6533b86dfe1ef96bd12cabc8

RESEARCH PRODUCT

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

subject

description

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 [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.