Clinical NeuroscienceFRET based ratiometric Ca2+ imaging to investigate immune-mediated neuronal and axonal damage processes in experimental autoimmune encephalomyelitis

- We report the use of a genetically encoded FRET-based Ca2+ (TN-XXL) sensor for intravital ratiometric Ca2+ imaging in the brainstem of living anesthetized mice.
- Intraaxonal free [Ca2+] in axons is increased under inflammatory conditions, representing a sign of axonal dysfunction, and can be monitored by intravital microscopy.
- Immune-neuronal interaction that leads to neuronal [Ca2+] increases in axons/neurons in inflammatory lesions, which is also a sign of neuronal injury.

BackgroundIrreversible 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 methodWe propose a method to specifically analyze the neurodegenerative aspects of experimental autoimmune encephalomyelitis by Förster Resonance Energy Transfer (FRET) imaging of neuronal and axonal Ca2+ dynamics by two-photon laser scanning microscopy (TPLSM).ResultsUsing 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 methodsIn 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.ConclusionsThus, we describe a method to analyze and monitor early neuronal damage processes in the brain in vivo.