IL-17 crosses the blood-brain barrier to trigger neuroinflammation: a novel mechanism in nitroglycerin-induced chronic migraine

Abstract

Background: Chronic migraine places a disabling burden on patients, which is extensively modeled by the nitroglycerin (NTG)-treated animal model. Although the NF-κB pathway is involved in an increase in CGRP levels and activation of the trigeminal system in the NTG model, the relationship between NTG and neuroinflammation remains unclear. This study aimed to optimize a chronic NTG rat model with hyperalgesia and the ethological capacity for estimating migraine therapies and to further explore the underlying mechanism of NTG-induced migraine.

Methods: Rats were administered different doses of NTG s.c. daily or every 2 d; 30 min and 2 h later, the mechanical threshold was tested. After 9 d, the rats were injected with EB or Cy5.5 for the permeability assay. The other animals were sacrificed, and then, brainstem and caudal trigeminal ganglion were removed to test CGRP, c-Fos and NOS activity; Cytokines levels in the tissue and serum were measured by ELISA; and NF-κB pathway and blood-brain barrier (BBB)-related indicators were analyzed using western blotting. Immunohistochemistry was performed to observe microglial polarization and IL-17A⁺ T cell migration in the medulla oblongata.

Results: NTG (10 mg/kg, s.c., every 2 d for a total of 5 injections) was the optimal condition, resulting in progressive hyperalgesia and migraine behavior. TNC neuroinflammation with increases in cytokines, CGRP and c-Fos and activation of the NF-κB pathway was observed, and these changes were alleviated by ibuprofen. Furthermore, NTG administration increased BBB permeability by altering the levels functional proteins (RAGE, LRP1, AQP4 and MFSD2A) and structural proteins (ZO-1, Occludin and VE-cadherin-2) to increase peripheral IL-17A permeation into the medulla oblongata, activating microglia and neuroinflammation, and eventually causing hyperalgesia and migraine attack.

Conclusions: This study confirmed that NTG (10 mg/kg, s.c., every 2 d for a total of 5 injections) was the optimal condition to provoke migraine, resulting in mechanical hyperalgesia and observable migraine-like behavior. Furthermore, IL-17A crossed the blood-brain barrier into the medulla oblongata, triggering TNC activation through microglia-mediated neuroinflammation. This process was a novel mechanism in NTG-induced chronic migraine, suggesting that IL-17A might be a novel target in the treatment of migraine.

Keywords: Blood–brain barrier; IL-17; Migraine; Neuroinflammation; Nitroglycerin.

Fig. 1

Time schedule of NTG administration in rats

Fig. 2

Analysis of the mechanical threshold of rats with NTG-induced migraine. Multiple NTG administrations evoked and sustained mechanical hyperalgesia. Rats were treated every 2 d with varying doses of NTG (0, 2.5, 5 and 7.5 mg/kg, s.c., daily or 0, 5, 10 and 15 mg/kg, s.c., every 2 d) for 9 d. (A) Basal mechanical threshold of all groups during NTG administration. (B) Posttreatment NTG mechanical threshold of all groups during NTG administration. (C-E) Mechanical threshold of the rats with migraine administered NTG s.c. daily for 9 d. (F) Mechanical threshold of the rats administered consecutive NTG s.c injections on the 9th day. (G-I) Mechanical threshold of the rats with migraine administered NTG s.c. every 2 d for 9 d. (J) Mechanical threshold of the rats s.c. administered intermittent NTG on the 9th day. Each dose group was significantly different from the vehicle group (p < 0.05), and each dose also produced significantly different results from the other doses (p < 0.05). NTG produces dose-dependent and persistent hyperalgesia in rats. Statistical analysis was performed using one–way ANOVA, n = 6, *p < 0.05 compared to the basal mechanical threshold

Fig. 3

Migraine-like behaviors of rats with NTG-induced migraine. (A) Latency of ear redness in rats with NTG-induced migraine. (B) Frequency of head scratching in rats with NTG-induced migraine. (C) Number of cage climbing instances recorded for rats with NTG-induced migraine. The statistical analysis was performed using one–way ANOVA, n = 6, *p < 0.05 compared to the control group, and ^#p < 0.05 compared to the indicated group

Fig. 4

Migrainous mediators of the TNC and TG in rats with NTG-induced migraine. (A) The expression of the CGRP mRNA in the TNC of rats with NTG-induced migraine. (B) The expression of the CGRP mRNA in the TG of rats with NTG-induced migraine. (C) CGRP contents in the TNC of rats with NTG-induced migraine. *p < 0.05 compared to control group. (D) The variation in NOS levels in the TNC of rats with NTG-induced migraine. *p < 0.05 compared to the 2.5 mg/kg daily group; ^#p < 0.05 compared to the 5 mg/kg every 2 d group. (E) The protein expression of the c-Fos in the TNC of rats with NTG-induced migraine. Statistical analysis was performed using one–way ANOVA, n = 3

Fig. 5

NTG increased the levels of inflammatory cytokines and activated the NF-κB signaling pathway in the TNC. (A-C) Content of TNF-α, IL-6 and IL-1β in the TNC of rats with NTG-induced migraine. (D and E) The expression of the NF-кB p65 mRNA in the TNC and TG of rats with NTG-induced migraine. (F-H) The levels of NF-κB p65, NF-κB p-p65 and the ratio of NF-κB p-p65/NF-κB p65 in the TNC of rats with NTG-induced migraine. Statistical analysis was performed using one–way ANOVA, n = 3, *p < 0.05 compared to the control group

Fig. 6

NTG promoted microglial inflammatory polarization in the TNC. (A) Images of Iba1 and CD68 costaining in the TNC of rats with NTG-induced migraine. (D) The ratio of Iba⁺/CD68⁺ cells. (B) Images of Iba1 and NF-κB p-p65 costaining in the TNC of rats with NTG-induced migraine. (E) The ratio of Iba⁺/ NF-κB p65⁺ cells. (C) Images of Iba1 and iNOS costaining in the TNC of rats with NTG-induced migraine. (F) The ratio of Iba⁺/ iNOS⁺ cells. Statistical analysis was performed using one–way ANOVA, n = 3, *p < 0.05 compared to the control group

Fig. 7

Inhibiting TNC neuroinflammation alleviated hyperalgesia and migraine-like behaviors in NTG-administered rats. (A) The expression of NF-κB p65 and the ratio of NF-κB p-p65/NF-κB p65 in the TNC after ibuprofen treatment. (B and C) The expression of the NF-кB p65 and CGRP mRNAs after lbu treatment. (D) Mechanical threshold recorded in NTG-treated rats after ibuprofen treatment. (E-G) Latency of ear redness, frequency of head scratching and number of cage climbing instances after ibuprofen treatment. Statistical analysis was performed using one-way ANOVA, n = 6, *p < 0.05 compared to the control group; ^#p < 0.05 compared to the NTG group

Fig. 8

NTG administration increased BBB permeability. (A) NTG enhanced the transportation function of BBB. (C-F) The expression of RAGE, LRP1, AQP4 and MFSD2A in the TNC of rats with NTG-induced migraine. (B) NTG altered the structure of BBB. (G-I) The expression of ZO-1, Occludin and VE-cadherin-2 in the TNC of rats with NTG-induced migraine. (J-L) NTG increased the permeability of peripheral dye. After Cy5.5 circulation for 30 min, NTG was subcutaneously injected. A live imaging system recorded the fluorescence signals at 695 nm for 4 h at 5 min intervals. Statistical analysis was performed using one-way ANOVA, n = 3, *p < 0.05 compared to the control group

Fig. 9

NTG-induced BBB alterations increased peripheral IL-17A access to the TNC. NTG administration increased IL-17A levels, while its transcription was not initiated in the TNC. (A and B) Content of IL-17A in the TNC and serum of rats with NTG-induced migraine. (C and D) The Ct value of the IL-17A mRNA in the TNC and TG of rats with NTG-induced migraine. NTG administration altered BBB permeability. (E) Bright-field image of EB permeation in rats with NTG-induced migraine. (F) EB permeability in the TNC of rats with NTG-induced migraine. (G) Images of EB staining in the TNC of rats with NTG-induced migraine. NTG administration did not induce CD₄⁺/IL-17A⁺ T cell migration into the TNC. (H) Images of CD₄ and IL-17A co-staining in rats with NTG-induced migraine. Statistical analysis was performed using one-way ANOVA, n = 3, *p < 0.05 compared to the control group

Fig. 10

Peripheral IL-17A crosses BBB into MO with TNC to trigger neuroinflammation in rats with NTG-induced migraine