Imaging of Myelin by Coherent Anti-Stokes Raman Scattering Microscopy - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 276

objective, spectrally separated from the excitation source by a
dichroic mirror (670dcxr, Chroma Technology Corp.,
Rockingham, VT, USA), transmitted through a bandpass fi lter
(42-7336, Ealing Catalog Inc., Rocklin, CA), and detected by
a photomultiplier tube mounted at the back port of the
microscope.

7. Mechanistic
Study of
Demyelination

The advantages of CARS microscopy allow the mechanistic study
of myelin damage in real time at the resolution of single cell level.
As the fi rst attempt to apply CARS microscopy to the study of
demyelination, the mechanism of a toxin lysophosphatidylcholine
(lyso-PtdCho)-induced demyelination was investigated ( 17 ). By
injecting a lyso-PtdCho solution into the fresh spinal tissue through
a microsyringe, the process of myelin swelling starting from the
outer layer of myelin sheath was recorded. Myelin swelling charac-
terized by the decrease of CARS intensity and loss of excitation
polarization dependence corresponded to myelin vesiculation
observed by electron microscopy. Treating spinal tissues with Ca 2+
ionophore A23187 resulted in the same kind of myelin degrada-
tion as lyso-PtdCho. Moreover, the demyelinating lesion size was
signifi cantly reduced upon pre-incubation of the spinal tissue with
Ca 2+ -free Krebs' solution or a cytosolic phospholipase A 2 (cPLA 2 )
inhibitor or Calpain inhibitor. These results suggest that lyso-
PtdCho induces myelin degradation via Ca 2+ infl ux into myelin and
subsequent activation of cPLA 2 and calpain which break down the
myelin lipids and proteins.
The goal of such study is to reveal the mechanism of myelin
degradation in the demyelinating diseases and also help to design
the therapeutic strategy for the diseases. Glutamate excitotoxicity
is a common pathway in the pathology of neurological disorders.
However, whether and how glutamate infl icts myelin remains
unknown. By CARS imaging of myelin sheath in fresh spinal tissue,
signifi cant paranodal myelin splitting and retraction were observed
following glutamate application both ex vivo and in vivo ( 20 ).
Multimodal NLO imaging further showed that glutamate applica-
tion broke axo-glial junctions and exposed juxtaparanodal K + chan-
nels, resulting in axonal conduction defi cit. Using CARS imaging
as a quantitative readout of nodal length to diameter ratio, a mech-
anism of NMDA and kainate receptor-mediated Ca 2+ overloading
was revealed accounting for high level of glutamate-induced dis-
ruption of paranodal myelin.