Two Stage Intravital Imaging Mouse Model to Assess Venous Thromboembolism in Sickle Cell Disease

Sickle cell disease (SCD) patients have an increased risk of venous thromboembolism (VTE). Population-based studies demonstrated that VTE has a cumulative incidence rate of approximately 25% in adult SCD patients and is associated with higher risk of mortality. VTE in SCD is most commonly manifested as deep vein thrombosis (DVT) with associated pulmonary embolism (PE). Although autopsy studies have regularly discovered pulmonary thromboembolic lesions in SCD patients, the pathophysiology of VTE in SCD remains largely unknown mostly due to the lack of relevant animal VTE model. Understanding the mechanisms that promote VTE in SCD is imperative to identify its prevention and treatment measures.

To elucidate the cellular, molecular and biophysical mechanisms of VTE in SCD we developed an innovative two stage intravital imaging analysis experimental model in SCD mice. DVT in SCD mice was induced by surgical ligation of femoral vein. Venous thrombus formation was visualized using intravital multi-photon-excitation (MPE) microscopy. Venous thrombus took the form of a large mass of elliptical shape which extended in the long-axis direction of the femoral vein. It was composed of fibrin, erythrocytes and sparse platelets. Over time, thrombus was infiltrated by migrating neutrophils. To trigger acute PE, femoral vein ligation was removed and the venous thrombus was observed to spontaneously detach and travel to the SCD mouse lung. This method allowed real time visualization of acute PE in vivo using MPE microscopy of intact lung in live breathing mice. Acute PE involved embolization of the pulmonary arterioles and the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries. The embolization of arteriolar circulation led to loss of blood flow in the arterioles and the down-stream capillaries.

Herein we introduce an intravital microscopy approach to probe VTE in SCD live mouse. Our model has potential application in investigating the molecular determinants of VTE associated with SCD as well as evaluating efficacy of new antithrombotic drugs.