Pharmacological Characterization of Orofacial Nociception in Female Rats Following Nitroglycerin Administration

Abstract

Rodent models of human disease can be valuable for understanding the mechanisms of a disease and for identifying novel therapies. However, it is critical that these models be vetted prior to committing resources to developing novel therapeutics. Failure to confirm the model can lead to significant losses in time and resources. One model used for migraine headache is to administer nitroglycerin to rodents. Nitroglycerin is known to produce migraine-like pain in humans and is presumed to do the same in rodents. It is not known, however, if the mechanism for nitroglycerin headaches involves the same pathological processes as migraine. In the absence of known mechanisms, it becomes imperative that the model not only translates into successful clinical trials but also successfully reverse translates by demonstrating efficacy of current therapeutics. In this study female rats were given nitroglycerin and nociception was evaluated in OPADs. Estrous was not monitored. Based on the ED₅₀ of nitroglycerin a dose of 10 mg/kg was used for experiments. Sumatriptan, caffeine, buprenorphine and morphine were administered to evaluate the reverse translatability of the model. We found that nitroglycerin did not produce mechanical allodynia in the face of the rats, which is reported to be a consequence of migraine in humans. Nitroglycerin reduced the animals' participation in the assay. The reduced activity was verified using an assay to measure exploratory behavior. Furthermore, the effects of nitroglycerin were not reversed or prevented by agents that are effective acute therapies for migraine. Two interesting findings from this study, however, were that morphine and nitroglycerin interact to increase the rats' tolerance of mechanical stimuli on their faces, and they work in concert to slow down the central motor pattern generator for licking on the reward bottle. These interactions suggest that nitroglycerin generated nitric oxide and mu opioid receptors interact with the same neuronal circuits in an additive manner. The interaction of nitroglycerin and morphine on sensory and motor circuits deserves additional examination. In conclusion, based on the results of this study the use of nitroglycerin at these doses in naïve female rats is not recommended as a model for migraine headaches.

Keywords: female; migraine; nitroglycerin; operant assay; orofacial pain; rodent.

FIGURE 1

The Orofacial Pain Assessment Device (OPAD) for Mechanical Nociception. (A) Spiked bars that the rats must press their faces against to obtain a reward solution. (B) Example of a rat performing the OPAD assay. (C) Examples of raw licking and stimulus contact data collected in the OPAD during a 10-min testing session.

FIGURE 2

Effect of nitroglycerin on rat behavior. (A) Nitroglycerin dose response relationship on reward bottle licking in the OPAD assay. ED50 = 9.68 ± 1.00 mg/kg, One-way ANOVA: F (3, 56) = 6.79, p = 0.0006. #p < 0.05 Dunnett’s test when compared to 0 mg/kg. (B) Nitroglycerin dose response relationship on stimulus contacts in the OPAD assay. ED50 = 9.11 ± 0.85 mg/kg, One-way ANOVA: F (3, 56) = 12.99, p < 0.0001. #p < 0.05 Dunnett’s test when compared to 0 mg/kg. (C) Nitroglycerin dose response relationship on the ratio of lick to stimulus contacts in the OPAD assay. One-way ANOVA: F (3, 54) = 3.490, p = 0.0217. *p < 0.05 Holm-Sidak's multiple comparisons test when compared to 0 mg/kg. (D) Effect of nitroglycerin (10 mg/kg, i.p.) on licking in the OPAD assay 4 h following injection. (N = 7, Baseline vs NTG paired t-test: t = 1.812 df = 6, p = 0.120). (E) Effect of nitroglycerin on stimulus contacts in the OPAD assay 4 h following injection. (N = 7, Baseline vs. NTG paired t-test: t = 1.580 df = 6, p = 0.165). (F) Effect of nitroglycerin (10 mg/kg, i.p.) in the rearing behavioral assay (N = 14 nitroglycerin, N = 15 PBS). Dashed lines are standard errors for the curves. (G) Nitroglycerin reduces total time rearing. * t-test: t = 2.758 df = 27, p = 0.010.

FIGURE 3

Influence of anti-nociceptive agents on the ratio of licks on the reward bottle to stimulus contacts. (A) Agents tested in the presence of 10 mg/kg nitroglycerin (i.p.). The low dose of sumatriptan (0.3 mg/kg) was given as a rescue. All other drugs were given preemptively. One-way ANOVA: F (6, 107) = 3.25, p = 0.0056, #p < 0.05 Dunnett’s test when compared to PBS. (B) Agents tested in the absence of nitroglycerin. One-way ANOVA: F (7, 150) = 1.55, p = 0.15.

FIGURE 4

Effect of 10 mg/kg morphine (i.p.) and nitroglycerin on average stimulus contact duration in the OPAD assay (Mean ± SEM). One-way ANOVA: F (4, 45) = 6.297, p = 0.0004, #p < 0.05 Dunnett’s test when compared to PBS + NTG.

FIGURE 5

Nitroglycerin and morphine increase the interval between licks. (A) Representative licking bouts from animals treated with PBS, morphine (10 mg/kg), PBS + nitroglycerin (10 mg/kg), or morphine (10 mg/kg) + nitroglycerin (10 mg/kg) i.p. (B) Averaged interval between licks (Mean ± SEM). T-test PBS vs. morphine: t = 2.807 df = 14, p = 0.014. T-test PBS + NTG vs morphine + NTG: t = 2.819 df = 13, p = 0.015. N = 7 to 8 rats per treatment group. (C) Frequency distribution of intervals between licks. The data demonstrate the increased variability of the intervals in the presence of morphine and/or nitroglycerin. Data was fitted with Gaussian curves using PRISM statistical software.

FIGURE 6

Repeated administration of nitroglycerin does not induce mechanical allodynia in the OPAD assay. Nitroglycerin (10 mg/kg i.p.) was administered on days 1–5 immediately following the rats’ session on the OPAD. (A) Effect of repeated nitroglycerin on licks on the reward bottle. One-way ANOVA: F (1, 28) = 0.4614, p = 0.50. (B) Effect of repeated nitroglycerin on the number of contacts with the mechanical stimulus. Two-way ANOVA: F (1, 28) = 0.07417, p = 0.79. (C) Effect of repeated nitroglycerin on the ratio of licks to stimulus contacts. Two-way ANOVA: F (1, 28) = 0.2838, p = 0.60. N = 15 rats per treatment group.