Effect of dural inflammatory soup application on activation and sensitization markers in the caudal trigeminal nucleus of the rat and the modulatory effects of sumatriptan and kynurenic acid.

BACKGROUND: The topical inflammatory soup can model the inflammation of the dura mater causing hypersensitivity and activation of the trigeminal system, a phenomenon present in migraineurs. Calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase are important in the sensitization process there. 5-HT1B/1D receptor agonists, triptans are used as a treatment of migraine. Kynurenic acid an NMDA antagonist can act on structures involved in trigeminal activation. AIM: We investigated the effect of inflammatory soup induced dural inflammation on the calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase levels in the caudal trigeminal nucleus. We also tested whether pretreatment with a well-known antimigraine drug, such as sumatriptan and kynurenic acid, a compound with a different mechanism of action, can affect these changes and if their modulatory effects are comparable. MATERIAL AND METHODS: After subcutaneous sumatriptan or intraperitoneal kynurenic acid the dura mater of adult male Sprague-Dawley rats (n = 72) was treated with inflammatory soup or its vehicle (synthetic interstitial fluid). Two and a half or four hours later perfusion was performed and the caudal trigeminal nucleus was removed for immunohistochemistry. RESULTS AND CONCLUSION: Inflammatory soup increased calcitonin gene-related peptide, transient receptor potential vanilloid-1 receptor, and neuronal nitric oxide synthase in the caudal trigeminal nucleus compared to placebo, which was attenuated by sumatriptan and kynurenic acid. This suggests the involvement of 5-HT1B/1D and NMDA receptors in neurogenic inflammation development of the dura and thus in migraine attacks.

Trigeminal activation patterns evoked by chemical stimulation of the dura mater in rats.

BACKGROUND: Although migraine is one of the most common primary headaches, its therapy is still limited in many cases. The use of animal models is crucial in the development of novel therapeutic strategies, but unfortunately, none of them show all aspects of the disease, therefore, there is a constant need for further improvement in this field. The application of inflammatory agents on the dura mater is a widely accepted method to mimic neurogenic inflammation in rodents, which plays a key role in the pathomechanism of migraine. Complete Freund's Adjuvant (CFA), and a mixture of inflammatory mediators, called inflammatory soup (IS) are often used for this purpose. METHODS: To examine the activation pattern that is caused by chemical stimulation of dura mater, we applied CFA or IS over the right parietal lobe. After 2 h and 4 h (CFA groups), or 2.5 h and 4 h (IS groups), animals were perfused, and c-Fos immunoreactive cells were counted in the caudal trigeminal nucleus. To explore every pitfall, we examined whether our surgical procedure (anesthetic drug, stereotaxic apparatus, local lidocaine) can alter the results under the same experimental settings. c-Fos labeled cells were counted in the second-order neuron area based on the somatotopic organization of the trigeminal nerve branches. RESULTS: We could not find any difference between the CFA and physiological saline group neither 2 h, nor 4 h after dural stimulation. IS caused significant difference after both time points between IS treated and control group, and between treated (right) and control (left) side. Stereotaxic frame usage had a substantial effect on the obtained results. CONCLUSIONS: Counting c-Fos immunoreactive cells based on somatotopic organization of the trigeminal nerve helped to examine the effect of chemical stimulation of dura in a more specific way. As a result, the use of IS over the parietal lobe caused activation in the area of the ophthalmic nerve. To see this effect, the use of lidocaine anesthesia is indispensable. In conclusion, application of IS on the dura mater induces short-term, more robust c-Fos activation than CFA, therefore it might offer a better approach to model acute migraine headache in rodents.

Chronic 17ß-estradiol pretreatment has pronociceptive effect on behavioral and morphological changes induced by orofacial formalin in ovariectomized rats.

BACKGROUND: The prevalence of craniofacial pain disorders show sexual dimorphism with generally more common appearance in women suggesting the influence of estradiol, but the exact cause remains unknown. The common point in the pathogenesis of these disorders is the activation of trigeminal system. One of the animal experimental models of trigeminal activation is the orofacial formalin test, in which we investigated the effect of chronic 17ß-estradiol pretreatment on the trigeminal pain-related behavior and activation of trigeminal second-order neurons at the level of spinal trigeminal nucleus pars caudalis (TNC). METHODS: Female Sprague Dawley rats were ovariectomized and silicone capsules were implanted subcutaneously containing cholesterol in the OVX group and 17ß-estradiol and cholesterol in 1:1 ratio in the OVX+E2 group. We determined 17ß-estradiol levels in serum after the implantation of capsules. Three weeks after operation, 50 µL of physiological saline or 1.5% of formalin solution was injected subcutaneously into the right whisker pad of rats. The time spent on rubbing directed to the injected area and c-Fos immunoreactivity in TNC was measured as the formalin-induced pain-related behavior, and as the marker of pain-related neuronal activation, respectively. RESULTS: The chronic 17ß-estradiol pretreatment mimics the plasma levels of estrogen occurring in the proestrus phase and significantly increased the formalin-induced pain-related behavior and neuronal activation in TNC. CONCLUSION: Our results demonstrate that the chronic 17ß-estradiol treatment has strong pronociceptive effect on orofacial formalin-induced inflammatory pain suggesting modulatory action of estradiol on head pain through estrogen receptors, which are present in the trigeminal system.

Nitroglycerin increases serotonin transporter expression in rat spinal cord but anandamide modulated this effect.

Migraine is one of the most prevalent neurological diseases, which affects 16% of the total population. The exact pathomechanism of this disorder is still not well understood, but it seems that serotonin and its transporter have a crucial role in the pathogenesis. One of the animal models of migraine is the systemic administration of nitroglycerin (NTG), a nitric oxide (NO) donor. NO can initiate a central sensitization process in the trigeminal system, which is also present in migraineurs. Recent studies showed that the endocannabinoid system has a modulatory role on the trigeminal activation and sensitization. Our aim was to investigate the effect of an endogenous cannabinoid, anandamide (AEA) on the NTG-induced changes on serotonin transporter (5-HTT) expression in the upper cervical spinal cord (C1-C2) of the rat, where most of the trigeminal nociceptive afferents convey. The animals were divided into four groups. Rats in the first group, called placebo, received only the vehicle solution as treatment. In the second group, they were treated with an intraperitoneal (i.p.) injection of NTG (10mg/kg). Rats in the third and fourth groups received i.p. AEA (2×5mg/kg) half hour before and one hour after the placebo or NTG treatment. Four hours after placebo/NTG injection, the animals were perfused and the cervical spinal cords were removed for immunohistochemistry and Western blotting. Our results show that both NTG and AEA alone are able to increase 5-HTT expression in the C1-C2 segments. Combination of NTG and AEA has an opposing effect on this marker, nullifying the effects of non-combined administration, probably by negative feedback mechanisms.

The modulatory effect of anandamide on nitroglycerin-induced sensitization in the trigeminal system of the rat.

BACKGROUND: One of the human and animal models of migraine is the systemic administration of the nitric oxide donor (NO) nitroglycerin (NTG). NO can provoke migraine-like attacks in migraineurs and initiates a self-amplifying process in the trigeminal system, probably leading to central sensitization. Recent studies suggest that the endocannabinoid system is involved in nociceptive signal processing and cannabinoid receptor (CB) agonists are able to attenuate nociception in animal models of pain. AIM: The purpose of the present study was to investigate the modulatory effects of a CB agonist anandamide (AEA) on the NTG-induced expression of transient receptor potential vanilloid type 1 (TRPV1), neuronal nitric oxide synthase (nNOS), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) and kynurenine aminotransferase-II (KAT-II) in the upper cervical spinal cord (C1-C2) of the rat, where most of the trigeminal nociceptive afferents convey. METHODS: A half hour before and one hour after NTG (10 mg/kg) or placebo injection, adult male Sprague-Dawley rats (n = 44) were treated with AEA (2 × 5 mg/kg). Four hours after placebo/NTG injection, the animals were perfused and the cervical spinal cords were removed for immunohistochemistry and Western blotting. RESULTS AND CONCLUSION: Our results show that NTG is able to increase TRPV1, nNOS, NF-κB and COX-2 and decrease KAT-II expression in the C1-C2 segments. On the other hand, we have found that AEA modulates the NTG-induced changes, thus it influences the activation and central sensitization process in the trigeminal system, probably via CBs.