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Experimental Animal Studies of Migraine Triggering Factors: The Role of NO, CGRP and Stress

Tina Zinck

Migraine is a common disease with lifetime prevalence around 16%. Stimulation of blood vessels within the meninges dura and pia mater gives rise to a throbbing unilateral migraine-like pain, indicating that these tissues are involved in migraine pathophysiology. Stress is the leading precipitating factor for migraine attacks but the underlying mechanisms are currently unknown. Migraine attacks can be triggered experimentally, e.g. by infusion of either donors of the lipophilic, short-lived gas nitric oxide (NO) or the sensory neuropeptide calcitonin gene-related peptide (CGRP). Both substances can be found endogenously. NO is synthesized by the enzymes neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) and all three enzymes are expressed in dura mater and pia mater. Among others, the potent vasodilator CGRP is located perivascularly in dural and pial trigeminal sensory nerves.

In the present thesis, it was investigated in rats whether NO is able to induce CGRP release into the external jugular vein, which drains dura mater and extracranial tissue. Over a period of 30 min, anaesthetized rats were administered one of four different treatments through the carotid artery: vehicle (0.9% NaCl), 2 µg/kg/min glyceryl trinitrate (GTN), 50 µg/kg/min GTN or 25 nmol/kg/min diethylamine NONOate. Blood samples were drawn 5, 15, 35 min and 4 h after the start of the infusion from the external jugular vein. The collected blood samples were analysed using a radioimmunoassay. No significant difference was found between the CGRP levels in blood samples from rats administered vehicle and rats infused with NO donors. Infusion of capsaicin on the other hand, significantly enhanced the CGRP release, thus verifying that the assay was functional. These results combined with those obtained by other groups, indicate that migraine attacks evoked by NO donors do not depend on CGRP release.

We hypothesized, that stress might increase the expression of NOS in dura and pia mater and thereby the formation of NO. This was investigated by exposing rats to 1 h of air-stress per day (in a small box with two air tubes attached) for three consecutive days. Subsequently, the rats were sacrificed 1, 2, 3 and 4 h after air-stress exposure and their dura and pia mater were dissected out. The NOS mRNA and protein expression in the investigated tissues was analysed using real-time PCR and Western blotting, respectively. The dural eNOS protein expression was significantly increased 1, 2 and 3 h after air-stress, while the mRNA level remained unaltered. The mRNA expression for nNOS in dura mater was significantly augmented 2 and 3 h after air-stress exposure but no significant increase in the corresponding nNOS protein was detected. The expression of iNOS mRNA and protein in dura mater were not altered by stress. In pia mater, the eNOS protein expression was significantly augmented 2 h and 4 h after air-stress, while no alteration in the mRNA expression could be detected. In the same manner, the nNOS protein expression in pia mater was significantly increased 2 h after stress exposure, whereas no significant change in the nNOS mRNA level was found. iNOS mRNA and protein expression in pia mater were not significantly affected by stress. The increased expression of eNOS in dura mater and eNOS and nNOS in pia mater seen 2 h after stress could not be antagonised by pretreatment with the migraine drug sumatriptan.

The obtained results indicate that the transiently increased eNOS and nNOS protein expression seen after air-stress may be due to a translational or posttranslational regulation mechanism but the exact mechanism remains to be elucidated. Furthermore, iNOS does not seem to be a part of the stress response in the investigated tissues. Sumatriptan´s lack of effect on the enhanced eNOS and nNOS expression seen after stress suggests two possibilities: 1) the increase is located upstream from the site of action of sumatriptan in the pathway leading to migraine or 2) the increased expression is not directly linked to the pathway leading to migraine pain.

It was investigated, whether CGRP affects the expression of NOS mRNA in dura and pia mater. Freely moving, catheterised rats were subjected to intravenous infusion of either vehicle (0.9% NaCl) or 2.5 µg/kg rat a-CGRP and subsequently sacrificed 1, 2, 4 and 6 h after the infusion. The infusion of CGRP had no significant impact on the mean arterial blood pressure or heart rate of the rats. No significant differences could be detected between the NOS mRNA expression in rats receiving vehicle and rats receiving CGRP in neither dura nor pia mater. In conclusion, CGRP has no effect on the NOS mRNA expression in dura and pia mater.

Ingen Dansk version