Stress during lactation: a hidden link to offspring bone health
Early life resource limitation is one factor that could have a major impact on child health and development. Thus, using a rat model of limited bedding and nesting (LBN), we investigated the postnatal bone development, mineralization, and microarchitecture. Pregnant Sprague-Dawley rats were subjected to a LBN model to induce chronic early life stress (CES), while a control group was maintained under standard conditions. The offspring were assessed at postnatal day (PND) 10, 21, and 35. Tibial length was measured, and tibial and lumbar vertebral bone mineral density (BMD), content (BMC), and area (BMA) were assessed using dual-energy X-ray absorptiometry (DXA). Bone microarchitecture was examined using microcomputed tomography (µCT). Changes in gene expression from the lumbar vertebrae were analyzed by transcriptome. At PND 10, there were no significant differences in BMD and BMC between the treatment groups, but tibial length was significantly decreased by CES. By PND 21, tibial BMC and BMA were significantly reduced in the CES group, indicating impaired bone mineral accumulation. At PND 35, tibial length remained significantly reduced by CES, while BMD and BMC differences were less affected. Vertebral BMA and BMC were reduced by CES. µCT analysis of tibial cortical bone showed significant changes in cortical thickness and bone volume at PND 10 and 21, respectively. For the lumbar vertebrae, µCT data indicated significant increases in the degree of anisotropy and structural model index at PND 21 and 35, respectively. Transcriptome analyses revealed significant differential expression of genes involved in immune response, cellular repair, and stress adaptation at PND 21 but not at PND 10 and PND 35. CES significantly disrupts BMC, BMD, length and microarchitecture differently at various stages of postnatal development. Transcriptome analyses suggest that these changes are mediated by alterations in gene expression related to immune function and cellular repair. Future research should focus on tracking the longitudinal impacts of CES on bone health from infancy into adulthood, and exploring nutritional interventions, stress reduction programs, and molecular studies that can mitigate the negative effects of CES on bone. Overall design: Animal experiments were performed at Arkansas Children's Research Institute as approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences (Little Rock, AR). Rats were housed in 12-hour light/dark conditions with ad libitum access to a standard rodent chow (Lab diets, Prolab RMH1800, St. Louis. MO) and water.  Timed pregnant Sprague-Dawley rats (Charles River, Wilmington, MA) were delivered to the vivarium on gestational day 18.  Dams delivered pups without intervention on PND 0. On PND 2, the dams were randomly transferred to cages containing a standard plexiglass cage with ample sawdust and 3 paper towels to use as nesting material (STD), or an impoverished cage (CES) which had limited sawdust which was inaccessible to the dam by a wire mesh platform which sat 2cm above the cage floor and one half of one paper towel was provided as nesting material (Figure 1). Pups were randomly distributed to dams until each litter contained 12 pups with equal male: female ratio. These cages then remained undisturbed for 7 days. On  PND 10, all litters were transferred to and maintained in standard cages until offspring were weaned on PND 21. On PND 10, 21 and 35, equal numbers of offspring were anesthetized with isoflurane and euthanized by decapitation. The bilateral tibia and lumbar vertebra were collected and stored at -80°C until analyses.