The effect of orofacial complete Freund’s adjuvant treatment on the expression of migraine-related molecules

Abstract

Background: Migraine is a neurovascular primary headache disorder, which causes significant socioeconomic problems worldwide. The pathomechanism of disease is enigmatic, but activation of the trigeminovascular system (TS) appears to be essential during the attack. Migraine research of recent years has focused on neuropeptides, such as calcitonin generelated peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP1-38) as potential pathogenic factors and possible therapeutic offensives. The goal of present study was to investigate the simultaneous expression of CGRP and precursor of PACAP1-38 (preproPACAP) in the central region of the TS in a time-dependent manner following TS activation in rats. Methods: The right whisker pad of rats was injected with 50 μl Complete Freund's Adjuvant (CFA) or saline. A mechanical allodynia test was performed with von Frey filaments before and after treatment. Transcardial perfusion of the animals was initiated 24, 48, 72 and 120 h after injection, followed by the dissection of the nucleus trigeminus caudalis (TNC). After preparation, the samples were stored at − 80°C until further use. The relative optical density of CGRP and preproPACAP was analyzed by Western blot. One-way ANOVA and Kruskal-Wallis followed by Tukey post hoc test were used to evaluate the data. Regression analysis was applied to explore the correlation between neuropeptides expression and hyperalgesia. Results: Orofacial CFA injection resulted in significant CGRP and preproPACAP release in the TNC 24, 48, 72 and 120 h after the treatment. The level of neuropeptides reached its maximum at 72 h after CFA injection, corresponding to the peak of facial allodynia. Negative, linear correlation was detected between the expression level of neuropeptides and value of mechanonociceptive threshold. Conclusion: This is the first study which suggests that the expression of CGRP and preproPACAP simultaneously increases in the central region of activated TS and it influences the formation of mechanical hyperalgesia. Our results contribute to a better understanding of migraine pathogenesis and thereby to the development of more effective therapeutic approaches.

Neurotransmitter and tryptophan metabolite concentration changes in the complete Freund’s adjuvant model of orofacial pain

“…The current study draws attention to the limited time interval for therapies targeting glutamatergic pathways as well, as based on our previous experiments, a clear shift to dominantly peptide-mediated pain processing can be seen even from 24 h after CFA application [9]. This time point corresponds to the onset of peripheral and central sensitization of the TS as well in this model [10,11,14].…”

“…1). Similar to the previous experiment on PACAP and CGRP in the same model [9], only sham-injected rats processed 24 h following the injection were used as CO, as a pilot study conducted on naïve and shaminjected (processed 24 and 48 h following injection) rats demonstrated that there is no difference in the level of the metabolites of interest, in neither TNC, nor ssCX (n = 3 in each group, data not shown). The rats were anesthetized with intraperitoneal 4% chloral hydrate solution mainly based on its safe application (CAS ID: 302-17-0, Sigma-Aldrich, St. Louis, MO, USA; 10 ml/kg body weight dose) in the morning and 50 μl of CFA was injected into the right whisker pad.…”

“…For the induction of this peripheral inflammation, the application of Complete Freund's adjuvant (CFA) into the whisker pad or the dural parietal surface is a widely used method [6,8,9] as it is able to enhance local reaction at the injection site and then to evoke the release of inflammatory cytokines, alongside with hyperalgesia/allodynia on the face via the activation/sensitization of the TS [7]. Regarding the delay of the development of peripheral and central sensitization, indirect data from studies with CFA injection to the paw demonstrated that pain hypersensitivities were observed 24 h after the injection [9][10][11][12][13], whereas data from studies with orofacial CFA model, more precisely from the temporomandibular joint induced inflammation model, suggest that both thermal and mechanical allodynia peak at 24 h as well [14]. The orofacial CFA model has been thoroughly studied regarding gene expression characteristics [6,[15][16][17][18][19][20].…”

“…The orofacial CFA model has been thoroughly studied regarding gene expression characteristics [6,[15][16][17][18][19][20]. Recently, in relation to two migraine-related biomarkers, the pituitary adenylate cyclase-activating peptide (PACAP) and calcitonin gene-related peptide (CGRP), their increasing levels were detected starting even 24 h after the administration of CFA in the trigeminal nucleus caudalis (TNC) [9]. However, there are no studies which aimed at the investigation of the small molecule neurotransmitters and neuromodulators and some of their precursors (glutamate (Glu), γaminobutyric acid (GABA), setotonin (5-hydroxy-tryptamine; 5-HT), noradrenaline (NA), tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA)) in this model with established or presumed role in the development of peripheral and central sensitization during headache.…”

“…Based on the observed gradually increasing levels of PACAP and CGRP from 24 h following CFA injection in our previous experiment [9], the aim of the current study was to find the shift point of concentration changes of small molecule neurotransmitters and neuromodulators and the above-mentioned peptides. This may yield substantial information for the selection between different therapeutic paradigms regarding diseases involving the activation of the TS, such as primary headache disorders, including migraine.…”