A model of epileptogenesis in rhinal cortex-hippocampus organotypic slice cultures

Copyright © 2021 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported LicenseOrganotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug's neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation. Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement Nº 952455, Fundação para a Ciência e Tecnologia (FCT) through Project PTDC/MEDFAR/30933/2017, and Faculdade de Medicina da Universidade de Lisboainfo:eu-repo/semantics/publishedVersio

Helminth component community of the loggerhead sea Turtle,Caretta caretta from Madeira Archipelago, Portugal

The helminth fauna of pelagic-stage loggerhead sea turtles, Caretta caretta, is still poorly known. Here, we describe the helminth-component community of healthy, free-ranging juvenile loggerhead sea turtles captured in the waters around Madeira Island, Portugal. Fifty-seven were used in this study. The esophagus, stomach, intestine, liver, gallbladder, spleen, kidneys, trachea, bronchi, urinary bladder, heart, left and right aortas, and coelomic cavity were macroscopically inspected; organs and tissues were removed and washed through a sieve. A search for parasites was made using a stereoscopic microscope; recovered parasites were fixed and stored in 70% alcohol until staining and identification. Prevalence, mean intensity, and mean abundance values were recorded. In total, 156 parasite specimens belonging to 9 species were found: nematodes included Anisakis simplex s.l. (larvae) and an unidentified species; digenetic trematodes present were Enodiotrema megachondrus, Rhytidodes gelatinosus, Pyelosomum renicapite, and Calycodes anthos; acanthocephalans included Bolbosoma vasculosum and Rhadinorhynchus pristis; a single cestode, Nybelinia sp., was present. Parasite infections were found to have both low prevalences and intensities. Possible reasons for this include the oligotrophic conditions of the pelagic habitat around Madeira; a 'dilution effect' because of the vastness of the area; and the small size, and thus ingestion rate, of the turtles. Results are discussed in terms of the various turtle populations that may use the waters surrounding Madeira. This work provides valuable information on the parasite fauna of a poorly known stage in the life of loggerhead sea turtles, thereby filling a fundamental gap with regard to features of the parasite fauna in this species.info:eu-repo/semantics/publishedVersio

Caracterização ao corte da ligação entre conector de GFRP e betão auto-compactável reforçado com fibras de aço

A utilização de compósitos poliméricos reforçados com fibras (FRP) mostra-se bastante competitiva em aplicações específicas de engenharia civil. No caso de elementos estruturais mistos, de que são exemplo os painéis sandwich constituídos por duas lâminas de betão separadas por uma camada de isolamento ou as vigas mistas com perfil em FRP e laje de betão, é necessário utilizar conectores capazes de garantir o funcionamento conjunto dos elementos que constituem a secção, de forma a assegurar a transferência de esforços entre eles. Os conectores metálicos são a solução mais corrente em painéis sandwich, mas o uso de conectores de FRP apresenta potenciais vantagens que resultam das propriedades mais relevantes deste material: baixa condutibilidade térmica, baixo peso específico, imunidade à corrosão, bom amortecimento ao choque e bom isolamento eletromagnético. Neste trabalho, apresenta-se um novo tipo de conector de polímero reforçado com fibras de vidro (GFRP) a ser empregue em painéis de betão estruturais do tipo sandwich, e descrevem-se os ensaios experimentais preliminares realizados na Universidade do Minho para caracterizar o comportamento ao corte da respetiva ligação. Nos ensaios apresentados são utilizados conectores planos perfurados, embebidos em camadas de betão auto-compactável reforçado com fibras de aço (BACRFA). A utilização de BACRFA tem como objetivo dispensar a utilização de armaduras convencionais e a vibração do betão, o que se torna particularmente vantajoso quando a camada de betão tem uma espessura reduzida. De acordo com ensaios anteriormente realizados pelos autores, a utilização de BACRFA contribui ainda para aumentar a ductilidade da ligação. A configuração de ensaio utilizada baseia-se nas recomendações relativas ao ensaio de push-out descrito na NP EN1994-1-1, com algumas adaptações específicas. Por fim, o comportamento do conector testado é caracterizado pelos modos de rotura que lhe estão associados, pela curva carga versus escorregamento obtida no decorrer do ensaio e pela avaliação de parâmetros relevantes como a carga máxima.Fundação para a Ciência e a Tecnologia (FCT) e QREN - Quadro de Referência Estratégico Naciona

Failure modes in filled hole GFRP laminates for connections between FRP and concrete

The PERFOFRP connector consists on a perforated glass fibre reinforce polymer (GFRP) rib that is specially designed to perform the connection between thin concrete layers, which can be also useful in the context of structural rehabilitation. It stays embedded in the concrete layers, avoiding the use of complementary connecting techniques, like special gluing or bolting. This type of connection is similar to the Perfobond steel connectors proposed for steel and concrete composites structures. Perfobond shear connector failure depends on the concrete dowels load capacity, since the steel connector failure does not occur in ribs with adequate thickness. However, in the case of the PERFOFRP connectors, the load capacity can be limited by the failure of the connector itself. To achieve a better understanding on the behaviour of the GFRP connector, an experimental study, based on tests with perforated GFRP laminates submitted to pin-bearing tests, is carried out. The tests are performed with four different rectangular GFRP plates containing a circular hole of 30 mm diameter, and differing from each other in terms of stacking and amount of fibre plies. Two different geometries of specimens are studied to enhance the evaluation of two expected failure modes. Finally, the failure modes, the peak loads and the evolution of strain in the vicinity of the holes are presented and discussed.QREN-ADI, Fundação para a Ciência e a Tecnologia (FCT

Changes in adenosine receptors and neurotrophic factors in the SOD1G93A mouse model of amyotrophic lateral sclerosis: modulation by chronic caffeine

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of corticospinal tract motor neurons. Previous studies showed that adenosine-mediated neuromodulation is disturbed in ALS and that vascular endothelial growth factor (VEGF) has a neuroprotective function in ALS mouse models. We evaluated how adenosine (A1R and A2AR) and VEGF (VEGFA, VEGFB, VEGFR-1 and VEGFR-2) system markers are altered in the cortex and spinal cord of pre-symptomatic and symptomatic SOD1G93A mice. We then assessed if/how chronic treatment of SOD1G93A mice with a widely consumed adenosine receptor antagonist, caffeine, modulates VEGF system and/or the levels of Brain-derived Neurotrophic Factor (BDNF), known to be under control of A2AR. We found out decreases in A1R and increases in A2AR levels even before disease onset. Concerning the VEGF system, we detected increases of VEGFB and VEGFR-2 levels in the spinal cord at pre-symptomatic stage, which reverses at the symptomatic stage, and decreases of VEGFA levels in the cortex, in very late disease states. Chronic treatment with caffeine rescued cortical A1R levels in SOD1G93A mice, bringing them to control levels, while rendering VEGF signaling nearly unaffected. In contrast, BDNF levels were significantly affected in SOD1G93A mice treated with caffeine, being decreased in the cortex and increased in spinal the cord. Altogether, these findings suggest an early dysfunction of the adenosinergic system in ALS and highlights the possibility that the negative influence of caffeine previously reported in ALS animal models results from interference with BDNF rather than with the VEGF signaling molecules.info:eu-repo/semantics/publishedVersio

Immersive training weeks in doctoral education

Ph.D. training worldwide, including Doctoral education in Marketing or Engineering fields, has been in trouble for some time. These last turbulent times (pandemic, energy, inflation, and war crises) have only increased the problems previously reported by the 3rd cycle students and early career researchers, including chronic lack of support and poor-quality supervision, with senior researchers rarely trained in mentorship. It is also reported that Ph.D. candidates are inadequately prepared for the cross-disciplinary working and large teams that characterize cutting-edge science today. In the last two decades, opposite decisions took place in Europe concerning the curricula of Doctoral programs. In the 2010s, a large number of classes was added to the Ph.D.s, contributing to almost residual time for thesis research in the first year of the programs. However, ten years later, an abrupt change took place and almost all classes were removed from the Ph.D. curricula, creating a void in (hard and soft skills) training and leaving all the responsibility of training to the supervisor. Ph.D. students reported guidance and isolation issues in the first year. Moreover, today’s little Ph.D. training is fully dedicated to the obtention of their Ph.D. and not to their role in society after the Ph.D. defense. This work discusses a new approach to doctoral education which started first at a professional doctorate implemented at University of Aveiro, Portugal, where few classes take place. This approach considers a novel Ph.D. training, both hard and soft skills development, through special intensive weeks, called Immersive Weeks. In these, distributed during the first year, Ph.D. students exclusively participate in several workshops, acquiring the tools for accomplishing both a successful Ph.D. and a future job. Pilots of this approach took place at the University of Aveiro with large success, while some improvement suggestions have also been pointed out by students.publishe

Age-related impact of social isolation in mice: Young vs middle-aged

Social isolation is a chronic mild stressor and a significant risk factor for mental health disorders. Herein we explored the impact of social isolation on depression- and anxiety-like behaviours, as well as spatial memory impairments, in middle-aged male mice compared to post-weaning mice. We aimed to quantify and correlate social isolation-induced behaviour discrepancies with changes in hippocampal glial cell reactivity and pro-inflammatory cytokine levels.Post-weaning and middle-aged C57BL7/J6 male mice were socially isolated for a 3-week period and behavioural tests were performed on the last five days of isolation. We found that 3 weeks of social isolation led to depressive-like behaviour in the forced swim test, anxiety-like behaviour in the open field test, and spatial memory impairment in the Morris water maze paradigm in middle-aged male mice. These behavioural alterations were not observed in male mice after post-weaning social isolation, indicating resilience to isolation-mediated stress.Increased Iba-1 expression and NLRP3 priming were both observed in the hippocampus of socially isolated middle-aged mice, suggesting a role for microglia and NLRP3 pathway in the detrimental effects of social isolation on cognition and behaviour. Young socially isolated mice also demonstrated elevated NLRP3 priming compared to controls, but no differences in Iba-1 levels and no significant changes in behaviour. Ageing-induced microglia activation and enhancement of IL-1β, TNF-α and IL-6 proinflammatory cytokines, known signs of a chronic low-grade inflammatory state, were also detected.Altogether, data suggest that social isolation, in addition to inflammaging, contributes to stress-related cognitive impairment in middle-aged mice

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

The sphingosine 1‐phosphate analogue, FTY720, modulates the lipidomic signature of the mouse hippocampus

The small‐molecule drug, FTY720 (fingolimod), is a synthetic sphingosine 1‐phosphate (S1P) analogue currently used to treat relapsing–remitting multiple sclerosis in both adults and children. FTY720 can cross the blood–brain barrier (BBB) and, over time, accumulate in lipid‐rich areas of the central nervous system (CNS) by incorporating into phospholipid membranes. FTY720 has been shown to enhance cell membrane fluidity, which can modulate the functions of glial cells and neuronal populations involved in regulating behaviour. Moreover, direct modulation of S1P receptor‐mediated lipid signalling by FTY720 can impact homeostatic CNS physiology, including neurotransmitter release probability, the biophysical properties of synaptic membranes, ion channel and transmembrane receptor kinetics, and synaptic plasticity mechanisms. The aim of this study was to investigate how chronic FTY720 treatment alters the lipid composition of CNS tissue in adolescent mice at a key stage of brain maturation. We focused on the hippocampus, a brain region known to be important for learning, memory, and the processing of sensory and emotional stimuli. Using mass spectrometry‐based lipidomics, we discovered that FTY720 increases the fatty acid chain length of hydroxy‐phosphatidylcholine (PCOH) lipids in the mouse hippocampus. It also decreases PCOH monounsaturated fatty acids (MUFAs) and increases PCOH polyunsaturated fatty acids (PUFAs). A total of 99 lipid species were up‐regulated in the mouse hippocampus following 3 weeks of oral FTY720 exposure, whereas only 3 lipid species were down‐regulated. FTY720 also modulated anxiety‐like behaviours in young mice but did not affect spatial learning or memory formation. Our study presents a comprehensive overview of the lipid classes and lipid species that are altered in the hippocampus following chronic FTY720 exposure and provides novel insight into cellular and molecular mechanisms that may underlie the therapeutic or adverse effects of FTY720 in the central nervous system