Induction of calcitonin gene-related peptide expression in rats by cortical spreading depression

Abstract

Objective: The neuropeptide calcitonin gene-related peptide (CGRP) has now been established as a key player in migraine. However, the mechanisms underlying the reported elevation of CGRP in the serum and cerebrospinal fluid of some migraineurs are not known. A candidate mechanism is cortical spreading depression (CSD), which is associated with migraine with aura and traumatic brain injury. The aim of this study was to investigate whether CGRP gene expression may be induced by experimental CSD in the rat cerebral cortex.

Methods: CSD was induced by topical application of KCl and monitored using electrophysiological methods. Quantitative PCR and ELISA were used to measure CGRP mRNA and peptide levels in discrete ipsilateral and contralateral cortical regions of the rat brain 24 hours following CSD events and compared with sham treatments.

Results: The data show that multiple, but not single, CSD events significantly increase CGRP mRNA levels at 24 hours post-CSD in the ipsilateral rat cerebral cortex. Increased CGRP was observed in the ipsilateral frontal, motor, somatosensory, and visual cortices, but not the cingulate cortex, or contralateral cortices. CSD also induced CGRP peptide expression in the ipsilateral, but not contralateral, cortex.

Conclusions: Repeated CSD provides a mechanism for prolonged elevation of CGRP in the cerebral cortex, which may contribute to migraine and post-traumatic headache.

Keywords: CGRP; gene expression; migraine; post-traumatic headache; traumatic brain injury.

Figure 1

Cortical spreading depression (CSD) induction and propagation in the rat cortex. (A) CSD was induced with topical application of 1 μl 3 M KCl (or artificial cerebrospinal fluid for sham) onto the dura via a posterior burr hole. An anterior hole was used for CSD recording. A total of 36 rats were used. Of these, 26 rats were used for multiple CSD experiments including 14 for CSD induction and 12 for sham. To minimize the animal use, three of 14 rats in the CSD group and three of 12 in the sham group were also used for measuring calcitonin gene-related peptide (CGRP) levels in addition to CGRP mRNA. In the single CSD group, 10 rats were used with five for CSD and sham, respectively. (B) A representative trace showing CSD propagation and magnitude (indicated as area under the curve, AUC, grey lines).

Figure 2

Ipsilateral calcitonin gene-related peptide (CGRP) mRNA is upregulated 24 hours post-multiple cortical spreading depression (CSD). (a) Absolute levels of CGRP mRNA were significantly elevated in the ipsilateral (ipsi), but not contralateral (contra), cortex post-CSD. There was no significant increase in the sham-treated rats. Data for individual rats are shown with lines connecting the paired cortices. (b) Comparison of CGRP mRNA levels between contralateral and ipsilateral cortices shown for individual rats (left panel) and as the fold change (right panel). Increased relative expression of CGRP mRNA in the ipsilateral normalized to contralateral cortex. In both (a) and (b), sham (n=6), CSD (n=8), **p<0.01; ***p<0.001.

Figure 3

Ipsilateral calcitonin gene-related peptide (CGRP) mRNA is not altered 24 hours post-single cortical spreading depression (CSD). Comparison of CGRP levels between ipsilateral (ipsi) cortices of CSD rats (n=5) normalized to sham rats (n=5). There was no significant difference (p=0.11).

Figure 4

Multiple cortical spreading depression (CSD)-induced calcitonin gene-related peptide (CGRP) mRNA expression shows regional specificity. (a) Top view of rat brain cortical regions used for dissections. Cortical regions are designated as 1=frontal, 2=cingulate, 3=motor, 4=somatosensory, and 5=visual. The CSD recording and induction sites are also indicated. (b) Absolute levels of CGRP mRNA were significantly elevated in ipsilateral (ipsi) frontal, motor, somatosensory (SS), and visual cortices, but not in the cingulate (Cing) cortex, or the contralateral (contra) hemispheres, 24 hours post-multiple CSD. (c) Significant increases in relative ipsilateral CGRP mRNA were observed in the frontal, motor, somatosensory, and visual cortices, but not in the cingulate cortex, at 24 hours post-multiple CSD. In both panels, sham (n=6), CSD (n=6), *p<0.05; **p<0.01; ***p<0.001.

Figure 5

Ipsilateral calcitonin gene-related peptide (CGRP) peptide levels are upregulated post-multiple cortical spreading depression (CSD). CGRP peptide levels are significantly elevated in ipsilateral (ipsi) cortex 24 hours post-CSD (n=3), but not the contralateral (contra) hemispheres, nor in sham rats (n=3), *p<0.05.