Assessing neurodegeneration throughout the entire brain at a single cell resolution after TBI in mice
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
Traumatic brain injury (TBI) accounts for one third of injury-related deaths and it is estimated that 10 million people, mostly the young (15-25 years old) and elderly (> 75 years old) are affected by TBI annually. Recently, researchers have provided more evidence on post TBI changes that form the basis of a variety of chronic illnesses. These include sleep disorders, epilepsy, neuropsychiatric disorders, dementia, metabolic disorders, musculoskeletal and sexual dysfunctions, suggesting that tissues beyond the lesion might also be affected over time. Yet, there are no effective disease-modifying drugs to treat TBI and its chronic complications. Thus, it is essential to learn how a localized brain injury impacts the rest of the central nervous system (CNS) and the body. Towards this end, in this project we sought to develop technologies that would enable mapping the structural connections of neurons from the injury site to their neuronal cell bodies at a single cell resolution and to reveal farreaching changes in neuronal projections and inflammatory processes following TBI. In the first part of the project , we developed a new whole-body nanobody labeling method, named vDISCO (nanobody(VHH)-boosted 3D imaging of solvent-cleared organs), in conjunction with wholebody tissue clearing. This technology enhanced fluorescent signals more than 100 times and allowed head-to-toe light sheet microscopy scanning of transparent mice (panoptic imaging) and quantification of subcellular details throughout centimeters-thick tissues of intact mouse bodies. Using panoptic imaging, we constructed a neuronal projection map for the Thy1-GFP-M transgenic line, showing subcellular details of long-range neuronal connections from the CNS to the distal extremities. The effects of a localized brain lesion on the rest of the body have been poorly understood, mainly due to the technical challenges in studying long-range neuronal projections. As vDISCO fluorescent signal boosting allows both imaging and quantifications of light-sheet microscopy images of transparent mouse bodies, we sought to investigate TBI-induced changes at the whole CNS and peripheral axonal projections innervating the skeletal musculature of the torso. Imaging the whole CNS, we found extensive neurodegeneration of the descending motor axons in the brainstem and spinal cord upon TBI over the right somatosensory and motor cortice. In intact cleared mouse bodies we demonstrated that the complexity of nerve terminals at the neuromuscular junctions was reduced, especially in the upper torso, compared with unlesioned control. Nerve endings were reduced in complexity at the contralateral body regions, with fewer axonal ramifications, sugesting partial degeneration of axon terminals. In this project we developed a technology (vDISCO) to image entire transparent mouse bodies and constructed a neuronal projection map for the Thy1-GFP-M mouse line. We also identified TBI-induced changes at the CNS and peripheral axonal projections innervating the skeletal musculature of the torso. Altogether, our data contributes to a more holistic understanding of the pathogenesis of TBI and provides a platform to facilitate discovery of hitherto unknown biological and pathophysiological processes.
