5-HT1AR agonist alleviates presurgical prolonged sleep deprivation-induced postsurgical pain in adolescent mice.

Objectives: Sleep deprivation (SD) is a risk factor for the development of chronic pain in adolescents, significantly affecting pain management and prognosis; however, the mechanisms by which SD influences postoperative pain outcomes remain unclear. This study aims to investigate the molecular mechanism through which the spinal 5-hydroxytryptamine 1_A receptor (5-HT1_AR) regulates the excitation/inhibition (E/I) balance in the dorsal horn to modulate postoperative chronic pain induced by SD in adolescent mice.

Methods: A pain model combining 4.5 days of SD and a plantar incision was established in adolescent C57BL/6J mice, which were randomly assigned to 4 groups: control (C), SD, hind toe incision (I), and SD combined with hind toe incision (SI). The effects of a single intrathecal injection of the 5-HT1_AR agonist 8-hydroxy-2-dipropylamino-tetralin (8-OH-DPAT) and both single and continuous intrathecal injections of the extracellular signal-regulated kinase (ERK) inhibitor U0126 on SD-induced postoperative chronic in mice (SI+8-OH-DPAT group, SI+U0126 group, and SI+Vehicle group) were observed. Paw withdrawal mechanical threshold (PWMT) was measured on the 1st, 3rd, 5th, 7th, 10th, and 14th day before and after model induction. On the 7th day after surgery, immunofluorescence was used to assess 5-HT1_AR expression in the spinal dorsal horn, and Western blotting was employed to measure protein expression levels of 5-HT1_AR, postsynaptic density protein-95 (PSD95), N-methyl-D-aspartic acid receptor 1 (NR1), phosphorylated NR1 (p-NR1), vesicular glutamate transporter 1 (VGLUT1), gephyrin, glutamic acid decarboxylase 67 (GAD67), vesicular gamma-aminobutyric acid transporter (VGAT), ERK, p-ERK, calmodulin-dependent protein kinase II (CaMKⅡ), phosphorylated CaMKⅡ (p-CaMKⅡ), cAMP- response element-binding protein (CREB), and phosphorylated CREB (p-CREB) in the dorsal horn.

Results: 1) Behavioral pain analysis. Compared with group C, the PWMT in groups I and SD decreased significantly on the 1st, 3rd, 5th and 7th day after surgery (all P<0.05), and returned to baseline on the 10th day. Compared with group I, the PWMT in group SI was significantly lower on the 5th, 7th and 10th day (all P<0.05). The SI+8-OH-DPAT group exhibited higher PWMT values at the 1st, 2nd, and 4th hour postoperatively, as well as on the 3rd, 5th, 7th,10th and 14th day compared to the solvent control group (all P<0.05). Similarly, both single and continuous intrathecal injections of U0126 resulted in higher PWMT values at 1, 2, and 4 hours after surgery postoperatively and on the 3rd, 5th and 7th day compared to the SI+Vehicle group (all P<0.05). 2) Immunofluorescence. On the 7th day after surgery, group SI showed decreased 5-HT1_AR expression in the spinal dorsal horn, while a single intrathecal injection of 8-OH-DPAT upregulated 5-HT1_AR expression (all P<0.05). 3) Western blotting. On the 7th day after surgery, group SI exhibited decreased 5-HT1_AR expression and increased ratios of p-CaMKⅡ/CaMKⅡ, p-CREB/CREB, and p-ERK/ERK (all P<0.05). Compared with group C, group SI showed increased expression of PSD95, VGLUT1, and the p-NR1/NR1 ratio, and decreased expression of 5-HT1_AR, gephyrin, and VGAT in the dorsal horn (all P<0.05). Compared with group I, group SI demonstrated upregulated 5-HT1_AR and PSD95 expression and downregulated gephyrin, GAD67, and VGAT expression (all P<0.05). In the SI+8-OH-DPAT group, the levels of PSD95, VGLUT1, p-NR1/NR1, p-CaMKⅡ/CaMKⅡ, p-CREB/CREB, and p-ERK/ERK on the 7th day after surgery were lower than those in the SI+Vehicle group, while the expression levels of 5-HT1_AR, GAD67, and VGAT were upregulated (all P<0.05). Additionally, 2 hours after a single intrathecal injection of U0126, the SI+U0126 group exhibited significantly lower levels of p-CaMKⅡ/CaMKⅡ, p-CREB/CREB, and p-ERK/ERK compared with the SI+Vehicle group (all P<0.05).

Conclusions: The 5-HT1_AR agonist alleviates postoperative chronic pain induced by SD in adolescent mice by inhibiting the CaMKⅡ-ERK-CREB signaling pathway, thereby correcting the E/I imbalance in the spinal dorsal horn.