Fig. 4

In vivo two-photon imaging shows differential microvascular remodeling at different cortical depths in the TBI model. A Schematic of 2P structural imaging of blood vessels in the mouse brain in vivo through thin skull preparation. B 2P laser scanning microscopy in conjunction with labeling of the blood plasma with Fluorescein isothiocyanate-dextran (FITC-dextran) was used to form maps of the healthy control angioarchitecture. C Damaged cortical angioarchitecture following TBI at 25 dpi corresponding to the same control region. D Vascular topology at different cortical depths visualized at 25 dpi after focal cortical injury. Left panel is a maximal projection image encompassing 250 µm of cortex that is color-coded for depths (top: red/pink; bottom: blue). The middle and right panel are images from the same brain injured mouse at different cortical depths, with more superficial cortical layers showing increased newly generated microvessels. E Vessel density is quantified from 2P angioarchitectural maps, revealing varying vascular density with depth. At 23 dpi there is an increase in vascular density at more superficial cortical layers (0–100 μm relative to the imaging start position) but appeared similar to pre-injury densities at deeper layers (101–200 µm relative to the imaging start position). The right panel shows averaged vessel density into 100 μm bins further documenting a significant increase at 23 dpi compared to 19 and 39 dpi but not pre-injury values (*p < 0.05, repeated 2-way ANOVA). (Left panel: (n = 3) normalized to the first 20 µm cortical vessel density to facilitate comparisons across time)