Férricos modelos murinos trombosis inducida por cloruro
Summary
Se presenta un procedimiento de refinado del cloruro férrico (FeCl3) modelos de trombosis inducidas en la carótida y la arteria mesentérica, así como la vena, que se caracteriza de manera eficiente mediante microscopía intravital para monitorear el tiempo de oclusiva formación de trombos.
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
La trombosis arterial (coágulos de sangre) es una complicación común de muchas enfermedades sistémicas asociadas con la inflamación crónica, incluyendo la aterosclerosis, la diabetes, la obesidad, el cáncer y los trastornos autoinmunes reumatológicas crónicas. Los trombos que se presentan en el tronco circulación arterial de la activación plaquetaria inadecuada, agregación y mecanismos procoagulantes posteriores, y están implicados en los ataques cardíacos, accidentes cerebrovasculares y la pérdida de la extremidad. La pared del vaso es un sistema complejo que incluye múltiples tipos de células y está influenciada por una multitud de factores extrínsecos como el estrés de cizallamiento, las células de la sangre, hormonas y citoquinas, así como la expresión de proteínas antioxidantes que circula en la pared del vaso. Experimentos in vitro no pueden replicar este entorno complejo y por lo tanto en estudios in vivo utilizando modelos animales son críticos para permitir una mejor comprensión de los mecanismos implicados en los trastornos trombóticos.
Los ratones han demostrado tener simmecanismos ILAR a los seres humanos en términos de la trombosis, aterosclerosis, inflamación y diabetes 1,2. Además, los ratones transgénicos y knockout pueden ser creados para probar la función de los productos génicos específicos en un complejo fisiológico o patológico ambiente. Tales estudios imitan la patología humana y pueden proporcionar información sobre el mecanismo importante relacionada con el descubrimiento de nuevas vías y terapias, así como proporcionar detalles importantes en la caracterización de los efectos de la droga sobre la trombosis.
Trombos arteriales patológicas se producen debido a la lesión endotelial capa o disfunción y la exposición de la corriente de la sangre a la matriz subendotelial 3,4. Varios modelos de trombosis se han desarrollado para inducir este daño endotelial tales como la lesión mecánica, lesión oxidativa a base de Bengala compuesto fotorreactivo Rose y lesiones láser 5. En este espectro, cloruro férrico (FeCl3) inducida por la lesión vascular es un modelo ampliamente utilizado de trombosis. Este reactivo cuandose aplica a la cara externa de los vasos induce daño oxidativo a las células vasculares 6-8, con pérdida de la protección de las células endoteliales a partir de plaquetas y componentes de la cascada de coagulación circulantes. El modelo de FeCl 3 es simple y sensible tanto a anticoagulante y anti-plaquetas medicamentos, y se ha realizado en arterias carótida y femoral, vena yugular, y mesentérica y arteriolas y vénulas cremastéricos en ratones, ratas, cobayas y conejos 6-15.
Un parámetro medible en este modelo es el tiempo transcurrido desde la lesión hasta completar la oclusión del vaso, medida como cese el flujo de sangre con un medidor de flujo Doppler o bajo la observación directa con 6,7,9 microscopía intravital. Una gama de tiempos entre 5 a 30 min se ha informado en diferentes estudios en ratones C57Bl6 7-10,16, lo que sugiere que FeCl 3 concentraciones, tipos de anestesia, las técnicas quirúrgicas, la edad del ratón, fondo genómico, método de medición de bLood flujo, y otras variables ambientales tienen efectos significativos en este modelo. Esta amplia variabilidad hace que sea difícil comparar los estudios de diferentes grupos de investigación y puede hacer la detección de diferencias sutiles difícil.
Con una visión para minimizar tales variabilidades y establecer un reproducible uniformemente en el sistema de modelo in vivo, hemos refinado el modelo de arteria carótida inducida por FeCl3 que hace que los datos altamente reproducibles con una variación mínima 6-10,16-19. En este trabajo se describen y compartir las habilidades y presenta varios ejemplos experimentales representativos que pueden beneficiarse de este modelo.
Protocol
Representative Results
Discussion
El FeCl3 inducida modelo es uno de los modelos de trombosis más ampliamente usados, que no sólo pueden proporcionar información valiosa acerca de las modificaciones genéticas sobre la función plaquetaria y trombosis 7,8,16,19,31-33, pero también puede ser una herramienta valiosa para la evaluación de compuestos terapéuticos y estrategias para el tratamiento y prevención de enfermedades aterotrombóticos 11,17,34-37. Aquí hemos demostrado que nuestros modificaciones y refinamien…
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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