TLR3 mediates central sensitization in a chronic migraine model induced by repeated nitroglycerin through the ERK signaling pathway

Background: Studies have demonstrated that Toll-like Receptor 3 (TLR3) plays a crucial role in neuropathic pain. However, there have been no relevant reports regarding the role of TLR3 in migraine chronification. This study aims to investigate the molecular mechanisms of TLR3 in the central sensitization of chronic migraine (CM).

Methods: C57BL/6 male mice were used as models for chronic migraine (CM) disease, receiving an intraperitoneal injection of nitroglycerin (NTG) every Other day. Calibrated von Frey filaments were employed to measure the pain threshold in the hind paw sole and periorbital region, enabling the assessment of mechanical allodynia. Western blot was employed to detect the expression changes of TLR3, TRAF6, TAK1, c-Fos, Calcitonin gene-related peptide (CGRP), and the extracellular signal-regulated kinase (ERK) signaling pathway. Immunofluorescence was used to detect the cellular localization of TLR3 and the expression changes of central sensitization-related indicators, such as c-Fos and CGRP. In addition, we investigated the effects of TLR3 Inhibitor (CU CPT4a), MEK Inhibitor(PD98059), TRAF6 inhibitor(C25-140), and TAK1 inhibitor (Takinib) on chronic migraine-like behavior, and activation of the ERK pathway in the Trigeminal nucleus caudalis (TNC).

Results: Recurrent injections of NTG resulted in a significant increase in the expression of TLR3, TRAF6, TAK1, CGRP, and c-Fos proteins, as well as the activation of the ERK signaling pathway. Concurrent inhibition of TLR3 function, TRAF6, TAK1, and the ERK pathway counteracted these changes and alleviated hyperalgesia in CM mice.

Conclusions: Our findings suggest that TLR3 may play a role in central sensitization in CM mice by TRAF6-TAK1 axis modulating the ERK signaling pathway.

ERK; TLR3; central sensitization; chronic migraine; neuron.