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Behavioral study of a rat model of migraine induced by CGRP
Neuroscience Letters 651 (2017) 134–139
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Behavioral study of a rat model of migraine induced by CGRP Gang Yao a , Qian Huang b , Min Wang a , Chun-Li Yang a , Cai-Fen Liu a , Ting-Min Yu a,∗ a b
Department of Neurology, The Second Hospital of Jilin University, Changchun, Jilin, China Department of Radiology, The Second Hospital of Jilin University, Changchun, Jilin, China
h i g h l i g h t s • A new animal model for migraine induced by calcitonin gene-related peptide (CGRP). • Behaviors featured in migraine attacks were observed in our CGRP induced animal model. • CGRP elicits migraine related behaviors such as climbing, grooming, and immobility.
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Article history: Received 13 February 2017 Received in revised form 25 April 2017 Accepted 27 April 2017 Available online 4 May 2017 Keywords: Migraine Animal model CGRP Behaviors
a b s t r a c t Migraine is a debilitating disorder characterized by recurrent headache arising from neurovascular dysfunction. Despite recent progress in migraine research, the exact mechanisms underpinning migraine are poorly understood. Furthermore, it is difficult to develop an animal model of migraine that resembles all symptoms of patients. In this study, we established a novel animal model of migraine induced by epidural injection of calcitonin gene-related peptide (CGRP), and examined climbing hutch behavior, facial-grooming behavior, body-grooming behavior, freezing behavior, resting behavior, and ipsilateral hindpaw facial grooming behavior of rats following CGRP injection. CGRP significantly reduced climbing hutch behavior, and face-grooming behavior, and increased immobile behavior. We also found that the P15 and P85 percentile range of behavioral data exhibited a high positive rate (83.3%) for establishing the model with less false positive rate. Our results verified that the rat model of migraine induced by CGRP featured many behaviors of migraine patients demonstrated during migraine attacks. Our findings suggest that this new model can be a useful tool for understanding the pathophysiology of migraine and studying novel therapeutic strategies for the treatment of migraine. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Migraine is a chronic and complex disorder, characterized by recurrent unilateral headache as well as a variety of symptoms, such as aura, phonophobia, photophobia, vomiting and nausea [1]. However, despite intense study, the pathophysiology of migraine remains unknown due to the complex nature of the condition. Several hypotheses such as the nitroglycerin (NTG) migraine model, cortical spreading depression model, and trigeminovascular model [2,3] have been utilized to attempt to explain the pathogenesis of migraine. Given the variety of models developed, it remains challenging to establish an animal model that represents all aspects of migraine. One common approach to induce migraine in animals is to inject nitroglycerin (NTG) peripherally,
∗ Corresponding author. E-mail address: ytm [email protected] (T.-M. Yu). http://dx.doi.org/10.1016/j.neulet.2017.04.059 0304-3940/© 2017 Elsevier B.V. All rights reserved.
which can induce attacks similar to spontaneous migraine attacks [4]. A recent study has reported that repeated NTG in rats elicits responses that are clinically relevant to behavioral endpoints of migraine [5]. The establishment of nitroglycerin induced migraine model mainly relies on the expansion effect of nitrogen oxide on cerebrovascular and meningeal vessels [6], which wasn’t involved in the major pathological mechanism of migraine that neurogenic inflammation caused by mast cell degranulation [7]. Cortical spreading depression (CSD) is a depolarization wave that can suppress brain activity and may be associated with dramatic changes in the neural and vascular function [8]. Emerging clinical, neurophysiological and neuroimaging evidence has shown that CSD is associated with migraine aura [9–11]. CSD is believed to contribute to migraine, since the spread rate of the migraine aura corresponds to a propagating velocity of the CSD of 2–6 mm/min [12]. CSD may, therefore, represent a new target for anti-migraine agents [13]. The cortical spreading depression (CSD) migraine model is often used for the study of migraine aura and pathogene-
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sis, which is rarely used for migraine behavioral studies and has a higher requirement for instrumentation [14,15]. For several decades, it has been speculated that the mechanisms of migraine pathogenesis is in the trigeminocervical complex, which is responsible for most of the symptoms of migraine. The stimuli factors including capsaicin and inflammatory soup for the establishment of trigeminal neurovascular model can further induce CGRP-based inflammatory mediators, and then cause the activation of trigeminal vascular system [16,17]. Drugs targeting the trigeminovascular system modulate the nociceptive pathway and relieve migraine [18,19]. The trigeminovascular nociceptive pathway may anatomically explain for the referral of pain to the back of the head in migraine [20]. However, although these theories help understand migraine, the pathophysiology remains unclear. It is, therefore, important to establish animal models that resemble clinical manifestations in humans. Calcitonin gene-related peptide (CGRP) has been hypothesized to be involved in the pathophysiology of migraine [21,22]. CGRP can dilate peripheral and cerebral blood vessels [23–25], and transmit nociceptive information from intracranial blood vessels to the nervous system [26]. CGRP concentrations are increased in the external jugular venous blood during migraine, suggesting that the CGRP may cause spontaneous migraine attacks [27]. Moreover, as CGRP is expressed in trigeminal ganglia, the activation of the trigeminal nerve results in the release of CGRP from perivascular nerve endings [22]. Thus, epidural injection of CGRP may be used to build a novel animal model of migraine. The aim of this study was to establish a new animal model of migraine through epidural injection of CGRP. The characteristic behaviors of migraine attacks were observed in this animal model, and the criteria for establishing a successful model were set up. This animal model may be useful for understanding the pathophysiology of migraine. 2. Material and methods 2.1. Animals The Institutional Animal Care and Use Committee of Jilin University approved all experimental protocols, and all procedures were conducted in accordance with National Institutes of Health guidelines. Adult male Sprague-Dawley rats were obtained from the Animal Care Center of Jilin University, China. The rats were housed individually at room temperature (25 ◦ C) with a 12/12-h light/dark cycle, and fed standard rat chow and water ad libitum. Sixty rats were randomly assigned to 6 groups (n = 10 per group): the control group, the normal saline (NS) group, the 1.5 g CGRP group, the 3 g CGRP group, 6 g CGRP group, and 9 g CGRP group. Rats in the control group received surgery without epidural injection. Rats in the NS group received epidural injection of normal saline (20 L) following surgery. Rats in the 1.5, 3, 6, and 9 g CGRP groups received an epidural injection of 1.5, 3, 6, and 9 g CGRP (20 L) following surgery.
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recovered for 3–5 days before testing. On the day of behavioral testing, the catheter was flushed with 5 L saline solution, followed by epidural injection of normal saline or CGRP (20 L). Ten minutes after epidural injection, the catheter and the needle were removed. 2.3. Behavioral recordings The behaviors of each rat were recorded using a video camera located in front of the cage. The behaviors and symptoms of migraine induced by CGRP injection were recorded and analyzed every 30 min after epidural injection, including redness of the ears, climbing hutch, facial grooming, body-grooming, freezing behavior, resting behavior, left hindpaw facial grooming. These behaviors were described previously [29]: 1. Climbing hutch behavior: front paws seizing the cage. 2. Facial-grooming behavior: movement patterns in which paws contact facial areas. 3. Body-grooming behavior: paws, tongue or incisors are brought in contact with a body area other than the face or the forepaws. 4. Freezing behavior: immobile posture, with the four paws in contact with the floor and no movement of the vibrissae. 5. Resting behavior: head resting on flexed forepaws with eyes opened or closed. 6. Ipsilateral hindpaw facial grooming: facial grooming performed with the hindpaw, ipsilateral to the place of cannula implantation and CGRP infusion. 2.4. Statistical analysis Statistical analyses were performed using SPSS 13.0 software (SPSS, Chicago, USA). All data are presented as means ± SEM. Twoway ANOVA was used to compare differences among groups, followed by post hoc Bonferroni test or Rank sum test for nonparametric test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Climbing hutch behavior Climbing hutch behavior has been considered as a natural reaction for new environment. For all groups, the climbing behavior started to appear within the first 30 min, and decreased within 60 min after behavioral testing. Compared with the control group, the climbing behavior within 30 min was significantly reduced in the NS group (p < 0.05). The mean climbing behavior within 30 min was significantly reduced in the CGRP groups (14.9 ± 2.86, 11.5 ± 4.01, 7.3 ± 1.65, and 5.4 ± 1.34 for the 1.5, 3, 6, and 9 g CGRP groups, respectively) compared with the NS group (25.2 ± 4.83) (F(35, 360) = 4.834, two-way ANOVA followed by Bonferroni test, p < 0.05) (Fig. 1). The effect of CGRP on climbing hutch behavior exhibited a dose-dependent relationship.
2.2. Surgery and dural cannulation 3.2. Facial grooming behavior Rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate. Then, rats were fixed in a stereotaxic frame. After the skull was exposed, a 1 mm hole was drilled at 2 mm lateral to the sagittal suture and 2 mm anterior to the lambdoid sutures described previously [28]. A guide cannula (22 GA, PlasticsOne) was affixed to the skull and sealed into the opening with glue. Two stainless-steel screws were used to fix the cannula. A dummy cannula (PlasticsOne) was inserted into the guide. A catheter was placed into the epidural space. After surgery, rats were housed separately and
The mean of facial grooming behavior within the first 30 min was significantly decreased in the CGRP groups (85.9 ± 22.54 s, 77 ± 13.86 s, 59 ± 12.92 s, and 61 ± 13.08 s for the 1.5, 3, 6, and 9 g CGRP groups, respectively) compared with the NS group (235 ± 30.61 s) (F(28, 228) = 1.950, two-way ANOVA followed by Bonferroni test p < 0.05). There were no significant differences in the mean of facial grooming behavior among CGRP groups (p > 0.05). There were no differences in the mean of facial grooming
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Fig. 1. Climbing hutch behavior of rats during the 240 min experiment. The mean climbing times were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. *p < 0.05, two-way ANOVA followed by Bonferroni test.
Fig. 4. Ipsilateral hindpaw facial grooming behavior of rats during the 240 min experiment. The mean ipsilateral hindpaw facial grooming time were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. p > 0.05, Student–Newman–Keuls test.
behavior (Fig. 4) over the 240 min time period of testing among groups (Student–Newman–Keuls test p > 0.05). 3.4. Freezing behavior and resting behavior
Fig. 2. Facial grooming behavior of rats during the 240 min experiment. The mean facial grooming time were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. *p < 0.05, two-way ANOVA followed by Bonferroni test.
The mean of resting behavior (Fig. 5A) and freezing behavior (Fig. 5B) within the time period of 0–60 min was most prominent and abundant as compared to the resting 60-min time periods from 60 to 240 min, while no significant difference was found among groups (Student–Newman–Keuls test p > 0.05). The mean of immobile behavior including the freezing and resting behaviors (Fig. 5C), was significantly higher within the first 30 min in the 3 g CGRP (798.7 ± 143.46 s), 6 g CGRP-6 (787.3 ± 129.71 s), and 9 g CGRP-9 (886.3 ± 124.54 s) groups compared with the control (148.3 ± 45.62 s), NS (162.8 ± 99.63 s), and 1.5 g CGRP (402.8 ± 111.3 s) groups (F(28, 228) = 1.158, two-way ANOVA followed by Bonferroni test, p < 0.05). There were no differences in the mean of immobile behavior within the time period of 30–240 min among groups. 3.5. Analysis of behavior during the first 30 min
Fig. 3. Body grooming behavior of rats during the 240 min experiment. The mean body grooming times were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. p > 0.05, Student–Newman–Keuls test.
behavior within each 30 min time period over the 30–240 min time period among groups (Fig. 2). 3.3. Body grooming behavior and ipsilateral hindpaw facial grooming behavior There were no significant changes in the means of body grooming behavior (Fig. 3) and ipsilateral hindpaw facial grooming
Table 1 shows the percentile data (P10, P90) of behaviors in different groups during the first 30 min. As redness of the ears was not observed in the 1.5 g CGRP group, rats in the 3 g, 6 g, and 9 g CGRP groups were chosen for establishing standards for successful modeling. In the three groups, the highest value of P90 was 34.5, and 149.6 for climbing behavior and facial grooming behavior, respectively, and the lowest value was 165.10 for immobile behavior. Criteria for successful modeling based on percentile distribution (P10 , P90 ) during the first 30 min were as follows: 1) The frequency of climbing ≤34 times; 2) The time of facial grooming ≤149 s; and 3) The time of immobile behaviors ≥166 s. Based on the criteria, the success rate of establishing the model was 90%. However, two rats in the control group, one rat in the NS group, and five rats in the 1.5 g CGRP groups met the criteria. Table 2 shows the percentile data (P15, P85) of behaviors in different groups during the first 30 min. In the 3 g, 6 g, and 9 g CGRP groups, the highest value of P85 was 31.7 and 125.4 for climbing behavior and facial grooming behavior, respectively, and the lowest value was 215.15 for immobile behavior. Criteria for successful modeling based on percentile distribution (P15 , P85 ) during the first 30 min were as follows: 1) The frequency of climbing ≤31; 2) The time of facial grooming ≤125 s; and 3) The time of immobile behaviors ≥215 s. Based on the criteria, the success rate of estab-
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Fig. 5. Resting, freezing and immobile behaviors of rats during the 240 min experiment. The mean resting time (A), freezing time (B), and immobile timer (C) were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. p > 0.05, Student–Newman–Keuls test. *p < 0.05, two-way ANOVA followed by Bonferroni test.
Table 1 Percentile value (P10 , P90 ) of behaviors measured in different groups. Group
n
Climbing
Facial grooming
Immobile behavior behabehavior
Control NS 1.5 g CGRP 3 g CGRP 6 g CGRP 9 g CGRP-F
10 10 10 10 10 10
13.10, 74.30 9.30, 56.30 3.20, 32.70 0.00, 34.50 0.30, 15.00 0.10, 13.5
83.20, 344.90 99.60, 381.60 13.70, 205.20 10.80, 149.60 13.80, 115.70 09.00, 135.00
3.00, 407.40 0.00, 922.40 96.50, 1185.70 65.10, 1387.30 211.20, 1448.30 344.30, 1496.70
Table 2 Percentile value (P15 , P85 ) of behaviors measured in different groups. Group
n
Climbing
Facial grooming
Immobile behavior behabehavior
Control NS 1.5 g CGRP 3 g CGRP 6 g CGRP 9 g CGRP-F
10 10 10 10 10 10
19.15, 70.45 10.95, 46.95 4.30, 25.55 0.00, 31.75 1.95, 15.00 0.65, 10.75
100.80, 316.85 124.90, 351.9 17.55, 178.80 20.70, 125.40 18.20, 114.05 14.50, 124.00
19.50, 349.10 00.00, 589.10 121.25, 821.05 215.15, 1300.95 333.30, 1323.45 345.95, 1440.05
lishing the model was 83.3%. No rats in the control and NS groups and only four rats in the 1.5 g CGRP groups met the criterion. Table 3 shows the percentile data (P20, P80) of behaviors in different groups during the first 30 min. In the 3 g, 6 g, and 9 g CGRP groups, the highest value of P80 was 27.6 and 111.40 for climbing behavior and facial grooming behavior, respectively, and the lowest value was 260.20 for the immobile behavior. Criteria for successful modeling based on percentile distribution (P20 , P80 ) during the first 30 min were as follows: 1) The frequency of climbing ≤27; 2) The time of facial grooming ≤111 s; and 3) The time of immobile behaviors ≥261 s. Based on the criteria, the success rate of establishing the model was 73.3%. No rats in the control and NS group and five rats in the 1.5 g CGRP group met the criterion. Taken together, based on the P15 and P85 value of behaviors of rats receiving CGRP, the success rate of CGRP-induced rat model of migraine is 83.3%, and all rats in control groups were excluded. Therefore, the criteria set by the P15 and P85 behavioral data should be used for evaluating the effectiveness of CGRP-induced rat model of migraine. 4. Discussion Establishing a reproducible and stable animal model of migraine is useful in studying the mechanisms underlying migraine, and important for identifying new drugs for the treatment of migraine. In the present study, we established a new rat model of migraine by epidural injection of CGRP. We observed the behaviors of rats after CGRP injection, including climbing hutch behavior, facial-grooming behavior, body-grooming behavior, freezing behavior, resting behavior, and ipsilateral hindpaw facial grooming behavior. We found that epidural injection of CGRP significantly reduced climb-
ing hutch behavior, and facial-grooming behavior, and increased immobile behavior. We then analyzed the data of climbing hutch behavior, facial-grooming behavior, and immobile behavior. Based on the P15 and P85 value of these behaviors, we set up criteria, which could be used for evaluating the effectiveness of CGRP-induced rat model of migraine in the future studies. We examined the effect of epidural injection of CGRP on a series of behaviors associated with migraine, such as climbing, grooming and immobile behaviors. We found that climbing behavior was increased during the first 30 min and decreased within 60 min, which reflected the nature of rodents exploring the new environment. We further found that the mean of climbing behavior within 30 min was significantly reduced in rats receiving CGRP than control rats, suggesting that CGRP reduced exploration time in rats. These findings are consistent with a previous study showing reduced climbing behavior in an animal model of migraine induced by repeated meningeal nociception [29]. The pain provoked by migraine has been considered as the main reason of decreased routine physical activity and movement [1,11]. It has been reported that head and facial grooming behaviors are induced by administration of capsaicin into the cerebellomedullary cistern in alert rats [30,31]. Although it has been reported that ipsilateral hindpaw facial grooming behavior is increased during migraine attacks [11], in this study, we did not found any significant differences in ipsilateral hindpaw facial grooming behavior and body grooming behaviors between the control groups and the CGRP groups. Different behaviors in a variety of animal models may be due to different pathophysiology and mechanisms of migraine, such as CGRP in our study, and capsaicin, nitric oxide (NO) and trigeminovascular pain utilized in previous studies [30,31].
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Table 3 Percentile value (P20 , P80 ) of behaviors measured in different groups. Group
n
Climbing
Facial grooming
Immobile behavior behabehavior
Control NS 1.5 g CGRP 3 g CGRP 6 g CGRP 9 g CGRP-F
10 10 10 10 10 10
24.00, 66.40 12.20, 39.80 5.60, 20.80 0.20, 27.60 3.00, 14.40 1.40, 8.80
116.00, 291.60 142.00, 326.0 20.80, 161.20 32.20, 109.80 22.40, 111.40 21.00, 109.60
32.00, 306.60 00.00, 345.20 145.20, 581.00 260.20, 1237.40 416.80, 1202.80 408.20, 1338.20
Freezing and resting behaviors are believed to be associated with migraine. Patients with migraine have less routine activities or movements which further aggravate the pain. In our study, freezing behaviors were often observed during the first 0–60 min. Akcali et al. [32] reported that freezing is the most significant behavioral change in cortical suppression, and only occurs one hour after drug administration. Although we found no significant difference in the freezing and resting behavior between the CGRP and control groups, immobile behaviors were significantly lower in rats treated with high doses of CGRP (3–9 g) compared with the control, NS and 1.5 g CGRP groups. The finding that CGRP induced an increase in immobile behaviors in rats correlates to clinic symptoms in migraine patients, who exhibit a reduction in physical activities and movement [33–35]. Moreover, determination of the lethal dose of CGRP is warranted in future studies, combining with measurement of the levels of pain related molecules and inflammatory factors in periphery blood to determine the correct dose for CGRP. This would aid in establishing a stable migraine animal model to investigate the underlying mechanism and potential target for migraine. In addition, we analyzed the behavioral data to set up the criteria for evaluating the effectiveness of a CGRP-induced rat model of migraine. We examined three percentile ranges (p10, and p90, p15 and p85, and p20 and p80) of behavioral data, and found that P15 and P85 percentile range of behavioral data exhibited a high positive rate (83.3%) for establishing the model with less false positive rate (no rats in the control group were included). 5. Conclusions In this study, we established a rat model of migraine by epidural injection of CGRP, which shared some characteristic features that resemble the clinical symptoms of migraine patients. This novel model may be used as a new technique for studying and understanding new drug candidates for the treatment of migraine. Fundings This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 31372267, 81500953), the Program of Jilin Provincial Development and Reform Commission (Grant Nos. 2014N151, 2015Y034-1), the Program of Jilin Provincial Science & Technology Department (Grant No. 20150520143JH), and the Norman Bethune Program of Jilin University (Grant No. 2015215). Acknowledgement Medjaden Bioscience Limited provided the language help. References [1] P.J. Goadsby, R.B. Lipton, M.D. Ferrari, Migraine-current understanding and treatment, N. Engl. J. Med. 346 (2002) 257–270. [2] E.A. Bates, T. Nikai, K.C. Brennan, Y.H. Fu, A.C. Charles, A.I. Basbaum, L.J. Ptacek, A.H. Ahn, Sumatriptan alleviates nitroglycerin-induced mechanical and thermal allodynia in mice, Cephalalgia 30 (2010) 170–178.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Behavioral study of a rat model of migraine induced by CGRP Gang Yao a , Qian Huang b , Min Wang a , Chun-Li Yang a , Cai-Fen Liu a , Ting-Min Yu a,∗ a b
Department of Neurology, The Second Hospital of Jilin University, Changchun, Jilin, China Department of Radiology, The Second Hospital of Jilin University, Changchun, Jilin, China
h i g h l i g h t s • A new animal model for migraine induced by calcitonin gene-related peptide (CGRP). • Behaviors featured in migraine attacks were observed in our CGRP induced animal model. • CGRP elicits migraine related behaviors such as climbing, grooming, and immobility.
a r t i c l e
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Article history: Received 13 February 2017 Received in revised form 25 April 2017 Accepted 27 April 2017 Available online 4 May 2017 Keywords: Migraine Animal model CGRP Behaviors
a b s t r a c t Migraine is a debilitating disorder characterized by recurrent headache arising from neurovascular dysfunction. Despite recent progress in migraine research, the exact mechanisms underpinning migraine are poorly understood. Furthermore, it is difficult to develop an animal model of migraine that resembles all symptoms of patients. In this study, we established a novel animal model of migraine induced by epidural injection of calcitonin gene-related peptide (CGRP), and examined climbing hutch behavior, facial-grooming behavior, body-grooming behavior, freezing behavior, resting behavior, and ipsilateral hindpaw facial grooming behavior of rats following CGRP injection. CGRP significantly reduced climbing hutch behavior, and face-grooming behavior, and increased immobile behavior. We also found that the P15 and P85 percentile range of behavioral data exhibited a high positive rate (83.3%) for establishing the model with less false positive rate. Our results verified that the rat model of migraine induced by CGRP featured many behaviors of migraine patients demonstrated during migraine attacks. Our findings suggest that this new model can be a useful tool for understanding the pathophysiology of migraine and studying novel therapeutic strategies for the treatment of migraine. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Migraine is a chronic and complex disorder, characterized by recurrent unilateral headache as well as a variety of symptoms, such as aura, phonophobia, photophobia, vomiting and nausea [1]. However, despite intense study, the pathophysiology of migraine remains unknown due to the complex nature of the condition. Several hypotheses such as the nitroglycerin (NTG) migraine model, cortical spreading depression model, and trigeminovascular model [2,3] have been utilized to attempt to explain the pathogenesis of migraine. Given the variety of models developed, it remains challenging to establish an animal model that represents all aspects of migraine. One common approach to induce migraine in animals is to inject nitroglycerin (NTG) peripherally,
∗ Corresponding author. E-mail address: ytm [email protected] (T.-M. Yu). http://dx.doi.org/10.1016/j.neulet.2017.04.059 0304-3940/© 2017 Elsevier B.V. All rights reserved.
which can induce attacks similar to spontaneous migraine attacks [4]. A recent study has reported that repeated NTG in rats elicits responses that are clinically relevant to behavioral endpoints of migraine [5]. The establishment of nitroglycerin induced migraine model mainly relies on the expansion effect of nitrogen oxide on cerebrovascular and meningeal vessels [6], which wasn’t involved in the major pathological mechanism of migraine that neurogenic inflammation caused by mast cell degranulation [7]. Cortical spreading depression (CSD) is a depolarization wave that can suppress brain activity and may be associated with dramatic changes in the neural and vascular function [8]. Emerging clinical, neurophysiological and neuroimaging evidence has shown that CSD is associated with migraine aura [9–11]. CSD is believed to contribute to migraine, since the spread rate of the migraine aura corresponds to a propagating velocity of the CSD of 2–6 mm/min [12]. CSD may, therefore, represent a new target for anti-migraine agents [13]. The cortical spreading depression (CSD) migraine model is often used for the study of migraine aura and pathogene-
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sis, which is rarely used for migraine behavioral studies and has a higher requirement for instrumentation [14,15]. For several decades, it has been speculated that the mechanisms of migraine pathogenesis is in the trigeminocervical complex, which is responsible for most of the symptoms of migraine. The stimuli factors including capsaicin and inflammatory soup for the establishment of trigeminal neurovascular model can further induce CGRP-based inflammatory mediators, and then cause the activation of trigeminal vascular system [16,17]. Drugs targeting the trigeminovascular system modulate the nociceptive pathway and relieve migraine [18,19]. The trigeminovascular nociceptive pathway may anatomically explain for the referral of pain to the back of the head in migraine [20]. However, although these theories help understand migraine, the pathophysiology remains unclear. It is, therefore, important to establish animal models that resemble clinical manifestations in humans. Calcitonin gene-related peptide (CGRP) has been hypothesized to be involved in the pathophysiology of migraine [21,22]. CGRP can dilate peripheral and cerebral blood vessels [23–25], and transmit nociceptive information from intracranial blood vessels to the nervous system [26]. CGRP concentrations are increased in the external jugular venous blood during migraine, suggesting that the CGRP may cause spontaneous migraine attacks [27]. Moreover, as CGRP is expressed in trigeminal ganglia, the activation of the trigeminal nerve results in the release of CGRP from perivascular nerve endings [22]. Thus, epidural injection of CGRP may be used to build a novel animal model of migraine. The aim of this study was to establish a new animal model of migraine through epidural injection of CGRP. The characteristic behaviors of migraine attacks were observed in this animal model, and the criteria for establishing a successful model were set up. This animal model may be useful for understanding the pathophysiology of migraine. 2. Material and methods 2.1. Animals The Institutional Animal Care and Use Committee of Jilin University approved all experimental protocols, and all procedures were conducted in accordance with National Institutes of Health guidelines. Adult male Sprague-Dawley rats were obtained from the Animal Care Center of Jilin University, China. The rats were housed individually at room temperature (25 ◦ C) with a 12/12-h light/dark cycle, and fed standard rat chow and water ad libitum. Sixty rats were randomly assigned to 6 groups (n = 10 per group): the control group, the normal saline (NS) group, the 1.5 g CGRP group, the 3 g CGRP group, 6 g CGRP group, and 9 g CGRP group. Rats in the control group received surgery without epidural injection. Rats in the NS group received epidural injection of normal saline (20 L) following surgery. Rats in the 1.5, 3, 6, and 9 g CGRP groups received an epidural injection of 1.5, 3, 6, and 9 g CGRP (20 L) following surgery.
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recovered for 3–5 days before testing. On the day of behavioral testing, the catheter was flushed with 5 L saline solution, followed by epidural injection of normal saline or CGRP (20 L). Ten minutes after epidural injection, the catheter and the needle were removed. 2.3. Behavioral recordings The behaviors of each rat were recorded using a video camera located in front of the cage. The behaviors and symptoms of migraine induced by CGRP injection were recorded and analyzed every 30 min after epidural injection, including redness of the ears, climbing hutch, facial grooming, body-grooming, freezing behavior, resting behavior, left hindpaw facial grooming. These behaviors were described previously [29]: 1. Climbing hutch behavior: front paws seizing the cage. 2. Facial-grooming behavior: movement patterns in which paws contact facial areas. 3. Body-grooming behavior: paws, tongue or incisors are brought in contact with a body area other than the face or the forepaws. 4. Freezing behavior: immobile posture, with the four paws in contact with the floor and no movement of the vibrissae. 5. Resting behavior: head resting on flexed forepaws with eyes opened or closed. 6. Ipsilateral hindpaw facial grooming: facial grooming performed with the hindpaw, ipsilateral to the place of cannula implantation and CGRP infusion. 2.4. Statistical analysis Statistical analyses were performed using SPSS 13.0 software (SPSS, Chicago, USA). All data are presented as means ± SEM. Twoway ANOVA was used to compare differences among groups, followed by post hoc Bonferroni test or Rank sum test for nonparametric test. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Climbing hutch behavior Climbing hutch behavior has been considered as a natural reaction for new environment. For all groups, the climbing behavior started to appear within the first 30 min, and decreased within 60 min after behavioral testing. Compared with the control group, the climbing behavior within 30 min was significantly reduced in the NS group (p < 0.05). The mean climbing behavior within 30 min was significantly reduced in the CGRP groups (14.9 ± 2.86, 11.5 ± 4.01, 7.3 ± 1.65, and 5.4 ± 1.34 for the 1.5, 3, 6, and 9 g CGRP groups, respectively) compared with the NS group (25.2 ± 4.83) (F(35, 360) = 4.834, two-way ANOVA followed by Bonferroni test, p < 0.05) (Fig. 1). The effect of CGRP on climbing hutch behavior exhibited a dose-dependent relationship.
2.2. Surgery and dural cannulation 3.2. Facial grooming behavior Rats were anaesthetized by intraperitoneal injection of 10% chloral hydrate. Then, rats were fixed in a stereotaxic frame. After the skull was exposed, a 1 mm hole was drilled at 2 mm lateral to the sagittal suture and 2 mm anterior to the lambdoid sutures described previously [28]. A guide cannula (22 GA, PlasticsOne) was affixed to the skull and sealed into the opening with glue. Two stainless-steel screws were used to fix the cannula. A dummy cannula (PlasticsOne) was inserted into the guide. A catheter was placed into the epidural space. After surgery, rats were housed separately and
The mean of facial grooming behavior within the first 30 min was significantly decreased in the CGRP groups (85.9 ± 22.54 s, 77 ± 13.86 s, 59 ± 12.92 s, and 61 ± 13.08 s for the 1.5, 3, 6, and 9 g CGRP groups, respectively) compared with the NS group (235 ± 30.61 s) (F(28, 228) = 1.950, two-way ANOVA followed by Bonferroni test p < 0.05). There were no significant differences in the mean of facial grooming behavior among CGRP groups (p > 0.05). There were no differences in the mean of facial grooming
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Fig. 1. Climbing hutch behavior of rats during the 240 min experiment. The mean climbing times were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. *p < 0.05, two-way ANOVA followed by Bonferroni test.
Fig. 4. Ipsilateral hindpaw facial grooming behavior of rats during the 240 min experiment. The mean ipsilateral hindpaw facial grooming time were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. p > 0.05, Student–Newman–Keuls test.
behavior (Fig. 4) over the 240 min time period of testing among groups (Student–Newman–Keuls test p > 0.05). 3.4. Freezing behavior and resting behavior
Fig. 2. Facial grooming behavior of rats during the 240 min experiment. The mean facial grooming time were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. *p < 0.05, two-way ANOVA followed by Bonferroni test.
The mean of resting behavior (Fig. 5A) and freezing behavior (Fig. 5B) within the time period of 0–60 min was most prominent and abundant as compared to the resting 60-min time periods from 60 to 240 min, while no significant difference was found among groups (Student–Newman–Keuls test p > 0.05). The mean of immobile behavior including the freezing and resting behaviors (Fig. 5C), was significantly higher within the first 30 min in the 3 g CGRP (798.7 ± 143.46 s), 6 g CGRP-6 (787.3 ± 129.71 s), and 9 g CGRP-9 (886.3 ± 124.54 s) groups compared with the control (148.3 ± 45.62 s), NS (162.8 ± 99.63 s), and 1.5 g CGRP (402.8 ± 111.3 s) groups (F(28, 228) = 1.158, two-way ANOVA followed by Bonferroni test, p < 0.05). There were no differences in the mean of immobile behavior within the time period of 30–240 min among groups. 3.5. Analysis of behavior during the first 30 min
Fig. 3. Body grooming behavior of rats during the 240 min experiment. The mean body grooming times were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. p > 0.05, Student–Newman–Keuls test.
behavior within each 30 min time period over the 30–240 min time period among groups (Fig. 2). 3.3. Body grooming behavior and ipsilateral hindpaw facial grooming behavior There were no significant changes in the means of body grooming behavior (Fig. 3) and ipsilateral hindpaw facial grooming
Table 1 shows the percentile data (P10, P90) of behaviors in different groups during the first 30 min. As redness of the ears was not observed in the 1.5 g CGRP group, rats in the 3 g, 6 g, and 9 g CGRP groups were chosen for establishing standards for successful modeling. In the three groups, the highest value of P90 was 34.5, and 149.6 for climbing behavior and facial grooming behavior, respectively, and the lowest value was 165.10 for immobile behavior. Criteria for successful modeling based on percentile distribution (P10 , P90 ) during the first 30 min were as follows: 1) The frequency of climbing ≤34 times; 2) The time of facial grooming ≤149 s; and 3) The time of immobile behaviors ≥166 s. Based on the criteria, the success rate of establishing the model was 90%. However, two rats in the control group, one rat in the NS group, and five rats in the 1.5 g CGRP groups met the criteria. Table 2 shows the percentile data (P15, P85) of behaviors in different groups during the first 30 min. In the 3 g, 6 g, and 9 g CGRP groups, the highest value of P85 was 31.7 and 125.4 for climbing behavior and facial grooming behavior, respectively, and the lowest value was 215.15 for immobile behavior. Criteria for successful modeling based on percentile distribution (P15 , P85 ) during the first 30 min were as follows: 1) The frequency of climbing ≤31; 2) The time of facial grooming ≤125 s; and 3) The time of immobile behaviors ≥215 s. Based on the criteria, the success rate of estab-
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Fig. 5. Resting, freezing and immobile behaviors of rats during the 240 min experiment. The mean resting time (A), freezing time (B), and immobile timer (C) were recorded in rats in the control, NS, 1.5 g, 3 g, 6 g and 9 g CGRP groups. p > 0.05, Student–Newman–Keuls test. *p < 0.05, two-way ANOVA followed by Bonferroni test.
Table 1 Percentile value (P10 , P90 ) of behaviors measured in different groups. Group
n
Climbing
Facial grooming
Immobile behavior behabehavior
Control NS 1.5 g CGRP 3 g CGRP 6 g CGRP 9 g CGRP-F
10 10 10 10 10 10
13.10, 74.30 9.30, 56.30 3.20, 32.70 0.00, 34.50 0.30, 15.00 0.10, 13.5
83.20, 344.90 99.60, 381.60 13.70, 205.20 10.80, 149.60 13.80, 115.70 09.00, 135.00
3.00, 407.40 0.00, 922.40 96.50, 1185.70 65.10, 1387.30 211.20, 1448.30 344.30, 1496.70
Table 2 Percentile value (P15 , P85 ) of behaviors measured in different groups. Group
n
Climbing
Facial grooming
Immobile behavior behabehavior
Control NS 1.5 g CGRP 3 g CGRP 6 g CGRP 9 g CGRP-F
10 10 10 10 10 10
19.15, 70.45 10.95, 46.95 4.30, 25.55 0.00, 31.75 1.95, 15.00 0.65, 10.75
100.80, 316.85 124.90, 351.9 17.55, 178.80 20.70, 125.40 18.20, 114.05 14.50, 124.00
19.50, 349.10 00.00, 589.10 121.25, 821.05 215.15, 1300.95 333.30, 1323.45 345.95, 1440.05
lishing the model was 83.3%. No rats in the control and NS groups and only four rats in the 1.5 g CGRP groups met the criterion. Table 3 shows the percentile data (P20, P80) of behaviors in different groups during the first 30 min. In the 3 g, 6 g, and 9 g CGRP groups, the highest value of P80 was 27.6 and 111.40 for climbing behavior and facial grooming behavior, respectively, and the lowest value was 260.20 for the immobile behavior. Criteria for successful modeling based on percentile distribution (P20 , P80 ) during the first 30 min were as follows: 1) The frequency of climbing ≤27; 2) The time of facial grooming ≤111 s; and 3) The time of immobile behaviors ≥261 s. Based on the criteria, the success rate of establishing the model was 73.3%. No rats in the control and NS group and five rats in the 1.5 g CGRP group met the criterion. Taken together, based on the P15 and P85 value of behaviors of rats receiving CGRP, the success rate of CGRP-induced rat model of migraine is 83.3%, and all rats in control groups were excluded. Therefore, the criteria set by the P15 and P85 behavioral data should be used for evaluating the effectiveness of CGRP-induced rat model of migraine. 4. Discussion Establishing a reproducible and stable animal model of migraine is useful in studying the mechanisms underlying migraine, and important for identifying new drugs for the treatment of migraine. In the present study, we established a new rat model of migraine by epidural injection of CGRP. We observed the behaviors of rats after CGRP injection, including climbing hutch behavior, facial-grooming behavior, body-grooming behavior, freezing behavior, resting behavior, and ipsilateral hindpaw facial grooming behavior. We found that epidural injection of CGRP significantly reduced climb-
ing hutch behavior, and facial-grooming behavior, and increased immobile behavior. We then analyzed the data of climbing hutch behavior, facial-grooming behavior, and immobile behavior. Based on the P15 and P85 value of these behaviors, we set up criteria, which could be used for evaluating the effectiveness of CGRP-induced rat model of migraine in the future studies. We examined the effect of epidural injection of CGRP on a series of behaviors associated with migraine, such as climbing, grooming and immobile behaviors. We found that climbing behavior was increased during the first 30 min and decreased within 60 min, which reflected the nature of rodents exploring the new environment. We further found that the mean of climbing behavior within 30 min was significantly reduced in rats receiving CGRP than control rats, suggesting that CGRP reduced exploration time in rats. These findings are consistent with a previous study showing reduced climbing behavior in an animal model of migraine induced by repeated meningeal nociception [29]. The pain provoked by migraine has been considered as the main reason of decreased routine physical activity and movement [1,11]. It has been reported that head and facial grooming behaviors are induced by administration of capsaicin into the cerebellomedullary cistern in alert rats [30,31]. Although it has been reported that ipsilateral hindpaw facial grooming behavior is increased during migraine attacks [11], in this study, we did not found any significant differences in ipsilateral hindpaw facial grooming behavior and body grooming behaviors between the control groups and the CGRP groups. Different behaviors in a variety of animal models may be due to different pathophysiology and mechanisms of migraine, such as CGRP in our study, and capsaicin, nitric oxide (NO) and trigeminovascular pain utilized in previous studies [30,31].
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Table 3 Percentile value (P20 , P80 ) of behaviors measured in different groups. Group
n
Climbing
Facial grooming
Immobile behavior behabehavior
Control NS 1.5 g CGRP 3 g CGRP 6 g CGRP 9 g CGRP-F
10 10 10 10 10 10
24.00, 66.40 12.20, 39.80 5.60, 20.80 0.20, 27.60 3.00, 14.40 1.40, 8.80
116.00, 291.60 142.00, 326.0 20.80, 161.20 32.20, 109.80 22.40, 111.40 21.00, 109.60
32.00, 306.60 00.00, 345.20 145.20, 581.00 260.20, 1237.40 416.80, 1202.80 408.20, 1338.20
Freezing and resting behaviors are believed to be associated with migraine. Patients with migraine have less routine activities or movements which further aggravate the pain. In our study, freezing behaviors were often observed during the first 0–60 min. Akcali et al. [32] reported that freezing is the most significant behavioral change in cortical suppression, and only occurs one hour after drug administration. Although we found no significant difference in the freezing and resting behavior between the CGRP and control groups, immobile behaviors were significantly lower in rats treated with high doses of CGRP (3–9 g) compared with the control, NS and 1.5 g CGRP groups. The finding that CGRP induced an increase in immobile behaviors in rats correlates to clinic symptoms in migraine patients, who exhibit a reduction in physical activities and movement [33–35]. Moreover, determination of the lethal dose of CGRP is warranted in future studies, combining with measurement of the levels of pain related molecules and inflammatory factors in periphery blood to determine the correct dose for CGRP. This would aid in establishing a stable migraine animal model to investigate the underlying mechanism and potential target for migraine. In addition, we analyzed the behavioral data to set up the criteria for evaluating the effectiveness of a CGRP-induced rat model of migraine. We examined three percentile ranges (p10, and p90, p15 and p85, and p20 and p80) of behavioral data, and found that P15 and P85 percentile range of behavioral data exhibited a high positive rate (83.3%) for establishing the model with less false positive rate (no rats in the control group were included). 5. Conclusions In this study, we established a rat model of migraine by epidural injection of CGRP, which shared some characteristic features that resemble the clinical symptoms of migraine patients. This novel model may be used as a new technique for studying and understanding new drug candidates for the treatment of migraine. Fundings This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 31372267, 81500953), the Program of Jilin Provincial Development and Reform Commission (Grant Nos. 2014N151, 2015Y034-1), the Program of Jilin Provincial Science & Technology Department (Grant No. 20150520143JH), and the Norman Bethune Program of Jilin University (Grant No. 2015215). Acknowledgement Medjaden Bioscience Limited provided the language help. References [1] P.J. Goadsby, R.B. Lipton, M.D. Ferrari, Migraine-current understanding and treatment, N. Engl. J. Med. 346 (2002) 257–270. [2] E.A. Bates, T. Nikai, K.C. Brennan, Y.H. Fu, A.C. Charles, A.I. Basbaum, L.J. Ptacek, A.H. Ahn, Sumatriptan alleviates nitroglycerin-induced mechanical and thermal allodynia in mice, Cephalalgia 30 (2010) 170–178.
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