Intravital Imaging of Pulmonary Neutrophils in Sickle Cell Anemia

Sickle cell disease (SCD) is a genetic disorder usually caused by a homozygous point mutation in the b subunit of hemoglobin. In sickle patients, hemoglobin polymerizes in low oxygen environments, resulting in red cell shape change, increased adhesiveness and fragility. Individuals with SCD experience periodic bouts of painful vaso-occlusive crises (pVOC), generalized damage to most tissues and sometimes life-threatening acute chest syndrome. Although pVOC is triggered by red blood cell sickling, it is associated with an inflammatory response that further reduces blood flow. pVOC is due in part to adhesion of red blood cells to leukocytes and adhesion of red blood cells and leukocytes to the endothelium. Our team has demonstrated that iNKT cells trigger an innate immune response in sickle cell patients. iNKT cells activation is followed by secondary activation of immune cells such as neutrophils and monocytes. We have developed procedures to image neutrophils in the lung, liver and spleen in living mice. In this study we sought to investigate in vivo neutrophil rolling and adherence to endothelial cells that occurs in multiple tissues including the lung during pVOC.

To visualize vascular neutrophils in situ we used a Leica SP5 RS confocal/two-photon microscope. The microscope can be operated to a depth of 50-80 µm with resonant scanning in the confocal mode. Mice are anesthetized by intra-peritoneal injection of a mixture ketamine/xylazine in PBS. Anesthesia is maintained during the experiment by applying half-doses periodically. The depth of the anesthesia is verified by paw pinching before starting the surgical dissection and during imaging. Extra care is taken to minimize inflammation that results from invasiveness of the surgery. Before lung imaging mice are tracheostomized to enable mechanical ventilation and the chest is opened to allow for access to the lung left lobe through a window of few millimeters in diameter. Spleen and liver are accessed by a small incision in the abdomen wall. Organs are kept moist with a drop of PBS. A custom built suction device covered with a No 1.5 cover glass 12 mm in diameter (Electron Microscopy Sciences, Hatfield USA) is placed onto the organ of interest. At the same time, minimal working pressure is exerted to seal the organ and the cover glass together. Throughout the procedure, mice are maintained at 37°C by a heating pad. Once surgery is completed, a mixture of antibodies coupled to fluorophores is given by retro-orbital injection using a U-100 28 1/2 gauge BD insulin syringe. At the end of the experiment, mice are sacrificed by cervical dislocation while still anesthetized.

To induce vaso-occlusion we have developped a hypoxia/reoxygenation protocol with 8% O2 for 12h followed by 4h of reoxygenation. We quantify the number of adherent and rolling neutrophils and their velocity in sickle Townes mice subjected to hypoxia/reoxygenation compared to normoxic Townes mice. By intravital confocal microscopy experiments we were able to detect a higher number of infiltrating neutrophils in the lung in sickle mice versus littermate controls in the normoxic state. Moreover, the infiltration of neutrophils was increased in sickle mice subjected to hypoxia/reoxygenation. Interstingly, the infiltration of neutrophils noted in sickle Townes mice subjected to hypoxia/reoxygenation is abrogated by prior NKT-14 antibody-mediated depletion of iNKT cells whereas mice treated with isotype control Abs were not affected. Our results agree with prior results suggesting that iNKT cell activation occurs prior to neutrophil infiltration in the lung. Furthermore, intravital imaging experiments allowed us to demonstrate that neutrophil infiltration and NKT cells are involved in vaso-occlusives crises in vivo. Intravital microscopy may be helpful for the evaluation of novel treatments for reducing vaso-occlusive events in SCD.