Ana Dias-Carvalho

Methylphenidate (MPH) is first-line pharmacotherapy for Attention Deficit Hyperactivity Disorder (ADHD). Misdiagnosis and misuse raise concerns about exposing children and adolescents to MPH. This study aimed to assess how clinically relevant oral doses of MPH influence the expression of brain proteins involved in synaptic plasticity and neuronal growth in both sexes. Thirty-seven Wistar-Kyoto (WKY) rats (18 males and 19 females) were divided into an MPH group (daily oral dose of 5 mg/kg MPH in a 5% sucrose solution) and a control group (equivalent volume of 5% sucrose solution). Daily gavage administration started on postnatal day (PND) 15 and lasted for 15 days, with sacrifice at PND 30. In five brain regions [prefrontal cortex (PFC), striatum, hippocampus, cerebellum, and diencephalon], GAP43, GAPDH and PSD-95 levels were measured by Western blot. Additionally, MAP2 and synaptophysin levels were assessed in the PFC, motor cortex, ventral and dorsal striatum, and hippocampus (including CA1, CA3, hilus, and dentate gyrus) using immunohistochemistry. In MPH-treated males, GAP43 and synaptophysin levels were reduced in the cerebellum and CA1 region, respectively, while PSD-95 and GAPDH levels increased in the striatum and diencephalon. MPH-treated females showed only a significant decrease in PSD-95 levels in the PFC. Regarding MAP2 levels, no significant changes were observed in any of the analyzed regions or sexes. In control animals, males exhibited higher MAP2 levels in the striatum compared to females. In conclusion, MPH in healthy rats can alter proteins associated with synaptic plasticity differently, highlighting the importance of sex as a variable.

Life expectancy has increased immensely over the past decades, bringing new challenges to the health systems as advanced age increases the predisposition for many diseases. One of those is the burden of neurologic disorders. While many hypotheses have been placed to explain aging mechanisms, it has been widely accepted that the increasing pro-inflammatory status with advanced age or “inflammaging” is a main determinant of biological aging. Furthermore, inflammaging is at the cornerstone of many age-related diseases and its involvement in neurologic disorders is an exciting hypothesis. Indeed, aging and neurologic disorders development in the elderly seem to share some basic pathways that fundamentally converge on inflammation. Peripheral inflammation significantly influences brain function and contributes to the development of neurological disorders, including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. Understanding the role of inflammation in the pathogenesis of progressive neurological diseases is of crucial importance for developing effective treatments and interventions that can slow down or prevent disease progression, therefore, decreasing its social and economic burden.

Doxorubicin (DOX) is an anthracycline used to treat a wide range of tumours. Despite its effectiveness, it is associated to a long range of adverse effects of which cognitive deficits stand out. The present study aimed to assess the neurologic adverse outcome pathways of two clinically relevant cumulative doses of DOX. Adult male CD-1 mice received biweekly intraperitoneal administrations for 3 weeks until reaching cumulative doses of 9 mg/kg (DOX9) or 18 mg/kg (DOX18). Animals were euthanized one week after the last administration, and biomarkers of oxidative stress and brain metabolism were evaluated in the whole brain. Coronal sections of fixed brains were used for specific determinations on the prefrontal cortex (PFC) and hippocampal formation (HF). In the whole brain, DOX18 tended to disrupt the antioxidant defences, affecting glutathione levels and manganese superoxide dismutase (MnSOD) expression. Considering the regional analysis, DOX18 increased the volume of all brain areas evaluated, while GFAP-immunoreactive astrocytes decreased in the dentate gyrus (DG) and increased in the CA3 region of HF both in a dose-dependent manner. Concerning apoptosis pathway, whereas Bax increased in the DOX9 group, it decreased in the DOX18 group, but only in the latter group, Bcl-2 levels also decreased. While p53 only increased in the CA3 region of the DOX9 group, AIF increased in the PFC and DG of DOX18. Finally, phosphorylation of Tau decreased with the highest DOX dose in DG and CA3, while TNF-α levels increased in CA1 of DOX18. Our results indicate new pathways not yet described that could be responsible for the cognitive impairments observed in treated patients.

Mitoxantrone (MTX) is a topoisomerase II inhibitor used to treat a wide range of tumors and multiple sclerosis but associated with potential neurotoxic effects mediated by hitherto poorly understood mechanisms. In adult male CD-1 mice, the underlying neurotoxic pathways of a clinically relevant cumulative dose of 6 mg/kg MTX was evaluated after biweekly administration for 3 weeks and sacrifice 1 week after the last administration was undertaken. Oxidative stress, neuronal damage, apoptosis, and autophagy were analyzed in whole brain, while coronal brain sections were used for a closer look in the hippocampal formation (HF) and the prefrontal cortex (PFC), as these areas have been signaled out as the most affected in ‘chemobrain’. In the whole brain, MTX-induced redox imbalance shown as increased endothelial nitric oxide synthase and reduced manganese superoxide dismutase expression, as well as a tendency to a decrease in glutathione levels. MTX also caused diminished ATP synthase β expression, increased autophagic protein LC3 II and tended to decrease p62 expression. Postsynaptic density protein 95 expression decreased in the whole brain, while hyperphosphorylation of Tau was seen in PFC. A reduction in volume was observed in the dentate gyrus (DG) and CA1 region of the HF, while GFAP-ir astrocytes increased in all regions of the HF except in the DG. Apoptotic marker Bax increased in the PFC and in the CA3 region, whereas p53 decreased in all brain areas evaluated. MTX causes damage in the brain of adult CD-1 mice in a clinically relevant cumulative dose in areas involved in memory and cognition.

Cognitive dysfunction has been one of the most reported and studied adverse effects of cancer treatment, but, for many years, it was overlooked by the medical community. Nevertheless, the medical and scientific communities have now recognized that the cognitive deficits caused by chemotherapy have a strong impact on the morbidity of cancer treated patients. In fact, chemotherapy-induced cognitive dysfunction or ‘chemobrain’ (also named also chemofog) is at present a well-recognized effect of chemotherapy that could affect up to 78% of treated patients. Nonetheless, its underlying neurotoxic mechanism is still not fully elucidated. Therefore, this work aimed to provide a comprehensive review using PubMed as a database to assess the studies published on the field and, therefore, highlight the clinical manifestations of chemobrain and the putative neurotoxicity mechanisms. In the last two decades, a great number of papers was published on the topic, mainly with clinical observations. Chemotherapy-treated patients showed that the cognitive domains most often impaired were verbal memory, psychomotor function, visual memory, visuospatial and verbal learning, memory function and attention. Chemotherapy alters the brain’s metabolism, white and grey matter and functional connectivity of brain areas. Several mechanisms have been proposed to cause chemobrain but increase of proinflammatory cytokines with oxidative stress seem more relevant, not excluding the action on neurotransmission and cellular death or impaired hippocampal neurogenesis. The interplay between these mechanisms and susceptible factors makes the clinical management of chemobrain even more difficult. New studies, mainly referring to the underlying mechanisms of chemobrain and protective measures, are important in the future, as it is expected that chemobrain will have more clinical impact in the coming years, since the number of cancer survivors is steadily increasing.

Doxorubicin (DOX) and mitoxantrone (MTX) are two chemotherapeutic drugs, inhibitors of topoisomerase II that are used against several types of cancer. Regardless of their efficacy in cancer treatment, these drugs can cause long-term toxicity in several organs. The chemotherapy-induced cognitive impairment or ‘chemobrain’ has been widely demonstrated in neuropsychological testing in cancer patients, even several years after treatment. Nonetheless, the underlying mechanisms of chemobrain are still not fully understood. Our work aimed to evaluate the effects of clinically relevant cumulative doses of DOX or MTX on the brain of male CD-1 mice at 3 different ages [juvenile (4 weeks), adult (3 months) and old (18-20 months)]. Mice received intraperitoneal injections of the drugs or a saline solution (control groups), twice a week, for 3 weeks. Between different aged groups, MTX-groups received a total cumulative dose of MTX 6 mg/kg. Regarding DOX, infant and adults received a cumulative dose of DOX 18 mg/kg, while the old group received a total cumulative dose of DOX 9 mg/kg. During the entire experimental procedure mice well-being was monitored, as well as food and water consumption. Mice were euthanized one week (adults and old mice) or seventeen days (juvenile mice) after the last injection and several determinations were done. Concerning the brain’s redox status, total glutathione (GSHt), reduced glutathione (GSH) and GSH/oxidized glutathione (GSSG) ratio decreased in DOX adults, but in DOX infant brains despite receiving an equal cumulative dose no changes were seen. Nevertheless, DOX increased brain ATP levels in juvenile mice. MTX did not cause significant changes neither in glutathione nor in ATP brain levels in the groups tested. An extra experimental procedure was done with adult mice, where the left brain hemisphere was fixated in 4% paraformaldehyde, mounted in a vibratome and sectioned in the coronal plane throughout its extent. A systematic random sampling of the coronal sections was done at 480 µm interval. The sections obtained are being used for immunofluorescent detection of phosphorylated Tau, glial fibrillary acidic protein, BAX and p53 proteins in the hippocampus formation. In conclusion, our work showed that DOX and MTX differentially impact the brain and that DOX evoked damage might be dependent on animals’ age. Ackowledgments: ARM and VMC acknowledge Fundação da Ciência e Tecnologia (FCT) for their grants (SFRH/BD/129359/2017 and SFRH/BPD/110001/2015). Work supported by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by national funds by the FCT within the project “PTDC/DTP-FTO/1489/2014 – POCI-01-0145-FEDER-016537”.