The World is Not Flat - Leveraging 3D whole brain imaging and analysis to investigate the pathophysiology of murine Traumatic Brain Injury

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Abstract:

Traumatic brain injury (TBI) induces a myriad of pathological processes resulting in brain-wide damage to cellular activity, axonal connectivity, and vasculature integrity. Despite the fundamental importance of understanding impaired brain activity exhibited in post-traumatic epilepsy and other neurological impairments associated with TBI, knowledge of how brain injury affects neuronal activity remains remarkably incomplete. We developed a whole-brain imaging and analysis approach to identify alterations in neuronal activity after TBI as a complementary method to conventional two-dimensional histological approaches. We established an easy-to-follow experimental pipeline to quantify changes in the whole mouse brain using tissue clearing, light sheet microscopy and an optimised open-access atlas registration workflow.

Using the CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) TBI model, TRAP2 mice were subjected to repeated mild TBI or sham treatment followed by tamoxifen injection to lock c-Fos activity after TBI. Brains were SHIELD fixed and passively cleared for imaging of c-Fos+ cells throughout the rostro-caudal axis of the brain using a light sheet microscope equipped with a specialized whole-brain imaging chamber. Volumetric images were stitched and 3D rendered using Zeiss arivis Pro image analysis software. For quantitative analysis, 2D image stacks were exported to segment c-Fos+ cells and register them to the Allen Mouse Brain Atlas using the BrainQuant3D python package. As a result, c-Fos+ cell counts were estimated throughout the brain and heatmaps were generated. A brain-wide reduction in c-Fos cell density was identified in the TBI group compared to sham controls, indicative of TBI-induced changes in whole brain neuronal activity. Further studies using multi-dimensional imaging of axonal and vascular damage coupled with 3D analysis tools will deepen our understanding of post-TBI brain-wide dynamics.

Read more about this topic in a preprint here.