Prolene

This model provides a total stasis environment and results in the most severe vein wall reaction to thrombosis of the models discussed.^{8 (link),17 (link),20 (link),23} Studies in rats suggest that after IVC ligation a combination of stasis-induced vein wall injury and enhanced tissue factor expression in endothelial cells and leukocytes produce thrombosis.^{42 (link)} In this model, mice are anesthetized and a midline laparotomy is performed. The small bowel is exteriorized and placed on a moistened gauze pad to the animal’s left. The infrarenal IVC is identified and all side branches are ligated with nonreactive 7-0 Prolene suture. Posterior venous branches are cauterized.²³ A 7-0 Prolene suture is tied down on the IVC, caudal to the left renal vein. This model has been widely used by our group for the study of venous thrombosis.^{8 (link),17 (link),20 (link),21 (link)} It provides reproducible thrombus weights beginning at 3 hours and extending to 21 days, for most mouse strains. It has proven valuable in the study of interactions between the vein wall and thrombus during the progression from acute to chronic inflammation and remodeling of the vein wall. Disadvantages include the lack of blood flow. A technical pitfall unique to this procedure is the potential to induce initial hypotension. However, compensation by vertebral veins is observed and the survival rate for this model is around 95%, based on our laboratory’s observations. In addition, the IVC cannot reopen because of the ligature. This model cannot reproduce the clinical scenario where a thrombus is nonocclusive, but it can mimic complete occlusion (Figure 2 and Table). As a guideline, data from our laboratories in C57BL/6 mice shows, approximate thrombus weights (IVC+thrombus at harvest), of 33 mg at day 2, 29 mg at day 6, and 18 mg at day 14.

Female severe combined immunodeficient (SCID) mice 2 months of age (Frederick Animal Production Program, Frederick, MD) were allowed to acclimate for 4 weeks before use. Strict adherence to the UAB Institutional Animal Care and Use Committee (IACUC) approved protocol was maintained throughout the study. Orthotopic cell implantation followed the procedure in DeRosier et al. (21 (link)). Briefly, mice were anesthetized with i.p. xylazine and ketamine. A 1-cm incision was made in the left upper abdominal quadrant in a sterile hood. The spleen was located and used to indirectly position the tail of the pancreas, carefully avoiding direct pancreatic manipulation. Mice (8/group) received S2VP10L, S2VP10L-CP, S2VP10L-KFMΔSS1, or S2VP10L-KISS1 cells. The cellular suspension was stored on ice in a sterile tube and then drawn up using a 28-gauge needle to aliquot 1 × 10⁵ cells/40 μl which was injected into the tail of the pancreas. A sterile cotton tipped applicator was used to cover the injection site for 30 s to prevent peritoneal leakage. The organs were returned to the abdomen and the skin and peritoneum were closed in a single layer closure with 5-0 Prolene sutures. Animals recovered on a warming blanket and received liquid acetaminophen for 24 h with food and water ad libitum.

After the induction of anesthesia with tribromoethanol (0.25 mg/g IP), mice were intubated and supported with a MiniVent Mouse Ventilator (Harvard Apparatus) and anesthesia was maintained with 1% isoflurane. Under sterile conditions, the heart was exposed via a left thoracotomy in the 4^th intercostal space. An 8.0 prolene ligature was passed and tied around the proximal left coronary artery (HF group). In sham animals, the suture was passed but not tied. The chest was then closed using 5.0 silk. The total mice used were: C57BL/6 n = 90; TNFR1−/− n = 46; TNFR2−/− n = 39. Mice were followed for 4 weeks following operation. All TNFR1−/− and TNFR2−/− ligated mice and ∼50% WT ligated mice with premature death underwent autopsy to assess for blood in the chest cavity as an indicator of LV rupture.