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Effect of genetic deletion and pharmacological antagonism of P2X7 receptors in a mouse animal model of migraine.

Gölöncsér F, Sperlágh B - J Headache Pain (2014)

Bottom Line: Purine receptors participate in peripheral and central sensitization and are associated with migraine headache.The effect of genetic deletion of P2X7 and the selective P2X7 antagonist Brilliant Blue G (BBG) were examined on hyperalgesia and c-fos induction.NTG decreased the paw withdrawal threshold in both wild-type and P2X7 knockout mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083, Budapest, Szigony u,, 43, Hungary. sperlagh@koki.hu.

ABSTRACT

Background: Purine receptors participate in peripheral and central sensitization and are associated with migraine headache. We investigated the role of P2X7 receptor (P2X7) in a nitroglycerin (NTG)-induced mouse model of migraine.

Methods: Intraperitoneal NTG injection (15 mg/kg) triggered thermal hyperalgesia in the hindpaws of wild-type C57BL/6J mice, followed by the induction of c-fos in upper cervical spinal cord and trigeminal nucleus caudalis. The effect of genetic deletion of P2X7 and the selective P2X7 antagonist Brilliant Blue G (BBG) were examined on hyperalgesia and c-fos induction.

Results: NTG decreased the paw withdrawal threshold in both wild-type and P2X7 knockout mice. Nevertheless, subacute BBG treatment (50 mg/kg/day i.p.) completely prevented the effect of NTG in wild-type, but not in knockout mice. Whereas P2X7 deficiency differentially affected the expression of c-fos, the average number of fos-immuno-reactive neurons in trigeminal nucleus caudalis, but not in upper cervical spinal cord was lower in BBG-treated wild-type mice after NTG treatment.

Conclusions: Our results show that P2X7 receptors might participate in the pathogenesis of migraine, although upregulation of other P2X receptors probably compensate for the loss of its action in knockout mice. The data also suggest the therapeutic potential of P2X7 antagonists for the treatment of migraine.

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NTG-induced fos expression in wild-type and P2X7 knockout mice. A. Quantification of fos-immuno-reactive nuclei in upper cervical spinal cord (C1-2) and TNC 2 h after 15 mg/kg NTG or vehicle. We counted 10 hemi-sections from each hemisphere for upper cervical spinal cord, and 30 hemi-sections from each hemisphere that included TNC from both sides (n = 5-6 animal/group; Kruskal-Wallis nonparametric ANOVA followed by Fischer LSD post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). F. The subacute BBG treatment (50 mg/kg i.p.) decreased the number of c-fos immuno-reactive cells in TNC, but not in C1-2 in wild-type mice (n = 6-8 animal/group; Student t test **P < 0.01). B,C,D,E,G,H Representative examples of c-fos immuno-reactivity in the TNC 2 h after i.p. administration of vehicle or 15 mg/kg NTG in P2X7+/+ (B,C) and P2X7-/- mice (D,E), and after subacute BBG (50 mg/kg i.p.) or saline + 15 mg/kg NTG treatment in P2X7 +/+ mice (G,H), shown here with a 10× objective.
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Figure 5: NTG-induced fos expression in wild-type and P2X7 knockout mice. A. Quantification of fos-immuno-reactive nuclei in upper cervical spinal cord (C1-2) and TNC 2 h after 15 mg/kg NTG or vehicle. We counted 10 hemi-sections from each hemisphere for upper cervical spinal cord, and 30 hemi-sections from each hemisphere that included TNC from both sides (n = 5-6 animal/group; Kruskal-Wallis nonparametric ANOVA followed by Fischer LSD post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). F. The subacute BBG treatment (50 mg/kg i.p.) decreased the number of c-fos immuno-reactive cells in TNC, but not in C1-2 in wild-type mice (n = 6-8 animal/group; Student t test **P < 0.01). B,C,D,E,G,H Representative examples of c-fos immuno-reactivity in the TNC 2 h after i.p. administration of vehicle or 15 mg/kg NTG in P2X7+/+ (B,C) and P2X7-/- mice (D,E), and after subacute BBG (50 mg/kg i.p.) or saline + 15 mg/kg NTG treatment in P2X7 +/+ mice (G,H), shown here with a 10× objective.

Mentions: As an index of activation of the nociceptive fibres implicated in migraine, changes in the average number of c-fos-immuno-reactive nuclei in the upper cervical dorsal horn and TNC 2 h after 15 mg/kg NTG injection were quantified. In line with previous findings [14,15], NTG profoundly increased c-fos expression in both areas, when compared to vehicle-treated animals (Figure 5). In knockout mice, NTG caused the same elevation in c-fos expression in TNC, compared with vehicle, whereas the c-fos level was slightly lower in the spinal cord (Figure 5A). Two-way ANOVA of the c-fos data in the C1-2 and TNC indicated a significant effect of NTG treatment, without an interaction effect of genotype with NTG (C1-2: F1,19 = 13.92, p = 0.0014; TNC: F1,19 = 48.14, p < 0.0001). Due to unequal variance, a Kruskal-Wallis nonparametric test was used for comparison of groups. It indicated that NTG significantly increased c-fos levels in the C1-2 of wild-type (WT: p = 0.0374; KO: p = 0.0679) and TNC of wild-type and knockout mice, respectively (WT: p = 0.0039; KO: p = 0.0062). Further, post hoc testing by Fischer LSD test revealed that NTG significantly increased fos level in the C1-2 and TNC of wild-type and knockout mice (Figure 5A).


Effect of genetic deletion and pharmacological antagonism of P2X7 receptors in a mouse animal model of migraine.

Gölöncsér F, Sperlágh B - J Headache Pain (2014)

NTG-induced fos expression in wild-type and P2X7 knockout mice. A. Quantification of fos-immuno-reactive nuclei in upper cervical spinal cord (C1-2) and TNC 2 h after 15 mg/kg NTG or vehicle. We counted 10 hemi-sections from each hemisphere for upper cervical spinal cord, and 30 hemi-sections from each hemisphere that included TNC from both sides (n = 5-6 animal/group; Kruskal-Wallis nonparametric ANOVA followed by Fischer LSD post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). F. The subacute BBG treatment (50 mg/kg i.p.) decreased the number of c-fos immuno-reactive cells in TNC, but not in C1-2 in wild-type mice (n = 6-8 animal/group; Student t test **P < 0.01). B,C,D,E,G,H Representative examples of c-fos immuno-reactivity in the TNC 2 h after i.p. administration of vehicle or 15 mg/kg NTG in P2X7+/+ (B,C) and P2X7-/- mice (D,E), and after subacute BBG (50 mg/kg i.p.) or saline + 15 mg/kg NTG treatment in P2X7 +/+ mice (G,H), shown here with a 10× objective.
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Figure 5: NTG-induced fos expression in wild-type and P2X7 knockout mice. A. Quantification of fos-immuno-reactive nuclei in upper cervical spinal cord (C1-2) and TNC 2 h after 15 mg/kg NTG or vehicle. We counted 10 hemi-sections from each hemisphere for upper cervical spinal cord, and 30 hemi-sections from each hemisphere that included TNC from both sides (n = 5-6 animal/group; Kruskal-Wallis nonparametric ANOVA followed by Fischer LSD post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001). F. The subacute BBG treatment (50 mg/kg i.p.) decreased the number of c-fos immuno-reactive cells in TNC, but not in C1-2 in wild-type mice (n = 6-8 animal/group; Student t test **P < 0.01). B,C,D,E,G,H Representative examples of c-fos immuno-reactivity in the TNC 2 h after i.p. administration of vehicle or 15 mg/kg NTG in P2X7+/+ (B,C) and P2X7-/- mice (D,E), and after subacute BBG (50 mg/kg i.p.) or saline + 15 mg/kg NTG treatment in P2X7 +/+ mice (G,H), shown here with a 10× objective.
Mentions: As an index of activation of the nociceptive fibres implicated in migraine, changes in the average number of c-fos-immuno-reactive nuclei in the upper cervical dorsal horn and TNC 2 h after 15 mg/kg NTG injection were quantified. In line with previous findings [14,15], NTG profoundly increased c-fos expression in both areas, when compared to vehicle-treated animals (Figure 5). In knockout mice, NTG caused the same elevation in c-fos expression in TNC, compared with vehicle, whereas the c-fos level was slightly lower in the spinal cord (Figure 5A). Two-way ANOVA of the c-fos data in the C1-2 and TNC indicated a significant effect of NTG treatment, without an interaction effect of genotype with NTG (C1-2: F1,19 = 13.92, p = 0.0014; TNC: F1,19 = 48.14, p < 0.0001). Due to unequal variance, a Kruskal-Wallis nonparametric test was used for comparison of groups. It indicated that NTG significantly increased c-fos levels in the C1-2 of wild-type (WT: p = 0.0374; KO: p = 0.0679) and TNC of wild-type and knockout mice, respectively (WT: p = 0.0039; KO: p = 0.0062). Further, post hoc testing by Fischer LSD test revealed that NTG significantly increased fos level in the C1-2 and TNC of wild-type and knockout mice (Figure 5A).

Bottom Line: Purine receptors participate in peripheral and central sensitization and are associated with migraine headache.The effect of genetic deletion of P2X7 and the selective P2X7 antagonist Brilliant Blue G (BBG) were examined on hyperalgesia and c-fos induction.NTG decreased the paw withdrawal threshold in both wild-type and P2X7 knockout mice.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083, Budapest, Szigony u,, 43, Hungary. sperlagh@koki.hu.

ABSTRACT

Background: Purine receptors participate in peripheral and central sensitization and are associated with migraine headache. We investigated the role of P2X7 receptor (P2X7) in a nitroglycerin (NTG)-induced mouse model of migraine.

Methods: Intraperitoneal NTG injection (15 mg/kg) triggered thermal hyperalgesia in the hindpaws of wild-type C57BL/6J mice, followed by the induction of c-fos in upper cervical spinal cord and trigeminal nucleus caudalis. The effect of genetic deletion of P2X7 and the selective P2X7 antagonist Brilliant Blue G (BBG) were examined on hyperalgesia and c-fos induction.

Results: NTG decreased the paw withdrawal threshold in both wild-type and P2X7 knockout mice. Nevertheless, subacute BBG treatment (50 mg/kg/day i.p.) completely prevented the effect of NTG in wild-type, but not in knockout mice. Whereas P2X7 deficiency differentially affected the expression of c-fos, the average number of fos-immuno-reactive neurons in trigeminal nucleus caudalis, but not in upper cervical spinal cord was lower in BBG-treated wild-type mice after NTG treatment.

Conclusions: Our results show that P2X7 receptors might participate in the pathogenesis of migraine, although upregulation of other P2X receptors probably compensate for the loss of its action in knockout mice. The data also suggest the therapeutic potential of P2X7 antagonists for the treatment of migraine.

Show MeSH
Related in: MedlinePlus