Additional file 1: of Tissue transglutaminase in astrocytes is enhanced by inflammatory mediators and is involved in the formation of fibronectin fibril-like structures

TG2 and fibronectin protein expression in rat astrocytes and microglia. Untreated (Ctrl) and 48 h LPS-treated primary rat astrocytes (A) expressed more fibronectin (FN) and more TG2 compared to untreated (Ctrl) and LPS-treated primary rat microglia (M). Representative blot of three independent experiments is shown. (TIFF 195 kb

Effects of mifepristone given at d18 only or during ds18–21 of 3-weeks corticosterone treatment period.

<p><b>A.</b> Mifepristone treatment during ds 18–21 fully normalizes the CORT-induced reduction in number of BrdU⁺ cells (p<0.05 compared to 21 ds CORT+d18–21 VEHM). Surprisingly, mifepristone treatment only on d18 also normalized the CORT-induced reduction (p<0.01). No significant difference was found between these two treatment groups (21ds CORT+ds18–21 MIF versus 21ds CORT+d18 MIF; p>0.05). Mifepristone treatment was entirely ineffective in the vehicle (VEHC) control groups. <b>B1.</b> Mifepristone treatment either during ds 18–21 (p<0.05) or only on d18 (p<0.05) normalized the CORT-induced reduction in DCX⁺ cells. These two treatment groups (21ds CORT+ds18–21 MIF versus 21ds CORT+d18 MIF) yielded comparable numbers of DCX⁺ cells. <b>B2.</b> Mifepristone treatment either during ds 18–21 (p<0.05) or only on d18 (p<0.05) was effective in normalizing the CORT-induced reduction in immature DCX⁺ cells. <b>B3.</b> Mifepristone was ineffective in restoring the CORT-induced reduction in the number of mature DCX⁺ cells (p>0.05 in both treatment groups). CORT-treated animals receiving mifepristone for 4 days (21ds CORT+ds18–21 MIF) still had a significantly lower number of mature DCX⁺ cells than the corresponding vehicle group (p<0.05 vs 21ds VEHC+ds18–21 MIF). <b>C.</b> Mifepristone administration either on d18 alone (p<0.05) or during ds18–21 (p<0.01) normalized CORT-induced reduction in PCNA⁺ cells. Again, mifepristone treatment had no effect in the VEHC groups. <b>D.</b> No significant overall effect of treatment was found on GFAP⁺ cells number (p>0.05). Data are presented as mean ± SEM (n = 6 animals per group). For each marker, the groups were first subjected to an ANOVA, followed by a post-hoc Tukey multiple comparison of the means. * p<0.05; ** p<0.01; ***p<0.005; **** p<0.001.</p

Percentual change in body weight.

<p>Corticosterone (CORT) compared to vehicle treatment (VEHC) reduced the percentual change in body weight, at 17 and 21 ds. This was not consistently affected by treatment with mifepristone (MIF).</p>a<p>: significantly different from the corresponding VEHC group (p<0.001).</p>b<p>: significantly different from the 21 ds VEHC+ds 18–21 VEHM group (p<0.05).</p>c<p>: significantly different from the 21 ds VEHC+d18 MIF group (p<0.001).</p>d<p>: significantly different from the 21 ds CORT group (p<0.05).</p

Effects of 17 versus 21 days treatment with corticosterone.

<p><b>A.</b> Both 17 ds (p<0.05) and 21 ds CORT (p<0.001) exposure significantly reduced the number of BrdU⁺ cells compared to the respective vehicle control groups. <b>B1.</b> Likewise, both 17 ds and 21 ds CORT exposure significantly (p<0.001) reduced the total number of DCX⁺ cells compared to the control groups. <b>B2.</b> A significant reduction in immature DCX⁺ cells was found after 17 ds CORT (p<0.05) as well as after 21 ds CORT exposure (p<0.05) compared to the respective control groups. <b>B3.</b> The number of mature DCX⁺ cells was significantly reduced after 17 ds CORT (p<0.005) and after 21 ds CORT exposure (p<0.001) compared to the respective control groups. <b>C.</b> Both 17 ds and 21 ds CORT exposure groups showed a significant (p<0.001 and p<0.005 respectively) reduction in the number of PCNA⁺ cells compared to the corresponding control groups. <b>D.</b> Treatment with corticosterone did not affect the number of GFAP⁺ astrocytes at all (p>0.05). Data are presented as mean+SEM (n = 6 animals per group). For each marker, the groups were first subjected to an ANOVA, followed by a post-hoc Tukey multiple comparison of the means. * p<0.05; ***p<0.005; **** p<0.001.</p

Distribution of BrdU, DCX, PCNA and GFAP positive cells in the rat dentate gyrus.

<p><b>A.</b> BrdU⁺ cells are mainly located in the subgranular zone (SGZ, arrowhead) but can also be found in the hilus. Considerably lower numbers are encountered in the granular cell layer (GCL). Calibration bar: 50 µm. <b>B.</b> Overview of the rat dentate gyrus with vehicle treatment showing strong immunoreactivity of DCX in the SGZ and GCL, with dendrites extending through the GCL into the molecular layer (ML). Calibration bar: 100 µm. <b>C.</b> Arrowhead points to a relatively immature type 1 DCX⁺ cell, without dendrites and/or with horizontally oriented dendrites. Calibration bar: 10 µm. <b>D.</b> Arrowhead points to a type 2 DCX⁺ cell, with dendrites with an oblique orientation, growing into the GCL but not ML. Calibration bar: 10 µm. <b>E.</b> Arrow points to a relatively mature type 3 DCX⁺ cell, characterized by a primary dendrite orientated perpendicularly to the SGZ and with protrusions extending into the ML. Calibration bar: 10 µm. <b>F.</b> Clustered PCNA-labeled cells prevail in the SGZ (arrowhead) but can also be found in the hilar region; these cells are less prevalent within the GCL. Calibration bar: 50 µm. <b>G.</b> GFAP⁺ astrocytes are mainly located in the hilus and ML but not in the GCL. These cells show brown DAB-staining in their processes and cytoplasm whereas the nucleus is devoid of staining (arrow). Calibration bar: 10 µm. H. Double immunofluorescent staining (orthogonal planes) of a DCX-Ki67 double immunopositive cell in the SGZ of a 21ds CORT+d18 MIF treated animal, demonstrating that at least a subset of the DCX⁺ cells can re-engage in cell cycle. Arrow indicates red Ki-67 signal in the nucleus of a green DCX⁺ cell. 40× magnification; GCL: granule cell layer of the hippocampal dentate gyrus.</p

Schematic representation of experimental groups.

<p>In experiment #1, we compared animals treated subcutaneously with corticosterone (CORT) or vehicle (VEHC) daily for 17 days with animals treated for 21 days; the latter groups also received the vehicle of mifepristone (VEHM) on days 18–21. In experiment #2, the 21 ds CORT/VEHC+ds 18–21 VEHM were used as statistical reference groups in a comparison with experimental groups receiving mifepristone on d18 only (21ds CORT+ds18 MIF and 21ds VEHC+ds18 MIF) or on 4 days from 18–21 (21ds CORT+ds18–21 MIF and 21ds VEHC+ds18–21 MIF). All animals were treated with BrdU on day 1 and sacrificed on the morning after the last treatment.</p

Combinations of primary and secondary antibodies used for immunoreactive labeling.

<p>FN: fibronectin.</p

Secondary antibodies used.

<p>Secondary antibodies used.</p

Quantification of TG2 and Iba-1 positive cells in early/late active versus inactive white matter lesions.

<p>The number of TG2 (A) and Iba-1 positive cells (B) per white matter sample area of 0.1 mm² is significantly decreased in inactive lesions compared to (early/late) active lesions. Data are shown as mean + SEM, n = 8 for early/late active lesions, n = 12 for inactive lesions, *P<0.001.</p

Characterization of marmoset EAE lesions and TG2 immunoreactivity.

<p>The normal appearing white matter (NAWM) shows an intact LFB myelin staining (A) and few MRP14 positive macrophages (G). Early active (EA) lesions display myelin degradation (B) and foamy macrophages (H). Late active (LA) lesions show degradation of myelin (C) combined with less MRP14 positive macrophages (I). Inactive (IA) lesions are characterized by an absence of both myelin staining (D) and MRP14 positive macrophages (J). The normal appearing grey matter (NAGM) shows intact myelin fibers (E) and very few MRP14 positive macrophages (K). Cortical grey matter lesions (cGML) show an absence of myelin fibers (F) and presence of MRP14 positive microglia (L). TG2 immunoreactivity is present in endothelium of the vessel walls in NAWM (M). Early active and late active lesions display additional TG2 positive cells (N and O respectively). Inactive lesions show less additional TG2 immunoreactivity (P). Cortical grey matter lesions also show additional TG2 positive cells (R) compared to the endothelial staining in normal appearing grey matter (Q). Scale bar is 20 µm.</p