Intravital Two-Photon Microscopy of the Native Thymus

Abstract

The thymus, a key organ involved in the adaptive immune system, is damaged by a variety of insults including cytotoxic preconditioning. This damage can lead to atrophy and potentially to changes in the hemodynamics of the thymic blood vascular system. Although the thymus has an innate ability to regenerate, the production of T cells relies on the trafficking of lymphoid progenitors from the bone marrow through the altered thymic blood vascular system. Our understanding of thymic blood vascular hemodynamics is limited due to technical challenges associated with accessing the native thymus in live mice. To overcome this challenge, we developed an intravital two-photon imaging method to visualize the native thymus in vivo and investigated functional changes to the vascular system following sublethal irradiation. 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. Ex vivo whole organ imaging of optically cleared thymus lobes confirmed a disruption of the thymus vascular structure, resulting in an increase in blood vessel diameter and vessel area, and concurrent thymic shrinkage. This novel two-photon intravital imaging method enables a new paradigm for directly investigating the thymic microenvironment in vivo.

Two-photon endomicroscopy of the native thymus

“…The mice were intubated and placed on a heated pad to maintain body temperature. The surgical operation to expose the thymus was performed as previously described [7]. To conduct the thoracotomy, the skin and pectoral muscle above the second intercostal space was cut back.…”

“…Intravital imaging near the heart and lungs has long proven difficult due to the presence of major blood vessels and significant biomechanical motion [5], [6]. Previously, our lab utilized adhesive-stabilized intravital two-photon microscopy to quantify changes in the thymus vasculature after sublethal irradiation [7]. However, this method was limited, due to the permanent attachment of a support structure to the thymus.…”