Uncovering system-specific stress signatures in primate teeth with multimodal imaging

Abstract

Early life stress can disrupt development and negatively impact long-term health trajectories. Reconstructing histories of early life exposure to external stressors is hampered by the absence of retrospective time-specific biomarkers. Defects in tooth enamel have been used to reconstruct stress but the methods used are subjective and do not identify the specific biological systems impacted by external stressors. Here we show that external physical and social stressors impart biochemical signatures in primate teeth that can be retrieved to objectively reconstruct the timing of early life developmental disruptions. Using teeth from captive macaques, we uncovered elemental imprints specific to disruptions of skeletal growth, including major disruptions in body weight trajectory and moderate to severe illnesses. Discrete increases in heat shock protein-70 expression in dentine coincided with elemental signatures, confirming that elemental signals were associated with activation of stress-related pathways. To overcome limitations of conventional light-microscopic analysis, we used high resolution Raman microspectral imaging to identify structural and compositional alterations in enamel and dentine that coincided with elemental signatures and with detailed medical and behavioural data. Integrating these objective biochemical markers with temporal mapping of teeth enables the retrospective study of early life developmental disruptions and their ensuing health sequelae.

Figure 1. Biochemical signatures in MMU401 permanent first molar during multiple stress events.

(a) Ba distribution map (as Ba/Ca) shows multiple bands of increased concentration. Nursing signal is also evident (MM). Timing of Ba bands was determined by histological analysis in (c). (b) Area highlighted in (a). Discrete bands of high Ba are shown by arrowheads numbered 1 to 8 corresponding to this animal’s medical history (f,g). (c) Light micrograph with accentuated lines labelled according to g. The neonatal line is labelled 0. (d) Grey scale Raman microspectroscopic map showing variation in the organic biomolecular matrix (874 cm⁻¹ band intensity). Raman analysis was performed on the opposing face tooth block of the thin section in (a–c). Elemental analysis was also performed on this block and the Ba/Ca map has been overlaid to show correlation of chemical bands. (e) Area highlighted in (d). Discrete bands are shown by arrowheads corresponding to events in medical history. Additional bands not observed in the light microscopy map are visible, for example band H. (f) Percent weight change over consecutive measurements as proxy for skeletal growth trajectory. Severe disruptions in normal weight gain trajectory are indicated by numbers coincident with medical events (g), bands of increased Ba (b), accentuated lines apparent under light microscopy (c), and accentuated lines in the Raman map (e). Weight measurements were not available before 150 days of age. (g) Summary of animal’s medical history. Event numbers correspond to data in (b,c,e).

Figure 2. Biochemical signatures in MMU336 permanent third molar during multiple stress events.

(a) Ba distribution map (as Ba/Ca) shows multiple bands of increased concentration. Timing of Ba bands was determined by histological analysis in (c). (b) Area highlighted in (a). Discrete bands of high Ba are shown by arrowheads numbered 1 to 9 corresponding to this animal’s medical history (f,g). (c) Light micrograph of third molar showing accentuated lines (developmental timing of these lines is shown in f). The section of enamel analysed by Raman microspectroscopy is overlaid and shows close agreement with accentuated lines in enamel. (d) Enlarged section of Raman microspectroscopic map shown in (c) with discrete bands shown by arrowheads corresponding to events in medical history (f). (e) Overlay of Ba/Ca map from a and Raman map from d showing correlation between the two techniques. Raman maps shows additional bands due to finer resolution. (f) Summary of macaque’s medical history. Event numbers correspond to data in panels (b−d). (g) Percent weight change over consecutive measurements as proxy for skeletal growth trajectory. Severe disruptions in normal weight gain trajectory are indicated by numbers coincident with medical events (f), bands of increased Ba concentration (b), accentuated lines apparent under light microscopy (c), and accentuated lines in the Raman map (d).

Figure 3. Imprint of stress events in dentine confirmed by heat shock protein (HSP70) expression.

(a) HSP70 distribution in dentine of MMU401 molar shown in Fig. 1. Seven increases in HSP70 content coincide with events shown in Fig. 1b,e. Variations in intensity of HSP expression are apparent; low expression, blue rectangle, and high expression, red rectangle. (b) High-resolution micrograph of dentine tubules in area highlighted in blue box in a. Structure of dentinal tubules shows minor disruptions with appearance of lateral strands (one such area is indicated by arrowhead). (c) Oblique view of area showing moderate expression of HSP70. Lateral strands can be seen in areas of stress lines (arrowheads). (d) High-resolution micrograph of dentine tubules in area highlighted in red in a. Complete disruption of tubular structure in an area of high HSP70 content corresponding to a stress event (bounded by arrowheads), with normal tubular structure before and after the event. Scale bars = 20 μm.

Figure 4. Conceptual framework of biochemical imprints in dentine.

(a) Three discrete hypothetical exposures to external stressors (labelled 1–3) which cause bodily systems to depart from homeostasis (dashed lines). (b) Disruption of skeletal homeostasis in response to stress events. Bone and blood elemental levels are in equilibrium under homeostasis. When external stressors disrupt bone remodelling, a net increase in elemental transfer to blood results in increased elemental deposition at the mineralizing front of dentine as discrete bands associated with the timing of stress events (e). (c) Disruption of odontoblastic homeostasis results in increased HSP70 content in dentine matrix. Higher levels of HSP70 during stress events results in a banding pattern evident in dentine. (d) Disruption of odontoblast function in response to external stressors will result in the deposition of an altered biomineral matrix composition. Changes in carbonate substitution or protein conformation will imprint a molecular signature in discrete regions of dentine formed during the stress exposure. (e) Representation of biochemical imprints in dentine that can be directly linked to stress events 1–3 shown in (a). Enamel (E) and pulp (P) are not depicted.