Development of a zebrafish platform for modeling reperfusion injury in neonatal Hypoxic-Ischemic encephalopathy:...

Reperfusion injury is a complex pathological process that exacerbates conditions such as myocardial infarction, stroke, and neonatal hypoxic-ischemic encephalopathy (HIE). In HIE, the reoxygenation phase amplifies the initial hypoxic insult by inducing oxidative stress, vascular dysfunction, and neuronal death via apoptosis and ferroptosis. Despite its clinical importance, the multifaceted nature of reperfusion injury poses challenges for experimental modeling and therapeutic screening. Here, we developed and refined a zebrafish hypoxia-reoxygenation platform that recapitulates key features of reperfusion injury associated with neonatal HIE, enabling both mechanistic studies and therapeutic validation with S-nitrosoglutathione (GSNO). Zebrafish larvae at 5 or 6 days post-fertilization (dpf) were subjected to varying hypoxia durations (5-20 min) followed by reoxygenation, with 6 dpf larvae exposed to 15 min hypoxia yielding a reproducible ∼50% survival at 48 h post-reoxygenation, providing an optimal baseline for intervention testing. This platform reproduces core pathophysiological outcomes including reduced survival, impaired motor behavior, neuronal loss, vasoconstriction, and diminished cerebral blood flow. GSNO (5-20 μM) was administered during early reoxygenation to assess dose-dependent effects. Treatment with 15 μM GSNO significantly improved survival, restored behavioral function, and mitigated neuronal and vascular dysfunction. Mechanistic analyses revealed that GSNO reduced caspase-3 expression, malondialdehyde (MDA) levels, and intracellular Fe²⁺ accumulation. These protective effects likely reflect GSNO's multifaceted mechanisms, including NO-mediated vasodilation, inhibition of apoptotic pathways, augmentation of glutathione to counter oxidative stress, and facilitation of S-nitrosation, a key post-translational modification regulating protein function and cellular signaling. Overall, this work establishes a physiologically relevant zebrafish platform for modeling reperfusion injury in neonatal HIE, demonstrating its utility for therapeutic screening and underscoring GSNO's potential as a multi-target protective agent in this setting.