Neuronal plasticity of trigeminal ganglia in mice following nerve injury

Abstract

Background: Nerve injury may induce neuropathic pain. In studying the mechanisms of orofacial neuropathic pain, attention has been paid to the plastic changes that occur in the trigeminal ganglia (TGs) and nucleus in response to an injury of the trigeminal nerve branches. Previous studies have explored the impact of sciatic nerve injury on dorsal root ganglia (DRGs) and it has shown dramatic changes in the expression of multiple biomarkers. In large, the changes in biomarker expression in TGs after trigeminal nerve injury are similar to that in DRGs after sciatic nerve injury. However, important differences exist. Therefore, there is a need to study the plasticity of biomarkers in TGs after nerve injury in the context of the development of neuropathic pain-like behaviors.

Aim: The aim of this study was to investigate the plasticity of biomarkers associated with chronic persistent pain in TGs after trigeminal nerve injury.

Materials and methods: To mimic the chronic nature of the disorder, we used an intraoral procedure to access the infraorbital nerve (ION) and induced a nerve injury in mice. Immunohistochemistry and quantification were used for revealing the expression level of each biomarker in TGs after nerve injury.

Results: Two weeks after partial ION injury, immunohistochemistry results showed strongly upregulated expressions of activating transcription factor 3 and neuropeptide Y (NPY) in the ipsilateral TGs. Microglial cells were also activated after nerve injury. In regard to positive neuronal profile counting, however, no significant difference in expression was observed in galanin, substance P, calcitonin gene-related peptide, neuronal nitric oxide synthase, phosphorylated AKT, or P2X3 in ipsilateral TGs when compared to contralateral TGs.

Conclusion: In this study, the expression and regulation of biomarkers in TGs have been observed in response to trigeminal nerve injury. Our results suggest that NPY and Iba1 might play crucial roles in the pathogenesis of orofacial neuropathic pain following this type of injury. Further investigations on the relevance of these changes may help to target suitable treatment possibilities for trigeminal neuralgia.

Keywords: animal model; infraorbital nerve; neuropeptides; orofacial pain; sensory neurons.

Figure 1

Expression of ATF3-LI in TGs 2 weeks after partial ION transection. Notes: (A) ATF3-LI is mainly seen in the infraorbital neurons (red box) in ipsilateral TGs. (B and C) High magnification micrographs show the expression of ATF3-IR neurons in ipsilateral (B) and contralateral (C) TGs. Scale bars indicate 250 μm (A) and 50 μm (B and C). Abbreviations: ATF3, activating transcription factor 3; ION, infraorbital nerve; IR, immunoreactive; LI, like immunoreactivity; TGs, trigeminal ganglia.

Figure 2

Percentage of positive NPs in contralateral and ipsilateral TGs 2 weeks after partial ION transection. Note: The columns represent contralateral (blue) and ipsilateral (red) TGs, respectively (n=5 in each group, **p<0.01, ***p<0.001). Abbreviations: ATF3, activating transcription factor 3; CGRP, calcitonin gene-related peptide; GAL, galanin; ION, infraorbital nerve; nNOS, neuronal nitric oxide synthase; NPs, neuronal profiles; NPY, neuropeptide Y; pAKT, phosphorylated AKT; SP, substance P; TGs, trigeminal ganglia.

Figure 3

Immunofluorescent micrographs show the expression of biomarkers in contralateral (top panel) and ipsilateral (bottom panel) TGs 2 weeks after partial ION transection. Notes: Sections were incubated with antisera of Iba1, CGRP, SP, GAL, NPY, P2X3, nNOS, and pAKT, respectively. Scale bar indicates 100 μm. Abbreviations: CGRP, calcitonin gene-related peptide; ION, infraorbital nerve; nNOS, neuronal nitric oxide synthase; NPY, neuropeptide Y; pAKT, phosphorylated AKT; SP, substance P; TGs, trigeminal ganglia.

Figure 4

Expression of NPY and Iba1 in ipsilateral and contralateral TGs 2 weeks after partial ION transection. Notes: NPY-LI is upregulated in ipsilateral TG (B) compared with the contralateral one (A). Immunofluorescence micrographs show a hypertrophic morphology of Iba1-LI in ipsilateral (D and F) vs. contralateral TGs (C and E), counterstaining with PI (red) (E and F). Arrows indicate the NPY-positive neurons (B) and Iba1-positive microglial cells (E and F), respectively. Scale bars indicate 50 μm (A and B), 30 μm (C and D), and 25 μm (E and F). Abbreviations: ION, infraorbital nerve; LI, like immunoreactivity; NPY, neuropeptide Y; PI, propidium iodide; TGs, trigeminal ganglia.

Figure 5

Immunofluorescent micrographs show the expression of biomarkers in contralateral TGs 2 weeks after partial ION transection. Notes: CGRP-LI (A), SP-LI (B), and pAKT-LI (C) are present in the TG neurons. Arrows indicate positive neurons. Scale bar indicates 50 μm. Abbreviations: CGRP, calcitonin gene-related peptide; ION, infraorbital nerve; LI, like immunoreactivity; SP, substance P; TGs, trigeminal ganglia; pAKT, phosphorylated AKT.