Background
Migraine is among the leading causes of disability, having a huge impact on public health [
1,
2]. Studies show that each year 2.5% of episodic migraine disease converts into chronic migraine [
3] which appears as a distinct entity in the classification of the International Headache Society. Although numerous studies have been performed aiming to understand the pathophysiology of migraine and the chronification process, however this is still enigmatic. The trigeminal system plays a pivotal role in the genesis of migraine headache [
4,
5]. The pseudo-unipolar nerve cells of the trigeminal ganglion (TG), provide sensory innervation of cranial structures and meningeal vessels while central projections terminate in trigeminal nucleus caudalis (TNC) and C
1-C
2 region of the spinal cord [
6]. This trigeminovascular complex transmits pain signals from meningeal and cerebral vessels to the brainstem and second-order neurons terminate in the thalamus and cortical regions, where further transmission and modulation of pain sensation occur [
6‐
8]. Following continous and repeated stimulation peripheral and central sensitisation of the primary-neurons might occur, leading to reduced activation treshold, represented clinically by allodynia [
4,
9,
10].
Previous studies have shown that application of inflammatory substances on the dura mater causes central sensitisation of the neurons in TNC and at C
1-C
2 levels of the spinal cord [
6,
10,
11]. Recent studies have demonstrated lower levels of kynurenic acid (KYNA) in serum of patients suffering from chronic migraine compared to controls [
12,
13]. KYNA could be a new therapeutic line in migraine chronification, but KYNA can poorly cross the blood–brain barrier (BBB), while newer KYNA analogues have better BBB penetration characteristics [
14,
15]. We have recently developed an animal model for long-term trigeminovascular activation following application of Complete Freund’s Adjuvant (CFA) onto the surface of the dura mater [
16]. We found activation of satellite glial cells and neurons of the trigeminal ganglion (IL-1, pERK1/2) that were abolished by the KYNA-analogue, SZR72 [
17] possibly acting on peripheral and central gluatamate receptors (30). The present study was designed to examine whether dural application of CFA can cause activation of the central part of the trigeminalvascular system,: the TNC and C
1-C
2 regions of the spinal cord. We asked the question whether the CFA-induced activation might be mitigated by use of SZR72 intraperitoneally.
Methods
Synthesis of novel KYNA derivative
The KYNA amide reported here was designed in the Pharmaceutical Chemistry and Research Group for Stereochemistry, University of Szeged Hungary. The synthesis procedure has previously been presented [
15,
17]. The KYNA analogue (SZR72, N-(2-N,N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride) has the following structural properties: the presence of a water-soluble side-chain, the inclusion of a new cationic centre, and side-chain substitution in order to enhance brain penetration [
17].
Animals
Adult male Sprague–Dawley rats (220–300 g) (n = 30) were used. The animals were raised and maintained under standard laboratory conditions with free access to food and tap water. The study followed the guidelines of the European Communities Council (86/609/ECC) and was approved by the Ethics Committee of The Faculty of Medicine, University of Szeged, Hungary (I-74-12/2012).
Treatments
The animals were divided into 7 groups: (i) CFA + saline application to the dura, (ii) saline application to the dura, (iii) pre-treatment KYNA (KYNA analog, 300 mg/kg body weight dissolved in 1 ml saline, 1 h before CFA administration), (iv) pre-treatment saline (saline, 1, ml 1 h before CFA), (v) repeated treatment (KYNA analog, 300 mg/kg body weight dissolved in 1 ml saline every 12 h, for 7 days), (vi) repeated saline (saline 1 ml every 12 h, for 7 days) and (vii) fresh (intact, control rats) (Table
1).
Table 1
Groups of animals
CFA | CFA | - | - | 6 |
Saline | saline | - | - | 3 |
CFA + KYNA pre-treatment | CFA | KYNA | - | 6 |
CFA + saline pre-treatment | CFA | saline | - | 3 |
CFA + KYNA repeated | CFA | KYNA | KYNA | 6 |
CFA + saline repeated | CFA | saline | saline | 3 |
Fresh, control rats | - | - | - | 3 |
Operation
The operation has been described in details earlier [
16,
17]. Briefly, animals were deeply anesthetized and a handheld drill was used to remove a 3x3 mm large portion of the parietal bone, cooled by saline irrigation to avoid local healing. The hole was made postero-laterally to the bregma (5 mm), on the left side, care being taken not to penetrate the dura mater. Ten μl of CFA (Sigma-Aldrich, St. Louis, MO, USA) or saline was applied on the dural surface, and washed with saline after 20 min.
Both treated and control animals were transcardially fixation-perfused with 4% paraformaldehyde in buffer after 7 days. As fresh control, intact rats were used.
Tissue analysis
After the perfusion-fixation the TNC brainstem region and C1-C2 region of the spinal cord were removed (−1, +5 mm from the obex). Specimens were frozen on dry ice, stored at −80 °C and prepeared for immunohistochemistry. To encompass TNC, sections were collected from 6 different levels from the central canal was visualized to the C1 segment of the spinal cord. (100–120 sections in total per animal).
Immunohistochemistry and microscopic analysis
Immunohistochemical staining was performed to demonstrate the localization of glutamate, c-fos, TNF-α, IL-1β, IL-6, substance P and PACAP. Details of the primary and secondary antibodies are given in Table
2 and
3.
Table 2
Details of primary antibodies used for immunohistochemistry
Anti c-fos | PC38 | Rabbit | 1:100 | Merck Millipore, Darmstadt, Germany |
Anti PACAP-38 | B57–1 | Rabbit | 1:100 | Europroxima, Arnhem, Netherlands |
Anti Glutamate | G9282 | Mouse | 1:100 | Sigma-Aldrich, St-Luis, MO, USA |
Anti Glutamate | AB5018 | Rabbit | 1:100 | Merck Millipore, Darmstadt, Germany |
Anti Substance P | B 45–1 | Rabbit | 1:200 | Europroxima, Arnhem, Netherlands |
Anti IL-1β | ab 9787 | Rabbit | 1:100 | Abcam, Cambridge, UK |
Anti IL-6 | ab6672 | Rabbit | 1:200 | Abcam, Cambridge, UK |
Anti-TNF α | ab66579 | Rabbit | 1:400 | Abcam, Cambridge, UK |
Table 3
Details of secondary antibodies used for immunohistochemistry
FITC (goat) | anti-rabbit | 1:100 | Cayman Chemical, Ann Arbor, MI, USA |
Alexa 488 (goat) | anti-mouse | 1:100 | Invitrogen, CA, USA |
Alexa 594 (donkey) | anti-rabbit | 1:100 | Jackson Immuno Research, West Baltimor, PA, USA |
The immunohistochemical method and the microscopic analysis have been described earlier [
16]. The areas of the brainstem were identified using rat brain atlas (Paxinos and Watson, second edition, 1986). Each procedure was repeated a minimum of three times to validate the results and minimize any experimental errors using the same antibody stock. Negative controls were performed for each set by omitting the primary antibody. One examinator was blinded. Any resulting immunofluorescence would suggest unspecific binding of the secondary antibodies.
Discussion
In this study we present the immunostaining pattern of several neuronal messengers and cytokines in the TNC/C1-C2 spinal region (11) that are indicated in migraine pathophysiology. CFA is a potent immun- potentiator, used in various peripheral pain model. (Spinal distribution of c-Fos activated neurons expressing enkephalin in acute and chronic pain models, 1st manu) We asked the question whether application of CFA on a defined area of the dura mater could cause activation of second-order neurons in the TNC and whether this activation can be mitigated by systemic adminstration of a KYNA analogue.
Gluatamate, the major excitatory neurotransmitter in CNS plays a key role in the trigeminovascular activation, especially in central sensitisation via activation of NMDA receptors [
18,
19]. Glutamate appears to be involved in nociception since glutamate is expressed in the trigeminal ganglion and other sensory ganglia [
20,
21]. Glutamate can be released from neurons following nociceptive stimuli putatively acting on satellite glial cells (SGC) [
22] but is also expressed in the sensory Aδ – fibers (19).
The kynurenine pathway, the major route of tryptophan metabolism to nicotinamide, has an important role in several diseases of the CNS [
23‐
25]. Kynurenic acid (KYNA) is one of the neuroactive metabolits of the kynurenine pathway in human astrocytes [
26] protecting against neuronal cell-death [
27]. KYNA in low concentration enhances AMPA receptor activity [
28,
29], while in high concentrations blocks the NR1 subunit of the NMDA receptors [
19,
30]. NMDA receptor consists of NR1, NR2 and NR3 subunits, where NR1 subunit has a glycine- binding domain. Glycine is essential for the functioning of the NMDA receptor and KYNA acts as an antagonist on the gylcine-binding site (NR1) (18). High level of KYNA might have a neuroprotective effect and could act on glutamate receptors, exerting an inhibitory effect on glutamate release [
23].
Here we asked the question whether the KYNA analogue might act on the fibers and cells in the TNC/C
1-C
2. Previous work has shown a positive effect in TG following CFA injection into the temporomandibular joint [
31] while SZR 72 decreased c-fos activation in the TNC in nitroglycerin induced trigeminal activation [
32]. We have reported that one dose of SZR 72 is able to reduce dura mater applied CFA induced activation in the TG [
16]. C-fos immunoreactivity is a widely used marker of neuronal activity in the TNC [
33,
34]. In the present study we report increased c-fos immunoreactivity following dura mater application of CFA as a sign of neuronal activity of TNC neurons. This effect is attenuated by SZR72. Glutamate activation as a sign of central sensitization can be observed in the second-order neurons after use of CFA, this effect that is also mitigated by the KYNA analogue. Surprisingly repeated-treatment of SZR 72 was not seen to be more effective than pre-treatment with one dose prior CFA application neither in the TG [
16] nor in the TNC. Therefore we postulate that early KYNA derivate intervention can block the development of central sensitization, whereas late, repeated treatment might not be able to further moderate mechanisms of central sensitization. Consequently, we assume that the action of the KYNA analogue seems to be exerted on the periphery that is conveyed to neurons of TNC, but an effect on central mechanisms cannot be surely excluded. Further studies are needed to elucidate the possible site of actions of the KYNA derivate.
In this study we examine a fair number of molecules suggested to play a role in migraine. Among these CGRP and PACAP 38 (PACAP) is currently of particular interest. CGRP plays an important role in migraine pathophysiology and localization of CGRP and its receptors (CLR and RAMP1) has already been described in TNC and C
1 region of the spinal cord [
35,
36]. PACAP is a neuromodulator that has some common actions with CGRP, sharing the same receptors RAMP1 subunit [
37]. PACAP might play a role in migraine having various neurobiological functions such as inhibitory effect on neurogenic inflammation [
38]. PACAP has shown to be involved in trigeminovascular activation as PACAP-38 infusion caused headache in healthy volunteers [
39] and PACAP-38-like immunoreactivity has proved to be altered in ictal compared to interictal phase of migraine and in cluster headache [
40,
41]. It is suggested that the effect of PACAP is biphasic: lower concentration increasing, higher concentration inhibiting the NMDA receptor activation [
42]. We have found PACAP positive but no change in TNC and C
1-C
2.
In addition, we examined several other molecules putatively involved in migraine pathophysiology: SP, IL-1β, IL-6 and TNF-α which have all been shown to be associated with activation of the trigeminovascular system [
43‐
46]. While we could document their presesnce in the TNC and C
1-C
2 of the spinal cord, we did not observe a difference in expression between saline vehicle or CFA administration.
Acknowledgements
This work was supported by the Swedish Medical Research Council (5889), and the Hungarian Brain Research Programme (NAP, Grant No. KTIA_13_NAP-A-III/9.); by EUROHEADPAIN (FP7-Health 2013-Innovation; Grant No.602633), by the GINOP-2.3.2–15–2016–00034 grant and by the MTA-SZTE Neuroscience Research Group of the Hungarian Academy of Sciences and the University of Szeged.