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J Neurosci Methods. 2015 Jul 15;249:8-15. doi: 10.1016/j.jneumeth.2015.04.005. Epub 2015 Apr 10.

FRET based ratiometric Ca(2+) imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis.

Author information

1
Neurology Department, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany. Electronic address: siffrinv@gmx.de.
2
Neurology Department, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany.
3
Central Research Animal Facility, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany.
4
German Rheumatism Research Center, 10117 Berlin, Germany.
5
Max Planck Institute of Neurobiology, 82152 Martinsried, Germany.

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.

KEYWORDS:

Ca(2+) imaging neurodegeneration; EAE/MS; FRET; Intravital microscopy; Two-photon laser scanning microscopy

PMID:
25864804
DOI:
10.1016/j.jneumeth.2015.04.005
[Indexed for MEDLINE]

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