Combined Omega-3 Fatty Acid and Folic Acid Supplementation Reduces Neonatal Hypoxic-Ischemic Brain Injury via Anti-inflammatory and Anti-apoptotic Mechanisms

Abstract

Background: Neonatal hypoxic ischemia (HI) injury results in neuronal cell death, which remains clinically challenging to mitigate. Omega-3 polyunsaturated fatty acids (PUFAs) are known for their antioxidative and anti-inflammatory effects. Folic acid (FA) correlates with apoptosis in neural stem cells and neurons. Objectives: This study aimed to evaluate whether combined PUFA and FA supplementation mitigates neonatal HI brain injury by reducing apoptosis, inflammation, neurotransmitter imbalance, and electrophysiological dysfunction, thereby offering enhanced neuroprotection and functional recovery. Methods: Brain tissue damage, orthodromic population spike (OPS), and hypoxic injury potential (HIP) were measured. Amino acid neurotransmitter concentration in the hippocampus sections was measured. Markers of inflammation and apoptosis were assayed from HI-induced rat brains and lipopolysaccharide (LPS)-induced microglia BV-2 cells. Results: The HI caused severe damage to brain tissues that were potentially prevented by PUFA-FA by reducing the infarct size by 88%. The PUFA-FA treatment decreased the latency time (51 and 43 s) and increased swimming velocity (152 and 170 mm/s) on training days 3 and 5. The PUFA-FA showed an improved OPS decay time of 327 s, OPS recovery rate (62 s), and recovery amplitude (58 s). Whereas it caused an average 57% HIP incidence with a notably delayed onset (564 s) and duration (182 s). The PUFA-FA treatment also decreased the HI-induced release of amino acid neurotransmitters (Asp, Glu, and Gly) and GABA. The PUFA-FA suppressed the levels of proinflammatory cytokines and chemokines (iNOS, COX-2, TNF-α, IL-1β, and IL-6) and might mediate the inhibition of the NF-κB signaling pathway. The PUFA-FA reduced apoptosis as evidenced by lowered expression of AIF, caspase-3, and PARP genes. Conclusions: The PUFA and FA reduced HI-induced brain infarct size, with the combination showing greater protection compared to individual effects. Both improved cognitive performances, decreasing latency times and enhancing swimming velocity. The PUFA-FA supplementation synergistically restored memory, learning, and motor functions, highlighting strong neuroprotective effects against HI-induced neuronal degeneration and cognitive impairments.