Fig 1: Autophagy flux is impaired during the maintenance of neuropathic pain. (A) The thresholds of mechanical allodynia (left) and thermal hyperalgesia (right) after spinal nerve ligation (SNL) were performed before catheter implantation (baseline value, BL), and on days 3, 7, 14 and 28 postsurgery. Each test was repeated 3 times. Data are presented as the mean ± SD. **p < 0.01 vs sham; n = 6 mice/group. (B) Reactive oxygen species (ROS) levels in the dorsal horn (DH) of the spinal cords reflected by 8-hydroxydeoxyguanosine (8-OHdG) immunofluorescence intensity at different time points after SNL. Bar: 50 µm. (C) Statistical results of intensity of 8-OHdG staining in Figure 1B. Data are presented as the mean ± SD. **p < 0.01 vs sham; n = 3 mice/group, 6 slices per mouse were employed. (D) Pro-inflammatory factor levels of IL1B, TNF, CCL7, and MMP2 in spinal cords from a sham 14 d group and SNL 14 d group. **P < 0.01 vs sham; n = 4 mice/group. (E) Double immunofluorescence staining of 8-OHdG (green) with neuron marker RBFOX3/NeuN (red, upper) or astrocyte marker glial fibrillary acidic protein (GFAP, red, down) in the dorsal horn of the spinal cords, SNL 14 d (SNL postoperative 14 d), respectively, n = 4 mice/group, 6 slices per mice were employed. Bar: 50 µm. (F) Western blots of autophagy proteins, including LC3, ATG5, and SQSTM1. ACTB was used as loading control. Data are presented as the mean ± SD (N = 3). *p < 0.05, **p < 0.01 vs Sham; ns: no significance. n = 3 mice/group. (G) The mRNA level of Sqstm1 in spinal cords on day 7, 14 and 28 after SNL. Data are presented as the mean ± SD. ns: no significance; n = 4 mice/group. (H) Representative immunofluorescence images of spinal dorsal cord staining LC3 or SQSTM1 from sham 14 d and SNL 14 d groups. n = 3 mice/group, 6 slices per mouse were employed. Data are presented as the mean ± SD, **p < 0.01 vs Sham
Fig 2: MT1 and MT2 were expressed in the neurons of spinal cord. (A) Representative immunoblots of MT1/MT2 in the spinal cord of mice subjected to cuff implantation with ß-actin as an internal standard. The baseline represented the 2 days before the implantation. Expression of MT1 and MT2 in the spinal cord was not affected after cuff implantation. (***P<0.001 vs. baseline; n=8 for each group). (B) DAB staining of MT1 and MT2 in the spinal cord in control C57BL/6J mice. Both MT1 and MT2 were expressed in the neurons of spinal cord. (bar=60 µm in the middle panel; bar=18 µm in the right panel). (C) Immunofluorescence staining of MT1/2 and NeuN in the spinal cord of normal C57BL/6J mice. The right panel is a magnified section of pictures in the left panel. (bar=30 µm in the left panel; bar=9 µm in the right panel).
Fig 3: IL-6 receptor-alpha (IL-6Ra) was present in NeuN-positive cells in the central amygdala and IL-6 stimulated the firing rate of neurones residing in the capsular part of the central amygdala. IL-6Ra immunoreactivity (green) and NeuN immunoreactivity (red) partially co-localised in the central amygdala (CeA). Cell nuclei were stained with 4',6-diamidino-2-phenylindole (blue). Yellow arrowheads show examples of cells where IL-6Ra and NeuN immunoreactivity co-localised, whereas green and red arrowheads show examples of cells with only IL-6Ra and NeuN immunoreactivity, respectively (A, B). Approximately 50% of IL-6Ra-immunoreactive cells also showed NeuN immunoreactivity. Conversely, approximately 50% of NeuN immunoreactive cells also showed IL-6Ra immunoreactivity (C, D). These results indicate that a substantial proportion of the neurones in the CeA could be responsive to IL-6. Loose-patch recording showed that IL-6 (1 nmol L-1) could rapidly increase the firing rate of the targeted neurones. The zoomed periods and frequency distribution graph under the recording also show this profound elevation (E). A cocktail of IL-6 and its neutralising antibody (IL-6 ab) evoked no significant change in the firing rate (F). Arrows show the onset of administration of IL-6 or the IL-6 + IL-6ab cocktail. Scale bars: overview = 80 µm, zoom = 10 µm. BLA, basolateral amygdala
Fig 4: Quantification of Ki67, DCX+ and NeuN+ cells in ipsilateral and contralateral hippocampi in the radiated relative to sham control. The numeric densities of Ki67+ and DCX+ cells are expressed as number (#) of cells per mm of the GCL length of DG, NeuN+ cells, expressed as number (#) of cells per mm2 of the GCL and the stratum pyramidale (s.p.) of CA1 in the dorsal hippocampus in each groups. (A,B) Plot the mean densities of Ki67+ and DCX+ cells in the ipsilateral and contralateral sides in the sham group, and the groups with radiation after survival for 30 and 60 days, as indicated. (C,D) Illustrate the densities of NeuN+ cells in the GCL and the s.p. in CA1 in the control and radiation groups. Abbreviations are as defined in Figure 2. All data are represented as mean ± SD. Statistical analysis results are as indicated **p < 0.01 compared with sham, ***p < 0.001 compared with sham.
Fig 5: NeuN antibodies validation using confocal microscopy. (A–C) Representative confocal images of human brain slices stained using anti-NeuN antibody (ABN91; left): SWITCH (A), SHIELD (B), and CLARITY (C) samples, respectively. White arrows highlight three different stained neuronal bodies. On the right, autofluorescence signal of lipofuscin (white arrows). Excitation light, 568 nm; laser power, 1 mW. Objective lens, 60×; NA, 1.4. Scale bar = 50 µm. (D,E) Representative confocal images of SWITCH (D) and SHIELD (E) clarified human brain slices stained using ABN91, ARG 10712, and 26975-1-AP anti-NeuN antibodies. Scale bar = 50 µm.
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