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Publication
Evaluating the effects of cannabinoids on an organoid-based model of prenatal exposure
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Authors
Abstract(s)
Nos últimos anos, a prevalência do uso de canabinóides tem aumentado rapidamente, inclusive em mulheres grávidas. Fitocanabinóides, substâncias extraídas da Cannabis sativa, atuam principalmente pela ligação aos recetores canabinóides 1 e 2 (CB1R e CB2R), exercendo efeitos psicoativos e não psicoativos. Os canabinóides sintéticos são uma classe de Novas Substâncias Psicoativas, e apresentam altas afinidades de ligação aos CB1/2R. Visto que o sistema endocanabinóide é altamente relevante para o desenvolvimento embrionário, temos como hipótese que o consumo de canabinóides durante a gravidez pode causar defeitos no neurodesenvolvimento. Assim, foi utilizado um modelo tridimensional que recapitula os passos iniciais do neurodesenvolvimento. Organoides corticais com 46 dias foram gerados e expostos aos fitocanabinóides Δ9-tetrahidrocanabinol (Δ9-THC) e canabidiol (CBD), e aos canabinóides sintéticos THJ-018 e EG-018 por 9 dias, mimetizando a exposição pré-natal aos mesmos. Estes foram também administrados na presença de um antagonista dos CB1R, para determinar se os seus efeitos são mediados por estes recetores. Análises de imunofluorescência, RNA e Western Blot foram realizadas para avaliar alterações em marcadores de diferenciação neuronal, e na expressão dos recetores canabinóides. Foi efetuado single-cell calcium imaging para avaliar a funcionalidade celular. Embora preliminares, estes resultados sugerem que o Δ9-THC pode retardar a diferenciação neuronal e afetar a funcionalidade celular por mecanismos mediados pelos CB1R. O CBD induziu mínimas alterações nestes parâmetros, mas evidenciou a interação complexa entre CB1R e CB2R. Os canabinóides sintéticos THJ-018 e EG-018 apresentaram um impacto mais negativo comparativamente aos fitocanabinóides. Estes induziram uma redução de progenitores neurais e neurónios maduros, e um aumento da apoptose, sugerindo efeitos neurotóxicos. Algumas destas alterações não foram revertidas pelo antagonista dos CB1R, indicando a interação com outros recetores. No entanto, os nossos resultados não obtiveram significância estatística, evidenciando a necessidade de validação adicional. Contudo, este projeto demonstra o potencial dos canabinóides para impactar o neurodesenvolvimento embrionário, evidenciando a importância da investigação científica sobre exposição pré-natal a canabinóides.
In the last years, the use prevalence of cannabinoids has been rapidly rising, including among pregnant women. Phytocannabinoids, substances extracted from Cannabis sativa, act mainly by binding to cannabinoid receptors 1 and 2 (CB1R and CB2R), exerting a range of psychoactive and nonpsychoactive effects. Synthetic cannabinoids are a class of New Psychoactive Substances, and also present high affinities to CB1/2R. Since the endocannabinoid system is highly relevant for embryonic development, we hypothesise that cannabinoids consumption during pregnancy can lead to neurodevelopmental defects. For this purpose, a three dimensional model, recapitulating the first steps of neurodevelopment, was used. 46 days-old dorsal forebrain organoids were generated and exposed to phytocannabinoids Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), and to synthetic cannabinoids THJ-018 and EG-018 for 9 days, to mimic prenatal exposure to these compounds. They were also administrated in the presence of a CB1R antagonist, to determine if the effects are CB1R-mediated. Immunofluorescence, RNA analysis and Western Blot were performed to evaluate alterations in neuronal differentiation markers, and in CB1/2R expression levels. Single-cell calcium imaging was performed to evaluate cell functionality. Although preliminary, the results suggest that Δ9-THC might delay neuronal differentiation and affect neuronal functionality by mechanisms mediated by CB1R. CBD induced minimal effects on these parameters, but highlighted the complex interaction between CB1R and CB2R. Synthetic cannabinoids THJ-018 and EG-018 had a more negative impact compared to phytocannabinoids. Their administration led to a reduction of neural progenitors and mature neurons, and increased apoptosis, suggesting neurotoxic effects. Some of these alterations were not reverted by the CB1R antagonist, indicating interation with other receptors. However, our results lack statistical significance, highlighting the need for further confirmation. Nonetheless, this project demonstrates the potential of cannabinoids to impact embryonic neurodevelopment, highlighting the importance of research on prenatal exposure to cannabinoids.
In the last years, the use prevalence of cannabinoids has been rapidly rising, including among pregnant women. Phytocannabinoids, substances extracted from Cannabis sativa, act mainly by binding to cannabinoid receptors 1 and 2 (CB1R and CB2R), exerting a range of psychoactive and nonpsychoactive effects. Synthetic cannabinoids are a class of New Psychoactive Substances, and also present high affinities to CB1/2R. Since the endocannabinoid system is highly relevant for embryonic development, we hypothesise that cannabinoids consumption during pregnancy can lead to neurodevelopmental defects. For this purpose, a three dimensional model, recapitulating the first steps of neurodevelopment, was used. 46 days-old dorsal forebrain organoids were generated and exposed to phytocannabinoids Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), and to synthetic cannabinoids THJ-018 and EG-018 for 9 days, to mimic prenatal exposure to these compounds. They were also administrated in the presence of a CB1R antagonist, to determine if the effects are CB1R-mediated. Immunofluorescence, RNA analysis and Western Blot were performed to evaluate alterations in neuronal differentiation markers, and in CB1/2R expression levels. Single-cell calcium imaging was performed to evaluate cell functionality. Although preliminary, the results suggest that Δ9-THC might delay neuronal differentiation and affect neuronal functionality by mechanisms mediated by CB1R. CBD induced minimal effects on these parameters, but highlighted the complex interaction between CB1R and CB2R. Synthetic cannabinoids THJ-018 and EG-018 had a more negative impact compared to phytocannabinoids. Their administration led to a reduction of neural progenitors and mature neurons, and increased apoptosis, suggesting neurotoxic effects. Some of these alterations were not reverted by the CB1R antagonist, indicating interation with other receptors. However, our results lack statistical significance, highlighting the need for further confirmation. Nonetheless, this project demonstrates the potential of cannabinoids to impact embryonic neurodevelopment, highlighting the importance of research on prenatal exposure to cannabinoids.
Description
Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz
Keywords
Fitocanabinóides Canabinóides sintéticos Recetores CB1 Organoides corticais Diferenciação neuronal