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Two-Photon In Vivo Microscopy

Using two-photon microscopy, we can observe cellls and fine structures in the deep tissue of live animals. In our lab, we have extensive experience in advanced microscopy. We have recently adapted a thinned skull method that allows us to image neurons and other cell types over time in the brain of living mice without creating neural inflammation. Below is a 3D reconstructed image of fluorescently-labeled neurons in the cerebral cortex. The dynamics of the dendrites and synapses can be observed over weeks or even months.

Following brain injury, injured neurites usually cannot regenerate past the lesion. The failure of neural regeneration is at least partially due to the "scar" that forms at the injury site. The scar is formed as a result of a series of cellular and molecular events, which occurs over days to weeks upon injury. The major cell types involved in the scar formation include microglia, macrophages, oligodendrocyte precursors, meningeal cells and astrocytes. Resident micoglia and macrophages from the blood stream are the first cells to arrive at the injury site, within hours of injury. The final structure of the scar mainly consists of astrocytic processes and microglia. Many molecules, such as transforming growth factor b (TGF-b), interleukin 1, and basic fibroblast growth factors, have been implicated as mediators of glial scar formation. Although glial scar may be important for restoring a stable environment for the neurons when injury or local bleeding occurs, it presents a physical barrier to regenerating neurites. Furthermore, many molecules in the scar, including chondroitin sulfate proteoglycans and tenascins, prevent neural regeneration. Despite the important role of microglial cells in various pathological situations such as brain injury, Alzheimer's disease and stroke, there has been no direct observation of microglial accumulation at lesions in spinal cord injury and the relationship between microglial accumulation and neural regeneration remains unclear. Therefore, it is essential to understand the sequence of events leading to scar formation and to prevent or even reverse the detrimental effects of the scar on the regenerating neurites.

Recently, we have achieved major advances in the investigation of the dynamic properties of microglia and the mechanisms underlying their response upon injury in the living animals by taking advantage of transgenic mice in which all microglia are fluorescently labeled by homologous recombination in embryonic stem cells. Using intravital two-photon laser scanning microscopy, the behavior of fluorescent microglia under normal and traumatic injury conditions can be readily imaged in the cerebral cortex of living mice. Using this unique model system, we are now monitoring microglial migration and accumulation at lesions after brain injury and stroke. We have been dedicated to the further determination of the possibility of inducing microglia migration away from glial scar. Our studies can not only provide a first glimpse at microglial response, scar formation, and neural regeneration following brain injury and test treatments aimed at enhancing neural regeneration, but also form a solid basis for future testing of other therapeutic strategies.

  • Chakraborty S, Karmenyan A, Tsai JW, Chiou A (2017) Inhibitory effects of curcumin and cyclocurcumin in 1-methyl-4-phenylpyridinium (MPP+) induced neurotoxicity in differentiated PC12 cells. Sci Rep, 7, 16977.

  • Ma L*, Qiao Q*, Tsai JW*, Yang G, Li W, Gan WB (2016) Experience-dependent plasticity of dendritic spines of layer 2/3 pyramidal neurons in the mouse cortex. Dev Neurobiol, 76, 277-286. (*equal contribution)

  • Qiao Q, Ma L, Li W, Tsai JW, Yang G, Gan WB (2016) Long-term stability of axonal boutons in the mouse barrel cortex. Dev Neurobiol, 76, 252-261.

  • Chakraborty S, Nian FS, Tsai JW, Karmenyan A, Chiou A (2016) Quantification of the metabolic state in cell-model of Parkinson's disease by fluorescence lifetime imaging microscopy. Sci Rep, 6, 19145.

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Last updated 6/13/2013. Copyright© 2013 Jin-Wu Tsai. All rights reserved.