SUMMARY The prevalence of Neonatal Opioid Withdrawal Syndrome (NOWS) has risen 5-fold in the past decade, and is a well-recognized consequence of perinatal opioid exposure (POE). By contrast, the long-term damage to the developing brain from opioid medications is just beginning to be recognized as a serious concern. No study has directly addressed repair of the developing brain secondary to opioid-induced neural injury. Thus, in response to this immense gap in knowledge and significant need for therapeutic strategies that improve brain health and function in vulnerable infants with opioid exposure, we propose to test a clinically-viable therapeutic regimen of the pleiotropic, endogenous neuroreparative molecules, erythropoietin (EPO) plus melatonin (MLT), for the treatment of perinatal opioid-induced brain injury. EPO and MLT are both endogenous developmentally- regulated neurorestorative agents with pluripotent mechanisms, including established anti-inflammatory properties. Our published data suggest that opioid exposure commencing in utero negatively affects the maturation of the neural-immune system, and trajectory of central nervous system (CNS) development. Specifically, methadone induces peripheral immune hyper-reactivity (SPIHR), lasting structural and microstructural brain injury, and significant deficits in executive function and cognitive control in adult animals following in utero exposure. Our central hypothesis is that a neonatal combination therapy with EPO plus MLT will mitigate cognitive and executive function deficits, structural and functional connectivity abnormalities, and neural-immune dysregulation in a validated preclinical model of perinatal methadone exposure. To investigate this hypothesis we will: 1) test that neonatal combined EPO+MLT therapy attenuates deficits of cognitive control and executive function in adult animals following perinatal methadone exposure; 2) test whether combined EPO+MLT therapy mitigates structural and functional brain injury in adulthood following perinatal methadone exposure; and 3) test that combined EPO+MLT therapy diminishes a sustained systemic pro-inflammatory microenvironment propagated by perinatal methadone exposure. Using state-of-the-art preclinical magnetic resonance imaging (MRI), including diffusion tensor (DTI) and functional connectivity (FcMRI), in concert with translational touchscreen cognitive assessment, we will rigorously test structure-function relationships secondary to perinatal opioid exposure and potential repair/recovery with EPO+MLT treatment. We will focus on neural networks and major white matter tracts essential to cognition and executive function. A clinical biomarker platform, multiplex electrochemiluminescent immunoassay (MECI), will be used to assay proinflammatory cytokines and chemokines over the lifespan and in response to EPO+MLT treatment. These investigations will be the first to examine a clinically-viable, mechanistically-targeted intervention for the neural injury associated with perinatal opioid exposure. Our long-term goal is to improve the brain health and functional, long-term outcome of children with perinatal opioid exposure.

NARRATIVE Infants exposed to opioids prenatally are prone to neurodevelopmental issues later in life. We propose that a neonatal combination therapy of erythropoietin plus melatonin will be an effective neurorepair strategy for perinatal opioid induced brain injury. We will validate this combination therapy as a clinically viable means of preventing white matter injury, aberrant functional connectivity, and executive function deficits secondary to perinatal opioid exposure in a translational preclinical model.