Proliferation, apoptosis, and fractal dimension analysis for the quantification of intestinal trophism in sole (Solea solea) fed mussel meal diets

Abstract

Background: The evaluation of intestinal trophism, mainly the mucosal layer, is an important issue in various conditions associated with injury, atrophy, recovery, and healing of the gut. The aim of the present study was to evaluate the kinetics of the proliferation and apoptosis of enterocytes by immunohistochemistry and to assess the complexity of intestinal mucosa by fractal dimension (FD) analysis in Solea solea fed different experimental diets.

Results: Histomorphological evaluation of all intestinal segments did not show signs of degeneration or inflammation. Cell proliferation index and FD were significantly reduced with a diet high in mussel meal (MM; p = 0.0034 and p = 0.01063, respectively), while apoptotic index did not show any significant difference for the same comparison (p = 0.3859). Linear regression analysis between apoptotic index (independent variable) and FD (dependent variable) showed a statistically significant inverse relationship (p = 0.002528). Linear regression analysis between cell proliferation index (independent variable) and FD (dependent variable) did not show any significant correlation (p = 0.131582).

Conclusions: The results demonstrated that diets containing increasing levels of mussel meal in substitution of fishmeal did not incite a hyperplastic response of the intestinal mucosa. The mussel meal, which is derived from molluscs, could mimic the characteristics of the sole's natural prey, being readily digestible, even without increasing the absorptive surface of intestinal mucosa. Interestingly, from this study emerged that FD could be used as a numeric indicator complementary to in situ quantification methods to measure intestinal trophism, in conjunction with functional parameters.

Figure 1

Immunohistochemistry with anti-PCNA antibody and TUNEL assay. (A) PCNA-positive nuclei of enterocytes are located mainly in the basal area and along the intestinal folds. (B) TUNEL-positive apoptotic cells (arrows) and apoptotic bodies (arrow heads) are located at the apex of intestinal folds. The intrinsic nonspecific binding is also evident (asterisks) (Bars = 25 μm).

Figure 2

Diet effect on PCNA index. Pooled data of PCNA index (9 anterior + 9 intermediate + 9 posterior intestinal tracts = 27 for each group) for each diet displayed PCNA index of MM75 group significantly lower than MM50 and MM0 (Post-hoc Kruskal-Wallis Multiple Comparison n = 108; H = 13.64672, significance p < 0.05) (letters mark significant differences in pairwise comparison).

Figure 3

Diet effect on intestinal fractal dimension. Pooled data of fractal dimension (respectively, MM0 = 8 anterior + 7 intermediate + 8 posterior intestinal tracts = 23; MM25 = 4 anterior + 5 intermediate + 6 posterior intestinal tracts = 15; MM 50 = 6 anterior + 8 intermediate + 7 posterior intestinal tracts = 21 and MM75 = 6 anterior + 8 intermediate + 7 posterior intestinal = 21) for each diet displayed an increase of FD with the reduction of mussel meal (MM) diet content (Anova Post hoc Fisher LSD test n = 80; mean ± 1.96*SE, significance p < 0.05) (letters mark significant differences in pairwise comparison).