Mechanisms involved in extraterritorial facial pain following cervical spinal nerve injury in rats

Abstract

Background: The aim of this study is to clarify the neural mechanisms underlying orofacial pain abnormalities after cervical spinal nerve injury. Nocifensive behavior, phosphorylated extracellular signal-regulated kinase (pERK) expression and astroglial cell activation in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal dorsal horn (C1-C2) neurons were analyzed in rats with upper cervical spinal nerve transection (CNX).

Results: The head withdrawal threshold to mechanical stimulation of the lateral facial skin and head withdrawal latency to heating of the lateral facial skin were significantly lower and shorter respectively in CNX rats compared to Sham rats. These nocifensive effects were apparent within 1 day after CNX and lasted for more than 21 days. The numbers of pERK-like immunoreactive (LI) cells in superficial laminae of Vc and C1-C2 were significantly larger in CNX rats compared to Sham rats following noxious and non-noxious mechanical or thermal stimulation of the lateral facial skin at day 7 after CNX. Two peaks of pERK-LI cells were observed in Vc and C1-C2 following mechanical and heat stimulation of the lateral face. The number of pERK-LI cells in C1-C2 was intensity-dependent and increased when the mechanical and heat stimulations of the face were increased. The decrements of head withdrawal latency to heat and head withdrawal threshold to mechanical stimulation were reversed during intrathecal (i.t.) administration of MAPK/ERK kinase 1/2 inhibitor PD98059. The area of activated astroglial cells was significantly higher in CNX rats (at day 7 after CNX). The heat and mechanical nocifensive behaviors were significantly depressed and the number of pERK-LI cells in Vc and C1-C2 following noxious and non-noxious mechanical stimulation of the face was also significantly decreased following i.t. administration of the astroglial inhibitor fluoroacetate.

Conclusions: The present findings have demonstrated that mechanical allodynia and thermal hyperalgesia occur in the lateral facial skin after CNX and also suggest that ERK phosphorylation of Vc and C1-C2 neurons and astroglial cell activation are involved in orofacial extraterritorial pain following cervical nerve injury.

Figure 1

Time-course change in head withdrawal threshold to mechanical stimulation of the lateral facial skin ipsilateral to CNX or Sham operation (A) and head withdrawal latency to heat stimulation of the lateral facial skin ipsilateral to CNX or Sham-operation (B) in CNX or Sham rats. *: p < 0.05, **: p < 0.01 (vs. Sham).

Figure 2

Camera-lucida drawings (A: Vc, B: C2) and photographs (C-F) of pERK-LI cells in Vc (A, C and E) and C2 (B, D and F). C: Vc ipsilateral to CNX, D: C2 ipsilateral to CNX, E: Vc contralateral to CNX, F: C2 contralateral to CNX. G: pERK-LI cells in C2, H: NeuN-labeled cells in C2, I: merge G with H. Arrows indicate pERK and NeuN double labelled cells.

Figure 3

Rostro-caudal distribution of pERK-LI cells in the Vc and C1-C2 following mechanical stimulation of the facial skin. A: low-intensity (6 g), B: medium-intensity (15 g), C: high-intensity (60 g), D: mean number of pERK-LI cells in Vc induced by noxious mechanical stimulation (60 g) of the lateral facial skin in day 7 CNX and Sham rats, E: mean number of pERK-LI cells in C1-C2 induced by 6 g, 15 g or 60 g stimulation of the lateral facial skin in CNX or Sham rats. *: p < 0.05, **: P < 0.01 #: p < 0.05, ##: p < 0.01 (vs. Sham).

Figure 4

Rostro-caudal distribution of pERK-LI cells in the Vc and C1-C2 following thermal stimulation of the facial skin. A: low-intensity (35°C), B: medium-intensity (40°C), C: high-intensity (50°C), D: mean number of pERK-LI cells in Vc induced by thermal stimulation (35°C, 40°C and 50°C) of the lateral facial skin in day 7 CNX and Sham rats, E: mean number of pERK-LI cells in C1-C2 induced by 35°C, 40°C or 50°C stimulation of the lateral facial skin in CNX or Sham rats. **: P < 0.01, #: p < 0.05, ##: p < 0.01 (vs. Sham).

Figure 5

Photograph of pERK-LI cells following mechanical (60 g) stimulation of the lateral facial skin in day 5 CNX rats with 5 days successive (A) i.t. administration of vehicle (saline) (A) or PD98059 (B). C: mean number of pERK-LI cells in Vc and C1-C2 following mechanical stimulation (60 g) of the lateral facial skin in CNX rats with vehicle or PD98059 i.t. administration. *: p < 0.05.

Figure 6

Time-course change in head withdrawal threshold to mechanical stimulation (A) or head withdrawal latency to heat stimulation (B) of lateral facial skin with i.t. administration of PD98059 or vehicle in CNX rats. *: p < 0.05, **: p < 0.01.

Figure 7

GFAP-labeled cells in C2 in day 5 CNX (A) and Sham (B) rats. C: mean percent area occupied by GFAP-labeled cells in C1-C2 on day 5 CNX and Sham rats. Aa: low magnification photomicrograph of C2 dorsal horn. Three square boxes were placed on the dorsal portion of C2. The low magnification photomicrograph of A corresponds to the area indicated by the arrow with box in Aa. *: p < 0.05.

Figure 8

The effect of 5 days successive i.t. vehicle or FA administration on nocifensive behavior in day 5 CNX rats. A: head withdrawal threshold to mechanical stimulation of the lateral facial skin, B: head withdrawal latency to heat stimulation of lateral facial skin. **: p < 0.01.

Figure 9

The effect of 5 days successive i.t. vehicle or FA administration on pERK-LI cells in Vc and C1-C2 in CNX rats. Photomicrographs of pERK cells following mechanical stimulation (60 g) of the lateral facial skin with i.t. administration of vehicle (A) and FA (B). C: mean number of pERK-LI cells following 15 g and 60 g mechanical stimulation of the lateral facial skin in CNX rats. *: p < 0.05, **: p < 0.01.