Combined Maternal Immune Activation and Prenatal Intermittent Hypoxic Stress Lead to Developmental Motor Deficits in Rats: a Two-hit Animal Model for Cerebral Palsy Across Different Age Trajectories.

Cerebral palsy (CP) is a prevalent neurodevelopmental disorder associated with motor and cognitive impairments. Inflammation and hypoxia are key contributors to CP pathogenesis, often acting synergistically to disrupt brain development. However, an understanding of the interplay between prenatal inflammation and perinatal brain hypoxic stress in animal models of the disease remains unclear. Hence, we designed an experimental approach to investigate the pathophysiological mechanisms of combined maternal immune activation and intermittent hypoxic stress (IHS) in rats, aiming to mimic the clinical conditions of CP. Pregnant rats received lipopolysaccharide (0.1 mg/kg, i.p.) on gestational day 15 and were exposed to IHS (10 min, twice daily) from day 17 to delivery. Offspring were divided into four groups: saline control, LPS, IHS, and LPS + IHS. Behavioral assessments across infancy, adolescence, and adulthood consisted of open-field, negative geotaxis, grip strength, beam walk, and pole tests. Biochemical mechanisms of oxidative/nitrergic stress, neuroinflammation, neurotrophic factors, cholinergic function, and hypoxia-inducible factor 1-alpha (HIF-1α) were analyzed in the prefrontal cortex, striatum, and cerebellum. Male offspring exhibit severe impairments, including elevated TNF-α, nitrite, lipid peroxidation indicated by increased malondialdehyde, reduced cholinergic system, and suppressed IL-4 and antioxidant enzymes. Females showed delayed region-specific impairments, with relative preservation of antioxidant capacity. Both offspring show altered BDNF levels, showing reductions in the PFC and STR as well as in the cerebellum, while dopamine was depleted in the STR and CER across both sexes. HIF-1α expression revealed interactive hypoxic-inflammatory signaling. Overall, the dual-hit model induced sex- and region-dependent neurodevelopmental deficits, with males more prone to CP-like outcomes via converging oxidative, inflammatory, and hypoxic mechanisms.