Received Date : 13-Aug-2016

Revised Date : 19-Nov-2016

Accepted Article
Accepted Date : 21-Nov-2016

Article type : Research Report

Chronic ghrelin treatment reduced photophobia and anxiety-like behaviors in NTG-

induced migraine: Role of pituitary adenylate cyclase-activating polypeptide

Fereshteh Farajdokht a,b, Shirin Babri a, Pouran Karimi a, Mohammad Reza Alipour c, Ramin

Bughchechi a, Gisou Mohaddes a*

Affiliations:

a
Neurosciences Research Center (NSRC), Tabriz University of Medical Sciences, Tabriz,

Iran.

b
Student Research Committee of Tabriz University of Medical Sciences, Tabriz, Iran.

c
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Corresponding author: Dr. Gisou Mohaddes

Neurosciences research center (NSRC), Tabriz University of Medical Sciences, Tabriz, Iran

Tele/fax number: +98-41-33364664

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/ejn.13486

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 Email: mohaddesg@yahoo.com, gmohades@yahoo.com
Accepted Article
Running title: Ghrelin reduces anxiety and photophobia in migraine through blood PACAP

attenuation

Key words: Elevated Plus Maze, Light/dark box, Satellite cells, Neuropeptides, Cortisol

Abstract

Chronic migraine is a debilitating disorder that has a significant impact on patients and

society. Nearly all migraineurs frequently reported light sensitivity during a headache attack.

Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the

activation of trigeminal system and migraine pain. To identify the effect of chronic ghrelin

treatment on endogenous PACAP and associated symptoms of migraine, an experimental

chronic migraine model was induced by intermittent intraperitoneal (i.p) injection of

nitroglycerin (NTG). Photophobia and anxiety-like behaviors were determined in the

modified elevated plus maze on days 2, 4, 6, 8, and 10 and in the light/dark box on days 3, 5,

7, 9, and 11. Blood levels of PACAP and cortisol were assessed by enzyme-linked

immunosorbent (ELISA) kits. Chronic injection of NTG evoked photophobia and anxiety-

like behaviors and treatment with ghrelin (150μg/kg) for 11 days effectively attenuated

photophobia and anxiety-like behaviors in the both paradigms. We further found that NTG

increased the blood levels of PACAP and cortisol, which was significantly reduced by ghrelin

treatment. Additionally, staining with Hematoxylin and Eosin (H&E) revealed that ghrelin

reduced NTG-induced increase in the number of satellite glial cells in the trigeminal

ganglion. Furthermore, for the first time we showed that repeated administrations of NTG

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 increased white blood cell (WBC) counts and mean platelet volume (MPV), and decreased

platelet counts. These results indicated that ghrelin decreased migraine associated symptoms
Accepted Article
possibly through attenuating endogenous PACAP and cortisol levels. Therefore, ghrelin may

hold therapeutic potentialities in managing the chronic migraine.

Introduction

Migraine is a highly prevalent neurological disorder with a wide spectrum of clinical

symptoms (Goadsby, 2005). Photophobia, abnormal hypersensitivity to light, is one of the

key symptoms of migraine that is present in >80% of migraineurs (Olesen, 2005, Noseda and

Burstein, 2011). The precise mechanism of migraine and photophobia yet remains to be

elucidated; however, activation of trigeminal ganglia plays pivotal role in the

pathophysiology of migraine pain and associated symptoms of the migraine attack such as

photophobia (Kaiser et al., 2012).

Chemical activation of trigeminovascular system (TS) by the administration of nitroglycerin

is a reliable and standard model for studying migraine (Tassorelli et al., 2001, Tassorelli et

al., 2003, Pradhan et al., 2014). Also, several neuropeptides, such as calcitonin gene-related

peptide (CGRP), substance P, vasoactive intestinal peptide (VIP) and pituitary adenylate

cyclase-activating polypeptide (PACAP), have crucial roles in the activation of the TS

(Juhasz et al., 2005, Edvinsson, 2013, Durham, 2006, Schytz et al., 2009).

PACAP belongs to the VIP/secretin/glucagon peptide superfamily (Vaudry et al., 2009),

present in the trigeminal system (Tajti et al., 2001). Recent studies have pointed out the

involvement of PACAP in the activation of trigeminal system and migraine pain (Schytz et

al., 2009, Schytz et al., 2010,Tuka et al., 2012). Intravenous administration of PACAP-38

induces migraine-like attacks in healthy subjects and migraineurs without aura (Schytz et al.,

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 2009). Tuka et al. have shown that PACAP concentration in the trigeminal nucleolus caudalis

(TNC) elevates in response to the chemical or electrical activation of the trigeminal system in
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rats (Tuka et al., 2012). Markovics et al. (2012) showed that light aversion was much less in

the PACAP-deficient mice than the intact animals after NTG-induced TS activation

(Markovics et al., 2012). In addition, Tuka et al. have reported that the plasma levels of

PACAP-38 in migraineurs increased during migraine attacks, but decreased in the interictal

period in comparison with healthy volunteers (Tuka et al., 2013). These results suggest that

PACAP is an important mediator in pathophysiology of migraine and its associated

symptoms.

Several studies have reported that migraine headaches often coexist with psychiatric

disorders such as anxiety and depression (Hamelsky and Lipton, 2006, Ligthart et al., 2013).

Zwart et al. found that chronic migraine sufferers were 6-7 times more likely to show anxiety

and depression disorder than non-migraine sufferers (Zwart et al., 2003). Data from several

sources also have identified the increased plasma levels of cortisol associated with migraine

(Van Hilten et al., 1991, Patacchioli et al., 2006, Strittmatter et al., 2005).

Impairment of platelet functions is also involved in the pathophysiology of migraine (Kitano

et al., 1994). Some studies have suggested that migraineurs exhibited increased platelet

activation and platelet aggregation (Lechner et al., 1985). Few studies have investigated

mean platelet volume (MPV), an indicator of platelet activation, in the migraine (Pradalier et

al., 1992, Varol et al., 2013). Pradalier et al. reported that there was no significant difference

in platelet count, and mean platelet volume between healthy controls and migraineurs with or

without aura (Pradalier et al., 1992). However, Varol et al. reported lower platelet levels in

the migraineurs (Varol et al., 2013).

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 It has also been established that satellite glial cells (SGCs) surrounding trigeminal neurons

are involved in the inflammatory response and contribute to migraine-related neurochemical

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events (Woodham P, 1989, Hanani, 2005, Capuano et al., 2009). Activation of the trigeminal

satellite cells by pro-inflammatory mediators promotes neuronal sensitization (Capuano et al.,

2009).

Ghrelin is an orexigenic peptide mainly secreted from the stomach, shows a variety of

physiological functions (Kojima et al., 1999, Wren et al., 2000, Ferrini et al., 2009). Ghrelin

has also been shown to have antinociceptive and anti-inflammatory properties (Waseem et

al.,2008, Sibilia et al., 2012). Ghrelin and its receptor, growth hormone secretagogue

receptor-1a (GHSR-1a), were found in the areas that involved in controlling pain

transmission, such as the hypothalamus, the midbrain and spinal cord (Zigman et al., 2006,

Vergnano et al., 2008, Ferrini et al., 2009). Effect of ghrelin in modulating anxiety-related

behaviors is confusing. Several studies have proposed that ghrelin has anxiogenic effect

(Asakawa et al., 2001, Carlini et al., 2002); however, there are other reports, which

demonstrate that ghrelin has anxiolytic effect (Lutter et al., 2008, Spencer et al., 2012).

Spencer et al. showed that ghrelin has a dual role in anxious behaviors and increases anxiety

under non-stressed conditions and decreases anxiety after acute stress (Spencer et al., 2012).

Recently we found that administration of ghrelin is effective in attenuating hyperalgesia and

associated symptoms of acute migraine attacks in rats (Farajdokht et al., 2016). However,

there is no research about the effects of chronic administration of ghrelin on migraine

symptoms and its mechanisms in chronic model of migraine. In the present study, we

determined the effects of ghrelin on clinically relevant behaviors in migraine and endogenous

PACAP levels in a chronic migraine model produced by repeated nitroglycerin (NTG)

administrations.

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 Material and methods

Animals
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Experiments were performed on fifty male Wistar rats about 90 days old, weighing 220-250 g

obtained from laboratory animal center of Tabriz University of Medical Sciences, Iran.

Animals were housed three per cage at 20–22 °C on a 12-hour light/dark cycle. Food and

water were available ad libitum. All procedures were carried out in accordance with the

National Institutes of Health Guide for the Care and Use of Laboratory Animals. In addition,

animal treatment and experiments were conducted in accordance with the health guide for the

care and use of laboratory animals of Tabriz University of Medical Sciences and Ethics

Committee of Animal Research of Tabriz University of Medical Sciences approved all

experiments (No. TBZmed.REC.1394.924). All efforts were made to reduce animal suffering

and reduce the number of animals used.

Drugs and treatments

A stock of 5.0 mg/ml NTG (Caspian Tamin Pharmaceutical Company, Iran) dissolved in

30% alcohol, 30% propylene glycol and water, was freshly diluted each day in 0.9% saline to

a dose of 10 mg/kg. After a week of acclimatization, animals were randomly divided into five

groups (n=10): untreated control group with no manipulation, vehicle control group received

i.p injections of saline (0.9%) every day for 11 days; NTG group received NTG (10 mg/kg,

i.p) every second day (on days 3, 5, 7, 9) for 9 days (Pradhan et al., 2014) which treated with

saline every day for 11 days, ghrelin group which received ghrelin 150 µg/kg/day for 11

days, and NTG + ghrelin group were treated with i.p injections of ghrelin (150 µg/kg) every

day for 11 days in addition to receiving NTG. The vehicle used in these experiments was

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 0.9% saline. Fig. 1 shows our experimental design in this study. The dose of ghrelin was

chosen according to our previous study (Farajdokht et al., 2016). All drugs were freshly
Accepted Article
prepared on the day of use and all injections were administered as a 10 ml/kg volume. As

there were no significant differences between the results of the untreated control and vehicle

control groups, data for the untreated control group are not shown.

Behavioral tests

All animals were transferred to the behavioral test room at least 30 min before the beginning

of the experiments and a blind experimenter performed all the tests. Behavioral tests were

carried out between 8:00 A.M. and 2:00 P.M. in a quiet room. Photophobia and anxiety-like

behaviors were quantified using a modified elevated plus maze on days 2, 4, 6, 8 and 10 and

a modified light/dark box on days 3, 5, 7, 9 and 11.

Modified elevated plus maze (EPM)

The EPM apparatus consists of two opposite open arms (50 × 10 cm) without walls

surrounded by a 0.5 cm-high edge and two arms of the same dimensions enclosed by 40 cm

high walls that are elevated to a height 50 cm from the floor. Two and a half hours after

injection of ghrelin or saline, animals were placed onto the center platform of the maze facing

an open arm and allowed to search the maze for 5 min then returned to their cages. The

behavioral parameters including the percentage of entries into open arms (%OAE) and the

percentage of time spent in the open arms (%OAT) were assessed. The percentage of entries

into open arms and time spent in the open arms were investigated in one of the two different

conditions: Room Light On (RL-On) and Bright Closed Arms. RL-On condition was used for

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 investigating the anxiety-like behaviors and Bright Closed Arms was used for investigating

the photophobia. In the Bright Closed Arms, closed arms were entirely covered by
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fluorescence bulbs (1600 lx) placed on the top and entirely covered the closed arms, and

room lights were off. An entry into an open arm was defined as all four paws crossing the

center of the maze. After each test, the apparatus was thoroughly cleaned with ethanol 70%

(Chanda et al., 2013).

Light/dark box

Two and a half hours after injection of NTG or saline, rats were individually tested in a

light/dark box (26×26×26 cm) with two similar chambers: one clear and brightly lit [1000 lx]

without a top, the other one black and fully enclosed. A small opening door (12×12cm)

connected the two chambers. Animals were placed in the middle of the light compartment of

the box and allowed to access the entire apparatus for 10 min (Recober et al., 2009).

Behaviors of rats in the apparatus were monitored by a video tracking system and multiple

variables were analyzed, including total time spent in the light box, transitions between two

chambers, the latency to enter the dark box for the first time, and the latency to re-enter the

light box after the first entry to the dark box. The apparatus was cleaned with 70% ethanol

after each test.

Measurement of blood levels of PACAP and cortisol

The blood samples (4 ml) were collected from the heart and mixed with EDTA. Then blood

samples were centrifuged at 4000 rpm for 10 min at 4 °C, and plasma aliquots were stored at

−20 °C for further analysis and determination of plasma levels of PACAP. The plasma levels

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 of PACAP and serum levels of cortisol were measured using rat-specific PACAP enzyme-

linked immunosorbent assay (ELISA) kit (Zellbio, Germany) and cortisol enzyme-linked
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immunosorbent assay (ELISA) kit (IBL-international, Germany) according to manufacturer’s

protocols, respectively. In addition, the values of white blood cells (WBC), platelets (plt),

mean platelet volume (MPV), hemoglobin (Hb), and hematocrit (Htc) in the blood samples

were evaluated.

Measurement of blood glucose

Tail blood samples were used for blood glucose measurement by a glucometer (Accu-chek

Active, Roche, Germany) both at the first and the last days of experiment.

Tissue Preparation

Animals were anesthetized with a combination of 80 mg/kg ketamine and 12 mg/kg xylazine

and then perfused transcardially through the ascending aorta with 10–20 ml saline followed

by 200 ml of 4% paraformaldehyde. The trigeminal ganglia was removed and post fixed in

the same solution. Paraffin embedded TG were cut longitudinally at 7µm thickness using a

microtome and processed for histological assay and then sections were stained with

Hematoxylin-Eosin (H&E) using a standard protocol. The number of satellite glial cells of

the entire ganglion was counted, by a blind person to the treatment group, using a Nikon light

microscope (Nikon, Tokyo, Japan) at final magnification 400X. The analysis was performed

on at least seven sequential trigeminal ganglia sections from each animal.

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 Statistical analysis

Data are reported as mean ± SEM. Data were analyzed by SPSS version 16.0 statistic
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software package. One-way ANOVA analysis followed by post-hoc Tukey's test was used for

multiple comparisons. A two-way repeated measure ANOVA was used to determine the

significance of differences in the behaviors of animals in the EPM and light/dark box. The

difference between the means for blood glucose levels were analyzed statistically by paired

sample t-test. Differences were considered significant if P< 0.05.

Results

Chronic ghrelin treatment reduced photophobia and anxiety-like behaviors induced by

NTG

Our results in the RL-On condition revealed that %OAT and %OAE (Fig. 2A1 and A2) in the

vehicle control group showed no significant changes during the experimental period. %OAT

and %OAE in the NTG group started to reduce on day 4 and continued all over study period,

indicating the development anxiety-like behaviors. Interestingly, chronic injection of ghrelin,

in the animals that did not receive NTG, decreased %OAT and %OAE under RL-On

condition when compared to the vehicle control group. Nevertheless, administration of

ghrelin in the NTG + ghrelin group significantly increased %OAT and %OAE under RL-On

condition. On the other hand, multiple NTG administrations significantly decreased

percentage of time spent in the Bright closed arms and increased %OAT and %OAE (Fig.

2B1 and B2) in the Bright closed arms condition, indicating animals showed symptoms of

photophobia by preferring to remain in the dark open arms. Though administration of ghrelin

in the ghrelin group did not alter %OAT and % OAE in the Bright closed arms condition,

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 every day treatment with ghrelin for 11 days in the NTG+ ghrelin group attenuated

photophobia by decreasing %OAT and %OAE in the Bright closed arms condition.
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The results of light/dark box revealed that NTG-received animals took shorter time to enter

the dark compartment for the first time (Fig. 3A) and had fewer transition numbers between

two sides of the chambers than vehicle control animals (Fig. 3B). Also NTG group spent

significantly shorter time in the light side of the modified light-dark box (Fig. 3C) and had

longer latency to re-enter the light chamber after the first entry to the dark box (Fig. 3D).

Furthermore, animals that only received ghrelin for 11 days had lower transitions and latency

to enter dark box as compared to the vehicle control group, although none of them was

significant. Conversely, chronic treatment with ghrelin in the NTG + ghrelin group

significantly increased total time spent in the light box, transition numbers, latency to enter

the dark box for the first time, and decreased latency to re-enter the light box.

Chronic ghrelin treatment reduced blood levels of PACAP and cortisol in NTG-received

group

To determine the effects of chronic intermittent injection of NTG on endogenous PACAP,

plasma concentration of PACAP were determined (Fig. 4A). In addition, we measured the

plasma PACAP levels after single dose of NTG. Our results showed that single dose of NTG

increased PACAP plasma levels (1.55 ± 0.10). The results of One-way ANOVA also showed

that chronic injection of NTG dramatically increased plasma levels of PACAP compared to

the vehicle control group (F4,38 = 4.57, P = 0.01). Although chronic injection of ghrelin

insignificantly decreased PACAP levels (1.23 ± 0.17), chronic treatment with ghrelin (150

μg/kg for 11 days) in the NTG group markedly reduced PACAP levels in the plasma

compared to the NTG group (1.41 ± 0.06, P = 0.02).

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 As shown in Fig.4B, there was a significant difference in serum cortisol levels between

vehicle control and NTG groups (F3, 37 = 7.73, P = 0.01). However, administration of ghrelin
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significantly (F3, 37 = 6.43, P = 0.03) increased the serum levels of cortisol. By contrast,

chronic administration of ghrelin for 11 days in the NTG + ghrelin group considerably

decreased cortisol levels (4.27 ± 1.41, P = 0.004) when compared to the NTG group.

Effect of chronic ghrelin treatment on NTG-induced hyperglycemia

The results of paired-sample t-test analysis revealed that glucose levels of animals in NTG

group significantly (99.3 ± 3.26 for before and 115 ± 2.78 for after, P = 0.01, Fig. 4C)

increased at the end of the experiment. However, chronic treatment with ghrelin did not

change blood glucose levels in the NTG + ghrelin and ghrelin groups. In addition, One-way

ANOVA analysis showed no statistically significant difference in the blood glucose levels

among different groups.

Effect of chronic ghrelin treatment on NTG-induced changes in blood parameters

The results of the chronic intermittent administration of NTG on different blood parameters

are shown in Table 1. The NTG-received animals showed a significant (F3, 38 = 3.92, P =

0.03) increase in white blood cell (WBC) counts. The results also showed a significant

decrease in total platelet count (F3, 38 = 4.32, P = 0.03) and a significant (F3, 38 = 14.89, P =

0.025) increase in the mean platelet volume (MPV) in the NTG group when compared to the

vehicle control group. In addition, ghrelin group had higher platelet count than NTG+ ghrelin

group (F 3, 38 = 3.57, P = 0.017), and higher hemoglobin (P = 0.02) and hematocrit (P= 0.003)

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 than vehicle control group. However, chronic treatment with ghrelin in the NTG + ghrelin

group did not significantly change the blood parameters.

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Chronic ghrelin treatment decreased the number of satellite glial cells in TG induced by

NTG

We further investigated the number of SGCs in TG. The structure and morphology of

trigeminal neurons in the NTG + ghrelin group were similar to the vehicle control group

(Fig.5A). One-way ANOVA followed by Tukey’s post-hoc test analysis showed a

statistically significant increase in the number of SGCs around trigeminal neurons in the

NTG group compared to the vehicle control group (F3, 38 = 15.57, P<0.001, Fig. 5B). Chronic

administration of ghrelin for 11 days remarkably (F3, 38 = 15.57, P = 0.012) decreased the

number of SGCs in the TG compared to the NTG group. In addition, administration of

ghrelin without NTG injection did not significantly alter the number of satellite cells around

TG neurons.

Discussion

In the present study, we established that NTG evoked photophobia and anxiety-like

behaviors, as migraine associated symptoms. These results were associated with elevation of

blood levels of PACAP and cortisol. In addition, for the first time we showed that ghrelin

treatment reduced blood PACAP and cortisol levels accompanied by the amelioration of

photophobia and anxiety-like behaviors.

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 Most of the patients with chronic migraine have symptoms of depression and anxiety and

higher levels of disability than the migraineurs without mood disorders (Zwart et al., 2003,
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Ligthart et al., 2013). A limited number of studies have investigated the effect of NTG on

anxiety-like behaviors in rodents. The elevated plus maze, has primarily been used to

determine anxiety in animals (Hogg, 1996), was modified by Chanda et al. for examining

photophobia behavior in transgenic Cacna1a mutant mice (Chanda et al., 2013). In this study,

NTG group displayed anxiety-like behaviors in the modified EPM and light/dark box

paradigms, including less %OAT and %OAE in RL-On condition of the EPM apparatus, and

less total time spent in the light chamber and transitions between two chambers in the

light/dark box. Interestingly, animals in NTG + ghrelin group showed less anxiety-like

behaviors in the EPM in RL-On condition, and modified light/dark box. There are some

inconsistencies in the literature about ghrelin's effect on anxiety. Several studies indicated

that acute central or peripheral injection of ghrelin increased anxiety-like behaviors in the

elevated plus maze (Carlini et al., 2002, Asakawa et al., 2001, Carvajal et al., 2009).

Similarly, Carlini et al. found injection of ghrelin into the ventricles or distinct structures of

the brain, decreased duration and number of entries into the open arms of an elevated plus

maze (Carlini et al., 2004). Hansson and colleagues also found that chronic central injection

of ghrelin, 4 weeks, increased anxiety- as well as depression-like behaviors in rats (Hansson

et al., 2011). Consistent with this study, we also observed an increase in anxiety-like

behaviors in the ghrelin group. In contrast to these finding, Lutter et al. showed that

subcutaneous injections of ghrelin or starvation resulted in anxiolytic and antidepressant

effects in mice (Lutter et al., 2008). Recently we also showed that a single dose of ghrelin

attenuated anxiety-like behaviors in the acute migraine model in rat (Farajdokht et al., 2016).

This diversity in the results is possibly due to different dose, route of administration, chronic

or acute administration, different experimental paradigms, and the interval of behavioral test

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 following the ghrelin administration. In the current study, chronic ghrelin treatment in the

NTG + ghrelin group attenuated anxious behaviors and cortisol levels induced by chronic
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intermittent injection of NTG; however, animals that only received chronic injection of

ghrelin displayed mild anxious behaviors in the EPM and light/dark box. Even though the

documents concerning the relationship between ghrelin and anxiety disorders seem

contradictory, taken together, these results revealed that the effect of ghrelin on anxiety

behaviors is context-dependent. Therefore, depending on the experimental conditions, ghrelin

can either decrease or increase anxiety-like behaviors in animals.

A number of researchers have reported the increased levels of cortisol in the migraineurs

(Van Hilten et al., 1991, Patacchioli et al., 2006). In our previous study a single dose of NTG

(10 mg/kg) increased serum levels of cortisol (Farajdokht et al., 2016). In the current

investigation, chronic intermittent injection of NTG also increased serum levels of cortisol.

Anxiety disorder has been proposed to be linked to the activity of HPA axis (Vreeburg et al.,

2010) and previous studies also demonstrated that anxiety is associated with elevated

cortisol levels (Mantella et al., 2008, Chaudieu et al., 2008, Vreeburg et al., 2010). Chronic

pain is a well-accepted source of stress (Chapman et al., 2008, Vierck et al., 2010, Vachon-

Presseau et al., 2013). Therefore, it seems that chronic stimulation of TS by NTG activates

the hypothalamus-pituitary-adrenal axis (HPA) resulting in elevation of adrenocorticotrophic

hormone (ACTH) and serum cortisol levels (Mason, 1968, Jain et al., 1991). As reported

previously, administration of ghrelin increases the activation of HPA axis (Giordano et al.,

2004, Korbonits et al., 2004) and increased corticosterone/cortisol levels (Asakawa etal.,

2001). In agreement with these findings, we observed an increase in cortisol levels in the

ghrelin group. However, Spencer et al. have shown that ghrelin attenuated anxious behavior

and HPA axis activation under stressed conditions. They showed that ghrelin knockout

(ghr−/−) mice had more anxious behavior after acute stress and enhanced paraventricular

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 nucleus (PVN) activation compared to the wild type mice, and exogenous ghrelin reversed

these effects (Spencer et al., 2012). In the present study, cortisol levels were significantly
Accepted Article
attenuated by chronic treatment with ghrelin in the NTG + ghrelin group.

In this study chronic treatment with ghrelin decreased plasma levels of PACAP. PACAP and

its specific PAC1 receptor have been implicated in the physiology of stress, anxiety and

depression disorders (Roman et al., Gaszner et al., 2012, Mustafa et al., 2015). It has been

demonstrated that PACAP contributes to increased anxiety behaviors (Hammack et al.,

2010). Moreover, recently Kormos et al. found that lack of PACAP in mice was associated

with blunted HPA axis activity (Kormos et al., 2016). Taken together, we suggest that ghrelin

decreased anxiety-like behaviors through attenuation of blood levels of cortisol and/or

PACAP.

Photophobia is a prominent feature of migraine which is commonly reported by migraineurs,

but the underlying mechanisms are not fully understood (Olesen, 2005, Markovics et al.,

2012). There is a lack of well-characterized research tools for investigating photophobia in

animals. Moreover, photophobia is not easy to study in rodents, because they have a natural

tendency to avoid light (Ferrari et al., 2015). In our study, NTG elicited remarkable

photophobia behaviors in rats, which was markedly attenuated by chronic ghrelin treatment.

Some mechanisms are proposed for explanation of migraine-related photophobia.

Convergence of retinal and nociceptive pathways in the thalamus is proposed as a neural

mechanism for the exacerbation of migraine headache by light (Burstein et al., 2000, Dodick

and Silberstein, 2006, Noseda et al., 2010). Markovics et al. also found that single dose of

NTG increased photophobia behaviors through vasodilation and trigeminal sensitization

(Markovics et al., 2012). The role of Pituitary adenylate cyclase activating polypeptide

(PACAP), as a mediator of trigeminovascular activation, has been extensively investigated

(Schytz et al., 2010, Tuka et al., 2012, Markovics et al., 2012, Tuka et al., 2013). Markovics

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 et al. found that PACAP plays a role in nitroglycerin-triggered trigeminovascular activation

in mice (Markovics et al., 2012). Tuka et al. also demonstrated that levels of PACAP
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increased in the blood following electrical stimulation of TG or nitroglycerin injection (Tuka

et al., 2012). Similarly, we found that plasma levels of PACAP increased by acute and

chronic intermittent injection of NTG. On the other hand, in our study administration of

ghrelin for 11 days, decreased %OAT and %OAE in the open dark arms of EPM and

increased total time spent in the light chamber and latency to enter the dark chamber and

decreased latency to re-enter the light chamber in the light/dark box. These results indicate

attenuation of the photophobia by chronic ghrelin treatment, which is consistent with our

recent findings (Farajdokht et al., 2016). The major finding of the present research was that

chronic ghrelin treatment significantly decreased plasma levels of PACAP in NTG + ghrelin

group; however, chronic administration of ghrelin in the ghrelin group insignificantly

decreased PACAP plasma levels. Furthermore, Markovics et al. demonstrated that NTG-

induced light aversion in the PACAP-deficient mice was less than intact animals (Markovics

et al., 2012). Our findings suggest that decreased photophobia in ghrelin-treated rats could be

due to potential of ghrelin in decreasing levels of endogenous PACAP.

Present study also indicated that animals in the NTG group had high blood glucose levels at

the end of experiments. It is likely that NTG-induced hyperglycemia is through activation of

TS and release of PACAP, which increases HPA axis activity (Halter et al., 1984, Jankord

and Herman, 2008) to provide the energy necessary to cope with stress. Although ghrelin

increases blood glucose levels and insulin (Sangiao-Alvarellos and Cordido, 2010), chronic

ghrelin treatment in this study insignificantly increased blood glucose levels. Similar to our

result, Asakawa et al. found repeated administration of ghrelin increased insulin levels, but

blood glucose concentration increased insignificantly (Asakawa et al., 2003). It seems that

ghrelin through inhibition of the release of cortisol attenuated NTG-induced hyperglycemia.

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 On the other hand, previous studies found that administration of PACAP induces

hyperglycemia through elevation of plasma glucagon and adrenalin (Sekiguchi et al., 1994,
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Green et al., 2006). Therefore, it seems that the effect of ghrelin on regulation of blood

glucose levels is complicated.

Neurogenic inflammation in the dura mater has been proposed as a possible mechanism in

migraine (Malhotra, 2016). Earlier studies showed that activation of trigeminovascular

system results in mast cells degranulation, vasodilation and neurogenic inflammation

(Moskowitz, 1992, Theoharides et al., 2005, Levy et al., 2007). We observed that NTG

significantly increased white blood cells count in animals. Administration of NTG through

activation of TS increases secretion of neuropeptides such as PACAP and CGRP, which

activate mast cells and increase WBC count (Levy et al., 2007). Several studies have shown

that ghrelin has an anti-inflammatory effect and suppresses inflammatory cytokines such as

IL-1, IL-6, and TNF-α (Cunha et al., 2000, Sibilia et al., 2006, Waseem et al., 2008, Guneli

et al., 2010). It seems that ghrelin through its anti-inflammatory effects inhibited the increase

in WBC counts in NTG-received animals.

Several researchers found a potential relationship between platelet biology and migraine

(Lechner et al., 1985, Pradalier et al., 1992, Kitano et al., 1994). Previous evidence indicated

that during and between migraine attacks platelet aggregation and activation increased in

migraineurs (Lechner et al., 1985). In the current study, repeated injection of ghrelin

increased platelet counts, hemoglobin concentration and Hct, but had no effect on MPV. In

contrast to our findings, some studies showed that ghrelin had no effect on Hb concentration

and Hct in rats (Narin et al., 2010) or in broiler chicken (Shahryar et al. 2012). Whereas,

Mirzaei Bavil et al. showed that administration of ghrelin, for two weeks, increased red blood

cells count (RBC), hemoglobin concentration and Hct (Mirzaie Bavil et al., 2014).

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 Differences between the animal's type, dose of ghrelin, and time interval between ghrelin

administration and sampling may explain the inconsistent results.

Accepted Article
Interestingly, NTG group had lower platelet counts and higher MPV than vehicle control

group. Findings of the present study are in line with Varol et al. study that reported lower

platelet levels in the migraineurs (Varol et al., 2013). Nevertheless, Pradalier et al. found no

significant differences in platelet count, and mean platelet volume between healthy controls

and migraineurs with or without aura (Pradalier et al., 1992). According to Mathur et al.

study, increased MPV is related to the platelet activation and aggregation (Mathur et al.,

2001). Therefore, it seems that increase of MPV, as an indicator of platelet activation and

aggregation, results in decrease of platelet counts in the NTG group. To our knowledge,

decreased number of platelets in NTG-received animals has not been reported in the rats

previously. The plausible biological link between platelet biology and migraine

pathophysiology is based on this fact that activation of platelets and formation of platelet-

leukocyte aggregates increase pro-inflammatory cytokines release that contributes to the

neuroinflammation and aggravates migraine pain (Danese et al., 2014).

There are increasing evidence that satellite glial cells have a role in the transmission of pain

signals (Pomonis et al., 2001, Ren and Dubner, 2008, Romero-Sandoval et al., 2008) and are

actively involved in the modulation of trigeminal neuronal activity (Capuano et al., 2009).

Capuano et al. showed that pro-inflammatory-activated trigeminal satellite cells promote

central neural sensitization (Capuano et al., 2009). Satellite cells activated and proliferated

following nerve injury and/or inflammation, and pain development (Donegan et al., 2013,

Ohara et al., 2009). However, the exact mechanisms leading to these morphological and

biochemical changes in SGCs are elusive. In our study, chronic injection of NTG increased

the number of SGCs in TG. It is likely that following activation of TS by injection of NTG,

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 trigeminal neurons release neuropeptides, such as PACAP and CGRP (Tuka et al., 2012,

Edvinsson, 2013, Capuano et al., 2014). These neuropeptides activate satellite glial cells to
Accepted Article
produce pro-inflammatory mediators and induce neurogenic inflammation, which can further

lead to proliferation of SGCs. On the other hand, chronic treatment with ghrelin effectively

decreased the number of SGCs around trigeminal neurons possibly through its effects on

blood PACAP, and pro- and anti-inflammatory cytokines levels (Sibilia et al., 2006, Waseem

et al., 2008, Guneli et al., 2010).

Conclusion

The present study demonstrates that NTG administration evoked photophobia and increased

anxiety-like behaviors as behavioral emotional states associated with migraine in rats;

however ghrelin treatment attenuated these behaviors possibly through attenuation of blood

PACAP and cortisol levels. Ghrelin also diminished NTG-induced increase in the number of

satellite cells in the trigeminal ganglion. Therefore ghrelin may hold therapeutic potential in

management of chronic migraine.

Acknowledgments

This study was supported by grants (No. 94/5-4/9) from Neurosciences research center

(NSRC) and Student research Committee of Tabriz University of medical Science. Authors

would like to express their gratitude to Dr Abbas Ebrahimi Kalan for his assistance in

histological study.

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 Author contributions

Fereshteh Farajdokht and Gisou Mohaddes designed the study; Fereshteh Farajdokht, Pouran
Accepted Article
Karimi and Ramin Bughchechi performed data collection; Gisou Mohaddes, Fereshteh

Farajdokht, and Mohammad Reza Alipour analyzed data; Gisou Mohaddes, Shirin Babri,

Fereshteh Farajdokht wrote and edited the paper.

Conflict of interest

The authors state that there are no conflicts of interest.

Abbreviations

CGRP: calcitonin gene-related peptide; ELISA: enzyme-linked immunosorbent assay; EPM:

elevated plus maze; GHSR-1a: growth hormone secretagogue receptor-1a; HPA:

hypothalamus-pituitary-adrenal axis; IL-1: interleukin 1; IL-6: interleukin 6; NTG:

nitroglycerin; OAT: open arms time; OAE: open arms entries; PACAP: pituitary adenylate

cyclase-activating polypeptide; RL-On: room light on; SGCs: satellite glial cells; TG:

trigeminal ganglion; TNC: trigeminal nucleus caudalis; TNF-α: tumor necrosis factor alpha;

TS: trigeminovascular system; VIP: vasoactive intestinal peptide.

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Table. 1. Comparison of blood factors among different groups.

Groups WBC(10^3/uL Hg (g/dL) Htc (%) Plt (10^3/uL) MPV (fL)

Vehicle 5.23 ± 0.56 13.22 ± 0.19 39.33 ± 0.54 761.2 ± 32.22 7.16 ± 0.13

ghrelin 5.33 ± 0.87 14.68 ± 0.68* 45.45 ± 2.16** # 7.23 ± 0.24

868.71 ± 71.28

NTG (10mg/kg) 6.52 ± 0.24* 13.85 ± 0.39 43.31 ± 1.68 622.8 ± 30.98* 7.68 ± 0.15*

NTG + ghrelin 5.90 ± 0.34 14.56 ± 0.41 43.16 ± 0.65 818.15 ± 20.42 7.50 ± 0.04

Data are shown as mean± SEM for 10 animals per group. (NTG: nitroglycerin; WBC: white

blood cell; Hg: hemoglobin; Htc: hematocrit; Plt: platelet; MPV: mean platelet volume).

*P<0.05, **P< 0.01 vs. vehicle control group, #P< 0.05 vs. NTG group.

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Accepted Article

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Accepted Article

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Accepted Article

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