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Doctoral Thesis
DOI
https://doi.org/10.11606/T.17.2021.tde-07022022-180334
Document
Author
Full name
Willian Lazarini Lopes
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2021
Supervisor
Committee
Cairasco, Norberto Garcia (President)
Calcagnotto, Maria Elisa
Santos, José Eduardo Peixoto
Velasco, Tonicarlo Rodrigues
Title in Portuguese
Efeitos do tratamento com canabidiol (CBD) em modelos genéticos de epilepsias: estudos comportamentais, farmacológicos e eletrofisiológicos
Keywords in Portuguese
Canabidiol
Efeitos anticrises
Epilepsia
Modelos genéticos
Receptores CB1
Tratamento farmacológico
Abstract in Portuguese
Introdução: Epilepsias são desordens neurológicas caracterizadas pela presença de crises epilépticas e, embora o número de fármacos anticrises tenha aumentado significativamente nas últimas décadas, 1/3 dos pacientes ainda são farmacorresistentes. O canabidiol (CBD) vem recebendo grande destaque devido a seus efeitos anticrises em humanos e modelos pré-clínicos. Porém, o conhecimento sobre os efeitos do CBD em modelos genéticos de epilepsias ainda é bastante restrito. Portanto, o presente estudo teve como objetivo caracterizar os efeitos do tratamento com CBD em três modelos genéticos de epilepsias, as linhagens Wistar Audiogenic Rats (WAR), Genetically Epilepsy Prone Rats (GEPR-3s) e Wistar Albino Glaxo from Rijswijk (WAG/Rij). Métodos: WARs foram submetidos ao protocolo de crise audiogênicas (AGS) crônicas, o kindling audiogênico (KA) e receberam CBD (25 mg/kg/ml) 1 h antes de cada estímulo acústico. Imuno-histoquímica para FosB foi realizada em áreas associadas à expressão das AGS agudas e crônicas: colículo inferior, substância cinzenta periaquedutal, núcleo basolateral da amígdala (BLA) e córtex piriforme. Ademais, a expressão de receptores CB1 no BLA e no hipocampo dorsal também foi avaliada após as AGS. Em GEPR-3s e WAG/Rijs foi realizada uma curva dose-resposta e um estudo farmacocinético (dose-response-time) dos efeitos do CBD (1, 10, 50 e 100 mg/kg/ml). Para avaliar os efeitos do CBD nas AGS agudas (generalizadas tônico-clônicas) e crônicas (crises límbicas), GEPR-3s receberam CBD 2, 4 e 6 h antes de um estímulo acústico. Os efeitos do CBD na epilepsia de ausência foram avaliados em WAG/Rijs submetidos à 6 h de registro eletrográfico (EEG) contínuo imediatamente após a administração do CBD. Resultados: O tratamento crônico com CBD atenuou a gravidade das crises mesencefálicas, impediu o desenvolvimento de crises límbicas durante o KA em WARs e reduziu o número de neurônios FosB+ em todas as áreas avaliadas. Embora o KA tenha intensificado a imunomarcação de CB1 em áreas límbicas, o tratamento crônico com CBD atenuou tais alterações. Em GEPR-3s, CBD apresentou efeito dose-dependente: CBD 50 e 100 mg/kg atenuaram as crises generalizadas tônico-clônicas e reduziu a duração das crises. Em GEPR-3s com recrutamento límbico estabelecido, a dose de 10 mg/kg bloqueou a expressão das crises límbicas em 6/8 GEPR-3s. Em WAG/Rijs, CBD 1 e 100 mg/kg reduziram a duração das crises generalizadas ponta-onda (spike-wave discharges; SWDs), mas a frequência das crises não foi alterada. Tanto em GEPR-3s quanto em WAG/Rijs os efeitos anticrises mais potentes foram observados 2 h após a administração do CBD. Conclusão: O tratamento crônico com CBD teve efeitos anticrises e antiepileptogênicos em WARs, prevenindo o recrutamento límbico e atenuando a hiperatividade neuronal crônica derivada do KA. Os efeitos antiepileptogênicos do CBD foram associados às alterações neuroplásticas em receptores CB1 em áreas límbicas, sugerindo que o sistema endocanabinóide possa estar envolvido nestes efeitos. O tratamento agudo com CBD em GEPR-3s, atenuou as AGS agudas e as crises límbicas em animais com recrutamento límbico pré-estabelecido, sugerindo que o CBD também possa controlar crises associadas às redes neurais com alterações epileptogênicas já estabelecidas. O CBD reduziu a gravidades das SWDs em WAG/Rijs, demonstrando eficácia contra crises corticais generalizadas e sugerindo potencial efeito contra as epilepsias de ausência infantil. Portanto, a eficácia do CBD em diferentes modelos genéticos de epilepsias foi demonstrada por meio de análises comportamentais, histológicas, farmacológicas e eletrofisiológicas, sugerindo potenciais resultados translacionais.
Title in English
Cannabidiol (CBD) effects in genetic models of epilepsies: behavioral, pharmacological, and electrophysiological studies
Keywords in English
Antiseizure effects
Cannabidiol
CB1 receptors
Epilepsy
Genetic models
Pharmacological treatment
Abstract in English
Introduction: Epilepsies are neurological disorders characterized by the presence of epileptic seizures and, although the number of antiseizure medication has significantly increased during the last decades, 1/3 of patients are still pharmacoresistant. In that scenario, cannabidiol (CBD) has been receiving especial attention due to its antiseizure effects in humans and pre-clinical models, however, CBD effects in genetic models of epilepsies are very limited. Therefore, the present study aimed to characterize CBD effects in 3 genetic models of epilepsies: Wistar Audiogenic Rats (WAR), Genetically Epilepsy Prone Rats (GEPR-3s) e Wistar Albino Glaxo from Rijswijk (WAG/Rij). Methods: WARs were submitted to the audiogenic kindling (AK), a protocol of chronic audiogenic seizures (AGS) and CBD (25 mg/kg/ml) was intraperitoneally (ip.) administered 1 h before every AGS. To analyze chronic neuronal hyperactivity, immunocytochemistry for FosB was performed in brainstem (inferior colliculus and periaqueductal gray matter) and forebrain (amygdala and piriform cortex) structures associated with AGS expression. Also, CB1 receptor expression was measured in the amygdala and dorsal hippocampus after acute and chronic AGS. CBD dose-response curve (1, 10, 50, and 100 mg/kg/ml) and pharmacokinetic analysis (dose-response-time) were performed in GEPR-3s and WAG/Rijs. To assess CBD effects in acute (generalized tonic-clonic) and chronic AGS (limbic seizures) in GEPR-3s, CBD was ip. administered 2, 4, and 6 h before AGS test session. To analyze CBD effects in a genetic model of absence epilepsy, immediately after ip. CBD administration, WAG/Rijs were submitted to a 6 h recording session of bilateral cortical electroencephalography (EEG). Results: Chronic CBD administration during the AK in WARs attenuated brainstem seizure severity, prevented limbic seizure expression, and reduced the number of FosB+ neurons in all analyzed areas. Additionally, although the AK increased CB1 immunostaining in limbic brain areas, the chronic treatment with CBD attenuated the AK-induced changes in CB1 expression. In GEPR-3s submitted to the acute AGS protocol, CBD presented dose-dependent antiseizure effects, with CBD 50 and 100 mg/kg reducing tonic-clonic seizures severity and seizure duration. Differently, in GEPR-3s with limbic recruitment previously established (kindled GEPR-3s), CBD 10 mg/kg prevented limbic seizure expression in 6/8 animals. In WAG/Rijs, CBD 1 and 100 mg/kg reduced generalized spike-wave discharges (SWDs) mean duration, but no change in SWDs frequency was detected. In both GEPR-3s and WAG/Rijs, antiseizure effects were mostly observed 2 h after ip. CBD administration. Conclusion: Chronic CBD administration induced antiseizure and antiepileptogenic effects in WARs, prevented the limbic recruitment and attenuated chronic neuronal hyperactivity induced by the AK in brainstem and limbic areas. CBD antiepileptogenic effects were associated with neuroplastic changes in CB1 receptors in limbic brain areas, suggesting that the endocannabinoid system may be involved with these protective effects. Acute CBD administration in GEPR-3s attenuated generalized tonic-clonic seizures in the acute AGS protocol, as well as prevented limbic seizures in previously kindled GEPR-3s, suggesting that CBD can control seizures associated with neuronal networks with epileptogenic alterations previously established. Additionally, CBD was effective against absence epilepsy in WAG/Rijs, suggesting potential effects against childhood absence epilepsy. Therefore, the present study demonstrated CBD efficacy in different genetic models of epilepsies, using behavioral, histological, pharmacological, and electrophysiological analyses, indicating potential translational results.
 
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Publishing Date
2022-02-16
 
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