Caesarean section birth Is associated with sex-dependent alterations in prefrontal cortex glial development at Wweaning

Mode of delivery influences early-life exposure to the microbiome and immune system, with potential consequences for neurodevelopment. Differences in microbial colonisation between mice born by Caesarean section (CS) or vaginal birth (VB) can alter the priming of both the immune system and the central nervous system. Weaning represents a critical developmental window during which the immune system and brain undergo coordinated maturation. The transition to solid food reshapes the microbiome and activates immune responses essential for long-term immunity; however, the impact of this period on brain development remains unclear. Whether early-life microbial signatures differentially affect the prefrontal cortex (PFC), a region involved in the regulation of social behaviour and responsive to immune signals, has yet to be determined.

Using a mouse model of Caesarean section, we assessed the impact of altered microbial colonisation at birth on the PFC at weaning (postnatal day 21; p21) in male and female mice, compared with VB controls. Following perfusion, single-cell suspensions were generated and sorted for CD11b⁺ (microglia) and ACSA2⁺ (astrocytes). Cell populations were quantified by flow cytometry across delivery mode and sex. RNA was extracted from sorted cells, and gene expression was analysed using qRT-PCR to assess markers of microglial and astrocytic function. In parallel, immunohistochemistry at p21 and p28 was performed to quantify Iba1⁺ microglial number and morphology, as well as astrocyte-associated function via aquaporin‑4 (Aqp4).

At p21, microglial number did not differ between CS and VB mice. However, at p28, male CS mice exhibited a significant reduction in microglial number compared to VB males (p<0.02). Morphological analyses revealed marked reductions in branch number (p<0.01), branch length (p<0.04), connecting endpoints (p = 0.02), and overall complexity (p<0.03) in CS males compared with VB males. In contrast, microglia from CS females showed no significant changes in complexity, size, or shape. Gene expression analyses of sorted PFC microglia at p21 demonstrated increased expression of Tnfα, Fos, and Lamp1 in CS males, while CS females showed elevated Il1β and Fos expression, indicating sex-specific immune activation profiles compared with their VB counterparts.

Astrocyte numbers in the PFC at p21 were unaffected by delivery mode in either sex. Immunohistochemical analyses revealed that CS females exhibited reduced Aqp4 coverage and decreased perivascular sheet thickness around blood vessels, an effect not observed in males. Consistent with these findings, Aqp4 gene expression was significantly reduced in sorted astrocytes from CS females. Markers associated with synaptic function (Agt, Ndrg4, and Slc7a10) were altered by delivery mode in both sexes. Agt expression was increased in CS females, whereas Ndrg4 and Slc7a10 were significantly reduced in CS males, with similar but non-significant trends observed in females. Together, these findings demonstrate sex-specific effects of altered early-life microbial colonisation on glial development and function within the PFC. Understanding interactions between the microbiome, immune system, and brain during critical developmental windows may provide insight into mechanisms underlying susceptibility to neurodevelopmental disorders.

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