Calcitonin gene-related peptide promotes cellular changes in trigeminal neurons and glia implicated in peripheral and central sensitization

Abstract

Background: Calcitonin gene-related peptide (CGRP), a neuropeptide released from trigeminal nerves, is implicated in the underlying pathology of temporomandibular joint disorder (TMD). Elevated levels of CGRP in the joint capsule correlate with inflammation and pain. CGRP mediates neurogenic inflammation in peripheral tissues by increasing blood flow, recruiting immune cells, and activating sensory neurons. The goal of this study was to investigate the capability of CGRP to promote peripheral and central sensitization in a model of TMD.

Results: Temporal changes in protein expression in trigeminal ganglia and spinal trigeminal nucleus were determined by immunohistochemistry following injection of CGRP in the temporomandibular joint (TMJ) capsule of male Sprague-Dawley rats. CGRP stimulated expression of the active forms of the MAP kinases p38 and ERK, and PKA in trigeminal ganglia at 2 and 24 hours. CGRP also caused a sustained increase in the expression of c-Fos neurons in the spinal trigeminal nucleus. In contrast, levels of P2X3 in spinal neurons were only significantly elevated at 2 hours in response to CGRP. In addition, CGRP stimulated expression of GFAP in astrocytes and OX-42 in microglia at 2 and 24 hours post injection.

Conclusions: Our results demonstrate that an elevated level of CGRP in the joint, which is associated with TMD, stimulate neuronal and glial expression of proteins implicated in the development of peripheral and central sensitization. Based on our findings, we propose that inhibition of CGRP-mediated activation of trigeminal neurons and glial cells with selective non-peptide CGRP receptor antagonists would be beneficial in the treatment of TMD.

Figure 1

Increased number of trigeminal ganglia neurons expressing P-p38 in response to CGRP injection into the TMJ capsule. Sections of the posterolateral portion of the ganglion (V3) were obtained from untreated animals (CON), and animals receiving bilateral injections of CGRP after 2 and 24 hours. (A) Images of neuron-satellite glial regions stained for P-p38 are shown in the top panels. Magnification bar = 50 μm. The bottom panels are the same sections co-stained for P-p38 and DAPI. (B) The number of P-p38 positive cells ± SEM for each condition is reported (n = 3 independent experiments) ^#P < 0.01 when compared to control levels, while ^† P < 0.01 when compared to CGRP stimulated levels at 2 hours.

Figure 2

CGRP stimulates increased P-ERK expression in trigeminal ganglia neurons and satellite glia. Sections of the posterolateral portion of the ganglion (V3) were obtained from untreated animals (CON), or animals injected with CGRP in each TMJ capsule. (A) Images of neuron-satellite glial regions stained for P-ERK are shown in the top panels. Magnification bar = 50 μm. The bottom panels are the same sections co-stained for P-ERK and DAPI. (B) The average fold change ± SEM of P-ERK staining intensity from control values, whose mean was made equal to one, is reported (n = 3 independent experiments) ^# P < 0.01 when compared to control levels, while ^† P < 0.01 when compared to CGRP stimulated levels at 2 hours.

Figure 3

Peripheral injection of CGRP induces a sustained increase in expression of neuronal c-Fos in spinal trigeminal nucleus. (A) An image (40×) of a section of spinal cord 4 mm from the obex stained with the nuclear dye DAPI is shown. The white box represents the area encompassing the spinomedullary junction (Vc/C1-C2) transition zone. (B) Sections of spinal cord within the Vc/C1-2 region of the spinal trigeminal nucleus were obtained from control animals (CON), animals 2 hours post CGRP injections, or animals 24 hours post CGRP injection. Images of spinal cord tissues stained for c-Fos are shown in the top panels. Magnification bar = 50 μm. The same sections costained for c-Fos and DAPI are displayed in the bottom panels. (C) The average fold change ± SEM of c-Fos staining intensity from control values is reported (n = 3 independent experiments). * P < 0.05 when compared to control values.

Figure 4

PKA levels are elevated in the spinal trigeminal nucleus in response to CGRP. Spinal cord sections were obtained from control animals (CON), animals 2 hours post CGRP injections, or animals 24 hours post CGRP injection. (A) Images of spinal cord tissues stained for PKA are shown in the top panels. Magnification bar = 50 μm. The same sections costained for PKA and DAPI are displayed in the bottom panels. (B) The average fold change ± SEM of PKA staining intensity from control values is reported (n = 3). * P < 0.05 when compared to control values.

Figure 5

CGRP causes a transient increase in P2X₃ expression in the spinal trigeminal nucleus at 2 and 24 hours. Sections of spinal cord were obtained from control animals (CON), animals 2 hours post CGRP injections or animals 24 hours post CGRP injection. (A) Images of spinal cord tissues stained for P2X₃ are shown in the top panels. Magnification bar = 50 μm. The same sections costained for P2X₃ and DAPI are displayed in the bottom panels. (B) The average fold change ± SEM of P2X₃ staining intensity from control values is reported (n = 3). * P < 0.05 when compared to control values.

Figure 6

Elevated expression of OX-42 in microglia. Sections of spinal cord were obtained from control animals (CON), animals 2 hours post CGRP injections, or animals 24 hours post CGRP injection. (A) Images of spinal cord tissues stained for OX-42 are shown in the top panels. Magnification bar = 50 μm. The same sections costained for OX-42 and DAPI are shown in the bottom panels. (B) The average fold change ± SEM of OX-42 staining intensity from control values is reported (n = 3). * P < 0.05 when compared to control values.

Figure 7

CGRP induces a prolonged increase in expression of GFAP in astrocytes. Spinal cord sections were obtained from control animals (CON), animals 2 hours post CGRP injections, or animals 24 hours post CGRP injection. (A) Images of spinal cord tissues stained for GFAP are shown in the top panels. Magnification bar = 50 μm. The same sections costained for GFAP and DAPI are displayed in the bottom panels. (B) The average fold change ± SEM of GFAP staining intensity from control values is reported (n = 3). ^# P < 0.01 when compared to control values.