Neonatal Procedural Pain Disrupts Phosphorylation of KCC2 in the Spinal Cord

Abstract

Neonatal procedural pain experienced in the neonatal intensive care unit can lead to long-lasting remodeling of the central nervous system and, in particular, of the spinal nociceptive network. Preclinical studies indicate a disrupted inhibitory versus excitatory balance in the spinal cord due to reduced γ-aminobutyric acid (GABA) ergic neurotransmission. During neonatal development a GABAergic shift occurs, which is regulated by the potassium-chloride co-transporter 2 (KCC2) and its oxytocin receptor (OXTR)-dependent phosphorylation at the serine 940 residue (pKCC2). As DNA methylation of Oxtr is sensitive to early life adversity, such as neonatal procedural pain, we hypothesized that neonatal procedural pain reduces Oxtr methylation in the lumbar spinal cord and subsequently prevents the developmental increase in KCC2 and pKCC2. Using a rat model of repetitive neonatal procedural pain, four needle pricks were applied to the left hind paw every day from postnatal day (P)0 to P7. Spinal cord samples were collected at P0 and P10 to assess the levels of KCC2 and pKCC2 via Western blot analysis. Additionally, spinal Oxtr methylation was quantified using bisulfite pyrosequencing. The results indicated that neonatal procedural pain downregulates spinal pKCC2 levels, while KCC2 levels remain unchanged. These findings suggest a disrupted KCC2-dependent chloride outflow and support the hypothesis that neonatal procedural pain disrupts the GABAergic shift. A developmental decrease in pKCC2/KCC2 levels was also observed in the ipsilateral spinal cord of P10 animals, indicating the involvement of other post-translational mechanisms in the developmental regulation of spinal KCC2. Methylation of the Oxtr does not seem to be related to the disturbed GABAergic shift, given that no significant changes in Oxtr promoter methylation were detected. Overall, this study demonstrates that neonatal procedural pain disrupts spinal KCC2 phosphorylation and supports the hypothesis that neonatal procedural pain alters the GABAergic shift in the spinal cord.