Effects of methylphenidate on mitochondrial dynamics and bioenergetics in the prefrontal cortex of juvenile rats are sex-dependent.

Methylphenidate (MPH) is a central nervous system stimulant drug and a first order prescription in the treatment of Attention Deficit Hyperactivity Disorder (ADHD). Although MPH biochemistry in neurodevelopment is not completely understood, studies showed it alters energy metabolism in rat brains. ADHD prevalence during neurodevelopment is related to males and the investigation has been mainly done in these subjects, therefore, little is known about MPH action in females and, consequently, about sexual dimorphism. In the present study we evaluated markers of mitochondrial dynamics (DRP1 and MFN2, fission and fusion, respectively), biogenesis (mtTFA) and bioenergetics (respiratory chain complexes) in prefrontal cortex of male and female juvenile rats submitted to exposure to MPH to better understand MPH effect during postnatal neurodevelopment. ATP and oxidative stress levels were also evaluated. Wistar rats received intraperitoneal injection of MPH (2.0 mg/kg) or control (saline), once a day, from 15th to 45th day of age. Results showed that MPH increased DRP1 and decreased MFN2, as well as increased mtTFA in prefrontal cortex of male rats. In female, MPH decreased NRF1 and increased Parkin, which are mitochondrial regulatory proteins. Respiratory chain complexes (complex I, SDH, complexes III and IV), ATP production and oxidative stress parameters were altered and shown to be sex-dependent. Taken together, results suggest that chronic MPH exposure at an early age in healthy animals changes mitochondrial dynamics, biogenesis and bioenergetics differently depending on the sex of the subjects.

Mild hyperhomocysteinemia alters oxidative stress profile via Nrf2, inflammation and cholinesterases in cardiovascular system of aged male rats.

Homocysteine (Hcy) is an independent cardiovascular disease (CVD) risk factor, whose mechanisms are poorly understood. We aimed to explore mild hyperhomocysteinemia (HHcy) effects on oxidative status, inflammatory, and cholinesterase parameters in aged male Wistar rats (365 days old). Rats received subcutaneous Hcy (0.03 µmol/g body weight) twice daily for 30 days, followed by euthanasia, blood collection and heart dissection 12 h after the last injection. Results revealed increased dichlorofluorescein (DCF) levels in the heart and serum, alongside decreased antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase), reduced glutathione (GSH) content, and diminished acetylcholinesterase (AChE) activity in the heart. Serum butyrylcholinesterase (BuChE) levels also decreased. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) protein content decreased in both cytosolic and nuclear fractions, while cytosolic nuclear factor kappa B (NFκB) p65 increased in the heart. Additionally, interleukins IL-1ß, IL-6 and IL-10 showed elevated expression levels in the heart. These findings could suggest a connection between aging and HHcy in CVD. Reduced Nrf2 protein content and impaired antioxidant defenses, combined with inflammatory factors and altered cholinesterases activity, may contribute to understanding the impact of Hcy on cardiovascular dynamics. This study sheds light on the complex interplay between HHcy, oxidative stress, inflammation, and cholinesterases in CVD, providing valuable insights for future research.

Homocysteine May Decrease Glucose Uptake and Alter the Akt/GSK3ß/GLUT1 Signaling Pathway in Hippocampal Slices: Neuroprotective Effects of Rivastigmine and Ibuprofen.

Homocysteine (Hcy) is a risk factor for neurodegenerative diseases, such as Alzheimer's Disease, and is related to cellular and tissue damage. In the present study, we verified the effect of Hcy on neurochemical parameters (redox homeostasis, neuronal excitability, glucose, and lactate levels) and the Serine/Threonine kinase B (Akt), Glucose synthase kinase-3ß (GSK3ß) and Glucose transporter 1 (GLUT1) signaling pathway in hippocampal slices, as well as the neuroprotective effects of ibuprofen and rivastigmine alone or in combination in such effects. Male Wistar rats (90 days old) were euthanized and the brains were dissected. The hippocampus slices were pre-treated for 30 min [saline medium or Hcy (30 µM)], then the other treatments were added to the medium for another 30 min [ibuprofen, rivastigmine, or ibuprofen + rivastigmine]. The dichlorofluorescein formed, nitrite and Na+, K+-ATPase activity was increased by Hcy at 30 µM. Ibuprofen reduced dichlorofluorescein formation and attenuated the effect of Hcy. The reduced glutathione content was reduced by Hcy. Treatments with ibuprofen and Hcy + ibuprofen increased reduced glutathione. Hcy at 30 µM caused a decrease in hippocampal glucose uptake and GLUT1 expression, and an increase in Glial Fibrillary Acidic Protein-protein expression. Phosphorylated GSK3ß and Akt levels were reduced by Hcy (30 µM) and co-treatment with Hcy + rivastigmine + ibuprofen reversed these effects. Hcy toxicity on glucose metabolism can promote neurological damage. The combination of treatment with rivastigmine + ibuprofen attenuated such effects, probably by regulating the Akt/GSK3ß/GLUT1 signaling pathway. Reversal of Hcy cellular damage by these compounds may be a potential neuroprotective strategy for brain damage.

Zika Virus affects neurobehavioral development, and causes oxidative stress associated to blood-brain barrier disruption in a rat model of congenital infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with several neurodevelopmental outcomes after in utero infection. Here, we studied a congenital ZIKV infection model with immunocompetent Wistar rats, able to predict disabilities and that could pave the way for proposing new effective therapies. We identified neurodevelopmental milestones disabilities in congenital ZIKV animals. Also, on 22nd postnatal day (PND), blood-brain barrier (BBB) proteins disturbances were detected in the hippocampus with immunocontent reduction of ß_Catenin, Occludin and Conexin-43. Besides, oxidative stress imbalance on hippocampus and cortex were identified, without neuronal reduction in these structures. In conclusion, even without pups' microcephaly-like phenotype, congenital ZIKV infection resulted in neurobehavioral dysfunction associated with BBB and oxidative stress disturbances in young rats. Therefore, our findings highlighted the multiple impact of the congenital ZIKV infection on the neurodevelopment, which reinforces the continuity of studies to understand the spectrum of this impairment and to provide support to future treatment development for patients affected by congenital ZIKV.

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.

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.

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-1ß, 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 offspringGraphical abstract 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 folic acid supplementation did not impair the mother-pup relationship. We showed that supplemented diet with 4 mg/kg diet of folic acid during pregnancy impairs memory and motricity of the offspring when compared with standard diet. It was also observed an increase in anxiety- and depression-like behavior in this group. Nitrative stress and neuroinflammation parameters were increased in the cerebral cortex of the offspring. ROS, reactive oxygen species.

Nanoemulsion Improves the Neuroprotective Effects of Curcumin in an Experimental Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor dysfunction. Recent studies have shown that curcumin (CUR) has neuroprotective effects in PD experimental models. However, its efficacy is limited due to low water solubility, bioavailability, and access to the central nervous system. In this study, we compared the effects of new curcumin-loaded nanoemulsions (NC) and free CUR in an experimental model of PD. Adult Swiss mice received NC or CUR (25 and 50 mg/kg) or vehicle orally for 30 days. Starting on the eighth day, they were administered rotenone (1 mg/kg) intraperitoneally until the 30th day. At the end of the treatment, motor assessment was evaluated by open field, pole test, and beam walking tests. Oxidative stress markers and mitochondrial complex I activity were measured in the brain tissue. Both NC and CUR treatment significantly improved motor impairment, reduced lipoperoxidation, modified antioxidant defenses, and prevented inhibition of complex I. However, NC was more effective in preventing motor impairment and inhibition of complex I when compared to CUR in the free form. In conclusion, our results suggest that NC effectively enhances the neuroprotective potential of CUR and is a promising nanomedical application for PD.

Hyperhomocysteinemia alters cytokine gene expression, cytochrome c oxidase activity and oxidative stress in striatum and cerebellum of rodents.

AIMS: Homocysteine has been linked to neurodegeneration and motor function impairments. In the present study, we evaluate the effect of chronic mild hyperhomocysteinemia on the motor behavior (motor coordination, functional performance, and muscular force) and biochemical parameters (oxidative stress, energy metabolism, gene expression and/or protein abundance of cytokine related to the inflammatory pathways and acetylcholinesterase) in the striatum and cerebellum of Wistar male rats. MAIN METHODS: Rodents were submitted to one injection of homocysteine (0.03 µmol Hcy/g of body weight) between 30th and 60th postnatal days twice a day. After hyperhomocysteinemia induction, rats were submitted to horizontal ladder walking, beam balance, suspension, and vertical pole tests and/or euthanized to brain dissection for biochemical and molecular assays. KEY FINDINGS: Chronic mild hyperhomocysteinemia did not alter motor function, but induced oxidative stress and impaired mitochondrial complex IV activity in both structures. In the striatum, hyperhomocysteinemia decreased TNF-α gene expression and increased IL-1ß gene expression and acetylcholinesterase activity. In the cerebellum, hyperhomocysteinemia increased gene expression of TNF-α, IL-1ß, IL-10, and TGF-ß, while the acetylcholinesterase activity was decreased. In both structures, hyperhomocysteinemia decreased acetylcholinesterase protein abundance without altering total p-NF-κB, NF-κB, Nrf-2, and cleaved caspase-3. SIGNIFICANCE: Chronic mild hyperhomocysteinemia compromises several biochemical/molecular parameters, signaling pathways, oxidative stress, and chronic inflammation in the striatum and cerebellum of rats without impairing motor function. These alterations may be related to the mechanisms in which hyperhomocysteinemia has been linked to movement disorders later in life and neurodegeneration.

In vivo toxicity evaluation of nanoemulsions for drug delivery.

Lipid nanocarriers (LNs), for example nanoemulsions (NE), are an emerging tool for drug delivery due to their ability to incorporate drugs, protect the drug from degradation, improve bioavailability, and control release. Although LNs are widely studied and applied, especially in the pharmaceutical field, knowledge about their toxicity is scarce. Moreover, the majority of studies focus on their efficiency rather than safety. Thus, the aim of this study was to evaluate the possible toxic effects of NE in vivo. Male Wistar rats (2 months old, 250 g) were treated once daily for 21 days with NE via oral or intraperitoneal delivery at 200, 400 or 800 mg lipid/kg body weight. At the end of the experiment, biochemical, hematological, oxidative stress, and genotoxicity parameters were analyzed. Our results showed that treatment with NE did not modify organ weight or biochemical parameters when compared to controls. The highest NE dose (800 mg/kg) via intraperitoneal injection caused changes in hematological parameters, namely increased plasma proteins, platelets, total leukocytes, and neutrophils, findings that suggest an inflammatory reaction. Further, the same dose evoked lipid peroxidation in the liver. Taken together, the results from this study suggest that NEs can be considered safe for oral administration, but high doses via the parenteral route can cause toxic effects. This study contributes to knowledge about NE toxicity and provides important data about their safe use in the pharmaceutical field.

Multimarker approach to assess the exposure of the wild rodent Calomys laucha to a simulated crude oil spill.

A mysterious oil spill occurred in the ocean near Brazil in 2019, which affected coastal areas in northeastern Brazil. When oil pollution occurs in coastal zones, organisms such as small mammals can suffer deleterious effects to their health. This study aimed to evaluate the effects of exposure to contaminated sandy soil with different crude oil concentrations in males of the species Calomys laucha. The exposure to crude oil resulted in multiple health issues for the subjects in the very first days of exposure. Furthermore, the exposure resulted in mutagenic damage to bone marrow blood cells and behavioral and morphological alterations, which were almost always in a dose-dependent form. The present study demonstrates the sensibility of the biomarkers used and highlights that small wild mammals such as C. laucha are useful for predicting environmental damage caused by the exposure to crude oil.