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  • Title: FRET based ratiometric Ca(2+) imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis.
    Author: Siffrin V, Birkenstock J, Luchtman DW, Gollan R, Baumgart J, Niesner RA, Griesbeck O, Zipp F.
    Journal: J Neurosci Methods; 2015 Jul 15; 249():8-15. PubMed ID: 25864804.
    Abstract:
    BACKGROUND: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca(2+)] is a common upstream event of many neuronal and axonal damage processes and could represent an early and potentially reversible step. NEW METHOD: We propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca(2+) dynamics by two-photon laser scanning microscopy (TPLSM). RESULTS: Using the genetically encoded Ca(2+) sensor TN-XXL expressed in neurons and their corresponding axons, we confirm the increase of cytoplasmic free [Ca(2+)] in axons and neurons of autoimmune inflammatory lesions compared to those in non-inflamed brains. We show that these relative [Ca(2+)] increases were associated with immune-neuronal interactions. COMPARISON WITH EXISTING METHODS: In contrast to Ca(2+)-sensitive dyes the use of a genetically encoded Ca(2+) sensor allows reliable intraaxonal free [Ca(2+)] measurements in living anesthetized mice in health and disease. This method detects early axonal damage processes in contrast to e.g. cell/axon morphology analysis, that rather detects late signs of neurodegeneration. CONCLUSIONS: Thus, we describe a method to analyze and monitor early neuronal damage processes in the brain in vivo.
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