Efeitos neurotóxicos da hiper-homocisteinemia leve experimental : papel neuroprotetor do ibuprofeno e da rivastigmina

A homocisteína (Hcy) é um aminoácido sulfurado derivado do metabolismo da metionina. Os níveis plasmáticos de Hcy dependem de vários fatores, sendo que os valores normais variam de 5 a 15 μmol/L e acima deste é definido como hiper-homocisteinemia (HHcy). A HHCy leve, 16-30 μmol/L, é um fator de risco para doenças neurodegenerativas. Estudos têm demonstrado que HHcy leve induz estresse oxidativo, neuroinflamação e alteração na atividade da acetilcolinesterase (AChE), no hipocampo de ratos. Essas alterações também têm sido evidenciadas na patogênese da doença de Alzheimer. O presente estudo tem como objetivo avaliar os efeitos e mecanismos da HHcy leve experimental em ratos Wistar, bem como o papel neuroprotetor do ibuprofeno e da rivastigmina sobre parâmetros comportamentais motores e cognitivos (campo aberto, trave de equilíbrio, rotarod e pole teste vertical, Y-maze) e parâmetros bioquímico-moleculares no hipocampo [homeostase redox, proteína sinapsina 1, parâmetros do perfil inflamatório, molécula adaptadora de ligação ao cálcio 1 (Iba1), expressão do gene que codifica para iNOS, níveis de fator de crescimento endotelial vascular (VEGF), fator de crescimento epidérmico (EGF), quimiocina ligante 5 (CCL5/RANTES), quimiocina ligante CX3C 1 (CX3CL1) e NGF/p75NTR/tropomiosina quinase B (TrkB)], por meio da análise imuno-histoquímica de marcadores gliais (Iba-1 e GFAP) e a via anti-inflamatória colinérgica. Também investigamos, em modelo ex vivo, possíveis alterações bioquímico-molecular em fatias de hipocampo [excitabilidade neuronal, níveis de glicose, lactato, a proteína Serina/Treonina quinase B (Akt), glicose sintase quinase-3β (GSK3β) e o transportador de glicose 1 (GLUT1)]. No capítulo I e III, o modelo crônico de HHcy leve foi induzido em ratos Wistar através da administração subcutânea de Hcy (0,03 μmol/g de peso corporal), duas vezes ao dia, do 30º a 60º dia de vida. Ibuprofeno (40 mg/kg) e rivastigmina (0,5 mg/kg) foram administrados via intraperitoneal uma vez ao dia. No capítulo II, fatias de hipocampo de ratos Wistar de 90 dias foram pré-tratadas por 30 minutos [meio salino ou Hcy (30 μM)], depois os outros tratamentos foram adicionados ao meio por mais 30 minutos [ibuprofeno (100 μM), rivastigmina (0,5 μM) ou ibuprofeno+rivastigmina]. Os resultados do capítulo I mostraram que a HHcy leve causou déficits cognitivos na memória de trabalho e a coordenação motora prejudicada, reduziu a quantidade da proteína sinapsina 1, alterou o quadro neuroinflamatório e causou alterações na atividade das enzimas catalase e AChE. Ambos os tratamentos, rivastigmina e ibuprofeno, foram capazes de mitigar esses danos causados pela HHcy leve. No capítulo II, os níveis fosforilados de GSK3β e Akt foram reduzidos em Hcy (30 μM) e o co-tratamento com Hcy+rivastigmina+ibuprofeno reverteu esses efeitos. A associação do tratamento com rivatigmina+ibuprofeno atenuou tais efeitos, provavelmente pela regulação da via de sinalização Akt/GSK3β/GLUT1. No capítulo III, os resultados mostraram aumento nos níveis de CCL5/RANTES e redução nos níveis de VEGF, EGF e TrkB no hipocampo de ratos submetidos à HHcy. A rivastigmina reverteu o efeito neurotóxico da HHcy com aumento de TrkB e VEGF. Já o ibuprofeno atenuou os níveis de CCL5/RANTES frente ao efeito neurotóxico da HHcy, reduzindo significativamente os níveis dessa quimiocina. Em conjunto, esses achados fornecem uma nova base para a compreensão dos resultados neuroquímicos e comportamentais associados à níveis aumentos de Hcy. Além disso, a reversão dos danos de HHcy por ibuprofeno e rivastigmina pode ser uma estratégia neuroprotetora potencial para danos cerebrais.Homocysteine (Hcy) is a sulfur amino acid derived from the metabolism of methionine. Plasma levels of Hcy depend on several factors, and normal values range from 5 to 15 μmol/L and above this is defined as hyperhomocysteinemia (HHcy). Mild HHCy, 16-30 μmol/L, is a risk factor for neurodegenerative diseases. Studies have shown that mild HHcy induces oxidative stress, neuroinflammation and changes in acetylcholinesterase (AChE) activity in the hippocampus of rats. These alterations have also been evidenced in the pathogenesis of Alzheimer's disease. The present study aims to evaluate the effects and mechanisms of experimental light HHcy in Wistar rats, as well as the neuroprotective role of ibuprofen and rivastigmine on motor and cognitive behavioral parameters (open field, balance beam, rotarod and vertical pole test, Y -maze) and biochemical-molecular parameters in the hippocampus [redox homeostasis, synapsin 1 protein, parameters of the inflammatory profile, calcium-binding adapter molecule 1 (Iba1), expression of the gene that encodes for iNOS, levels of vascular endothelial growth factor ( VEGF), epidermal growth factor (EGF), chemokine ligand 5 (CCL5/RANTES), chemokine ligand CX3C 1 (CX3CL1) and NGF/p75NTR/tropomyosin kinase B (TrkB)], through immunohistochemical analysis of glial markers (Iba-1 and GFAP) and the cholinergic anti-inflammatory pathway. We also investigated, in an ex vivo model, possible biochemical-molecular changes in hippocampus slices [neuronal excitability, levels of glucose, lactate, protein Serine/Threonine kinase B (Akt), glucose synthase kinase-3β (GSK3β) and the transporter glucose 1 (GLUT1)]. In chapters I and III, the chronic model of mild HHcy was induced in Wistar rats through the subcutaneous administration of Hcy (0.03 μmol/g of body weight), twice a day, from the 30th to the 60th day of life. Ibuprofen (40 mg/kg) and rivastigmine (0.5 mg/kg) were administered intraperitoneally once a day. In chapter II, slices of hippocampus from 90-day-old Wistar rats were pre-treated for 30 minutes [saline or Hcy (30 μM) medium], then the other treatments were added to the medium for another 30 minutes [ibuprofen (100 μM), rivastigmine (0.5 μM) or ibuprofen+rivastigmine]. The results of chapter I showed that mild HHcy caused cognitive deficits in working memory and impaired motor coordination, reduced the amount of synapsin 1 protein, altered the neuroinflammatory picture and caused alterations in the activity of catalase and AChE enzymes. Both treatments, rivastigmine and ibuprofen, were able to mitigate this damage caused by mild HHcy. In chapter II, phosphorylated levels of GSK3β and Akt were reduced in Hcy (30 μM) and co-treatment with Hcy+rivastigmine+ibuprofen reversed these effects. The combination of treatment with rivatigmine+ibuprofen attenuated these effects, probably by regulating the Akt/GSK3β/GLUT1 signaling pathway. In chapter III, the results showed an increase in the levels of CCL5/RANTES and a reduction in the levels of VEGF, EGF and TrkB in the hippocampus of rats submitted to HHcy. Rivastigmine reversed the neurotoxic effect of HHcy with increased TrkB and VEGF. Ibuprofen, on the other hand, attenuated CCL5/RANTES levels against the neurotoxic effect of HHcy, significantly reducing the levels of this chemokine. Taken together, these findings provide a new basis for understanding the neurochemical and behavioral outcomes associated with increased Hcy levels. Furthermore, reversal of HHcy damage by ibuprofen and rivastigmine may be a potential neuroprotective strategy for brain damage

Quinolinic acid impairs redox homeostasis, bioenergetic, and cell signaling in rat striatum slices : prevention by coenzyme Q10

Quinolinic acid (QUIN) is an important agonist of NMDA receptors that are found at high levels in cases of brain injury and neuroinflammation. Therefore, it is necessary to investigate neuroprotection strategies capable of neutralizing the effects of the QUIN on the brain. Coenzyme Q10 (CoQ10) is a provitamin that has an important antioxidant and anti-inflammatory action. This work aims to evaluate the possible neuroprotective effect of CoQ10 against the toxicity caused by QUIN. Striatal slices from 30-day-old Wistar rats were preincubated with CoQ10 25–100 μM for 15 min; then, QUIN 100 μM was added to the incubation medium for 30 min. A dose–response curve was used to select the CoQ10 concentration to be used in the study. Results showed that QUIN caused changes in the production of ROS, nitrite levels, activities of antioxidant enzymes, glutathione content, and damage to proteins and lipids. CoQ10 was able to prevent the effects caused by QUIN, totally or partially, except for damage to proteins. QUIN also altered the activities of electron transport chain complexes and ATP levels, and CoQ10 prevented totally and partially these effects, respectively. CoQ10 prevented the increase in acetylcholinesterase activity, but not the decrease in the activity of Na+,K+-ATPase caused by QUIN. We also observed that QUIN caused changes in the total ERK and phospho-Akt content, and these effects were partially prevented by CoQ10. These findings suggest that CoQ10 may be a promising therapeutic alternative for neuroprotection against QUIN neurotoxicity

Rivastigmine reverses the decrease in synapsin and memory caused by homocysteine : is there relation to inflammation?

Elevated levels of homocysteine (Hcy) in the blood, called hyperhomocysteinemia (HHcy), is a prevalent risk factor for it has been shown that Hcy induces oxidative stress and increases microglial activation and neuroinflammation, as well as causes cognitive impairment, which have been linked to the neurodegenerative process. This study aimed to evaluate the effect of mild hyperhomocysteinemia with or without ibuprofen and rivastigmine treatments on the behavior and neurochemical parameters in male rats. The chronic mild HHcy model was chemically induced in Wistar rats by subcutaneous administration of Hcy (4055 mg/kg body weight) twice daily for 30 days. Ibuprofen (40 mg/kg) and rivastigmine (0.5 mg/kg) were administered intraperitoneally once daily. Motor damage (open field, balance beam, rotarod, and vertical pole test), cognitive deficits (Y-maze), neurochemical parameters (oxidative status/antioxidant enzymatic defenses, presynaptic protein synapsin 1, inflammatory profile parameters, calcium binding adapter molecule 1 (Iba1), iNOS gene expression), and cholinergic anti-inflammatory pathway were investigated. Results showed that mild HHcy caused cognitive deficits in working memory, and impaired motor coordination reduced the amount of synapsin 1 protein, altered the neuroinflammatory picture, and caused changes in the activity of catalase and acetylcholinesterase enzymes. Both rivastigmine and ibuprofen treatments were able to mitigate this damage caused by mild HHcy. Together, these neurochemical changes may be associated with the mechanisms by which Hcy has been linked to a risk factor for AD. Treatments with rivastigmine and ibuprofen can effectively reduce the damage caused by increased Hcy levels

Folic acid supplementation during pregnancy alters behavior in male rat offspring : nitrative stress and neuroinflammatory implications

Pregnancy diet can impact offspring’s neurodevelopment, metabolism, redox homeostasis, and inflammatory status. In pregnancy, folate demand is increased due to the requirement for one-carbon transfer reactions. The present study was proposed to investigate the effect of folic acid supplementation throughout pregnancy on a battery of behavior tests (olfactory preference, motor activity, exploratory capacity, habituation, memory, anxiety- and depression-like behavior). Redox homeostasis and neuroinflammatory status in cerebral cortex were also investigated. After pregnancy confirmation, the pregnant rats were randomly divided into two groups, according to the diet: group 1, (control) standard diet (2 mg/kg diet of folic acid) and group 2, supplemented diet with 4 mg/kg diet of folic acid. Throughout the gestational period, the pregnant rats received experimental diets. Results show that the supplemented diet with 4 mg/kg diet of folic acid throughout pregnancy impaired memory and motricity of the offspring when compared with control (standard diet). It was also observed an increase in anxiety- and depression-like behavior in this group. Nitrite levels increased in cerebral cortex of the offspring, when compared to control group. In contrast, iNOS expression and immunocontent were not altered. Moreover, we identify an increase in TNF-α, IL-1β, IL-6, IL-10, and MCP-1 gene expression in the cerebral cortex. In conclusion, our study showed that the supplemented diet with 4 mg/kg diet of folic acid throughout pregnancy may cause behavioral and biochemical changes in the male offspring