300 - Role of sildenafil in neuronal plasticity after neonatal hypoxic-ischemic encephalopathy: Linking morphological recovery with proteomics profiling

Publication Number: 4294.300

Shubhendra Kumar. Mishra, Mcgill University, montreal, PQ, Canada; Seline Vancolen, McGill University, Montreal, PQ, Canada; Math Chevin, research institute of the McGill University Health Center, Montreal, PQ, Canada; Guillaume Sebire, McGill University Faculty of Medicine and Health Sciences, Montreal, PQ, Canada; Pia Wintermark, McGill University Faculty of Medicine and Health Sciences, Montreal, PQ, Canada

Background: Perinatal asphyxia is a major cause of neonatal brain injury (~3/1000 live births) leading to hypoxic-ischemic encephalopathy (HIE). Despite therapeutic hypothermia (TH), 40–60% of treated infants die or develop long-term neurodisability, underscoring the need for adjunctive neurorestorative strategies. Sildenafil citrate (SDF), a phosphodiesterase-5 inhibitor that elevates cGMP–PKG signaling, shows neuroprotective promise, but neuronal mechanisms remain insufficiently defined.

Objective: To elucidate how sildenafil promotes neurorestoration after neonatal hypoxia-ischemia (HI), linking morphological recovery with proteomics profiling

Design/Methods: Primary rat cortical neurons (E18) were cultured and exposed to HI (1% O₂/glucose deprivation, 6h) at DIV8, followed by SDF (150 nM, 48h). Neuronal structure was quantified via MAP2/Tuj1 immunocytochemistry and IMARIS (n=6/group: control, HI, HI+SDF). Label-free proteomics (n=5/group) and IPA/GO enrichment examined synaptogenesis, cytoskeleton, mitochondrial metabolism, and AMPK–mTOR–autophagy signaling.

Results: HI induced severe structural degeneration vs control, reducing dendrite length (42.6±29.6 µm vs 122.9±42.4 µm, p< 0.005), dendrite number (0.66±0.80 vs 3.0±0.83, p< 0.005), neurite length (40.8±17.3 µm vs 216.3±81.9 µm, p< 0.005), and branch number (1.5±1.0 vs 6.9±2.8, p< 0.005). SDF significantly reversed HI-induced deficits, restoring or exceeding control levels: dendrite length (162.7±45.6 µm), dendrite number (3.5±1.1), neurite length (220.3±80.1 µm), and branch number (6.0±2.4; all p< 0.005 vs HI). Proteomics showed HI suppressed synaptogenesis, actin nucleation, and exocytic machinery, while activating stress metabolism. SDF reinstated synaptogenesis and vesicle-exocytosis programs, enhanced cytoskeletal protein-binding and Arp2/3-mediated actin remodeling, and restored mitochondrial pathways (import machinery, L-carnitine shuttle). SDF shifted AMPK–mTOR signaling toward growth-permissive protein translation and reduced autophagy/CLEAR pathway activity. Key plasticity-associated candidates included GAP43 (axonal/dendritic growth), WASF1/Arp2/3 (spine remodeling), EEF1A2 (translation), and LC3/TFEB (autophagy balance), suggesting coordinated structural–metabolic reprogramming

Conclusion(s): : SDF drives robust neuronal recovery after HI and reprograms proteomic networks toward synaptic, cytoskeletal, and mitochondrial restoration, favoring mTOR-driven growth. These findings identify mechanistic biomarkers of SDF-mediated neurorestoration and support SDF as a feasible adjunct to TH, including relevance for resource-limited settings.