Fig 1.
Distribution of Arc positive cells along segments of the spinal cord.
Number of Arc positive cells relative to spinal cord segmental location. Vertical segmented line represents the position between the L3 and L4 segments with the highest number of Arc positive cells. Data points represent means ± SEM of four consecutive sections (400μm) from each of three rats (N = 3 animals). The control graph shows the average number of cells in control, unstimulated rats (N = 3).
Fig 2.
Distribution of Arc, Zif268 and c-Fos positive cells along the spinal cord 1, 2, 3, 6 and 12 h after CS.
For each time point, for each IEGP the mean of four consecutive sections (400μm) from each animal (N = 3) was calculated, and the data points represent the means ± S.E.M. of these values. Control lines represent the number of positive cells in non- stimulated animals (sham operated) calculated in the same way Two-way ANOVA with Sidaks post-hoc test showed high significant/ significant effect of stimulation for all positions and time points, except: 12 h time point for all IEGPs; 2,65 mm; 3,85 mm at 6 h post CS for Arc; 0,25–1,45 mm at 6 h for c-Fos; 2,25mm, 3.05–3,85 mm at 6 h post CS for Zif268.
Fig 3.
Arc protein immunoblot analysis 2 h post CS.
Immunoblot analysis (Fig 3A), 2h post CS, Arc IEGP, stimulated side dorsal horn (SD), non-stimulated side dorsal horn (NSD) and control, 2mm caudal from midpoint. Data given as % of mean control values, means ± S.E.M (N = 5)(**p<0.01, unpaired Student’s T-tests vs control). Immunoblot scan (Fig 3B), for Arc protein: Ctr1,2—Sham controls; SD- Stimulated side; NSD- Non stimulated side (Production of western blot spinal cord sections described in material and methods). Immunoblot scan (Fig 3C), for GAPDH- used as a quantifiable loading control.
Fig 4.
Arc and c-Fos co-localization Fluorescent micrographs.
Arc was found expressed in a subpopulation of cells in the superficial dorsal horn laminae together with C-Fos. Fluorescent micrographs showing the superficial dorsal horn laminar neurons (mamina I and II, L3/L4 spinal segment)(63x; 2h post-CS) expressing Arc (red- Dsred) and C-Fos (green- FITC)(DAPI- blue- nuclear staining). Arrows indicate position of co-localization. Lamina I and II are marked with their roman numerals. Scale bar: 20 μm.
Fig 5.
Arc protein nucleo-cytoplasmic localization.
Confocal micrographs (Fig 5A) using a Leica SP5 laser scanning microscope (Leica Microsystems, Germany), with a resonant scanner, 40x oil immersion objective and 1.7x or 7-10x electronic zoom for low and high magnifications, respectively. Images are showing the superficial dorsal horn laminar cells, nucleo-cytoplasmic Arc localization from 1–12 h post CS. Arc (red- Dsred) and NeuN (green- FITC)(DAPI- blue- nuclear staining). Scale bars 50 μm and 10 μm for low and high magnifications, respectively. Co- localization of Arc and GFAP (2 h post- CS) (Fig 5B) shows no noticeable Arc staining that co- localized with glia cells. Scale bars 50 μm and 10 μm for low and high magnifications, respectively.