Análisis de la reacción inflamatoria y los procesos degenerativos provocados por la inyección intracerebroventricular de neuraminidasa en rata

Las infecciones víricas y bacterianas del sistema nervioso central (SNC) y la neuroinflamación han sido relacionadas en los últimos años con en el desarrollo de ciertas enfermedades neurodegenerativas, tales como la esclerosis múltiple, la enfermedad de Alzheimer, la esclerosis lateral amiotrófica o trastornos neurocognitivos como la demencia asociada al VIH o la esquizofrenia. Muchas de estas enfermedades continúan hoy sin tratamiento, incluyendo aquellas que son sospechosas de ser consecuencia a largo plazo de infecciones víricas o bacterianas. Resulta importante, por tanto, procurar una mejor comprensión de los procesos neuroinflamatorios provocados por los patógenos (y sus componentes), así como sus consecuencias a largo plazo. Algunos de los agentes patógenos que causan neuroinflamación contienen neuraminidasa (NA) en sus cubiertas. Es esta enzima la que podría estar desencadenando algunos de los procesos lesivos mencionados anteriormente. El presente trabajo de Tesis Doctoral investiga los efectos lesivos e inflamatorios acontecidos después de la inyección intracerebroventricular (ICV) de neuraminidasa de origen bacteriano en ratas. Tales efectos implican la lesión y muerte de parte del epitelio ependimario, la inflamación ventricular y meníngea, y la degeneración vacuolar de ciertos tractos mielínicos. Además, el estudio aborda la cuestión de si el sistema de inmunidad innato, concretamente el sistema del complemento, está implicado en tales efectos. La Tesis plantea dos objetivos principales: 1) describir el daño, la inflamación y los eventos degenerativos que ocurren en diferentes momentos después de una única inyección ICV de NA; y 2) estudiar el papel del sistema del complemento en el desarrollo de los citados procesos, con especial atención al contraste de la siguiente hipótesis: ¿la muerte de las células ependimarias como consecuencia de la inyección de NA en la cavidad ventricular está causada por la acción del sistema del complemento sobre dicho epitelio¿. Los resultados del estudio establecieron el curso temporal de los efectos inducidos por la inyección ICV de NA en el ventrículo lateral, mostrando que la desialización y destrucción del epitelio ependimario están asociadas con la activación de la microglía, los astrocitos y los macrófagos periventriculares, así como al aumento local en la expresión de ICAM1 por parte de los vasos sanguíneos afectados y la infiltración de células inflamatorias desde la periferia. El volumen de esta infiltración varía entre las distintas regiones cerebrales en función de su cercanía al ventrículo inyectado, pero el orden en el que llegan las distintas poblaciones celulares y el perfil inflamatorio que definen fue muy similar tanto en los plexos coroideos, el espacio perivascular y las meninges, con afluencia de granulocitos y macrófagos dentro de las primeras 24 horas, seguidos por linfocitos T y B desde los 7 a los 15 días posinyección. Un proceso de degeneración vacuolar transitoria de algunos tractos mielínicos, sin pérdida de oligodendrocitos, se produjo a partir de las 24-48 horas. Dicha degeneración presentó un máximo entre los 4 y 7 días, y se resolvió en gran parte entre los 15 y 30 días posteriores a la inyección de NA. Interesantemente algunos tractos mielínicos, como por ejemplo la estría medular, tenían una cierta tendencia a estar siempre afectados. El trabajo concluye evaluando la implicación del sistema del complemento, presente en el SNC de forma completa y funcional, en los procesos lesivos e inflamatorios descritos. Se encontraron evidencias inmunocitoquímicas de la actuación del sistema del complemento sobre las células ependimarias dañadas del ventrículo lateral inyectado. Además, los resultados indicaron que NA activa al sistema del complemento de rata a través de la vía alternativa, pero no se encontraron indicios de la activación de las vías clásica o de las lectinas. Así mismo, se constató que la formación de los complejos de ataque a la membrana resultantes de la activación de la vía lítica del complemento no se requiere para la lesión del epitelio ependimario ni la degeneración de tractos mielínicos provocados por la NA, a pesar de que su activación agrava los efectos lesivos. No se descartó, sin embargo, que los componentes activados de la cascada del complemento puedan contribuir al inicio de la reacción inflamatoria posterior

FGF2, but not EGF, Induces multiciliated ependymal cells to dedifferentiate and adopt radial glial features in vitro

Multiciliated ependymal cells form an epithelium lining most of the ventricular cavities of vertebrates brain. Although considered postmitotic and completely differentiated, ependymal cells maintain some phenotypic characteristics of neural stem cells. Thereby, under specific conditions they behave as neural stem cells, developing radial glia characteristics, and undergoing asymmetric division. Our group is searching for factors that promote dedifferentiation of ependymal cells in vitro. We developed a simple method to obtain pure cultures of non-adherent multiciliated ependymal cells from adult rats. These cultures were used to investigate the effect of FGF2 on the differentiation state and the aggregation of ependymal cells. Thus, FGF2 treated ependymal cells lose cilia and hence mobility, and after 7 days they aggregate to form irregular spheres (diameter ≥ 20-30 μm). Such changes were not observed when EFG was used instead of FGF2. To assess the specificity of FGF2 action on cell aggregation, the FGF receptor inhibitor PD166866 and an anti-FGF2 neutralizing antibody were used. In both conditions the aggregative effect of FGF2 was abolished. No cell proliferation was observed during sphere formation, at least in such experimental conditions. Spheres were analyzed by immunocytochemistry using radial glia markers. They were positive for GFAP, vimentin, BLBP and GLAST. These data suggest that FGF2 promotes the identity loss in multiciliated ependymal cells in vitro, which are transformed into cells with radial glia features.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A model of neuroinflammation and demyelination by intracerebroventricular injection of microbial neuraminidase

Comunicación en forma de poster al congresoNeuraminidase from Clostridium perfringens, which cleaves terminal sialic acid from carbohydrate chains, was injected in the lateral ventricle of rats. It diffused in the ipsilateral ventricle, the third ventricle, and also towards the periventricular brain parenchyma. Soon after, the complement system activated, and some ependymal cells detached and died. In the affected zones, there was an increased expression of GFAP in astrocytes, IBA1 in microglia, and ICAM1 in the endothelial cells of blood vessels. Cytokines, such as IL1β secreted by activated macrophages and microglia, provoked the extravasation of leucocytes from about 4 h post-injection. The main sources of cells were large venules located in the choroid plexus, the meninges and the subependyma around the foramen interventricularis. Invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last lymphocytes (mainly CD8α-positive T-lymphocytes). Leucocytes invaded the ventricle and the meninges, and also penetrated the brain parenchyma, sometimes passing through the ependyma and the glia limitans. As a result, some myelinated tracts suffered vacuolar degeneration, being the stria medullaris consistently affected. Oligodendrocytes in the damaged tracts were not affected. Vacuolated myelin recovered with time. Thus, the intracerebroventricular injection of neuraminidase may represent a novel reversible animal model to study experimental neuroinflammation and myelin vacuolization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia e Innovación, SAF2010-19087; Junta de Andalucía, Consejería de Sanidad, SAS 08-0029; Junta de Andalucía, Proyecto de excelencia P-11-CVI-07637

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

Background In 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. Methods The 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. Results The 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. Conclusions These 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

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Ultralight vector dark matter search using data from the KAGRA O3GK run

Among the various candidates for dark matter (DM), ultralight vector DM can be probed by laser interferometric gravitational wave detectors through the measurement of oscillating length changes in the arm cavities. In this context, KAGRA has a unique feature due to differing compositions of its mirrors, enhancing the signal of vector DM in the length change in the auxiliary channels. Here we present the result of a search for U(1)B−L gauge boson DM using the KAGRA data from auxiliary length channels during the first joint observation run together with GEO600. By applying our search pipeline, which takes into account the stochastic nature of ultralight DM, upper bounds on the coupling strength between the U(1)B−L gauge boson and ordinary matter are obtained for a range of DM masses. While our constraints are less stringent than those derived from previous experiments, this study demonstrates the applicability of our method to the lower-mass vector DM search, which is made difficult in this measurement by the short observation time compared to the auto-correlation time scale of DM

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 pointsMicroglia 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 ependymal detach due to neuraminidase provokes hydrocephalus

Background: In many cases there seems to exist a tight relationship between the integrity of the ependymal barrier and the onset of hydrocephalus (see 1). We wanted to investigate this by means of a repetitive model that could be easily raised in the laboratory from normal rats. Material and methods: We observed that the, easily available, enzyme neuraminidase from Clostridum perfringens destroys the ependyma (2). So we proved different amounts of the drug at different times and explored by means of morphological techniques, at structural and ultra-structural levels, the events occurring after the intracerebroventricular administration of the drug into the normal rat brain Results: The administration provoked the detachment of a great part of the ciliated cubic ependyma but not other types of ependyma, particularly those attached by tight junctions, as is the notable case of the choroid plexus. The effect is dose-dependent and led to hydrocephalus when the detachment is massive and affects the brain aqueduct. The absence of ependyma marks a zone of great permeability where the immuno-responsible cells proceeding from the vessels, mainly from those venules located in the choroid plexus, easily penetrated the brain parenchyma. With time, these zones are covered by impermeable astrocytic glial scars (3, 4). We think that this could represent a good model for study the relationships between the ependymal detach and the onset of hydrocephalus. Conclusions: The microbial neuraminidase broke the intercellular junctions of the ependymal line, non sealed by tight junctions. The dissapearance of this barrier led to an increased permeability or/and a stenosis of the cerebral acueduct that in turn provokes hydrocephalus. References: 1. Jiménez AJ, et al. Structure and function of the ependymal barrier and diseases associated with ependyma disruption. Tissue Barriers 2014 Mar 19;2:e28426. 2. Grondona JM et al. Ependymal denudation, aqueductal obliteration and hydrocephalus after a single injection of neuraminidase into the lateral ventricle of adult rats. J Neuropathol Exp Neurol. 1996 55:999-1008

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 pointsMicroglia 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