Fig 1.
Experimental setup and thymus holder design.
(A) Experimental schematic of intravital two-photon imaging of the native mouse thymus. (B) Image showing the exposed thymus through the 2nd intercostal space after surgery. Green outline: thymus. (C) 3D model of thymus adhesion stabilization holder (top) and image of 3D-printed prototype (bottom). (D) Image showing adhesion stabilization holder attached to the exposed thymus after surgery. (E) Image showing the Evans blue perfused thymus after intravital imaging, indicating intact blood flow in the thymus at the time of injection. Green outline: thymus.
Fig 2.
Intravital imaging of blood flow in the native thymus.
(A) Representative maximum intensity projection of the untreated thymus from a UBC-GFP mouse in vivo. Red: blood vessels (Evans Blue)/thymus capsule; Green: GFP; Blue: autofluorescence. Scale bars ~ 50 μm. (B) Example average intensity projection of the untreated thymus from a UBC-GFP mouse demonstrating the ability to visualize individual GFP+ cells in the thymus in vivo. Red: blood vessels (Evans Blue); Green: GFP; Blue: autofluorescence. Scale bars ~ 25 μm. (C) Example negative contrast labeled blood flow in the untreated thymus from a UBC-GFP mouse showing the movement of RBCs through blood vessels over time. Red: blood vessels (Evans Blue); Blue Arrow: negative contrast labeled RBC; White Arrow: direction of blood flow. Scale bars ~ 25 μm.
Fig 3.
In Vivo comparison of native thymus vasculature in SL-TBI and untreated mice.
(A) Quantification of thymus area from untreated and SL-TBI mice. (B) Example negative contrast labeled blood flow in the SL-TBI thymus from a UBC-GFP mouse showing the movement of RBCs through blood vessels over time. Red: blood vessels (Evans Blue); Blue Arrows: negative contrast labeled RBCs. Scale bars ~ 25 μm. Quantification of blood flow velocity (C), vessel shear rate (D), vessel diameter (E), and vessel leakage (F) in the thymus of untreated and SL-TBI UBC-GFP mice.
Fig 4.
Changes to the thymus vasculature observed ex vivo.
(A) Representative average intensity projection of the untreated thymus. Grey: blood vessels (labeled with Alexa647 conjugated antibodies against CD31, CD144, and Sca-1); Red Box: cropped FOV. Scale bar ~ 500 μm. (B) Cropped FOV of the untreated thymus. Grey: blood vessels (labeled with Alexa647 conjugated antibodies against CD31, CD144, and Sca-1). Scale bar ~ 100 μm. (C) Representative average intensity projection of the SL-TBI thymus. Grey: blood vessels (labeled with Alexa647 conjugated antibodies against CD31, CD144, and Sca-1); Red Box: cropped FOV. Scale bar ~ 500 μm. (D) Cropped FOV of the SL-TBI thymus. Grey: blood vessels (labeled with Alexa647 conjugated antibodies against CD31, CD144, and Sca-1). Scale bar ~ 100 μm. Quantification of blood vessel diameter (E), blood vessel frequency (F) and blood vessel area (G) in the untreated and SL-TBI thymus ex vivo.