Hypoxic-ischaemic encephalopathy is a leading cause of neonatal neurological injury, with neuroinflammation contributing to both disease progression and long-term impairment (Tetorou et al., 2021). Neonatal hypoxia induces peripheral immune infiltration and alters glial activation within the developing brain (Li et al., 2017). However, it remains unclear whether early-life hypoxic injury reprograms the neuroimmune response to subsequent inflammatory challenges later in life. This study characterised acute peripheral immune infiltration following neonatal hypoxia and assessed whether prior hypoxic injury alters inflammatory signalling, myeloid recruitment, and microglial phenotype after a subsequent peripheral immune challenge.

C57BL/6J pups were subjected to global hypoxia (5% O2/95% N2) or normoxia for 15 mins on postnatal day 7. Brains were collected at 3, 6, 24, or 72 hours post-hypoxia, and CD45+ immune populations were quantified by flow cytometry (n = 4–6 per group per time point). Additional cohorts also received lipopolysaccharide (0.3 mg/kg , i.p.) or saline at P21. Cortex, hippocampus, and dura mater were collected 3 hours post-injection for flow cytometry, ELISA, and qPCR (n = 3–6 per group), and 24 hours post-injection for immunohistochemistry (n = 3–4 per group). Microglial morphology was quantified by Iba1 immunofluorescence and analysed using FIJI with unsupervised clustering. All procedures were approved by the Trinity College Dublin Animal Research Ethics Committee and performed under authorisation from the Health Products Regulatory Authority in accordance with European Union Directive 2010/63/EU and S.I. No. 543 of 2012; animals were culled by transcardial perfusion under terminal pentobarbital anaesthesia. Data are presented as mean ± S.E.M. and were compared by unpaired t-test or two-way ANOVA with Tukey’s multiple comparisons test.

Neonatal hypoxia increased brain neutrophils at 6 hours, CD8+ T cells at 6 and 24 hours, CD4+ T cells at 24 hours, and B cells at 6 and 24 hours (all P < 0.05). Following lipopolysaccharide administration at P21, hypoxia-exposed mice exhibited fewer cortical monocytes (P < 0.01) and macrophages (P < 0.05) compared to challenged controls, while dura mater populations remained unchanged. This attenuated recruitment occurred despite elevated cortical IL-1β (P < 0.001), markedly lower CCL2 (P < 0.01), and reduced expression of the endothelial activation marker Sele (P < 0.0001). Morphometric clustering identified homeostatic, hyper-ramified, and reactive-like microglia. After challenge, hypoxia-exposed mice had fewer homeostatic microglia in the cortex (P < 0.05) and a shift toward a hyper-ramified phenotype (P = 0.068).