Ultrastructural characterization of human oligodendrocytes and their progenitor cells by pre-embedding immunogold.

Oligodendrocytes are the myelinating cells of the central nervous system. They provide trophic, metabolic, and structural support to neurons. In several pathologies such as multiple sclerosis (MS), these cells are severely affected and fail to remyelinate, thereby leading to neuronal death. The gold standard for studying remyelination is the g-ratio, which is measured by means of transmission electron microscopy (TEM). Therefore, studying the fine structure of the oligodendrocyte population in the human brain at different stages through TEM is a key feature in this field of study. Here we study the ultrastructure of oligodendrocytes, its progenitors, and myelin in 10 samples of human white matter using nine different markers of the oligodendrocyte lineage (NG2, PDGFRα, A2B5, Sox10, Olig2, BCAS1, APC-(CC1), MAG, and MBP). Our findings show that human oligodendrocytes constitute a very heterogeneous population within the human white matter and that its stages of differentiation present characteristic features that can be used to identify them by TEM. This study sheds light on how these cells interact with other cells within the human brain and clarify their fine characteristics from other glial cell types

Wnt-Dependent Oligodendroglial-Endothelial Interactions Regulate White Matter Vascularization and Attenuate Injury.

Recent studies have indicated oligodendroglial-vascular crosstalk during brain development, but the underlying mechanisms are incompletely understood. We report that oligodendrocyte precursor cells (OPCs) contact sprouting endothelial tip cells in mouse, ferret, and human neonatal white matter. Using transgenic mice, we show that increased or decreased OPC density results in cognate changes in white matter vascular investment. Hypoxia induced increases in OPC numbers, vessel density and endothelial cell expression of the Wnt pathway targets Apcdd1 and Axin2 in white matter, suggesting paracrine OPC-endothelial signaling. Conditional knockout of OPC Wntless resulted in diminished white matter vascular growth in normoxia, whereas loss of Wnt7a/b function blunted the angiogenic response to hypoxia, resulting in severe white matter damage. These findings indicate that OPC-endothelial cell interactions regulate neonatal white matter vascular development in a Wnt-dependent manner and further suggest this mechanism is important in attenuating hypoxic injury.M.C. acknowledges fellowship awards from the American Heart Association and The Children’s Heart Foundation and funding support from a Career Development Grant awarded by Cerebral Palsy Alliance Research Foundation. J.M.G.V is funded by Red deTerapia Celular (TerCel-RD16/0011/0026) and the Valencian Council for Innovation, Universities Science and Digital Society (PROMETEO/2019/075). M.J.U.N was supported by a McDonald Fellowship from the Multiple Sclerosis International Federation. This work was supported by funding from the National Multiple Sclerosis Foundation (to D.H.R.), the Adelson Medical Research Foundation (D.H.R), the European Research Council (D.H.R.) and the National Institutes of Health, NINDS (1K99NS117804 to M.C; P01- NS083513 to D.H.R., E.J.H and P.S.M)

Hallazgos histológicos y ultraestructurales en áreas de displasia cortical focal de pacientes pediátricos intervenidos por epilepsia refractaria

Las malformaciones del desarrollo cortical (MDC) comprenden un amplio grupo de enfermedades malformativas del desarrollo de la corteza cerebral, que pueden conducir a crisis epilépticas, habitualmente refractarias a tratamiento médico, en la infancia o la juventud. La displasia cortical focal (DCF) es reconocida como una de las MDC más epileptogénica y se clasifica en tres subtipos dependiendo del tipo de diseminación cortical (radial o tangencial) y la presencia o ausencia de alteraciones citológicas (neuronas dismórficas y/o células en balón). Muchas proteínas relacionadas con la migración neuronal y neurogénesis, como la Doblecortina (DCX) se han estudiado en este tipo de patologías. La DCX es una proteína asociada a microtúbulos ampliamente utilizada para marcar neuronas migratorias (radiales y tangenciales) durante el desarrollo cortical, así como neuronas inmaduras en nichos neurogénicos postnatales en roedores y humanos. En pacientes epilépticos, se ha descrito la presencia de células DCX positivas en casos de DCF y de epilepsia del lóbulo temporal. Sin embargo, debido a la dificultad de una fijación óptima de las muestras humanas, la ultraestructura de estas células y el medio que les rodea sigue siendo una incógnita. El objetivo de nuestro estudio es analizar y describir la estructura, contactos específicos y medio que envuelve a células neuronales y gliales en un grupo de once pacientes pediátricos diagnosticados de DCF y esclerosis tuberosa (ET) con epilepsia parcial compleja e intervenidos mediante craneotomía y exéresis de la lesión. Las muestras fueron fijadas inmediatamente después de la cirugía y se procesaron para estudios histológicos, de inmunohistoquímica, marcaje mediante inmuno-oro y examen al microscopio electrónico de transmisión (MET). En la IHQ se utilizaron marcadores de estirpe neuronal: DCX, NeuN, Parvalbúmina (PV), sinaptopfisina y neurofilamentos (SMI32); y marcadores de estirpe glial: GFAP, Vimentina, Olig2, Iba1. Las técnicas de inmuno-oro se realizaron con los marcadores DCX, GFAP, Olig2 e Iba1 para un análisis más exhaustivo al MET. Además, realizamos un recuento de conexiones sinápticas en las muestras de displasia y control, con el objetivo de encontrar diferencias entre ellos. Nuestros resultados sugieren que la ultraestructura de las células DCX positivas es bastante heterogénea y engloba a neuronas inmaduras, maduras y dismórficas, así como también células en balón y algunos astrocitos. De este modo, mediante el estudio de todos estos hallazgos bajo visión al MET podemos esclarecer cómo la expresión anómala de DCX por ciertos tipos celulares puede estar relacionada con la epilepsia.Malformations of cortical development (MCDs) comprise a set of malformative diseases of the cortical brain development, that can result into refractory epilepsy seizures in the childhood. Focal cortical dysplasia (FCD) is recognized as one of the most epileptogenic MCD and is classified in three subtypes depending of the type of dyslamination of the cortex (radial or tangential) and the presence or absence of cytologic alterations (dysmorphic neurons and/or ballon cells). Many proteins related to neuronal migration and neurogenesis such as Doublecortin (DCX) have been studied in these pathologies. DCX is a microtubule associated protein, which has been used widely to label radially and tangentially migrating neurons in development and immature neurons in the postnatal neurogenic niches in rodents and humans. In epilepsy patients, the presence of DCX- positive cells has been reported in FCD and in temporal lobe epilepsy. However, due to the lack of optimal fixation in human samples, the ultrastructure of these cells and the environment surrounding them remains elusive. The objective of our study is to analyze and describe the fine structure, specific contacts and surrounding environment of neuronal and glial cells in a group of eleven pediatric patients who presented FCD and tuberous sclerosis (TE) with partial complex epilepsy and underwent through surgery by craniotomy and lesion scission. Samples were fixed immediately after surgery and processed for histological analysis, immunohistochemistry, immunogold labeling and transmission electron microscopy (TEM). Immunohistochemistry was performed for neuronal lineage markers: DCX, NeuN, Parvalbumin (PV), synaptophysin and neurofilament (SMI32); and glial lineage markers: GFAP, Vimentin, Olig2, Iba1. Immunogold labelling was performed for DCX, GFAP, Olig2 and Iba1 for further TEM analysis. Moreover we counted synaptic contacts in dysplastic and control samples, in order to find differences between them. Our results suggest that the ultrastructure of DCX cells is quite heterogeneous and involves immature, mature and dysmorphic neurons, as well as balloon cells and astrocytes. Thus, exploring all these features through TEM can shed light on how the abnormal expression of DCX in certain cell types can be related to epilepsy