Introduction: Infantile spasms (IS), now classified as infantile epileptic spasm syndrome (IESS), is a rare, severe epileptic encephalopathy of infancy with substantial neurodevelopmental morbidity. Its interictal EEG hallmark, hypsarrhythmia, is a chaotic, high-amplitude pattern of irregular slow waves in the delta range (<3 Hz), and hypoxic-ischemic encephalopathy (HIE) is among the most common identifiable causes of IESS, accounting for up to 12% of cases. We propose that HIE-induced stress elevates corticotrophin-releasing factor (CRF) signalling, disrupting GABAergic inhibitory networks and triggering the age-specific onset of IESS; suppression of this pathway likely underlies the efficacy of first-line ACTH therapy. No existing preclinical model combines a clinically realistic HIE aetiology with CRF sensitivity. We therefore developed a novel ex vivo model combining neonatal hypoxic injury, pharmacologically induced delta oscillations as a hypsarrhythmia proxy, and CRF challenge, to test whether hypoxia-exposed networks develop IESS-like excitability and stress-sensitivity.

Aims/objectives: (1) Establish a reproducible ex vivo model of delta-frequency oscillations in developing mouse cortex, (2) determine whether neonatal hypoxic-ischemic injury alters cortical network activity and parvalbumin-positive (PV+) interneuron density, and (3) test whether hypoxia-exposed networks show increased CRF sensitivity across the postnatal window relevant to IESS (P10–P14).

Methods: All procedures accorded with EU Directive 2010/63/EU and were approved by the Trinity College Dublin Animal Welfare Body, licensed by the Health Products Regulatory Authority (AE19136/I775, AE19136/P140). Neonatal hypoxia was induced in C57BL/6J pups (P7) by exposure to 5% O2/95% N2 for 15 min at 34°C; littermate controls received a room-air sham exposure. Motor function was assessed at 24 h and 72 h post-insult. Pups were killed by decapitation for preparation of 400 μm somatosensory cortex slices. Local field potentials were recorded from layer IV/V of the barrel field; delta oscillations were induced with carbachol (2 μM) and SCH23390 (10 μM), followed by CRF (20 nM), and quantified by fast Fourier transform (area under the curve, AUC; peak amplitude; 0.5–4 Hz). Separate cohorts underwent PV immunohistochemistry (P10, P12, P14) and cFos/Arc mRNA quantification (P14). Data are mean ± SEM; comparisons used Mann–Whitney U tests, mixed-effects models, or two-way ANOVA with Šídák’s post hoc test; p<0.05 was significant.

Results: By 72 h, hypoxia-exposed pups (n=6–16 per test) showed reduced ambulation (p=0.0286), increased hindlimb foot angle (p=0.0007), impaired negative geotaxis (p=0.0129) and reduced grip strength (p=0.0122) versus controls. Hypoxia slices (N=4–5/group) showed increased delta-band AUC and amplitude at P10–P14, at baseline and following CRF (p<0.05), with CRF unmasking large, hypsarrhythmia-like discharges in hypoxia-exposed but not control tissue. PV+ interneuron counts in S1BF (N=3/group/age) were reduced at P12 (p<0.05) and P14 (p<0.0001), with layer-specific loss at P14 in layers II/III, IV and V (p<0.01–p<0.001). cFos and Arc mRNA in P14 cortex did not differ between groups (n=5/group).

Conclusions: Neonatal hypoxia produces persistent behavioural deficits, layer-specific PV+ interneuron loss, and enduring cortical hyperexcitability with heightened CRF sensitivity across a developmental window relevant to IESS. This is the first ex vivo model linking a clinically realistic HIE aetiology to CRF-driven, IESS-like network dysfunction, offering a platform for testing CRF-targeted and interneuron-directed interventions.