Background: Preterm infants born before 32 weeks frequently experience chronic intermittent hypoxia (CIHH) due to cardiorespiratory instability and are at heightened risk of nosocomial gram-positive infection. Both stressors independently compromise blood-brain barrier (BBB) integrity and activate neuroinflammation, yet their combined hypothalamic effects in early life remain poorly understood.
The paraventricular nucleus (PVN) is a critical integrator of the stress response, coordinating HPA axis activation via corticotrophin-releasing hormone (CRH), sympathetic output via tyrosine hydroxylase (TH)-positive, and release of oxytocin (OXT) and arginine vasopressin (AVP).
AIM: Using a neonatal rat model of chronic oxygen dysregulation and subsequent acute gram-positive bacterial challenge (LTA+PGN), we sought to characterise the model examining the potential autonomic changes associated with the stressors.
Methods: Study was conducted following HRPA approval and in line with European guidelines. Dams and litters were either raised at room air or placed in an environmental chamber where FiO2 was manipulated to generate intermittent hypoxia (8% FiO2)/hyperoxia (30% FiO2)/normoxia (21% FiO2) (CIHH) between postnatal day (PND) 3 and 12. On PND13, pups received intraperitoneal saline or LTA+PGN. Animals were assigned to different experimental assessments a) plasma hormone analysis (multiplex assay), b) plasma cytokine analysis (multiplex assay), c) BBB permeability (Fitc-dextran extravasation) e) ultrasonic vocalisation monitoring in vivo (Ultravox and DeepSqueak). Brains from group (e) were harvested for neural characterisation by immunofluorescence for OXT, CRH, AVP, TH and c-Fos. Data was analysed using 3-way ANOVA.
Results: LTA+PGN challenge increased plasma inflammatory cytokines, with IL-1β, IL-6, and IL-10 showing significant synergistic effects on concentrations when pre-exposed to CIHH. LTA+PGN elevated circulating progesterone and cortisol concentrations. LTA+PGN increased the extra/intravascular fluorescence ratio in the magnocellular and intermediocellular regions of the PVN (Gas × LTA+PGN, p=0.02 and p=0.01 respectively). Preliminary histological data (n=3) illustrate that OXT⁺ and CRH⁺ cell numbers were unaffected by treatment or sex. In the magnocellular region, c-Fos⁺ cell counts and percentage of c-Fos⁺ OXT⁺ neurons were increased with CIHH exposure (gas; ). In the intermediocellular region, c-Fos⁺ cell counts are increased with CIHH () and LTA+PGN (), while the percentage of c-Fos⁺ CRH⁺ neurons were increased in males LTA+PGN (interaction LTA+PGN x Sex ) in sex and LTA+PGN conditions. USV features did not differ between experimental groups.
Conclusions: Increased PVN permeability may facilitate cytokine access to the hypothalamus and contribute to increased neuronal activation observed. Yet, despite evidence of inflammatory, vascular, and neuronal responses, data suggest that these interventions did not alter USV behaviour, suggesting that these stressors may activate hypothalamic pathways without producing measurable autonomic changes at this developmental stage.