Microglial cells in astroglial cultures: a cautionary note

Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970's a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions

La fitxa molecular: una eina d'autoaprenentatge i d'avaluació continuada de Biologia Molecular

Podeu consultar la Vuitena trobada de professorat de Ciències de la Salut completa a: http://hdl.handle.net/2445/66524Tradicionalment la principal font de nou coneixement en Biologia Molecular han estat els articles científics. Cada vegada més, però, una part important d’aquesta nova informació es troba dipositada en bases de dades. Amb l’objectiu de familiaritzar l’estudiant tant amb els articles científics com amb les bases de dades de Biologia Molecular hem dissenyat una activitat d’avaluació continuada, anomenada Fitxa Molecular, pels estudiants de “Biologia Molecular” del grau de Medicina i de “Bioquímica” del grau d’Enginyeria Biomèdica. El treball consisteix en trobar una sèrie de dades sobre DNA, mRNA, proteïna i funció d’un gen assignat. El treball progressa durant el curs paral·lelament als continguts de les classes teòriques mitjançant una sessió inicial de presentació, 4 sessions de seguiment i introducció de noves tasques i una sessió final d’avaluació. El treball es realitza en grups de 3-4 estudiants, la presentació es fa de forma oral i la qualificació obtinguda suposa un 20% de la nota final de l’assignatura. En l’activitat es treballen les bases de dades NCBI..

Team Based Learning (TBL): una metodología de aprendizaje y evaluación continuada en Bioquímica

Podeu consultar la Vuitena trobada de professorat de Ciències de la Salut completa a: http://hdl.handle.net/2445/66524La estrategia de Aprendizaje Basado en Equipos (Team Based Learning, TBL) es una metodología estructurada de aprendizaje basada en el concepto de aula invertida. Los equipos están formados por 4-5 estudiantes agrupados al azar al inicio del curso. Los estudiantes trabajan los contenidos autónomamente y son evaluados en clase de forma individual (iRAT) y en equipo (tRAT) mediante pruebas de tipo test. El aprendizaje es aplicado a la resolución conjunta de problemas en equipo durante las clases. Al finalizar el curso los equipos se autoevalúan vía rúbrica. Esta estrategia de TBL se ha implementado en las asignaturas de Bioquímica (grado de Ingeniería Biomédica) y la asignatura optativa de Terapia personalizada y genómica (grados de Medicina y de Ciencias Biomédicas). Las actividades TBL han consistido en seminarios (visionado e interpretación de videos y en la resolución de dudas después de los bloques de teoría), en evaluación de contenidos de teoría (clases de repaso de conceptos adquiridos en otras asignaturas) y en la autoevaluación de los miembros del equipo respecto a los trabajos orales en grupo (ficha molecular, resolución de problemas clínicos). Estas actividades representan entre el 10 y el 20% de la nota final de las asignaturas. Esta nueva metodología TBL ha sido evaluada a través de una encuesta de satisfacción de los alumnos, siendo el resultado muy satisfactorio (7.7/10) y recomendado por la mayoría de los alumnos (60/64). Algunos aspectos remarcables de esta metodología son: • Participación activa de todos los alumnos resultando en clases más dinámicas. • Trabajo individual inicial con la posterior auto-revisión de conceptos por parte del equipo. • Mejores resultados en el aprendizaje por la acción del trabajo en equipo. • Autoevaluación y análisis crítico del trabajo realizado por los miembros del grupo. • Las actividades se adecuan al ritmo y objetivos de la evaluación continuada. Conclusión: Los resultados obtenidos y el interés del alumnado nos animan a mantener esta metodología de aprendizaje e intentar ampliarla a otras asignaturas

Alterations in CD200-CD200R1 System during EAE Already Manifest at Presymptomatic Stages

In the brain of patients with multiple sclerosis, activated microglia/macrophages appear in active lesions and in normal appearing white matter. However, whether they play a beneficial or a detrimental role in the development of the pathology remains a controversial issue. The production of pro-inflammatory molecules by chronically activated microglial cells is suggested to contribute to the progression of neurodegenerative processes in neurological disease. In the healthy brain, neurons control glial activation through several inhibitory mechanisms, such as the CD200-CD200R1 interaction. Therefore, we studied whether alterations in the CD200-CD200R1 system might underlie the neuroinflammation in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. We determined the time course of CD200 and CD200R1 expression in the brain and spinal cord of an EAE mouse model from presymptomatic to late symptomatic stages. We also assessed the correlation with associated glial activation, inflammatory response and EAE severity. Alterations in CD200 and CD200R1 expression were mainly observed in spinal cord regions in the EAE model, mostly a decrease in CD200 and an increase in CD200R1 expression. A decrease in the expression of the mRNA encoding a full CD200 protein was detected before the onset of clinical signs, and remained thereafter. A decrease in CD200 protein expression was observed from the onset of clinical signs. By contrast, CD200R1 expression increased at EAE onset, when a glial reaction associated with the production of pro- and anti-inflammatory markers occurred, and continued to be elevated during the pathology. Moreover, the magnitude of the alterations correlated with severity of the EAE mainly in spinal cord. These results suggest that neuronal-microglial communication through CD200-CD200R1 interaction is compromised in EAE. The early decreases in CD200 expression in EAE suggest that this downregulation might also occur in the initial phases of multiple sclerosis, and that this early neuronal dysfunction might facilitate the development of neuroinflammation. The increased CD200R1 expression in the EAE model highlights the potential use of targeted agonist molecules as therapeutic tools to control neuroinflammation. In summary, the CD200-CD200R1 system is a potential therapeutic target in multiple sclerosis, and CD200R1 agonists are molecules that may be worth developing in this context

The CD200R1 Microglial inhibitory receptor as a therapeutic target in the MPTP model of Parkinson's disease

Background It is suggested that neuroinflammation, in which activated microglial cells play a relevant role, contributes to the development of Parkinson's disease (PD). Consequently, the modulation of microglial activation is a potential therapeutic target to be taken into account to act against the dopaminergic neurodegeneration occurring in this neurological disorder. Several soluble and membrane-associated inhibitory mechanisms contribute to maintaining microglial cells in a quiescent/surveillant phenotype in physiological conditions. However, the presence of activated microglial cells in the brain in PD patients suggests that these mechanisms have been somehow overloaded. We focused our interest on one of the membrane-associated mechanisms, the CD200-CD200R1 ligand-receptor pair. Methods The acute MPTP experimental mouse model of PD was used to study the temporal pattern of mRNA expression of CD200 and CD200R1 in the context of MPTP-induced dopaminergic neurodegeneration and neuroinflammation. Dopaminergic damage was assessed by tyrosine hydroxylase (TH) immunoreactivity, and neuroinflammation was evaluated by the mRNA expression of inflammatory markers and IBA1 and GFAP immunohistochemistry. The effect of the modulation of the CD200-CD200R1 system on MPTP-induced damage was determined by using a CD200R1 agonist or CD200 KO mice. Results MPTP administration resulted in a progressive decrease in TH-positive fibres in the striatum and TH-positive neurons in the substantia nigra pars compacta, which were accompanied by transient astrogliosis, microgliosis and expression of pro- and anti-inflammatory markers. CD200 mRNA levels rapidly decreased in the ventral midbrain after MPTP treatment, while a transient decrease of CD200R1 mRNA expression was repeatedly observed in this brain area at earlier and later phases. By contrast, a transient increase in CD200R1 expression was observed in striatum. The administration of a CD200R1 agonist resulted in the inhibition of MPTP-induced dopaminergic neurodegeneration, while microglial cells showed signs of earlier activation in CD200-deficient mice. Conclusions Collectively, these findings provide evidence for a correlation between CD200-CD200R1 alterations, glial activation and neuronal loss. CD200R1 stimulation reduces MPTP-induced loss of dopaminergic neurons, and CD200 deficiency results in earlier microglial activation, suggesting that the potentiation of CD200R1 signalling is a possible approach to controlling neuroinflammation and neuronal death in PD

Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

Exposure to pesticides such as rotenone is a risk factor for Parkinson's disease. Dopaminergic neurons are especially sensitive to the toxicity of compounds that inhibit the mitochondrial respiratory chain such as rotenone and 1-methyl-4-phenylpyridinium (MPP+). However, there is scarce information on their effects on glia. To evaluate whether these neurotoxicants affect the immune response of glia, primary mouse mixed glial and microglial cultures were treated with interleukin (IL) 4 in the absence and presence of MPP+ or rotenone. Using qRTPCR or western blot, we determined the expression of anti-inflammatory markers, the CD200R1 microglial receptor and its ligand CD200, and genes regulating glycolysis and oxidative metabolism. ATP and lactate levels were additionally determined as an index of cell metabolism. Microglial phagocytosis was also evaluated. MPP+ and rotenone clearly abrogated the IL4-induced expression of anti-inflammatory markers in mixed glial cultures. CD200 and CD200R1 expression and microglia phagocytosis were also affected by the neurotoxicants. Changes in the mRNA expression of the molecules regulating glycolysis and oxidative metabolism, as well as in ATP levels and lactate release suggested that metabolic reprogramming in response to MPP+ and rotenone differs between microglial and mixed glial cultures. These findings support the hypothesis that parkinsonian neurotoxicants may impair brain immune response altering glial cell metabolism

RNA-Seq transcriptomic profiling of primary murine microglia treated with LPS or LPS+IFNγ

Microglia, the main resident immune cells in the CNS, are thought to participate in the pathogenesis of various neurological disorders. LPS and LPS + IFNγ are stimuli that are widely used to activate microglia. However, the transcriptomic profiles of microglia treated with LPS and LPS + IFNγ have not been properly compared. Here, we treated murine primary microglial cultures with LPS or LPS + IFNγ for 6 hours and then performed RNA-Sequencing. Gene expression patterns induced by the treatments were obtained by WGCNA and 11 different expression profiles were found, showing differential responses to LPS and LPS + IFNγ in many genes. Interestingly, a subset of genes involved in Parkinson's, Alzheimer's and Huntington's disease were downregulated by both treatments. By DESeq analysis we found differentially upregulated and downregulated genes that confirmed LPS and LPS + IFNγ as inducers of microglial pro-inflammatory responses, but also highlighted their involvement in specific cell functions. In response to LPS, microglia tended to be more proliferative, pro-inflammatory and phagocytic; whereas LPS + IFNγ inhibited genes were involved in pain, cell division and, unexpectedly, production of some inflammatory mediators. In summary, this study provides a detailed description of the transcriptome of LPS- and LPS + IFNγ treated primary microglial cultures. It may be useful to determine whether these in vitro phenotypes resemble microglia in in vivo pathological conditions

Gender analysis of the frequency and course of depressive disorders and relationship with personality traits in general population: a prospective cohort study

Depressió; Epidemiologia; PersonalitatDepresión; Epidemiología; PersonalidadDepression; Epidemiology; PersonalityBackground: We aimed to determine the prevalence and course of subthreshold depressive symptomatology (sDS) and probable major depressive episode (MDE) and to examine their association with personality traits among men and women. Methods: A community-based sample aged 35 years or older was examined in two waves (median follow-up of 6.9 years). The Patient Health Questionnaire-9 (PHQ-9) was used to assess sDS and MDE. The 10-item version of the Big Five Inventory was used to assess personality traits. Prevalence was assessed at baseline (n=5,557) and incidence and persistence-recurrence rates were computed at follow up (n=3,102). Logistic regression models were adjusted to explore the association of personality traits with prevalence and course of depressive disorders. Results: The prevalence of sDS and MDE was 14.04% (95% CI = 17.04-19.08) and 8.54 (95% CI=7.82-9.31), the incidence was 14.30 per 1,000 person-years (95% CI=12.49-16.31) and 4.34 per 1,000 person-years (95% CI=3.46-5.36), and the persistence-recurrence was 35.04 per 1,000 person-years (95% CI=29.00-41.96) and 28.8 per 1,000 person-years (95% CI=20.49-38.14). The gender gap was higher for MDE. Personality traits were differentially associated with the prevalence and course of depressive disorders between men and women. Limitations: Because this study used questionnaires to assess depressive disorders and personality traits, information bias could not be ruled out. Conclusions: The gender gap was higher for the prevalence and course of the probable MDE. There were more personality traits related with the course of the sDS and they had a major role in the course of the probable MDE in women.This study was supported by research grant STL006/17/00234 from the Strategic Plan for Health Research and Innovation (PERIS) 2016-2020 of the Department of Health. Government of Catalunya

Myeloid C/EBPβ deficiency reshapes microglial gene expression and is protective in experimental autoimmune encephalomyelitis

Background CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPβ show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPβ-expressing cell types is not solved. Since C/EBPβ-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPβ deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPβ deficiency. Methods Mice with myeloid C/EBPβ deficiency were generated by crossing LysMCre and C/EBPβfl/fl mice. Primary microglial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPβ deletion were analyzed in vivo in microglia isolated from the brains of C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPβfl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used. Results LysMCre-C/EBPβfl/fl mice showed an efficiency of C/EBPβ deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPβ deficiency. Transcriptomic analysis of C/EBPβ-deficient primary microglia revealed C/EBPβ-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPβ deletion. CNS expression of C/EBPβ was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPβfl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis. Conclusion This study provides new data that support a central role for C/EBPβ in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPβ inhibition in multiple sclerosis