Headache Medicine, v.2, n.2, p.41-45, Apr./May/Jun. 2011 41

Orexin-A CSF levels correlate with anxiety but not

excessive daytime sleepiness in chronic migraine

Orexina-A se correlaciona com ansiedade mas não sonolência diurna

no líquido cefalorraquiano de pacientes com enxaqueca crônica

ABSTRACTABSTRACT

ABSTRACT

Background: Background:

Background: The hypothalamus is a key brain region in the

control of energy metabolism, sleep and circadian rhythms,

stress and anxiety, food intake, sexual and reproductive

behaviors. Orexin-A (hypocretin-1) is a neuropeptide,

synthesized in the hypothalamus extensively linked to sleep/

wake states, particularly excessive daytime sleepiness. Chronic

migraine is comorbid with several conditions but little is known

about its mechanisms. We aimed to study the role of orexin-A

in the mechanism of chronic migraine and comorbid

conditions.

Methods:Methods:

Methods: We studied orexin-A levels in the CSF

of 60 chronic migraine patients, comparing with age and sex

matched controls, and comorbidity with anxiety, depression

and excessive daytime sleepiness, using appropriate scales.

Results:Results:

Results: Orexin-A levels were inversively correlated with anxiety

levels (r=-308, p=0,03), but not depression and excessive

daytime sleepiness. Orexin levels in CM patients were not

different than controls.

Discussion/Conclusion:Discussion/Conclusion:

Discussion/Conclusion: Anxiety in

CM may be due to a decrease in orexin-A or may be the

cause of its depletion. The orexinergic system may be implicated

in anxiety comorbid with migraine.

Keywords:Keywords:

Keywords: Migraine; Anxiety; Orexin

ORIGINAL ARTICLEORIGINAL ARTICLE

ORIGINAL ARTICLE

Mario Fernando Prieto Peres

1,2,3

, Domingos Savio Vieira

1,2

, Marcelo Rodrigues Masruha

1,2

André Leite Gonçalves

1,2,3

, Juliane Prieto Peres Mercante

, Maria da Graça Naffah-Mazzacoratti

Instituto Israelita de Ensino e Pesquisa - Hospital Albert Einstein, Sao Paulo, Brazil

UNIFESP, Escola Paulista de Medicina, Department of Neurology, Sao Paulo, Brazil

FMABC, Department of Neurology, Sao Paulo, Brazil

Peres MF, Vieira DS, Masruha MR, Gonçalves AL, Mercante JP, Naffah-Mazzacoratti MG

Orexin-A CSF levels correlate with anxiety but not excessive daytime sleepiness in chronic migraine.

Headache Medicine. 2011;2(2):41-45

RESUMORESUMO

RESUMO

O hipotálamo é uma estrutura chave para o controle do ciclo

sono-vigília, estresse e ansiedade, comportamento alimentar

e sexual. A orexina-A (hipocretina-1) é um peptídeo hipo-

talâmico ligado ao ciclo de sono, especialmente sonolência

diurna excessiva e narcolepsia. Enxaqueca crônica é comór-

bida com diversas doenças, mas pouco se sabe sobre os

seus mecanismos. Estudamos o papel da orexina-A na

fisiopatologia da enxaqueca crônica e comorbidades dosando

seus níveis no líquido cefalorraquiano de 60 pacientes com

diagnóstico de enxaqueca crônica, comparados com controles

pareados por idade e sexo. Escalas apropriadas foram usadas

para mensurar ansiedade, depressão e sonolência diurna

associadas. Os níveis de orexina-A foram inversamente

correlacionados com os de ansiedade (r=308, p=0,03), mas

não houve diferença com as escalas de depressão e sonolência

excessiva. Os níveis de orexina-A não foram diferentes em

pacientes e controles.

Conclusão:Conclusão:

Conclusão: Ansiedade em enxaqueca

crônica pode ser devido a uma diminuição da orexina-A ou

ser a causa da sua depleção. O sistema orexinérgico pode

estar implicado na comorbidade da ansiedade com enxa-

queca.

alavrasalavras

alavras

chaves:chaves:

chaves: Migrânea; Ansiedade; Orexina

42 Headache Medicine, v.2, n.2, p.41-45, Apr./May/Jun. 2011

PERES MF, VIEIRA DS, MASRUHA MR, GONÇALVES AL, MERCANTE JP, NAFFAH-MAZZACORATTI MG

INTRODUCTION

The hypothalamus is a key region in the central

nervous system (CNS) responsible for the regulation of

several physiological functions, such as control of energy

metabolism, sleep and circadian rhythms, stress and

anxiety, food intake, sexual and reproductive behaviors.

Orexin (hypocretin) is a neuropeptide, synthesized in a

small set of neurons in the perifornical area of the

hypothalamus. Recent studies have implicated the orexin

system as a critical regulator of sleep/wake states as well

as feeding behavior and reward processes.

Orexin has

been linked to many neurological conditions not only in

sleep disorders such as narcolepsy, but also in

neurological diseases with associated sleep

symptomatology, mainly excessive daytime sleepiness

(EDS) including traumatic brain injury, neurodegenerative,

neuromuscular and neuroimmunological disorders.

Orexin deficiency also cause abnormalities in energy

homeostasis and reward systems. Orexin activates waking

active monoaminergic and cholinergic neurons in the

hypothalamus and brainstem receiving significant input

from the limbic system.

Migraine is a chronic and debilitating condition

affecting a significant proportion of the population.

Migraine pathophysiology is multifactorial, genetic,

hormonal, environmental aspects are involved, the

hypothalamus has been considered to play an important

role in migraine mechanisms.

The orexin system may be

a potentially important target for both mechanisms and

treatment of headache disorders.

A number of studies in

experimental animals showed that orexin is involved in

pain modulation within the CNS, and suggested the

presence of a link between these peptides and nociceptive

phenomena observed in primary headaches.

7-9

Cluster

headache has been linked to the orexin system, several

polymorphisms were studied in 109 patients, the 1246

G>A polymorphism of the hypocretin receptor 2 (HCRTR2)

gene was significantly different between cases and

controls. Homozygosity for the G allele was associated

with an increased disease risk.

Migraine has been studied

genetically, but the HCRTR2 G1246A polymorphism has

not found to be associated.

11,12

The orexinergic system

has been implicated in the comorbidity with migraine and

obesity.

13-15

Orexin-A was studied in the cerebrospinal fluid

(CSF) of chronic migraine (CM) and medication-overuse

headache (MOH) patients showing higher levels in these

patients.

Orexin has been linked to anxiety,

17,18

stress,

addiction,

but to date it has never been linked to migraine

psychiatric comorbidity. We therefore aimed to study the

role of orexin-A in chronic migraine patients and comorbid

conditions.

PATIENTS AND METHODS

Sixty-two patients (10 men and 52 women, ages 15-

69 years, mean age 37.9 years) were diagnosed with

CM according to International Headache Society criteria

2004

and Appendix 2006,

and were consecutively

enrolled. All patients attending at the Brain Research

Institute-Hospital Israelita Albert Einstein, São Paulo, Brazil.

Two patients were excluded: a woman who had chronic

meningitis due to cysticercosis, and another woman with

idiopathic intracranial hypertension with papilloedema,

both of whom had migrainous features. Sixty patients were

analyzed. All these patients had a history of episodic

migraine, but had subsequently developed frequent

migraine. The patients were referred from a basic health

programme, in a community near the hospital,

representative of the general population. Patients were

not exposed to previous migraine prevention treatments.

All 60 patients suffered from daily headaches at the time

of the study. Clinical history was obtained by the authors.

Interviews and neurological examinations were performed

by two neurologists (D.S.S.V. and M.R.M.), supervised by

the senior author (M.F.P.P.). Body mass index (BMI; weight

in kilograms divided by the square of height in meters)

was recorded in all patients.

COMORBIDITY MEASURES

Patients were interviewed and diagnosed if any

psychiatric comorbidity was present according to the

DSM-IV. All the patients were submitted to a detailed

headache questionnaire and answered the following

questionnaires: Epworth sleepiness scale, in order to

measure excessive daytime sleepiness which was

considered when levels were equal or higher than 10.

STAI (State-Trait Anxiety Inventory) measured anxiety levels,

20 questions ranging from 1 to 4 each one, total score

would range from 20 to 80, a cutt-off on 40 was

stablished for the dicotomous analysis. The BDI-II (Beck

Depression Inventory) was used to assess depression

severity, the score has 21 questions, each one scoring 0

to 3, the total score ranges from 0 to 63, depression

diagnosis has been correlated in the literature with a score

higher than 16.

Headache Medicine, v.2, n.2, p.41-45, Apr./May/Jun. 2011 43

CSF PROCEDURES

All patients had a lumbar puncture (LP), which was

performed with the patient positioned in the lateral

decubitus position on a level surface. A standard 22-G

spinal needle was used. The opening pressure (OP) was

recorded by using a manometer positioned at a 90° angle

to the spinal canal with the patient's knees and hip in the

extended position and neck straightened. CSF pressure

was recorded until the patient was relaxed and the pressure

values had stabilized. All LPs were performed by the same

investigator (D.S.S.V.). Increase in CSF was considered

when the OP was > 200 mmH2O.

Controlled CSF specimens were also obtained from

60 age- and sex-matched subjects who underwent lumbar

puncture for others diagnostic purposes. Their CSF and

blood tests were normal. When necessary, instrumental

investigations including neuroimaging also excluded CNS

diseases (multiple sclerosis, vasculitis, and other

autoimmune diseases affecting the CNS) or systemic

diseases (diabetes, renal or hepatic dysfunction,

inflammatory diseases). Neurodegenerative diseases,

mood, and anxiety disorders were also excluded. Orexin-

A was quantified in the CSF using a ultra-sensitive,

commercially available ELISA Kit (Peninsula Laboratories,

San Carlos, CA).

The protocol was approved by the local Ethics

Committee and all patients gave written consent to these

study. The subjects' consent was obtained according to

the Declaration of Helsinki. All patients were in pain at the

time of lumbar puncture.

Statistical analysisStatistical analysis

Statistical analysis

The values in µmol/l were expressed as mean ±

SD. The chi-square test (χ2) (without Yates correction) was

used for categorical data comparisons. Mean differences

of continuous measurements were tested by the one-way

analysis of variance - ANOVA (F). Whenever the ANOVA

showed significant differences, the Bonferroni's multiple

comparison test was used to verify between which groups

the differences were found. The Pearson's product-moment

correlation coefficient (r) was used to assess the relationship

between two continuous variables, and point-biserial

correlation coefficients (rpb) were used to assess the

relationship between orexin levels and dichotomic

categorical variables. A p value of less than 0.05 was

considered to indicate statistical significance; all tests were

two-tailed. Ninety-five percent confidence intervals (CI)

were calculated for the difference between means and

the regression coefficients. All statistical analyses were

performed on a personal computer with the statistical

package SPSS 11.5.1 for Windows.

RESULTS

The mean age of patients was 42.9 years, SD 11.6,

mean frequency of headache 28.9 days per month,

SD 2.43 days; mean baseline pain intensity (0 to 10)

was 5.1, SD 1.3; mean exacerbations pain intensity

9.2, SD 0.8.

All CSF studied presented normal levels of protein,

glucose, lactate, as well as the cell count.

Orexin levels were not signifficantly different in CM

patients versus controls (p=0.278), no other demographic

variable was statistically significant.

Orexin-A levels were inversively correlated with anxiety

levels (r=-308,p=0.03). Depression levels (BDI-II) and

Epworth Sleepiness scale levels were not significantly

correlated with orexin-A levels (r=-0.215; p=0.139,

r=0.105; p=0.245, respectively). Body Mass index was

not correlated with orexin-A levels.

No significant correlation with analgesic intake and

orexin levels were determined.

DISCUSSION

The more anxious the chronic migraine patient the

less orexin-A levels were found in the CSF in our study,

suggesting a role of hypothalamic orexinergic neurons in

migraine comorbidity, particulatly in anxious behaviour.

We can hypothesised a hypoactive orexinergic system

underlying anxiety in chronic migraine patients.

In a study in CM and MOH patients, significantly higher

levels of orexin-A were found in the CSF of MOH and to a

lesser extent in patients with CM compared with control

subjects. A significant positive correlation was also found

between CSF orexin-A values and monthly drug intake

group (R=0.39; P < 0.03) and scores of the Leeds

Dependence Questionnaire (LDQ), an instrument to

measure dependence of substances, in the MOH group

(R=0.68; P < 0.0003). The authors interpreted the higher

orexin-A levels found in CM and MOH as a compensatory

response to pain or a hypothalamic overexpression to stress

16. In this study anxiety was not specifically assessed.

Intracerebroventricular injection of orexin-A in two major

anxiety tests, the light-dark exploration test (mouse) and the

elevated plus-maze test (mouse, rat) suggested an

anxiogenic effect of orexin.

OREXIN-A CSF LEVELS CORRELATE WITH ANXIETY BUT NOT EXCESSIVE DAYTIME SLEEPINESS IN CHRONIC MIGRAINE

44 Headache Medicine, v.2, n.2, p.41-45, Apr./May/Jun. 2011

PERES MF, VIEIRA DS, MASRUHA MR, GONÇALVES AL, MERCANTE JP, NAFFAH-MAZZACORATTI MG

In contrast, a recent study in combat related

postraumatic stress disorder showed the opposite. CSF

and plasma orexin-A concentrations were significantly

lower in the patients with PTSD as compared with healthy

subjects, as wel as strongly and negatively correlated with

PTSD severity as measured by the Clinician-Administered

PTSD Scale (CAPS) in patients with PTSD.

These findings,

in the same direction as our study, suggest a low orexin-A

activity in anxiety.

The hypothalamus is a key region in the brain

regulating important aspects of defense mechanisms and

survival such as pain, sleep-wake cycle, appetite, hormonal

and metabolism balance, reproduction. Although limited

evidence is found in the literature, it is not surprising that

an important defense mechanism such as anxiety might

be regulated in the hypothalamus and possibly in

orexinergic neurons. One can hypothesize orexin could

play a role in regulating anxiety in CM, and migrainous

patients with lowered orexin-A levels could develop anxiety

because orexin is deficient. On the other hand, the excess

of anxiety commonly experienced by CM patients could

lead to a depletion in orexin levels.

Lowered orexin-A levels have been extensively linked

to excessive daytime sleepiness in narcolepsy and other

conditions due to a loss of orexinergic neurons.

We have

not found a difference in patients versus controls, nor a

correlation with BMI and EDS in our sample. It is less likely

that a loss in orexinergic neurons happen in this chronic

migraine context.

We could not find a difference between patients and

controls, these could be due to the lack of an adequate

control population, our controls were not absolutely free

of symptoms; they underwent a LP to rule out a

neurological condition and they could have experienced

pain and anxiety during the procedure.

This orexinergic system has been considered as a

potential target for the treatment of sleep disorders, it may

be also a potentially important therapeutic agent for

migraine and anxiety in the future.

We hope to encourage further studies focusing the

orexinergic system and the hypothalamus in migraine and

anxiety.

REFERENCES

1. Eriksson KS, Sergeeva OA, Haas HL, Selbach O. Orexins/

hypocretins and aminergic systems. Acta Physiol (Oxf). 2010;

198(3):263-75.

2. de Lecea L. A decade of hypocretins: past, present and future of the

neurobiology of arousal. Acta Physiol (Oxf). 2010;198(3):203-8.

3. Arias-Carrion O, Bradbury M. The sleep-wake cycle, the

hypocretin/orexin system and narcolepsy: advances from

preclinical research to treatment. CNS Neurol Disord Drug

Targets. 2009;8(4):232-4.

4. Queiroz LP, Peres MF, Piovesan EJ, Kowacs F, Ciciarelli M, Souza

J, et al. A nationwide population-based study of migraine in

Brazil. Cephalalgia. 2009;29(6):642-9.

5. Peres MF, Sanchez del Rio M, Seabra ML, Tufik S, Abucham J,

Cipolla-Neto J, et al. Hypothalamic involvement in chronic

migraine. J Neurol Neurosurg Psychiatry. 2001;71(6):747-51.

6. Rainero I, De Martino P, Pinessi L. Hypocretins and primary

headaches: neurobiology and clinical implications. Expert Rev

Neurother. 2008;8(3):409-16.

7. Holland PR, Akerman S, Goadsby PJ. Modulation of nociceptive

dural input to the trigeminal nucleus caudalis via activation of

the orexin 1 receptor in the rat. Eur J Neurosci. 2006;24

(10):2825-33.

8. Holland PR, Akerman S, Goadsby PJ. Orexin 1 receptor activation

attenuates neurogenic dural vasodilation in an animal model of

trigeminovascular nociception. J Pharmacol Exp Ther. 2005;

315(3):1380-5.

9. Bartsch T, Levy MJ, Knight YE, Goadsby PJ. Differential modulation

of nociceptive dural input to [hypocretin] orexin A and B receptor

activation in the posterior hypothalamic area. Pain. 2004;

109(3):367-78.

10. Rainero I, Rubino E, Valfre W, Gallone S, De Martino P, Zampella

E, et al. Association between the G1246A polymorphism of the

hypocretin receptor 2 gene and cluster headache: a meta-analysis.

J Headache Pain. 2007;8(3):152-6.

11. Pinessi L, Binello E, De Martino P, Gallone S, Gentile S, Rainero

I, et al. The 1246G-->A polymorphism of the HCRTR2 gene is

not associated with migraine. Cephalalgia. 2007; 27 (8):945-

12. Schurks M, Limmroth V, Geissler I, Tessmann G, Savidou I,

Engelbergs J, et al. Association between migraine and the

G1246A polymorphism in the hypocretin receptor 2 gene.

Headache. 2007;47(8):1195-9.

13. Peterlin BL, Rosso AL, Rapoport AM, Scher AI. Obesity and

migraine: the effect of age, gender and adipose tissue

distribution. Headache. 2010;50(1):52-62.

14. Bigal ME. Correlation of increase in phosphene threshold with

reduction of migraine frequency: a comment. Headache. 2009;

49(5):783-4.

15. Peterlin BL, Rapoport AM, Kurth T. Migraine and obesity:

epidemiology, mechanisms, and implications. Headache.

2010;50(4):631-48. Comment in Headache. 2010; 50(4):649.

16. Sarchielli P, Rainero I, Coppola F, Rossi C, Mancini M, Pinessi L,

et al. Involvement of corticotrophin-releasing factor and orexin-

A in chronic migraine and medication-overuse headache: findings

from cerebrospinal fluid. Cephalalgia. 2008;28(7):714-22.

17. Suzuki M, Beuckmann CT, Shikata K, Ogura H, Sawai T. Orexin-

A (hypocretin-1) is possibly involved in generation of anxiety-

like behavior. Brain Res. 2005;1044(1):116-21.

18. Johnson PL, Truitt W, Fitz SD, Minick PE, Dietrich A, Sanghani S,

et al. A key role for orexin in panic anxiety. Nat Med. 2010;

16(1):111-5.

Headache Medicine, v.2, n.2, p.41-45, Apr./May/Jun. 2011 45

Correspondence

DrDr

. Mario F. Mario F

. Mario F

ernando Pernando P

ernando P

rieto Prieto P

rieto P

ereseres

eres

Joaquim Eugenio de Lima, 881cj 708

Sao Paulo, SP, Brazil

http://cefaleias.com.br/

marioperes@cefaleias.com.br

19. Strawn JR, Pyne-Geithman GJ, Ekhator NN, Horn PS, Uhde

TW, Shutter LA, et al. Low cerebrospinal fluid and plasma orexin-

A (hypocretin-1) concentrations in combat-related post-

traumatic stress disorder. Psychoneuroendocrinology. 2010;

35(7):1001-7.

20. Martin-Fardon R, Zorrilla EP, Ciccocioppo R, Weiss F. Role of

innate and drug-induced dysregulation of brain stress and arousal

systems in addiction: Focus on corticotropin-releasing factor,

nociceptin/orphanin FQ, and orexin/hypocretin. Brain Res.

2010;1314:145-61.

21. Olesen J, Steiner TJ. The International classification of headache

disorders, 2nd edn (ICDH-II). J Neurol Neurosurg Psychiatry.

2004;75(6):808-11.

22. Olesen J. The International Classification of Headache Disorders,

2nd edition: application to practice. Funct Neurol. 2005;

20(2):61-8.

23. Niimi M. Centrally administered neuropeptide S activates orexin-

containing neurons in the hypothalamus and stimulates feeding

in rats. Endocrine. 2006;30(1):75-9.

24. Tsujino N, Sakurai T. Orexin/hypocretin: a neuropeptide at the

interface of sleep, energy homeostasis, and reward system.

Pharmacol Rev. 2009;61(2):162-76.

OREXIN-A CSF LEVELS CORRELATE WITH ANXIETY BUT NOT EXCESSIVE DAYTIME SLEEPINESS IN CHRONIC MIGRAINE