Jernklorid-indusert Murine Trombose Modeller
Summary
Vi rapporterer en raffinert prosedyre av jernklorid (FeCl3) indusert trombose modeller på carotis og mesenteric arterie samt vene, preget effektivt ved hjelp av intravital mikroskopi for å overvåke tid til okklusiv tromber formasjon.
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
Arteriell trombose (blodpropp) er en vanlig komplikasjon ved mange systemiske sykdommer assosiert med kronisk betennelse, inkludert aterosklerose, diabetes, fedme, kreft og kroniske autoimmune revmatologiske lidelser. Tromber som oppstår i den arterielle sirkulasjonen stammer fra upassende plateaktivering, aggregering og påfølgende coagulatory mekanismer, og er innblandet i hjerteinfarkt, slag og ekstremiteten tap. Karveggen er et komplekst system som inneholder flere celletyper og er påvirket av en rekke av ytre faktorer, inkludert skjærspenning, sirkulerende blodceller, hormoner og cytokiner, så vel som ekspresjon av antioksidant proteiner i karveggen. In vitro forsøk kan ikke replikere dette komplekse miljø og derfor in vivo-studier som anvender dyremodeller er kritiske for å gi bedre forståelse av mekanismene som er involvert i trombotiske lidelser.
Mus har vist seg å ha simILAR mekanismer til mennesker i form av trombose, aterosklerose, betennelser og diabetes 1,2. Videre kan transgene mus og knockout opprettes for å teste funksjonen av spesifikke genprodukter i et komplekst fysiologisk eller patologisk miljø. Slike studier etterligne menneskelig patologi og kan gi viktig mekanistisk informasjon knyttet til oppdagelsen av nye stier og terapi, samt gi viktige opplysninger for å karakterisere virkninger av rusmidler på trombose.
Patologiske arterielle tromber oppstå på grunn til endotele lag skade eller dysfunksjon og eksponering av blodstrømmen til subendotel-matriksen 3,4. Ulike trombose modeller har blitt utviklet for å stimulere denne endothelial skader som mekanisk skade, fotoreaktive forbindelse Rose Bengal-baserte oksidativ skade og laser skade fem. I dette spektrum, Ferroklorid (FeCl3) indusert vaskulær skade er en mye brukt modell av trombose. Denne reagensen nårpåført den ytre del av fartøyer induserer oksidativ skade på vaskulære celler 6-8, med tap av endotel celle beskyttelse fra sirkulerende blodplater og komponenter i koagulasjonskaskaden. Det FeCl3-modellen er enkel og følsomme for både antikoagulant og anti-blodplater stoffer, og har vært utført på arteria carotis og femorale arterier, vena jugularis, og mesenterisk og cremasteric arterioler og venuler i mus, rotter, marsvin og kaniner 6-15.
En målbar parameter i denne modellen er den tiden som går fra skade til å fullføre fartøy okklusjon, målt som blodstrømmen opphør med en Doppler strømningsmåler eller under direkte observasjon med intramikros 6,7,9. En rekke ganger mellom 5 til 30 minutter er blitt rapportert i forskjellige studier på C57BL6 mus 7-10,16, noe som tyder på at FeCl3 konsentrasjoner typer anestesi, kirurgiske teknikker, mus alder, genomisk bakgrunn, fremgangsmåte for måling blood flyt, og andre miljøvariabler har betydelige effekter i denne modellen. Denne brede variasjonen gjør det vanskelig å sammenligne studier fra ulike fagmiljøer og kan gjøre deteksjon av små forskjeller vanskelig.
Med en visjon om å minimere slike variabilities og etablere en enhetlig reproduserbar in vivo modell system, har vi videreutviklet FeCl3 indusert halspulsåren modell som gjør dataene svært reproduserbare med minimal variasjon 6-10,16-19. I denne artikkelen beskriver vi og dele kompetanse og rapportere flere representative eksperimentelle eksempler som kan dra nytte av denne modellen.
Protocol
Representative Results
Discussion
Det FeCl3-indusert modell er en av de mest brukte tromboser modeller, som ikke bare kan gi verdifull informasjon om genetiske modifikasjoner på blodplatefunksjon og trombose 7,8,16,19,31-33, men kan også være et verdifullt verktøy for evaluering av terapeutiske forbindelser og strategier for behandling og forebygging av sykdommer aterotrombotiske 11,17,34-37. Her har vi vist våre modifikasjoner og forbedringer av denne modell og viste ytterligere bevis på anvendeligheten av denne t…
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).
