Fig 2: Astrogliosis in the rat hippocampus following pilocarpine-induced SE. (a) Immunohistochemistry targeting glial fibrillary acidic protein (GFAP) was used for the detection of astrocytes. Representative images of hippocampal sections with GFAP-positive cells and their corresponding black–white masks, which were obtained with ImageJ software and used for area counting (upper panels: ×5 magnification and lower panels: ×40 magnification). The averaged GFAP-positive areas in different hippocampal regions: CA1 (b), CA3 (c), hilus (d), and DG (e). Five to seven animals were in each group. The diamonds show the individual values for each rat. The columns indicate average values, and error bars show standard errors of the means. Two-way ANOVA was performed to determine the effects of SE, time after SE, and the interaction of these factors: CA1 (SE: F1, 28 = 48, p < 0.001; time: F2, 28 = 7.6; p < 0.01; SE × time: F2, 28 = 6.1, p < 0.01); CA3 (SE: F1, 29 = 17.4, p < 0.001; time: F2, 29 = 0.82; p = 0.45; SE × time: F2, 29 = 1.4, p = 0.26); hilus (SE: F1, 29 = 7.3, p < 0.05; time: F2, 29 = 0.32; p = 0.73; SE × time: F2, 29 = 2.7, p = 0.09); DG (SE: F1, 29 = 4.6, p < 0.05; time: F2, 29 = 1.1; p = 0.36; SE × time: F2, 29 = 2.0, p = 0.16). * p <0.05, ** p < 0.01, *** p < 0.001 (Tukey’s post hoc test).

Fig 3: Western blotting data of proteins production in the dorsal hippocampus 7 days after pilocarpine-induced SE. For inserts, the upper part shows the chemiluminescent signal, the lower part shows the Ponceau S. For bands c is sample calibrator, 1 is Ctrl, 2 is Pilo, and 3 is MTEP group. Each dot represents one animal; the columns indicate average values and error bars show standard deviations. One-way ANOVA, GFAP: F2,17 = 29, p < 0.001; EAAT2: F2,17 = 4, p < 0.05. Asterisks indicate significant differences between groups according to Tukey’s post hoc tests: * p < 0.05, *** p < 0.001.

Fig 4: Mlxipl was upregulated in ipsilateral SDH of SNI-induced mechanical allodynia rats. (A and B) The Von Frey test were carried out before and after surgery (Sham or SNI). Mechanical allodynia was induced by SNI surgery in the ipsilateral paw. N = 5. ***P < 0.001 vs. SNI BL Ipsi; $$$P < 0.001, $$P < 0.01 vs. SNI Contra; ###P < 0.001 vs. Sham Ipsi. QPCR (C) and western blot (E) were performed before and after surgery. Mlxipl in the ipsilateral SDH was upregulated after SNI surgery. Quantification of the western blot (F). N = 5. ***P < 0.001, **P < 0.01 vs. SNI BL Ipsi; $$$P < 0.001, $$P < 0.01 vs. SNI BL Contra. QPCR (D), western blot (G) and immunofluorescence (I) were performed at day 7 after Sham or SNI surgery. Mlxipl was significantly upregulated in the ipsilateral SDH. Quantification of the western blot (H) and immunofluorescence (J). N = 5. $$$P < 0.001, $$P < 0.01 vs. SNI Contra; ###P < 0.001, ##P < 0.01 vs. Sham Ipsi. (K) Double immunofluorescence staining showed that Mlxipl was co-localized with Iba1 (white arrows) and NeuN (yellow arrows) but not GFAP. BL, baseline (before surgery); ipsi, ipsilateral; Contra, contralateral; SNI, spare nerve injury; SDH, spinal dorsal horn.

Fig 5: Trimetazidine (TMZ) protects from motor neuron loss and neuroinflammation. (a and b) Representative lumbar spinal cord sections (L3-L5) stained with cresyl violet (scale bar 100 µm) and quantification of motor neuron number (histograms) in 120- (a) and 150-day old (b) wild-type mice treated without TMZ (WT) and SOD1G93A mice treated without (G93A) or with TMZ (G93A TMZ) from disease onset (70 days of age). Data are presented as means and ± SEM, *P < 0.05, compared with wild-type; # P < 0.05, compared with untreated G93A, n = 4 per group (n = 4 for WT, n = 6 for G93A, G93A TMZ). (c) Representative immunofluorescence images and analysis of astrocytes (GFAP) in lumbar spinal cord sections (L3-L5) of 120- and 150-day old wild-type mice treated without TMZ (WT) and SOD1G93A mice treated without (G93A) or with TMZ (G93A TMZ) from disease onset (70 days of age). (d and e) Representative immunofluorescence images and analysis of activated microglia (CD68) and resting microglia (P2T12) in lumbar spinal cord sections (L3-L5) of 120- and 150-day old wild-type mice treated without TMZ (WT) and SOD1G93A mice treated without (G93A) or with TMZ (G93A TMZ) from disease onset (70 days of age). Scale bar 100 µm. Data are presented as means and ± SEM, *P < 0.05 compared with wild-type; # P < 0.05, compared with untreated G93A, n = 4 per group (n = 4 for WT, n = 6 for G93A, G93A TMZ). P values were obtained using parametric one-way ANOVA with Bonferroni post hoc test (a–e)