How demyelination is initiated is a standing question for pathology of multiple sclerosis. By label-free coherent anti-Stokes Raman scattering (CARS) imaging of myelin lipids, we investigate myelin integrity in the lumbar spinal cord tissue isolated from naïve SJL mice, and from mice at the onset, peak acute, and remission stages of relapsing experimental autoimmune encephalomyelitis (EAE). Progressive demyelinating disease is initially characterized by the retraction of paranodal myelin both at the onset of disease and at the borders of acute demyelinating lesions. Myelin retraction is confirmed by elongated distribution of neurofascin proteins visualized by immunofluorescence. The disruption of paranodal myelin subsequently exposes Kv1.2 channels at the juxtaparanodes and lead to the displacement of Kv1.2 channels to the paranodal and nodal domains. Paranodal myelin is partially restored during disease remission, indicating spontaneous myelin regeneration. These findings suggest that paranodal domain injury precedes formation of internodal demyelinating lesions in relapsing EAE. Our results also demonstrate that CARS microscopy is an effective readout of myelin disease burden.
In vivo imaging of white matter is important for the mechanistic understanding of demyelination and evaluation of remyelination therapies. Although white matter can be visualized by a strong coherent anti-Stokes Raman scattering (CARS) signal from axonal myelin, in vivo repetitive CARS imaging of the spinal cord remains a challenge due to complexities induced by the laminectomy surgery. We present a careful experimental design that enabled longitudinal CARS imaging of de- and remyelination at single axon level in live rats. In vivo CARS imaging of secretory phospholipase A2 induced myelin vesiculation, macrophage uptake of myelin debris, and spontaneous remyelination by Schwann cells are sequentially monitored over a 3 week period. Longitudinal visualization of de- and remyelination at a single axon level provides a novel platform for rational design of therapies aimed at promoting myelin plasticity and repair.
We report in vivo molecular imaging of mouse sciatic nerve by epi-detected coherent anti-Stokes Raman Scattering (E-CARS) microscopy with vibrational selectivity, high signal-to-background ratio, 3D spatial resolution, and real-time imaging capability. The large CARS signal from the CH2 stretch vibration allows highly sensitive and selective imaging of the myelin membrane which possesses a high lipid to protein ratio. The underlying contrast mechanism of in vivo CARS is explored by 3D imaging of fat cells that surround the nerve as well as dermal adipocytes in the mouse ear. Simultaneous E-CARS imaging of myelinated axons and second harmonic generation imaging of the surrounding collagen fibers were performed in vivo without any labeling. Finally, we show that CARS microscopy is able to distinguish between healthy myelin and disintegrated myelin induced by lysolecithin based on decrease in E-CARS intensity as well as loss of dependence on excitation polarization. Our system provides a multimodality in vivo imaging tool for studying neurodegenerative disorders.
Plasmon-resonant gold nanorods have outstanding potential as multifunctional agents for image-guided therapies.
Nanorods have large absorption cross sections at near-infrared (NIR) frequencies, and produce two-photon
luminescence (TPL) when excited by fs-pulsed laser irradiation. The TPL signals can be detected with single-particle
sensitivity, enabling nanorods to be imaged in vivo while passing through blood vessels at subpicomolar concentrations.
Furthermore, cells labeled with nanorods become highly susceptible to photothermal damage when irradiated at
plasmon resonance, often resulting in a dramatic blebbing of the cell membrane. However, the straightforward
application of gold nanorods for cell-specific labeling is obstructed by the presence of CTAB, a cationic surfactant
carried over from nanorod synthesis which also promotes their nonspecific uptake into cells. Careful exchange and
replacement of CTAB can be achieved by introducing oligoethyleneglycol (OEG) units capable of chemisorption onto
nanorod surfaces by in situ dithiocarbamate formation, a novel method of surface functionalization. Nanorods with a
dense coating of methyl-terminated OEG chains are shielded from nonspecific cell uptake, whereas nanorods
functionalized with folate-terminated OEG chains accumulate on the surface of tumor cells overexpressing their cognate
receptor, with subsequent delivery of photoinduced cell damage at low laser fluence.
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