Fetal exposure to elevated levels of glucocorticoids (GCs) during pregnancy, whether due to maternal stress or the use of antenatal corticosteroid therapy (often prescribed to women at risk of preterm birth), can disrupt normal brain development and increase susceptibility to psychiatric disorders later in life. In male offspring, this has been associated with behaviors relevant to schizophrenia, while in females, it is linked to depression-like symptoms. Previous studies have shown that prenatal exposure to synthetic GCs, such as dexamethasone (DEX), impairs neurogenesis and dendritic spine development, however the impact of prenatal stress on the gliovascular interface remains poorly understood. This interface, the interaction between astrocytes and blood vessels, is critical for brain development and function. Our research aims to explore how prenatal GC exposure impacts gliovascular maturation and potential sex differences across brain regions.

 

To investigate the effects of prenatal stress on this interface, we administered either saline (control, n=5) or 50 μg/kg of DEX (n=5) subcutaneously to pregnant mice from gestational days 16 to 18. At postnatal day 14 (P14), we collected brains from male and female offspring and label astrocyte endfoot processes using aquaporin-4 (AQP4), a water channel protein that plays a crucial role in maintaining brain homeostasis by regulating water and ion balance. Brain microvessels were labelled with lectin (LEC). Brain slices were then imaged using a slide scanner, confocal and high-resolution Stimulated Emission Depletion (STED) microscopes. Protein expression and vessel tracing were analyzed with Fiji software. A two-way ANOVA (p<0.05) tested the effects of treatment (CTR vs DEX) and sex (female vs male).

 

Physiologically, we observed sex-specific differences in regional brain volume, with males displaying a wider somatosensory cortex and cerebellum at P14. However, there were no significant differences between sexes in AQP4 expression or vascular endfoot coverage. When examining the vasculature, the only notable difference was in the cerebellum, where males had shorter vessel segments compared to females. Our findings also highlighted a more pronounced impact of prenatal stress on female offspring. In the hippocampus of DEX-treated females, we detected an increased expression of AQP4 within astrocytic endfeet, suggesting alterations in astrocyte function due to prenatal stress. In the prefrontal cortex of DEX-treated females, AQP4 showed both increased expression and greater co-localization with blood vessels compared to controls, indicating a shift in localization. Interestingly, this shift occurred without changes in vascular density or segment length. Furthermore, we observed an increased frequency of vessel tortuosity in nearly all brain regions of female offspring, a phenomenon that was not seen in males.

 

In conclusion, this study underscores the importance of understanding how prenatal GC exposure, a known consequence of maternal stress and medical treatments, disrupts the gliovascular interface, with particularly pronounced effects on female offspring. These alterations could have lasting implications for brain function and may contribute to sex-specific vulnerabilities to psychiatric disorders.

 

Animal procedures were approved by the Animal Welfare Committee (iCBR, ORBEA 03/2021) and performed by FELASA-licensed users, following European (2010/63/EU) and Portuguese law (Decreto-lei nº 113/2013).