UC Merced

Hematopoietic Cell Transplantation (HCT) is a common treatment for patients suffering from a variety of malignant or benign diseases, reconstituting the hematopoietic system after preconditioning treatment. In patients undergoing HCT, the capacity of the thymus to produce functional T cells is inhibited due to damage from cytotoxic preconditioning. Endogenous thymus regeneration depends on the complex relationship between thymus stromal cells (including vascular endothelial cells (EC)) and the recruitment of de novo “seeding” early thymic progenitors (ETPs) from the regenerated bone marrow (BM). However; functional damage to the vascular network (ECs compartment) may alter the hemodynamics and negatively impact ETP homing and thymus regeneration. Traditionally, flow cytometry, immunohistochemistry, ex vivo imaging, and other molecular biology techniques have been applied to study the thymus in preclinical mouse models since direct visualization of the native thymus in live mice was deemed impossible. In my project, we developed a new method for intravital two-photon microscopy of the native thymus to study functional changes to the vascular system after cytotoxic preconditioning. We hypothesize that cytotoxic preconditioning causes functional and anatomical changes in blood vessel architecture, especially cortical vasculature, that negatively impacts ETP homing and leads to long-term changes in the thymus microenvironment. Using our methodology, we were able to quantify the changes to the blood vessel network after sublethal irradiation (4.5 Gy). We were able to quantify blood flow velocity and shear rate in cortical blood vessels and identified a subtle but significant increase in vessel diameter and barrier function ~24 hrs post-sublethal irradiation. We validated this result using tissue clearing and ex vivo imaging. In addition, most cortical blood velocity is <500 μm/s in both control and one day after sublethal irradiation, although no significant changes were observed in blood velocity and shear rate between the groups at this time point. Taken together, our study suggests that native intravital thymus imaging is a powerful technique enabling functional and anatomical characterization of the thymus vascular network. We believe further work will help clarify the changes to the vascular system at later time points and in the context of higher irradiation doses. This method enables a whole new paradigm for studying thymus biology not previously possible. In the second project, we developed whole organ imaging based on a modified tissue clearing method and investigated the performance based on clearing capability, fluorescence preservation, imaging depth, and size deformation. An optical clearing technique is a powerful tool to reduce light scattering for deep-tissue imaging and enable 3-D imaging of thick tissue samples. We hypothesize that by modifying the temperature and pH of the ultimate 3D imaging of solvent-cleared organs (uDISCO) clearing method, we can improve the retention of GFP fluorescence over time without sacrificing the clearing capability. We developed a modified uDISCO clearing method named a-ucDISCO (alkaline-ultimate chilled DISCO) by adjusting the PH and temperature and performed ex vivo imaging of vascular networks of the murine thymus using two-photon microscopy. Our results revealed a significant increase in GFP fluorescence retention over time compared to the standard uDISCO method. This modified clearing method, therefore, represents an alternative approach for three-dimensional whole-organ imaging of samples with endogenous GFP fluorescence.