María Dolores López-Ávalos

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies.

Bovine Reissner′s fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen‐mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF‐glycoproteins. Twenty‐three Mabs recognizing the bovine RF‐ and SCO‐glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non‐mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti‐RF serum. It is concluded that all Mabs raised in our laboratory are directed against non‐repeated sequences of RF‐glycoproteins that have not been conserved in vertebrate phylogeny. Microsc. Res. Tech. 52:510–519, 2001.

The subcommissural organ and the development of the posterior commissure.

Growing axons navigate through the developing brain by means of axon guidance molecules. Intermediate targets producing such signal molecules are used as guideposts to find distal targets. Glial, and sometimes neuronal, midline structures represent intermediate targets when axons cross the midline to reach the contralateral hemisphere. The subcommissural organ (SCO), a specialized neuroepithelium located at the dorsal midline underneath the posterior commissure, releases SCO-spondin, a large glycoprotein belonging to the thrombospondin superfamily that shares molecular domains with axonal pathfinding molecules. Several evidences suggest that the SCO could be involved in the development of the PC. First, both structures display a close spatiotemporal relationship. Second, certain mutants lacking an SCO present an abnormal PC. Third, some axonal guidance molecules are expressed by SCO cells. Finally, SCO cells, the Reissners fiber (the aggregated form of SCO-spondin), or synthetic peptides from SCO-spondin affect the neurite outgrowth or neuronal aggregation in vitro.

Microglia Morphological Categorization in a Rat Model of Neuroinflammation by Hierarchical Cluster and Principal Components Analysis

It is known that microglia morphology and function are closely related, but only few studies have objectively described different morphological subtypes. To address this issue, morphological parameters of microglial cells were analyzed in a rat model of aseptic neuroinflammation. After the injection of a single dose of the enzyme neuraminidase (NA) within the lateral ventricle (LV) an acute inflammatory process occurs. Sections from NA-injected animals and sham controls were immunolabeled with the microglial marker IBA1, which highlights ramifications and features of the cell shape. Using images obtained by section scanning, individual microglial cells were sampled from various regions (septofimbrial nucleus, hippocampus and hypothalamus) at different times post-injection (2, 4 and 12 h). Each cell yielded a set of 15 morphological parameters by means of image analysis software. Five initial parameters (including fractal measures) were statistically different in cells from NA-injected rats (most of them IL-1β positive, i.e., M1-state) compared to those from control animals (none of them IL-1β positive, i.e., surveillant state). However, additional multimodal parameters were revealed more suitable for hierarchical cluster analysis (HCA). This method pointed out the classification of microglia population in four clusters. Furthermore, a linear discriminant analysis (LDA) suggested three specific parameters to objectively classify any microglia by a decision tree. In addition, a principal components analysis (PCA) revealed two extra valuable variables that allowed to further classifying microglia in a total of eight sub-clusters or types. The spatio-temporal distribution of these different morphotypes in our rat inflammation model allowed to relate specific morphotypes with microglial activation status and brain location. An objective method for microglia classification based on morphological parameters is proposed. Main points Microglia undergo a quantifiable morphological change upon neuraminidase induced inflammation. Hierarchical cluster and principal components analysis allow morphological classification of microglia. Brain location of microglia is a relevant factor.

The subcommissural organ and the development of the posterior commissure in chick embryos

The subcommissural organ (SCO) is an ependymal differentiation located in the diencephalon under the posterior commissure (PC). SCO-spondin, a glycoprotein released by the SCO, belongs to the thrombospondin superfamily and shares molecular domains with axonal pathfinding molecules. Several lines of evidence suggest a relationship between the SCO and the development of the PC in the chick: (1) their close location to each other, (2) their differentiation at the same developmental stage in the chick, (3) the abnormal PC found in null mutants lacking an SCO and (4) the release by the SCO of SCO-spondin. By application of DiI crystals in the PC of chick embryos, we have identified the neurons that give rise to the PC. Labelling is confined to the magnocellular nucleus of the PC (MNPC). To gain insight into the role of the SCO in PC development, coculture experiments of explants of the MNPC region (MNPCr) from embryos at embryonic day 4 (E4) with SCO explants from E4 or E13 embryos have been performed and the neurite outgrowth from the MNPCr explants has been analysed. In the case of coculture of E4 MNPCr with E4 SCO, the number of neurites growing from the MNPCr is higher at the side facing the SCO. However, when E4 MNPCr and E13 SCO are cocultured, the neurites grow mostly at the side opposite to the SCO. These data suggest that, at early stages of development, the SCO releases some attractive or permissive molecule(s) for the growing of the PC, whereas at later stages, the SCO has a repulsive effect over neurites arising from MNPCr.

Neuroinflammation induced by intracerebroventricular injection of microbial neuraminidase

In the present paper, we describe the facts that took place in the rat brain after a single injection of the enzyme neuraminidase from Clostridium perfringens into the right lateral ventricle. After injection, it diffused through the cerebrospinal fluid of the ipsilateral ventricle and the third ventricle, and about 400 μm into the periventricular brain parenchyma. The expression of ICAM1 in the endothelial cells of the periventricular vessels, IBA1 in microglia, and GFAP in astrocytes notably increased in the regions reached by the injected neuraminidase. The subependymal microglia and the ventricular macrophages begun to express IL1β and some appeared to cross the ependymal layer. After about 4 h of the injection, leukocytes migrated from large venules of the affected choroid plexus, the meninges and the local subependyma, and infiltrated the brain. The invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last CD8α-positive T-lymphocytes and B-lymphocytes. Leukocytes in the ventricles and the perivascular zones penetrated the brain parenchyma passing through the ependyma and the glia limitans. Thus, it is likely that a great part of the damage produced by microorganism invading the brain may be due to their neuraminidase content.

Complement system activation contributes to the ependymal damage induced by microbial neuraminidase

BackgroundIn the rat brain, a single intracerebroventricular injection of neuraminidase from Clostridium perfringens induces ependymal detachment and death. This injury occurs before the infiltration of inflammatory blood cells; some reports implicate the complement system as a cause of these injuries. Here, we set out to test the role of complement.MethodsThe assembly of the complement membrane attack complex on the ependymal epithelium of rats injected with neuraminidase was analyzed by immunohistochemistry. Complement activation, triggered by neuraminidase, and the participation of different activation pathways were analyzed by Western blot. In vitro studies used primary cultures of ependymal cells and explants of the septal ventricular wall. In these models, ependymal cells were exposed to neuraminidase in the presence or absence of complement, and their viability was assessed by observing beating of cilia or by trypan blue staining. The role of complement in ependymal damage induced by neuraminidase was analyzed in vivo in two rat models of complement blockade: systemic inhibition of C5 by using a function blocking antibody and testing in C6-deficient rats.ResultsThe complement membrane attack complex immunolocalized on the ependymal surface in rats injected intracerebroventricularly with neuraminidase. C3 activation fragments were found in serum and cerebrospinal fluid of rats treated with neuraminidase, suggesting that neuraminidase itself activates complement. In ventricular wall explants and isolated ependymal cells, treatment with neuraminidase alone induced ependymal cell death; however, the addition of complement caused increased cell death and disorganization of the ependymal epithelium. In rats treated with anti-C5 and in C6-deficient rats, intracerebroventricular injection of neuraminidase provoked reduced ependymal alterations compared to non-treated or control rats. Immunohistochemistry confirmed the absence of membrane attack complex on the ependymal surfaces of neuraminidase-exposed rats treated with anti-C5 or deficient in C6.ConclusionsThese results demonstrate that the complement system contributes to ependymal damage and death caused by neuraminidase. However, neuraminidase alone can induce moderate ependymal damage without the aid of complement.

Pharmacological blockade of fatty acid amide hydrolase (FAAH) by URB597 improves memory and changes the phenotype of hippocampal microglia despite ethanol exposure

Graphical abstract Figure. No Caption available. ABSTRACT Changes in endogenous cannabinoid homeostasis are associated with both ethanol‐related neuroinflammation and memory decline. Extensive research is still required to unveil the role of endocannabinoid signaling activation on hippocampal microglial cells after ethanol exposure. Either microglial morphology, phenotype and recruitment may become notably altered after chronic alcohol‐related neurodegeneration. Here, we evaluated the pharmacological effects of fatty‐acid amide‐hydrolase (FAAH) inhibitor URB597 (0.3 mg/kg), oleoylethanolamide (OEA, 10 mg/kg), arachidonoylethanolamide (AEA, 10 mg/kg), the CB1 receptor agonist ACEA (3 mg/kg) and the CB2 receptor agonist JWH133 (0.2 mg/kg) administered for 5 days in a rat model of subchronic (2 weeks) ethanol diet (11% v/v) exposure. URB597 turned to be the most effective treatment. URB597 increased microglial (IBA‐1+) cell population, and changed morphometric features (cell area and perimeter, roughness, fractal dimension, lacunarity) associated with activated microglia in the hippocampus of ethanol‐exposed rats. Regarding innate immune activity, URB597 specifically increased mRNA levels of toll‐like receptor 4 (TLR4), glial fibrillary acidic protein (Gfap) and the chemokine stromal cell‐derived factor 1 (SDF‐1&agr;/CXCL12), and elevated the cell population expressing the chemokine receptors CX3CR1, CCR2 and CCR4 in the ethanol‐exposed rat hippocampus. Contrary to ethanol effect, URB597 reduced mRNA levels of Iba‐1, Tnf&agr;, IL‐6 and the monocyte chemoattractant protein‐1 (MCP‐1/CCL2), as well as cell population expressing iNOS. URB597 effects on hippocampal immune system were accompanied by changes in short and long‐term visual recognition memory. These results suggest that FAAH inhibition may modulates hippocampal microglial recruitment and activation that can be associated with improved hippocampal‐dependent memory despite ethanol exposure.

Microbial Neuraminidase Induces a Moderate and Transient Myelin Vacuolation Independent of Complement System Activation

Aims Some central nervous system pathogens express neuraminidase (NA) on their surfaces. In the rat brain, a single intracerebroventricular (ICV) injection of NA induces myelin vacuolation in axonal tracts. Here, we explore the nature, the time course, and the role of the complement system in this damage. Methods The spatiotemporal analysis of myelin vacuolation was performed by optical and electron microscopy. Myelin basic protein-positive area and oligodendrocyte transcription factor (Olig2)-positive cells were quantified in the damaged bundles. Neuronal death in the affected axonal tracts was assessed by Fluoro-Jade B and anti-caspase-3 staining. To evaluate the role of the complement, membrane attack complex (MAC) deposition on damaged bundles was analyzed using anti-C5b9. Rats ICV injected with the anaphylatoxin C5a were studied for myelin damage. In addition, NA-induced vacuolation was studied in rats with different degrees of complement inhibition: normal rats treated with anti-C5-blocking antibody and C6-deficient rats. Results The stria medullaris, the optic chiasm, and the fimbria were the most consistently damaged axonal tracts. Vacuolation peaked 7 days after NA injection and reverted by day 15. Olig2+ cell number in the damaged tracts was unaltered, and neurodegeneration associated with myelin alterations was not detected. MAC was absent on damaged axonal tracts, as revealed by C5b9 immunostaining. Rats ICV injected with the anaphylatoxin C5a displayed no myelin injury. When the complement system was experimentally or constitutively inhibited, NA-induced myelin vacuolation was similar to that observed in normal rats. Conclusion Microbial NA induces a moderate and transient myelin vacuolation that is not caused either by neuroinflammation or complement system activation.

Las ratas tratadas con el suplemento dietético Vitamix® (Ceregumil® con vitaminas) muestran mayor resistencia física y capacidad antioxidante

Objetivo Vitamix® es un producto dietetico compuesto por un extracto hidroalcoholico de cereales y leguminosas con miel, glicerofosfato de calcio, vitaminas B y D selenio y fluor. El producto base, Ceregumil®, patentado en 1912, ha sido muy popular como reconstituyente, y los usuarios refieren una sensacion de salud, resistencia a enfermedades o mayor predisposicion para el trabajo o el ejercicio. Material y metodo En el presente trabajo se analiza el efecto de Vitamix®, utilizado como suplemento alimenticio en ratas de laboratorio, en diversos parametros fisiologicos y pruebas fisicas. Periodicamente se realizaron hemogramas y se midieron la ingesta y el peso de los animales, asi como las concentraciones sanguineas de glucosa, trigliceridos, colesterol, transaminasas y malondialdehido, un producto de la lipoperoxidacion. Se realizaron pruebas de resistencia fisica y se llevo a cabo un estudio histoquimico del higado. Resultados Los animales que tomaron Vitamix® tenian menor peso e ingesta en edades avanzadas, mostraban mayor capacidad antioxidante, mayor resistencia en la prueba del alambre y menor fatiga en la piscina de Morris. En este ultimo caso, la mejoria era notable en los animales considerados de mal desempeno suplementados con Vitamix®. El resto de los parametros medidos se mantuvieron estadisticamente similares a los de los controles y no se observaron alteraciones hepaticas de ningun tipo. Conclusiones Este estudio supone una base cientifica y experimental para conocer el efecto de dichos complementos en los parametros fisiologicos.