Figure 1.
FluoroGold (FG) staining delineates the demyelination zone.
Representative fluorescence photomicrographs from a longitudinal section of tibial nerve six days after injection with a lysophosphatidyl choline (LPC)/FG mixture and doubly immunostained for myelin basic protein (MBP; green, C) and β-III tubulin (red, D). Note: FG was taken up by cells in the injection site region and diffused beyond the injection site. There was good register between the most intensely stained region of FG (blue, A, E) and the area of demyelination as defined by the punctate immunostaining for MBP and a lack of the normally uniform linear MBP staining (A, C). The presence of positive linear β-III tubulin IF indicates that axons within the demyelination site persist (A, D). The demyelination site was identified by the joint presence of FG, axonal (β-III tubulin) and paucity of myelin marker (MBP) (boxed areas A, E) in contrast to the robust MBP immunostaining outside the zone of demyelination (asterisks, A). Thus, FG serves to identify the regions of interest (ROIs; similar to that outlined by dashed lines in (C)), where alterations in various markers impacted by the LPC and electrical stimulation were quantified (see Figures 2, 4, 5, 7). Scale bar = 100 µm.
Figure 2.
Increased myelin basic protein (MBP) expression following 1hr ES delivered 5 d post-LPC.
Representative photomicrographs from FG-positive areas on longitudinal sciatic nerve sections processed for MBP immunofluorescence (IF) reveal extensive demyelination 5d post lysophosphatidyl choline (LPC)/FG demyelinating injection (B) as compared to contralateral normal control nerves (A). Temporal analysis of LPC) focal demyelination +/- electrical stimulation (ES) as indicated in days post-LPC: 6d (D), 6d+ES (E), 8d (G), 8d+ES (H), 10d (J) 10d+ES (K) and 12d (M) 12d+ES (N) post-LPC. Note: In the LPC only group, MBP IF was localized in distinct patches consistent with uptake by phagocytosing cells (D, G, J) with faint linear regions similar to periaxonal MBP localization observed by 12 days post-LPC only injection (M). In contrast, in the LPC + ES group faint regions of MBP peri-axonal-like IF were already apparent 8d post-LPC (3d post-ES; H) and consistently stronger in the 10d post-LPC (5d post-ES; K) and 12d post-LPC (7d post-ES; N). Summary bar graphs of relative changes in immunofluorescence signal for MBP in regions of demyelination (C, F, I, L, O). Note: values obtained from individual slides at each time point were normalized to the mean value of the Average Gray per micron2 readings for the nerves ipsilateral to LPC treatment for the LPC only animal on that slide and for that time point. N = 4-6 animals analyzed per condition; regions quantified per condition: 49 ipsi and 38 contra (5d, LPC only); 128 ipsi and 76 contra (6d, LPC only); 81 ipsi and 54 contra (6d LPC+Stim); 83 ipsi and 49 contra (8d, LPC only); 60 ipsi and 58 contra (8d, LPC+Stim); 62 ipsi and 52 contra (10d, LPC only); 54 ipsi and 48 contra (10d, LPC+Stim); 155 ipsi and 73 contra (12d, LPC only); 119 ipsi and 104 contra (12d LPC+Stim). Asterisks indicate significant differences between experimental groups; *P<0.05, **P<0.01, ***P<0.001. Scale bar = 100 µm.
Figure 3.
Accelerated node of Ranvier reorganization in focally demyelinated nerves subjected to 1hr ES.
Representative photomicrographs of FG-positive (i.e focally demyelinated) regions of longitudinal tibial nerve sections dually immunostained for the paranodal protein Caspr (red) and the juxtaparanodal Kv1.2 ion channel (green). Contralateral control nerves display well-organized nodes of Ranvier with Caspr IF in the paranodal region and Kv1.2 IF at the juxtaparanodal region (A, insert reveals nodal staining at higher magnification) and an average of 24.1 visible nodes per field of view as defined by a 1300×900 pixel rectangle superimposed on the photomicrograph (I). A marked loss of nodal organization was observed 5d post-lysophosphatidyl choline (LPC)/FG injection, with an average of 1.5 nodal regions per field of view (B, I). Temporal analysis of LPC +/- ES delivered 5 d post-LPC and indicated in days post-LPC: 8d (C), 8d+ES (D), 10d (E), 10d+ES (F) and 12d (G) 12d+ES (H) post-LPC, revealed that ES delivered 5 d post_LPC resulted in nodal reorganization apparent as early as 8d post-LPC (8d+ES - 3d post-ES; D), with a mean of 13.28 nodes per field of view, as compared to 5.19 in the non-stimulated nerves (8d post-LPC only; C, I). The reorganization continued at 10d post-LPC in the ES nerves, approaching contralateral control nerve levels (10d+ES - 5d post-ES; F), with a mean of 21.53 nodes per field of view in the stimulated nerves (I), compared to 9.96 in the non-stimulated (10d post-LPC; E, I) that did not discernibly change at the 12d post-LPC (12d+ES - 7d post-ES; H and insert). Tissue from the LPC only group displayed modest nodal re-organization (G)12d post-LPC consistent with the appearance of only faint remyelination (see Figure 2M). Scale bar = 100 µm.
Figure 4.
Decrease in number of activated macrophages in demyelinated nerves subjected to 1hr ES.
Representative photomicrographs from FG-positive (i.e focally demyelinated) areas of longitudinal tibial nerve sections processed for ED-1 IF to detect activated macrophages. There was marked macrophage infiltration into the demyelination zone 5d post- lysophosphatidyl choline (LPC)/FG injection (B), while contralateral control nerves displayed only minimal ED-1 IF (A). Temporal analysis of LPC focal demyelination +/- ES delivered at 5 d post-LPC as indicated in days post-LPC: 6d (D), 6d+ES (E), 8d (G), 8d+ES (H), 10d (J), 10d+ES (K), 12d (M) or 12d+ES (N) post-LPC. Note: ES results in a significant decrease in the ED-1 IF 10d post-LPC (5d post-ES; K) and 12d post-LPC (7d post-ES; N) relative to LPC only in a pattern suggestive of movement toward lateral edges and egress (arrow). Summary bar graphs of relative changes in IF signal for ED-1 in the zone of demyelination (C, F, I, L, O). Note: for each time point examined, all values were normalized to the mean value of the Average Gray per micron2 readings for the nerves ipsilateral to LPC treatment for the LPC Only animal on that slide and for that time point. N = 4-6 animals analyzed per condition; regions quantified per condition: 35 ipsi and 36 contra (5d, LPC only); 89 ipsi and 69 contra (6d, LPC only); 52 ipsi and 53 contra (6d, LPC+Stim); 74 ipsi and 50 contra (8d, LPC only); 66 ipsi and 70 contra (8d, LPC+Stim); 79 ipsi and 64 contra (10d, LPC only); 87 ipsi and 61 contra (10d, LPC+Stim); 122 ipsi and 52 contra (12d, LPC only); 98 ipsi and 71 contra (12d, LPC+Stim). Asterisks indicate significant differences between experimental groups; *P<0.05, ***P<0.001. Scale bar = 100 µm.
Figure 5.
Increased neurofilament phosphorylation in demyelinated nerves subjected to ES.
Representative photomicrographs from FG-positive (i.e focally demyelinated) areas of longitudinal tibial nerve sections processed for phosphorylated neurofilament (SMI-31) IF reveal extensive loss of SMI-31 IF (B) as compared to contralateral control nerves which displayed intense SMI-31 immunoreactivity (A). Temporal analysis of LPC focal demyelination +/- ES as indicated in days post-LPC. Note: In the LPC+Stim group there was dramatically increased SMI-31 IF signal apparent as early as 8d post-LPC (3d post-ES; D), which was increasingly stronger in the 10d (5d post-ES; F) and 12d (7d post-ES; H) tissue. In contrast, in the LPC only group there was just a slight increase in SMI-31 IF signal beginning 10d post-LPC. Note: for each time point examined, all values obtained were normalized to the mean value of the Average Gray per micron2 readings for the nerves ipsilateral to LPC treatment for the LPC only animal on that slide and for that time point. N = 4-6 animals analyzed per condition; regions quantified per condition: 45 ipsi and 59 contra (5d, LPC only); 57 ipsi and 82 contra (8d, LPC only); 83 ipsi and 72 contra (8d, LPC+Stim); 72 ipsi and 70 contra (10d, LPC only); 83 ipsi and 72 contra (10d, LPC+Stim); 75 ipsi and 82 contra (12d, LPC only); 50 ipsi and 41 contra (12d, LPC+ES). Scale bar = 100 µm.
Figure 6.
Neurofilament expression increases coincident with reappearance of myelinated axons in demyelinated nerves subjected to ES.
Representative photomicrographs from FluoroGold (FG)-positive (i.e.focally demyelinated) areas of longitudinal tibial nerve sections immunostained for neurofilament proteins (NF – brown/black) then stained for the presence of myelin (Luxol Fast Blue [LFB] - blue) and nuclei (nuclear fast red [NFR] – purple/darkblue). Uninjured (contralateral) control tibial nerves displayed intense uniform LFB staining, indicative of abundant myelin with prominent linear NF immunostaining (A) and elongated dark blue Schwann cell nuclei. Five days post tibial nerve injection of lysophosphatidyl choline (LPC)/FG, extensive NF and myelin loss is observed coupled with infiltration by numerous macrophages filled with largely unprocessed myelin debris (B,C arrow). Temporal analysis of LPC-focally demyelinated nerves +/- ES as indicated in days post-LPC. Note: In the LPC+ES group there was increasing linear NF immunoreactivity detected (F,H) with higher levels of uniform LFB staining consistent with myelinated axons, and the appearance of macrophages devoid of myelin debris apparent as early as 8d post-LPC (3d post-ES; D, arrow). The LFB staining was even stronger in the 10d (5d post-ES) animals where presumptive Schwann cells now display elongated nuclei (F, arrow) and 12d (7d post-ES; H) post-ES tissue. In contrast, in the LPC only group there was just a slight increase in NF immunoreactivity beginning 10d post-LPC (E), but it was much less robust than that observed in the stimulated as well as the contralateral control nerves. Notably in the nonstimulated nerves the immune cell infiltration is still largely unresolved at the 12d post-LPC time point. Scale bar = 5 µm.
Figure 7.
Decreased reactive gliosis following ES delivered 5d post-demyelination.
Representative immunofluorescence photomicrographs from FluoroGold (FG) positive areas of tibial nerve sections processed for glial fibrillary acidic protein (GFAP) immunofluorescence (IF) to detect reactive Schwann cells. Contralateral intact control nerves displayed only minimal GFAP IF (A). However 5d post-lysophosphatidyl choline (LPC)/FG injection there was a marked increase in GFAP IF (B). Temporal analysis of LPC focal demyelination +/- ES as indicated in days post-LPC: 6d (D), 6d+ES (E), 8d (G), 8d+ES (H), 10d (J) 10d+ES or 12d (M) 12d+ES (N) post-LPC. Note: ES resulted in a significant decrease in the GFAP IF signal detected at 12d post-LPC (7 days post-ES; N) relative to 12d LPC only (M). Summary bar graphs of relative changes in immunofluorescence signal for GFAP from sections of tibial nerve bridging the site of demyelination (C, F, I, L, O). N = 4-6 animals analyzed per condition; regions quantified per condition: 49 ipsi and 32 contra (5d, LPC only); 73 ipsi and 64 contra (6d, LPC only); 60 ipsi and 48 contra (6d, LPC+Stim); 84 ipsi and 68 contra (8d, LPC only); 53 ipsi and 60 contra (8d, LPC+Stim); 89 ipsi and 74 contra (10d, LPC only); 86 ipsi and 76 contra (10d, LPC+Stim); 142 ipsi and 74 contra (12d, LPC only); 108 ipsi and 80 contra (12d, LPC+Stim). Asterisks indicate significant differences between experimental groups; *P<0.05, ***P<0.001. Scale bar = 100 µm.
Figure 8.
Increased BDNF protein in demyelination zone following 1 hr ES.
Representative immunofluorescence (IF) photomicrographs from FG-positive focally demyelinated areas of tibial nerve sections immunostained for BDNF. Contralateral control nerves displayed only minimal BDNF IF (A). There was a marked increase in BDNF observed 5d post- lysophosphatidyl choline (LPC)/FG injection (B), largely within cells identified as ED-1 positive macrophages (data not shown). Temporal analysis of LPC focal demyelination +/- ES delivered 5 d post_LPC and as indicated in days post-LPC: 8d (D), 8d+ES (E), 10d (G), 10d+ES (H). Note: ES results in an increase in the BDNF IF signal detected 8 and 10 d post-LPC (3 and 5 days post-ES; E,H) relative to LPC only nerves (D,G). Summary bar graphs of BDNF protein levels measured by ELISA in samples of sciatic nerve bridging the site of demyelination are in agreement with BDNF IF (C, F, I). Asterisks indicate significant differences between experimental groups; **P<0.01. Scale bar = 100 µm.