Effects of CGRP-Primed Dental Pulp Stem Cells on Trigeminal Sensory Neurons

Dental pulp stem cells (DPSCs) are important in tooth physiology, contributing to development, repair, regeneration, and immunomodulatory processes. However, their role in inflammatory mechanisms underlying pulpitis is not well understood. We evaluated the influence of DPSCs stimulated with calcitonin gene-related peptide (CGRP), a proinflammatory neuropeptide, on the expression of mediators released from DPSCs and the effect of these mediators on sensory neuron activity. Human DPSCs were treated with either control media or media containing CGRP (10−8 M) for 7 d, and the conditioned media (CM) containing DPSC-released mediators was collected. The expression of cytokines and chemokines from DPSCs was evaluated by reverse transcription quantitative polymerase chain reaction. The effects of the CM from CGRP-primed DPSCs (primed DPSC-CM) were evaluated on sensory afferents by using primary cultures of mouse trigeminal neurons and an organotypic model of cultured human pulp slices. Mouse trigeminal neurons and human pulp explants were pretreated for 24 h with control or primed DPSC-CM and then stimulated with capsaicin. Afferent activity was measured by quantifying the response to capsaicin via live cell calcium imaging in mouse neurons and CGRP released from pulp explants. Gene expression analysis showed that primed DPSCs overexpressed some proinflammatory cytokines and chemokines, including chemokines CXCL1 and CXCL8, which are both agonists of the receptor CXCR2 expressed in sensory neurons. Primed DPSC-CM increased human pulp sensory afferent activity as compared with control DPSC-CM. Similarly, primed DPSC-CM increased the intensity of calcium responses in cultured mouse trigeminal neurons. Furthermore, the CXCR2 antagonist SB225002 prevented trigeminal neuron sensitization to capsaicin induced by primed DPSC-CM. In conclusion, mediators released by DPSCs, primed with the proinflammatory mediator CGRP, induce neuronal sensitization through CXCR2 receptor. These data suggest that DPSCs might contribute to pain symptoms that develop in pulpitis.

Estrogen receptors α, β and GPER in the CNS and trigeminal system - molecular and functional aspects

Background Migraine occurs 2–3 times more often in females than in males and is in many females associated with the onset of menstruation. The steroid hormone, 17β-estradiol (estrogen, E2), exerts its effects by binding and activating several estrogen receptors (ERs). Calcitonin gene-related peptide (CGRP) has a strong position in migraine pathophysiology, and interaction with CGRP has resulted in several successful drugs for acute and prophylactic treatment of migraine, effective in all age groups and in both sexes. Methods Immunohistochemistry was used for detection and localization of proteins, release of CGRP and PACAP investigated by ELISA and myography/perfusion arteriography was performed on rat and human arterial segments. Results ERα was found throughout the whole brain, and in several migraine related structures. ERβ was mainly found in the hippocampus and the cerebellum. In trigeminal ganglion (TG), ERα was found in the nuclei of neurons; these neurons expressed CGRP or the CGRP receptor in the cytoplasm. G-protein ER (GPER) was observed in the cell membrane and cytoplasm in most TG neurons. We compared TG from males and females, and females expressed more ER receptors. For neuropeptide release, the only observable difference was a baseline CGRP release being higher in the pro-estrous state as compared to estrous state. In the middle cerebral artery (MCA), we observed similar dilatory ER-responses between males and females, except for vasodilatory ERβ which we observed only in female arteries. Conclusion These data reveal significant differences in ER receptor expression between male and female rats. This contrasts to CGRP and PACAP release where we did not observe discernable difference between the sexes. Together, this points to a hypothesis where estrogen could have a modulatory role on the trigeminal neuron function in general rather than on the acute CGRP release mechanisms and vasomotor responses.

Trigeminal satellite cells modulate neuronal responses to triptans: relevance for migraine therapy

In the present paper, we have further developed an in vitro model to study neuronal–glial interaction at trigeminal level by characterizing the effects of conditioned medium (CM) collected from activated primary cultures of satellite glial cells (SGCs) on calcitonin gene-related peptide (CGRP) release from rat trigeminal neurons. Moreover, we investigated whether such release is inhibited by a clinically relevant anti-migraine drug, sumatriptan. CM effects were tested on trigeminal neuronal cultures in different conditions of activation and at different time points. Long-term exposures of trigeminal neurons to CM increased directly neuronal CGRP release, which was further enhanced by the exposure to capsaicin. In this framework, the anti-migraine drug sumatriptan was able to inhibit the evoked CGRP release from naïve trigeminal neuron cultures, as well as from trigeminal cultures pre-exposed for 30 min to CM. On the contrary, sumatriptan failed to inhibit evoked CGRP release from trigeminal neurons after prolonged (4 and 8 h) pre-exposures to CM. These findings were confirmed in co-culture experiments (neurons and SGCs), where activation of SGCs or a bradykinin priming were used. Our data demonstrate that SGCs activation could influence neuronal excitability, and that this event affects the neuronal responses to triptans.

Targeted Regulation Of Cgrp Gene Expression

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent regulator of cerebral vascular tone and contributes to neurogenic inflammation. Clinical studies have shown that CGRP levels are elevated during the painful phase of migraine headache, then restored to baseline by antimigraine 5-HT1 drugs. Conversely, CGRP is depleted in perivascular nerve terminals from patients who have suffered vasospasm following subarachnoid hemorrhage. We have investigated the mechanisms controlling CGRP expression in the trigeminal ganglia neurons, which provide virtually all of the CGRP innervation to the cerebral vasculature. We found that nerve depolarization, inflammatory compounds, and nitric oxide can increase CGRP synthesis and secretion. Using both adenoviral vectors and transfection approaches, we have shown that the increased synthesis is due to activation of a cell-specific MAP kinase-responsive enhancer upstream of the CGRP gene. Interestingly, the 5-HT1 migraine drugs are able to block this up-regulation by a mechanism that involves a very prolonged elevation of calcium. We have shown that the duration of the calcium signal is a key determinant for whether a MAP kinase responsive gene will be stimulated or repressed by calcium-activated pathways. This observation supports the importance of a finely tuned balance of calcium in the trigeminal neuron, which is intriguing in light of genetic evidence for calcium channel mutations in a rare form of inherited migraine. These studies suggest that modulation of MAP kinase control of the cell-specific CGRP gene enhancer may be a useful therapeutic strategy for neurovascular disorders.