Avaliação da teneurina-2 em astrócitos reativos no modelo experimental de epilepsia induzida com cloreto de lítio-cloridrato de pilocarpina em ratos adultos. Análises imunoistoquímica, histoquímica e de expressão gênica
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Data
2019-12-02
Autores
Tessarin, Gestter Willian Lattari [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As teneurinas (Tens) são proteínas transmembrana do tipo II, constituídas de quatro membros homólogos (Ten-1-4). Estas proteínas são expressas principalmente durante a neurogênese do sistema nervoso central (SNC) e estão envolvidas primariamente no estabelecimento dos circuitos neuronais. Tens apresentam vários sítios de clivagens intracelular e extracelular que resultam em peptídeos bioativos, destacando-se os peptídeos associados aos terminais carboxila das teneurinas (Teneurin C-terminal-Associated Peptides, TCAPs). As latrofilinas (LPHN1-3) representam receptores associados à proteína G, sendo os principais receptores endógenos das Tens. A interação da Ten-2 com a LPHN-1 resulta na modulação nos níveis de cálcio intracelular, fato este que pode estar desbalanceado durante episódios epileptogênicos. O principal propósito deste estudo foi verificar possíveis alterações na imunorreatividade e na expressão gênica da Ten-2 no SNC em um modelo de epilepsia induzida por cloreto de lítio-cloridrato de pilocarpina em ratos adultos. Adicionalmente, as expressões gênicas do TCAP-2 e LPHN1 também foram analisadas, visto que são as principais proteínas correlacionadas à Ten-2. Para isto, ratos adultos (Rattus norvegicus; n=49) foram submetidos a indução de status epilepticus (SE) com cloreto de lítio (127 mg/kg) e cloridrato de pilocarpina (40 mg/kg) e divididos em grupos controles, grupos 2, 5 e 14 dias após SE e grupos epilepsia crônica (35 e 75 dias). Amostras do SNC destes animais foram submetidos à técnica imunoistoquímica para identificação da imunorreatividade semelhante à Ten-2 (teneurin-2- like-immunoreactive, Ten-2-LI), assim como combinado com a técnica histoquímica com o corante Fluoro-Jade C para evidenciação de degeneração neuronal e analisados em microscopias de campo claro ou confocal à laser para mapeamento e avaliações semi-quantitativa e quantitativa. Amostras do RNA total da área somatossensorial primária e do hipocampo também foram avaliadas pela técnica da reação em cadeia da polimerase em tempo real (qPCR). Os dados quantitativos destas analises foram submetidos aos testes de normalidade e homocedasticidade, seguido da análise de variância simples (ANOVA) e pós-teste de Tukey, considerando p < 0,05 como significante. A análise imunoistoquímica revelou significante aumento na expressão da Ten-2-LI em perfis de astrócitos reativos na área somatossensorial primária, córtex piriforme, hipocampo, amígdala, tálamo, além de outras áreas telencefálicas e diencefálicas, principalmente nos grupos de 2 e 5 dias após SE. Os perfis de astrócitos reativos Ten-2-LI estavam presentes nas mesmas regiões encefálicas exibindo degeneração neuronal. Além disso, significante aumento nas expressões gênicas da Ten-2, TCAP-2 e LPHN1 também foram observadas na área somatossensorial primária e hipocampo, especialmente nos grupos de 2 e 5 dias após SE. O presente estudo demonstrou um significante aumento na Ten-2 e de suas proteínas correlatas no SNC de ratos adultos, após a indução do SE ou epilepsia crônica.
Teneurins (Tens) are a type II transmembrane protein family composed of four homologous members (Ten-1-4). These proteins are primarily present in the central nervous system (CNS) during neurogenesis and exert an important role in the development and establishment of neuronal circuits. Tens have several intra- and extracellular cleavage sites, originating bioactive peptides, such as the carboxyl-terminal peptides named Teneurin C-terminal-Associated Peptides (TCAPs). Latrophilins (LPHN1-3) represent G protein-coupled receptors and are considered the main endogenous receptors for Tens. The Ten-2-LPHN-1interaction results in intracellular calcium modulation in neurons and this system can be changed during epilepsy induction. The main purpose of this study was to verify possible alterations in immunoreactivity and gene expression of Ten-2 in the CNS from an adult rat model of lithium chloridepilocarpine-induced epilepsy. In addition, TCAP-2 and LHPN1 gene expressions were also analyzed, as they are the main Ten-2 related proteins. For this, adult male (Rattus norvegicus; n = 49) were submitted to status epilepticus (SE) induced by intraperitoneal administration of lithium chloride (127 mg/kg) and pilocarpine hydrochloride (40 mg/kg). Subsequently, the animals were divided into control groups, 2-, 5- and 14-day groups after SE, as well as chronic epilepsy group (35-75 days). Samples were submitted to immunohistochemistry technique to identify Teneurin-2-like immunoreactive (Ten-2-LI) cells and histochemical technique for evidence of neuronal degeneration using Fluoro-Jade C. The histologic sections were analyzed by light microscope or confocal fluorescence microscopy for neuroanatomic mapping, semiquantitative and quantitative evaluations. Additionally, total RNA was purified from samples collected from the primary somatosensory area and hippocampus and submitted to real time polymerase chain reaction (qPCR). The quantitative data from immunohistochemistry and gene expressions were evaluated for normal distribution and homoscedasticity, followed by analysis of variance and Tukey’s post-test, considering p < 0.05 as significant. Immunohistochemistry analysis showed a significant increase in Ten-2-LI reactive astrocyte profiles present mostly in the cerebral cortex, hippocampus, piriform cortex, thalamic nuclei and amygdala, besides other areas from telencephalon and diencephalon, mainly in groups 2 and 5 days after SE induction. Ten-2-LI reactive astrocyte profiles were collocated in brain regions where neuronal degeneration was evidenced by the Fluoro-Jade C staining method. The gene expression data for Ten-2, TCAP-2 and LPHN1 showed significant up-regulation in the primary somatosensory area and hippocampus, particularly in groups 2 and 5 days after SE induction. The present study demonstrated that SE or chronic epilepsy induced a significant increase in Ten-2 and its related proteins in the CNS of adult rats.
Teneurins (Tens) are a type II transmembrane protein family composed of four homologous members (Ten-1-4). These proteins are primarily present in the central nervous system (CNS) during neurogenesis and exert an important role in the development and establishment of neuronal circuits. Tens have several intra- and extracellular cleavage sites, originating bioactive peptides, such as the carboxyl-terminal peptides named Teneurin C-terminal-Associated Peptides (TCAPs). Latrophilins (LPHN1-3) represent G protein-coupled receptors and are considered the main endogenous receptors for Tens. The Ten-2-LPHN-1interaction results in intracellular calcium modulation in neurons and this system can be changed during epilepsy induction. The main purpose of this study was to verify possible alterations in immunoreactivity and gene expression of Ten-2 in the CNS from an adult rat model of lithium chloridepilocarpine-induced epilepsy. In addition, TCAP-2 and LHPN1 gene expressions were also analyzed, as they are the main Ten-2 related proteins. For this, adult male (Rattus norvegicus; n = 49) were submitted to status epilepticus (SE) induced by intraperitoneal administration of lithium chloride (127 mg/kg) and pilocarpine hydrochloride (40 mg/kg). Subsequently, the animals were divided into control groups, 2-, 5- and 14-day groups after SE, as well as chronic epilepsy group (35-75 days). Samples were submitted to immunohistochemistry technique to identify Teneurin-2-like immunoreactive (Ten-2-LI) cells and histochemical technique for evidence of neuronal degeneration using Fluoro-Jade C. The histologic sections were analyzed by light microscope or confocal fluorescence microscopy for neuroanatomic mapping, semiquantitative and quantitative evaluations. Additionally, total RNA was purified from samples collected from the primary somatosensory area and hippocampus and submitted to real time polymerase chain reaction (qPCR). The quantitative data from immunohistochemistry and gene expressions were evaluated for normal distribution and homoscedasticity, followed by analysis of variance and Tukey’s post-test, considering p < 0.05 as significant. Immunohistochemistry analysis showed a significant increase in Ten-2-LI reactive astrocyte profiles present mostly in the cerebral cortex, hippocampus, piriform cortex, thalamic nuclei and amygdala, besides other areas from telencephalon and diencephalon, mainly in groups 2 and 5 days after SE induction. Ten-2-LI reactive astrocyte profiles were collocated in brain regions where neuronal degeneration was evidenced by the Fluoro-Jade C staining method. The gene expression data for Ten-2, TCAP-2 and LPHN1 showed significant up-regulation in the primary somatosensory area and hippocampus, particularly in groups 2 and 5 days after SE induction. The present study demonstrated that SE or chronic epilepsy induced a significant increase in Ten-2 and its related proteins in the CNS of adult rats.
Descrição
Palavras-chave
Astrócitos reativos, Epilepsia, Cloridrato de Pilocarpina, Ratos adultos, Teneurina, Reactive astrocytes, Epilepsy, Pilocarpine, Adult rats, Teneurin