Abstract
BackgroundNitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Infusion of the nitrovasodilator glyceroltrinitrate (nitroglycerin, GTN), which mobilizes NO in the organism, is an approved migraine model in humans. Calcitonin gene-related peptide (CGRP) is regarded as another key mediator in migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. The nociceptive processes and interactions underlying the NO and CGRP mediated headache are poorly known but can be examined in animal experiments. In the present study we examined changes in immunofluorescence of CGRP receptor components (CLR and RAMP1) and soluble guanylyl cyclase (sGC), the intracellular receptor for NO, in rat trigeminal ganglia after pretreatment with GTN.MethodsIsoflurane anaesthetised rats were intravenously infused with GTN (1 mg/kg) or saline for four hours and two hours later the trigeminal ganglia were processed for immunohistochemistry. Different primary antibodies recognizing CLR, RAMP1, CGRP and sGC coupled to fluorescent secondary antibodies were used to examine immunoreactive cells in serial sections of trigeminal ganglia with epifluorescence and confocal laser scanning microscopy. Several staining protocols were examined to yield optimized immunolabeling.ResultsIn vehicle-treated animals, 42% of the trigeminal ganglion neurons were immunopositive for RAMP1 and 41% for CLR. After GTN pretreatment CLR-immunopositivity was unchanged, while there was an increase in RAMP1-immunopositive neurons to 46%. RAMP1 and CLR immunoreactivity was also detected in satellite cells. Neurons immunoreactive for sGC were on average smaller than sGC-immunonegative neurons. The percentage of sGC-immunopositive neurons (51% after vehicle) was decreased after GTN infusion (48%).ConclusionsProlonged infusion of GTN caused increased fractions of RAMP1- and decreased fractions of sGC-immunopositive neurons in the trigeminal ganglion. The observed alterations are likely immunophenotypic correlates of the pathophysiological processes underlying nitrovasodilator-induced migraine attacks and indicate that signalling via CGRP receptors but not sGC-mediated mechanisms may be enhanced through endogenous NO production.
Highlights
Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine
The anti human receptor activity-modifying protein 1 (RAMP1) 844 antibody coupled to Alexa 488 showed clear and constant neuronal staining throughout the whole ganglia (Figure 2B); some neurons exhibited very intense fluorescence, while others were less intensely stained; both were evaluated as immunopositive
We found that after GTN pretreatment the proportion of trigeminal ganglion neurons immunopositive for the smaller Calcitonin gene-related peptide (CGRP) receptor component, RAMP1, was increased whereas the proportion of neurons immunofluorescent for the large receptor component, Calcitonin receptor-like receptor Rabbit (CLR), did not change
Summary
Nitric oxide (NO) is thought to play an important role in the pathophysiology of migraine. Increased plasma levels of CGRP have been found during spontaneous as well as nitrovasodilator-induced migraine attacks. Calcitonin gene related peptide (CGRP), which is expressed in a major part of primary afferent neurons, is known to play an important role in migraine and other primary headaches [1,2,3]. Increased plasma levels of CGRP have been found in the jugular vein of migraineurs during spontaneous and nitrovasodilator-induced migraine attacks [4,5,6]. Spontaneous, heatevoked and nitrovasodilator-induced activity of rat spinal trigeminal neurons with meningeal afferent input was reduced when CGRP receptor antagonists were intravenously infused but local administration of olcegepant onto meningeal receptive fields was ineffective [13,14]. The activity of neurons in the upper cervical dorsal horn with meningeal afferent input was modulated by microiontophoretic application of CGRP and CGRP receptor antagonists, which indicates a role for central CGRP receptors in the control of spinal trigeminal activity [15]
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