Teeth and Dental Development as a Peripheral Marker of Early Life Stress Exposure in Mice

Abstract

Introduction: Early life stress (ELS) has been shown to have long-lasting effects on human and murine development. In humans, ELS has been shown to increase the risk of anxiety, depression, and substance abuse later on in life. In mice, ELS in the form of limited nesting and bedding material has been shown to have persistent effects on adult murine behavior, with more persistent effects observed in males. Compared to females, males show an accelerated development of fear conditioning and significant depression and anxiety-like behavior. Conversely, females show delayed sexual maturation and cognitive function; these manifestations seemingly resolve with age. Objectives: We asked whether dental development tracks the deviations in development and allows for the use of teeth as a potential biomarker of early life adversity. We tested whether the expression of receptors that link neural development with dental development can be used as mechanistic markers of altered tooth mineralization. Material and Methods: Eighty-seven C57BL/6J mice from ELS and control groups were sacrificed at postnatal days 12 (n=49) and 78-83 (n=38). Whole heads were fixed in 10% zinc-formalin and, after rinsing, stored in 50% ethanol. To analyze craniofacial and dental development, whole heads and mandibles were scanned at 6 μm voxel size (Scanco μCT-40). Image stacks were analyzed using ImageJ, Imaris, and Amira software. For immunohistochemistry (IHC) and immunofluorescence (IF) procedures, soft tissue was removed, and fixed mandibles were dehydrated, embedded in paraffin, and sectioned. Kallikrein-related peptidase 4 (KLK4), androgen receptor (AR), and gamma-aminobutyric acid alpha one (GABA-α1) receptor expression were evaluated. Primary antibodies used were: rabbit polyclonal anti-GABA A receptor alpha 1 antibody, rabbit monoclonal recombinant anti-androgen receptor antibody, and rabbit polyclonal anti-KLK4 receptor antibody. Secondary antibodies used for IHC and IF, respectively, were biotinylated goat anti-rabbit IgG and goat anti-rabbit Alexa Fluor 594. For elemental maps of mature, erupted enamel and dentin, fixed mandibles were embedded in Epo-Tek 301-1 epoxy resin and polished to the sagittal midplane of the incisor. Laser ablation-inductively coupled mass spectrometry (LA-ICP-MS) analysis was performed. Semi-quantitative analyses of sodium, potassium, magnesium, phosphorous, sulfur, chloride, iron, nitrogen, and carbon normalized to calcium were performed using Iolite 4 software (Elemental Scientific, Inc.). Results: Our μCT findings show 6.9% (p=0.01) thinner incisor enamel in adult males exposed to ELS. No significant differences in molar or incisor enamel thickness are seen in adult females. On day 12, ELS males show 59% thinner molar enamel (p.0001), and females show 49.9% (p.0001) thinner molar enamel than controls. Moreover, differences in mandibular first molar mineral density are observed between male and female ELS groups compared to controls (n=49)(p.05). To our knowledge, this is the first study to show the presence of GABA- α1 receptors on mature ameloblasts of the continuously developing adult mouse incisor. We find differences in the timing of GABA- α1 expression between ELS and controls, with a slightly accelerated expression in ELS males. We also find a more robust expression of AR in the dental epithelium of ELS and control males compared to females. Our LA-ICP-MS analyses show differences in the hardness of enamel and dentin between ELS groups and controls. However, no differences in elemental composition were observed between groups. Conclusion: The results of this study are consistent with the overall findings of sex-dependent responses to stress and an altered pace of development. To our knowledge, this is the first study to demonstrate the connection between ELS and GABA- α1 expression in dental tissues. Ultimately, our findings support the role of dental tissues as a possible biomarker of early life adversity.