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ABSTRACT
RESUMO
Descritores: Melatonina, CGRP, Modelo Animal.
ORIGINAL ARTICLE
Melatonin reverts CGRP expression induced by capsaicin
Melatonina reverte a expressão de CGRP induzida pela
capsaicina
Fabiano da Cunha Tanuri
1
Debora Amado
1
Eliangela de Lima
1
Iron Dangoni Filho
2
Mario Fernando Prieto Peres
2,3
1
Departamento de Neurologia/Neurocirurgia,
Universidade Federal de São Paulo, Escola
Paulista de Medicina, São Paulo, Brazil
2
Hospital Israelita Albert Einstein, São Paulo,
Brazil
3
Instituto de Psiquiatria, Faculdade de
Medicina da Universidade de São Paulo
*Correspondence
Mario Fernando Prieto Peres
E-mail: marioperes@usp.br
Received: January 20, 2019.
Accepted: January 31, 2019.
Introduction: CGRP, a neuropeptide synthetized and released in the central
nervous system and potent vasodilator, has been implicated in migraine
physiopathology. Because of that, there are CGRP targeted therapies that
decrease CGRP levels. Melatonin, a pineal gland secretion, has already proved
its analgesic effect. We aimed to study CGRP expression in an animal model
comparing capsaicin, CGRP and melatonin. Methods: We used in our study
male animal rats and separated them into groups based in the kind of received
solution (control group, capsaicin only and melatonin plus capsaicin). It was
prepared brain stem slices and measured the CGRP levels in the trigemino
nucleus caudalis (TNC). Results: Capsaicin group (N = 5) presented low intensity
of GCRP expression and animals that received capsaicin plus melatonin (N =
5) showed high intensity of CGRP expression compared to capsaicin group.
Conclusion: Melatonin decreases CGRP in an experimental model in rats
induced by capsaicin, reducing its inammatory action in cerebral vessels.
Keywords: Melatonin, CGRP, Animal Model.
Introdução: CGRP, um peptídeo produzido e liberado no sistema nervoso
central e potente vasodilatador, tem sido implicado na siopatologia da
Migrânea. Devido a isso, tem surgido diversas terapias direcionadas ao CGRP
que reduzem seus níveis. A melatonina, substância produzida pela glândula
pineal, já possui seu efeito analgésico comprovado. Nós objetivamos estudar
a expressão do CGRP em um modelo animal comparando capsaicina, CGRP e
melatonina. Métodos: Foi utilizado em nosso estudo ratos machos adultos e
estes foram separados em grupos baseados na solução que recebiam (grupo
controle, apenas capsaicina e melatonina mais capsaicina). Foram preparadas
fatias dos cérebros dos animais e então medidos os níveis de CGRP no núcleo
caudal trigeminal. Resultados: Grupo da Capsaicina (N = 5) apresentou baixa
intensidade da expressão de CGRP, enquanto aqueles animais que receberam
capsaicina mais melatonina (N = 5) mostraram altos níveis de expressão de
CGRP quando comparados ao grupo do CGRP. Conclusão: No nosso estudo
experimental com ratos induzidos por capsaicina notou-se que a melatonina
reduz os níveis de CGRP, diminuindo a ação inamatória nos vasos cerebrais.
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Tanuri FC, et al.
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Headache Medicine, v.10, n.1, p.24-28, 2019
INTRODUCTION
CGRP (Calcitonin Gene-Related Protein) is a
37-amino acid neuropeptide that belongs to a family
of structurally related peptides (e.g. calcitonin, amylin,
adrenomodulin). This neuropeptide is synthesized and
released from sensory nerves in the central nervous
system and gastrointestinal system, where it acts as a
potent vasodilator
1,2
.
It has been implicated in the mechanisms of
migraine, acting along trigeminovascular pathways as a
vasodilator and nociceptive initiator
3
.
CGRP targeted therapies have been studied for both
acute (gepants) and preventive (anti-CGRP monoclonal
antibodies) treatment
4
.
Decrease CGRP levels contribute to migraine
treatment. Other strategies may also improve migraine
control by reducing CGRP levels, such as the use triptans,
coenzyme Q10, serotonin reuptake inhibitors, exercise,
acupuncture and some kinds of food, such as grape
pomace, cocoa and ginger extracts
4-9
.
Melatonin is the primary secretory product of the
pineal gland, an indoleamine derivate of the essential
amino acid tryptophan
9
. It has been extensively linked
to migraine pathophysiology, due to its capacity of
membrane stabilization, anti-inammatory properties,
modulation of serotonin, inhibition of dopamine release,
gamma amino butyric acid (GAMA) and glutamate
neurotransmission, scavenging toxic free radicals
and cerebrovascular regulation
10,11
. Besides, melatonin
plays important roles in antinociceptive mechanisms.
It has been reported that patients suffer less pain and
prolonged latencies thresholds during nighttime. These
observations were attributed to high melatonin levels
through night and its analgesic effect
12
.
Melatonin has been studied as a prophylaxis
headache treatment in cluster headache and migraine,
but its underlying mechanisms has yet to be determined
11
.
We aimed to study the pattern of CGRP expression in an
experimental model of headache, comparing capsaicin,
melatonin and CGRP levels.
METHODS
Animals
The ethical committee of the Universidade Federal
de São Paulo (UNIFESP) approved all experimental
protocols. All efforts were made to minimize animal
suffering following the proposal of International Ethical
Guideline for Biomedical Research
13
. Wistar adult
male rats (250–300 g) housed under environmentally
controlled conditions in a 12 hours light/dark cicle and
granted free access to food and water were used. These
animals were separated into four groups.
Groups
VEI (n = 5): animals that received vehicle solution
only; CAP (n = 5): animals that received capsaicin solution
(200 nM) only; and CAP + MEL (n = 5) animals that
received capsaicin solution (200 nM) and intraperitoneal
melatonin (Sigma, 10 mg/kg) 20 min after capsaicin
injection.
Drugs
Capsaicin solution was prepared with 3.05 mg
capsaicin (Merck) per 1 ml of vehicle (saline–ethanol–
Tween 80, 8:1:1) and diluted 1:50 (200 nM) with saline.
Vehicle was diluted 1:50 in saline.
Surgical procedures
Capsaicin stimulation
For this procedure, all rats were anesthetized with
pentobarbital (40 mg/kg i.p.) and a surgical opening
was made in the region between the scalp and C1 (rst
cervical vertebra). An amount of 10 ml of capsaicin
solution (see ‘‘Drugs’’) was injected into the cisterna
magna (over 15 min) using a Hamilton syringe with the
aid of a stereotaxic frame
14
. To avoid capsaicin outow,
the needle was only removed 10 min after injection.
Perfusion and immuno-histochemistry
The rats were anesthetized with pentobarbital
overdose (120 mg/kg) after two hours infusion,
followed by perfusion via the ascending aorta with 0.1
M phosphate saline buffer (PBS, 200 ml, pH 7.4) and
4% paraformaldehyde (200 ml) in 0.1 M phosphate
buffer (PB, pH 7.4). Brain stem with attached cervical
cord was stored overnight in the same xative and then
placed in a cryoprotectant (30% sucrose in 0.1 M PB,
pH 7.4). Coronal serial sections (40 ml) were prepared
on a cryostat microtome at -20ºC and collected in PBS
with sodium azide (0.1%) to Nissl staining and immuno-
histochemistry. Sections were rinsed three times 5 min
in PBS, pre-treated with 0.3% H2O2 in PBS for 15 min,
rinsed three times 5 min in PBS and pre-incubated in
10% bovine serum albumin (Calbiochem) and 2% normal
serum (Vector) in PBS for 2 hours at room temperature.
Sections were incubated for 48 hours at 4ºC in PBS
solution containing 2% BSA, 2% normal serum and 0.3%
Triton X-100 in PBS. Following three washes in PBS, the
sections were incubated in a PBS solution contained
biotinylated rabbit IgG (1:200) (Vector) for 2 h at room
temperature. Sections were rinsed three times 5 min in
PBS and incubated with the avidin–biotin–peroxidase
complex (Vector) in PBS for 1 h and 30 min at room
temperature. Sections were rinsed twice 5 min in PBS
and 5 min in Tris–HCl (pH 7.6) and revealed with 0.06%
3,30-diaminobenzidine tetrahydrochloride (Sigma)
and with 0.002% H2O2. Sections were then mounted
in slides and dehydrated through alcohol to xylene and
coverslipped with Entellan (Merck).
Nissl staining
Brain stem slices (40 ml) were hydrated in alcohol
solutions of decreased concentration followed by
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Tanuri FC, et al.
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26
staining in 0.5% cresyl Violet acetate (Sigma) diluted
in 0.1 M acetate buffer pH 4.0. Slices were dehydrated,
coverslipped and analyzed by light microscopy optic
Zeiss Axiolab.
Quantications
The CGRP expression sections in TNC layer I/II were
counted at 0 to - 1 mm caudal to obex. Representative
images of the brainstem slices were digitalized using the
Image 1.61 system. The images were transformed into
black and White. The image analysis were performed
in the anterior region of the TNC, which presented the
same area analyzed in all the cuts. It was quantied the
optic density from the negative obteined of the images,
through the grayscale analysis of the Image Tool program
in “pixels” unit. The white color “pixels” were quantied
and the results were expressed as mean ± standard
deviation.
Statistical analysis
Data were analyzed using one-way analyses of
variance (ANOVA) followed by Tukey’s Q test. A value of
p < 0.05 was accepted as signicant.
RESULTS
The studied groups presented difference in the
CGRP expression analyzed through the densitometry.
Control group that received vehicle into the cisterna
magna showed high intensity of CGRP expression
(VEI: 733,95 ± 144,08) in TNC (layer I/II). In contrast,
we observed that animals submitted to trigeminal
stimulation of intracisternal capsaicin presented low
intensity of CGRP expression (CAP: 295,1 ± 49,93). This
number is signicantly different when we compare
CAP x VEI (p < 0.001). On the other hand, animals
that received intraperitoneal melatonin 20 min before
the capsaicin stimulation presented high intensity of
CGRP expression (CAP + MEL: 584,02 ± 133,59) when
compared to animals that received capsaicin only (p <
0.05) and similar to VEI group.
The results of immunohistochemical and
quantication through CGRP expression optic density
were observable in gures 1, 2 and 3.
DISCUSSION
We found in this experimental study direct relation of
the levels of Melatonin and Capsaicin in rats model, which
were exposed to capsaicin and melatonin injection. After
that, it was measured the CGRP density and it showed
decrease of its density when associated to capsaicin.
However, when we measure melatonin and capsaicin
both together, its levels increase and almost normalize.
A similar study, has already evidenced data about the
relation of melatonin and pineal gland in neurovascular
headaches’ pathophysiology
15
.
Melatonin reverts CGRP alteration induced by
capsaicin due to the inhibition of CGRP-induced increase
Figure 1. Photomicrographs of CGRP expression in TNC
(layer I/II). A: Tissue from animal that received vehicle.
B: Tissue from capsaicin-injected animal. C: Tissue from
animal that received capsaicin and melatonin. Detail
shows the area used to quantify by optic density. x200,
scale bar 55 µc.
Figure 2. Photomicrographs of CGRP expression in TNC
(layer I/II). A: Tissue from animal that received vehicle.
B: Tissue from capsaicin-injected animal. C: Tissue from
animal that received capsaicin and melatonin. Detail
shows the area used to quantify by optic density. x200,
scale bar 55 µc. A’, B’ and C’ represents the negative of
the cut images used for quantication by density optic.
in adenylate cyclase. It has already been proposed that
CGRP and melatonin may share an active role in the
maintenance of arterial tone in cerebral vasculature.
Several studies have demonstrated that melatonin
causes constriction of rat cerebral arteries
16-18
.
We hypothesized melatonin could revert capsaicin
effect owing to its ability of avoiding capsaicin. These
data are in accordance with previous studies showing
melatonin is able to produce a signicant inhibition
against the neurogenic pain caused by capsaicin. It has
been reported the melatonin antinociceptive effect by
central administration of it, showing its high lipid solubility,
capacity to penetrate the blood-brain-barrier and
produce a signicant inhibition against the neurogenic
pain by activating supraspinal sites. In another study, it
was presented that melatonin has the ability of avoid
capsaicin effect of initiation and limit the development of
“central sensibilization”
19
.
Clinical implications
It is an exciting moment to migraine specialists
and patients. There is one approved CGRP receptor
antagonist and some others being studied. Thus, some
doubts about it and its interaction to other established
drugs will need to be answered in the next years.
Melatonin has been showed as a potential candidate
for migraine treatment, including a Brazilian study that
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Tanuri FC, et al.
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Headache Medicine, v.10, n.1, p.24-28, 2019
Figure 3. Quantication of CGRP expression through
optic density in rats that received: VEI – vehicle (n = 5);
CAP – rats that received capsaicin (n = 5); CAP + MEL –
rats that received capsaicin and melatonin (n = 5). Rats
were killed 60 min after injection. Cells were counted in
40 µm sections sampled in the TNC layers I and II at 0
to – 1 mm caudal to the obex (3 secctions). *p < 0.05
compared with capsaicin-treated animals; **p <0.001
compared with vehicle only.
found signicant headache response with melatonin as a
migraine prevention
20
.
Our study brings new questions and challenges
to headache societies: may migraine patients taking
melatonin still respond to CGRP antagonists? Is the
decrease of CGRP the real explanation for melatonin
improvement in headache disorders? Or do these drugs
have synergistic effect?
Melatonin has been associated to CGRP decreased in
patients with pure menstrual migraine
21
. It was investigated
the melatonin capability of reduce inammation through
decreasing CGRP and inducible nitric oxide synthase.
At the beginning of the 2000s, it was proposed that
melatonin could inhibit CGRP vasodilatation effect and
increases cAMP in rats’ arteries
22
.
LIMITATIONS
CGRP should be measured in other brain structures
besides trigeminal nucleus caudalis, such as trigeminal
ganglion, cerebral ventricles, meningeal afferents and
even medullary components of the TNC, as well as
structures outside the central nervous system (CNS), like
skin
23
, gastrointestinal tract, lymphocytes
24
and thymus
25
.
CONCLUSION
Melatonin decreases CGRP in an experimental model
in rats induced by capsaicin, reducing its inammatory
action in cerebral vessels.
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