Evidence for leakage across structurally altered endothelium.

<p>In addition to the observation of a dramatic increase of the vesicle density, we often observed a disintegration of the whole endothelial layer. In areas exhibiting BBB breakdown the cellular surface of the endothelium was frequently found to be ruffled or discontinuous. Therefore, the vascular wall was often shown to consist of endothelial debris and basement membranes, only (brackets). Thus, in contradiction to a variety of studies our data strongly suggest a transendothelial leakage pattern of affected vessels. DAB grains indicating extravasation of FITC-albumin are demarked by arrow heads. E = endothelial cells; L = vascular lumen; asterisk = cellular debris in the lumen of the vessel; arrow heads = DAB grains; arrow = tight junction.</p

Expression of claudin-5 and occludin in areas of FITC-albumin extravasation.

<p>Double fluorescence labeling of claudin-5 (blue) and occludin (red), both being transmembrane proteins critical for tight junction formation, demonstrates extravasation of FITC-albumin (green) in the vicinity of vessels expressing both markers. Please also note the presence of discontinuities in the staining pattern of control vessels with an ‘intact’ endothelial barrier (arrow heads).</p

Ultrastructural evidence for transcellular, not paracellular leakage.

<p>(A) Vessels in control areas on the contralateral hemisphere do not show signs of a transcellular, vesicle-mediated extravasation of FITC-albumin. (B–D) In contrast to alterations within the belts of tight junctions, ultrastructural examination regularly revealed signs for a transcellular leakage of the tracer. The endothelial cytoplasm exhibits a remarkable increase in vesicle density (white arrows) across the whole vascular circumference. Again, tight junctions (black arrow) are found to be intact. DAB grains are indicated by arrow heads. Control = contralateral hemisphere, E = endothelial cells; L = vascular lumen.</p

Evidence for FITC-albumin leakage across disintegrated endothelium in the early stroke phase.

<p>Finally, the same pattern of extravasation and endothelial damage was found at 5 hours after ischemia induction (A–D), as shown at 25 hours before. While the tight junctions remain, the rest of the endothelial cells may be constituted of debris, only (A and B). Often, the vascular basement membrane is exposed to the vascular lumen (B). Furthermore, electron dense vesicles carrying FITC-albumin can often be observed in the endothelium and the adjacent basement membrane (C) where the content is found to be deployed (D). E = endothelial cells; L = vascular lumen; arrow heads = DAB-filled vesicles; arrow = tight junction.</p

Ratio of occludin-positive vessels in areas of FITC-albumin leakage.

<p>(A) Quantitative analysis of differences in the expression of occludin in areas of FITC-albumin extravasation and their corresponding control areas was performed using low power (10× objective) magnification. Here, laminin-immunolabeling revealed the total number of vessels, whereas occludin-immunoreactivity visualized a critical tight junction constituent. The ratio of occludin-positive vessels to the total number of vessels was determined in 5 animals. (B) The ratio of occludin-positive vessels to the total number of vessels did not differ significantly in areas of FITC-albumin leakage and their corresponding control areas. Bars represent means and added lines indicate standard errors.</p

Vascular leakage in areas of ultrastructurally intact tight junctions.

<p>(A) Ultrastructural analysis of a control area located on the contralateral hemisphere shows a smooth endothelial layer (E) with an intact tight junction complex (arrow). The surrounding basement membrane is clearly visible and the adjacent neuropil does not show any structural alterations. (B–D) In areas of FITC-albumin extravasation the tight junction complexes (arrows) regularly appear to be established. The adjacent neuropil often displays cellular edema and cellular debris. Extravasated FITC-albumin and its product of conversion (black DAB grains, arrow heads) can constantly be found in the adjacent brain parenchyma proper. E = endothelial cells; L = vascular lumen.</p

Detection of FITC-albumin leakage following experimental ischemic stroke.

<p>(A) Double fluorescence labeling of laminin (color-coded in blue) and GFAP (red) in combination with applied FITC-albumin (green) reveals areas of ischemia-related BBB breakdown. By application of both, an antibody detecting astrocytes (GFAP, red) and an antibody for pan-laminin (blue) which visualizes vascular as well as glial basement membranes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056419#pone.0056419-Sixt1" target="_blank">[47]</a> the observed leakage can clearly be demonstrated to reach the brain parenchyma proper. Therefore, FITC-albumin is detectable within all the three compartments of the neurovascular unit, represented by the vascular wall (1^st compartment), the perivascular space (2^nd compartment) and the adjacent neuropil (3^rd compartment), delineated by astocytic endfeet (red) forming the glia limitans <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056419#pone.0056419-Bechmann1" target="_blank">[54]</a>. (B) To prove specificity of the applied reagent used for conversion of extravasated FITC-albumin into a permanent labeling by DAB for ultrastructural analysis, we exemplarily performed control stainings on vibratome sections, which were not further processed for electron microscopy. The low magnification of a coronary section clearly demonstrates the specificity of the applied reagent and its reaction product (DAB, brown). These sections were counterstained with hemalaun (blue). Areas of FITC-albumin leakage are clearly confined to the striatum. Higher magnification reveals a general leakage of FITC-albumin into the neuropil (brown background). In perivascular and juxtavascular areas the DAB staining appears to be more intense. (C) To confine ultrastructural analysis to areas with BBB breakdown, we identified areas of FITC-albumin extravasation after embedding in resin on coated microscope slides. These areas were selectively processed for ultrastructural analysis by electron microscopy. In corresponding control areas no FITC-albumin extravasation was observed. Please note, the general brown tissue background is a consequence of the embedding procedure using osmium tetroxide and uranyl acetate, which clearly can be distinguished from DAB staining as indicated on the left.</p

Movement ecology of coastal fishes in a marine protected area: implications for management and conservation

Animal movement is a key biological process for the maintenance of ecosystem services, and a major concern for the conservation of biodiversity. The aim of movement ecology is to understand the causes and consequences of these movements, including the effect of internal and external factors and its ecological implications. This research field has rapidly grown in the last decades fostered by recent technological and analytical developments, and is making substantial contributions to conservation biology, such as allowing the incorporation of the spatial and temporal scales of movements into management policies to enhance their scope and efficiency. Marine protected areas (MPAs) are the most used tools to face the effects of anthropogenic impacts on marine ecosystems. They play a major role in restoring and conserving overfished fish populations, and also potentially enhancing fisheries yield in adjacent areas through the direct spillover of juvenile and adult individuals. However, in order to be effective and generate such benefits, MPAs must be designed according to the movement attributes of targeted species, but this information is still rarely available, specially in temperate regions. In the last decades, passive acoustic telemetry techniques have demonstrated to be a valuable tool to study the movements and behavior of fishes covering large spatial and temporal scales, but the acoustic nature of the signals and the large amounts of data that they provide entail a series of challenges for their analysis and interpretation. The main objective of this thesis is to characterize the movement ecology of coastal fishes in relation to MPAs. This constitutes a basic information on the biology of species, which is required to understand changes in populations and ecosystems driven by natural or human induced impacts, as well as to correctly evaluate the outcomes of management actions. Specifically, the movements of two species, the white seabream (Diplodus sargus) and the common dentex (Dentex dentex), were monitored using acoustic telemetry in the Medes Islands MPA (Catalonia, NW Mediterranean Sea). Both species play important ecological and economical roles, shaping the structure and functioning of biological communities through top-down controls, and being an important resource for local artisanal and recreational fisheries. Nevertheless, they present a contrasting biology (omnivorous vs. predator), and therefore, different conservation needs. Firstly, we characterized the general movement attributes of the two species, including their habitat requirements and space use and activity patterns, within zones with different protection levels of the MPA. Secondly, we studied the behavioral responses of the two species to environmental fluctuations, by adding environmental information (seawater temperature and wave height) to movement analysis. These behavioral responses provide essential information on the ecology of the species such as their resistance to perturbations. Thirdly, we characterized their movement behavior during the spawning season, describing, for the first time, the formation of spawning aggregations for both species. Finally, this thesis also has an important computational and numerical component. A special effort has been done to adapt and develop new methods to visualize and analyze acoustic telemetry data, specially to improve the space use estimations by incorporating the vertical dimension, in order to provide a more comprehensive view of complex movement patterns. By studying the movement ecology of these species, we provide general mechanistic insights to understand the effects of protection on coastal fish species, as well as to predict future changes in their populations derived from climate change. We specially highlight the importance of studying the movement ecology of diverse species in order to propose integrative and more efficient management actions.Las áreas marinas protegidas (AMPs), son las herramientas de gestión más utilizadas para contrarrestar los impactos antropogénicos sobre los ecosistemas marinos y juegan un papel fundamental en la protección y restauración de las poblaciones de peces afectadas por la sobrepesca. Sin embargo, para ser efectivas y generar los beneficios que se esperan de ellas, las AMPs deben ser diseñadas en concordancia con los atributos de los movimientos de las especies de peces que se pretenden proteger, pero esta información no suele estar disponible. El principal objetivo de esta tesis es caracterizar, mediante técnicas de telemetría acústica, la ecología del movimiento de especies de peces costeros en relación a AMPs, mediante el estudio del movimiento de dos especies: el sargo común (Diplodus sargus) y el dentón (Dentex dentex), en la reserva marina de las islas Medas (Catalunya, Mediterráneo NO). Específicamente, se han caracterizado los patrones de movimiento y actividad generales de las dos especies, la presencia de respuestas comportamentales a fluctuaciones ambientales (temperatura y oleaje), y su comportamiento reproductor. Además, se ha hecho un esfuerzo especial en adaptar y desarrollar nuevas técnicas de análisis y visualización para datos de telemetría acústica, con el objetivo de mejorar las estimaciones del uso del espacio, incorporando la dimensión vertical, y de proporcionar una visión más exhaustiva de los complejos patrones de movimiento. Toda esta información es de gran aplicabilidad para la gestión de estas y otras especies costeras, así como para entender los cambios en las poblaciones y en los ecosistemas derivados del cambio global

Examples of diffusion-weighted (left), corresponding perfusion-weighted images (middle), and collateralization grades on 4D MR angiograms (right) in anterior cerebral artery (ACA) occlusion.

<p>A. Grade 0 in a case of right anterior cerebral artery (ACA) occlusion. B. Grade 3 in a case of left ACA occlusion. C. Grade 4 in a case of right ACA occlusion.</p

Two examples of FVH in the distal branches of the anterior cerebral artery (ACA, arrows) on FLAIR images in acute ischemic stroke due to ACA occlusion.

<p>Two examples of FVH in the distal branches of the anterior cerebral artery (ACA, arrows) on FLAIR images in acute ischemic stroke due to ACA occlusion.</p