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Electrophysiological Methods to Assess Peripheral Pain Block in an Anesthetized Rat
Published on: November 21, 2025
Involvement of Spinal PKMζ Expression and Phosphorylation in Remifentanil-Induced Long-Term Hyperalgesia in Rats
Qi Zhao1,2, Linlin Zhang1,2, Ruichen Shu1,2
1Department of Anesthesiology, Tianjin Medical University General Hospital, Tianjin, 300052, China.
Abstract:
Up-regulation of GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) expression and trafficking is the key mechanism for remifentanil-induced hyperalgesia (RIH), nevertheless, the signaling pathway and pivotal proteins involved in RIH remain equivocal. PKMζ, an isoform of protein kinase C (PKC), maintains pain memory storage in neuropathic pain and inflammatory pain, which plays a parallel role regulated by NMDARs in long-term memory trace. In the present study, Zeta Inhibitory Peptide (ZIP), a PKMζ inhibitor, and a selective GluN2B antagonist Ro-256981 are injected intrathecally before remifentanil infusion (1 μg kg-1 min-1 for 1 h, iv) in order to detect whether GluN2B contributes to RIH through affecting synthesis and activity of PKMζ in spinal dorsal horn. Nociceptive tests are measured by Paw withdrawal mechanical threshold (PWT) and paw withdrawal thermal latency (PWL). The L4-L6 segments of dorsal horn taken from rats with RIH are for determining expression of PKMζ and pPKMζ by Western blot and immunohistochemistry. Our data suggest that remifentanil infusion causes an increase of PKMζ in expression and phosphorylation in rats with nociceptive sensitization, beginning at 2 h, peaked at 2 days, and returned to basal level at 7 days. ZIP (10 ng) could block behavioral sensitization induced by remifentanil. Ro25-6981 dosage-dependently attenuated mechanical and thermal hyperalgesia and reversed expression of PKMζ and pPKMζ, indicating that GluN2B-containing NMDA receptor facilitates development of RIH through mediating expression and activity of spinal PKMζ in rats. Although detailed mechanisms require further comprehensive study, the preventive role of Ro25-6981 and ZIP provide novel options for the effective precaution of RIH in clinics.
Insights
Remifentanil-induced hyperalgesia (RIH) involves GluN2B-containing N-methyl-D-aspartate receptors (NMDARs) and protein kinase M zeta (PKMζ). Blocking PKMζ or NMDARs with ZIP or Ro25-6981 prevents this pain sensitization.
Area of Science:
- Neuroscience
- Pharmacology
- Pain Research
Background:
- Remifentanil-induced hyperalgesia (RIH) is a clinical concern, yet its underlying molecular mechanisms, particularly the involvement of specific signaling pathways and proteins, remain unclear.
- N-methyl-D-aspartate receptors (NMDARs), specifically those containing the GluN2B subunit, and protein kinase M zeta (PKMζ), known for its role in pain memory, are implicated in pain sensitization.
Purpose of the Study:
- To investigate the role of GluN2B-containing NMDARs in the development of remifentanil-induced hyperalgesia (RIH).
- To determine if GluN2B contributes to RIH by modulating the synthesis and activity of PKMζ in the spinal dorsal horn.
Main Methods:
- Rats received intrathecal injections of Zeta Inhibitory Peptide (ZIP), a PKMζ inhibitor, or a selective GluN2B antagonist (Ro25-6981) prior to remifentanil infusion.
- Nociceptive responses were assessed using paw withdrawal mechanical threshold (PWT) and paw withdrawal thermal latency (PWL).
- Expression and phosphorylation of PKMζ in the spinal dorsal horn were analyzed using Western blot and immunohistochemistry.
Main Results:
- Remifentanil infusion significantly increased PKMζ expression and phosphorylation in the spinal dorsal horn, correlating with nociceptive sensitization.
- Intrathecal administration of ZIP blocked the development of behavioral sensitization induced by remifentanil.
- Ro25-6981 dose-dependently attenuated mechanical and thermal hyperalgesia and reversed the elevated expression and phosphorylation of PKMζ.
Conclusions:
- The findings indicate that GluN2B-containing NMDARs play a critical role in the development of RIH.
- This process is mediated through the regulation of PKMζ expression and activity in the spinal dorsal horn.
- Targeting GluN2B-containing NMDARs or PKMζ may offer effective strategies for preventing RIH in clinical settings.
