UNIVERSIDADE ESTADUAL DE CAMPINAS
INSTITUTO DE BIOLOGIA
MARIA JULIA CEOLIN GIOIA
EFEITO DOS Β-ADRENOCEPTORES NA REDUÇÃO DA ATIVIDADE DO
CONTROLE NOCICEPTIVO ASCENDENTE INDUZIDA PELA
HIPERALGESIA PERSISTENTE
THE EFFECT OF Β-ADRENOCEPTORS IN THE ASCENDING
NOCICEPTIVE CONTROL ACTIVITY’S REDUCTION INDUCED BY
PERSISTENT HYPERALGESIA
CAMPINAS 2018
MARIA JULIA CEOLIN GIOIA
EFEITO DOS Β-ADRENOCEPTORES NA REDUÇÃO DA ATIVIDADE DO
CONTROLE NOCICEPTIVO ASCENDENTE INDUZIDA PELA
HIPERALGESIA PERSISTENTE
THE EFFECT OF Β-ADRENOCEPTORS IN THE ASCENDING
NOCICEPTIVE CONTROL ACTIVITY’S REDUCTION INDUCED BY
PERSISTENT HYPERALGESIA
Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Mestra em Biologia Funcional e Molecular, na área de Fisiologia.
Dissertation presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Functional and Molecular Biology, in Physiology area.
Orientadora: Profa. Dra. Cláudia Herrera Tambeli
CAMPINAS 2018
ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA MARIA JULIA CEOLIN GIOIA E ORIENTADA PELA PROFª DRª CLAUDIA HERRERA TAMBELI.
Campinas, 13 de Julho de 2018.
COMISSÃO EXAMINADORA
Profa. Dra. Cláudia Herrera Tambeli
Prof. Dr. César Renato Sartori
Prof. Dr. Alexandre César Santos de Rezende
Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno.
RESUMO
Na dor crônica, circuitos de modulação de dor, como o Controle Nociceptivo Ascendente (ANC), estão prejudicados. O ANC é um sistema endógeno inibitório de dor que é ativado por um estímulo nociceptivo (como uma injeção de capsaicina) e desencadeia uma antinocicepção heterosegmental que dura por mais de uma hora. A dor persistente está associada a uma redução na atividade do ANC e este efeito parece depender do sistema simpático. A medula da adrenal produz e libera epinefrina que atua principalmente em receptores β-adrenérgicos. Entretanto, se a ativação destes receptores diminui a duração da analgesia do ANC durante a hiperalgesia persistente é desconhecido. Sendo assim, nós formulamos a hipótese de que o bloqueio dos receptores β-adrenérgicos previne a diminuição – induzida pela hiperalgesia persistente - da duração da analgesia do ANC. Para testar essa hipótese, nós induzimos a hiperalgesia persistente com 14 injeções diárias de Prostaglandina E2 (PGE2) na pata direita traseira do rato. Sete dias após
o término das injeções, nós injetamos Propranolol ou seu veículo sistemicamente e depois de 10 minutos nós ativamos o ANC com uma injeção de Capsaicina na pata direita dianteira do rato. Então nós avaliamos a resposta nociceptiva da pata traseira imediatamente após a injeção na pata dianteira e a cada 15 minutos durante 1 hora. Nós verificamos que a hiperalgesia persistente reduziu a duração da analgesia induzida por Capsaicina de 60 para 30 minutos em ratos e para 15 minutos em ratas. O bloqueio dos receptores β-adrenérgicos pela injeção de Propranolol antes da injeção de Capsaicina restaurou a duração do ANC durante a hiperalgesia persistente. Esse efeito foi completo em ratos (analgesia de 60 minutos) e parcial em ratas (analgesia de 45 minutos). Nós concluímos que o bloqueio dos receptores β-adrenérgicos previne a diminuição da duração da analgesia do ANC induzida pela hiperalgesia persistente em ratos e ratas.
Palavras-chave: Hiperalgesia Persistente; Controle Nociceptivo Ascendente; Propranolol; Antinocicepção
ABSTRACT
In chronic pain syndromes, endogenous pain modulation circuits, such as the
Ascending Nociceptive Control (ANC), are impaired. The ANC is an inhibitory endogenous pain
system that is activated by a nociceptive stimulus (e.g. a capsaicin injection) and triggers a heterosegmental antinociception that lasts for more than one hour. Persistent pain is associated to ANC’s activity reduction and this effect seems sympathoadrenal dependent. The adrenal medulla produces and releases epinephrine which acts mainly on β- adrenergic receptors. However, whether the activation of these receptors decreases the analgesia duration of the ANC during persistent hyperalgesia is unknown. Therefore, we hypothesized that the blockade of β-adrenergic receptors prevents the decreased analgesia duration – induced by persistent hyperalgesia - of the ANC induced by. To test this hypothesis, we induced the persistent hyperalgesia with 14 daily Prostaglandin E2 (PGE2) injections in the rat’s right hind paw. Seven days after the injections
cessation, we injected Propranolol or its vehicle systemically and after 10 minutes, we activated the ANC by a Capsaicin injection in the rat’s right forepaw. Then we evaluated the hind paw nociceptive response immediately after the forepaw injection and every 15 minutes for 1 hour. We found that the persistent hyperalgesia reduced the duration of capsaicin-induced analgesia from 60 to 30 minutes on male rats and to 15 minutes on female rats. The blockade of β-adrenergic receptors by a Propranolol injection before the Capsaicin injection restored the analgesia duration of the ANC during the persistent hyperalgesia. This effect was complete in male rats (60 minutes analgesia) and partial in female rats (45 minutes analgesia). We concluded that the blockade of β-adrenergic receptors prevents the decreased analgesia duration of the ANC induced by persistent hyperalgesia in male and female rats.
Keywords: Persistent Hyperalgesia; Ascending Nociceptive Control; Propranolol; Antinociception.
LISTA DE ABREVIATURAS E SIGLAS
ANC = Controle Nociceptivo Ascendente (do inglês Ascending Nociceptive Control) h = hora i.p. = intraperitoneal kg = quilograma mg = miligrama mL = mililitro ng = nanograma
NAc = Nucleus Accumbens PGE2 = Prostaglandina E2
RVM= Bulbo rostral ventromedial (do inglês Rostral Ventromedial Medulla) μg = micrograma
SUMÁRIO
INTRODUÇÃO ... 9
CAPÍTULO 1- β-adrenoceptor blockade prevents the attenuation of activity in an endogenous pain modulation circuit induced by persistent pain in male and female rats ...13
CONCLUSÕES ...32
REFERÊNCIAS BIBLIOGRÁFICAS ...33
INTRODUÇÃO
A dor é uma experiência sensorial e emocional complexa desagradável cuja percepção é variável, pois é influenciada tanto por fatores cognitivos quanto emocionais (Bushnell et al., 2013). A dor de caráter agudo é caracterizada por ocorrer após dano ao tecido e ser delimitada no tempo; por isso, possui importante papel de proteção (Cox et al., 2010). Entretanto, sob condições persistentes, este sistema nociceptivo pode tornar-se sensibilizado e a dor passa a ser crônica. Isso significa que a dor permanece apesar da ausência de estímulo nociceptivo, sendo, por isso, tida como patológica (Millan, 1999; Merskey & Bogduk, 1994; Zeilhofer, 2005).
A dor crônica é um problema de saúde pública significativo; estimativas sugerem que 20% dos adultos sofrem de dor crônica globalmente (Goldberg & McGee, 2011; Bushnel et al., 2013). No Brasil, estima-se que a dor crônica acometa cerca de 40% da população de adultos e idosos (Souza et al., 2017). A dor crônica é associada a prejuízo nas atividades físicas e aumento no sofrimento emocional, prejudicando a qualidade de vida das pessoas acometidas por essa moléstia (Bushnel et al., 2013; Driscoll & Kerns, 2016). É sabido que a persistência da hiperalgesia é associada com mudanças expressas em todos os componentes nociceptivos e a alterações anatômicas e funcionais nos sistemas endógenos de modulação da dor (Bolay & Moskowitz, 2002; Bushnell et al., 2013; Dias et al., 2015). Esse estado patológico ainda permanece pobremente tratado na clínica – as terapias utilizadas atualmente (como anti-inflamatórios não-esteroidais, opioides e gabapentinoides) provêm um alívio de dor satisfatório em apenas 30% a 40% dos pacientes, o que demonstra a falta de conhecimento sobre esses estados dolorosos (Burgess & Williams, 2010; Tsantoulas & Mcmahon, 2014).
Considerando que, muitas vezes, a dor crônica resulta a partir de um episódio inflamatório (Woolf, 2011), Ferreira e colaboradores (1990) desenvolveram um modelo de hiperalgesia persistente em ratos. Neste modelo, a administração de Prostaglandina E2 (PGE2) na
pata do rato por 14 dias consecutivos (chamado de “Período de indução”) leva a uma hiperalgesia que persiste por até 30 dias após o término das injeções de PGE2 (chamado de “Período de
manutenção”). A PGE2 é o mediador final inflamatório, estando envolvido nos processos que levam
à vermelhidão, inchaço e dor – sinais clássicos da inflamação (Ricciotti & FitzGerald, 2011). É sabido que, na dor crônica mecanismos endógenos de modulação da dor estão alterados. Isso ocorre tanto na clínica (Nasri-Heir et al., 2015; Mills et al., 2018) quanto em
modelos animais (Vanegas & Schaible, 2004). Foi demonstrado que, dentre os mecanismos endógenos da modulação da dor que estão prejudicados em modelos animais de dor crônica está o Controle Nociceptivo Ascendente (ANC, do inglês Ascending Nociceptive Control) (Ferrari et al., 2010; Miranda et al., 2015). Ele leva este nome porque ascende da Medula Espinal para os Nucleus
Accumbens (NAc), onde ocorre liberação de opioides e dopamina (Schimidt et al., 2002) e ao Bulbo
Rostral Ventromedial (RVM), onde ocorre a ativação de um mecanismo colinérgico (Gear & Levine, 2009).
A ativação do ANC se dá através de um estímulo nociceptivo intenso (como a injeção de Capsaicina ou inserção da pata em água quente) que desencadeia uma antinocicepção heterosegmental, ou seja, o efeito analgésico é percebido em regiões distantes da em que o estímulo foi aplicado (Gear et al., 1999). Isso foi demonstrado pela administração de Capsaicina na pata traseira de ratos, refletindo na atenuação do reflexo de abertura bucal (Gear et al., 1999; Schimidt
et al., 2002; Tambeli et al., 2009) e também pela administração de Capsaicina na pata dianteira de
ratos gerando antinocicepção na pata traseira (Gear et al., 1999; Ferrari et al., 2010; Miranda et al., 2015).
Foi observado que a analgesia desencadeada pela ativação do ANC independe da duração do estímulo nociceptivo inicial, ou seja, a partir do momento em que houve a ativação desse mecanismo a analgesia persiste (Gear et al., 1999). Isso foi demonstrado através do bloqueio neural periférico no local da ativação do ANC com anestésicos: quando há a administração do anestésico antes da estimulação nociceptiva, o efeito antinociceptivo do ANC é bloqueado; entretanto, se o anestésico é administrado após o estímulo, o efeito antinociceptivos do ANC não é alterado (Gear et al., 1999).
Posteriormente, foram avaliados os mecanismos espinais envolvidos na ativação do ANC. Primeiramente observou-se que a administração intratecal (na região lombar da medula espinal) de antagonistas dos receptores excitatórios AMPA ou mGluR1 gerava antinocicepção heterosegmental (Tambeli et al., 2002). Também foi demonstrado que a administração intratecal de antagonistas de receptores inibitórios (como o µ-opioide, GABAA e GABAB) bloqueia a
analgesia desencadeada pela ativação do ANC (Tambeli et al., 2003). Com isso, foi proposto que o mecanismo de ação do ANC é que, em condições fisiológicas basais há uma liberação tônica de glutamato na medula espinal, que excita um neurônio de projeção espino-supraespinal. Este, por
sua vez, inibe neurônios que medeiam a antinocicepção no NAc. Na presença de um estímulo nociceptivo, a atividade do neurônio de projeção é inibida, induzindo à antinocicepção (Tambeli et
al., 2003). Posteriormente, foram estudos os mecanismos responsáveis pela longa duração da
analgesia desencadeada pela ativação do ANC. Foi demonstrado que a administração intratecal de antagonistas de receptores inibitórios µ-opioide e GABAB reverteu a analgesia desencadeada pela
ativação do ANC, indicando que o ANC é mantido na medula espinal por sinapses inibitórias (Tambeli et al., 2009).
Em animais intactos e na hiperalgesia aguda, a analgesia desencadeada pela ativação do ANC dura por pelo menos 1h (Gear et al., 1999; Ferrari et al., 2010; Miranda et al., 2015), mas em modelos animais de neuropatia e de hiperalgesia persistente induzida por PGE2 a duração da
analgesia por ativação do ANC é diminuída (Ferrari et al., 2010; Miranda et al., 2015). Entretanto, na dor neuropática, com a retirada da medula da adrenal a duração da analgesia induzida pelo ANC foi restaurada (Ferrari et al., 2010).
Na medula da adrenal é produzido o neurohormônio epinefrina (também chamado de “adrenalina”), o qual é rapidamente secretado na circulação em resposta ao stress, atuando principalmente sobre os receptores do tipo β-adrenérgicos (Wong et al., 2012). Foram identificados e descritos três subtipos de receptores β-adrenérgicos, nomeados de receptores β1, β2 e β3-adrenérgicos (Bylund et al., 1994).
Diversos estudos têm mostrado o papel antinociceptivo de antagonistas dos receptores β-adrenérgicos (chamados de “betabloqueadores”) em diferentes modelos animais de dor, como na dor inflamatória aguda (Khasar et al., 1999; Pelegrini-da-Silva et al., 2008; Zhang et al., 2014; Zanelatto et al., 2017); dor crônica (Gil et al., 2015) e inclusive na clínica (Light et al., 2009; Bunevicius et al., 2013; Härkänen et al., 2015). Dentre os diferentes antagonistas de receptores β-adrenérgicos está o Propranolol (Bortolotto & Consolim-Colombo, 2009). O Propranolol é classificado como um betabloquador não-seletivo, pois bloqueia tanto os receptores adrenérgicos do tipo β1 quanto os do tipo β2 (Helfand et al., 2009). Além disso, possui alta lipossolubilidade, o que permite que ele penetre na barreira hematoencefálica (Dimsdale et al., 1989).
Apesar de se saber que em modelos animais de dor neuropática e hiperalgesia persistente induzida por PGE2 a duração da analgesia induzida pela ativação do ANC é diminuída
restaura a duração da analgesia do ANC (Ferrari et al., 2010), não se sabe se o mesmo ocorre na hiperalgesia persistente induzida por PGE2. Considerando que a medula da adrenal secreta
epinefrina, a qual atua sobre receptores β-adrenérgicos (Wong et al., 2012), surge a hipótese de que a diminuição da analgesia induzida pela ativação do ANC em modelos animais de dor neuropática e dor crônica se dá pela ativação dos receptores β-adrenérgicos. Sendo assim, o objetivo desse trabalho foi avaliar se o β-bloqueador Propranolol é capaz de restaurar a duração da analgesia induzida pela ativação do ANC em ratos e ratas durante hiperalgesia persistente induzida por PGE2.
O presente estudo está em formato alternativo, conforme deliberação da Comissão Central
Capítulo 1
β-adrenoceptor blockade prevents the attenuation of activity in an endogenous pain modulation circuit induced by persistent pain in male and female rats
Maria Julia Ceolin Gioia César Renato Sartori Carlos Amilcar Parada Claudia Herrera Tambeli*
Department of Physiology and Biophysics, Institute of Biology, State University of Campinas - UNICAMP, Campinas, Sao Paulo, Brazil
* Corresponding author:
Rua Carl Von Linnaeus, s/n – Cidade Universitária, Campinas – SP – Brasil CEP 13083-864 – Brazil
Tel.: +55-19-3521-6195
Abstract
In chronic pain syndromes, endogenous pain modulation circuits, such as the
Ascending Nociceptive Control (ANC), are impaired. The ANC is an inhibitory endogenous pain
system that is activated by a nociceptive stimulus (e.g. a capsaicin injection) and triggers a heterosegmental antinociception that lasts for more than one hour. Persistent pain is associated to ANC’s activity reduction and this effect seems sympathoadrenal dependent. The adrenal medulla produces and releases epinephrine which acts mainly on β- adrenergic receptors. However, whether the activation of these receptors decreases the analgesia duration of the ANC during persistent hyperalgesia is unknown. Therefore, we hypothesized that the blockade of β-adrenergic receptors prevents the decreased analgesia duration – induced by persistent hyperalgesia - of the ANC induced by. To test this hypothesis, we induced the persistent hyperalgesia with 14 daily Prostaglandin E2 (PGE2) injections in the rat’s right hind paw. Seven days after the injections
cessation, we injected Propranolol or its vehicle systemically and after 10 minutes, we activated the ANC by a Capsaicin injection in the rat’s right forepaw. Then we evaluated the hind paw nociceptive response immediately after the forepaw injection and every 15 minutes for 1 hour. We found that the persistent hyperalgesia reduced the duration of capsaicin-induced analgesia from 60 to 30 minutes on male rats and to 15 minutes on female rats. The blockade of β-adrenergic receptors by a Propranolol injection before the Capsaicin injection restored the analgesia duration of the ANC during the persistent hyperalgesia. This effect was complete in male rats (60 minutes analgesia) and partial in female rats (45 minutes analgesia). We concluded that the blockade of β-adrenergic receptors prevents the decreased analgesia duration of the ANC induced by persistent hyperalgesia in male and female rats.
Keywords: Persistent Hyperalgesia; Ascending Nociceptive Control; Propranolol; Antinociception.
Introduction
Chronic pain is a public health problem that affects billions of people worldwide. It is associated to physical activities impairment and emotional suffering increase (Bushnel et al., 2013; Driscoll & Kerns, 2016). This pathological state remains poorly treated: the therapies used nowadays (e.g. Nonsteroidal anti-inflammatory drugs, opioids and gabapentinoids) alleviates the pain on a satisfactory way of only 30% to 40% of the patients (Burgess & Williams, 2010; Tsantoulas & Mcmahon, 2014).
Although neuropathic pain is a kind of chronic pain, this kind of hyperalgesia often raises from an inflammatory episode (Woolf, 2011). To mimic this, Ferreira et al. (1990) developed a persistent hyperalgesia model in rats, in which 14 daily subcutaneous Prostaglandin E2 (PGE2)
injections induce a hyperalgesia that persists for at least 30 days after the injections’ ceased. It has been shown that in chronic pain syndromes the endogenous inhibitory pain systems are impaired in both humans (Nasri-Heir et al., 2015; Mills et al., 2018) and animal studies (see Vanegas & Schaible, 2004, for a review). Among the endogenous inhibitory pain systems is the Ascending Nociceptive Control (ANC) (Gear & Levine, 1995; Tambeli et al., 2003). The ANC is activated by a nociceptive stimulus (e.g. a subcutaneous capsaicin injection far from the nociceptive testing) (Gear et al., 1999; Tambeli et al., 2003; Tambeli et al., 2009) and triggers a heterosegmental antinociception similar to a high dose of morphine (10mg/kg), which lasts for more than an hour (Gear et al., 1999; Schmidt et al., 2002). However, the duration of the analgesia induced by ANC activation is shortened in animal models of neuropathic pain (Ferrari et al., 2010; Kimura et al., 2015) and persistent hyperalgesia induced by PGE2 (Miranda et al., 2015).
Nevertheless, in neuropathic pain animals, the ablation of the sympathoadrenal axis by adrenal medullectomy was capable of restoring the duration of the analgesia induced by the activation ANC (Ferrari et al., 2010). The adrenal medulla produces and releases epinephrine which act mainly on adrenergic receptors (Wong et al., 2012). However, it is not known whether the activation of β-adrenergic receptors decreases the analgesia duration of the ANC during persistent hyperalgesia. Therefore, we hypothesized that the blockade of β-adrenergic receptors prevents the decreased analgesia duration of the ANC induced by persistent hyperalgesia. To test this hypothesis, we investigated whether the blockade of the β-adrenergic receptors by a systemic administration of
Propranolol restores the duration of the analgesia of the ANC in a rat model of persistent hyperalgesia.
Materials and Methods Animals
Experiments were performed with male and female Wistar rats (180-200g; about 6 weeks old), obtained from the Multidisciplinary Center for Biological Investigation (CEMIB) at the State University of Campinas (UNICAMP). The rats were kept under a 12/12-h light–dark cycle, controlled humidity (60–80%) and temperature (22–25 °C). Food and water were available
ad libitum. Rats were taken to the testing area at least 30 minutes before behavioral testing and all
the experiments were performed between 08:00 and 17:00h. It was used 5-6 rats/experimental group. Animal care and handling procedures were approved by the institutional Committee for Ethics in Animal Experimentation at UNICAMP (CEUA/IB-UNICAMP), São Paulo, Brazil, protocol number 4390-1, which conformed to the guidelines set by IASP for the study of pain in animals (Zimmermann, 1983).
Drugs and doses
The drugs used in this study were: PGE2, 100ng/50µL/hind paw (Ferreira et al., 1990);
Capsaicin, 125ug/50µL/fore-paw (Gear et al., 1999); Propranolol, 9mg/kg, i.p. (Kline et al., 2015). Capsaicin was obtained from Tocris Bioscience; PGE2 and Propranolol were obtained from
Sigma-Aldrich.
PGE2 first stock solution was made diluting the drug in ethanol to an initial
concentration of 10µg/µL. Then it was diluted in Phosphate Buffer (PB) 0,1M (0,1µg/µL). The usage concentration of 2ng/µL (dose of 100ng/50µL) was obtained adding 0.9% NaCl (Saline) just before the injection. Capsaicin stock solution was made diluting the drug in ethanol and Tween 80 on a 1:1 proportion to a concentration of 25ug/µL. Just before the use it was diluted in saline (2.5ug/µL). Propranolol was dissolved in saline to a concentration of 3mg/ml.
Experimental Design
First, the persistent hyperalgesia protocol was reproduced in male and female rats by injecting PGE2 or Saline on the right hind paw for 14 consecutive days. Their nociceptive responses
were evaluated every day before the injections during the induction period and on the 3rd and 7th days of the maintenance period. The PWT was quantified using Randall-Sellito nociceptive test. It was also reproduced the ANC activation protocol in male and female rats by injecting Capsaicin or its vehicle on the right fore paw. Their nociceptive responses were evaluated immediately after the forepaw injection and every 15 minutes for 1 hour. On the experimental groups, the persistent hyperalgesia was induced and the PWT was evaluated on days 1, 7 and 14, before and 3h after the injection, in order to make sure the nociceptive response was the expected. On the 21st day of experiment (i.e., the 7th day of the maintenance period) the treatments were evaluated as it follows:
it was done a PWT measure, Propranolol (9mg/kg) or saline was injected (i.p.) with a 30G ½ ‘’ needle; immediately after this it was done another PWT measure, after 10 minutes of the i.p. injection it was injected Capsaicin or its vehicle on the right fore paw and the nociceptive response of the right hind paw was evaluated immediately (0),15, 30, 45 and 60 minutes after the fore paw injection.
Nociceptive testing
The mechanical hyperalgesia was quantified using the Randall-Sellito nociceptive paw-withdrawal test (Randall & Sellito, 1957) (Ugo BasileAlgesymeter, Stoelting). This equipment has a two items compression device: a flat surface in which the paw plant rests and a conical part that exerts pressure on the paw dorsum. At the time of the test the experimenter presses a foot pedal that transmits a force that increases linearly on the conical part of the appliance, pressing the rat’s paw. This pressure is quantified by a mobile cursor that slides over a scale and shows, in grams, the pressure being exerted at the time. When the rat withdrawals the paw, the experimenter releases the foot pedal to stop the force indicator and reads the scale. The Paw Withdrawal Threshold (PWT) was defined as the average of three measures alternately performed.
Persistent hyperalgesia model
The persistent hyperalgesia was induced as described by Ferreira et al., 1990. In this protocol, daily subcutaneous injections of PGE2 (100ng/50µL) into the rat’s hind paw for 14 days
(which is called “Induction period”) induces a hyperalgesia that persists for at least 30 days (period defined as “Maintenance period”).
Subcutaneous Injections
Concerning subcutaneous injections, drugs or their vehicle were subcutaneously injected in the dorsum of the rat paw with a 29G needle connected to an insulin syringe (BD ultra-fine 30U). PGE2 or its vehicle was injected in the hind paw and Capsaicin or its vehicle in the fore
paw. The total volume administered was 50µL.
Statistical analysis
To analyze if there were differences in the nociceptive response among the groups it was done a two-way repeated measures analysis of variance (ANOVA), followed by the Bonferroni multiple comparison test. Data are presented as mean ± SEM. The level accepted for statistical significance was p<0.05.
Results
Induction of persistent hyperalgesia on male and female rats
PGE2 (100ng/50µL) injection on the right hind paw of male (A) and female (B) rats for
14 consecutive days induced a paw-withdrawal threshold significantly lower than that induced by the saline injection (50µL) from the 6th to the 21st day of the experimental protocol (Fig.1).
Fig 1. Development and maintenance of the persistent hyperalgesia on male (A) and female rats (B). The symbol " * " indicates a paw-withdrawal threshold significantly lower than that induced by the saline injection. Data are presented as mean ± S.E.M. of 6 rats. Two-way repeated measures ANOVA, Bonferroni test, p<0.05.
Capsaicin-induced analgesia on male and female rats
The activation of the ANC by a Capsaicin injection (125ug/50µL) into the rat’s fore paw induced a hind paw-withdrawal threshold significantly higher than that induced by its vehicle injection for 60 minutes in males and female rats (Fig. 2). Therefore, the duration of capsaicin-induced analgesia was 60 minutes in both male and female rats.
Fig. 2. Capsaicin-induced analgesia on rats. The symbol " * " indicates that the subcutaneous injection of capsaicin (125ug) into the rat's fore paw induced a hind paw-withdrawal threshold
significantly higher than that induced by its vehicle injection for 60 minutes in males (A) and female (B) rats. (repeated measures two-way ANOVA, Bonferroni test, p<0.05). Data are presented as mean +- S.E.M. of 5 rats.
Capsaicin-induced analgesia on male and female rats with persistent hyperalgesia
Capsaicin injection (125ug/50µL) into the rat’s fore paw induced a hind paw-withdrawal threshold significantly higher than that induced by the saline injection for 30 minutes in male (A) and 15 minutes in female (B) rats. Therefore, the persistent hyperalgesia reduced the duration of capsaicin-induced analgesia from 60 minutes (naive rats) to 30 and 15 minutes in males and female rats, respectively. Capsaicin-induced analgesia was significantly greater in male than in female rats (Fig.3, C).
Fig. 3. Capsaicin-induced analgesia on rats with persistent hyperalgesia induced by PGE2. A) In male rats, the subcutaneous fore paw injection of capsaicin (125ug) significantly increased the hind paw-withdrawal threshold recorded immediately (0), 15, and 30 min after the capsaicin injection (indicated by the symbol “ * ”). B) In female rats, the capsaicin injection also significanlty increased the hind paw-withdrawal threshold at 0 and 15 minutes (*). C) Capsaicin-induced analgesia was significantly greater in male than in female rats nas indicated by the symbol “#” . There was no difference between the vehicle's groups. Data are presented as mean ± S.E.M. of 6 rats. Two-way repeated measures ANOVA; Bonferroni test, p<0.05. Abreviation: SAL: saline (0,9%); CAP: capsaicin; VEI: capsaicin vehicle.
Effect of Propranolol on Capsaicin-induced analgesia on male and female rats with persistent hyperalgesia
The blockade of β-adrenergic receptors by a Propranolol injection (9mg/kg, i.p.) 10 minutes before the Capsaicin injection in the right fore paw (125ug/50µL) restored the duration of capsaicin-induced analgesia during the persistent hyperalgesia to 60 minutes in male (Fig.4A) and 45 minutes in female rats (Fig. 4B).
Fig 4. Effect of Propranolol on Capsaicin-induced analgesia in rats with persistent hyperalgesia induced by PGE2. The symbol “#” indicates a PWT significantly higher than that of other experimental groups. When PROP was injected before CAP, the analgesia was sustained until 60 min on male rats (A) and 45 min on female rats (B) In both cases PROP by itself didn’t
significantly change the PWT. Data are presented as mean ± S.E.M. of 6 rats. Two-way repeated measures ANOVA; Bonferroni test, p<0.05. Abreviation: PROP: Propranolol (9mg/kg); SAL: saline (0,9% NaCl); CAP: capsaicin; VEI: capsaicin vehicle; PWT: Paw-withdrawal threshold.
Discussion
In this study we showed that systemic administration of Propranolol can restore the duration of the Capsaicin-induced analgesia, which is shortened on chronic pain syndromes, on male and female rats on the 7th day of maintenance of the persistent hyperalgesia. The analgesia induced by capsaicin on rats with persistent hyperalgesia lasted for 30 minutes on male rats and for 15 minutes on female rats (Fig. 2). The Capsaicin-induced analgesia is impaired in neuropathic rats (Ferrari et al., 2010; Kimura et al., 2015) and in persistent hyperalgesia rats (Miranda et al., 2015). It was shown that in male rats on the 7th day of maintenance of the persistent hyperalgesia the
Capsaicin-induced analgesia lasted for 30 minutes (Miranda et al., 2015), but this was the first time that the analgesia induced by ANC was tested on female rats with persistent hyperalgesia. We showed that the capsaicin-induced analgesia lasted more on male than on female rats. One explanation for the Capsaicin-induced analgesia have lasted longer on male rats may be on their higher concentration of testosterone, given that in a recent study the antinociceptive effect of a forepaw capsaicin injection was greater on male than on female rats, and gonadectomized male rats’ nociceptive response was equal to the female’s (Da Silva et al., 2018). However, it is soon to conclude this, since this study was performed with a much lower dose of capsaicin, which activates the mechanism of “Diffuse noxious inhibitory controls
”
.Another hypothesis is that the analgesia lasted longer on male rats due fact that male rodents exhibit a greater analgesia induced by opioids than female (see Kest et al., 2000, for a review), and the analgesia induced by ANC depends on opioids and dopamine (DA) release on
Nucleus Accumbens (NAc) (Gear et al., 1999). Although it is not known for sure why the analgesia
induced by opioids is greater on male than on female rodents, this effect doesn’t seem related to gonadal hormones (Stoffel et al., 2005).
Also, it must be considered that the analgesia induced by ANC depends on a cholinergic mechanism on Rostral Ventromedial Medulla (RVM) (Gear & Levine, 2009), which communicates with the midbrain Periaqueductal Gray (PAG), making the PAG-RVM circuit,
which is anatomically and functionally different in male and female rats (Loyd & Murphy, 2006). Although the PAG-RVM circuit is involved in the opioid analgesia, the Capsaicin-induced analgesia doesn’t depend on the release of opioids neither on PAG, neither on RVM (Gear & Levine, 1995), which means another system but the opioidergic should be involved on the analgesia induced by ANC on these brain parts.
The great novelty of this study is that the pre-administration of Propranolol increased the capsaicin-induced analgesia for 30 minutes, resulting in 60 minutes of analgesia on male rats and 45 minutes of analgesia on female rats (Fig.3). Many studies have shown the antinociceptive role of β-adrenoceptors antagonists in different pain animal models, such as temporomandibular joint hyperalgesia (Pelegrini-da-Silva et al., 2008; Zanelatto et al., 2017), visceral hypersensitivity (Zhang et al., 2014); in vitro (Kanno et al., 2010), and also in clinical trials (Light et al., 2009; Bunevicius et al., 2013; Härkänen et al., 2015). However, this was the first time that Propranolol was used to enhance the analgesia induced by ANC, which was diminished by persistent hyperalgesia induced by PGE2 on male and female rats.
It was shown that the duration of the analgesia induced by ANC was shortened in models of neuropathic (Ferrari et al., 2010; Kimura et al., 2015) and persistent pain (Miranda et
al., 2015), and that the removal of the adrenal medulla restored the normal duration (Ferrari et al.,
2010), pointing to the possible role of the sympathetic axis on this mechanism. It was shown in a study that previous injections of a DA antagonist or an opioid antagonist into the NAc blocks the antinociceptive effects of ANC activation, which means that the analgesia induced by ANC depends on the release of opioids and DA on NAc (Gear et al., 1999). The dopaminergic system on NAc was known as antinociceptive on acute nociceptive behavior (Chudler & Dong, 1995; Haghparast et al., 2012); this was demonstrated by a dopamine reuptake inhibitor injection in the NAc blocking PGE2 acute hyperalgesia (Dias et al., 2015). However, when the dopamine reuptake
inhibitor was injected in the NAc over 7 days of the PGE2-induced persistent hyperalgesia protocol,
the induction was facilitated (Dias et al., 2015). These results led the authors to suggest that, on PGE2-induced persistent hyperalgesia, the chronification of pain switches the dopaminergic system
on NAc from antinociceptive to pronociceptive. Finally, it was shown that the synergistic activation of NAc β1 and β2-adrenoceptors enhanced the NAc DA efflux, which was suppressed by Propranolol injection (Aono et al., 2013). It was hypothesized that these β-adrenoceptors are
located on the postsynaptic neurons of the dopaminergic terminals. Considering these studies, it may be that, on rats with persistent hyperalgesia induced by PGE, Propranolol restored the duration
of Capsaicin-induced analgesia (which is shortened on chronic pain syndromes) by inhibiting DA release on NAc, which it was shown to be pronociceptive on persistent hyperalgesia induced by PGE2.
Another possible mechanism for Propranolol enhancing the duration of the Capsaicin-induced analgesia on rats with persistent hyperalgesia may be on Propranolol acting on β2
-adrenoceptors co-expressed with a 6-transmembrane u-opioid receptor (MOR). The 6TM-MOR, when expressed alone, is retained in the intracellular compartments, but translocates to the plasma membrane when co-expressed with β2-adrenoceptors. This co-expression was
demonstrated at nervous ganglia. It was shown that the co-administration of β2-adrenoceptors
blockers and 6TM-MOR agonists leads to an analgesic synergy (Samoskhin et al., 2015). However, further studies are required for clarify the mechanisms underlying the effect of Propranolol on the analgesia induced by ANC on chronic pain models.
In summary, we provide evidence that the blockade of the β-adrenergic receptors can partially restore the duration of the analgesia induced by the ANC in a rat model of persistent hyperalgesia induced by PGE2. Therefore, the β-blockers may be used to treat chronic pain in association with treatments that activate the endogenous inhibitory pain systems.
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CONCLUSÕES
A hiperalgesia persistente reduz a duração da analgesia induzida pelo ANC em ratos e ratas, sendo que a redução foi maior nas ratas, e o bloqueio sistêmico dos β-adrenoceptores restaurou a duração da analgesia induzida pela ativação do ANC. O bloqueio sistêmico dos β-adrenoceptores não foi capaz, por si só, de gerar analgesia. Nesse contexto, os resultados desse trabalho sugerem que os β-bloqueadores poderiam aumentar a duração da analgesia dos tratamentos usados no controle da dor crônica que ativam os mecanismos endógenos de modulação da dor.
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ANEXOS A. Parecer do Comitê de Ética
