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1.
Fig. 1

Fig. 1. From: Coherent Anti-Stokes Raman Scattering Imaging of Myelin Degradation Reveals a Calcium-Dependent Pathway in Lyso-PtdCho-Induced Demyelination.

Illustrations of live spinal tissue preparation, injection of lyso-PtdCho into a spinal tissue, and schematic of our CARS microscope. PDMS, polydimethylsiloxane; D, dichroic mirror.

Yan Fu, et al. J Neurosci Res. ;85(13):2870-2881.
2.
Fig. 3

Fig. 3. From: Coherent Anti-Stokes Raman Scattering Imaging of Myelin Degradation Reveals a Calcium-Dependent Pathway in Lyso-PtdCho-Induced Demyelination.

Real-time CARS imaging of myelin degradation. A: Time-lapse CARS images of myelin swelling in the spinal tissue incubated with a Krebs’ solution containing 10 mg/ml lyso-PtdCho. B: Diagram of measuring the g ratio of a partially swollen myelin fiber based on the remaining compact region. C: The increase of g ratio during the process of myelin swelling. Scale bar = 10 μm.

Yan Fu, et al. J Neurosci Res. ;85(13):2870-2881.
3.
Fig. 5

Fig. 5. From: Coherent Anti-Stokes Raman Scattering Imaging of Myelin Degradation Reveals a Calcium-Dependent Pathway in Lyso-PtdCho-Induced Demyelination.

CARS imaging of Ca2+ ionophore induced myelin degradation. A,B: CARS images of swollen myelin sheath with horizontal (horizontal arrows; A) and vertical (vertical arrows; B) excitation polarization, respectively. The myelin swelling was induced by injecting 10 μl of 20 μg/ml Ca2+ ionophore A23187 into the spinal tissue. The images were taken 6 hr after the injection. C,D: Real-time imaging of myelin degradation induced by incubation with 500 μg/ml Ca2+ ionophore at 5 min (A) and 315 min (B). Scale bar = 10 μm.

Yan Fu, et al. J Neurosci Res. ;85(13):2870-2881.
4.
Fig. 4

Fig. 4. From: Coherent Anti-Stokes Raman Scattering Imaging of Myelin Degradation Reveals a Calcium-Dependent Pathway in Lyso-PtdCho-Induced Demyelination.

In vivo epidetected CARS imaging of lyso-PtdCho-induced myelin degradation in a mouse sciatic nerve. A: Image of partially swollen myelin. Intensity profile along the red line in A is shown below the image. The PMT signal from the compact myelin region (flat peaks) had to be saturated to allow for clear visualization of the swollen region displayed as shoulders on the sides of the flat peaks. B,C: Swollen myelin sheath imaged with vertically and horizontally polarized excitation, respectively. Loss of myelin lamellar structure resulted in an excitation polarization-independent CARS signal as shown in the intensity profiles for the swollen myelin surrounding axon “1” in B and C. Scale bar = 5 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Yan Fu, et al. J Neurosci Res. ;85(13):2870-2881.
5.
Fig. 2

Fig. 2. From: Coherent Anti-Stokes Raman Scattering Imaging of Myelin Degradation Reveals a Calcium-Dependent Pathway in Lyso-PtdCho-Induced Demyelination.

Characterization of lyso-PtdCho-induced myelin swelling by CARS microscopy and EM. The laser beams were focused into the equatorial plane of axons. A: CARS image of normal myelin sheath wrapping two parallel axons acquired at a speed of 1.13 sec/frame. The same speed was used for other images. B: CARS image of partially swollen myelin sheath acquired at 5 min after injecting 2 μl of 10 mg/ml lyso-PtdCho into the tissue. C: CARS intensity profiles of normal and swollen myelin fibers. Green: taken along the green line in A. Red: taken along the red line in B. Note the decrease of CARS intensity in the swollen region. D: CARS images of normal myelin sheath with vertical (vertical arrows) and horizontal (horizontal arrows) excitation polarization. E: CARS images of totally swollen myelin sheath with vertical (vertical arrows) and horizontal (horizontal arrows) excitation polarization. F: The ordering degree characterized by I||/I of intramyelin lipids for normal and swollen myelin sheath. The CARS intensity from the swollen myelin showed no dependence on the excitation polarization. G,H: TEM images of normal myelin (G, × 30,000) and degraded myelin induced by incubating the spinal tissue with 10 mg/ml lyso-PtdCho for 90 min (H, × 6,300). In H, green stars indicate myelin vesiculation and red arrows indicate myelin splitting. Scale bars = 10 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Yan Fu, et al. J Neurosci Res. ;85(13):2870-2881.
6.
Fig. 6

Fig. 6. From: Coherent Anti-Stokes Raman Scattering Imaging of Myelin Degradation Reveals a Calcium-Dependent Pathway in Lyso-PtdCho-Induced Demyelination.

Lyso-PtdCho-induced demyelination is Ca2+, cPLA2, and calpain dependent. LPC represents lyso-PtdCho. A: The sizes of de-myelination lesion induced by lyso-PtdCho under different conditions. The inset illustrates a spinal cord sample with the lesion size defined by the longitudinal and transverse lengths of myelin swelling. The lesion size was measured by CARS microscopy at 30 min after injecting 10 μl of 10 mg/ml lyso-PtdCho into the tissues preincubated with indicated chemicals. The histogram data are presented as mean ± SD of three independent measurements. Statistical differences were calculated by ANOVA with Turkey’s test. **P < 0.01 compared with longitudinal dimension in normal Krebs’ solution. B,C: Effects of Ca2+ removal and cPLA2 and calpain inhibition on lyso-PtdCho-induced CAP reduction of spinal cord ventral white matter. B shows representative CAP tracings of the control spinal tissue under different conditions. C shows the statistical analysis of 10 independent measurements. Lyso-PtdCho in normal Krebs’ solution induced ~80% reduction of the mean CAP amplitude but only ~40% reduction in Ca2+-free Krebs’ solution or with MAFP and 51% reduction with MDL 28170. **P < 0.01 compared with the CAP reduction by lyso-PtdCho in normal Krebs’ solution.

Yan Fu, et al. J Neurosci Res. ;85(13):2870-2881.

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