A Novel Mouse Model of Acute Sickle Cell Vaso-Occlusion

Background: Sickle cell disease (SCD) is caused by a single-nucleotide mutation in the β globin gene which leads to production of short-lived and very dysfunctional red blood cells (RBC) that cause a wide variety of systemic complications. One of the most severe, vaso-occlusive crisis (VOC), results from microvascular occlusion, causing severe pain and organ damage and is associated with increased mortality. VOC is a complex process that involves endothelial activation and adhesion of RBCs, platelets, and leukocytes to the vessel wall in the microvasculature. Many of the underlying mechanisms of VOC have not been fully elucidated. The most common model to mimic VOC in mice is systemic administration of TNF-α. Although this model has been useful, the vaso-oclusive process take a long time to develop, usually from 2-24 hr of TNF- α injection. Thus, there is a critical need for an animal model of acute SCD-associated VOC for assessing new therapeutic approaches.

Aims: To establish a new model of acute VOC in SCD mice for the purpose of investigating the cellular and molecular mechanisms of VOC.

Methods: Townes SS and control Townes AA mice were used in these experiments. Circulating platelets, leukocytes, and RBCs were labeled with cell-specific fluorescently conjugated antibodies injected intravenously. Blood circulation and blood cell adhesion was monitored in the cremaster microvessels by intravital imaging. VOC in the Townes SS mice was induced by activating the endothelium systemically by intravenous injection of the protease-activated receptor-1 (PAR-1) agonist peptide TRAP-6, (10 mg/kg), which activates the endothelium but not platelets. Mouse platelets, unlike human platelets, do not express PAR-1.

Results: Intravital imaging of the cremaster microcirculation in Townes SS mice at baseline showed a significantly higher number of adherent single platelets and small platelet aggregates on the venular wall than in control mice. Small adherent platelet aggregates were unstable as they frequently embolized downstream. Furthermore, a significantly higher number of rolling and adherent leukocytes were observed in the in Townes SS mice but not in Townes AA control. At baseline, neither Townes SS mice nor control mice demonstrated RBC adhesion to the vascular wall. Within 2 to 3 minutes of the intravenous injection of TRAP-6, we observed a notable increase in platelet, leukocyte, and RBC adhesion to the venular endothelium. The number of adherent cells increased with time, leading to multicellular aggregates that eventually occluded the venules completely after 15 min. Confocal imaging revealed that there were many adherent multicellular aggregates with clearly sickled RBC within them. In contrast, the TRAP-6 challenge in Townes AA mice only resulted in transient increases in platelet and leukocyte adhesion, which resolved quickly without forming aggregates. We did not observe any RBC adhesion to the vessel wall.

Conclusion(s): We have succeeded in establishing a novel model of acute VOC in SCD mice that recapitulates the key pathophysiology of VOC. In this model, we are able to microscopically visualize the three major classes of circulating blood cells through fluorescent labeling. Acute VOC was induced by selective systemic endothelial activation, which allowed us to monitor blood cell-vascular wall interactions and assess the cellular composition of the occlusive thrombi. Importantly, this model allows us to determine the contribution of different cell types to the development of VOC, identify novel therapeutic targets, and assess novel treatments.

No relevant conflicts of interest to declare.