Abstract Repetitive neonatal painful procedures experienced in the neonatal intensive care unit (NICU) are known to alter the development of the nociceptive system and have long-lasting consequences, notably lower post-operative µ-opioid receptor levels in the spinal cord. Given the influence of the NICU on the epigenome, the present study hypothesized that neonatal procedural pain alters the DNA methylation status of the opioid receptor mu 1 encoding gene ( Mor-1 ) in the spinal cord and dorsal root ganglions (DRGs). To this end, the needle prick model of repetitive neonatal pain was used, and methylation of Mor-1 promotor was assessed in the spinal cord and the DRG using bisulfite pyrosequencing. Our findings demonstrated that neonatal procedural pain increased spinal cord Mor-1 promotor DNA methylation in the ipsilateral side as compared to the contralateral side, an effect that was not observed in the control animals, nor in the DRG. We also identified a behaviorally-associated CpG site following neonatal needle pricks. This study is the first to highlight a localized and noxious-stimuli-dependent effect of repetitive neonatal procedural pain on Mor-1 promotor methylation and emphasizes the need to explore the effects of repetitive neonatal procedural pain on the epigenome. Impact This study reveals that repetitive neonatal procedural pain increases DNA methylation of the Mor-1 promoter in the spinal cord of neonatal rats. This is the first study to identify an effect of neonatal procedural pain on DNA methylation, emphasizing the critical need for further investigation into the epigenetic consequences of neonatal procedural pain. These insights could lead to better management and treatment strategies to mitigate the long-term impacts of early pain exposure on neurodevelopment and behavior.

Repetitive neonatal pain increases spinal cord DNA methylation of the μ-opioid receptor