Intranasal insulin reduces ADHD-like behaviors and neurodevelopmental deficits following neonatal hypoxia-ischemia in juvenile rats.

Neonatal hypoxia-ischemia (HI) is a leading cause of long-term neurodevelopmental impairment and is increasingly associated with a heightened risk of attention-deficit/hyperactivity disorder (ADHD) and related behavioral abnormalities. Beyond its metabolic role, insulin functions as a neurotrophic and immunomodulatory factor in the developing brain. However, whether early enhancement of central insulin signaling can mitigate the neuroinflammatory and behavioral sequelae of HI remains unclear. Male and female Sprague-Dawley rats were subjected to HI (right common carotid artery ligation followed by 90 min of 8% oxygen) at P10 and randomized to Sham + Vehicle, Sham + Insulin, HI + Vehicle, or HI + Insulin groups (n = 12 males and 12 females/group). Recombinant human insulin (rhInsulin) (50 μg/day) was administered intranasally once daily from P10 to P12, and behavioral and histological outcomes were assessed at P21-P25. Neonatal HI produced persistent ADHD-like behavioral abnormalities and deficits in neurobiological outcomes. Notably, sex-specific responses were observed: males exhibited greater deficits in inattention, spatial working memory, impulsivity, adaptive social development, myelination and vascularization, whereas females showed more pronounced increases in repetitive and compulsive-like behaviors. Intranasal rhInsulin treatment significantly attenuated HI-induced behavioral deficits by 100% and increased myelination (MBP+) by 64% in cingulate white matter, restored dendritic expression (MAP2+) by 56%, and reduced astrocytes (GFAP+) by 70% in hippocampal regions, indicating suppression of chronic astrogliosis neuroinflammation. Furthermore, intranasal rhInsulin increased cerebral vascular volume by 49% and normalized vessel diameters as assessed by micro-computed tomography (microCT) imaging, suggesting enhanced neurovascular integrity. While our previous study demonstrated that intranasal rhInsulin attenuated acute brain injury, neuronal apoptosis, and short-term sensorimotor deficits following neonatal hypoxia-ischemia (HI), its effects on long-term neurodevelopmental outcomes remained unclear. The present study addresses this important knowledge gap by evaluating juvenile behavioral and neurobiological outcomes through P25, including ADHD-like behaviors, social deficits, repetitive behaviors, white matter integrity, astrogliosis, cerebrovascular development, and sex-specific treatment responses. Collectively, these findings identify central insulin signaling as a key regulator of post-HI neuroimmune and neurodevelopmental trajectories and support intranasal insulin as a promising, minimally invasive therapeutic approach to reduce the long-term neurobehavioral sequelae of neonatal brain injury.