Fig 2: VIP promoted the transformation from primordial follicles to early growing follicles in neonatal rat ovary. Four-day-old rat ovaries were cultured in basal medium without VIP (control group) or with VIP in different concentrations for three days. (A) The representative photos of all groups stained with hematoxylin and eosin: control (a), 10−6 mol/L (b), 10−7 mol/L (c), 10−8 mol/L (d). (B) and (C) The numbers of early-growing follicles and primordial follicles were counted, and their ratio was calculated. Scale bar: 50 μm. Values are mean ± s.d. of at least three experiments. (*P < 0.05 compared with the VIP−6 mol/L group, & P < 0.05 compared with the VIP−7 mol/L group, # P < 0.05 compared with the VIP−8 mol/L group).

Fig 3: Caspase-3, 3-Nitrotyrosine (3-NT) staining scores and nitrite measurements at 14 day post-infection.(A) Semi-quantification of immunohistochemical Caspase-3 staining in distal colon from C. rodentium infected mice with and without VIP treatment (B) Semi-quantification of immunohistochemical 3-NT in distal colon from infected mice with and without VIP treatment. (C) Nitrite concentration in the murine distal colon of infected and VIP treated mice (n = 2–7 mice/group). Of the mice harvested day 14 post infection, one mouse died in the group that was administered VIP day 5–10 and two in the group that were administered VIP day 10–14. Data about dead animals is not included in the graphs, and the group with only two mice remaining (VIP day 10–14) was omitted from the statistical analysis. (D) Nitrite concentration in the in vitro mucosal intestinal model infected with C. rodentium and treated with cytokines in the presence and absence of VIP. (E-I) Representative photos showing caspase-3 tissue localization in distal colon from C. rodentium infected mice with and without VIP treatment. Note that although many cells in the tissue from infected mice stains pale brown, a study that performed both Caspase-3 and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assays in parallel to detect DNA degradation, demonstrated that the proportion of cells that are in late stage apoptosis correspond to the strongly stained cells, not the light brown cells [58]. (J-N) Representative photos showing 3-NT tissue localization in distal colon from C. rodentium infected mice with and without VIP treatment. Scale bar 200 μm, magnification x400. Statistics: data are presented as mean ± S.E.M. and analysed by ANOVA with Student Newman-Keuls Multiple Comparison post hoc test: * P<0.05, ** P<0.01, *** P<0.001.

Fig 4: Effect of VIP on colitis during C. rodentium infection.A. The total colitis score is the sum of the scores present in A, B, C, D, E, G and H: The goblet cell depletion score (F) was not included, due to the fact that VIP is a mucus secretagogue. (B) crypt architecture, (C) tissue damage, (D) crypt length (the crypt lengths were translated into scores for incorporation into the total colitis score according to the numbers 1–3 on the y-axes), (E) crypt abscesses, (F) goblet cell depletion, (G) neutrophils in lamina propria and (H) infiltration of inflammatory cells. Statistics: data are presented as mean ± S.E.M. (n = 2–7 mice, as indicated by the individual symbols in the graph) and analyzed using ANOVA with Student Newman-Keuls Multiple Comparison post hoc test: * P<0.05, ** P<0.01, *** P<0.001 vs. control. Of the mice harvested day 14 post infection, one mouse died in the group that was administered VIP day 5–10 and two in the group that were administered VIP day 10–14. Data about dead animals is not included in the graphs, and the group with only two mice remaining (VIP day 10–14) was omitted from the statistical analysis.

Fig 5: The expression of vasoactive intestinal peptide (VIP) and its receptors in 4-day-old rat ovary. Four-day-old rat ovaries were freshly collected and cut into 5 μm sections, followed by immunohistochemistry for detecting VIP and its receptors – VPAC1 and VPAC2 (arrowheads, positive primordial follicle; arrows, positive early growing follicles). Scale bar: 50 μm.