Name: Inferior Vena Cava (IVC) Ligation for Venous Thrombosis Analysis in Mouse
Uploaded: 2024-01-05T13:30:00
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inferior vena cava (ivc) ligation for venous thrombosis analysis in mouse

Optimerad analys av venös trombos i en muscancermodell med hjälp av vaskulära klämmor

Cancer-associated venous thromboembolism (VTE) 
Venous thromboembolism (VTE) risk is 4 to 7 times higher in cancer survivors compared to the general population1,2,3. This condition proves fatal in one out of seven patients with cancer. The incidence of VTE varies depending on the type of cancer and the tumor burden and is highest among patients with pancreatic and gastric cancers4.
Cancer-associated VTE in cancer patients has prognostic significance. It is associated with unfavorable overall survival in the first year after a cancer diagnosis, even after adjusting for age, race, and stage of underlying cancer5. These findings highlight the importance of examining cancer associated VTE and the need to probe its mechanism using an animal model. The translational relevance of this area is further emphasized by the fact that VTE in cancer patients is preventable and treatable with thromboprophylaxis and antithrombotic therapy6.
Animal models of cancer and venous thrombosis 
Cancer models are conventionally termed xenografts, which entail the injection of cancer cells in mice. The injection of cancer cells at a site like its origin is referred to as an orthotopic model, while at a different site (subcutaneous plane over the flank) is known as a heterotopic model. The species of origin of cancer cells determines them as an allogeneic model, such as the HT-29 cell line (human colon cancer)7,8,9. On the contrary, syngeneic models use the murine cancer cell lines, including RenCa and MC-38 cell lines3,10.
The literature has described arterial, venous, and capillary thrombosis models in rodents. Venous thrombosis is induced in the inferior vena cava (IVC) by mechanical injury (guide wire) or complete IVC ligation, chemical (Ferric chloride), or electrolytic injury. Ferric chloride-induced thrombosis or IVC ligation represents complete occlusion models. The latter results in the stasis of blood and inflammatory infiltrates in veins11,12,13. The complete ligation model results in a high rate of thrombosis formation in 95% to 100% of mice. The partial IVC ligation model might include interruption of lateral iliolumbar branches, and the venous return is abrogated by applying suture ligations in the distal target points of IVC12. Sometimes, a space holder is used to interrupt the venous return partially. However, the thrombus weight is inconsistent in the current partial occlusion model, resulting in high variability in clot weights and heights12,14.
Both these large vein mechanical models (partial and complete) have limitations. First, IVC ligation (stasis model) often results in hypotension. The blood gets shunted through vertebral veins. Though in experienced hands, the mortality with this model ranges from 5%-30%, with the higher rate expected during the learning curve. Importantly, the complete occlusion model does not reproduce deep vein thrombosis (DVT) in humans, where a thrombus typically is nonocclusive. Complete occlusion is likely to alter hemorheological factors and pharmacodynamic parameters, altering the bioavailability of compounds at the local site. Due to these limitations, complete occlusion models may not be optimal for testing novel chemical compounds for therapeutic purposes and drug discoveries12.
It should be noted that to provide a more clinically relevant murine model of venous thrombosis with decreased flow with endothelial damage, a venous thrombosis model has been introduced, where DVT is triggered by the restriction of blood flow in the absence of endothelial disruption. The model was validated by scanning electron microscopy15. A preferred clinically relevant thrombosis model is one with near complete thrombosis that enables drug discoveries. The clot formation in the current partial occlusion models is inconsistent, resulting in high variability in the clot weight and heights12,16. Furthermore, the clot weight is variable with the conventional methods, requiring&#160;more&#160;mice per studies12.
Previous cancer-associated thrombosis models focused on colon, pancreatic, and lung cancer and were all complete occlusion models17,18,19. This manuscript modifies the partial occlusion thrombosis model to provide clots with lower variability and mouse mortality (Figure 1). Former studies used allogeneic cancer cell lines on immunocompromised athymic mice background19,20,21. This manuscript uses an MC-38 cell syngeneic xenograft in C57Bl6/J mice, which allows the use of immunocompetent mice and examination of immune components to thrombogenesis.

Författare i fokus: Främjar cancerassocierad trombosforskning i gnagarmodeller 

Venös trombosanalys i en musmodell av cancer

This research aims to enhance the rodents model for venous thrombosis focusing on cancer-associated thrombosis. It introduces a refined surgical technique using vascular clips in a syngeneic colon cancer xenograft mouse model to improve consistency and reproducibility, advancing cancer-associated thrombosis mechanistic understanding and drug discovery. Although the flow reduction model was introduced in the IVT stenotic models in 2012, no significant progress has been made in cancer-associated thrombosis models in the last 10 years.This was the rationale for this endeavor. In the field of cancer-associated thrombosis research, current technologies include advanced imaging techniques, like scanning, electron microscopic images, molecular biology tools, such as immunofluorescent staining, precision surgical instrument, data analysis software and data interpretation, and animal welfare. The current experimental challenges include variability in clot weights, a high mortality rate, and a prolonged learning curve in rodent models of venous thrombosis.Additionally, the venous flow direction and clot formation mechanisms differ from clinical cases. Our research protocol bridges gaps in rodent models of cancer-associated thrombosis. It introduces a novel surgical technique using vascular clips in xenograft models, yielding more consistent and reproducible thrombosis outcomes.This enhances our understanding of cancer-related thrombosis and drug discovery in translational research.

Watch this Scientific Journal Video about Inferior Vena Cava IVC Ligation for Venous Thrombosis Analysis in Mouse at JoVE.com

Inferior Vena Cava (IVC) Ligation for Venous Thrombosis Analysis in Mouse  

Inferior Vena Cava (IVC) ligering för venös trombosanalys hos mus

Börja med att använda en fin sax för att göra ett snitt i bukhuden på en sövd mus, med början från den xiphoida eminensen till urinblåsan. Med en atraumatisk pincett nyper du ihop bukhinnan halvvägs längs hudsnittet och ser till att det finns ett tillräckligt mellanrum mellan snittet och de överliggande tarmarna. Öppna sedan bukhinnan i längdriktningen längs avascular linea alba.Använd sedan våta bomullsspetsar för att försiktigt dra ut tarmarna. Droppa sedan 200 till 300 mikroliter normal saltlösning över tarmarna och utforska vägen för urinledarna inferior vena cava, eller IVC, aorta och njurvener. Kauterisera de bilaterala grenarna distalt om sammanflödet av vänster njurven och infrarenal inferior vena cava.Kontrollera urinledarna och kärlen för att se om det sipprar ut. För att förhindra potentiella komplikationer av spinal ischemi och claudicatio ska ländryggsgrenarna lämnas intakta. Kontrollera sidogrenarna, inklusive det bilaterala livmoderhornet och hypogastriska kärl.Använd sedan en atraumatisk pincett med stängd spets för att försiktigt passera en polypropensutur med sex nollor genom det avaskulära planet två till tre gånger. Applicera ett lätt tryck med en bomullsspets för att kontrollera eventuell vätska. Slutligen, passera en fem-noll polypropensutur genom planet med mjuka kuddle till kraniala rörelser för att vidga den.Placera kärlklämmorna över suturen för att garantera tillräckligt med utrymme. Blodproppsvikterna i den xenotransplanterade experimentgruppen visade en 1,5-faldig ökning jämfört med kontrollmössen. Immunofluorescensanalyser visade att IVC från kontrollgruppen hade intakt CD31 och fibrin som delvis upptog kärllumen.CD31-uttrycket var inte intakt i experimentgruppen, med väggen som visade fibrinfärgning. Kontrollgruppen hade signifikant lägre fibrinuttryck jämfört med experimentgruppen. Ultraljudsanalys av mössen visade att den ligerade infrarenala hålvenen hos en kontrollgruppsmus hade en partiell koagel.Experimentgruppen hade dock en stor propp som nästan täppte till infrarenal vena cava.

Research

JoVE Journal

Cancer Research

Inferior Vena Cava (IVC) Ligation for Venous Thrombosis Analysis in Mouse

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...