Toll-like receptor 4 signaling in neurons of trigeminal ganglion contributes to nociception induced by acute pulpitis in rats

Abstract

Pain caused by acute pulpitis (AP) is a common symptom in clinical settings. However, its underlying mechanisms have largely remained unknown. Using AP model, we demonstrated that dental injury caused severe pulp inflammation with up-regulated serum IL-1β. Assessment from head-withdrawal reflex thresholds (HWTs) and open-field test demonstrated nociceptive response at 1 day post injury. A consistent up-regulation of Toll-like receptor 4 (TLR4) in the trigeminal ganglion (TG) ipsilateral to the injured pulp was found; and downstream signaling components of TLR4, including MyD88, TRIF and NF-κB, and cytokines such as TNF-α and IL-1β, were also increased. Retrograde labeling indicated that most TLR4 positve neuron in the TG innnervated the pulp and TLR4 immunoreactivity was mainly in the medium and small neurons. Double labeling showed that the TLR4 expressing neurons in the ipsilateral TG were TRPV1 and CGRP positive, but IB4 negative. Furthermore, blocking TLR4 by eritoran (TLR4 antagonist) in TGs of the AP model significantly down-regulated MyD88, TRIF, NF-κB, TNF-α and IL-1β production and behavior of nociceptive response. Our findings suggest that TLR4 signaling in TG cells, particularly the peptidergic TRPV1 neurons, plays a key role in AP-induced nociception, and indicate that TLR4 signaling could be a potential therapeutic target for orofacial pain.

Figure 1. AP inflammation and behavioral change induced by dental pulp exposure.

(A) Hematoxylin and eosin staining of left pulp at different time points of AP in rat. (Arrows indicate infiltration of inflammatory cells at the surface of necrotic and normal tissue). In SHAM group, there is normal dental pulp without inflammatory cells or necrosis; In the AP-1 group, the intensively gathered inflammatory cells occupy the most part of the dental pulp cavity with necrosis at the top; only a minimal vital pulp is left at the bottom of the cavity. In AP-3, most of the pulp tissue has developed into necrosis with few inflammatory cells inside the cavity. (Bar = 100 μm) ^***P < 0.01; n = 8 for each group. (B) ELISA assay for serum IL-1βat different time points of the AP rats showing significant increase in serum IL-1β in AP-1 group as compared to that in SHAM and AP-3 groups. ^**P < 0.01 vs. SHAM; ^##P < 0.01 vs. AP-3; n = 8 for each group. (C) Mechanical and thermal HWTs at different time points in AP rats. There is no significant difference between the right and left TG of the SHAM and AP-3 groups. Only HWTs of the left side of tongue in AP-1 group is significantly decreased. ^**P < 0.01 vs. left side of tongue in the SHAM group; ^##P < 0.01 vs. right side of tongue in the AP-1 group; ^&&P < 0.01 vs. left side of tongue in the AP-1 group; n = 8 for each group. (D) Results of open-field tests of AP rats at different time points. There was no significant difference between SHAM and AP-3 groups. In AP-1, spontaneous activity time was decreased, and grooming time and rearing times were significantly increased as compared to other groups. ^**P < 0.01 vs. SHAM; ^##P < 0.01 vs. AP-3; n = 8 for each group.

Figure 2. AP induced up-regulation of TLR4 expression in the TG ipsilateral (left) to the injured pulp.

(A) and (B) Representative pictures of immunohistochemical labeling for TLR4 in TG at different time points of the AP rats with quantification analysis. There was no significant difference between the right and left TG of the SHAM group, and all the data are normalized by the left TG of the SHAM group. Compared to left TG of the SHAM group or right TG of the AP-1 group, the left TG of AP-1 group showed remarkable increase of TLR4 immunoreactivity. (Bar = 100 μm) ^**P < 0.01 vs. left TG in SHAM; ^##P < 0.01 vs. right TG in AP-1; ^&&P < 0.01 vs. left TG in AP-1; n = 8 for each group. (C) Representative immunoblots of samples from rat TGs subjected to AP model with quantitative densitometric analysis of TLR4 protein with β-actin as an internal standard. The immunoblots were obtained from the microgel running in the same experiment conditions. There is no significant difference between the right and left TG of the SHAM group; the pixels of the blots are normalized by the left TG of the SHAM group for comparison. TLR4 protein level is significantly increased in the left TG in AP-1 group as compared to the other groups. ^**P < 0.01 vs. left TG of SHAM; ^##P < 0.01 vs. right TG of AP-1; ^&&P < 0.01 vs. left TG of AP-1; n = 8 for each group. (D) Graphical representation of TLR4 mRNA expression in TGs. There is no significant difference between the right and left TG in the SHAM group, and all the data are normalized by the left TG of the SHAM group. There is a significant increase in left TG in AP-1, as compared to the other groups. ^**P < 0.01 vs. left TG of SHAM; ^##P < 0.01 vs. right TG of AP-1; ^&&P < 0.01 vs. left TG of AP-1; n = 8 for each group.

Figure 3. Immunofluorescent staining of TLR4 in TG at AP-1.

(A) Retrograde labeled FG and immunofluorescent staining of TLR4 in the neurons. Representative pictures show a similar number of FG-labeled neurons in the left and right TG; but the number with TLR4 immunostaining is significantly increased in the left TG, compared to the right TG. The number of FG labeled neurons that also express TLR4 is significantly increased in the left TG compared with the right TG in the AP-1 group. ^***P < 0.01 vs. right TG of AP-1; n = 8 for each group. (B) and (C) The stack of scanned pictures of confocal microscopy for TLR4 immunoreactivity in neurons from both TGs of AP-1 group, as well as the satellite cells. The TLR4 staining is significantly increased in the neurons of the left TG of AP-1, including cytoplasm (B section: Bar = 300 μm; C section: left: Bar = 40 μm, right: Bar = 450 μm). (D) Measurement of diameters of the TLR4 positive neurons. 54.59 ± 1.19% neurons are of 10–20 μm, 35.56 ± 1.49% are of 20–40 μm and 9.85 ± 1.56% are of 40–60 μm, indicating that most of the TLR4 immunoreactivity in the small and medium-sized neurons, including cytoplasm. ^###P < 0.01; n = 8 for each group.

Figure 4. Immunofluorescence staining of TLR4 with typical pain-associated molecules.

Immunofluorescent staining of TLR4 and TRPV1/CGRP/IB4 in both TGs from the AP-1 group with quantification analysis. The results showed that the number of TRPV1/CGRP/IB4 cells was significantly increased in the left TG, compared to the right TG of the AP-1 group. The double labeled staining revealed that increased expression of TLR4 was mainly in the TRPV1- or CGRP- positive neurons, but not in IB4-positive neurons of the left TGs of the AP-1 group (Bar = 40 μm). ^***P < 0.01; n = 8 for each group.

Figure 5. Expression of downstream molecules of TLR4 in the TGs.

(A) and (B) Representative immunoblots of samples from TGs of AP rat with quantitative densitometric analysis with β-actin/lamin B as an internal standard. The immunoblots were obtained from the microgel running in the same experimental conditions. There is no significant difference between the right and left TG of the SHAM group, and the pixels of the blots are normalized by the left TG of the SHAM group for comparison. The statistical analysis indicated that MyD88, TRIF and NF-κB (in the nucleus) protein levels were significantly increased in left TG of the AP-1 group, compared to the other groups. ^**P < 0.01 vs. left TG of SHAM; ^##P < 0.01 vs. right TG of AP-1; ^&&P < 0.01 vs. left TG of AP-1; n = 8 for each group. (C) Results of ELISA assay for IL-1β and TNF-α in the TGs at different time points of AP rats: There was no significant difference between the right and left TG of the SHAM group, and the data were normalized by the left TG of the SHAM group. The statistical analysis indicated that IL-1β and TNF-α are significantly increased in left TG of the AP-1 group, as compared to that in the other groups. ^**P < 0.01 vs. left TG of SHAM; ^##P < 0.01 vs. right TG of AP-1; ^&&P < 0.01 vs. left TG of AP-1; n = 8 for each group.

Figure 6. Effect of eritoran on behavior of nociceptive response at 1 day post-pulp exposure.

Results from the open-field test and HWTs test in different groups: compared to the other groups, the nociceptive response was significantly increased in left TG in AP-1 group, which was rescued by eritoran or ibuprofen administration. After eritoran or ibuprofen administration, the activity time was increased significantly while the grooming time was down-regulated in the AP-1 + E group compared with the AP-1 group. The rearing times were also increased in the AP-1 + E and AP-1 + I group compared with the AP-1 group. The level of the HWTs to mechanical and heat stimulation of the tongue ipsilateral to the injured pulp were significantly higher in the AP-1 + E/AP-1 + I group than AP-1 group, while it showed no difference between the AP-1 and AP-1 + V group. ^**P < 0.01 vs. SHAM; ^##P < 0.01 vs. AP-1; n = 8 for each group.

Figure 7. Effect of eritoran on the expression of downstream molecules of TLR4 in left TG.

(A) and (B) Representative immunoblots of samples from left TGs from different groups with quantitative densitometric analysis with β-actin/lamin B as an internal standard. The immunoblots were obtained from the microgel running in the same experimental conditions. There was no significant difference between the right and left TG of the SHAM group, and the pixels of the blots were normalized by the left TG of the SHAM group for comparison. Compared to the other groups, MyD88, TRIF and NF-κB (in the nucleus) protein levels were significantly increased in left TG of the AP-1 group, which were rescued by the eritoran.^**P < 0.01 vs. SHAM; ^##P < 0.01 vs. AP-1; n = 8 for each group. (C) ELISA assay for IL-1β and TNF-α in left TGs from different groups. Compared to the other groups, there was a significant increase in left TG of AP-1 rats, which was rescued by the eritoran. ^**P < 0.01 vs. SHAM; ^##P < 0.01 vs. AP-1; n = 8 for each group.