Inhibition of toll-like receptor 4 alleviates hyperalgesia induced by acute dural inflammation in experimental migraine

Abstract

Objective Although nociceptive sensitisation is an important pathophysiological process in migraine and migraine chronification, its underlying mechanisms remain unclear. Toll-like receptor 4 (TLR4), a pattern-recognition molecule, has a critical role in both neuropathic pain and morphine tolerance. The present study examined whether elements of the TLR4 pathway contribute to hyperalgesia induced by dural inflammation in rats. Methods A rat model of migraine was established by infusing a dural inflammatory soup. A group pretreated with TAK-242 was used to inhibit the activation of TLR4. The protein levels of TLR4 and its downstream molecules in the trigeminal pathway were examined by Western blot and immunofluorescence. The expression of activated microglia and astrocytes was also analysed. Levels of interleukin-1 beta, tumour necrosis factor-alpha, and brain-derived neurotrophic factor were measured by enzyme-linked immunosorbent assay. Results Acute inflammatory soup infusion induced time-dependent facial mechanical hyperalgesia, which was blocked by TAK-242 pretreatment. The inflammatory soup stimulus increased the production of TLR4 downstream molecules and interleukin-1 beta. Higher levels of microglia activation and brain-derived neurotrophic factor release were observed following the administration of the inflammatory soup but were alleviated by TAK-242. Conclusions These data suggest that the TLR4 signalling pathway promotes hyperalgesia induced by acute inflammatory soup delivery by stimulating the production of proinflammatory cytokines and activating microglia.

Keywords: Migraine; hyperalgesia; microglia; neuroinflammation; toll-like receptor 4.

Figures

Figure 1.

The mechanical (a) or thermal (b) withdrawal thresholds of rats during the 6-hour experiment. Data are presented as the mean ± SD, n = 9. Compared with the CON group, rats in IS group showed a time-dependent and reversible decline in periorbital withdrawal thresholds to mechanical stimulus (*P 

Figure 2.

The level of Fos in trigeminal ganglion (a), trigeminal nucleus caudalis (b), and upper cervical spinal cord (c). All values given are the mean ± SD, n = 4. *P 

Figure 3.

The protein levels of TLR4 and its downstream molecules in rats’ medullary and upper cervical spinal cord. Protein levels of TLR4 (a), MyD88 (b), and TRIF (c) were analysed by Western blot. Typical Western blot strips obtained were exhibited at the top of each image, and for all strips, each blot from left to right means sample from rat in group CON, IS, and TAK-242, respectively. All values given are the mean ± SD, n = 5. *P 

Figure 4.

The protein levels of NF-κB p65 and related molecules in rats’ medullary and upper cervical spinal cord. Protein levels of IκB (a), p-IκB (b), NF-κB p65 (c), and nuclear NF-κB p65 (d) were analysed by Western blot. Typical Western blot strips obtained were exhibited at the top of each image, and for all strips, each blot from left to right means sample from rat in group CON, IS, and TAK-242, respectively. All values given are the mean ± SD, n = 5. #P 

Figure 5.

Immunofluorescence of TLR4 and NF-κB p65 expression in the trigeminal pathway. The typical immunofluorescence samples were presented at the left part of the image, with the scale bar equalling to 50 μm. The right part showed the statistical results of NF-κB p65 expression in TG (a), TNC (b), and UCSC (c), respectively. All values given are the mean ± SD, n = 4. #P 

Figure 6.

The protein levels of IL-1β (a), TNF-α (b), and BDNF (c) in rat brain by enzyme-linked immunosorbent assay. All values given are the mean ± SD, n = 5. #P 

Figure 7.

Expression of microglia (a) and astrocytes (b) activating marker in rats’ medullary and upper cervical spinal cord. Protein levels of Iba-1 (ionised calcium-binding adapter molecule 1) and GFAP (glia fibrillary acidic protein) were analysed by Western blot. Typical Western blot strips obtained were exhibited at the top of each image, and for all strips, each blot from left to right means sample from rat in group CON, IS, and TAK-242, respectively. All values given are the mean ± SD, n = 5. #P 

Figure 8.

Immunofluorescence of Iba-1 and GFAP expression in the trigeminal pathway. The typical immunofluorescence samples were presented at the left part of the image, with the scale bar equalling to 50 μm. The right part showed the statistical results of Iba-1 expression in TNC (a) and UCSC (b), respectively. All values given are the mean ± SD, n = 4. #P