Comparative study on the physiological dynamics evoked by different profiles of lead exposure

Tese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2018Lead is a toxic metal which widespread use has resulted in environmental contamination and significant health problems. It is a cumulative toxicant that affects multiple body systems, including the cardiovascular, hematopoietic, reproductive, and renal systems. Lead is also a well-known neurotoxin, inducing changes in neurogenesis, neurodegeneration and changes on glial cells. These changes in the molecular and cellular processes lead to cognitive and behaviour alterations, particularly during developmental phases, persisting throughout the lifetime. Most of the studies that have been performed in both humans and animals were focused in a continuous chronic exposure to lead. This lead exposure causes behavioural changes, cognitive impairment and hypertension associated with sympathoexcitation, baroreceptor reflex hyposensitivity and increased chemoreceptor reflex sensitivity. But the effects of an intermittent lead exposure are scarse and standardized animal models are non-existent. This pattern of exposure has been increasing in the last years due to migrations, implementation of school exchange programs and/or residential changes. Therefore, the overall purpose of this work was to evaluate lead effects on mammal’s physiology along different profiles of lead exposure, including a new animal model of intermittent low-level lead exposure. Animal models of lead exposure were developed by replacing the tap water of seven-day pregnant Wistar females with 0.2% (p/v) solution of lead acetate. After being weaned at 21 days, rat pups, both sexes, were divided into 3 groups of lead exposure: long-term (exposure from foetal period until 28 weeks of age), short-term (exposure from foetal period until 12 weeks) and intermittent (exposure from foetal period until 12 weeks, lead-free period until 20 weeks and a second exposure between 20 and 28 weeks of age). At 12, 20 and 28 weeks of age, behavioural tests were performed for anxiety (Elevated Plus Maze Test), locomotor activity (Open Field Test), spatial working memory (Y-Maze) and episodic long-term memory (Novel Object Recognition test) assessment. Blood pressure (BP), electrocardiogram (ECG), heart rate (HR) and respiratory frequency (RF) were recorded at the same timepoints in the acute experiment. Baroreflex gain (BRG), chemoreflex sensitivity (ChS), cardiovascular variability were also evaluated. Immunohistochemistry studies for neuronal nuclear antigen (NeuN), Synaptophysin (Syn), ionized calcium binding adapter molecule-1 (Iba-1) and Glial fibrillary acidic protein (GFAP) stainings were performed in brain slices, and confocal imaging acquired and stainings quantified at dentate gyrus (DG) of the hippocampus. Blood lead levels were assessed by atomic absorption spectroscopy and metabolic evaluation of all groups was done using metabolic cages. A control group of Wistar rats without lead exposure, of both sexes, with the same number of individuals, underwent the same protocol and were evaluated in the same time points. Student T-test and one-way ANOVA with Tukey’s multiple comparison between means were used (significance p<0.05) for statistical analysis. Our data showed a clear association between lead exposure, hypertension and cardiorespiratory reflexes impairment, without heart rate changes, independently of the type of lead exposure profile. We also demonstrated for the first time that lead intermittent exposure causes adverse health effects, i.e, hypertension, sympathetic overactivity, increased chemoreflex sensitivity and baroreflex impairment, similar to a chronic exposure, however less pronounced. In fact, at 28 weeks, PbI group, the intermittent animal model of lead exposure developed, had a less severe hypertension when compared to the long-term exposure group (PbP), which might suggest that the duration of Pb exposure is more relevant than the time of exposure. Moreover, the effect on diastolic blood pressure produced by lead exposure was more evident than that of systolic blood pressure. Lead exposure from foetal period until 12 weeks of age causes long lasting hypertension and chemoreceptor reflex dysfunction even after a 16 weeks period without exposure. However, the clearance of lead promoted an improvement in baroreceptor reflex function, with repercussions on blood pressure values, since these values decreased, but did not reached the normotensive values. Regarding the autonomic data, in our study, the overactivity of the sympathetic nervous system, evaluated by the LF band, is concomitant with baroreceptor reflex impairment and/or hypertension. This means that the sympathetic nervous system may be involved in the modulation of the baroreceptor reflexes responses or in the hypertension development due to lead exposure. Concerning the effect of lead at behavioural level, all groups exposed to lead, evaluated in the three different time points, had behavioural changes, namely anxiety, hyperactivity and/or long-term memory impairment and molecular changes in the hippocampus region, more specifically, reactive astrogliosis and microgliosis were detected, indicating the presence of neuroinflammation. However, these alterations seem to reverse after lead abstinence for a certain period (single exposure) and are enhanced when a second exposure occurs (intermittent exposure), along with a synaptic loss. In summary, this study shows, that exposure to lead during the developmental phase can alter the normal course of development, with lifelong health consequences. Since all exposed Pb groups had the same route of exposure (i.e. exposure to lead by water) and the same dose and, despite the different time of exposure, all were exposed to lead since foetal period until adulthood, the most susceptible period to adverse health effects. Therefore, we can conclude that the different effects of lead toxicant between groups mainly depends on the total duration of lead exposure. This comparative study brings new insights on the environmental factors that influence nervous and cardiovascular systems during development, which can help creating public policy strategies to prevent and control the adverse effects of Pb toxicity.A identificação de agentes potencialmente tóxicos e a avaliação dos seus efeitos sobre o organismo humano constituem um tema importante de saúde pública. O chumbo encontra-se neste grupo de agentes, sendo bastante utilizado em todo o mundo, devido às suas propriedades únicas, como a alta maleabilidade, baixo ponto de fusão, suavidade, ductilidade e resistência à corrosão. O vasto uso deste metal pesado em indústrias, como a automóvel, cerâmica, de tintas e do plástico levou ao aumento da quantidade de chumbo livre no ambiente e a sua ocorrência nos sistemas biológicos, devido à sua natureza não biodegradável. A toxicidade do chumbo, como resultado da sua ingestão, inalação ou por contacto direto, mesmo em pequenas quantidades, pode evocar efeitos adversos irreversíveis em várias funções do corpo, afetando principalmente os sistemas cardiovascular (sendo uma das causas da hipertensão, promovendo aterosclerose, trombose, arteriosclerose e doenças cardiovasculares), hematopoiético, reprodutivo e renal. O chumbo é também uma neurotoxina já bem estudada, que induz alterações na neurogénese, nas células gliais e neurodegeneração. Estas alterações nos mecanismos celulares e moleculares, quando ocorrem durante as fases de desenvolvimento, provocam alterações cognitivas e comportamentais, que persistem durante toda a vida. Em termos de classificação, dois tipos de toxicidade de chumbo podem ser definidos: a toxicidade aguda, que geralmente ocorre pela exposição ocupacional a níveis elevados de chumbo, sendo esta bastante incomum, e a toxicidade crónica, uma exposição a níveis baixos de chumbo, mais comum no ambiente familiar. A maioria dos estudos realizados até à data em seres humanos e animais, focam-se na exposição crónica contínua e/ou permanente ao chumbo e nas consequências adversas na saúde deste tipo de exposição. Existem já, vários modelos animais descritos para a exposição contínua a níveis baixos de chumbo. No entanto, em determinadas situações, como nas migrações, nos programas de intercâmbio escolar e/ou nas mudanças residenciais, a exposição intermitente ao chumbo pode ocorrer, mas os estudos disponíveis em seres humanos são escassos e os modelos animais padronizados inexistentes para este tipo de exposição, que tem vindo a crescer exponencialmente nos últimos anos. Posto isto, o objetivo geral deste trabalho consistiu em avaliar os efeitos de diferentes perfis de exposição a níveis baixos de chumbo na fisiologia de ratos Wistar, incluindo o desenvolvimento de um novo modelo animal de exposição intermitente a chumbo. Os modelos animais de exposição ao chumbo foram desenvolvidos substituindo a água dos biberões das fêmeas Wistar grávidas de sete dias por uma solução de acetato de chumbo a 0,2% (p/v). Após os 21 dias de desmame, as crias, de ambos sexos, foram divididas em 3 grupos de exposição ao chumbo: de longo prazo (PbP - exposição do período fetal até às 28 semanas de idade), de curto prazo (Short-term PbS - exposição do período fetal até às 12 semanas, com abstinência ao chumbo até às 28 semanas) e intermitente (PbI - exposição do período fetal até 12 semanas, seguida por um período sem chumbo até 20 semanas e uma segunda exposição entre 20 e 28 semanas de idade). Em três diferentes pontos temporais (12, 20 e 28 semanas de idade), os diferentes grupos de animais foram sujeitos a testes comportamentais, para a avaliação dos níveis de ansiedade (EPM), da atividade locomotora (OFT), da memória espacial de trabalho (Y-Maze) e da memória episódica de longo prazo (NOR). Para avaliação dos parâmetros fisiológicos nos diferentes pontos temporais, os animais foram sujeitos a uma experiência aguda, onde foram registados os seguintes parâmetros: pressão arterial (PA), eletrocardiograma (ECG), frequência cardíaca (FC) e frequência respiratória (FR). Nesta experiência também se avaliaram os reflexos baro- e quimiorrecetores e obtiveram-se registos para a análise da variabilidade da FC e da PA sistólica. Após o término da experiência aguda, os animais foram sacrificados e os cérebros extraídos para estudos de imunohistoquímica em secções coronais, nas quais se analisou a morfologia das células e se quantificou a perda neuronal (neuronal nuclear antigen - NeuN), a astrogliose (Glial fibrillary acidic protein – GFAP) e a microgliose (ionized calcium binding adapter molecule-1 - Iba-1), bem como alterações na transmissão sináptica (Synaptophysin – Syn) no girus dentado do hipocampo. Os níveis de chumbo no sangue foram avaliados por espectroscopia de absorção atómica e a avaliação metabólica realizada através do uso de gaiolas metabólicas. Um grupo controlo de ratos Wistar sem exposição ao chumbo, de ambos os sexos e com o mesmo número de indivíduos, foi submetido ao mesmo protocolo e foi avaliado nos mesmos pontos temporais (12, 20 e 28 semanas de idade). Para a análise estatística foi utilizado o teste T de Student e a análise de Variância (ANOVA) unidirecional com o teste post-hoc de Tukey, considerando-se significativas diferenças com p < 0,05. Os resultados deste estudo mostram que, independentemente do tipo de perfil de exposição ao chumbo, existe uma associação clara entre exposição a chumbo, hipertensão e diminuição do ganho do barorreflexo, sem alterações de frequência cardíaca. Também demonstramos, pela primeira vez, que uma exposição intermitente a chumbo provoca efeitos adversos para a saúde, como hipertensão, hiperatividade simpática, aumento da sensibilidade quimiorreflexa e diminuição do ganho do barorreflexo, efeitos adversos semelhantes ao de uma exposição crónica permanente (PbP), porém menos pronunciada. De facto, às 28 semanas, o grupo PbI, o modelo animal intermitente de exposição ao chumbo desenvolvido, apresentou uma hipertensão menos grave em relação ao grupo de exposição de longo prazo (PbP), o que pode sugerir que a duração da exposição ao chumbo é mais relevante do que o tempo de exposição. Além disso, o efeito da exposição ao chumbo sobre a pressão arterial diastólica foi mais evidente do que sobre a pressão arterial sistólica. A exposição ao chumbo, desde o período fetal até as 12 semanas de idade, provoca hipertensão e disfunção quimiorreflexa duradoura, mesmo com um período de 16 semanas sem exposição. No entanto, a abstinência do chumbo promoveu uma melhoria na função barorreflexa, com repercussões nos valores da pressão arterial, uma vez que estes valores diminuíram, apesar de não atingirem os valores de normotensão. Em relação à avaliação autonómica, os dados indicam que quando existe um aumento do tónus simpático, avaliado pela banda LF, este é concomitante com disfunção barorreflexa e/ou hipertensão arterial. Isso significa que o sistema nervoso simpático deve estar envolvido na modulação da resposta barorreflexa ou no desenvolvimento da hipertensão decorrente da exposição ao chumbo. Relativamente ao efeito do chumbo a nível comportamental, todos os grupos expostos ao chumbo, avaliados nos diferentes pontos temporais, apresentaram alterações comportamentais, nomeadamente ansiedade, hiperatividade e / ou défices de memória a longo prazo, bem como alterações moleculares, mais especificamente, astrogliose e microgliose reativa, que indicam a presença de neuroinflamação. No entanto, estas alterações parecem reverter após a abstinência do chumbo durante um determinado período (PbS - exposição de curta duração), sendo mais evidentes quando ocorre uma segunda exposição a chumbo (PbI - exposição intermitente), levando mesmo a perda sináptica mais pronunciada. Em resumo, este estudo mostra, que exposições a chumbo durante as fases de desenvolvimento podem alterar o seu curso normal, com consequências adversas para a saúde que podem persistir para toda a vida. Uma vez que todos os grupos expostos a chumbo tiveram a mesma via de exposição (isto é, exposição ao chumbo através da água) e a mesma dose e, apesar do tempo de exposição diferente, todos foram expostos ao chumbo desde o período fetal até a idade adulta, período em que são mais suscetíveis a efeitos adversos na saúde. Portanto, podemos concluir que os diferentes efeitos tóxicos do chumbo entre os grupos dependem principalmente da duração total da exposição ao chumbo. As novas evidências obtidas por este estudo comparativo permitem-nos contribuir para o esclarecimento sobre os fatores ambientais que influenciam os sistemas nervoso e cardiovascular durante o desenvolvimento, o que pode ajudar a criar estratégias de políticas públicas para prevenir e controlar os efeitos adversos da toxicidade do chumbo

The Neuroprotective Action of Amidated-Kyotorphin on Amyloid β Peptide-Induced Alzheimer’s Disease Pathophysiology

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous dipeptide initially described to have analgesic properties. Recently, KTP was suggested to be an endogenous neuroprotective agent, namely for Alzheimer’s disease (AD). In fact, KTP levels were shown to be decreased in the cerebrospinal fluid of patients with AD, and recent data showed that intracerebroventricular (i.c.v.) injection of KTP ameliorates memory impairments in a sporadic rat model of AD. However, this administration route is far from being a suitable therapeutic strategy. Here, we evaluated if the blood-brain permeant KTP-derivative, KTP-NH2, when systemically administered, would be effective in preventing memory deficits in a sporadic AD animal model and if so, which would be the synaptic correlates of that action. The sporadic AD model was induced in male Wistar rats through i.c.v. injection of amyloid β peptide (Aβ). Animals were treated for 20 days with KTP-NH2 (32.3 mg/kg, intraperitoneally (i.p.), starting at day 3 after Aβ administration) before memory testing (Novel object recognition (NOR) and Y-maze (YM) tests). Animals were then sacrificed, and markers for gliosis were assessed by immunohistochemistry and Western blot analysis. Synaptic correlates were assessed by evaluating theta-burst induced long term potentiation (LTP) of field excitatory synaptic potentials (fEPSPs) recorded from hippocampal slices and cortical spine density analysis. In the absence of KTP-NH2 treatment, Aβ-injected rats had clear memory deficits, as assessed through NOR or YM tests. Importantly, these memory deficits were absent in Aβ-injected rats that had been treated with KTP-NH2, which scored in memory tests as control (sham i.c.v. injected) rats. No signs of gliosis could be detected at the end of the treatment in any group of animals. LTP magnitude was significantly impaired in hippocampal slices that had been incubated with Aβ oligomers (200 nM) in the absence of KTP-NH2. Co-incubation with KTP-NH2 (50 nM) rescued LTP toward control values. Similarly, Aβ caused a significant decrease in spine density in cortical neuronal cultures, and this was prevented by co-incubation with KTP-NH2 (50 nM). In conclusion, the present data demonstrate that i.p. KTP-NH2 treatment counteracts Aβ-induced memory impairments in an AD sporadic model, possibly through the rescuing of synaptic plasticity mechanisms.publishersversionpublishe

Comparative study on the physiological dynamics evoked by different profiles of lead exposure

Tese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2018Lead is a toxic metal which widespread use has resulted in environmental contamination and significant health problems. It is a cumulative toxicant that affects multiple body systems, including the cardiovascular, hematopoietic, reproductive, and renal systems. Lead is also a well-known neurotoxin, inducing changes in neurogenesis, neurodegeneration and changes on glial cells. These changes in the molecular and cellular processes lead to cognitive and behaviour alterations, particularly during developmental phases, persisting throughout the lifetime. Most of the studies that have been performed in both humans and animals were focused in a continuous chronic exposure to lead. This lead exposure causes behavioural changes, cognitive impairment and hypertension associated with sympathoexcitation, baroreceptor reflex hyposensitivity and increased chemoreceptor reflex sensitivity. But the effects of an intermittent lead exposure are scarse and standardized animal models are non-existent. This pattern of exposure has been increasing in the last years due to migrations, implementation of school exchange programs and/or residential changes. Therefore, the overall purpose of this work was to evaluate lead effects on mammal’s physiology along different profiles of lead exposure, including a new animal model of intermittent low-level lead exposure. Animal models of lead exposure were developed by replacing the tap water of seven-day pregnant Wistar females with 0.2% (p/v) solution of lead acetate. After being weaned at 21 days, rat pups, both sexes, were divided into 3 groups of lead exposure: long-term (exposure from foetal period until 28 weeks of age), short-term (exposure from foetal period until 12 weeks) and intermittent (exposure from foetal period until 12 weeks, lead-free period until 20 weeks and a second exposure between 20 and 28 weeks of age). At 12, 20 and 28 weeks of age, behavioural tests were performed for anxiety (Elevated Plus Maze Test), locomotor activity (Open Field Test), spatial working memory (Y-Maze) and episodic long-term memory (Novel Object Recognition test) assessment. Blood pressure (BP), electrocardiogram (ECG), heart rate (HR) and respiratory frequency (RF) were recorded at the same timepoints in the acute experiment. Baroreflex gain (BRG), chemoreflex sensitivity (ChS), cardiovascular variability were also evaluated. Immunohistochemistry studies for neuronal nuclear antigen (NeuN), Synaptophysin (Syn), ionized calcium binding adapter molecule-1 (Iba-1) and Glial fibrillary acidic protein (GFAP) stainings were performed in brain slices, and confocal imaging acquired and stainings quantified at dentate gyrus (DG) of the hippocampus. Blood lead levels were assessed by atomic absorption spectroscopy and metabolic evaluation of all groups was done using metabolic cages. A control group of Wistar rats without lead exposure, of both sexes, with the same number of individuals, underwent the same protocol and were evaluated in the same time points. Student T-test and one-way ANOVA with Tukey’s multiple comparison between means were used (significance p<0.05) for statistical analysis. Our data showed a clear association between lead exposure, hypertension and cardiorespiratory reflexes impairment, without heart rate changes, independently of the type of lead exposure profile. We also demonstrated for the first time that lead intermittent exposure causes adverse health effects, i.e, hypertension, sympathetic overactivity, increased chemoreflex sensitivity and baroreflex impairment, similar to a chronic exposure, however less pronounced. In fact, at 28 weeks, PbI group, the intermittent animal model of lead exposure developed, had a less severe hypertension when compared to the long-term exposure group (PbP), which might suggest that the duration of Pb exposure is more relevant than the time of exposure. Moreover, the effect on diastolic blood pressure produced by lead exposure was more evident than that of systolic blood pressure. Lead exposure from foetal period until 12 weeks of age causes long lasting hypertension and chemoreceptor reflex dysfunction even after a 16 weeks period without exposure. However, the clearance of lead promoted an improvement in baroreceptor reflex function, with repercussions on blood pressure values, since these values decreased, but did not reached the normotensive values. Regarding the autonomic data, in our study, the overactivity of the sympathetic nervous system, evaluated by the LF band, is concomitant with baroreceptor reflex impairment and/or hypertension. This means that the sympathetic nervous system may be involved in the modulation of the baroreceptor reflexes responses or in the hypertension development due to lead exposure. Concerning the effect of lead at behavioural level, all groups exposed to lead, evaluated in the three different time points, had behavioural changes, namely anxiety, hyperactivity and/or long-term memory impairment and molecular changes in the hippocampus region, more specifically, reactive astrogliosis and microgliosis were detected, indicating the presence of neuroinflammation. However, these alterations seem to reverse after lead abstinence for a certain period (single exposure) and are enhanced when a second exposure occurs (intermittent exposure), along with a synaptic loss. In summary, this study shows, that exposure to lead during the developmental phase can alter the normal course of development, with lifelong health consequences. Since all exposed Pb groups had the same route of exposure (i.e. exposure to lead by water) and the same dose and, despite the different time of exposure, all were exposed to lead since foetal period until adulthood, the most susceptible period to adverse health effects. Therefore, we can conclude that the different effects of lead toxicant between groups mainly depends on the total duration of lead exposure. This comparative study brings new insights on the environmental factors that influence nervous and cardiovascular systems during development, which can help creating public policy strategies to prevent and control the adverse effects of Pb toxicity.A identificação de agentes potencialmente tóxicos e a avaliação dos seus efeitos sobre o organismo humano constituem um tema importante de saúde pública. O chumbo encontra-se neste grupo de agentes, sendo bastante utilizado em todo o mundo, devido às suas propriedades únicas, como a alta maleabilidade, baixo ponto de fusão, suavidade, ductilidade e resistência à corrosão. O vasto uso deste metal pesado em indústrias, como a automóvel, cerâmica, de tintas e do plástico levou ao aumento da quantidade de chumbo livre no ambiente e a sua ocorrência nos sistemas biológicos, devido à sua natureza não biodegradável. A toxicidade do chumbo, como resultado da sua ingestão, inalação ou por contacto direto, mesmo em pequenas quantidades, pode evocar efeitos adversos irreversíveis em várias funções do corpo, afetando principalmente os sistemas cardiovascular (sendo uma das causas da hipertensão, promovendo aterosclerose, trombose, arteriosclerose e doenças cardiovasculares), hematopoiético, reprodutivo e renal. O chumbo é também uma neurotoxina já bem estudada, que induz alterações na neurogénese, nas células gliais e neurodegeneração. Estas alterações nos mecanismos celulares e moleculares, quando ocorrem durante as fases de desenvolvimento, provocam alterações cognitivas e comportamentais, que persistem durante toda a vida. Em termos de classificação, dois tipos de toxicidade de chumbo podem ser definidos: a toxicidade aguda, que geralmente ocorre pela exposição ocupacional a níveis elevados de chumbo, sendo esta bastante incomum, e a toxicidade crónica, uma exposição a níveis baixos de chumbo, mais comum no ambiente familiar. A maioria dos estudos realizados até à data em seres humanos e animais, focam-se na exposição crónica contínua e/ou permanente ao chumbo e nas consequências adversas na saúde deste tipo de exposição. Existem já, vários modelos animais descritos para a exposição contínua a níveis baixos de chumbo. No entanto, em determinadas situações, como nas migrações, nos programas de intercâmbio escolar e/ou nas mudanças residenciais, a exposição intermitente ao chumbo pode ocorrer, mas os estudos disponíveis em seres humanos são escassos e os modelos animais padronizados inexistentes para este tipo de exposição, que tem vindo a crescer exponencialmente nos últimos anos. Posto isto, o objetivo geral deste trabalho consistiu em avaliar os efeitos de diferentes perfis de exposição a níveis baixos de chumbo na fisiologia de ratos Wistar, incluindo o desenvolvimento de um novo modelo animal de exposição intermitente a chumbo. Os modelos animais de exposição ao chumbo foram desenvolvidos substituindo a água dos biberões das fêmeas Wistar grávidas de sete dias por uma solução de acetato de chumbo a 0,2% (p/v). Após os 21 dias de desmame, as crias, de ambos sexos, foram divididas em 3 grupos de exposição ao chumbo: de longo prazo (PbP - exposição do período fetal até às 28 semanas de idade), de curto prazo (Short-term PbS - exposição do período fetal até às 12 semanas, com abstinência ao chumbo até às 28 semanas) e intermitente (PbI - exposição do período fetal até 12 semanas, seguida por um período sem chumbo até 20 semanas e uma segunda exposição entre 20 e 28 semanas de idade). Em três diferentes pontos temporais (12, 20 e 28 semanas de idade), os diferentes grupos de animais foram sujeitos a testes comportamentais, para a avaliação dos níveis de ansiedade (EPM), da atividade locomotora (OFT), da memória espacial de trabalho (Y-Maze) e da memória episódica de longo prazo (NOR). Para avaliação dos parâmetros fisiológicos nos diferentes pontos temporais, os animais foram sujeitos a uma experiência aguda, onde foram registados os seguintes parâmetros: pressão arterial (PA), eletrocardiograma (ECG), frequência cardíaca (FC) e frequência respiratória (FR). Nesta experiência também se avaliaram os reflexos baro- e quimiorrecetores e obtiveram-se registos para a análise da variabilidade da FC e da PA sistólica. Após o término da experiência aguda, os animais foram sacrificados e os cérebros extraídos para estudos de imunohistoquímica em secções coronais, nas quais se analisou a morfologia das células e se quantificou a perda neuronal (neuronal nuclear antigen - NeuN), a astrogliose (Glial fibrillary acidic protein – GFAP) e a microgliose (ionized calcium binding adapter molecule-1 - Iba-1), bem como alterações na transmissão sináptica (Synaptophysin – Syn) no girus dentado do hipocampo. Os níveis de chumbo no sangue foram avaliados por espectroscopia de absorção atómica e a avaliação metabólica realizada através do uso de gaiolas metabólicas. Um grupo controlo de ratos Wistar sem exposição ao chumbo, de ambos os sexos e com o mesmo número de indivíduos, foi submetido ao mesmo protocolo e foi avaliado nos mesmos pontos temporais (12, 20 e 28 semanas de idade). Para a análise estatística foi utilizado o teste T de Student e a análise de Variância (ANOVA) unidirecional com o teste post-hoc de Tukey, considerando-se significativas diferenças com p < 0,05. Os resultados deste estudo mostram que, independentemente do tipo de perfil de exposição ao chumbo, existe uma associação clara entre exposição a chumbo, hipertensão e diminuição do ganho do barorreflexo, sem alterações de frequência cardíaca. Também demonstramos, pela primeira vez, que uma exposição intermitente a chumbo provoca efeitos adversos para a saúde, como hipertensão, hiperatividade simpática, aumento da sensibilidade quimiorreflexa e diminuição do ganho do barorreflexo, efeitos adversos semelhantes ao de uma exposição crónica permanente (PbP), porém menos pronunciada. De facto, às 28 semanas, o grupo PbI, o modelo animal intermitente de exposição ao chumbo desenvolvido, apresentou uma hipertensão menos grave em relação ao grupo de exposição de longo prazo (PbP), o que pode sugerir que a duração da exposição ao chumbo é mais relevante do que o tempo de exposição. Além disso, o efeito da exposição ao chumbo sobre a pressão arterial diastólica foi mais evidente do que sobre a pressão arterial sistólica. A exposição ao chumbo, desde o período fetal até as 12 semanas de idade, provoca hipertensão e disfunção quimiorreflexa duradoura, mesmo com um período de 16 semanas sem exposição. No entanto, a abstinência do chumbo promoveu uma melhoria na função barorreflexa, com repercussões nos valores da pressão arterial, uma vez que estes valores diminuíram, apesar de não atingirem os valores de normotensão. Em relação à avaliação autonómica, os dados indicam que quando existe um aumento do tónus simpático, avaliado pela banda LF, este é concomitante com disfunção barorreflexa e/ou hipertensão arterial. Isso significa que o sistema nervoso simpático deve estar envolvido na modulação da resposta barorreflexa ou no desenvolvimento da hipertensão decorrente da exposição ao chumbo. Relativamente ao efeito do chumbo a nível comportamental, todos os grupos expostos ao chumbo, avaliados nos diferentes pontos temporais, apresentaram alterações comportamentais, nomeadamente ansiedade, hiperatividade e / ou défices de memória a longo prazo, bem como alterações moleculares, mais especificamente, astrogliose e microgliose reativa, que indicam a presença de neuroinflamação. No entanto, estas alterações parecem reverter após a abstinência do chumbo durante um determinado período (PbS - exposição de curta duração), sendo mais evidentes quando ocorre uma segunda exposição a chumbo (PbI - exposição intermitente), levando mesmo a perda sináptica mais pronunciada. Em resumo, este estudo mostra, que exposições a chumbo durante as fases de desenvolvimento podem alterar o seu curso normal, com consequências adversas para a saúde que podem persistir para toda a vida. Uma vez que todos os grupos expostos a chumbo tiveram a mesma via de exposição (isto é, exposição ao chumbo através da água) e a mesma dose e, apesar do tempo de exposição diferente, todos foram expostos ao chumbo desde o período fetal até a idade adulta, período em que são mais suscetíveis a efeitos adversos na saúde. Portanto, podemos concluir que os diferentes efeitos tóxicos do chumbo entre os grupos dependem principalmente da duração total da exposição ao chumbo. As novas evidências obtidas por este estudo comparativo permitem-nos contribuir para o esclarecimento sobre os fatores ambientais que influenciam os sistemas nervoso e cardiovascular durante o desenvolvimento, o que pode ajudar a criar estratégias de políticas públicas para prevenir e controlar os efeitos adversos da toxicidade do chumbo

Autonomic Function Evaluation in an Intermittent Lead Exposure Animal Model

Lead (Pb) is a toxic metal, which widespread use has resulted in environmental contamination, human exposure and significant public health problems. The autonomic nervous system, being a homeostatic controller, is impaired in acute and chronic lead exposure. In fact, sympathoexcitation associated to hypertension and tachypnea has been described together with baroreflex and chemoreflex dysfunction. However, up to date, no studies described the autonomic effects of an intermittent low-level lead exposure. In the present work, we addressed in vivo, autonomic behaviour in rats under chronic Pb exposure (control) and in rats under intermittent Pb exposure. For that, arterial blood pressure (BP) and ECG were recorded in 28 weeks old animal and low frequencies (LF) and high frequencies (HF) were determined (to estimate sympathetic and parasympathetic activities) using FisioSinal software with Wavelet module. Preliminary results: Rats intermittently exposed to lead showed a significant decrease in systolic BP (126 ± 4 vs 144 ± 3 mmHg) with no significant changes in LF, HF and LF/HF bands (1.5 ± 0.3 vs 1.7 ± 0.5 mmHg2, 1.9 ± 0.7 vs 2.8 ± 1.2 bpm2 and 1.2 ± 0.4 vs 1.1 ± 0.3 mmHg2/bpm2, respectively) when compared to chronically Pb exposed rats. Our data suggests that the autonomic dysfunction induced by lead exposure is similar in a chronic and intermittent Pb exposure. Nevertheless, it seems that an intermittent exposure was no effect on systolic BP values.The present study brings new insights on the environmental factors that influence autonomic and cardiovascular systems during development, which can help apprise public policy strategies to prevent and control the adverse effects of Pb toxicity

Intermittent low-level lead exposure causes anxiety and cardiorespiratory impairment

Copyright © 2019 Ovid Technologies, Inc., and its partners and affiliates. All Rights Reserved.Aim: To characterize behavioural and cardiorespiratory changes in a new, intermittent low-level lead exposure animal model. Introduction: Lead (Pb) is a cumulative toxic metal affecting all body systems that are particularly vulnerable during developmental phase. Permanent lead exposure has been defined as a cause of behavioural changes, cognitive impairment, sympathoexcitation, tachycardia, hypertension and autonomic dysfunction. However, no studies have been performed to describe a new, intermittent low-level lead exposure profile, that has been increased in the past years. Methods: Foetuses were intermittently (PbI) exposed to water containing lead acetate (0.2%, w/v) throughout life until adulthood (28 weeks of age). A control group (without exposure, CTL), matching in age and sex was used. At 26 weeks, behavioural tests were performed for anxiety (Elevated Plus Maze Test) and locomotor activity (Open Field Test) assessment. Blood pressure (BP), electrocardiogram (ECG), heart rate (HR) and respiratory frequency (RF) rates were recorded at 28 weeks of age. Baroreflex gain (BRG) and chemoreflex sensitivity (ChS) were calculated. Student’s T-test was used (significance p < 0.05) for statistical analysis. Results: An intermittent lead exposure causes hypertension (increased diastolic and mean BP), increased RF, decreased baroreflex function and increased ChS, without significant changes in HR, when compared to CTL group. Regarding behavioral changes, the intermittent lead exposure model showed an anxiety-like behaviour without changes in locomotor activity. Conclusion: Intermittent low-level lead exposure induces changes on the cardiorespiratory profile characterized by hypertension, carotid chemosensitivity and baroreflex impairment. According to behavioural tests results, this study also shows that the exposure to lead during developmental phases causes anxiety in adult animals without locomotor activity impairment. In summary, this study brings new insights on the environmental factors that influence nervous and cardiovascular systems during development, which can help creating public policy strategies to prevent and control the adverse effects of Pb toxicity.info:eu-repo/semantics/publishedVersio

Intermittent low-level lead exposure provokes anxiety, hypertension, autonomic dyfunction and neuroinflammation

© 2018 Elsevier B.V. All rights reserved.Background: Exposures to lead (Pb) during developmental phases can alter the normal course of development, with lifelong health consequences. Permanent Pb exposure leads to behavioral changes, cognitive impairment, sympathoexcitation, tachycardia, hypertension and autonomic dysfunction. However, the effects of an intermittent lead exposure are not yet studied. This pattern of exposure has been recently increasing due to migrations, implementation of school exchange programs and/or residential changes. Objective: To determine and compare lead effects on mammal’s behavior and physiology, using a rat model of intermittent and permanent Pb exposures. Methods: Fetuses were intermittently (PbI) or permanently (PbP) exposed to water containing lead acetate (0.2% w/v) throughout life until adulthood (28 weeks of age). A control group (CTL) without any exposure to lead was also used. Anxiety was assessed by elevated plus maze (EPM) and locomotor activity and exploration by open field test (OFT). Blood pressure (BP), electrocardiogram (ECG), heart rate (HR), respiratory frequency (RF), sympathetic and parasympathetic activity and baro- and chemoreceptor reflex profiles were evaluated. Immunohistochemistry protocol for the assessment of neuroinflammation, neuronal loss (NeuN), gliosis and synaptic alterations (Iba-1, GFAP, Syn), were performed at the hippocampus. One-way ANOVA with Tukey’s multiple comparison between means were used (significance p < 0.05) for statistical analysis. Results: The intermittent lead exposure produced a significant increase in diastolic and mean BP values, concomitant with a tendency to sympathetic overactivity (estimated by increased low-frequency power) and without significant changes in systolic BP, HR and RF. A chemoreceptor hypersensitivity and a baroreflex impairment were also observed, however, less pronounced when compared to the permanent exposure. Regarding behavioral changes, both lead exposure profiles showed an anxiety-like behavior without changes in locomotor and exploratory activity. Increase in GFAP and Iba-1 positive cells, without changes in NeuN positive cells were found in both exposed groups. Syn staining suffered a significant decrease in PbI group and a significant increase in PbP group. Conclusion: This study is the first to show that developmental Pb exposure since fetal period can cause lasting impairments in physiological parameters. The intermittent lead exposure causes adverse health effects, i.e, hypertension, increased respiratory frequency and chemoreflex sensitivity, baroreflex impairment, anxiety, decreased synaptic activity, neuroinflammation and reactive gliosis, in some ways similar to a permanent exposure, however some are lower-grade, due to the shorter duration of exposure. This study brings new insights on the environmental factors that influence autonomic and cardiovascular systems during development, which can help in creating public policy strategies to prevent and control the adverse effects of Pb toxicity.Vera Geraldes acknowledges the post-doctoral fellow given by the Fundação para a Ciência e Tecnologia (FCT) (Ref: SFRH/BPD/119315/ 2016).info:eu-repo/semantics/publishedVersio

Persistent effects on cardiorespiratory and nervous systems induced by long-term lead exposure : results from a longitudinal study

© 2020 Springer Nature Switzerland AG. Part of Springer Nature.Long-term lead (Pb) exposure alters the normal development of the nervous system and physiology. It affects multiple organ systems, causing hypertension, cardiorespiratory dysfunction, being a well-known neurotoxin, inducing changes in neurogenesis, neurodegeneration, and glial cells. However, studies of the developmental effects of lead and its outcomes throughout life are lacking. Determine morphofunctional, behavioral, and cognitive developmental effects of long-term lead exposure at three different ages. Wistar rats were exposed to a Pb-acetate solution from fetal period until adulthood and compared to a non-exposed control group. General behavior and cognitive skills were evaluated by behavioral tests and physiological data and cardiorespiratory reflexes measured. Neurodegeneration, neuroinflammation, and synaptic activity were assessed by immunohistochemistry. Lead exposure caused long-lasting anxiety-like behavior and strong long-term memory impairment without changes in locomotor and exploratory activity. Hypertension was observed at all time points, concomitant with baroreflex impairment and increased chemoreflex sensitivity. Persistent neuroinflammation, transient synaptic overexcitation without neurodegeneration was observed. Long-term Pb exposure, since fetal period, causes long-lasting anxiety-like behavior, concomitant with hypertension, without general motor skills impairment. Synaptic overexcitation, reactive astrogliosis, and microgliosis could underlie behavioral and long-term memory changes, which might have been caused during developmental phases and consolidated during adulthood. Also, alterations observed in the cardiorespiratory reflexes can explain persistent hypertension. This longitudinal study identifies and characterizes lead toxicity nature and magnitude, important to devise and test potential interventions to attenuate the long-term harmful effects of lead on the nervous and cardiovascular systems.Vera Geraldes received the post-doctoral fellowship given by the Fundação para a Ciência e Tecnologia (FCT) (Ref: SFRH/BPD/119315/2016).info:eu-repo/semantics/publishedVersio

From Molecular to Functional Effects of Different Environmental Lead Exposure Paradigms

Lead is a heavy metal whose widespread use has resulted in environmental contamination and significant health problems, particularly if the exposure occurs during developmental stages. It is a cumulative toxicant that affects multiple systems of the body, including the cardiovascular and nervous systems. Chronic lead exposure has been defined as a cause of behavioral changes, inflammation, hypertension, and autonomic dysfunction. However, different environmental lead exposure paradigms can occur, and the different effects of these have not been described in a broad comparative study. In the present study, rats of both sexes were exposed to water containing lead acetate (0.2% w/v), from the fetal period until adulthood. Developmental Pb-exposed (DevPb) pups were exposed to lead until 12 weeks of age (n = 13); intermittent Pb exposure (IntPb) pups drank leaded water until 12 weeks of age, tap water until 20 weeks, and leaded water for a second time from 20 to 28 weeks of age (n = 14); and the permanent (PerPb) exposure group were exposed to lead until 28 weeks of age (n = 14). A control group (without exposure, Ctrl), matched in age and sex was used. After exposure protocols, at 28 weeks of age, behavioral tests were performed for assessment of anxiety (elevated plus maze test), locomotor activity (open-field test), and memory (novel object recognition test). Metabolic parameters were evaluated for 24 h, and the acute experiment was carried out. Blood pressure (BP), electrocardiogram, and heart (HR) and respiratory (RF) rates were recorded. Baroreflex gain, chemoreflex sensitivity, and sympathovagal balance were calculated. Immunohistochemistry protocol for NeuN, Syn, Iba-1, and GFAP staining was performed. All Pb-exposed groups showed hypertension, concomitant with a decrease in baroreflex gain and chemoreceptor hypersensitivity, without significant changes in HR and RF. Long-term memory impairment associated with reactive astrogliosis and microgliosis in the dentate gyrus of the hippocampus, indicating the presence of neuroinflammation, was also observed. However, these alterations seemed to reverse after lead abstinence for a certain period (DevPb) and were enhanced when a second exposure occurred (IntPb), along with a synaptic loss. These results suggest that the duration of Pb exposure is more relevant than the timing of exposure, since the PerPb group presented more pronounced effects and a significant increase in the LF and HF bands and anxiety levels. In summary, this is the first study with the characterization and comparison of physiological, autonomic, behavioral, and molecular changes caused by different low-level environmental lead exposures, from the fetal period to adulthood, where the duration of exposure was the main factor for stronger adverse effects. These kinds of studies are of immense importance, showing the importance of the surrounding environment in health from childhood until adulthood, leading to the creation of new policies for toxicant usage control

Therapeutic effects of IkB kinase inhibitor during systemic inflammation

Copyright © 2020 Elsevier B.V. All rights reserved.Animal models of inflammatory diseases support the idea that nuclear factor κB (NF-κB) activation plays a pathophysiological role and is widely implicated in multiple organ dysfunction (MOD). Indeed, the inhibition of the IκB kinase (IKK) complex, involved in the NF-κB pathway, can represent a promising approach to prevent MOD. The present work employed a rat model of systemic inflammation to investigate the preventive effects of Inhibitor of IKK complex (IKK16). In male Wistar rats, systemic inflammation was induced by a tail vein injection of lipopolysaccharides (LPS challenge; 12 mg/kg). Treatment with IKK16 (1 mg/kg body weight) was administered, by tail vein, 15 min post-LPS. Age- and sex-matched healthy rats and LPS rats without treatment were used as controls. At 24 h post-IKK16 treatment, serum enzyme levels indicative of liver, kidney, pancreas and muscle function were evaluated by biochemical analysis, and RT-PCR technique was used to analyze gene expression of pro-inflammatory cytokines. Hemodynamic parameters were also considered to assess the LPS-induced inflammation. IKK16 treatment yielded a strong therapeutic effect in preventing LPS-induced elevation of serological enzyme levels, attenuating hepatic, renal, pancreatic and muscular dysfunction after LPS challenge. Moreover, as expected, LPS promoted a significantly overexpression of TNF-α, IL-6 and IL-1β in the heart, kidney, and liver; which was diminished by IKK16 treatment. The present study provides convincing evidence that selective inhibition of the IκB kinase complex through the action of IKK16, plays a protective role against LPS-induced multiple organ dysfunction by reducing the acute inflammatory response induced by endotoxin exposure.info:eu-repo/semantics/publishedVersio

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo

A molecular hallmark in Parkinson's disease (PD) pathogenesis are a-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of alpha-synuclein. Here, we investigated the effects of CDNF on alpha-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with alpha-synuclein with a KD = 23 +/- 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of alpha-synuclein fibrils and induces the formation of insoluble phosphorylated alpha-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with a-synuclein fibrils, though it did not reduce the number of phosphorylated alpha-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with a-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.Peer reviewe