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


  • V. Siffrin
  • J. Birkenstock
  • D.W. Luchtmann
  • R. Gollan
  • J. Baumgart
  • R.A. Niesner
  • O. Griesbeck
  • F. Zipp


  • Journal of Neuroscience Methods


  • J Neurosci Methods 249: 8-15


  • 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.