Papel dos recptores TRPV1 periféricos e centrais na hiperalgesia térmica orofacial em modelo de diabetes em ratos

Orientadora : Profª Drª Juliana Geremias ChichorroCoorientadora : Profª Drª Joice Maria da CunhaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa: Curitiba, 17/02/2017Inclui referências : f. 58-72Resumo: O diabetes mellitus é um grupo heterogêneo de distúrbios metabólicos que apresenta em comum a hiperglicemia, resultado de defeitos na ação ou secreção da insulina. A complicação mais prevalente do diabetes é a neuropatia diabética periférica que ocasiona alterações sensoriais manifestadas inicialmente por sintomas positivos, tais como dor e alodinia, os quais podem ser seguidos por sintomas negativos, tais como a hipoalgesia e hipoestesia. Existem evidências clínicas de que pacientes com diabetes apresentam maior incidência de dor orofacial. Em adição, em modelos animais de diabetes induzido pela administração única intraperitoneal de estreptozotocina (STZ), há a descrição do desenvolvimento de hiperalgesia facial ao calor. Os mecanismos relacionados a fisiopatologia desta alteração sensorial, no entanto, ainda não estão totalmente esclarecidos. Assim, o objetivo do presente estudo foi avaliar a contribuição de receptores de potencial transitório vanilóide tipo 1 (TRPV1), principal receptor envolvido na transdução de estímulos térmicos de calor, a nível periférico e central na hiperalgesia térmica em ratos Wistar machos com diabetes induzido por injeção intraperitoneal de STZ (60 mg/kg). Para isto, a hiperalgesia térmica facial foi avaliada antes e semanalmente até a quarta semana após a indução do diabetes. Em ratos diabéticos foi avaliado o efeito do tratamento diário com insulina bem como do tratamento com o antagonista de receptores TRPV1 capsazepina, quando administrado no lábio superior, no gânglio do trigêmeo (TG) ou no subnúcleo caudalis (Sp5C) do núcleo sensorial trigeminal na quarta semana após indução do diabetes. Em ratos naive, o efeito da capsazepina administrada nos três diferentes sítios descritos acima foi avaliado na hiperalgesia facial térmica induzida pela administração de capsaicina no lábio superior dos animais. Alterações na expressão de receptores TRPV1 periféricos (i.e. nervo infraorbital e TG) e centrais (i.e Sp5C) foram avaliadas em amostras coletadas quatro semanas após a indução do diabetes. Os resultados mostram que os ratos desenvolvem hiperalgesia facial ao calor desde a primeira até a quarta semana após a injeção de STZ, a qual foi prevenida pelo tratamento diário com insulina. A injeção de capsazepina no lábio superior, TG ou Sp5C resultou em redução significativa da hiperalgesia térmica facial tanto em ratos diabéticos quanto em ratos naive que receberam uma injeção prévia de capsaicina no lábio superior. Em conjunto, os resultados sugerem os receptores TRPV1 periféricos e centrais contribuem na hiperalgesia facial ao calor em ratos diabéticos e que a hiperalgesia orofacial ao calor está relacionada ao estado hiperglicêmico. Palavras-chave: diabetes, dor neuropática, receptor TRPV1, nervo trigêmeo, gânglio do trigêmeo, subnúcleo caudalis, capsaicina.Abstract: Diabetes mellitus represent an heterogeneous group of metabolic disturbs which has in common hyperglycemia, as a result of defects in insulin action or release. The most frequent diabetes complication is diabetic peripheral neuropathy, which leads to sensory alteraltions manifested by positive symptons, such as pain and allodynia, which may be followed by negative symptons, including hypoalgesia and hypoesthesia. There is clinical evidence that diabetic patients present increased incidence of orofacial pain. Additionally, in animal models of diabetes the administration of streptozotocin (STZ) induces facial heat hyperalgesia, but the mechanisms remain to be elucidated. Thus, the use of animal models to investigate peripheral and central mechanisms and changes in the trigeminal system after diabetes induction by STZ is clearly warrantedIn the present study, diabetes was induced in male Wistar rats by STZ (60 mg/kg, i.p) and facial heat hyperalgesia was assessed once a week up to four weeks. In diabetic rats, it was assessed the effect of daily insulin treatment and of capsazepine injection into the upper lip, trigeminal ganglion (TG) or subnucleus caudalis (Sp5C) on facial heat hyperalgesia. In naïve rats, the effect of capsazepine injected in the three different sites was assessed in heat hyperalgesia induced by upper lip injection of capsaicin. Changes in peripheral (i.e. infraorbital nerve and TG) and central (i.e Sp5C) TRPV1 expression were evaluated four weeks after diabetes induction. Diabetic rats exhibited facial heat hyperalgesia from the first up to the fourth week after STZ injection, which was prevented by insulin treatment. Capsazepine injection into the upper lip, trigeminal ganglion or medullary subarachnoid space resulted in significant reduction of facial heat hyperalgesia in diabetic rats, as well as capsaicin-treated naïve rats. Compared to naïve rats, diabetic rats exhibited a significant decrease in TRPV1 expression in the infraorbital nerve, but increased expression in the trigeminal ganglion. Conclusion: Our results suggest that peripheral and central TRPV1 receptors participate in facial heat hyperalgesia in diabetic rats, which is related to the hyperglycemic state. Keywords: diabetes, neuropathic pain, TRPV1 receptores, trigeminal nerve, trigeminal ganglion, subnucleus caudalis, capsaicin

Editorial: Women in science: pharmacological treatment of pain

Chronic pain; Depression; KetamineDolor crònic; Depressió; KetaminaDolor crónico; Depresión; KetaminaThis study showed that both ketamine and lidocaine infusion was able to reduce mean daily morphine consumption by 28% and 23%, respectively. This finding provided a useful therapeutic approach to improve analgesia and decrease opioid consumption, which may be extremely relevant, especially for patients in need of long-term opioid use.JGC is recipient of a research productivity fellowship from CNPq (National Council for Scientific and Technological Development, Brazil)

An LPAR5-antagonist that reduces nociception and increases pruriception

IntroductionThe G-protein coupled receptor LPAR5 plays a prominent role in LPA-mediated pain and itch signaling. In this study we focus on the LPAR5-antagonist compound 3 (cpd3) and its ability to affect pain and itch signaling, both in vitro and in vivo.MethodsNociceptive behavior in wild type mice was induced by formalin, carrageenan or prostaglandin E2 (PGE2) injection in the hind paw, and the effect of oral cpd3 administration was measured. Scratch activity was measured after oral administration of cpd3, in mice overexpressing phospholipase A2 (sPLA2tg), in wild type mice (WT) and in TRPA1-deficient mice (Trpa1 KO). In vitro effects of cpd3 were assessed by measuring intracellular calcium release in HMC-1 and HEK-TRPA1 cells.ResultsAs expected, nociceptive behavior (induced by formalin, carrageenan or PGE2) was reduced after treatment with cpd3. Unexpectedly, cpd3 induced scratch activity in mice. In vitro addition of cpd3 to HEK-TRPA1 cells induced an intracellular calcium wave that could be inhibited by the TRPA1-antagonist A-967079. In Trpa1 KO mice, however, the increase in scratch activity after cpd3 administration was not reduced.ConclusionsCpd3 has in vivo antinociceptive effects but induces scratch activity in mice, probably by activation of multiple pruriceptors, including TRPA1. These results urge screening of antinociceptive candidate drugs for activity with pruriceptors

Estudo de alterações nos componentes sensorial e afetivo da dor orofacial em diferentes modelos animais

Orientadora: Prof(a). Dr(a). Juliana Geremias ChichorroTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia. Defesa : Curitiba, 25/05/2021Inclui referências: p. 105-120Resumo: A dor crônica está associada a redução na qualidade de vida dos pacientes. Em particular, a dor orofacial persistente promove transtornos emocionais, tal como a ansiedade, prejudica as atividades essenciais do dia-a-dia, como, por exemplo comer e beber, e resulta em diminuição nas interações sociais. Nesse estudo investigamos, em modelos de dor orofacial que incluem a formalina, a dor pós-operatória por incisão da mucosa oral e a dor neuropática trigeminal por constrição do nervo infraorbital (CION), a presença de alterações sensoriais, dor tônica, comportamento do tipo ansioso e mudanças na emissão de vocalizações ultrassônicas (USV) com o intuito de fornecer ferramentas adicionais para o estudo do componente afetivo da dor. No modelo de formalina orofacial, os animais desenvolveram hiperalgesia ao calor por 3 dias, em comparação com animais do grupo controle. Em adição, a formalina induziu um aumento imediato das emissões de USV aversivas (i.e. 22 kHz), no entanto no terceiro dia após a administração houve redução das USV apetitivas (i.e. 50 kHz, do subtipo flat). Além disso, no terceiro dia após a injeção foi detectado comportamento do tipo ansioso através da redução do número de entradas e do tempo de permanência dos animais nos braços abertos no teste do labirinto em cruz elevado (LCE). No modelo de dor pós-operatória, os animais operados desenvolveram hiperalgesia mecânica por 5 dias e térmica por 3 dias. Em adição, no dia 3 após incisão da mucosa, foi detectado aumento do grooming facial espontâneo, bem como preferência condicionada ao lugar induzida pelo tratamento sistêmico com morfina em ratos operados em comparação com o grupo falso-operado. Neste mesmo período houve redução das USV de 50 kHz flat e os animais apresentaram comportamento do tipo ansioso. No modelo de dor neuropática trigeminal foi observado a presença de hiperalgesia mecânica, comportamento do tipo ansioso e redução das USV de 50 kHz flat no décimo quinto dia após a CION. Adicionalmente, em ambos os modelos cirúrgicos, a análise de RT-PCR, Western blot e imunohistoquímica mostrou uma redução na expressão da tirosina hidroxilase no nucleo accumbens (NAc) dos animais operados, em relação aos respectivos controles, o que pode indicar uma diminuição na atividade dopaminérgica mesolímbica. Finalmente, houve uma maior expressão de c-Fos em áreas cerebrais implicadas no processamento de dor (i.e. córtex pré-frontal, amígdala, NAc, substância cinzenta periaqueductal e subnúcleo caudal do trigêmeo), evidenciando alterações plásticas resultantes da dor pós-operatória e da dor neuropática trigeminal. Em conjunto, os resultados corroboram evidências recentes de que a análise de USV pode ser usada como uma medida adicional do componente afetivo da dor, e sugere que as alterações sensoriais e afetivas estão relacionadas à redução da atividade dopaminérgica mesolímbica. Também demonstram que a dor orofacial persistente pode causar plasticidade neuronal, a qual está relacionada com a expressão comportamental e afetiva da dor. Palavras-chave: Dor trigeminal. Vocalização ultrassônica. Ansiedade. Formalina orofacial. Dor tônica. Dor pós-operatória.Abstract: Chronic pain is associated with a reduction in patients' quality of life. In particular, persistent orofacial pain promotes emotional disorders, such as anxiety, impairs essential daily activities, such as eating and drinking, and results in decreased social interactions. In this study, we investigated, in models of orofacial pain that include formalin, postoperative pain due to incision of the oral mucosa and trigeminal neuropathic pain due to constriction of the infraorbital nerve (CION), the presence of sensory changes, tonic pain, anxiety-like behavior and changes in the emission of ultrasonic vocalization (USV) in order to provide additional methods for the study of the affective component of pain. In the orofacial formalin model, animals developed heat hyperalgesia for 3 days compared to the control group. In addition, formalin induced an immediate increase in the emissions of aversive calls (i.e. 22 kHz), however on the third day after administration, there was a reduction in the emission of appetitive calls (i.e. 50 kHz, flat subtype). Moreover, on day 3 after injection anxiety-like behavior was observed by the reduction in the number of entries and by the time animals spent in the open arms of the elevated plus maze (EPM). In the postoperative pain model, the operated animals developed mechanical hyperalgesia for 5 days and thermal hyperalgesia for 3 days. In addition, on day 3 after incision of the intraoral mucosa, there was a significant increase in spontaneous facial grooming and induction of conditioned preference to the place to systemic treatment with morphine in operated, but not sham rats. At this timepoint, anxiety-like behavior was observed, as well as a reduction in 50 kHz flat USV emissions. In the model of trigeminal neuropathic pain, the presence of mechanical hyperalgesia, anxiety-like behavior and a reduction of 50 kHz flat calls were detected on day 15 after CION. Additionally, in both surgical models, analysis of RT-PCR, Western blot and immunohistochemistry showed a reduction in the expression of tyrosine hydroxylase in the nucleus accumbens (NAc) of the operated animals in relation to their respective controls, which may indicate a decrease in mesolimbic dopaminergic activity. Finally, there was a greater expression of c-Fos in brain areas involved in pain processing (i.e. prefrontal cortex, amygdala, NAc, gray periaqueductal area and caudal trigeminal subnucleus), showing plastic changes as a consequence of postoperative pain and trigeminal neuropathic pain. Taken together, the results corroborate recent evidence that USV analysis can be used as an additional measure of the affective component of pain and suggests that sensory and affective changes are related to the reduction of mesolimbic dopaminergic activity. They also demonstrate that persistent orofacial pain can cause neuronal plasticity, which is related to the expression of behavioral and affective components of pain. Keywords: Trigeminal pain. Ultrasonic vocalization. Anxiety. Orofacial formalin. Tonic pain. Postoperative pain

Comparison of antinociceptive effects of plain lidocaine versus lidocaine complexed with hydroxypropyl-beta-cyclodextrin in animal models of acute and persistent orofacial pain

Herein, it was investigated whether a complex of lidocaine with 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) would present a better antinociceptive profile in vivo when compared with plain lidocaine in models of orofacial pain. Plain lidocaine (LDC) and complexed lidocaine (LDC:HP-beta-CD) were initially evaluated in vitro to determine the release rate of the two formulations. Subsequently, the effect of both formulations was evaluated in independent groups of rats submitted to the orofacial formalin test, induction of facial heat hyperalgesia by capsaicin and carrageenan, and induction of facial heat and mechanical hyperalgesia by constriction of the infraorbital nerve. LDC:HP-beta-CD led to a reduction in the lidocaine release assessed in the in vitro release assay compared to plain LDC. Both formulations presented an antinociceptive effect in all models, but LDC:HP-beta-CD showed a better effect in the second phase of the formalin response, in carrageenan-induced heat hyperalgesia, and in the heat hyperalgesia associated to infraorbital nerve constriction. Our results show that complexation improved in vivo antinociceptive effects of LDC, but further studies are necessary to elucidate what properties contribute to the better effect of the complexed formulation on this models and/or what characteristics of the pain model facilitate the action of the complexed formulation3925573583CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível Superior00

Diversity of cultivable fungi associated with Antarctic marine sponges and screening for their antimicrobial, antitumoral and antioxidant potential

The diversity of sponge-associated fungi has been poorly investigated in remote geographical areas like Antarctica. In this study, 101 phenotypically different fungal isolates were obtained from 11 sponge samples collected in King George Island, Antarctica. The analysis of ITS sequences revealed that they belong to the phylum Ascomycota. Sixty-five isolates belong to the genera Geomyces, Penicillium, Epicoccum, Pseudeurotium, Thelebolus, Cladosporium, Aspergillus, Aureobasidium, Phoma, and Trichocladium but 36 isolates could not be identified at genus level. In order to estimate the potential of these isolates as producers of interesting bioactivities, antimicrobial, antitumoral and antioxidant activities of fungal culture extracts were assayed. Around 51 % of the extracts, mainly from the genus Geomyces and non identified relatives, showed antimicrobial activity against some of the bacteria tested. On the other hand, around 42 % of the extracts showed potent antitumoral activity, Geomyces sp. having the best performance. Finally, the potential of the isolated fungi as producers of antioxidant activity seems to be moderate. Our results suggest that fungi associated with Antarctic sponges, particularly Geomyces, would be valuable sources of antimicrobial and antitumoral compounds. To our knowledge, this is the first report describing the biodiversity and the metabolic potential of fungi associated with Antarctic marine sponges.This work was supported by FONDECYT grant 11090192, Instituto Antártico Chileno (INACH) and “Programa Bicentenario de Ciencia y Tecnología” (Chile) project PDA13.Peer Reviewe