Sex differences in behavior and expression of CGRP-related genes in a rodent model of chronic migraine

Abstract

Objective: The objectives of this study were to develop a preclinical rodent model that produces migraine-like behaviors based on International Headache Society diagnostic criteria, to determine whether sex differences are present, and to determine whether expression of calcitonin gene-related peptide (CGRP) and the genes encoding its receptor in trigeminal ganglion or medulla correlates with those behaviors.

Background: Few animal studies of migraine have tested behaviors associated with migraine diagnostic criteria. In this study, changes in activity and in mechanical sensitivity of facial regions following application of inflammatory soup (IS) or vehicle (phosphate-buffered saline [PBS]) to the dura were measured to model changes in routine activity and allodynia. CGRP, an important mediator of migraine pathogenesis, and the 3 components of its receptor, calcitonin-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP) mRNAs were quantified in the trigeminal ganglion and medulla to identify baseline sex differences and changes associated with application of IS or PBS to the dura.

Methods: Male and female Sprague-Dawley rats were implanted with a dural cannula. Groups of rats were treated with 10 or 20 µL volumes of IS or PBS. Baseline behavioral testing was conducted prior to surgery and again at 7 days postsurgery, and dural application of IS or PBS was performed repeatedly for a total of 8 applications. Locomotor activity was assessed using force plate actimetry during and following application to provide information on distance traveled, bouts of low mobility, spatial confinement, and focused energy. Periorbital and perimasseter sensory testing was performed 20 minutes post-application to measure allodynia. The rats were sacrificed 30 minutes following the final dural treatment, tissue was dissected and total RNAs were isolated from ipsilateral trigeminal ganglia and ipsilateral medulla. Quantitative real-time polymerase chain reactions were used to measure the expression of amplified constructs using gene-specific primers for CGRP, RAMP1, CLR, and RCP.

Results: Both males and females showed behavioral effects of IS application, but there were pronounced sex differences. Females showed effects at the lower dose, and activity changes were present for a longer duration, but males required fewer applications of IS to exhibit behavioral changes. Females showed increased withdrawal responses for periorbital and perimasseter mechanical testing (10 µL IS groups), and males showed increased perimasseter withdrawal responses (20 µL IS group). In the trigeminal ganglion, there were no baseline sex differences in CGRP-encoding mRNA, but females had lower baseline expression of RAMP1, CLR, and RCP-encoding mRNAs. In the medulla, females had higher baseline levels of CGRP-encoding mRNAs and lower baseline levels of RAMP1, CLR, and RCP-encoding mRNAs than males. Both IS and PBS increased expression of mRNAs encoding CGRP, RAMP1, RCP, and CLR in the trigeminal ganglion in males, but in females, only CLR and RCP were increased. In the medulla both IS and PBS increased expression of CGRP, CLR in males and CLR and RCP in females. Thus, expression of CGRP-related genes did not mirror the behavioral differences between IS and PBS groups. Instead, CGRP-related genes were upregulated by both IS and PBS applications.

Conclusions: This study demonstrates significant changes in locomotor activity and facial allodynia associated with application of IS to the dura as well as significant sex differences, demonstrating that International Headache Society diagnostic criteria can be used to design a rodent behavioral model of migraine. In addition, there were prominent baseline sex differences in expression of CGRP and its receptor in both the trigeminal ganglion and medulla, but the majority of changes in expression of CGRP and its receptor were present in both the IS and PBS treated rats. This suggests that the CGRP pathway responds to changes in intracranial pressure or meningeal stretch, while migraine-like behaviors occur after meningeal inflammation.

Figure 1. Inflammatory soup decreases locomotor activity in onset phase

A) Distance traveled measurement: millimeters of distance traveled between t=0 and t=5 minutes. B) Bouts of low mobility: number of ten-second periods of inactivity between t=0 and t=5 minutes. C) Spatial confinement score: unit measure with inverse relation to area explored between t=0 and t=5 28 minutes. D) Focused energy: unit measure of activity while animal remains stationary between t=0 and t= 5 minutes. X-axes show number of treatments. For each animal, 10µl or 20µ PBS (closed circles) or IS (open circles) was delivered to the surface of intact dura via cannula. Male 10µl PBS group, n = 5; male 10µl IS group, n=5; female 10µl PBS group, n=9; female 10µl IS group, n=7; male 20µl PBS group, n= 3; male 20µl IS group, n=5; female 20µl PBS group, n = 3; female 20µl IS group, n=5. Standard error is shown in error bars. # indicates a statistical significance with P < .05, * indicates P < .005.

Figure 2. Inflammatory soup decreases locomotor activity in persistence phase

A) Distance traveled measurement: millimeters of distance traveled between t=10 and t=15 minutes. B) Bouts of low mobility: number of ten-second periods of inactivity between t=10 and t=15 minutes. C) Spatial confinement score: unit measure with inverse relation to area explored between t=10 30 and t= 15 minutes. D) Focused energy: unit measure of activity while animal remains stationary between t =10 and t=15 minutes. X-axes show number of treatments. For each animal, 10µl or 20 µl PBS (closed circles) or IS (open circles) was delivered to the surface of intact dura via cannula. Male 10µl PBS group, n=5; male 10µl IS group, n= ; female 10µl PBS group, n=9 ; female 10µl IS group, n=7; male 20µl PBS group, n=3; male 20µl IS group, n=5; female 20µl PBS group, n=3, female 20µl IS group, n=5. Standard error is shown in error bars. # indicates a statistical significance with P < .05, * indicates P < .005.

Figure 3. Inflammatory soup induces facial allodynia

A) Primary division allodynia: Withdrawal scores in response to monofilament (4g) stimulation of the periorbital region. B) Secondary division allodynia. Withdrawal scores in response to monofilament (4g) stimulation of the perimasseter region. Over the course of testing the withdrawal scale measures the maximum withdrawal as a score of 15 and no response as a score of 0. X-axis shows number of treatments. For each animal, 10µl or 20µl PBS (closed circles) or IS (open circles) was delivered to the surface of intact dura via cannula. Male 10µl PBS group, n=5; male 10µl IS group, n=5; female 10µl PBS group, n= 9; female 10µl IS group, n=7; male 20µl PBS group, n=3; male 20µl IS group, n= ; female 20µl PBS group, n=3, female 20µl IS group, n=5. Standard error is shown in error bars. # indicates a statistical significance with P < .05, * indicates P < .005.

Figure 4. Inflammatory soup decreases routine activity interictally

A) Distance traveled measurement: difference in millimeters of distance traveled between post-surgical baseline and treatment day 8. B) Bouts of low mobility: difference in number of 10 second periods of inactivity between post-surgical baseline and treatment day 8. C) Change in spatial confinement score between post-surgical baseline and treatment day 8. D) Change in focused energy score between post-surgical baseline and treatment day 8. For each animal, 10µl or 20µl PBS (closed circles) or IS (open circles) was delivered to the surface of intact dura via cannula. Male 10µl PBS group, n=5; male 10µl IS group, n=5; female 10µl PBS group, n=9; female 10µl IS group, n = 7; male 20µl PBS group, n=3; male 20µl IS group, n=5; female 20µl PBS group, n= 3, female 20 µl IS group, n=5. Data are shown as a box plot with a line within the box representing the 50th percentile or median, while the boundary of the box closest to zero indicates the 25th percentile and the boundary of the box farthest from zero indicates the 75th percentile. The whiskers mark the 10th and 90th percentiles. Outlying data points are shown.

Figure 5. Inflammatory soup does not illicit interictal allodynia

A) Primary division allodynia: Withdrawal scores in response to monofilament (4g) stimulation of the periorbital region. B) Secondary division allodynia. Withdrawal scores in response to monofilament (4g) stimulation of the perimasseter region. Change calculated as difference between post-surgical baseline and treatment day 8. Over the course of testing the withdrawal scale measures the maximum withdrawal as a score of 15 and no response as a score of 0. For each animal, 10µl or 20µl PBS (closed circles) or IS (open circles) was delivered to the surface of intact dura via cannula. Male 10µL PBS group, n=5; male 10µL IS group, n=5 ; female 10µl PBS group, n=9 ; female 10µl IS group, n=7 ; male 20µl PBS group, n=3 ; male 20µl IS group, n=5; female 20µl PBS group, n=3, female 20µl IS group, n=5. Standard error is shown in error bars. # indicates a statistical significance with P < .05, * indicates P < .005. Data are shown as a box plot with a line within the box representing the 50th percentile or median, while the boundary of the box closest to zero indicates the 25th percentile and the boundary of the box farthest from zero indicates the 75th percentile. The whiskers mark the 10th and 90th percentiles. Outlying data points are shown.

Figure 6. In female rats, onset phase distance traveled is highly correlated with interictal periorbital mechanical facial allodynia

X-axis shows interictal periorbital withdrawal scores in response to monofilament (4g) stimulation of the periorbital region during the interictal period on Day 8. Y-axis shows distance traveled on Day 7. Female 10µl PBS group, n = 5; female 10µl IS group, n = 6; female 20µl PBS group, n = 3, female 20µl IS group, n = 5. The data are shown with a general linear regression with a goodness of fit analysis performed using ANOVA F-test. The correlation is statistically significant (P = .0003).

Figure 7. Sex Differences in baseline expression of CGRP-related Genes in Rat Trigeminal Ganglion and Medulla

Relative expression of CGRP-related genes in the trigeminal ganglia and medulla as measured by qRT-PCR using ΔΔCt analysis to calculate relative expression. GAPDH was used as the internal control. Average male expression levels were used as comparison controls. Animals are naïve control male or naïve control female Sprague-Dawley rats. Male group, n=6, female group, n=6. Error bars indicate standard error. # indicates a statistically significant difference from male naïve with P < .05, * indicates P < .005.