(E-179) Intravital Microscopy and Endomicroscopy of the Native Thymus

Introduction:: The thymus is a primary lymphoid organ located above the heart and as the site of T cell maturation, the thymus plays a critical role in the functionality of the adaptive immune system^1,2. The thymus is susceptible to damage from acute and chronic insults, such as Sublethal Total Body Irradiation (SL-TBI) normally delivered as part of the preparative regimen for bone marrow transplants^3,4, although the thymus has the capacity to regenerate^5–8. The recovery of the thymus and the role that the thymus vasculature system plays in the thymus regeneration process is therefore of significant interest^5,7,8. Despite this, intravital microscopy techniques which can visualize the thymus in mice in vivo have historically been limited. In vivo microscopy methods have required the thymus to be transplanted to optically accessible organs^9,10, while ex vivo imaging^11,12 and non-mammalian animal models^13,14 may not accurately reflect the endogenous thymus microenvironment. To overcome these limitations, we have developed an adhesion-stabilized two-photon imaging technique¹⁵ and vacuum-stabilization endoscopic technique to study changes to hemodynamics in the thymus after SL-TBI.


Materials and Methods:: UBC-GFP mice were SL-TBI and then imaged one day after irradiation. WT mice were SL-TBI, transplanted with whole bone marrow and then imaged 7/14 days after irradiation. For imaging, all mice were anesthetized with isoflurane and intubated. A thoracotomy through the second intercostal space was performed to expose the thymus. For adhesion-stabilized imaging, an adhesion stabilization holder was inserted through the chest and attached to the thymus via Vetbond. For vacuum-stabilized endomicroscopy, a GRIN probe surrounded by a suction tube is placed directly on top of the thymus. After stabilization, mice were injected with Evans Blue to label the thymus vasculature and provide negative contrast labelling. Various hemodynamic perimeters, such as blood flow, vessel diameter and leakage were quantified in the thymus via Two-photon microscopy. The thymus was then dissected, immersed in PBS and imaged ex vivo. In addition, some thymus lobes were optically cleared with the uDISCO tissue clear protocol. Briefly, UBC-GFP mice were sublethally irradiated and then anesthetized with isoflurane and injected with fluorescent antibodies (Ly-6A/CD144/CD31) to label the vasculature. Mice were intracardially perfused with PBS and PFA and the thymus was dissected. The thymus lobe was then dehydrated with tert-butanol and delipidated with dichloromethane. Finally, the thymus was immersed in BABB-D4 and imaged. Blood vessel diameter, vessel frequency and density were quantified in the tissue cleared thymus.

Results, Conclusions, and Discussions:: We have developed two methods for intravital two-photon microscopy of the thymus, adhesion-stabilized imaging and vacuum-stabilized endomicroscopy. Via adhesion stabilization, we were able to stabilize the thymus to the point of being able to visualize individual cells. In addition, we managed to quantify blood flow in the thymus via negative contrast labelling. While we did not observe a significant change in blood flow one day after SL-TBI, we did observe a significant increase in blood vessel diameter and leakage. Examining the vasculature in the optically cleared thymus shows significant reorganization within one day of irradiation. The vessel frequency and density were both observed to be significantly increased in the thymus. These results support the observation that the vasculature undergoes significant reconfiguration, and that thymus-blood vessel barrier is most likely disrupted within one day of SL-TBI. It remains to be seen how these changes may affect the regeneration of the thymus at later timepoints after irradiation.

Further refinement and characterization of the vacuum-stabilized endoscope will allow for repeat imaging of the thymus at later timepoints. Presently, attempts to image the thymus 7/14 days after SL-TBI via the Adhesion-stabilization method have produced unreliable imaging results, due to the change in size of the thymus. Vacuum-stabilized endomicroscopy should allow for the imaging of the thymus under less ideal conditions.

Our development of intravital imaging techniques capable of visualizing the thymus in mice will allow for novel discoveries related to changes to the thymus vasculature and microenvironment. Using these techniques, we have already managed to characterize significant changes to the thymus hemodynamics following SL-TBI which may disrupt regeneration of the thymus.


Acknowledgements: : We would like to thank Drs. Jennifer Manilay and Katrina Hoyer of UC Merced for their very helpful input, suggestions, and encouragement throughout this work. We would like to thank Mr. Mario Muniz for his help in designing and testing the sample cooler system. We would like to thank Mr. Reagan Chan for his assistance with cleared thymus sample analysis. We would like to thank the staff of the Department of Animal Research Services at UC Merced for their continued assistance with the breeding and maintenance of our animal colony. We would like to thank Drs. Karin Gustafsson and David Scadden of Harvard University and Massachusetts General Hospital for encouraging us to develop intravital imaging of the native thymus. Schematic figures were created with BioRender.com. This work was funded by faculty startup funds provided to JAS by the University of California and through support of the NSF-CREST: Center for Cellular and Biomolecular Machines at the University of California, Merced (NSF-HRD-1547848 and NSF-HRD-2112675).

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