Figure 1.
Real-time CARS imaging of paranodal myelin splitting and retraction induced by glutamate in spinal cord white matter.
(A) CARS images of paranodal myelin after different periods of 1 mM glutamate treatment ex vivo. (B) CARS images of paranodal myelin in the control sample ex vivo without glutamate treatment. Little change was observed in paranodal myelin after 300 min. (C) Schematic nodal length and nodal diameter. (D) Changes of the nodal length-to-diameter ratio with time for the nodes in (A) and (B). (E) Temperature effect on the ratios of nodal length to diameter. * p<0.001 compared with the ‘Ctrl’ group at the corresponding temperature. ** p<0.001 between two indicated groups. (F) Typical CARS image of paranodal myelin in the rat spinal cord after 12 h of in vivo 1 mM glutamate treatment. (G) Typical CARS image of paranodal myelin in the rat spinal cord after 12 h of in vivo saline treatment. (H) With similar nodal diameters, the in vivo glutamate-treated group (Glut) shows larger nodal length than the in vivo saline-treated group (Ctrl). (I) The in vivo glutamate-treated group has 3 times larger ratios of nodal length to nodal diameter than the in vivo saline-treated group. Each column represents the mean ratio of nodal length to nodal diameter measured from one rat. Bar = 10 µm.
Figure 2.
Glutamate application breaks axo-glial junctions, disrupts paranodal myelin and subsequently exposes juxtaparanodal K+ channels.
(A) Time-lapse images showing the leakage of dextran-FITC into the split myelin after glutamate application. The myelin (red) and dextran-FITC (green) were monitored by CARS and TPEF, simultaneously. (B) and (C) CARS images of myelin sheath (red) and confocal fluorescence images of degraded MBP (green) in spinal tissues after incubation in 1 mM glutamate solution (Glut) or normal Krebs' solution (Normal). The curves on the left of the images are intensity profiles of the lines indicated in the images. (D) EM images in a normal tissue show the paranodal myelin held in tight contact with the axolemma. The right panel is the magnified image of the dash frame in the left panel. (E) EM images show that glutamate induces paranodal myelin splitting (arrow), disruption and retraction. The right panel is the magnified image of the dash frame in the left panel. An elongated node, detachment of paranodal myelin from axolemma (arrow head), and disrupted myelin debris (star) were observed. (F)–(H) CARS images of myelin sheath (red) and TPEF images of Kv1.2 channels (green) at the juxtaparanodes after normal Krebs' solution (F, Normal) and application of glutamate (G–H, Glut). Both the exposure of Kv1.2 channels (G) and displacement of Kv1.2 channels into paranodes and node (H) were observed. For (D) and (E), bar = 1 µm. For (A)–(C) and (F)–(H), bar = 10 µm.
Figure 3.
Effect of glutamate (Glut) and 4-AP on CAPs in ex vivo spinal ventral columns.
(A) Histogram showing an irreversible increase in mean peak CAP amplitudes (normalized to 100% of the CAP amplitude (Pre) prior to glutamate treatment) after 1 mM glutamate treatment. (B) Representative CAP recordings before and after glutamate treatment and washing. (C) Histogram showing a decrease in CAP widths at half amplitude after glutamate treatment. (D) Representative CAP recordings showing a width decrease after glutamate treatment. The CAP amplitudes were normalized to the same level of pre-glutamate treatment (Pre) in order to compare CAP widths at half amplitude. (E) Histogram showing an increase in mean peak CAP amplitudes (normalized to 100% of the CAP amplitude (1 mM Glut) after glutamate treatment and washing) after 100 µM 4-AP treatment. (F) Representative CAP recordings before and after 4-AP treatment. (G) Histogram showing an increase in CAP widths at half amplitude after 4-AP treatment. (H) Representative CAP recordings showing a width increase after 4-AP treatment. The CAP amplitudes were normalized to the same level of that before 4-AP treatment (1 mM Glut, after glutamate treatment and washing) in order to compare CAP widths at half amplitude. In all cases, a paired Student's t-test was used to compare measurements between two groups (n = 6).
Figure 4.
Ca2+ influx and calpain activation are involved in glutamate-induced paranodal myelin damage.
(A) Time-lapse CARS images of paranodal myelin after different periods of treatment with 250 µg/mL Ca2+ ionophore A23187. Bar = 10 µm. (B) Increase of the nodal length-to-diameter ratio with time for the node in (A). (C) Statistical analysis of ratios of nodal length to nodal diameter under five different conditions. ‘Ctrl’ represents samples incubated in Krebs' solution. ‘Ca2+-free’ represents samples incubated in Ca2+-free Krebs' solution. ‘Ca2+-free + Glut’ represents samples exposed to 1 mM glutamate in Ca2+-free Krebs' solution. ‘Glut’ represents samples exposed to 1 mM glutamate in normal Krebs' solution. ‘Calpain inhibitor’ represents samples exposed to calpain inhibitor III MDL 28170 and glutamate together. Five groups of samples were exposed to the different conditions for 5 h. * p<0.001 between the ‘Glut’ group and the ‘Ctrl’ group, and between the ‘Calpain inhibitor’ group and the ‘Glut’ group.
Figure 5.
Glutamate excitotoxicity in paranodal myelin is mediated by kainate and NMDA receptors.
(A) CARS images of paranodal myelin under NMDA (0.68 mM plus 27 µM glycine) treatment at different time periods. 27 µM glycine was used to maximize activation of NMDA receptors. (B) CARS images of paranodal myelin under kainate (1 mM) treatment at different time periods. (C) CARS images of paranodal myelin under AMPA (0.16 mM) treatment at different time periods. (D) NMDA, kainate, but not AMPA induced paranodal myelin retraction. Black bars represent the average ratios of nodal length to diameter under various treatments. White bars with patterns represent the percentages of paranodal myelin damage under various treatments. * p<0.001 compared with the control. (E) Glutamate-induced paranodal myelin retraction is partially diminished by antagonists of NMDA, kainate, but not AMPA receptor. MK-801 (a noncompetitive NMDA antagonist), GYKI52466 (a selective AMPA antagonist) or NS-102 (a selective kainate antagonist) were used. The spinal tissues were pre-incubated in the Krebs' solution supplemented with a specific glutamate receptor antagonist at 37°C for 0.5 h. Glutamate (1 mM) was then added into the incubating solution. After additional 1 h incubation, the nodal lengths and nodal diameters in the spinal tissue were measured by CARS. * p<0.001 compared with the ‘Glut’ group. Bar = 10 µm in (A–C).
Figure 6.
Paranodal myelin retraction precedes axonal injury.
(A) Simultaneously acquired CARS image of myelin sheath (red) and TPEF image of a calcium indicator, Oregon green 488 AM (green), including a node of Ranvier during glutamate treatment. The Ca2+ influx observed at 240 min was preceded by paranodal myelin splitting and retraction, which began at 60 min of glutamate treatment. Bar = 10 µm. (B) Ratios of nodal length to diameter for the node in (A) and intra-axonal TPEF signals recorded at different time points. FCa2+ represents the TPEF intensity from Ca2+ indicator Oregon green 488 AM. F0 is the background intensity. (C) The TPEF signal of Oregon green 488 AM at different positions marked in (A, 0 min) varied after glutamate application for 0 min (black), 240 min (red), 325 min (green) and 460 min (blue), respectively. The signal elevation trends at the four positions implicate that Ca2+ conceivably flew into axons via the exposed paranodes and juxtaparanodes and then spread into the internodal area.