp van intravitale microscopie tot tijd om trombi vorming occlusief bewaken.
Abstract
Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.
Introduction
Arteriële trombose (bloedstolsel) is een veel voorkomende complicatie van vele systemische ziekten verbonden met chronische ontsteking, waaronder atherosclerose, diabetes, obesitas, kanker en chronische auto reumatologische aandoeningen. Trombi die zich in de arteriële circulatie voort uit ongepaste activatie van bloedplaatjes, aggregatie en coagulatie volgende mechanismen, en zijn betrokken in hartaanvallen, beroertes en extremiteiten verlies. De vaatwand is een complex dat meerdere celtypen omvat en wordt beïnvloed door een groot aantal extrinsieke factoren afschuifspanning circulerende bloedcellen, hormonen en cytokinen, alsook expressie van antioxidant eiwitten in de vaatwand. In vitro experimenten kunnen niet repliceren deze complexe omgeving en daarom in vivo studies met diermodellen zijn essentieel voor een beter begrip van de mechanismen betrokken bij trombotische aandoeningen toe.
Muizen is aangetoond sim hebbenilar mechanismen mens in termen van trombose, atherosclerose, ontsteking en diabetes 1,2. Bovendien kunnen transgene en knockout muizen worden gecreëerd om de functie van specifieke genproducten te testen in een complexe fysiologische of pathologische omgeving. Dergelijke studies na te bootsen menselijke pathologie en kunnen belangrijke mechanistische informatie met betrekking tot de ontdekking van nieuwe wegen en therapieën, om te voorzien in en verstrekt belangrijke details in het karakteriseren van drugs effecten op trombose.
Pathologische arteriële trombi optreden als gevolg van laag verwonding of disfunctie en de blootstelling van de bloedstroom naar de endotheliale subendotheliale matrix 3,4. Verschillende trombose modellen zijn ontwikkeld om deze endotheliale beschadiging induceren zoals mechanische schade fotoreactieve verbinding Bengaals gebaseerde oxidatieve schade en verwonding laser 5. In dit spectrum ferrichloride (FeCl3) geïnduceerde vasculaire schade is een veel gebruikt model van trombose. Dit reagens wanneeraangebracht op het buitenste aspect van vaartuigen induceert oxidatieve beschadiging van vasculaire cellen 6-8, met verlies van endotheliale cel bescherming van circulerende bloedplaatjes en componenten van de coagulatie cascade. Het FeCl3 model is eenvoudig en gevoelig voor zowel anticoagulerende en anti-bloedplaatjes geneesmiddelen, en is uitgevoerd op carotis en femoralis, halsaders en mesenterische en cremasteric arteriolen en venulen bij muizen, ratten, cavia's en konijnen 6-15.
Een meetbare parameters in dit model is de verstreken tijd van de schade naar vat occlusie, gemeten als de bloedstroom stoppen met een Doppler stroommeter of onder directe observatie met intravitale microscopie 6,7,9 voltooien. Een reeks van tijden tussen 5 en 30 min werd gerapporteerd in verschillende studies bij muizen C57Bl6 7-10,16 suggereert dat FeCl3 concentraties vormen van anesthesie, chirurgische technieken, muis leeftijd, genomische achtergrond, meetmethode bLood stroming, en andere omgevingsvariabelen grote invloed in dit model. Deze grote variabiliteit bemoeilijkt studies vergelijken van verschillende onderzoeksgroepen en kan de detectie van kleine verschillen bemoeilijken.
Met een visie om deze variabiliteit te minimaliseren en om een uniform reproduceerbaar in vivo modelsysteem, hebben we de FeCl3-geïnduceerde halsslagader model dat de gegevens zeer reproduceerbare met minimale variatie 6-10,16-19 maakt verfijnd. In dit artikel beschrijven we en delen van de vaardigheden en het verslag een aantal representatieve experimentele voorbeelden die kunnen profiteren van dit model.
Protocol
Representative Results
Discussion
De FeCl3-geïnduceerde model is een van de meest gebruikte trombose modellen, die niet alleen waardevolle informatie over genetische modificatie op bloedplaatjesfunctie en trombose 7,8,16,19,31-33 kunnen verschaffen, maar kan ook een waardevol hulpmiddel voor de evaluatie van de therapeutische samenstellingen en strategieën voor de behandeling en preventie van ziekten atherothrombotische 11,17,34-37. Hier hebben we modificaties en verfijningen van het model getoond en toonde aanvullend …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta), R01 HL129179 (PI: Sen Gupta, Co-I: Li) and R01 HL098217 (PI: Nieman). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Surgical Scissors – Tungsten Carbide | Fine Science Tools | 14502-14 | cut and hold skin |
| Micro-Adson Forceps – Serrated/Straight/12cm | Fine Science Tools | 11018-12 | cut and hold skin |
| Metzenbaum Fino Scissors – Tungsten Carbide/Curved/Blunt-Blunt/14.5cm | Fine Science Tools | 14519-14 | to dissect and separate soft tissue |
| Ultra Fine Hemostat – Smooth/Curved/12.5cm | Fine Science Tools | 13021-12 | to dissect and separate soft tissue |
| Graefe Forceps – Serrated/Straight/10cm | Fine Science Tools | 11050-10 | to dissect and separate soft tissue |
| Dumont #5 Fine Forceps – Biology Tips/Straight/Inox/11cm | Fine Science Tools | 11254-20 | Isolate vessel from surounding tissue |
| Dumont #5XL Forceps – Standard Tips/Straight/Inox/15cm | Fine Science Tools | 11253-10 | Isolate vessel from surounding tissue |
| Blunt Hook- 12cm/0.3mm Tip Diameter | Fine Science Tools | 10062-12 | Isolate vessel from surounding tissue |
| Castroviejo Micro Needle Holders | Fine Science Tools | 12061-02 | Needle holders |
| Suture Thread 4-0 | Fine Science Tools | 18020-40 | For fix the incisors to the plate |
| Suture Thread 6-0 | Fine Science Tools | 18020-60 | For all surgery and ligation |
| Kalt Suture Needles | Fine Science Tools | 12050-03 | |
| rhodamine 6G | Sigma | 83697-1G | To lebel platelets |
| FeCl3 (Anhydrous) | Sigma | 12321 | To induce vessel injury |
| Papaverine hydrochloride | Sigma | P3510 | To inhibit gut peristalsis. |
| Medline Surgical Instrument Sterilization Steam Autoclave Tapes | Medline | 111625 | To fix the mouse to the plate |
| Fisherbrand™ Syringe Filters – Sterile 0.22µm | Fisher | 09-720-004 | For sterlization of solutions injected to mice |
| Fisherbrand™ Syringe Filters – Sterile 0.45µm | Fisher | 09-719D | To filter the FeCl3 solution |
| Sterile Alcohol Prep Pad | Fisher | 06-669-62 | To sterilize the surgical site |
| Agarose | BioExpress | E-3120-500 | To make gel stage |
| Leica DMLFS fluorescent microscope | Leica | Intravital microscope | |
| GIBRALTAR Platform and X-Y Stage System | npi electronic GmbH | http://www.npielectronic.de/products/micropositioners/burleigh/gibraltar.html | |
| Streampix version 3.17.2 software | NorPix | https://www.norpix.com/ |
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