Baudat Mathilde, Joosten Elbert A J, Simons Sinno H P, Hove Daniël L A van den, Riemens Renzo J M
Department of Anesthesiology and Pain Management, Maastricht University Medical Centre+, Maastricht, the Netherlands.
Department of Translational Neuroscience, Mental Health and Neuroscience Research Institute, Maastricht University, Maastricht, the Netherlands.
Dev Neurobiol. 2025 Oct;85(4):e22993. doi: 10.1002/dneu.22993.
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.
新生儿重症监护病房中经历的新生儿程序性疼痛可导致中枢神经系统，尤其是脊髓伤害感受网络的长期重塑。临床前研究表明，由于γ-氨基丁酸（GABA）能神经传递减少，脊髓中抑制性与兴奋性平衡被破坏。在新生儿发育过程中会发生GABA能转变，这由氯化钾共转运体2（KCC2）及其在丝氨酸940残基（pKCC2）处的催产素受体（OXTR）依赖性磷酸化调节。由于Oxtr的DNA甲基化对早期生活逆境敏感，如新生儿程序性疼痛，我们推测新生儿程序性疼痛会降低腰段脊髓中Oxtr的甲基化，进而阻止KCC2和pKCC2的发育性增加。使用重复性新生儿程序性疼痛大鼠模型，从出生后第（P）0天到P7天，每天对左后爪进行四次针刺。在P0和P10收集脊髓样本，通过蛋白质免疫印迹分析评估KCC2和pKCC2的水平。此外，使用亚硫酸氢盐焦磷酸测序法定量脊髓Oxtr甲基化。结果表明，新生儿程序性疼痛下调脊髓pKCC2水平，而KCC2水平保持不变。这些发现表明KCC2依赖性氯化物流出受到破坏，并支持新生儿程序性疼痛破坏GABA能转变这一假说。在P10动物的同侧脊髓中也观察到pKCC2/KCC2水平的发育性降低，表明其他翻译后机制参与脊髓KCC2的发育调节。鉴于未检测到Oxtr启动子甲基化的显著变化，Oxtr的甲基化似乎与GABA能转变紊乱无关。总体而言，本研究表明新生儿程序性疼痛会破坏脊髓KCC2磷酸化，并支持新生儿程序性疼痛改变脊髓GABA能转变这一假说。
