Tonabersat inhibits trigeminal ganglion neuronal-satellite glial cell signaling

Abstract

Background: Sensitization and activation of trigeminal neurons are implicated in the underlying pathology of migraine, acute sinusitis, and allergic rhinitis. Cell bodies of trigeminal neurons that provide sensory innervation of the dura and nasal mucosa reside in the trigeminal ganglion in association with satellite glial cells where they communicate via gap junctions. Gap junctions, channels formed by connexins, modulate the excitability state of both neurons and glia under pathological conditions. Tonabersat, a compound being tested as an antimigraine drug, is thought to block gap junction activity.

Objective: To investigate the cellular events within trigeminal ganglia that may account for the significant comorbidity of migraine and rhinosinusitis and determine the effect of tonabersat on neuron-satellite glia communication.

Methods: Sprague Dawley rats injected with True Blue were used to localize neuronal cell bodies in the ganglion and study neuron-glia signaling via gap junctions in the trigeminal ganglion. Dye coupling studies were conducted under basal conditions and in response to tumor necrosis factor-alpha injection into the whisker pad and/or capsaicin injection into the eyebrow. Changes in connexin 26 and active p38 levels were determined by immunohistochemistry. In addition, the effect of tonabersat prior to chemical stimulation on gap junction activity and expression of connexins and active p38 was investigated.

Results: Injection of tumor necrosis factor-alpha, a cytokine implicated in the pathology of acute sinusitis and allergic rhinitis, into the V2 region was shown to lower the amount of capsaicin required to stimulate neurons located in the V1 region of the ganglion. While injection of tumor necrosis factor-alpha into the whisker pad or capsaicin injection into the eyebrow alone did not cause increased dye movement, the combination of both stimuli greatly increased neuron-satellite glia communication via gap junctions in both V1 and V2 regions. The change in gap junction activity was accompanied by increased expression of connexin 26 and active p38 levels in both neurons and satellite glia in V1 and V2 regions. Pretreatment with tonabersat inhibited gap junction communication between neurons and satellite glia and blocked the increase in connexin 26 and active p38 levels in response to injection of both tumor necrosis factor-alpha (V2) and capsaicin (V1).

Conclusions: We propose that increased levels of tumor necrosis factor-alpha, as reported during acute sinusitis and allergic rhinitis, reduces the amount of capsaicin necessary to stimulate V1 neurons that leads to cellular changes in both V1 and V2 regions. The cellular events observed in this study may help to explain, in part, the significant comorbidity reported with migraine and rhinosinusitis. In addition, we have provided evidence to suggest that tonabersat can prevent increased neuron-satellite glia signaling and, thus, may be useful in the treatment of migraine, acute sinusitis, and allergic rhinitis.

Fig 1

Localization of neuronal cell bodies in trigeminal ganglia 5 days after injection of True Blue in the eyebrows or whisker pads of young male rats. The anterior portion of trigeminal ganglia obtained from animals injected in the eyebrow (A–C) or whisker pad (D–F) are shown at 40× magnification. Panels A and D are representative sections from the dorsal region of the ganglion, while B and E are from the medial region, and C and F are from the ventral region. Each ganglion section is oriented such that the top of the figure is the anterior most region and the right side is the lateral region. Dye-labeled neuronal cell bodies were seen in the V1 and V2 regions of the ganglion in the dorsal (A) and medial (B) regions of the ganglion, but not the ventral (C) region, following injection of True Blue into the eyebrow. After injection of True Blue into the whisker pad, labeled neuronal cell bodies not only were observed in the dorsal V2 (D) region, but were also located in both the V2 and V1 regions in the medial (E) and ventral (F) regions of the ganglion.

Fig 2

Tumor necrosis factor-alpha (TNF-α) treatment of V2 neurons causes sensitization of capsaicin-responsive neurons in the V1 region of the trigeminal ganglion that results in increased neuron-satellite glia cell signaling through gap junctions in V1 and V2 regions. The cellular localization pattern of True Blue in the anterior portion of the trigeminal ganglion in the V1 region is shown in the top panels and the V2 region is shown in the bottom panels. Under basal conditions (control, CON), the dye was primarily located in the cytoplasm of neuronal cell bodies (large arrows) in the V1 and V2 regions of the ganglion. A similar pattern was observed in the V1 and V2 regions in response to TNF-α (TNF) or capsaicin (CAP) treatment. However, treatment with both TNF-α and capsaicin resulted in increased dye coupling between neuronal cell bodies (large arrows) and satellite glial cells (small arrows). Images obtained at 400× magnification.

Fig 3

Expression of connexin 26 in trigeminal ganglion neurons and satellite glia is increased in both V1 and V2 regions of the trigeminal ganglion in response to tumor necrosis factor-alpha (TNF-α) and capsaicin treatment. A section of the anterior portion of the ganglia obtained from untreated animals (control, CON) or animals injected with TNF-α (TNF) for 2 hours in the whisker pad or capsaicin (CAP) for 15 minutes in the eyebrow or injection of both agents is shown in the top panel at 40× magnification (A). In control ganglion, low-level connexin 26 expression is visible within the V1 and V2 regions. Similarly, low-level expression of connexin 26 is observed in TNF-α- or capsaicin-treated animals. Increased connexin 26 expression was observed in V1 and V2 regions following treatment with both chemical stimuli. Panels B, C, and D correspond to staining at 400× magnification for connexin 26, nuclear dye 4′,6 diamidino-2-phenylindole (DAPI), or merged images of connexin 26 and DAPI, respectively. A small number of plaques were observed between neurons and satellite glial cells in ganglia obtained from untreated, TNF-, or capsaicin-treated animals. The number of connexin 26 plaques was greatly increased in response to treatment of TNF-α then capsaicin.

Fig 4

Tonabersat blocks increased neuron-satellite glia signaling through gap junctions in response to tumor necrosis factor-alpha (TNF-α) and capsaicin treatment. The cellular localization pattern of True Blue in the anterior portion of the trigeminal ganglion in the V1 region is shown in the top panels and the V2 region is shown in the bottom panels. Under basal conditions (control, CON), the dye was primarily detected in neuronal cell bodies (large arrows) in the V1 and V2 regions of the ganglion. A similar pattern was observed in the V1 and V2 regions in response to 2 hour treatment with TNF-α (TNF) or 60 minute treatment with capsaicin (CAP). The increased amount of dye coupling between neurons and satellite glia was greatly reduced in both V1 and V2 regions by pretreatment with tonabersat (TON). Neuronal cell bodies are indicated by large arrows while satellite glial cells are indicated by small arrows. Images were obtained at 400× magnification.

Fig 5

Tonabersat inhibits the number of connexin 26 plaques observed between trigeminal ganglion neurons and satellite glia in response to tumor necrosis factor-alpha (TNF-α) and capsaicin. A section of the anterior portion of the ganglia obtained from untreated animals (control, CON) or animals injected with TNF-α (TNF) for 2 hours in the whisker pad or capsaicin (CAP) for 60 minutes in the eyebrow or injection of both agents or pretreatment with tonabersat (TON) prior to injections is shown in the top panel at 40× magnification (A). Control ganglion or ganglion obtained from TNF-α- or capsaicin-treated animals exhibited low-level connexin 26 expression in the V1 and V2 regions. Similarly, low-level expression of connexin 26 was observed in TNF-α-or capsaicin-treated animals. Pretreatment of animals with tonabersat greatly decreased the level of connexin 26 staining in V1 and V2 regions compared with animals treated with both TNF-α and capsaicin. Panels B, C, and D correspond to staining at 400× magnification for connexin 26, nuclear dye 4′,6 diamidino-2-phenylindole (DAPI), or merged images of connexin 26 and DAPI, respectively. A small number of plaques were observed between neurons and satellite glial cells in ganglia obtained from untreated, TNF-α-, or capsaicin-treated animals. The increased number of connexin 26 plaques seen in response to treatment of TNF-α then capsaicin was reduced to untreated control levels by pretreatment with tonabersat.

Fig 6

Tonabersat inhibits the level of active p38 in trigeminal ganglion neurons and satellite glia in response to tumor necrosis factor-alpha (TNF-α) and capsaicin. A section of the anterior portion of the ganglia obtained from untreated animals (control, CON) or animals injected with TNF-α (TNF) for 2 hours in the whisker pad or capsaicin (CAP) for 60 minutes in the eyebrow or injection of both agents or pretreatment with tonabersat (TON) prior to injections is shown in the top panel at 40× magnification (A). Control ganglion and ganglion obtained from TNF-α- or capsaicin-treated animals exhibited low levels of active p38 in V1 and V2 regions. In contrast, levels of active p38 were increased in response to TNF-α and capsaicin. Pretreatment of animals with tonabersat greatly decreased the level of p38 staining in V1 and V2 regions compared with animals treated with both TNF-α and capsaicin. Panels B, C, and D correspond to staining at 400× magnification for active p38, nuclear dye 4′,6 diamidino-2-phenylindole (DAPI), or merged images of active p38 and DAPI, respectively. Increased nuclear p38 staining was observed in animals injected with TNF-α and capsaicin when compared with untreated control animals or animals injected with either TNF-α or capsaicin alone. The increased level of nuclear and cytoplasmic p38 staining seen in response to TNF-α then capsaicin treatment was reduced to control levels by pretreatment with tonabersat.