Expression of messenger molecules and receptors in rat and human sphenopalatine ganglion indicating therapeutic targets

Migraine and Cluster Headache (CH) are two primary headaches with severe disease burden. In particular, CH is an extremely painful disorder characterized by periods (clusters) of recurrent, unilateral attacks of excruciating pain with a retro-orbital maximum and attacks lasting from 15–180 min [1]. CH usually appears between 20 and 40 years of age and during its active phase the attacks occur from once every second day to 8 times a day. Most patients show ipsilateral symptoms such as conjunctival injection, lacrimation, nasal congestion, rhinorrhoea and forehead/facial sweating, indicating an ipsilateral parasympathetic dysfunction which has been proven by co-release of the parasympathetic messenger molecule VIP [2].

The mechanisms involved in migraine and CH are considered to differ but also share some aspects [3]. Cranial autonomic symptoms (CAS), i.e. parasympathetic symptoms, occur in both migraine and CH patients [4‐6], indicating involvement of the trigemino-autonomic reflex with increased parasympathetic outflow [5‐7], mediated through the sphenopalatine ganglion (SPG) [8, 9]. Markedly raised levels of VIP and, in particular, calcitonin gene-related protein (CGRP) have been measured during spontaneous CH attacks. These findings were considered as evidence of involvement of the cranial parasympathetic nervous system [2]. In human SPG the parasympathetic signaling transmitters in neural cell bodies consist mainly of VIP, PACAP, acetylcholine (ACh) and NOS [10].

Onabotulinumtoxin A (BoNT-A), more commonly known by the trade name Botox®, comes from Clostridium botulinum. It works by blocking the release of the neurotransmitter ACh, which has been found in SPG [11], by cleaving SNAP25, a protein necessary for ACh release from vesicles in nerve endings [12]. However, it is unknown if SNAP25, and SV-2A, are expressed in the SPG. A previous study from our group has shown the presence of SV-2A and SNAP25 in rat trigeminal ganglion (TG) [11]. Incubation with BoNT-A was shown to reduce the inflammation response elicited by organ culture of the TG.

Triptans are 5-hydroxytryptamine (5-HT) receptor agonists with a high affinity for the 5-HT_1B/1D/1F receptors, which generally are effective for aborting headache attacks of both migraine and CH. The multiple mechanism of action for 5-HT_1B/_1D receptors includes vasoconstriction, inhibition of the release of vasoactive neurotransmitters by trigeminal nerves as well as inhibition of nociceptive neurotransmission [13, 14]. 5-HT_1F receptors is characterized by lack of vasoconstrictive properties [15]. 5-HT_1B,_1D receptors have been localized in the human TG [14‐16]. Activation of those receptors seems to be one of the triptans modes of action. Early clinical studies showed effects in CH [2], thus triptans might have a direct effect on human SPG.

The aim of the present work was to examine if rat and human SPG contain the SV2-A and SNAP25 proteins and, by extension, if BoNT-A might have a mechanism of action in SPG. Secondly, we aimed at investigating the expression of 5-HT_1B,1D,1F receptors and the SPG neurotransmitters (PACAP-38, nNOS and VIP). This will provide novel and greater understanding of the action mechanisms in the SPG and could increase the possibility for future drug developments for CH.

Wistar or Sprague–Dawley male rats (n _Wistar = 9, n _{Spraque-Dawley} = 10, weight = approx. 250 g) were euthanized by CO₂ inhalation followed by decapitation. The SPG was carefully dissected out, close to the time of euthanasia, by initially making an incision over the zygomatic bone. The zygomatic bone was cut at both extremities and removed. The exposed deep masseter muscle was removed. The fifth cranial nerve trunk was revealed, carefully cut, and pulled posteriorly. The SPG, situated against the dorsal part of the maxillary bone, is thereby disclosed. The entire ganglion was carefully dissected and placed in 4 % paraformaldehyde for 2–4 h, followed by incubation overnight in Sörensen’s phosphate buffer (pH 7.2) containing 10 % and 25 % sucrose in turn. Thereafter, the tissue was embedded in Yazulla embedding medium (30 % egg albumin and 3 % gelatin in distilled water) and 10 μm cryosections were cut in a cryostat (Thermo Scientific Microm HM560). The sections were stored at −20 °C until use.

The human SPG was collected at autopsy, within 48 h post-mortem, from three patients. The patients were without disorders related to the central nervous system. The specimens were fixed in 4 % paraformaldehyde followed by sucrose-cryoprotection in 10 % sucrose Tyrode solution. The tissue samples were kept at −80 °C until embedding and cryo-sectioning. The study followed the guidelines of the European Communities Council (86/609/ECC) and was approved by the Committee of the Animal Research of University of Szeged (I-74-12/2012) and the Scientific Ethics Committee for Animal Research of the Protection of Animals Advisory Board (XI./352/2012). The rat study was approved by the Regional Ethical Committee on Animal Research, Malmö/Lund, Sweden. (M43-07).

The present study is the first to examine the co-expression of signalling molecules and receptor elements in human and rat SPG. It is well known that triptans have clinically positive effects on acute pain in CH [17]. Thus, we asked the question if 5-HT_1B, 5-HT_1D and 5-HT_1F receptors are expressed in neurons and SGCs in SPG. Importantly, we report that 5-HT_1B, 5-HT_1D and 5-HT_1F receptors are expressed on most neurons in the rat SPG, which correlates well with the clinical effectiveness of triptans in CH. Here we demonstrate that all three 5-HT receptor subtypes occur in neurons and SGCs of the rat SPG. However, the 5-HT_1F receptor was only found in rodent material, possibly due to the antigenic properties of the used antibody. In addition 5-HT_1B receptors occur in the intraganglionic blood vessels, putatively indicating a possible vasomotor role. Previous studies have revealed expression of the parasympathetic signaling transmitters VIP, PACAP and nNOS in rat [18] and human [19] SPG. The results in the present study are in concert with these earlier studies. We found that both species contain SV2-A and SNAP25, elements involved in ACh neurotransmission, which has not been described earlier, however with a mixed expression. In rat, SNAP25 was expressed in neurons and fibers, but with SV2-A in the SGCs. In humans, SNAP25 was expressed in the SGCs, but SV2-A in the neurons. SNAP25 was mainly seen in the SGCs, while in man the neurons expressed Botox receptors elements SV2-A (opposite in rat). This could indicate that some effect of BoNT-A could occur in SPG provided it reaches this structure. The anatomical proximity of facial/temporal injection sites of BoNT-A in the PREEMPT protocol is much closer to the SPG than to the TG. The significance of the differential expression of SNAP25 and SV2-A is unclear but perhaps the localization of the receptor elements might suggest a potential target site of botulinum toxin if it has access to the receptor site.

Treatment with BoNT-A in adults with chronic migraine (CM) has shown safety and efficacy [20, 21]. Pilot studies of SPG injection of BoNT-A for treatment of CM as well as in chronic CH (CCH) have showed promising results [22, 23]. A previous study has shown presence of SV-2A and SNAP25 protein with same location in the TG [11]. The present results illustrate a possible site/mechanism of action for BoNT-A in CH. There is however no data available for an effect of BoNT-A in CH. The work provides anatomical rationale for this possibility, and given the proximity of the SPG to injection sites used in BoNT-A therapy it might be considered at least. Recent work has focused on neuromodulation of the SPG using e.g. specific SPG stimulation [24, 25].

An issue for the present work is if BoNT-A has a theoretical possibility to work as medical prophylaxis in CH. Earlier studies have shown varying results regarding BoNT-A as prophylactic treatment for CH. Twelve CCH patients were included in an open study where BoNT-A was given as an add-on therapy, i.e. prior prophylactic medication was continued [26]. BoNT-A was injected according to a standardised protocol, ipsilateral to the pain. Four of the twelve patients showed improvement [26]. A pilot study where BoNT-A was injected towards the SPG in CCH patients showed at least 50 % reduction of attack frequency in five out of ten patients [27]. So far, no randomised, placebo-controlled study regarding BoNT-A and treatment of CH has been performed.

Triptans were early found to abort CH attacks [28‐30]. It was not until fairly recently that Ivanusic (2011) reported 5-HT_1D receptor immunoreactivity in nerve terminals around neurons in the rat SPG. These fibers were all CGRP positive and thus sensory in nature. They were traced back to the TG. Csati et al. (2012) showed CGRP positive fibers also in the human SPG, which agrees well with the present study.

Triptans are 5-HT_1B/_1D receptor agonists with high affinity for 5-HT_1B/_1D receptors, which are generally effective for aborting attacks in both migraine and CH. The multiple mechanism of action for 5-HT_1B/_1D receptors includes vasoconstriction, inhibition of the release of vasoactive neuropeptides by trigeminal nerves as well as inhibition of nociceptive neurotransmission [13, 14]. Both 5-HT_1B/1D receptors have been localized in the human TG [14, 16]. Activation of those receptors seems to be one of the triptans modes of action. Triptans might have a direct effect on human SPG. So far, the issue has to be answered. We showed 5-HT_1B and 5-HT_1D immunoreactivity in SPG neurons, which suggests a role in modifying the activity in SPG. The variability in the 5-HT_1F expression is likely due to low specificity of these antibodies species. This issue is under current development.

Some limitations of our study need to be addressed. The human material is restricted to three SPG obtained at autopsy, due to difficulties to obtain those structures. Further, although the material has been carefully processed, we cannot exclude postmortem changes. In addition, our findings are purely anatomical and the question as to function may be addressed in subsequent work.

Theoretically, our work provides anatomical indication, that both triptans and BoNT-A may have an effect on the SPG. Further randomised, placebo-controlled studies regarding especially BoNT-A treatment of CH are warranted. In addition this study also provides evidence for triptan effects in the SPG.