Recurrent administration of the nitric oxide donor,isosorbide dinitrate,induces a persistent cephalic cutaneous hypersensitivity: A model for migraine progression

Abstract

Background

A subgroup of migraineurs experience an increase in attack frequency leading to chronic migraine.

Methods

We assessed in rats the roles of dose and repeat administration of systemic isosorbide dinitrate (ISDN), a nitric oxide donor, on the occurrence and development of cephalic/face and extracephalic/hindpaw mechanical allodynia as a surrogate of migraine pain, and the effect of acute systemic sumatriptan and olcegepant and chronic systemic propranolol on these behavioral changes.

Results

A single high (H-ISDN) but not low (L-ISDN) dose of ISDN induces a reversible cephalic and extracephalic mechanical allodynia. However, with repeat administration, L-ISDN produces reversible cephalic but never extracephalic allodynia, whereas H-ISDN induces cephalic and extracephalic allodynia that are both potentiated. H-ISDN-induced cephalic allodynia thus gains persistency.

Sumatriptan and olcegepant block single H-ISDN-induced behavioral changes, but only olcegepant reduces these acute changes when potentiated by repeat administration. Neither sumatriptan nor olcegepant prevent chronic cephalic hypersensitivity. Conversely, propranolol blocks repeat H-ISDN-induced chronic, but not acute, behavioral changes.

Conclusions

Repeated ISDN administration appears to be a naturalistic rat model for migraine progression, suitable for screening acute and preventive migraine therapies. It suggests frequent and severe migraine attacks associated with allodynia may be a risk factor for disease progression.

Keywords

Headache allodynia trigeminal triptans CGRP antagonist propranolol

Introduction

Cutaneous hypersensitivity is a key feature of migraine (1), as it reflects central sensitization in the pain pathways subserving meningeal nociception (2). Cutaneous hypersensitivity may stay within the focal headache area (typically the periorbital region) or spread through the face and scalp to the body and limbs (1 –3). Cutaneous hypersensitivity is more prevalent and severe in chronic than episodic migraineurs (1), suggesting that allodynia is a risk factor for transition from episodic to chronic (1) or non-remission from chronic to episodic (4). Assessing cutaneous sensitivity in animal models of migraine could thus usefully help model migraine transformation (5 –8).

Systemic nitroglycerin (NTG) administration is a widely-used model of migraine in both humans and animals (9). Systemic NTG triggers headache in healthy subjects and induces delayed headache with migraine features in migraineurs (9). NTG-induced migraine-like headache is associated with thermal cephalic allodynia (10). In rodents, systemic NTG produces extracephalic thermal and mechanical allodynia (MA) (7,11 –13) as well as hyperalgesia at the hindpaw in the formalin test (13,14). Interestingly, this extracephalic mechanical hypersensitivity becomes part-persistent after chronic intermittent administration of NTG (7). However, on one hand, this result diverges from clinical studies reporting that migraine headache is not associated with cutaneous hypersensitivity below the C8 dermatome, and on the other, the surprisingly few studies that have investigated the effect of systemic NTG on cephalic cutaneous sensitivity come up with conflicting results. A decrease (15,16) or no change (17,18) in periorbital von Frey thresholds has been reported after systemic NTG, whereas animals pre-treated with NTG (14) or isosorbide dinitrate (ISDN) (19) and injected with formalin into the upper lip show an increase in face rubbing behavior. Likewise, NTG administration increases the face rubbing behavior evoked by subcutaneous injection of calcitonin gene-related peptide (CGRP) into the upper lip (20,21).

The experiments reported here were designed to assess the roles of both dose and repeat administration of systemic ISDN, a nitric oxide donor, on cutaneous cephalic and extracephalic mechanical sensitivity. The rationale for testing ISDN was that: a) it reliably produces headache in migraineurs but less often in healthy subjects (22,23), b) it is less hypotensive than NTG (24), c) it has an injectable form, deliverable to humans and animals, allowing comparison between preclinical and clinical results, and d) it does not need to be dissolved in alcohol and propylene glycol, thus reducing non-specific effects (25).

Methods

Animals

Male Sprague-Dawley rats weighing 250–275 g (Charles River, L’Arbresle, France) were housed at 22 ± 1℃ in plastic cages (size: 425 × 266 × 185 mm; 3–4 rats per cage) on soft bedding with ad libitum water and food under a 12 h/12 h light/dark cycle for at least one week before the experiment. Every effort was made to minimize the number of animals used. Numbers of animals were selected according to previous experience (5,26), that is, a trade-off between reaching routine sample sizes for field experiments while minimizing numbers of animals for pain experiments. The experiments ultimately used 55 animals (4–8 rats/group). All experimenters were blind to treatment conditions. Rats were randomized into treatment groups before assessment.

All experiments, analysis and reporting was ARRIVE-compliant (Animals in research: reporting in vivo experiments). Animal experiments were performed according to the ethical guidelines set by the International Association for the Study of Pain (27) and European Directive 2010/63/EU on the protection of animals used for scientific purposes. The protocols applied here for animal care and use were approved by the Clermont Auvergne University institutional review board and authorized by the French Ministry of Primary, Secondary and Higher Education and Research (No. #04648.02).

Behavioral sensory testing

Habituation

The behavioral sensory testing protocols are similar to those described in previous reports (5,26). Rats were first allowed to acclimatize to the 0.3 × 0.3 m² glass chambers used for behavioral testing as well as innocuous mechanical stimulation for three days before beginning behavioral sensory testing. Test sessions took place during the light phase, between 11 am and 7 pm, in a quiet, temperature-controlled (21–23℃) room.

Face and hindpaw sensory testing

Cephalic and extracephalic cutaneous mechanical sensitivity were assessed before and on the day of test (Days 1, 2, 3, 4 and 5: Before injection and at 30 min intervals for 3–5 h after injection) in three groups of animals receiving i.p. vehicle (0.9% saline), low-dose ISDN (5 mg/kg, L-ISDN, Sanofi-Aventis, France) or high-dose ISDN (10 mg/kg, H-ISDN). The volume of ISDN was 10 mL/kg. Cephalic and extracephalic withdrawal thresholds were determined using von Frey (VF) filaments (Bioseb, France) applied to the midline of the forehead (between the eyes; maximum filament strength: 8 g) and the plantar aspect of the hindpaw (maximum filament strength: 15 g), via the descending-ascending method. Each filament was applied to the skin for 5 times for 3 s (at 10 s intervals). The VF filament threshold (VFT, in grams) was equal to the filament evoking a head or hindpaw withdrawal response in three out of five trials. The experimenter was blinded to the experimental group.

The rats had no access to food or water during the tests. Each rat was used once only. At the end of the experiment, the rats were sacrificed with a lethal dose of pentobarbital sodium (150 mg/kg, i.p., CEVA). Death was confirmed by permanent cessation of cardio-respiratory functions.

Drug administration

Sumatriptan (0.3 mg/kg in a 1 mL/kg volume; Imiject, GlaxoSmithKline, France), olcegepant (0.9 mg/kg in a 3 mL/kg volume; Boehringer Ingelheim, Biberach, Germany) or vehicle (0.9% saline) were administered intraperitoneally 5 min after H-ISDN administration. Propranolol (10 mg/kg in a 6 mL/kg volume; Mano Pharma) or vehicle was administrated per os (oral gavage) every day during the five day injection protocol, at one minute before H-ISDN administration, under brief anesthesia (2% halothane; ≤3 min). The doses of each drug were selected on the basis of previous reports (28,29). All testing was performed by an experimenter blinded to treatment. Vehicle and drug treated rats were stored in different cages.

Statistical analysis

All data generated through the study was analyzed using SigmaStat for Windows version 13.0 (SPSS, Erkrath, Germany). Results are expressed as mean ±standard error of the mean (s.e.m.). Statistical analysis was performed using two-way repeated-measures (RM) ANOVA to determine the main effects of time and drug treatment or their interaction on the development of cephalic and extracephalic hypersensitivity. Duncan’s post-hoc test was used to assess differences between groups and across time points. The evolution of the baseline withdrawal responses to cephalic and extracephalic VF filament stimulation before each ISDN administration was analyzed using one-way RM ANOVA followed by a post-hoc Student-Newman-Keuls test (normally distributed data) or Friedman RM ANOVA on ranks with post-hoc Student-Newman-Keuls test (non-normally distributed data). The level of significance was set at P < 0.05.

Results

Effect of systemic administration of ISDN on cephalic and extracephalic cutaneous mechanical sensitivity

To study the roles of dose and repeat administration of ISDN on cutaneous allodynia development, we compared the effects of single and repeated injections of two ISDN doses, that is, a low dose (5 mg/kg, L-ISDN) and a high dose (10 mg/kg, H-ISDN), on both cephalic/face and extracephalic/hindpaw cutaneous mechanical sensitivity patterns. Before starting injections (Figure 1), there were no differences in baseline withdrawal responses to cephalic and extracephalic VF filament stimulation between rats treated with saline (n = 4), L-ISDN (n = 4) and H-ISDN (n = 7), referred to as saline, L-ISDN and H-ISDN rats in the remainder of the paper. The main results are summarized in Table 1.

Figure 1.

Effect of dose and repeat administration of systemic isosorbide dinitrate (ISDN), a nitric oxide donor, on the occurrence and development of cephalic/face and extracephalic/hindpaw mechanical allodynia (MA). Time–courses of von Frey withdrawal thresholds (VFT) of the face (upper panels) and hindpaw (lower panels) after Low doses ((a), L-ISDN: 5 mg/kg i.p., n = 4) and High doses ((b), H-ISDN: 10 mg/kg i.p., n = 7) of ISDN injections. Cutaneous mechanical sensitivity was assessed before and at 30 min intervals for 5 h after ISDN injection. In each panel, the time courses before and after the first (black circles) and fifth (red circles) ISDN injection in L-ISDN and H-ISDN rats and the fifth vehicle in control rats (green circles) are superimposed. Note that facial VFT are already reduced before the fifth H-ISDN, indicating a persistent cephalic MA ((b), upper panel) and that extracephalic MA only appears after the H-ISDN. Values are means ± s.e.m. Analysis using two-way RM ANOVA followed by Duncan’s post hoc test, with time and treatment as factors. # p < 0.05 and ### p < 0.01 indicate the level of significance between the first H-ISDN injection and vehicle (control) injection. *p < 0.05, **p < 0.01, ***p < 0.001 indicate level of significance between the first and fifth H-ISDN injection.

Table 1.

Effect of systemic administration of isosorbide dinitrate (ISDN) on cephalic and extracephalic cutaneous mechanical sensitivity.

	L-ISDN (5 mg/kg)		H-ISDN (10 mg/kg)
	Single	Repeated	Single	Repeated
Cephalic (face)	No effect	Reversible allodynia	Reversible allodynia	Persistent allodynia
Extracephalic (hindpaw)	No effect	No effect	Reversible allodynia	Reversible allodynia

L-ISDN: low dose of ISDN (5 mg/kg), H-ISDN: high dose of ISDN (10 mg/kg).

Single L-ISDN had no effect on cutaneous mechanical sensitivity (Figure 1(a)). However, single H-ISDN produced a reversible cephalic MA (two-way RM ANOVA followed by Duncan's post-hoc test: p < 0.001 on treatment, p < 0.001 on time and p = 0.024 on time × treatment, n = 7, Figure 1(b)) and extracephalic MA (two-way RM ANOVA followed by Duncan's post-hoc test: p < 0.001 on treatment, p < 0.001 on time and p = 0.002 on time × treatment, n = 7, Figure 1(b)). The MA onset, from just 30–60 min after H-ISDN administration, peaked at 1–1.5 h, but was no longer detectable after 2–2.5 h. At peak, cephalic VFT was 2.69 ± 0.47 g (vs. baseline: 7.71 ± 0.29 g; p < 0.001) and extracephalic VFT was 11.57 ± 1.25 g (vs. baseline: 15.00 ± 0.00 g; p < 0.001).

With repeat dose, L-ISDN produced cephalic MA (two-way RM ANOVA followed by Duncan’s post-hoc test: p < 0.022 on treatment, p < 0.001 on time and p < 0.001 on time × treatment, n = 4, Figure 1(a)). A fifth L-ISDN triggered reversible cephalic MA, as VFTs fell to 3.35 ± 0.65 g (vs. baseline, p < 0.001), 1 h after L-ISDN administration (Figure 1(a)). Note that there was still no change in extracephalic sensitivity after a fifth L-ISDN.

Compared to the first H-ISDN, the fifth H-ISDN induced a much stronger change in cutaneous mechanical sensitivity. Cephalic VFTs before this fifth H-ISDN were already lower than baseline VFTs in the same H-ISDN rats (5.43 ± 0.00 g vs. 7.71 ± 0.29 g, RM ANOVA followed by a post-hoc Student-Newman-Keuls test, p = 0.029), indicating persistent static cephalic mechanical hypersensitivity (Figure 1(b)). Moreover, this fifth H-ISDN injection produced a much stronger and longer acute cephalic MA than a single H-ISDN injection: 1 h after the fifth H-ISDN, cephalic VFTs were further reduced to 1.21 ± 0.55 g (vs. 7.71 ± 0.29 g before the first H-ISDN, p < 0.001, n = 7) and did not return to pre-H-ISDN levels until 4 h post-H-ISDN injection (Figure 1(b)). A fifth H-ISDN also elicited a much stronger and longer acute extracephalic cutaneous MA than a single H-ISDN injection. One hour after the fifth H-ISDN, extracephalic VFT were reduced to 9.57 ± 0.55 g (vs. 11.57 ± 1.25 g after the first H-ISDN, p = 0.003) and did not return to pre-H-ISDN levels until 3 h post H-ISDN injection. Extracephalic VFTs before this fifth H-ISDN were not different from VFTs in the same H-ISDN rats before the first injection or in saline rats before the fifth injection, indicating that there was no persistent static extracephalic mechanical hypersensitivity.

As rats only developed persistent cephalic and reversible extracephalic MA with repeat H-ISDN, all subsequent studies were performed with H-ISDN.

Effect of sumatriptan and olcegepant on ISDN-induced cephalic mechanical allodynia

We examined whether two antimigraine drugs, sumatriptan (0.3 mg/kg, i.p.) and olcegepant (0.9 mg/kg, i.p.), were able to reduce ISDN-induced cephalic MA. The main results are summarized in Table 2.

Table 2.

Effect of systemic administration of sumatriptan, olcegepant or propranolol on isosorbide dinitrate (ISDN)-induced cutaneous mechanical allodynia.

	Cephalic allodynia		Extracephalic allodynia
	Single H-ISDN	Repeated H-ISDN	Single H-ISDN	Repeated H-ISDN
Sumatriptan	Yes	No	NT	NT
Olcegepant	Yes	Yes	NT	NT
Propranolol	No	Yes	No	Yes

H-ISDN: high dose of ISDN (10 mg/kg).

No: no effect, Yes: anti-allodynic effect, NT: not tested.

Consistent with the above results, a single H-ISDN injection induced a cephalic MA that peaked at 1 h and lasted less than 2 h in vehicle-treated rats (Figure 2(a)) but not in sumatriptan- and olcegepant-treated (two-way RM ANOVA followed by Duncan's post-hoc test: p < 0.001 on time, treatment and time × treatment, n = 8/group, Figure 2(a)). One hour after ISDN injection, VFTs were higher in sumatriptan-treated rats (6.75 ± 0.75 g, n = 8; p < 0.001) and olcegepant-treated rats (5.93 ± 0.72 g, n = 8; p < 0.001) than in vehicle-treated rats (3.00 ± 0.73 g, n = 8). There was no difference in VFTs between sumatriptan- and olcegepant-treated rats.

Figure 2.

Effect of sumatriptan and olcegepant on ISDN-induced cephalic MA. Time–courses of von Frey withdrawal thresholds (VFT) of the face in vehicle-treated (black circles, n = 8), olcegepant-treated (red circles, 0.9 mg/kg, n = 8) and sumatriptan-treated (green triangles, 0.3 mg/kg, n = 8) rats after (a) single H-ISDN injection or (b) five H-ISDN injections. Antimigraine drugs were administered i.p. 5 min after H-ISDN administration. Values are means ± s.e.m. Analysis using two-way RM ANOVA followed by Duncan’s post hoc test, with time and treatment as factors. *p < 0.05, **p < 0.01, ***p < 0.01 indicate level of significance between vehicle and antimigraine drugs.

Repeat systemic ISDN in vehicle-treated rats induced a strong acute, superimposed on persistent, cephalic MA. Systemic administration of olcegepant, but not sumatriptan, significantly reduced the acute cephalic MA. The anti-allodynia effect of olcegepant was significant at 1 h post-ISDN administration and lasted for at least 4 h (two-way RM ANOVA followed by Duncan’s post-hoc test, p = 0.021 on treatment, p < 0.001 on time, p = 0.068 (non-significant) on time × treatment, n = 8/group, Figure 2(b)). At one hour post-administration, VFTs were lower in vehicle-treated rats (0.75 ± 0.28 g, n = 8) than olcegepant-treated rats (3.76 ± 0.88 g, n = 8; p = 0.019) but not sumatriptan-treated rats (2.49 ± 0.88 g, n = 8; p =0.124). Note that neither olcegepant nor sumatriptan was able to prevent the repeat-ISDN-induced persistent cephalic MA.

Effect of propranolol on ISDN-induced cephalic and extracephalic mechanical allodynia

The β-blocker propranolol is one of the most widely used preventive therapies for chronic migraine. We assessed the behavioral effects of the five-day ISDN injection protocol in rats treated per os with either propranolol (10 mg/kg/day, n = 8) or vehicle (0.9% saline, n = 8). The main results are summarized in Table 2. At this dose, propranolol had no motor effect, as tested by the accelerating rotarod (data not shown).

In vehicle-treated rats, a single H-ISDN exclusively produced a reversible cephalic MA (Figure 3(a)), whereas repeat H-ISDN produced both a persistent cephalic and a reversible extracephalic MA (Figure 3(b)). Preventive treatment with propranolol completely suppressed the repeat-H-ISDN-induced behavioral changes (two-way RM ANOVA followed by Duncan’s post-hoc test: p = 0.018 on treatment, p < 0.001 on day, p < 0.001 on treatment × day, n = 8/group, Figure 3(a)). The effect of propranolol on H-ISDN-induced cephalic MA was significant from the third administration (data not shown). Chronic propranolol treatment (n = 8) also prevented the repeat-H-ISDN-induced extracephalic MA (two-way RM ANOVA followed by Duncan’s post-hoc test: p =0.041 on treatment, p = 0.001 on day, p = 0.004 on treatment × day, n = 8/group, Figure 3(b)). The effect was significant only from the fourth administration (data not shown).

Figure 3.

Chronic propranolol treatment prevents ISDN-induced cephalic and extracephalic MA. Time courses of VF withdrawal thresholds (VFT) of the face (a) and hindpaw (b) after a single H-ISDN injection, in 1-treated vehicle (black circles, n = 8) and 1-treated propranolol rats (red circles, 10 mg/kg, n = 8), or five H-ISDN injections in 5-treated vehicle (green triangles, n = 8) and 5-treated propranolol rats (yellow triangles, n = 8). Propranolol or vehicle were administered daily, per os, 1 min before H-ISDN injection. There is neither reversible extracephalic nor persistent cephalic MA in propranolol-treated rats after repeat H-ISDN injection. Values are means ± s.e.m. Analysis using two-way RM ANOVA followed by Duncan’s post hoc test, with time and treatment as factors. **p < 0.01, ***p < 0.01 indicate level of significance between repeat vehicle-treated (green triangles) and propranolol-treated (yellow triangles) rats.

Discussion

This study compared the effects of single and repeat administrations of the nitric oxide donor isosorbide dinitrate (ISDN) on cephalic and extracephalic cutaneous mechanical sensitivity. We demonstrated that single H-ISDN but not L-ISDN injection induces a reversible cephalic and extracephalic allodynia. However, with repeat administration, L-ISDN induces a reversible cephalic MA and H-ISDN induces increasingly stronger and longer lasting reversible cephalic and extracephalic allodynia. Repeat H-ISDN also produces a chronic trigeminal cutaneous mechanical hypersensitivity. Both sumatriptan and olcegepant prevent single-H-ISDN-induced acute behavioral changes, but only olcegepant can also reduce these changes when they are potentiated by repeat H-ISDN. Note that neither sumatriptan nor olcegepant have an effect on repeat-H-ISDN-induced chronic trigeminal cutaneous hypersensitivity, but the persistent hypersensitivity selectively responds to chronic treatment with propranolol.

There are very few studies to date investigating the effect of systemic administration of nitric oxide donors on cephalic cutaneous mechanical sensitivity, and those that do report conflicting results, with either a decrease (15,16) or no change (17,18) in periorbital VFTs after systemic NTG. These discrepancies may stem from differences in doses or from difficulties in assessing mechanical responses to von Frey filaments in mice at cephalic level (17). However, it is worth noting that in several other studies in mice, responses to cephalic mechanical stimulation could be examined (16,30). Here we show that a single dose L-ISDN cannot induce cephalic MA. Likewise, a single low-concentration application of inflammatory soup (IS) on the dura—an inflammation model of migraine—also has no effect on cutaneous mechanical sensitivity (5,6). However, when repeated, low-concentration IS (5,6), like L-ISDN (as here), can induce the same reversible cephalic MA as single high-concentration IS (5,8), single H-ISDN (as here) and single high-dose NTG (16). Taken together, these results suggest that the behavioral expression of cephalic allodynia requires a minimum level of trigeminal central sensitization. This threshold can be reached after a single barrage of incoming signals from dural nociceptors if strong enough, or repeated barrage if not.

Another key finding here is that repeat H-ISDN produces a chronic state of cephalic cutaneous mechanical hypersensitivity. Thus, chronic changes, like acute changes, are dose- and repetition-dependent. Cephalic allodynia becomes persistent after the third systemic H-ISDN (as here) or local IS application on the dura (5). Consistent with this pattern, patients with frequent migraine attacks show interictal cephalic allodynia (31).

Extracephalic MA induction with ISDN (present results), as with NTG (7,11,16,32, but see 12), also appears to be dose-dependent. In addition, with repeat administration, the H-ISDN-induced extracephalic allodynia progressively potentiates, but nevertheless – and crucially – remains reversible after five H-ISDN injections. This runs counter to Pradhan et al. (7), who recently showed that chronic intermittent treatment with NTG produces a persistent extracephalic MA. Differences in species [mice (7) vs. rat (here)] or nitric oxide donors [NTG (7) vs. ISDN (here)] may account for this divergence. Finally, our conclusion that MA gradually potentiates with repeated H-ISDN is consistent with clinical studies showing that although most migraine patients do not exhibit allodynia during the early years of disease, they do eventually develop extracephalic MA as frequency of migraine attacks increases (1).

The mechanisms underlying ISDN-induced cutaneous hypersensitivity are still unknown. However, as the behavioral changes induced by repeat H-ISDN administration, sustained sumatriptan administration (7,34) or repeat dural stimulation with IS (5) look very similar, they may share similar mechanisms. Recent evidence on cephalic cutaneous hypersensitivity suggests that (i) its development may be due to sensitization of trigeminovascular nociceptive neurons (2,5) and activation of descending pain-facilitating processes from the rostral ventromedial medulla (8); (ii) its spread may be due to sensitization of thalamic nociceptive neurons (2), activation of descending pain-facilitating processes from the rostral ventromedial medulla (8) and/or sensitization of spinal dorsal horn neurons (5); and (iii) its persistence may be due to enduring sensitization of trigeminovascular nociceptive neurons and/or impairment of descending pain inhibitory controls (5).

To validate the model, we tested the effects of two specific acute antimigraine drugs, sumatriptan and olcegepant, on ISDN-induced cephalic MA. We demonstrated that both sumatriptan and olcegepant prevent the single-H-ISDN-induced allodynia. This is consistent with previous reports that sumatriptan and olcegepant inhibit NTG-induced cephalic MA (15,16) and Fos expression in the trigeminal nucleus caudalis (25,35, but see 16). It is worth noting, however, that there are differences in species, administered doses, NTG formulations and methods to dissolve NTG between studies (25,35,16). In addition, CGRP receptor antagonists have been shown to inhibit NTG-induced sensitization of trigeminal nociceptive neurons (36,37) and hyperalgesia at the hindpaw formalin test (38) and sumatriptan has been shown to reduce NTG-induced extracephalic MA in rats (12,39) and mice (1,7). Taken together, these results show that the nitric oxide donor models have predictive validity.

In human patients, pretreatment with the antimigraine drug sumatriptan also reduces immediate NTG-induced headache. However, when given after nitric oxide donor infusion, sumatriptan (40), zolmitriptan (41), and olcegepant (42) have no effect. Post-treatment with olcegepant also fails to inhibit NTG-induced Fos expression in the trigeminal nucleus caudalis (35). This suggests that the efficacy of the antimigraine drugs is related to time of treatment (early vs. late), and that they need to be given as a pretreatment before the NTG administration.

Interestingly, after repeat H-ISDN, sumatriptan failed to inhibit cephalic allodynia. Similarly, Pradhan et al. (7) found that the sustained extracephalic MA induced by repeat NTG administration is less sensitive to sumatriptan. These observations are consistent with clinical data showing that inadequate response to sumatriptan is associated with greater monthly frequency of headache days and cutaneous allodynia (43). This suggests that the total duration of trigeminal-system activation may be involved in the causal pathway that leads to migraine progression (43).

Unlike sumatriptan, olcegepant was also able to reduce acute H-ISDN-induced cephalic allodynia when potentiated by repeat administration. However, neither sumatriptan nor olcegepant had an effect on the chronic trigeminal hypersensitivity progressively developing with repeated H-ISDN. These results are consistent with clinical evidence that chronic migraineurs may show cutaneous allodynia symptoms both between and during migraine attacks (31). This suggests that the action of sumatriptan and olcegepant cannot inhibit an already-established trigeminal sensitization, and that migraine attacks and migraine progression are driven by different underlying mechanisms.

Propranolol is a first-line medication for migraine prophylaxis, successfully reducing headache frequency in nearly 70% of patients (44). Our observation that propranolol did not interact with the effects of single H-ISDN is consistent with clinical studies indicating that propranolol has no significant aborting effect on acute migraine attacks (45). However, propranolol treatment completely prevented all repeat-H-ISDN-induced cutaneous changes, i.e. both the extracephalic allodynia and the persistent cephalic allodynia. Similarly, chronic, but not acute, daily administration of propranolol dose-dependently inhibited cortical spreading depression induction (46). However, a recent study in mice reported that both acute and chronic treatment with high doses of propranolol (20 mg/kg, i.p.) effectively blocked the development of acute and chronic NTG-induced extracephalic hypersensitivity (47). Note that a clinical study failed to show any effect of propranolol against NTG-induced headache and migraine (48).

That propranolol has such specific effect is consistent with the idea that migraine attacks and migraine transformation have different underlying mechanisms. The role of propranolol in migraine prophylaxis is not completely understood. Though a peripheral action cannot be ruled out, the fact that neurogenic dural vasodilation (49) and plasma protein extravasation (50) fail to demonstrate a peripheral action of propranolol suggests that propranolol decreases attack frequency through central effects. Centrally mediated-specific actions might involve not only the β-adrenoreceptor but also serotonin receptor blockade (51). Repeated, but not single systemic injection of propranolol dose-dependently decreases locus coeruleus neuronal firing in rat brainstem (52). Propranolol also inhibits cortical spreading depression in rats by blocking glutamate release (53). Finally, propranolol strongly reduces trigeminovascular responses in the thalamic ventroposteromedial nucleus (54), suggesting an effect in migraine by blocking the thalamic β1-adrenoceptor. Taken together, the body of evidence suggests that noradrenergic descending pain modulatory pathways may have a specific role in migraine progression.

In summary, this study found evidence that frequent and severe migraine attacks associated with allodynia is a risk factor for disease progression. It demonstrates that, depending on dose and number of administrations, ISDN induces either acute or chronic behavioral changes that have different sensitivity to pharmacological treatment (preventive vs abortive) of migraine, that mimic many of the clinical features of migraine, and that offer another possibility to gain deeper insight into the pathophysiology of migraine as a step towards developing novel treatments.

Footnotes

Article highlights

Repeat administration of a nitric oxide donor (NO) induces acute reversible cephalic and extracephalic mechanical allodynia superimposed on a chronic state of trigeminal hypersensitivity.

Sumatriptan and olcegepant block NO-induced acute, but not chronic, mechanical hypersensitivity.

Conversely, propranolol prevents NO-induced chronic, but not acute, mechanical hypersensitivity.

These patterns point to an association between headache frequency and allodynia as a marker of risk for frequent and severe migraine attacks.

This model, which mimics many of the clinical features of migraine, can serve to help elucidate its driving mechanisms and design novel strategies for treatment.

Acknowledgments

We thank Pr A Artola for helpful comments on this manuscript and A-M Gaydier for secretarial assistance.

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