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

Abstract

BackgroundThe 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.ResultsHistomorphological 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).ConclusionsThe 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.

Retrospective study of pathology-based investigative techniques for the assessment of diet-induced changes in liver and intestine of flatfish

“…The in situ techniques (anti-PCNA immunohistochemistry and TUNEL method) were used to assess cellular turnover of enterocytes in flatfish fed mussel meal diets (Sirri et al 2014) (Table 2).…”

“…Cell counting. Cell counting was performed using the plugin colour segmentation of ImageJ software (http:// rsbweb.nih.gov/ij/plugins/index.html#segmentation) for PCNA-immunopositive and TUNEL-positive enterocytes (Sirri et al 2014). From each stained section, photomicrographs of five fields per gastrointestinal tract at 20 Â magnification were captured using a Nikon Digital Sight SD-MS camera (Nikon Corporation, Tokyo, Japan) connected to an optical microscope.…”

“…In the intestine, FD analysis was performed to assess diet-induced hyperplastic changes of the mucosa (Sirri et al 2014). Eighty photomicrographs of H&E-stained slides of intestinal tracts were obtained in a blinded fashion at 4 Â magnification using a Nikon Digital Sight SD-MS camera (Nikon Corporation, Tokyo, Japan) connected to an optical microscope.…”

“…Unfortunately, regarding morphometry, several different methodological approaches have been used in fish feeding trials, making almost impossible the comparison between them. Image analysis by segmentation has been used on common sole for quantification of PCNA-and TUNEL-positive enterocytes (markers of proliferation and apoptosis, respectively) (Sirri et al 2014). In this study the classic evaluation of cell turnover has been transposed from mammals to fish since hyperplasia of mucosal folds was an expected result in relation to a specific dietary ingredient.…”

“…Fractal geometry provides formal mathematical foundations to better understand and characterise a large number of phenomena where structural complexity, irregularity and seemingly random morphology are a common motif (Landini 2011); it is widely applied in biomedical sciences, from diagnostic imaging to histopathological fields (Tambasco and Magliocco 2008;Landini 2011). To the best of our knowledge, in fish tissues, only two studies have been performed using FD analysis (Bianco et al 2013;Sirri et al 2014). In the first one, FD analysis of liver texture given by the lipid storage pattern (microvesicular or macrovesicular) was compared with the area of lipid accumulation (empty area, EA) in sole.…”