Efeito do tratamento com melatonina sobre o comportamento, expressão gênica e neuroquímica em um modelo animal de autismo
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2023-05-10
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Universidade Estadual Paulista (Unesp)
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A ativação imunológica do organismo materno na gestação pode acarretar alterações no desenvolvimento fetal. A endotoxina proveniente da parede de bactérias gram-negativas, o LPS, é capaz de desencadear a produção de citocinas, mimetizando um quadro de inflamação pré-natal ao ser administrado na gestação. Sabe-se que em quadros inflamatórios pode ocorrer a redução na produção de melatonina (MEL) pela glândula pineal, com consequente redução na circulação. A MEL apresenta funções sincronizadora, antioxidante e neuroprotetora. Em estudo anterior do nosso grupo foi demonstrado que fêmeas prenhas injetadas com LPS, cuja prole apresenta problemas no neurodesenvolvimento, tem redução no conteúdo circulante de MEL. Entre os mais diversos quadros que podem resultar de alterações no neurodesenvolvimento está o transtorno do espectro autista (TEA). Dependendo do grau de acometimento é comum encontrar nos indivíduos com TEA problemas comportamentais, atencionais, de aprendizado e memória. Várias alterações comportamentais e em processos cognitivos foram observadas neste modelo animal caracterizando-o como um modelo animal cuja exposição a um quadro inflamatório pré-natal resulta em uma prole com comportamentos autísticos. Nossa hipótese no presente estudo é de que as alterações na síntese e liberação da MEL no organismo materno no decorrer da gestação tem relação com as alterações de neurodesenvolvimento da prole. Para testar esta hipótese o objetivo geral deste estudo foi avaliar se o tratamento com MEL associado à infecção com LPS em fêmeas prenhas reverteria algumas das alterações comportamentais, neuroquímicas e moleculares já demonstradas na prole de fêmeas prenhes expostas ao LPS, causadas pelo quadro inflamatório pré-natal nesse modelo animal experimental. Para isso, primeiramente foram avaliados parâmetros como peso, ingesta hídrica e alimentar das fêmeas prenhes em resposta aos diferentes tratamentos. As proles de ratas expostas ao LPS com ou sem associação ao tratamento com MEL foram avaliadas quanto à memória espacial e comportamento inflexível por meio de teste comportamental de alternância espontânea no labirinto em T. Quanto à neuroquímica foi avaliada a expressão da proteína ligante de cálcio Calretinina em neurônios das diferentes regiões do hipocampo, por já ter sido demonstrada estar alterada neste modelo. Quanto a parte molecular, foi avaliado se administração de LPS durante a gestação pode alterar a expressão gênica de receptores de melatonina MT1 e MT2 e do fator neurotrófico derivado do cérebro BDNF no hipocampo da prole e se o tratamento com MEL em associação ao LPS durante o período gestacional reverteria estas alterações. Os resultados mostraram que no período pós-tratamento o grupo de ratas prenhez injetadas com LPS apresentou menor ganho de peso (27,4 ± 5,6) que o grupo salina (59,3 ± 4,1, p = 0,001) e menor consumo de água (salina 55,4 ± 5,9 vs. LPS 44,5 ± 7,7, p< 0,001). Não houve diferença na ingesta alimentar das fêmeas prenhes nem no número de filhotes das ninhadas entre os grupos, no peso dos filhotes e no peso do encéfalo dos filhotes. No teste comportamental o número total de erros na escolha do braço durante o teste do labirinto em T foi menor no grupo salina (1,7 ± 0,9) em relação ao grupo LPS (4,1 ± 0,9; p < 0,01). Os grupos melatonina (2,3 ± 0,8, p=0,02) e LPS + melatonina (2,3 ± 1,4, p=0,02) apresentaram menor quantidade de erros em relação ao grupo LPS (4,12 ± 0,99, p=0,01).Na análise da neuroquímica das regiões do hipocampo para as células imunorreativas a calretinina foi possível observar que na região do giro denteado (GD) houve diferença entre os grupos salina (34,3 ± 11,8) e LPS (58,3 ± 1,1; p= 0,02) com maior expressão no grupo LPS. Essa diferença não foi encontrada quando comparados os grupos salina (34,3 ± 11,8) e LPS+Mel (44,6 ± 6,1) (p= 0,45). Na análise da expressão de RNAm do MT1 no hipocampo, não foi encontrada diferença na prole dos diferentes grupos. Quanto ao MT2, a prole do grupo LPS apresentou menor valor do RNAm relativo (0,16 ± 0,12) que a do grupo salina (1,18 ± 0,7) (p< 0,05). O grupo LPS+MEL não apresentou diminuição na expressão dos receptores MT2 em relação ao grupo salina. Na análise da expressão de RNAm do fator neurotrófico derivado do cérebro (BDNF) não foi observado diferença estatística entre os grupos salina (1,74 ± 1,90) e LPS (1,28 ± 0,99) (p= 0,98). Os resultados permitem concluir que: (1) O quadro inflamatório gestacional em ratas prenhes levou a alterações no peso corporal e ingesta hídrica, sem alterar a ingesta de alimento desses animais e o tratamento com MEL reverteu estas alterações. (2) A prole das ratas prenhes com quadro inflamatório gestacional mostrou alteração no teste de memória espacial, aumento na imunorreatividade da calretinina e diminuição na expressão dos receptores MT2 em neurônios do hipocampo. (3) A prole das ratas prenhes com quadro inflamatório gestacional não mostrou alteração na expressão dos receptores MT2 ou do BDNF em neurônios do hipocampo. (4) A MEL quando ofertada previamente e após o desafio imunológico durante o período gestacional promoveu a reversão do efeito do LPS no desempenho no teste de memória espacial da prole e não permitiu a diminuição na expressão do receptor MT2 e o aumento da imunorreatividade da calretinina em neurônios no hipocampo.
The immune activation of the maternal organism throughout pregnancy can lead to changes in fetal development. The endotoxin from the wall of gram-negative bacteria, LPS, is capable of triggering the production of cytokines, thus mimicking a picture of prenatal inflammation when administered during pregnancy. It is known that in inflammatory conditions there may be a reduction in the production of melatonin by the pineal gland, with a consequent reduction in circulation. Melatonin has important synchronizing, antioxidant, and neuroprotective functions during neurodevelopment. In a previous study by our group, it was demonstrated that pregnant females injected with LPS, whose offspring have neurodevelopmental problems, have a reduction in the circulating melatonin content. Among the most diverse conditions that may result from alterations in neurodevelopment is the autism spectrum disorder (ASD). Depending on the degree of involvement, it is common to find behavioral, attentional, learning and memory problems in individuals with ASD. Several changes in behavior and cognitive processes were observed in this animal model, characterizing it as an animal model whose exposure to a prenatal inflammatory condition results in offspring with autistic behaviors. Our hypothesis in the present study is that changes in the synthesis and release of melatonin in the mother's body during pregnancy are related to changes in the neurodevelopment of the offspring. To test this hypothesis, the general objective of this study was to evaluate whether treatment with melatonin associated with LPS infection in pregnant females would reverse some of the behavioral, neurochemical, and molecular changes already demonstrated in the offspring of pregnant females exposed to LPS, caused by the pre-inflammatory condition. natal in this experimental animal model. For this, parameters such as weight, water and food intake of pregnant females in response to different treatments were first evaluated. The offspring of rats exposed to LPS with or without association with melatonin treatment were evaluated for spatial memory and inflexible behavior using a behavioral test of spontaneous alternation in the T-maze. As for the neurochemistry, the expression of the calcium-binding protein Calretinin was evaluated in neurons from different regions of the hippocampus, as it has already been demonstrated to be altered in this model. As for the molecular part, it was evaluated whether the administration of LPS during pregnancy can alter the gene expression of melatonin receptors MT1 and MT2 and of the brain-derived neurotrophic factor BDNF in the hippocampus of the offspring and whether the treatment with melatonin in association with LPS during pregnancy gestational period could reverse these changes. The results showed that in the post-treatment period, the group of pregnant rats injected with LPS showed less weight gain (27.4 ± 5.6) than the saline group (59.3 ± 4.1, p = 0.001) and less water consumption (saline 55.4 ± 5.9 vs. LPS 44.5 ± 7.7, p< 0.001). There was no difference in the food intake of the pregnant females, nor in the number of pups in the litters between the groups, in the weight of the pups and in the weight of the brain of the pups. In the behavioral test, the total number of errors in choosing the arm during the T-maze test was lower in the saline group (1.7 ± 0.9) compared to the LPS group (4.1 ± 0.9; p < 0 ,01). The melatonin (2.3 ± 0.8, p=0.02) and LPS + melatonin (2.3 ± 1.4, p=0.02) groups had fewer errors than the LPS group (4. 12 ± 0.99, p=0.01). In the analysis of the neurochemistry of the hippocampal regions for calretinin-immunoreactive cells, it was possible to observe that in the dentate gyrus (DG) region there was a difference between the saline (34.3 ± 11.8) and LPS (58.3 ± 1. 1; p= 0.02) with greater expression in the LPS group. This difference was not found when comparing saline (34.3 ± 11.8) and LPS+Vehicle (44.6 ± 6.1) groups (p= 0.45). In the analysis of MT1 mRNA expression in the hippocampus, no difference was found in the offspring of different groups. As for MT2, the offspring of the LPS group had a lower relative mRNA value (0.16 ± 0.12) than the saline group (1.18 ± 0.7) (p< 0.05).The LPS+MEL group did not show a decrease in the expression of MT2 receptors compared to the saline group. In the analysis of brainderived neurotrophic factor (BDNF) mRNA expression, no statistical difference was observed between saline (1.74 ± 1.90) and LPS (1.28 ± 0.99) groups (p= 0.98). The results allow us to conclude that: (1) The gestational inflammatory condition in pregnant rats led to changes in body weight and water intake, without changing the food intake of these animals and melatonina treatment reversed these changes. (2) The offspring of pregnant rats with gestational inflammation showed alterations in the spatial memory test, an increase in calretinin immunoreactivity and a decrease in the expression of MT2 receptors in hippocampal neurons. (3) The offspring of pregnant rats with gestational inflammation showed no alteration in the expression of MT2 receptors or BDNF in hippocampus neurons. (4) Melatonin, when offered before and after the immunological challenge during the gestational period, promoted the reversal of the effect of LPS on the performance in the spatial memory test of the offspring and did not allow the decrease in the expression of the MT2 receptor and the increase in the immunoreactivity of calretinin in neurons in the hippocampus.
The immune activation of the maternal organism throughout pregnancy can lead to changes in fetal development. The endotoxin from the wall of gram-negative bacteria, LPS, is capable of triggering the production of cytokines, thus mimicking a picture of prenatal inflammation when administered during pregnancy. It is known that in inflammatory conditions there may be a reduction in the production of melatonin by the pineal gland, with a consequent reduction in circulation. Melatonin has important synchronizing, antioxidant, and neuroprotective functions during neurodevelopment. In a previous study by our group, it was demonstrated that pregnant females injected with LPS, whose offspring have neurodevelopmental problems, have a reduction in the circulating melatonin content. Among the most diverse conditions that may result from alterations in neurodevelopment is the autism spectrum disorder (ASD). Depending on the degree of involvement, it is common to find behavioral, attentional, learning and memory problems in individuals with ASD. Several changes in behavior and cognitive processes were observed in this animal model, characterizing it as an animal model whose exposure to a prenatal inflammatory condition results in offspring with autistic behaviors. Our hypothesis in the present study is that changes in the synthesis and release of melatonin in the mother's body during pregnancy are related to changes in the neurodevelopment of the offspring. To test this hypothesis, the general objective of this study was to evaluate whether treatment with melatonin associated with LPS infection in pregnant females would reverse some of the behavioral, neurochemical, and molecular changes already demonstrated in the offspring of pregnant females exposed to LPS, caused by the pre-inflammatory condition. natal in this experimental animal model. For this, parameters such as weight, water and food intake of pregnant females in response to different treatments were first evaluated. The offspring of rats exposed to LPS with or without association with melatonin treatment were evaluated for spatial memory and inflexible behavior using a behavioral test of spontaneous alternation in the T-maze. As for the neurochemistry, the expression of the calcium-binding protein Calretinin was evaluated in neurons from different regions of the hippocampus, as it has already been demonstrated to be altered in this model. As for the molecular part, it was evaluated whether the administration of LPS during pregnancy can alter the gene expression of melatonin receptors MT1 and MT2 and of the brain-derived neurotrophic factor BDNF in the hippocampus of the offspring and whether the treatment with melatonin in association with LPS during pregnancy gestational period could reverse these changes. The results showed that in the post-treatment period, the group of pregnant rats injected with LPS showed less weight gain (27.4 ± 5.6) than the saline group (59.3 ± 4.1, p = 0.001) and less water consumption (saline 55.4 ± 5.9 vs. LPS 44.5 ± 7.7, p< 0.001). There was no difference in the food intake of the pregnant females, nor in the number of pups in the litters between the groups, in the weight of the pups and in the weight of the brain of the pups. In the behavioral test, the total number of errors in choosing the arm during the T-maze test was lower in the saline group (1.7 ± 0.9) compared to the LPS group (4.1 ± 0.9; p < 0 ,01). The melatonin (2.3 ± 0.8, p=0.02) and LPS + melatonin (2.3 ± 1.4, p=0.02) groups had fewer errors than the LPS group (4. 12 ± 0.99, p=0.01). In the analysis of the neurochemistry of the hippocampal regions for calretinin-immunoreactive cells, it was possible to observe that in the dentate gyrus (DG) region there was a difference between the saline (34.3 ± 11.8) and LPS (58.3 ± 1. 1; p= 0.02) with greater expression in the LPS group. This difference was not found when comparing saline (34.3 ± 11.8) and LPS+Vehicle (44.6 ± 6.1) groups (p= 0.45). In the analysis of MT1 mRNA expression in the hippocampus, no difference was found in the offspring of different groups. As for MT2, the offspring of the LPS group had a lower relative mRNA value (0.16 ± 0.12) than the saline group (1.18 ± 0.7) (p< 0.05).The LPS+MEL group did not show a decrease in the expression of MT2 receptors compared to the saline group. In the analysis of brainderived neurotrophic factor (BDNF) mRNA expression, no statistical difference was observed between saline (1.74 ± 1.90) and LPS (1.28 ± 0.99) groups (p= 0.98). The results allow us to conclude that: (1) The gestational inflammatory condition in pregnant rats led to changes in body weight and water intake, without changing the food intake of these animals and melatonina treatment reversed these changes. (2) The offspring of pregnant rats with gestational inflammation showed alterations in the spatial memory test, an increase in calretinin immunoreactivity and a decrease in the expression of MT2 receptors in hippocampal neurons. (3) The offspring of pregnant rats with gestational inflammation showed no alteration in the expression of MT2 receptors or BDNF in hippocampus neurons. (4) Melatonin, when offered before and after the immunological challenge during the gestational period, promoted the reversal of the effect of LPS on the performance in the spatial memory test of the offspring and did not allow the decrease in the expression of the MT2 receptor and the increase in the immunoreactivity of calretinin in neurons in the hippocampus.
Descrição
Palavras-chave
Autismo, Aprendizagem, Hipocampo, Neurodesenvolvimento, Neuroinflamação, Autism, Learning, Hippocampus, Neurodevelopment, Neuroinflammation
Como citar
SPILLA, Caio Sergio Galina. Efeito do tratamento com melatonina sobre o comportamento, expressão gênica e neuroquímica em um modelo animal de autismo. Universidade Estadual Paulista (Unesp), 2023.