The effect of prenatal fumonisin B exposure on bone innervation in newborn Wistar rats

Abstract

Introduction: This study explored the effects of prenatal exposure to fumonisins B (FB) on bone innervation in newborn Wistar rats.

Material and methods: Pregnant dams (n = 6 per group) were assigned to either the control or one of two FB-exposed groups (60 mg or 90 mg/kg body weight) from the 7^th day of gestation until parturition. On the day of parturition, one male pup from each litter (n = 6 per group) was randomly selected and euthanised, and their femurs were dissected for analysis. Bone innervation was quantified by examining the morphology patterns of sympathetic, parasympathetic, sensory and cocaine- and amphetamine-regulated transcript (CART)-positive fibres. Prepared bone sections were analysed using immunohistochemistry staining for protein gene product 9.5, tyrosine hydroxylase, choline acetyltransferase, vasoactive intestinal peptide (VIP), substance P and CART-positive neurons.

Results: The group that received a higher dose of FB demonstrated an increase in both the size and complexity of the complete bone neuronal network together with heightened sympathetic and sensory innervation, and displayed a decrease in neuron density and sympathetic innervation. Fumonisin B exposure led to a decrease in galanin-positive and VIP-positive bone neuronal networks in both groups exposed to FB, while in the lower-dose group, there was also a decrease in CART-positive innervation.

Conclusion: Prenatal FB exposure significantly influences the neuronal bone network of rats, which is essential for maintaining bone homeostasis. These findings emphasise the necessity for further research to understand the lasting effects and underlying mechanisms of alterations induced by FB.

Keywords: bone innervation; mycotoxins; peripheral nervous system; prenatal exposure.

Fig. 1.

A diagram showing the graphical analysis steps for image segmentation and bone neuronal net analysis. Red arrows indicate an example of an identified neuronal element in subsequent processes of image transformation

Fig. 2.

Effects of fumonisin exposure on Wistar rat dams. a – post-parturition body weight; b – liver weight; c – relative liver weight (percentage of total body weight); d – serum aspartate aminotransferase (AST) activity; e – serum alanine aminotransferase (ALT) activity; f – litter size (number of live pups); (g) – offspring sex ratio (female-to-male); h – average pup body weight; i – female pup body weight; j – male pup body weight. Group I – control group; Group II – dams intoxicated with 60 fumonisins B (FB)/kg b.w. during gestation; Group III – dams intoxicated with 90 FB/kg b.w. during gestation. The data are presented as mean values ± standard error of the mean (n = 6), except for (f), where the line indicates the median, the box represents the interquartile range, and the whiskers show minimum and maximum value ranges. * – statistical significance at P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001

Fig. 3.

Effects of prenatal fumonisins exposure on general bone innervation: A – representative photomicrographs showing protein gene product 9.5 (PGP9.5)-positive neuronal nets (arrowheads) in compact bone, trabecular bone and bone marrow; B – general bone innervation pattern quantified by morphology of the PGP9.5-positive neurons: i – spatial distribution of PGP9.5-positive neurons; ii – average cross-sectional area of PGP9.5-positive neuronal protrusions; iii – neuronal protrusion cross-sectional density of PGP9.5-positive neurons; iv – average length of neuronal trees in PGP9.5-positive neurons; v – mean number of branches in PGP9.5-positive neurons. Group I – control group; Group II – newborns from dams intoxicated with 60 fumonisins B (FB)/kg b.w. during gestation; Group III – newborns from dams intoxicated with 90 FB/kg b.w. during gestation. The data are presented as mean values ± standard error of the mean (n = 6); * – statistical significance at P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001; scale bars – 20 μm

Fig. 4.

Effects of prenatal fumonisin exposure on bone innervation: A – sympathetic and B and C – parasympathetic patterns quantified by morphology of the immunoreactive (IR) neurons. A – tyrosine hydroxylase (TH)-positive; B – choline acetyltransferase (ChAT)-positive; C – vasoactive intestinal peptide (VIP)-positive neurons: i – spatial distribution of IR neurons; ii – average cross-sectional area of IR neuronal protrusions; iii – neuronal protrusion cross-sectional density of IR neurons; iv – average length of neuronal trees in IR neurons; v – mean number of branches in IR neurons. Group I – control group; Group II – newborns from dams intoxicated with 60 fumonisins B (FB)/kg b.w. during gestation; Group III – newborns from dams intoxicated with 90 FB/kg b.w. during gestation. The data are presented as mean values ± standard error of the mean (n = 6); * – statistical significance at P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001

Fig. 5.

Effects of prenatal fumonisins exposure on bone innervation: A and B – sensory and C – cocaine- and amphetamine-regulated transcript (CART)-positive patterns quantified by morphology of the immunoreactive (IR) neurons. A – SP-positive; B – GAL-positive; C – CART-positive neurons; i – spatial distribution of IR neurons; ii – average cross-sectional area of IR neuronal protrusions; iii – neuronal protrusion cross-sectional density of IR neurons; iv – average length of neuronal trees in IR neurons; v – mean number of branches in IR neurons. Group I – control group; Group II – newborns from dams intoxicated with 60 fumonisins B (FB)/kg b.w. during gestation; Group III – newborns from dams intoxicated with 90 FB/kg b.w. during gestation. The data are presented as mean values ± standard error of the mean (n = 6); * – statistical significance at P-value < 0.05; ** – P-value < 0.01; *** – P-value < 0.001