In Vivo Two-Photon Excitation Microscopy Reveals Pulmonary Arteriole Micro-Embolism in Sickle Cell Disease Mice
Authors: Bennewitz Margaret F. , Gladwin Mark T , Sundd Prithu 
Published: October 2019

Abstract
Introduction: Sickle cell disease (SCD) affects ~100,000 Americans and millions worldwide. SCD is the most commonly inherited hemolytic disorder that leads to red blood cell (RBC) sickling, hemolysis and presentation of adhesion molecules on sickle RBCs. Chronic hemolysis renders SCD as a hyper-inflammatory and hypercoagulable state, which is characterized by constitutive activation of circulating leukocytes, platelets and endothelial cells, and thrombin generation. Sepsis in SCD patients is a risk factor for acute chest syndrome (ACS), which is a form of lung injury and one of the leading causes of morbidity and mortality among SCD patients. Autopsies of ACS patients reveal the presence of lung infarction and congestion, which supports a role for pulmonary vaso-occlusion in ACS. However, the cellular and molecular events that enable pulmonary vaso-occlusion are not known.
Methods: Intravital two-photon excitation (TPE) video microscopy was used to study myeloid cell trafficking within the pulmonary microcirculation of live BERK SCD or non-sickle control mice. Mice were anesthetized and mechanically ventilated, and a thoracotomy was performed to expose the left lung. The lower zone of the left lung was stabilized using a vacuum-enabled imaging window and observations were made using TPE microscopy. Arterial blood pressure, heart rate, and blood oxygen saturation were continuously monitored. Fluorchrome-conjugated anti-mouse Ly-6G, Ter-119, and CD49b antibodies were administered intravascularly (iv) to label circulating neutrophils, RBCs and platelets, respectively. Cellular trafficking was first recorded at baseline and then at different time points following iv challenge with bacterial lipopolysaccharide (LPS). Vaso-occlusion was defined as cellular aggregation and stasis of blood flow within the lumen of pulmonary capillaries and arterioles.
Results: At baseline, vaso-occlusion was absent and cellular trafficking within the pulmonary microcirculation was comparable between the BERK SCD and control mice. RBC trafficking through pulmonary arterioles and capillaries was rapid. In contrast, neutrophil transit was a combination of fast moving cells that hopped from one capillary junction to the next and slowly transiting neutrophils. Remarkably, our initial findings revealed that iv LPS challenge induced nucleation of neutrophil-platelet-RBC micro-emboli in the pulmonary arteriole of BERK SCD mice, which eventually led to formation of micro-thrombi, complete occlusion of the pulmonary arteriole and death.
Conclusions: Initial findings indicate that neutrophil-platelet-RBC interactions are crucial in initiating pulmonary vaso-occlusion and ACS. Future studies will be aimed at determining the cellular and molecular events responsible for pulmonary arteriolar micro-embolism.
This study is supported by the 11SDG7340005 from the American Heart Association (P.S.) and the VMI startup account (P.S.). M.F.B is supported by the NIH Pulmonary T32 training grant 2T32HL007563-26.

Disclosures
No relevant conflicts of interest to declare.