Studying all the supraspinal projecting populations after spinal cord injury has been difficult because sectioning of tissue and analysis of 2D images of a whole brain is time consuming and laborious. We developed methods that can image the whole mouse brain without sectioning using tissue clearing methods and light sheet microscopy. The present study investigates, in more detail, most of the projecting neurons and mainly quantification using enhanced 3D imagining of most areas including those that been historically overlooked in the spinal cord injury field. This study incorporates newer versions of fluorescent proteins such, as mScarlet and mGreenLanternallow for greater and more precise detection in critical supraspinal areas such as the brainstem. Using 3D software such as Imaris allows us for more accurate, uniform, and overall accessible quantification and labelling of these areas. These advancements in detection and examination allow for contribution and generation of databases. This is especially important regarding closing the gap in dissimilarities and inconsistencies in literature and provide and educational resource for those lacking the expert knowledge for studying these brain areas. Furthermore, this type of approach will provide essential information and tools for those studying regenerative therapies.
