Journal
JOURNAL OF NEUROSCIENCE METHODS
Volume 249, Issue -, Pages 8-15Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jneumeth.2015.04.005
Keywords
EAE/MS; Two-photon laser scanning microscopy; Intravital microscopy; FRET; Ca2+ imaging neurodegeneration
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Funding
- Gemeinnutzige Hertie-Stiftung
- German Research Foundation (DFG) [SFB-TRR128]
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Background: Irreversible axonal and neuronal damage are the correlate of disability in patients suffering from multiple sclerosis (MS). A sustained increase of cytoplasmic free [Ca2+] 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 Forster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca2+ dynamics by two-photon laser scanning microscopy (TPLSM). Results: Using the genetically encoded Ca2+ sensor TN-XXL expressed in neurons and their corresponding axons, we confirm the increase of cytoplasmic free [Ca2+] in axons and neurons of autoimmune inflammatory lesions compared to those in non-inflamed brains. We show that these relative [Ca2+] increases were associated with immune-neuronal interactions. Comparison with existing methods: In contrast to Ca2+-sensitive dyes the use of a genetically encoded Ca2+ sensor allows reliable intraaxonal free [Ca2+] 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. (C) 2015 Elsevier B.V. All rights reserved.
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