Reactive oxygen species induce procalcitonin expression in trigeminal ganglia glia

Abstract

Objective: To examine calcitonin gene-related peptide (CGRP) gene expression under inflammatory conditions using trigeminal ganglia organ cultures as an experimental system. These cultures have increased proinflammatory signaling that may mimic neurogenic inflammation in the migraine state.

Background: The trigeminal nerve sends peripheral pain signals to the central nervous system during migraine. Understanding the dynamic processes that occur within the trigeminal nerve and ganglion may provide insights into events that contribute to migraine pain. A neuropeptide of particular interest is CGRP, which can be elevated and play a causal role in migraine. However, most studies have overlooked a second splice product of the Calca gene that encodes calcitonin (CT), a peptide hormone involved in calcium homeostasis. Importantly, a precursor form of CT called procalcitonin (proCT) can act as a partial agonist at the CGRP receptor and elevated proCT has recently been reported during migraine.

Methods: We used a trigeminal ganglion whole organ explant model, which has previously been demonstrated to induce pro-inflammatory agents in vitro. Quantitative polymerase chain reaction and immunohistochemistry were used to evaluate changes in messenger ribonucleic acid (mRNA) and protein levels of CGRP and proCT.

Results: Whole mouse trigeminal ganglia cultured for 24 hours showed a 10-fold increase in CT mRNA, with no change in CGRP mRNA. A similar effect was observed in ganglia from adult rats. ProCT immunoreactivity was localized in glial cells. Cutting the tissue blunted the increase in CT, suggesting that induction required the close environment of the intact ganglia. Consistent with this prediction, there were increased reactive oxygen species in the ganglia, and the elevated CT mRNA was reduced by antioxidant treatment. Surprisingly, reactive oxygen species were increased in neurons, not glia.

Conclusions: These results demonstrate that reactive oxygen species can activate proCT expression from the CGRP gene in trigeminal glia by a paracrine regulatory mechanism. We propose that this glial recruitment pathway may occur following cortical spreading depression and neurogenic inflammation to increase CGRP nociceptive actions in migraine.

Keywords: calcitonin gene-related peptide; migraine; procalcitonin; reactive oxygen species; trigeminal ganglion.

Figure 1. Organ culture induces CT mRNA in rat and mouse trigeminal ganglia

A. Schematic of the alternative mRNAs from the Calca locus, coding for either calcitonin (CT) or calcitonin gene-related peptide (CGRP). The precursor peptides proCT and proCGRP and mature CT and CGRP peptides from each mRNA are shown relative to their corresponding mRNA sequences. B. Organ culture (24 h, black bars) leads to an increase in CT mRNA, with no change in CGRP mRNA in both mouse and rat relative to fresh tissue (gray bars). Mean +/−SEM from 5 (for CT) and 6 (for CGRP) independent experiments for mouse and 3 for rat. *, p<0.05 vs. fresh (Mann Whitney test).

Figure 2. ProCT immunoreactivity in mouse glial cells

Fresh tissue (A-C) has only faint proCT immunoreactivity (red, B). Cultured ganglia (24 h, D-F) contain proCT immunoreactivity (red, E) that co-localizes with GFAP (green, D), a glial cell marker. GFAP immunoreactivity also increases after 24 h. Open and filled arrowheads indicate cells defined as faint and bright proCT positive cells, respectively. Inset regions of panels D-F are higher magnificent images of the region outlined with a white box. Inset scale bar: 5 μm. G-I: ProCT (red, H) does not co-localize with the neuronal marker beta tubulin III (green, G). J-L: CGRP immunoreactivity (red, K) is found in a subset of neurons (labeled with beta tubulin III, green, J). No change in staining intensity or relative amount of CGRP positive neurons was noted after 24 h. All panels: nuclei are labeled with DAPI. Panels C, F, I, L: merged images. Scale bar: 50 μM.

Figure 3. CT induction is diminished in organ slices

Levels of CT and VEGF (hypoxia marker) RNA were determined in fresh and cultured (24 h) mouse ganglia, as well as ganglia that were cut and then cultured at normoxia (21% O₂) or hypoxia (1% O₂). Mean +/−SEM from 3 independent experiments. *, p < 0.05 vs. fresh (Kruskal Wallis test with Dunn’s posttest).

Figure 4. Treatment with an anti-oxidant attenuates CT induction

Mouse trigeminal ganglia incubated with tempol (Temp, 1 mM) show a smaller increase in CT compared to untreated ganglia. Mean +/− SEM from 3 independent experiments. *, p < 0.05 vs. fresh (Kruskal Wallis test with Dunn’s posttest).

Figure 5. Reactive oxygen species accumulate in neurons following 24 h mouse organ culture

A-F: CellROX Deep Red staining demonstrates the presence of ROS in neurons (based on double labeling with NeuN, indicated with arrowheads) of cultured (24 h), but not fresh, ganglia. Images are representative of 3 independent experiments. Sections are stained with DAPI to detect nuclei. Scale bar: 50 μM. G and H: dihydroethidium (DHE) staining shows superoxide presence in neurons (based on size and location, indicated with arrowheads). Scale bar: 100 μM