Fig 1: ER stress responses after sciatic nerve crush.Wild-type mice were crush injured in the right sciatic nerve and sham operated in the left sciatic nerve. After 8 days, nerves were removed for histological analysis. (A) KDEL staining (red) was performed using indirect immunofluorescence and co-stained with S100 (Schwann cells), NF-M (axons), MBP (myelin), and a Cd11b (macrophages) in green. Cell nuclei were counter stained using DAPI (blue). Co-localization is denoted using white arrows and S100 negative KDEL positive cells with asterisk. Scale bar: 20 µm. (B) DRGs and were collected from animals described in A and KDEL staining (red) was performed together with the neuronal marker NF-M (green). Scale bars: 100 µm. (C) Thoracic spinal cord from animals described in A was analyzed for KDEL staining (red). Scale bars: 100 µm (low magnification) and 40 µm (high magnification).
Fig 2: Methylcobalamin accelerates the myelination of DRGs/SCs in coculture. We used cocultures of DRGs and SCs to evaluate the differentiation of axon-related SCs in the presence or absence of 100 μM MeCbl. Cocultures were visualized with an anti-MBP antibody red (A,B,G,H,M,N) and an anti-NF200 antibody green (C,D,I,J,O,P) and merged pictures (E,F,K,L,Q,R) were shown at 7 (A–F), 14 (G–L), and 21 days (M–R) days after the induction of differentiation. (S) The extent of myelination was quantified by measuring the number of MBP-positive segments. Values are means ± SEM of five independent experiments. *p < 0.05, **p < 0.01 compared with the control group.
Fig 3: Differentiated cells from IC. NSCs after 6 days on glass coverslips in differentiation medium (DIF). (a) ß-III-Tubulin (red) marked neuronal differentiated cells. The ß-III-tubulin-positive cells had neuron-typical spindled, slender somata with bipolar axon configuration. (b) Astrocytes were identified by the staining of glial fibrillary acidic protein (GFAP, red). These cells showed stellate-configured branches, which is typical for astrocytes. (c) Oligodendrocytes showed positive labelling of myelin basic protein (MBP, red). Typically for oligodendrocytes, they had a peripheral-starting myelination, which is characterized by MBP staining. The proximal portions are ß-tubulin-positive (green). The extensive, diversified growth of the branches is a morphological sign for oligodendrocytes. (d) Cells that were still in the undifferentiated progenitor cell stage were stained by Nestin (red). These cells had a very similar morphology to that of the GFAP-positive, astrocyte-differentiated cells. They have multipolar branches, which are labelled positively for Nestin. The cytoskeleton of all viable cells was stained by ß-tubulin (green). Cell nuclei were stained with DAPI (blue).
Fig 4: Axonal and myelin sheath status in the distal nerve stump after the 1st and 2nd surgery-I. Longitudinal sections of the distal part of the transplanted site were stained with NF200 (an axon marker) and MBP (a myelin sheath marker). (a–d) One month after the 1st surgery, there were small amounts of myelin debris and axons in the distal stump in the vehicle group. Meanwhile, NF200-positive axons appeared in the cell group and were partially wrapped by the MBP-positive myelin sheath. (e–h) Three months and (i–l) six months after the 1st surgery, the axonal and myelin debris had been gradually eliminated in the vehicle group, and abundant NF200-positive axons and MBP-positive myelin sheath were observed in the cell group. (m–p) Two weeks after the 2nd surgery-I, the vehicle group showed no positive staining. Meanwhile, the distal stump in the cell group reentered a denervated status, exhibiting discontinuous axons and myelin sheath debris. (u) The normalized intensities were determined in triplicate in sections taken from 1 rat. The trend in the expression intensity of NF200-positive axons was consistent with the MBP-positive myelin sheath in both groups. A sharp decline was observed in the expression of both markers after cell excision in the cell group. Scale bar = 100 µm.
Fig 5: Expression of CD300f and its ligand in normal and lesioned nerve. QPCR from lesioned nerve show time-dependent increased CD300f expression (a, relative to unlesioned nerve = 1). b-c, representative flow cytometry profiles showing the selection of macrophages in the crushed sciatic nerve at 3 dpl (b, CD45+CD11b+; c, CD45+CD11b+F480+), and d, expression of CD300f in CD45+CD11b+F480+ cells in the crushed sciatic nerve at 3 dpl (black, only secondary antibody-FITC; grey, hamster isotype control; green, hamster anti-CLM-1). e Quantification showing the percentage of macrophages expressing CD300f in the uninjured nerve and at 3, 10, and 28 dpl. Note that CD300f peaked at 3 dpl declining thereafter. Confocal images of teased fibres from adult sciatic nerves showed staining for the CD300f ligand (using CD300f-IgG2a), which did not co-localize with the axon (f, Thy1-YFP-H mice) nor with the MBP-positive myelin domain of myelinating Schwann cells (g). Partial co-localization could be observed with the S100-positive non-myelinating outer limit of the myelinating Schwann cells (h). The staining with CD300f-IgG2a in uninjured nerve (i) did not change at 10 dpl (j). IgG2a negative control did no show any staining (k). Scale bars: b: 30 µm; c, d: 100 µm; e-g: 20 µm
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