Mechanisms underlying ectopic persistent tooth-pulp pain following pulpal inflammation

Abstract

In order to clarify the peripheral mechanisms of ectopic persistent pain in a tooth pulp following pulpal inflammation of an adjacent tooth, masseter muscle activity, phosphorylated extracellular signal-regulated protein kinase (pERK) and TRPV1 immunohistochemistries and satellite cell activation using glial fibrillary acidic protein (GFAP) immunohistochemistry in the trigeminal ganglion (TG) were studied in the rats with molar tooth-pulp inflammation. And, Fluorogold (FG) and DiI were also used in a neuronal tracing study to analyze if some TG neurons innervate more than one tooth pulp. Complete Freund's adjuvant (CFA) or saline was applied into the upper first molar tooth pulp (M1) in pentobarbital-anesthetized rats, and capsaicin was applied into the upper second molar tooth pulp (M2) on day 3 after the CFA or saline application. Mean EMG activity elicited in the masseter muscle by capsaicin application to M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. The mean number of pERK-immunoreactive (IR) TG cells was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats. Application of the satellite cell inhibitor fluorocitrate (FC) into TG caused a significant depression of capsaicin-induced masseter muscle activity and a significant reduction of satellite cell activation. The number of TRPV1-IR TG cells innervating M2 was significantly larger in M1 CFA-applied rats compared with M1 vehicle-applied rats, and that was decreased following FC injection into TG. Furthermore, 6% of TG neurons innervating M1 and/or M2 innervated both M1 and M2. These findings suggest that satellite cell activation following tooth pulp inflammation and innervation of multiple tooth pulps by single TG neurons may be involved in the enhancement of the activity of TG neurons innervating adjacent non-inflamed teeth that also show enhancement of TRPV1 expression in TG neurons, resulting in the ectopic persistent tooth-pulp pain following pulpal inflammation of adjacent teeth.

Figure 1. Masseter muscle activity following capsaicin application to M2 in the rats with M1 CFA-applied or saline-applied rats.

Typical examples of EMG activities recorded from the masseter muscle following capsaicin application to M2 in the rats with saline (A) or CFA (Ba: large response, Bb: small response) application to M1 on day 3 after the treatments. C and D: The mean area under the curve of integrated EMG following capsaicin application to M2 in M1 CFA- or saline-applied rats (C), and that following Veh- or PD98059-administration in the rats with M1 CFA application (D). E and F: Low (E) and high (F) magnification photomicrographs of CFA-applied M1 sections. G: High magnification photomicrograph of the area inidicated by the square box with the solid arrow in E. The open arrow in A indicates the timing of capsaicin application to M2. Open arrows in D and E indicate the apex of M1. The inset diagrams in A and D indicate the tooth pulp with drug application in this and following figures. CFA: complete Freund’s adjuvant, pre: before capsaicin application, post-capsaicin: after capsaicin application. +, # : p<0.05, ##: p<0.01.

Figure 2. pERK-IR cells in TG following capsaicin application to M2 in the rats with CFA or saline-application to M1.

A: Low magnification photomicrograph of TG in M1 CFA-applied rats. B: pERK- and NeuN-IR cells in TG following capsaicin application to M2 in the rats with CFA application to M1. C: High magnification photomicrograph of pERK-IR cells in M1 CFA-applied rats. D: High magnification photomicrograph of pERK-IR cells in M1 saline-applied rats. E: The mean percentage of pERK-IR cells in TG of M2 untreated/M1 saline, M2 Veh/M1 saline, M2 Cap/M1 saline, M2 untreated/M1 CFA, M2 Veh/M1 CFA or M2 Cap/M1 CFA rats. White arrow heads indicate pERK- and NeuN-IR cells in B. Open arrow heads indicate NeuN-IR cells in B. The solid arrow heads indicate pERK-IR cells in C and D. Veh: vehicle for capsaicin, Cap: capsaicin. * : p<0.05, ***: p<0.001.

Figure 3. FG-labeled TG cells and pERK-IR TG cells following capsaicin application to M2 in M1 CFA- or saline-applied rats.

Fluorescent photomicrographs of FG(+) cells (A and D), pERK-IR cells (B and E) and FG merged with pERK-IR (C and F) in TG (M1 saline-applied rats: A, B and C; M1CFA-inected rats: D, E and F ). G and H: The mean percentages of FG (+) pERK-IR cells/FG (+) cells (G) and mean percentage of FG(–) pERK-IR cells/FG (–) cells (H). I: The photomicrograph of FG-applied M1 sections. FG can be seen as blue-stained product in M1. The solid arrow heads indicate FG(+) pERK-IR cells in A-F. The open arrow heads indicate FG(+) cells in D and F. The arrows indicate pERK-IR cells in B, C, E and F. The large white arrow in I indicates the apex of M1. The white star in I indicates coronal pulp cavity. * : p<0.05.

Figure 4. GFAP-IR cells in TG following capsaicin application to M2 in M1 CFA- or saline-applied rats.

A, B and C: GFAP-IR cells in naïve (A), M1 saline (B) or CFA-applied (C) rats. D: Photomicrograph of GFAP-IR cells (D), FG(+) cells (E) and FG(+) GFAP-IR cells (F) in TG. G: The mean relative number of TG neurons encircled with GFAP-IR cells (%). H: The mean relative number of FG(+) TG neurons encircled with GFAP-IR cells in FG(+) cells (%). I: The mean relative number of FG(–) TG neurons encircled with GFAP-IR cells in FG(–) cells (%). The open arrow heads indicate GFAP-IR cells. The white arrow heads indicate FG(+) cells encircled with GFAP-IR cells. *: p<0.05, **: p<0.01.

Figure 5. Effect of FC injection into TG on masseter muscle activity and GFAP and pERK-IR cell expression.

A and B: Typical example of masseter muscle EMG activities following capsaicin application to M2 in M1 CFA-applied rats with Veh (A) or FC (B) injection to TG. C: The mean area under the curve of integrated EMG following capsaicin application to M2 in M1 CFA-applied rats with Veh or FC-injection to TG (C). D and E: Photomicrographs of GFAP-IR cells in TG Veh-injected rats (D) and TG FC-injected rats (E). F: The mean relative number (%) of TG cells encircled with GFAP-IR cells following Veh- (solid bur) or FC- (open bur) administration in M1 CFA rats. G and H: Photomicrographs of pERK-IR cells following M2 capsaicin application in TG Veh- (G) and TG FC-injected rats (H). I: The mean relative number (%) of pERK-IR cells in TG following M2 capsaicin application in TG Veh- and TG FC-injected rats which received CFA in M1. Solid arrow in A indicates the timing of capsaicin application. White arrow heads in D and E indicate GFAP-IR cells and those in G and H are pERK-IR cells, respectively. Veh: vehicle for FC, FC: Fluorocitrate. #: p<0.05, +++: p<0.001, *: p<0.05, **: p<0.01.

Figure 6. Photomicrographs and percentage of double retrograde tracing from M1 and M2 afferents to TG neurons.

A, B and C: Low magnification photomicrographs of FG(+) TG cells at 3 days after FG application to M1 (A) and DiI application to M2 (B), and the photomicrograph of A merged with B (C). D, E and F: High magnification photomicrographs of FG(+) TG cells 3 days after FG application to M1 (D) and DiI application to M2 (E), and the photomicrograph of E merged with F (F). Ga: Percentage of FG and DiI double-labeled TG cells indicated by the yellow pie. White arrow heads indicate FG and DiI double-labeled TG cells, the white arrows are FG(+) TG cells and open arrow heads are DiI(+) TG cells.

Figure 7. TRPV1-IR cells labeled with FG applied into M2 following CFA application to M1.

A: TRPV1-IR cells (upper panel), NeuN-IR cells (middle panel) and merged photomicrographs (lower panel) in M2 capsaicin-applied rats which received CFA to M1. B-G: FG(+) TG cells expressing TRPV1-IR in M1saline-adminstrated (B, C and D) and M1 CFA-administrated (E, F and G) rats. H: The mean number of FG(+) TRPV1-IR cells/FG (+) following saline (open bur) or CFA (solid bur) application to M1. I: The mean number of FG(+) TRPV1-IR cells/FG(+) following Veh (solid bur) or FC (open bur) injection into TG in M1 CFA rats. *: p<0.05.

Figure 8. The diagram indicating proposed mechanisms of the present results.

The TG cell innervating M1 is indicated by red and TG cell innervating M2 is indicated by blue. Following CFA application to M1, the excitability of TG cell innervating M2 (blue cell) is enhanced via neuron-neuron or satellite cell-neuron interaction.