3.3. Effects of iAs and F-, alone or in combination, on pathological changesin the hippocampal CA1 region
H&E staining of neuron in the hippocampal CA1 region was observed at different optical microscope magnifications (Bar = 100 um 50 um in Fig. 2B & C respectively). In the control group, hippocampal CA1 neurons were tightly organized and had multiple cell layers, with healthy cell morphology and clearly stained nuclei and nucleoli. Compared with the control group, hippocampal CA1 neuron pathological changes were observed in iAs and/or F-exposure groups, including neurofibrillary degeneration, loosely and irregularly organized structure, fewer cell layers, cytoplasmic cavitation, deeper staining of cytoplasm and nuclear pyknosis. In the iAs exposure group the cytoplasm of the neuronal cells deepened markedly, and the neuronal fibers were swollen. In the F-exposure group, neuronal cells were loosely and disorderly organized, and some swollen neuronal fibers were observed. In the AsF combination group, the structure of neuronal cells was even looser and more irregular. The number of cell layers and cell quantities significantly decreased, along with increased swollen neuronal fibers and cells with cytoplasmic cavitation.
In summary, our present study evaluated neurotoxicity and gutmicrobiome disturbance as a result of exposure to high-dose of iAs andF-, alone or in combination, from early life (in utero and childhood) to puberty in rats. iAs and/or F-exposure led to neurobehavioral deficits in spatial learning and memory, most prominently in offspring rats co-exposed to iAs and F-. The H&E staining results confirmed that iAs and/or F-caused pathological changes in hippocampal neuron. 16S rRNAgene sequencing results demonstrated perturbation of gut microbiomecommunities at different taxonomic levels in response to iAs and/or F-exposure. Again, the effects were more noticeable in the AsF combination group. Furthermore, nine genera, which had significant difference of relative abundance between the control and AsF combination groups, were identified to have significantly strong correlation (r= 0.70–0.90) with spatial learning and memory performance. Collectively, these results suggest that concurrent iAs and F-exposureled to more prominent effects on neurodevelopment and gut micro-biome composition structures in rats, and the strong correlation between them indicates a high potential for the development of novel microbiome-based biomarkers of iAs and/or F-associated neurodevelopmental deficits.