Ferrico I modelli murini Trombosi indotta da cloruri
Summary
Riportiamo una procedura raffinata del cloruro ferrico (FeCl 3) modelli trombosi -indotta sulla carotide e dell'arteria mesenterica così come la vena, caratterizzato in modo efficiente usando la microscopia intravitale per monitorare il tempo di occlusiva la formazione di trombi.
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
trombosi arteriosa (coagulo di sangue) è una complicanza comune di molte malattie sistemiche associate a infiammazione cronica, tra cui l'aterosclerosi, il diabete, l'obesità, cancro e patologie reumatologiche autoimmuni croniche. Trombi che si verificano nel gambo circolazione arteriosa da piastrine di attivazione inadeguato, l'aggregazione e successive meccanismi coagulatoria, e sono implicati in attacchi di cuore, ictus e perdita di estremità. La parete del vaso è un sistema complesso che comprende diversi tipi di cellule ed è influenzata da molteplici fattori estrinseci compreso sollecitazione di taglio, le cellule del sangue, ormoni e citochine, così come espressione di proteine antiossidanti circolanti nella parete del vaso. In vitro esperimenti non possono replicarsi questo ambiente complesso e, pertanto, in studi in vivo su modelli animali sono fondamentali per consentire una migliore comprensione dei meccanismi coinvolti nei disordini trombotici.
I topi hanno dimostrato di avere simmeccanismi ili per gli esseri umani in termini di trombosi, arteriosclerosi, infiammazione e il diabete 1,2. Inoltre, i topi transgenici e knockout possono essere creati per testare la funzione di prodotti genici specifici in un fisiologico complesso o un ambiente patologico. Tali studi imitano patologia umana e possono fornire importanti informazioni relative al meccanicistico scoperta di nuovi percorsi e terapie, oltre a fornire dettagli importanti nel caratterizzare gli effetti della droga sulla trombosi.
Trombi arteriosi patologiche si verificano a causa di lesioni endoteliali strato o disfunzione e l'esposizione del flusso sanguigno alla matrice sottoendoteliale 3,4. Vari modelli trombosi sono stati sviluppati per indurre il danno endoteliale, come lesioni meccaniche, danno ossidativo composto fotoreattivo Rosa Bengala-based e lesioni a laser 5. In questo spettro, cloruro ferrico (FeCl 3) indotta danno vascolare è un modello ampiamente utilizzato di trombosi. Questo reagente quandoapplicata all'aspetto esterno delle navi induce danno ossidativo alle cellule vascolari 6-8, con perdita di protezione delle cellule endoteliali dalle piastrine e componenti della cascata della coagulazione circolante. Il modello di FeCl 3 è semplice e sensibile sia anticoagulanti e antipiastrinici farmaci, ed è stata eseguita su arterie carotidee e femorali, vene giugulari e mesenterici ed arteriole cremasterico e venule nei topi, ratti, cavie e conigli 6-15.
Un parametro misurabile in questo modello è il tempo trascorso da un infortunio per completare l'occlusione nave, misurata come la cessazione del flusso sanguigno con un misuratore di portata Doppler o sotto l'osservazione diretta con intravitale 6,7,9 microscopia. Una gamma di volte tra i 5 ei 30 minuti è stato riportato in diversi studi in topi C57Bl6 7-10,16, suggerendo che FeCl 3 concentrazioni, tipi di anestesia, tecniche chirurgiche, l'età del mouse, sfondo genomica, metodo di misurazione Bflusso lood, e di altre variabili ambientali hanno effetti significativi in questo modello. Questa ampia variabilità rende difficile il confronto tra gli studi di diversi gruppi di ricerca e può rendere la rilevazione di sottili differenze difficile.
Con una visione per ridurre al minimo tali variabilità e stabilire un modo uniforme e riproducibile nel sistema modello in vivo, abbiamo affinato il modello dell'arteria carotide FeCl 3 indotta che rende i dati altamente riproducibili con minime variazioni 6-10,16-19. In questo lavoro descriviamo e condividere le competenze e riportiamo alcuni esempi rappresentativi sperimentali che possono beneficiare di questo modello.
Protocol
Representative Results
Discussion
Il modello di FeCl 3 indotta è uno dei modelli più utilizzati trombosi, che possono non solo fornire preziose informazioni modificazioni genetiche sulla funzione piastrinica e la trombosi 7,8,16,19,31-33, ma può anche essere uno strumento prezioso per la valutazione di composti terapeutici e strategie per il trattamento e la prevenzione delle malattie aterotrombotici 11,17,34-37. Qui abbiamo dimostrato le nostre modifiche e perfezionamenti di questo modello e ha mostrato ulteriori pro…
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/ |
References
- Sachs, U. J., Nieswandt, B. In vivo thrombus formation in murine models. Circ Res. 100, 979-991 (2007).
- Libby, P., Lichtman, A. H., Hansson, G. K. Immune effector mechanisms implicated in atherosclerosis: from mice to humans. Immunity. 38, 1092-1104 (2013).
- Ruggeri, Z. M. Platelet adhesion under flow. Microcirculation. 16, 58-83 (2009).
- Watson, S. P. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis. Curr Pharm Des. 15, 1358-1372 (2009).
- Furie, B., Furie, B. C. Thrombus formation in vivo. J Clin Invest. 115, 3355-3362 (2005).
- Li, W., McIntyre, T. M., Silverstein, R. L. Ferric chloride-induced murine carotid arterial injury: A model of redox pathology. Redox Biol. 1, 50-55 (2013).
- Ghosh, A., et al. Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice. J Clin Invest. 118, 1934-1943 (2008).
- Chen, K., et al. Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state. Blood. , (2011).
- Li, W., et al. CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs. J Clin Invest. 120, 3996-4006 (2010).
- Chen, K., Febbraio, M., Li, W., Silverstein, R. L. A specific CD36-dependent signaling pathway is required for platelet activation by oxidized low-density lipoprotein. Circ Res. 102, 1512-1519 (2008).
- Kurz, K. D., Main, B. W., Sandusky, G. E. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res. 60, 269-280 (1990).
- Konstantinides, S., Schafer, K., Thinnes, T., Loskutoff, D. J. Plasminogen activator inhibitor-1 and its cofactor vitronectin stabilize arterial thrombi after vascular injury in mice. Circulation. 103, 576-583 (2001).
- Leadley, R. J., et al. Pharmacodynamic activity and antithrombotic efficacy of RPR120844, a novel inhibitor of coagulation factor Xa. J Cardiovasc Pharmacol. 34, 791-799 (1999).
- Marsh Lyle, E., et al. Assessment of thrombin inhibitor efficacy in a novel rabbit model of simultaneous arterial and venous thrombosis. Thromb Haemost. 79, 656-662 (1998).
- Farrehi, P. M., Ozaki, C. K., Carmeliet, P., Fay, W. P. Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice. Circulation. 97, 1002-1008 (1998).
- Robertson, J. O., Li, W., Silverstein, R. L., Topol, E. J., Smith, J. D. Deficiency of LRP8 in mice is associated with altered platelet function and prolonged time for in vivo thrombosis. Thromb Res. 123, 644-652 (2009).
- Gupta, N., Li, W., Willard, B., Silverstein, R. L., McIntyre, T. M. Proteasome proteolysis supports stimulated platelet function and thrombosis. Arterioscler Thromb Vasc Biol. 34, 160-168 (2014).
- Srikanthan, S., Li, W., Silverstein, R. L., McIntyre, T. M. Exosome poly-ubiquitin inhibits platelet activation, downregulates CD36 and inhibits pro-atherothombotic cellular functions. J Thromb Haemost. 12, 1906-1917 (2014).
- Li, W., et al. Thymidine phosphorylase participates in platelet signaling and promotes thrombosis. Circ Res. 115, 997-1006 (2014).
- Le Menn, R., Bara, L., Samama, M. Ultrastructure of a model of thrombogenesis induced by mechanical injury. J Submicrosc Cytol. 13, 537-549 (1981).
- Li, W., et al. CD36 participates in a signaling pathway that regulates ROS formation in murine VSMCs. J Clin Invest. 120, 3996-4006 (2010).
- Re-examining Acute Eligibility for Thrombolysis Task Force. Review, historical context, and clarifications of the NINDS rt-PA stroke trials exclusion criteria: Part 1: rapidly improving stroke symptoms. Stroke. 44, 2500-2505 (2013).
- Mumaw, M. M., de la Fuente, M., Arachiche, A., Wahl, J. K., Nieman, M. T. Development and characterization of monoclonal antibodies against Protease Activated Receptor 4 (PAR4). Thromb Res. 135, 1165-1171 (2015).
- Mumaw, M. M., de la Fuente, M., Noble, D. N., Nieman, M. T. Targeting the anionic region of human protease-activated receptor 4 inhibits platelet aggregation and thrombosis without interfering with hemostasis. J Thromb Haemost. 12, 1331-1341 (2014).
- Modery-Pawlowski, C. L., Kuo, H. H., Baldwin, W. M., Sen Gupta, A. A platelet-inspired paradigm for nanomedicine targeted to multiple diseases. Nanomedicine (Lond). 8, 1709-1727 (2013).
- Anselmo, A. C., et al. Platelet-like nanoparticles: mimicking shape, flexibility, and surface biology of platelets to target vascular injuries. ACS Nano. 8, 11243-11253 (2014).
- Modery, C. L., et al. Heteromultivalent liposomal nanoconstructs for enhanced targeting and shear-stable binding to active platelets for site-selective vascular drug delivery. Biomaterials. 32, 9504-9514 (2011).
- Woollard, K. J., Sturgeon, S., Chin-Dusting, J. P., Salem, H. H., Jackson, S. P. Erythrocyte hemolysis and hemoglobin oxidation promote ferric chloride-induced vascular injury. J Biol Chem. 284, 13110-13118 (2009).
- Ciciliano, J. C., et al. Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach. Blood. 126, 817-824 (2015).
- Barr, J. D., Chauhan, A. K., Schaeffer, G. V., Hansen, J. K., Motto, D. G. Red blood cells mediate the onset of thrombosis in the ferric chloride murine model. Blood. 121, 3733-3741 (2013).
- Dunne, E., et al. Cadherin 6 has a functional role in platelet aggregation and thrombus formation. Arterioscler Thromb Vasc Biol. 32, 1724-1731 (2012).
- Lockyer, S., et al. GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism. Thromb Res. 118, 371-380 (2006).
- Zhou, J., et al. The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis. J Clin Invest. , (2015).
- Eckly, A., et al. Mechanisms underlying FeCl3-induced arterial thrombosis. J Thromb Haemost. 9, 779-789 (2011).
- Day, S. M., Reeve, J. L., Myers, D. D., Fay, W. P. Murine thrombosis models. Thromb Haemost. 92, 486-494 (2004).
- Cooley, B. C. Murine models of thrombosis. Thromb Res. 129 Suppl 2, S62-S64 (2012).
- Gupta, N., Li, W., McIntyre, T. M. Deubiquitinases Modulate Platelet Proteome Ubiquitination, Aggregation, and Thrombosis. Arterioscler Thromb Vasc Biol. 35, 2657-2666 (2015).
- Konstantinides, S., et al. Distinct antithrombotic consequences of platelet glycoprotein Ibalpha and VI deficiency in a mouse model of arterial thrombosis. J Thromb Haemost. 4, 2014-2021 (2006).
- Versteeg, H. H., Heemskerk, J. W., Levi, M., Reitsma, P. H. New fundamentals in hemostasis. Physiol Rev. 93, 327-358 (2013).
- Yan, S. F., Mackman, N., Kisiel, W., Stern, D. M., Pinsky, D. J. Hypoxia/Hypoxemia-Induced activation of the procoagulant pathways and the pathogenesis of ischemia-associated thrombosis. Arterioscler Thromb Vasc Biol. 19, 2029-2035 (1999).
- Rahaman, S. O., Li, W., Silverstein, R. L. Vav Guanine nucleotide exchange factors regulate atherosclerotic lesion development in mice. Arterioscler Thromb Vasc Biol. 33, 2053-2057 (2013).
- Silverstein, R. L., Li, W., Park, Y. M., Rahaman, S. O. Mechanisms of cell signaling by the scavenger receptor CD36: implications in atherosclerosis and thrombosis. Trans Am Clin Climatol Assoc. 121, 206-220 (2010).
- Liu, J., Li, W., Chen, R., McIntyre, T. M. Circulating biologically active oxidized phospholipids show on-going and increased oxidative stress in older male mice. Redox Biol. 1, 110-114 (2013).
