CLARITY for High-resolution Imaging and Quantification of Vasculature in the Whole Mouse Brain

Abstract

Elucidating the normal structure and distribution of cerebral vascular system is fundamental for understanding its function. However, studies on visualization and whole-brain quantification of vasculature with cellular resolution are limited. Here, we explored the structure of vasculature at the whole-brain level using the newly developed CLARITY technique. Adult male C57BL/6J mice undergoing transient middle cerebral artery occlusion and Tie2-RFP transgenic mice were used. Whole mouse brains were extracted for CLARITY processing. Immunostaining was performed to label vessels. Customized MATLAB code was used for image processing and quantification. Three-dimensional images were visualized using the Vaa3D software. Our results showed that whole mouse brain became transparent using the CLARITY method. Three-dimensional imaging and visualization of vasculature were achieved at the whole-brain level with a 1-μm voxel resolution. The quantitative results showed that the fractional vascular volume was 0.018 ± 0.004 mm3 per mm3, the normalized vascular length was 0.44 ± 0.04 m per mm3, and the mean diameter of the microvessels was 4.25 ± 0.08 μm. Furthermore, a decrease in the fractional vascular volume and a decrease in the normalized vascular length were found in the penumbra of ischemic mice compared to controls (p < 0.05). In conclusion, CLARITY provides a novel approach for mapping vasculature in the whole mouse brain at cellular resolution. CLARITY-optimized algorithms facilitate the assessment of structural change in vasculature after brain injury.

Microglia exacerbate white matter injury via complement C3/C3aR pathway after hypoperfusion

“…Rat brain clearing procedures were performed according to an optimized CLARITY protocol 34, 35. For immunostaining, the 500-μm-thick brain slices were incubated with Rat anti-MBP (Abcam, ab7349, 1:200) and rabbit anti-Iba-1 primary antibodies (Wako, 019-19741, 1:200) for 3 days at 37 °C with shaking.…”

Automated analysis of whole brain vasculature using machine learning

“…Importantly, our findings on vasculature length are in line with the predictions in the literature obtained in small volumes, confirming the robustness of our method. For example, previous studies quantifying small patches estimated the density of cortical blood vessels as 0.922 m/mm³, 0.444 m/mm³ or 0.471 m/mm³ in the cortex 12,17,37 . Using VesSAP, calculating centerline density as described above, and accounting for the 30 % isotropic tissue shrinkage in DISCO clearing 38 , we found an average vascular density of 0.473 ± 0.161 m/mm³ over the whole mouse cortex.…”

“…However, such methods including local spatial regularization cannot segment large vascular networks across changing intensity distributions. Finally, the size of the acquired datasets poses a difficulty to assess the organization of the whole vascular network; therefore, such methods can only segment small volumes [15][16][17][18][19] .…”

On the existence of mechanoreceptors within the neurovascular unit of the rodent and rabbit brain

“…4a-d), olfactory bulb (not shown), and basal ganglia ( Fig. 1), where long arteries follow a straight trajectory (Zhang et al 2018). Elsewhere, in the cervical spinal cord (Online resource, Fig.…”