Fig 1.
Change in body weight following treatment of adult and newborn mice.
(a) In adult mice, controls continuously gained weight over 8 d, whilst after DSS treatment initial weight gain reversed into weight loss, significantly different from controls from the 5th experimental day on. (b) In neonatal mice, controls also continuously gained weight over 60 h. Animals from the LPS and DSS group gained weight in a similar matter for 36 h followed by a significant weight loss, which was more pronounced for DSS (p < 0.001). ** p < 0.01, *** p < 0.001.
Fig 2.
Normalized mRNA expression of Cxcl2 in total intestinal tissue.
(a) In adult mice (n = 6) treated with DSS, Cxcl2 expression significantly increased in colon samples only. (b) In neonatal mice (n = 6), Cxcl2 expression was significantly higher in the DSS group as compared to LPS (jejunum, ileum) and controls (jejunum, ileum, colon), respectively. LPS and controls did not differ. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3.
Histological changes of the small intestinal tissue after 72 h treatment.
(a) Representative ileal sections of neonatal mice from controls, the LPS, and DSS group (magnification 200x), showing healthy villi-structure versus injured intestinal segments (LPS: NEC score 2; DSS: NEC score 4) in the treatment groups. Severity of intestinal lesions and the incidence of NEC (b) as assessed by the NEC score was similar in both groups (n = 10). *** p < 0.001.
Fig 4.
Extension of intestinal lesions in neonatal mice after LPS and DSS treatment.
(a) Representative histological sections of intestinal lesions (magnification 100x) after LPS and DSS treatment. Extensive lesions were only detected in DSS treated animals, affecting up to three adjacent intestinal loops of the formed snail. Evaluation (b) revealed significantly larger lesions after application of DSS compared to LPS treatment. (c) Histological sample illustrating the method of length measurement of intestinal lesions. In the central region, the jejunum is located (beginning arrow), the outer loops represent the ileum. The rectangle illustrates the area shown in (a), the arrow indicates the course of length measurement. ** p < 0.01.
Fig 5.
FACS analysis of leukocytes in the lamina propria of the small intestine of neonatal mice.
(a) Gated living CD45+, CD11b+ cells were either further gated on SSCint/hi, Ly6-G and Ly6-C double positive cells to identify neutrophils or on SSClo, Ly6-G-, Ly6-C and MHC II to identify intestinal monocytes/macrophages (M/M) populations (n = 8 per group). The percentage (b) as well as the total number (c) of neutrophils was significantly higher in the LPS group than in the DSS group and in controls, which did not differ among each other. Conversely, the percentage (d) of Ly6-Chi “inflammatory monocytes” (P1 gate) was higher in the DSS group as compared to the LPS group and controls. The total number of SSClo monocytes/macrophages was significantly lower in both treatment groups compared to controls. The percentage of resident macrophages found in the “incl. P3, P4, P5” gate (f) was significantly decreased after treatment compared to controls. (g) Total numbers of living leukocytes was significantly decreased in both treatment groups. * p < 0.05 ** p < 0.01, *** p < 0.001; SI = small intestine.
Fig 6.
In vitro viability of neonatal intestinal epithelial cells exposed to DSS.
Viability of IECs (n = 8) remained unaltered by an exposure to 3% DSS for 72 h in SmBM full medium. 1% Triton-X served as dead cell controls. *** p < 0.001.
Fig 7.
Cleaved caspase-3 staining of the ileum in neonatal mice.
(a) Tissue of control animals at the age of 5.5 and 7 days did not show any apoptosis. In contrast, LPS treatment for 36 h led to rare apoptosis, whereas DSS treatment induced moderate apoptosis after the same period of time. After 72 h, increased apoptosis was detected in tissue of LPS treated animals, but to a significantly lesser extent than DSS, which caused a large amount of apoptotic cells. Magnification 200x, cleaved caspase-3: red, nuclei (DAPI): blue. (b) Grading of apoptosis after 72 h. ** p < 0.01, *** p < 0.001.