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

The FB utilised in this study were generated in vitro on maize kernels using F. verticillioides sourced from the biobank at the Institute of Veterinary Medicine of the National Academy of Agrarian Sciences, Kiev, Ukraine as described previously (34). Briefly, the cultures were grown on the maize substrates for a duration of four weeks. Contaminated grains underwent analysis for FB1 and FB2 content using liquid chromatography in accordance with the AOAC method #2001.04 (1); the National Veterinary Research Institute in Puławy, Poland, analysed the maize. The analysis confirmed a 3:1 ratio of FB1 to FB2 (73% and 27%, respectively). The FB were extracted from the ground maize using ethanol, quantified via a commercial ELISA kit (RIDASCREEN Fumonisin; R-Biopharm, Darmstadt, Germany), and the extract was then concentrated to a stock solution of 100 mg of FB1+FB2 per mL. Throughout the experiment, the stock solution was diluted with 0.5 mL of 0.9% saline to achieve the desired concentrations for administration based on the daily weight measurements of each pregnant dam. The median lethal dose (LD₅₀) value was established based on developmental studies previously conducted at the State Scientific Research Control Institute of Veterinary Medicinal Products and Feed Additives in Lviv, Ukraine (26).

The study involved eighteen pregnant, six-week-old Wistar rat dams. The dams were individually housed in polypropylene cages at a temperature of 21 ± 3°C, in relative humidity of 55 ± 5% and a 12h/12h light/dark cycle. The rats were randomly assigned to one of three groups (n = 6 each): to a control group (group I) not treated with FB, or to one of two groups intoxicated with FB, either at a dose of 60 mg FB/kg body weight (b.w.) (group II) or 90 mg FB/kg b.w. (group III). A dose of 60 mg FB/kg b.w., an equivalent to 1/15 of the established LD₅₀, has been shown not to lead to clinical or subclinical symptoms in adolescent rats, while a dose of 90 mg FB/kg b.w., constituting 1/10 of the LD₅₀, has been shown to induce subclinical intoxication in adolescent rats (26). Fumonisins B were administered via intragastric gavage in a volume of 0.5 mL of 0.9% saline solution, starting from the 7^th day of gestation until parturition, as previously described (35, 36, 37). Control animals were given an equivalent volume of saline solution using the same method. Dams were allowed free access to drinking water and fed standard laboratory rodent chow (Ssniff Spezialdiäten, Soest, Germany) which met the American Institute of Nutrition AIN-93 nutritional requirements. On the day of parturition, to exclude litter effects on the results, only one male pup from each litter was randomly selected to be euthanised via CO₂ inhalation for subsequent analyses. The sex of the selected pups was confirmed by inspecting primary sexual characteristics after opening the abdominal cavity. The right femora were dissected and the bones were then fixed in phosphate-buffered paraformaldehyde (4% v/v, pH 7.0). The selection of the male sex for analysis in this study was informed by previous research on bone innervation (31). To assess FB mother toxicity, after dams` euthanasia, the weight of dams and their livers, the aspartate aminotransferase (AST) and alanine aminotransferase activity, and the sex ratio in the litters were determined.

After fixation, paraffin-embedded, 4-μm-thick sections were rehydrated and treated with proteinase K for 30 min at room temperature (RT) to retrieve antigens. A 3% H₂O₂ solution was applied for 5 min to inhibit the endogenous peroxidase activity. To reduce nonspecific binding, sections were blocked for 30 min using UltraCruz Blocking Reagent (sc-516214; Santa Cruz Biotechnology, Dallas, TX, USA). For visualising the bone nervous system, a wide range of primary antibodies was used. For the general bone innervation pattern, it was protein gene product 9.5 (PGP9.5) monoclonal rabbit antibody (ab108986; Abcam, Cambridge, UK, at 1:1,000 dilution); for sympathetic bone innervation it was tyrosine hydroxylase (TH) monoclonal mouse antibody (22941; ImmunoStar, Hudson, WI, USA, at 1:1,000 dilution); for parasympathetic bone innervation the reagents were choline acetyltransferase (ChAT) monoclonal rabbit antibody (abl78850; Abcam, at 1:1,000 dilution) and vasoactive intestinal peptide (VIP) polyclonal rabbit antibody (ab22736; Abcam, at 1:1,200 dilution); the antibodies for sensory bone innervation were substance P (SP) monoclonal mouse (ab14184; Abcam, at 1: 1,000 dilution) and galanin (GAL) monoclonal rabbit immunoglobulins (ab254556; Abcam, at 1:4,000 dilution); and the CART-positive bone innervation antibody was a cocaine- and amphetamine-regulated transcript (CART) polyclonal rabbit product (H-003-61; Phoenix Pharmaceuticals, Burlingame, CA, USA, at 1:10,000 dilution). Immunohistochemical staining was performed overnight at 4°C. After primary antibody incubation, the sections were treated at RT with a polyclonal horseradish peroxidase goat anti-mouse/rabbit immunoglobulin G detection system (DPVB110HRP; Immunologic WellMed, Duiven, the Netherlands). Visualisation was achieved with a 3,3′-diaminobenzidine (DAB) chromogen (D5905; Sigma-Aldrich, St. Louis, MO, USA) followed by haematoxylin counterstaining (MHS32; Sigma-Aldrich).

Evaluation of changes in neuronal morphology and their spatial distribution was carried out with image material and was based on the methodology described by Marques et al. (20). Images were acquired using a CX43 light microscope (Olympus Life Science, Tokyo, Japan) equipped with a 20× S Plan Apo objective and SC50 4,912 × 3,684 pixel digital microscope camera (Olympus). Images were saved in the TIF format to ensure high-resolution data for subsequent analysis. Image analysis was performed using Fiji-ImageJ 1.54f (28) with its image processing pipeline of calibration for accurate measurements, noise reduction, and linking of dark pixels to improve the image quality. A key aspect of this analysis was the application of carefully designed colour thresholding using the MeanError filter, which allowed selective identification of DAB-stained neurons and effectively eliminated non-neuronal background elements. Following the thresholding step, a skeletonisation process was applied to streamline neuronal structures while preserving their essential morphological features. The Skeletonize (2D/3D) and Summarize Skeleton plugins for Fiji were used to extract relevant information from the images (Fig. 1).

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

The comprehensive image analysis approach allowed the quantification of critical parameters relevant to studies on neural morphology, connectivity or the studied substances’ toxicity to neuronal structures. The first parameter was the spatial distribution of neurons (%): a quantitative assessment of the spatial distribution of all elements of neurons within the studied region and insights into the arrangement, clustering or dispersion of these neurons in the bone. The second was the mean cross-sectional area of neuronal protrusions (μm²): a quantitative assessment of the cross-sectional area occupied by neuronal bodies and their protrusions including dendrites and axons. The cross-sectional density of neuronal protrusions (per mm²) was the next parameter: the number of intersected or cut neuronal protrusion cross-sections within a defined area. The mean length of the neuronal tree (μm) was also calculated: the mean length of the dendritic and axonal arbours, collectively referred to as “neuronal trees” in neurons. The final parameter was the mean number of branches (per neuron): the average count of branches in individual neurons, quantifying the branching complexity of neurons.

Analyses were conducted using GraphPad Prism software v. 10.0.2 (GraphPad Software, San Diego, CA, USA). A Shapiro–Wilk test validated that the data were normally distributed, and variance homogeneity was confirmed using the Brown–Forsythe test. A one-way analysis of variance was employed, with post-hoc analysis by Tukey’s honest significant difference test. Statistical significance was established at P-value < 0.05.

No changes in the behaviour or basal health state of the pregnant dams were observed during assessments by a veterinarian. Food and water consumption remained consistent across all groups of dams regardless of the treatment they received. In the current study, a decrease in the body weight of dams was only observed after exposure to 90 mg FB/kg b.w. (Fig. 2a). Both FB doses led to a reduction in the liver weight of dams (Fig. 2b and c) and an increase in the activity of AST (Fig. 2d), a non-organ-specific enzyme commonly associated with skeletal and heart muscles as well as with the liver. No FB effect on litter size (Fig. 2f) or sex ratio in the litter (Fig. 2g) was observed, but the terminal body weight of offspring of both sexes was reduced in the FB-administrated groups (Fig. 2h–j).

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

Protein gene product 9.5 was used to quantify the complete bone neuronal network, which innervates the periosteum, cortical and trabecular bones, and bone marrow. The network underwent a significant change in its spatial distribution (Fig. 3). Exposure to the lower dose of FB led to a significant extension of the spatial distribution of the neuronal network compared to that in both the control and higher-dose groups.

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

The mean cross-sectional area of the protrusions throughout the network expanded after exposure to FB, regardless of dose. However, the cross-sectional density of neuronal processes showed a nuanced response, decreasing after exposure to the lower dose and increasing after exposure to the higher dose. After exposure to the lower dose of FB, neuronal morphology and the mean length of neuronal trees and number of branches per neuron significantly increased compared to both the control and the higher-dose exposure groups.

Analysis of the TH-positive neuronal network (Fig. 4A), as demonstrated by the spatial distribution of TH-positive neurons, showed a significant reduction in TH-positive neurons in the femora of pups exposed to the higher dose of FB compared to the femora of control group pups and those of the group exposed to the lower dose. The smaller mean cross-sectional area of protrusions and shorter mean length of neuronal trees within the TH-positive neuronal network after exposure to either dose of FB indicated a diminished impact area on the bone tissue overall. The cross-sectional thickness of the TH-positive cell protrusions showed intricate regulatory mechanisms. Exposure to the higher dose of FB increased the cross-sectional density, in contrast to the control and lower-dose groups. The density was lower in the lower-dose group than in the control group. Similarly, there were fewer branches on average per TH-positive neuron in the bones of pups exposed to the higher dose of FB, implying a narrow impact zone.

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

The parasympathetic bone neuronal network was evaluated using ChAT (Fig. 4B) and VIP (Fig. 4C). The spatial distribution of ChAT-positive neurons in the higher-dose exposure group exceeded that of both the control group and the lower-dose exposure group, and this difference was considered statistically significant. The presence of more ChAT-positive neurons suggests amplified parasympathetic influence on bone regulation. Examination of ChAT-positive neuronal network parameters revealed a greater mean cross-sectional area of protrusions, length of neuronal trees and number of branches per neuron after the higher FB dose. Several parameters related to the VIP-positive neuronal network were lower because of exposure to FB, including the average area of VIP-positive neuronal protrusions, length of neuronal trees in the VIP-positive neuronal network and number of branches per VIP-positive neuron, which was accompanied by a rise in the crosssectional density of neuronal protrusions.

The neuronal network exhibiting SP-positive characteristics (Fig. 5A) was notably smaller in the lower-dosed group than in the control and higher-dosed groups. These findings indicated that the lower dose of FB resulted in a significant size reduction in the SP-positive neuronal network. The observed decrease implied a contraction of the affected area and emphasised the intricate relationship between bone cells and SP-positive neurons. The average cross-sectional area of SP-positive neuronal protrusions in the femora of the pups exposed to the higher dose of FB was larger than that in the femora of the pups exposed to the lower dose. The average length of the neuronal tree in the SP-positive neuronal network and the mean number of branches per SP-positive neuron were diminished following exposure to the lower dose of FB compared to this length and number following only saline solution administration. This, coupled with the absence of changes in cross-sectional density of neuronal protrusions, suggests a reduction in impact area. The components of the sensory bone neuron network were also examined using a GAL marker (Fig. 5B). The distribution of GAL-positive neurons in the spatial domain was sparser in the FB-exposed bones, irrespective of the administered dose. A decrease in GAL-innervation was observed as a simplification of the neuronal structure, which could have significant implications for the intricate relationship between GAL-positive neurons and bone cells. It is worth noting that the mean cross-sectional area of GAL-positive neuronal protrusions, mean length of neuronal trees in the GAL-positive neuronal network, and mean number of branches and end-points per GAL-positive neuron decreased after exposure to FB, with no observable dose dependency. Similarly to the SP-positive network, the GAL-positive neuronal network’s protrusion cross-sectional density was unaffected by exposure to FB, despite the simplified structure. These alterations suggest that the structure of GAL-innervation had been simplified, which could potentially impact the development and remodelling of the bone tissue. The present results demonstrated a very strong impact of FB on the GAL-positive network in the bones of newborn rats, irrespective of the dose used.

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

In the present investigation of the neural network exhibiting CART-positivity (Fig. 5C), a notably smaller spatial distribution of CART-expressing neurons was observed in bones exposed to the lower dose of FB, compared to unexposed bones and bones exposed to the higher dose. The results indicated a greater impact on the bone region at the lower dose, as evidenced by a significant reduction in the average cross-sectional area of CART-positive neuronal protrusions, mean length of neuronal trees in the CART-positive neuronal network and mean number of branches in CART-positive neurons after exposure to a lower dose of FB than after exposure to none and to the higher dose. In contrast, the cross-sectional density of neuronal protrusions noted in the femora of the more intoxicated pups significantly exceeded the density in the femora of both the control pups and the less intoxicated pups.