Additional file 1: of Interleukin-25 is detrimental for recovery after spinal cord injury in mice

Supplementary Materials. Detailed description of materials and methods used throghout the manuscript, provided as supplementary information. (DOCX 35 kb

Study design and liver weight following PHx or MCD diet or chemotherapy in combination with PHx.

<p>(A) Study design, for both male and female rats. (B/C) Mean liver weight for each experimental group at week 10 before PHx (left panels), at week 10 directly after PHx (middle panels) and at week 18 (end of the study, right panels); for male (B) and female (C). Data are expressed in gram ± standard error.</p

Biochemical Parameters for Longitudinal Monitoring of Liver Function in Rat Models of Partial Hepatectomy Following Liver Injury

<div><p>Background</p><p>While evaluation of liver function in preclinical animal studies is commonly performed at selected time-points by invasive determination of the liver/body weight ratio and histological analyses, the validation of longitudinal measurement tools for monitoring liver function are of major interest.</p><p>Aims</p><p>To longitudinally evaluate serum cholinesterase (CHE) and total serum bilirubin (TSB) levels as non-invasive markers to determine injury- and partial hepatectomy (PHx)-induced alterations of liver function in rats.</p><p>Methods</p><p>Male and female Lewis rats were subjected to either methionine/choline deficient (MCD) diet or treatment with FOLFOX chemotherapy prior to PHx. Body weight and CHE/TSB levels are determined weekly. Following PHx and at the study end, histological analyses of liver tissue are performed.</p><p>Results</p><p>Following MCD diet, but not after FOLFOX chemotherapy treatment, results indicate gender-specific alterations in serum CHE levels and gender-independent alterations in TSB levels. Likewise, histological analyses of resected liver parts indicate significant liver injury following MCD-diet, but not following FOLFOX treatment. While TSB levels rapidly recover following MCD diet/FOLFOX treatment combined with a PHx, serum CHE levels are subject to significant model- and gender-specific differences, despite full histopathological recovery of liver tissue.</p><p>Conclusions</p><p>Longitudinal measurements of serum CHE levels and TSB levels in rats are highly complementary as non-invasive parameters for evaluation of liver injury and/or recovery.</p></div

Recovery assessment following partial hepatectomy or MCD diet or chemotherapy in combination with PHx.

<p>(A/D) Evolution of body weight of male/female rats for each experimental group. Data are expressed in gram ± standard error. (B/E) Evolution of total serum bilirubin (TSB) of male/female rats for each experimental group. Data are expressed in milligram per deciliter ± standard error. (C/F) Evolution of serum cholinesterase (CHE) level of male/female rats for each experimental group. Data are expressed in units per liter ± standard error.</p

Additional file 2: Figure S1. of Interleukin-25 is detrimental for recovery after spinal cord injury in mice

IL-25 has no effect on mature oligodendrocyte, astrocyte, microglia, or primary neuron cell viability. (A) MO3.13 cells were differentiated to mature oligodendrocytes using PMA for 72 h and were treated for 48 h with selected concentrations of IL-25. (B, C) The astrocytic and microglial cell lines (CCF and BV2, respectively) were treated for 48 h with selected concentrations of IL-25. (D) Primary neurons were incubated with selected concentrations of IL-25 for 48 h in the presence or absence of B27. B27 deprivation induced a decreased cell viability, but IL-25 treatment had no effect on this. The selected concentrations of IL-25 used for all cell types were 5 ng/ml, 50 ng/ml, 500 ng/ml, and 1 μg/ml. Cell survival was measured using an MTT assay, and values are expressed as percentage of the control. (A-D) There was no significant effect observed on cell viability in all cell types tested. Data represent mean ± SEM of one representative experiment (from two to three independent experiments) ***p < 0.001. (PDF 316 kb

Histological analysis of liver tissue following PHx or MCD diet or chemotherapy in combination with PHx.

<p>Haematoxylin-eosin (H/E) staining (upper panel), Oil Red O staining (middle panel) and PAS/OX-1 staining (lower panel) (A/C) for each experimental group (which is subjected to PHx) of male/female rats at week 10, (B/D) for each experimental group of male/female rats at week 18. Representative images were chosen out of 3 rats analyzed per experimental group. Scale bars indicate 100 µm.</p

Histological analysis of liver tissue following intraportal administration of BM-MSC after MCD diet and PHx in rat.

<p>Haematoxylin-eosin (H–E) staining (upper panel), Oil Red O staining (middle panel) and PAS/OX-1 staining (lower panel). (i) after 13 weeks of control diet (GROUP A), (ii) after 4 weeks of MCD diet, followed by PHx and control diet for 9 weeks (GROUP B), (iii) after 4 weeks of MCD diet, followed by PHx, BM-MSC administration and control diet for 9 weeks (GROUP C). Representative images were chosen out of 3 mice analysed per experimental group. For H-E staining, scale bars in the main image indicate 500 μm, while scale bars in the inset images indicate 50 μm. For Oil Red O and PAS/OX-1 staining, scale bars indicate 100 μm.</p

Histological analysis of liver tissue following PHx and/or MCD.

<p>Haematoxylin-eosin (H–E) staining (upper panel), Oil Red O staining (middle panel) and PAS/OX-1 staining (lower panel). (i) after 4 weeks of control diet (GROUP A*), (ii) after 4 weeks of MCD diet (GROUP C*), (iii) after 13 weeks of control diet (GROUP A), (iv) after 13 weeks of control diet with PHx at week 4 (GROUP B), (v) after 4 weeks of MCD diet followed by 9 weeks of control diet (GROUP C), (vi) after 4 weeks of MCD diet followed by PHx and 9 weeks of control diet (GROUP D), (vii) after 13 weeks of MCD diet (GROUP E), (viii) after 13 weeks of MCD diet with PHx at week 4 (GROUP F). Representative images were chosen out of 3 mice analysed per experimental group. For H-E staining, scale bars in the main image indicate 500 μm, while scale bars in the inset images indicate 50 μm. For Oil Red O and PAS/OX-1 staining, scale bars indicate 100 μm.</p

Recovery assessment following partial hepatectomy (PHx) and/or methionine/choline-deficient (MCD) diet in rat.

<p>(A) Study design. (B) Mean liver weight for each experimental group at week 4 before PHx (left panel), at week 4 directly after PHx (middle panel) and at week 13 (end of study, right panel). (C) Evolution of body weight for each experimental group. Data are expressed in gram ± standard error. (D) Evolution of total serum bilirubin (TSB) level for each experimental group. Data are expressed in milligram per decilitre ± standard error. (E) Evolution of serum triglyceride (TG) level for each experimental group. Data are expressed in milligram per decilitre ± standard error. (F) Evolution of serum cholinesterase (CHE) level for each experimental group. Data are expressed in units per litre ± standard error. For each experimental group at each time point n=10.</p

<i>In</i><i>vivo</i> / <i>ex</i><i>vivo</i> bioluminescence imaging (BLI) following intravenous (IV) and intraportal (IP) administration of BM-MSC/eGFP-Luc.

<p>(A) Bright field phase contrast microscopic image of cultured rat BM-MSC. The scale bar indicates 100 μm. RT-PCR analysis of cultured BM-MSC: GAPDH, vimentin, AFP, albumin, CD45. A representative output was chosen from two independent measurements at multiple passages. Flow cytometric overlay histograms showing the expression pattern of membrane proteins on BM-MSC derived from Lewis rats (i.e. expression of CD27, CD73 and CD90.1, but no expression of CD45). Open histograms: unstained control. Filled histograms: specific antibody staining. A representative histogram overlay was chosen from three independent measurements at multiple passages. (B) Overlay histogram: level of eGFP expression by control BM-MSC (open black histogram) and transduced BM-MSC/eGFP-Luc (filled green histogram). A representative histogram overlay was chosen from four independent measurements at multiple passages. Right graph: In vitro luminescence assay using 1x10⁵ control BM-MSC and transduced BM-MSC/eGFP-Luc. Data are expressed as mean relative light units ± standard error (n=4 measurements at multiple passages). (C) Representative BLI images of healthy (upper panel) and MCD diet-fed animals (lower panel) subjected to PHx at week 4 with intravenous BM-MSC/eGFP-Luc administration at week 5 (n=2). (D) Representative BLI images of control animals (upper panel) with intraportal BM-MSC/eGFP-Luc administration (n=4) and BLI images of healthy (middle panel) and MCD diet-fed animals (lower panel) subjected to PHx at week 4 with intraportal BM-MSC/eGFP-Luc administration at week 5 (n=3). Left panel: <i>In vivo</i> BLI (left picture) and <i>ex vivo</i> BLI (right picture) at 2 hours post-implantation. Right panel: <i>In vivo</i> BLI (left picture) and <i>ex vivo</i> BLI (right picture) at 24 hours post-implantation. <i>Ex vivo</i> images show the following organs in clockwise direction: lung (upper), heart, spleen, kidney, liver.</p