Efeito da exposição à deprivação materna e/ou dieta hipercalórica em parâmetros de desenvolvimento neuromotor, ponderais, comportamentais e neuroquímicos em ratos

O consumo de dieta ocidental altamente palatável e hipercalórica expõe os indivíduos, desde a primeira infância, a uma alimentação obesogênica. Essa dieta aumenta o prazer relacionado ao consumo alimentar estimulando o sistema de recompensa e alterando vias de sinalização do sistema nervoso central (SNC). Esses eventos são relacionados à perda do controle adequado do apetite. Por outro lado, exposição ao estresse também tem importante relação com controle alimentar. Uma importante fonte de estresse é a deprivação materna (DM) no período neonatal, trazendo prejuízos ao indivíduo ao longo da vida. Estudos em animais têm demonstrado que o estresse induzido pela DM nesse período provoca alterações neuroquímicas. Essas alterações têm sido relacionadas a transtornos mentais como ansiedade e depressão, assim como, obesidade na idade adulta. Considerando a relevância do tema, nessa dissertação avaliamos o efeito da DM no período neonatal e/ou exposição à dieta hipercalórica (leite condensado) após o desmame sobre parâmetros de desenvolvimento neuromotor, ponderais, comportamentais e neuroquímicos em ratos Wistar adultos jovens machos e fêmeas. Foram utilizados 64 ratos Wistar machos e fêmeas distribuídos em quatro grupos: Controle (C) – sem DM e exposto à dieta padrão; Deprivação Materna (DM)- animais submetidos à DM (P1-P10) e exposto à dieta padrão; Dieta Hipercalórica (DH) - sem DM e exposto à dieta hipercalórica (alimento palatável/leite condensado); e Deprivação Materna e Dieta Hipercalórica (DMDH)- animais submetidos à DM (P1-P10) e expostos à dieta hipercalórica (alimento palatável/leite condensado). Os efeitos da DM nos padrões de reflexos iniciais nos filhotes foram avaliados por meio dos testes de geotaxia negativa e endireitamento postural, assim como seus efeitos sobre a resposta nociceptiva avaliada por meio do teste da Placa Quente (PQ) e do Tail-Flick (TFL). Avaliamos também o comportamento do tipo ansioso por meio do teste do Labirinto em Cruz Elevado - Elevated Plus Maze (EPM) e a atividade locomotora pelo teste de Campo Aberto (CA) antes do desmame (P21). A partir de P21, os animais dos grupos DH e DMDH foram expostos a uma dieta hipercalórica/alimento palatável (leite condensado) por três semanas e novamente avaliados a resposta nociceptiva, o comportamento do tipo ansioso e a atividade locomotora. Salientamos que foi analisado o efeito do gênero sobre todos os parâmetros analisados. Após a segunda leva de testes comportamentais, os animais foram mortos (P44) e retirados córtex cerebral, hipocampo e hipotálamo para a realização de dosagens bioquímicas. Foram dosados níveis do fator de desenvolvimento neuronal do encéfalo (BDNF), fator de crescimento neuronal 1 (NGF-1) e de citocinas pro-inflamatórias (IL6 e TNF-α) e anti-inflamatória (IL10). Nossos resultados demonstram que a exposição à DM induz, em curto prazo (entre P20 e P21), alterações no desenvolvimento motor inicial, resposta nociceptiva térmica (aumento do limiar nociceptivo –PQ e TFL-fêmeas), aumento da atividade locomotora de fêmeas devido ao maior número de cruzamentos externos e do tempo de grooming em machos, o que pode estar relacionado a alterações em sistema dopaminérgico (teste CA); aumento no tempo de permanência nos braços abertos dos animais deprivados sugerindo um efeito ansiolítico dessa intervenção, aumento no número de non-protected head dipping (NPHD) em machos, comportamentos relacionados à atividade exploratória e avaliação de risco (teste do EPM). Os efeitos tardios da DM, avaliados entre P42 e P43, foram observados na resposta nociceptiva (diminuição do limiar nociceptivo dos machos -TFL) e no teste EPM apenas os machos apresentaram maior número de rearings, indicativo de aumento na atividade exploratória dependente de gênero da DM. Quando expostos à DH, os machos apresentaram maior ganho de peso e índice de Lee. A DH induziu um aumento no peso relativo de tecido adiposo, independente do sexo. Foram observados efeitos tardios da DM e do gênero sobre os níveis de BDNF nas estruturas cerebrais avaliadas. Efeito da exposição à DH foi observado apenas nos níveis hipotalâmicos de NGF-1. Fêmeas apresentaram níveis mais elevados de BDNF e IL-6 em hipocampo e hipotálamo, e de TNF-α em hipotálamo. Em níveis corticais de IL10 houve interação entre gênero e DM. Concluindo, esses achados contribuem para um maior entendimento dos possíveis efeitos da exposição precoce a um estressor e/ou ao alimento palatável na infância/adolescência, demonstrando diferentes respostas de acordo com gênero às intervenções propostas. Além disso, diferenças entre o gênero per se foram observadas, sugerindo a importância de incluir ambos os sexos nas investigações pré-clínicas a fim de trazer maior translacionalidade aos estudos.The consumption of a highly palatable and hypercaloric Western diet exposes individuals, from early infancy, to an obesogenic diet. This diet increases the pleasure related to food consumption by stimulating the reward system and altering Central Nervous System (CNS) signaling pathways. These events are related to loss of adequate appetite control. On the other hand, stress exposure also has important relation with food control. An important source of stress is maternal deprivation (MD) in the neonatal period, causing lifetime impacts. Animal studies have shown that stress induced by MD causes neurochemical changes. These changes have been related to mental disorders such as anxiety and depression, as well as obesity in adulthood. Considering the relevance of this topic, in this dissertation we evaluated the effect of MD in neonatal period and / or exposure to hypercaloric diet (condensed milk) after weaning on neuromotor, ponderal, behavioral and neurochemical development parameters in young adult male and female Wistar rats. Sixty-four male and female Wistar rats were distributed into four groups: Control (C) - without MD and exposed to the standard diet; Maternal Deprivation (MD) - animals submitted to MD (P1-P10) and exposed to the standard diet; Hypercaloric (HD) diet - without MD and exposed to the hypercaloric diet (palatable food / condensed milk); and Maternal Deprivation and Hypercaloric Diet (MDHD) - animals submitted to MD (P1-P10) and exposed to the hypercaloric diet (palatable food / condensed milk). The effects of MD on the initial reflex patterns in the pups were evaluated by the negative geotaxis and righting reflex tests, as well as their effects on the nociceptive response assessed by the Hot Plate (HP) and Tail-Flick tests (TFL). We also evaluated the anxiety-like behavior through the Elevated Plus-Maze (EPM) test and the locomotor activity by the Open Field Test (OF) before weaning (P21). From P21, the animals from the HD and MDHD groups were exposed to a hypercaloric / palatable diet (condensed milk) for three weeks and the nociceptive response, anxious-like behavior and locomotor activity were evaluated once again. We emphasize that the effect of gender in all parameters was analyzed. After the second set of behavioral tests, the animals were killed (P44) and the cerebral cortex, hippocampus and hypothalamus were removed for biochemical measurements. Levels of neuronal brain development factor (BDNF), neuronal growth factor 1 (NGF-1) and proinflammatory cytokines (IL6 and TNF-α) and anti-inflammatory cytokine (IL10) were measured. Our results demonstrate that the exposure to MD induces, in the short term, changes in the initial motor development, thermal nociceptive response (increase of the nociceptive threshold - PQ and TFL-females), increases locomotor activity of females due to the greater number of outter crossings and grooming time in males, which may be related to changes in dopaminergic system (OF test); increases time spent in open arms of deprived animals suggesting an anxiolytic effect of this intervention, increases the number of non-protected head dipping (NPHD) in males, behaviors related to exploratory activity and risk assessment (EPM test). The late effects of DM, evaluated between P42 and P43, were observed in the nociceptive response (decrease males’ nociceptive thresholds in the TFL) and in the EPM test males presented a greater number of rearings, indicative of an increase in exploratory activity dependent on gender. When exposed to HD, males showed greater weight gain and Lee index. HD induced an increase in the relative adipose tissue weight, regardless of sex. Late effects of MD and gender on the BDNF levels were observed in the brain structures evaluated. Effect of HD exposure was observed only on the hypothalamic levels of NGF-1. Females presented higher levels of BDNF and IL-6 in hippocampus and hypothalamus, and TNF-α in the hypothalamus. At cortical levels of IL-10 there was interaction between gender and MD. In conclusion, these findings contribute to a better understanding of the possible effects of early exposure to a stressor and / or palatable food in childhood / adolescence, demonstrating different responses according to gender to the proposed interventions. In addition, gender differences per se were observed, suggesting the importance of including both genders in preclinical investigations in order to bring greater translationality to the studies

Estimulação magnética transcraniana repetitiva em modelo animal de dor neuropática

Dor neuropática (DN) está relacionada à presença de hiperalgesia, alodinia e dor espontânea, afetando de 7% a 10% da população geral. É uma dor de difícil tratamento, sendo necessário a busca de novas opções terapêuticas. Neste contexto, a estimulação magnética transcraniana repetitiva (EMTr) vem sendo aplicada para o alívio da DN, especialmente em pacientes com dor refratária. Considerando a relevância do tema, o objetivo desta tese foi avaliar o efeito do tratamento com EMTr sobre a resposta nociceptiva e a memória de longo prazo de ratos submetidos a um modelo de DN, além de avaliar seus efeitos nos níveis centrais de neurotrofinas e citocinas pró e anti-inflamatórias. Para tanto, foram utilizados 106 ratos Wistar machos adultos (60 dias de idade), divididos em 9 grupos experimentais: controle (C), controle + sham EMTr (C+s.EMTr), controle + EMTr ativo (C+EMTr), sham dor neuropática (s.DN), sham dor neuropática + sham EMTr (s.DN+s. EMTr), sham dor neuropática + EMTr ativo (s.DN+EMTr), dor neuropática (DN), dor neuropática + sham EMTr (DN+s. EMTr) e dor neuropática + EMTr ativo (DN+EMTr). O estabelecimento do modelo de DN ocorreu 14 dias após a cirurgia de constrição crônica do nervo isquiático (CCI). A partir disso, os ratos foram tratados com sessões diárias de 5 minutos de EMTr por 8 dias consecutivos. As respostas de hiperalgesia térmica e alodinia mecânica foram avaliadas antes do procedimento cirúrgico, após o estabelecimento da DN e 24h após o término do tratamento, por meio dos testes da Placa Quente (PQ) e von Frey (VF). A memória de longo prazo foi avaliada pelo teste de reconhecimento de objetos, 48h após o término do tratamento. Em seguida, os animais foram eutanasiados e estruturas foram coletadas para análises bioquímicas. Os ensaios bioquímicos (níveis de BDNF, TNF-α e IL-10) foram realizados em homogenatos de córtex pré-frontal (CPF), medula espinhal e hipocampo. O tratamento com EMTr promoveu reversão total da hiperalgesia térmica (Placa Quente) induzida pelo modelo de DN e reversão parcial da alodinia mecânica (Von-Frey). Além disso, a EMTr aumentou os níveis de BDNF e TNF-α no CPF e reverteu a diminuição dos níveis hipocampais de IL-10 nos animais com DN. Complementarmente, o tratamento com EMTr reverteu o déficit na memória de longo prazo induzido pela DN. Desta forma, a EMTr apresenta-se como uma ferramenta promissora para o manejo da DN, e as alterações induzidas pela estimulação parecem ser estado-dependente, ou seja, EMTr induz alterações em parâmetros neuroquímicos, modulando a neuroplasticidade, apenas nos animais com um estado mal adaptativo como a DN, aumentando o limiar nociceptivo e recuperando a memória de longo prazo desses animais.Neuropathic pain (NP) is related to the presence of hyperalgesia, allodynia, and spontaneous pain, affecting 7% to 10% of the general population. The management of NP is difficult, demanding new therapeutic options. In this context, repetitive transcranial magnetic stimulation (rTMS) has been applied for NP relief, especially in patients with refractory pain. Considering the relevance of this theme, the aim of the present thesis was to evaluate the effects of rTMS treatment on the nociceptive response and long-term memory of rats in a NP model, and to evaluate its effects on central levels of neurotrophins and pro and anti-inflammatory cytokines. For this purpose, 106 adult male Wistar rats (60 days old) were used, divided into 9 experimental groups: control(C), control + sham rTMS (C+s.rTMS), control + active rTMS (C+ rTMS), sham neuropathic pain (s.NP), sham neuropathic pain + sham rTMS (s.NP+s.rTMS), sham neuropathic pain + active rTMS (s.NP+rTMS), neuropathic pain (NP), neuropathic pain + sham rTMS (NP+s.rTMS) and neuropathic pain + active rTMS (NP+rTMS). The establishment of the NP model occurred 14 days after surgery for chronic constriction injury (CCI) of the sciatic nerve. Thereafter, the rats were submitted to daily 5-minute rTMS sections during 8 consecutive days. The responses of thermal hyperalgesia and mechanical allodynia were evaluated before the surgical procedure, after the establishment of NP and after treatment, respectively, by the hot plate and von-Frey tests, 24h after the end of the treatment. Long-term memory was assessed by the object recognition test, 48 hours after the end of treatment. After, animals were killed, and the structures collected for biochemical assays. BDNF, TNF-α and IL-10 levels were performed in prefrontal cortex (PFC), spinal cord and hippocampus homogenates. Treatment with rTMS promoted total reversal of thermal hyperalgesia (hot plate) and partial reversal of mechanical allodynia (von-Frey) induced by the NP model. Furthermore, rTMS increased BDNF and TNF-α levels in the PFC and reversed the decrease in hippocampal IL-10 levels in animals with NP. In addition, rTMS treatment reversed the long-term memory deficit induced by NP. Thus, rTMS presents itself as a promising tool for the management of NP; changes induced by the stimulation seem to be state-dependent, that is, rTMS induces changes in neurochemical parameters, modulating neuroplasticity, only in animals with a maladaptive state (i.e. NP), increasing nociceptive thresholds and long-term memory restoration in these animals

Cafeteria diet increases liquid intake and serum creatinine levels in rats

Introduction: Important changes in human dietary pattern occurred in recent decades. Increased intake of processed foods leads to obesity, which is related with the development of chronic diseases such as type 2 diabetes mellitus, hypertension, as well as cardiovascular and chronic kidney diseases. The prevalence of hypertension has also dramatically increased in recent years, and high sodium intake contributes to this scenario. In healthy individuals, kidneys are the primary end-organs that regulate sodium homeostasis. This study aims to evaluate renal function parameters and systolic blood pressure measurements in an animal model of obesity. Methods: Sixty-day-old male Wistar rats (n=30) were divided into two groups: standard (SD) and cafeteria diet (CD). Cafeteria diet was altered daily and was composed by crackers, wafers, sausages, chips, condensed milk, and soda. All animals had free access to water and chow and the experiment was carried out for 6 weeks. Weight gain, sodium and liquid intake control, systolic blood pressure measurements, and renal function parameters were evaluated.Results: Animals exposed to cafeteria diet had an increase of 18% in weight compared to the control group. Sodium intake was increased by cafeteria diet and time (F(1,28)= 773.666, P=0.001 and F(5,28)= 2.859, P=0.02, respectively) and by the interaction of both factors (F(6,28)= 2.859, P=0.02). On liquid intake occurred only effect of cafeteria diet and time (F(1,28)= 147.04, P=0.001 and F(5,28)=3.996, P=0.003, respectively). Cafeteria diet exposure also induced an increase on creatinine serum levels (P=0.002), however this effect was not observed on creatinine urine levels (P>0.05) nor on systolic pressure measurements (Students’ t test, P>0.05).Conclusions: Obesity induced by cafeteria diet exposure increases liquid intake and alters creatinine serum levels, an important renal function marker. Considering the high consumption of hypercaloric food currently in the world, further studies are required to elucidate the modifications on renal function triggered by this diet over time.Key-words: Hypertension; kidney; renal function; obesity; hypercaloric diet

Neonatal morphine exposure and maternal deprivation alter nociceptive response and central biomarkers’ levels throughout the life of rats

In the present study, we investigated the effect of repeated neonatal morphine exposure and/or maternal deprivation(MD) on the nociceptive response and central biomarkers’ BDNF, IL-1β, and IL-4 levels at postnatal days 16(PND16), 30(PND30), and 60(PND60). At birth, the litters were standardized to contain 8 pups/dam (n = 58). From PND1 to PND10, the pups of the deprived groups were separated daily from their mothers for 3 h and divided into 5 groups: control(C), saline(S), morphine(M), deprived-saline(DS), and deprived-morphine (DM). The pups received subcutaneous injections of saline/morphine (5 μg) in the mid-scapular area between PND8 and PND14. Nociceptive responses were assessed by hot plate(HP) and tail-flick(TFL) tests and biomarker levels by ELISA. Thermal hyperalgesia(HP) was found in all assessments for the M, DS, and DM groups, and a decrease in nociceptive threshold(TFL) was found in the DS group at PND16; M and DM groups at PND30; and M, DS, and DM groups at PND60. There were interactions between treatment/deprivation/timepoint in all central biomarkers’ levels. The current study indicates that neonatal exposure to morphine and MD, which occurs in the pediatric ICU, can alter the nociceptive and neuroinflammatory responses

Cafeteria diet increases liquid intake and serum creatinine levels in rats

Introduction: Important changes in human dietary pattern occurred in recent decades. Increased intake of processed foods leads to obesity, which is related with the development of chronic diseases such as type 2 diabetes mellitus, hypertension, as well as cardiovascular and chronic kidney diseases. The prevalence of hypertension has also dramatically increased in recent years, and high sodium intake contributes to this scenario. In healthy individuals, kidneys are the primary end-organs that regulate sodium homeostasis. This study aims to evaluate renal function parameters and systolic blood pressure measurements in an animal model of obesity. Methods: Sixty-day-old male Wistar rats (n=30) were divided into two groups: standard (SD) and cafeteria diet (CD). Cafeteria diet was altered daily and was composed by crackers, wafers, sausages, chips, condensed milk, and soda. All animals had free access to water and chow and the experiment was carried out for 6 weeks. Weight gain, sodium and liquid intake control, systolic blood pressure measurements, and renal function parameters were evaluated Results: Animals exposed to cafeteria diet had an increase of 18% in weight compared to the control group. Sodium intake was increased by cafeteria diet and time (F(1,28)=773.666, P=0.001 and F(5,28)=2.859, P=0.02, respectively) and by the interaction of both factors (F(6,28)=2.859, P=0.02). On liquid intake occurred only effect of cafeteria diet and time (F(1,28)=147.04, P=0.001 and F(5,28)=3.996, P=0.003, respectively). Cafeteria diet exposure also induced an increase on creatinine serum levels (P=0.002), however this effect was not observed on creatinine urine levels (P>0.05) nor on systolic pressure measurements (Students’ t test, P>0.05). Conclusions: Obesity induced by cafeteria diet exposure increases liquid intake and alters creatinine serum levels, an important renal function marker. Considering the high consumption of hypercaloric food currently in the world, further studies are required to elucidate the modifications on renal function triggered by this diet over time