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Psilodimitrakopoulos S.,ICFO - Institute of Photonic Sciences | Petegnief V.,Institute for Biomedical Research of Barcelona IIBB | Soria G.,Institute for Biomedical Research of Barcelona IIBB | De Vera N.,Institute for Biomedical Research of Barcelona IIBB | And 5 more authors.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE | Year: 2010

Polarization sensitive second harmonic generation (PSHG) imaging can provide useful information which is unreachable by intensity SHG imaging. Specifically, it can provide geometrical characteristics of the SHG source molecular architecture. The information is obtained by rotating the excitation linear polarization and by fitting the SHG intensity variation to a cylindrical symmetry biophysical model. As a result, the ratios of the non-vanishing χ2 tensor elements, responsible for the SHG conversion, are retrieved. In the end, by assuming a SHG source with dominant hyperpolarizability, its molecular orientation can be estimated. Here, we developed and used this approach to retrieve submicron structural information from cultured neurons and to provide estimation on the effective orientation of the molecular SHG source in axons. For that purpose, the PSHG images of axons were fitted pixel by pixel using an algorithm based on the above mentioned model. A coefficient of determination of r2>90% was chosen as a filtering mechanism. For a selected region of interest we then retrieved the pixels' values histogram of the harmonophores' effective orientations, θe. The distribution was centred at θe=34.93°, with σ=7.62°. These angle values correspond to the geometrical characteristics of the tubulin heterodimmers forming the microtubules. Modifications on tubulin dimers may alter θe, σ thus the PSHG optical technique suggests novel quantitative biomarkers able to characterize neurons' plasticity as well as disease progression. © 2010 SPIE.

Psilodimitrakopoulos S.,ICFO - Institute of Photonic Sciences | Petegnief V.,Institute for Biomedical Research of Barcelona IIBB | Soria G.,Institute for Biomedical Research of Barcelona IIBB | Amat-Roldan I.,ICFO - Institute of Photonic Sciences | And 4 more authors.
Progress in Biomedical Optics and Imaging - Proceedings of SPIE | Year: 2010

We use high resolution polarization second harmonic generation (PSHG) imaging microscopy in cultured neurons, and we provide estimation on the effective orientation of the SHG source in neuronal processes in vitro. We performed pixel by pixel analysis and we found a picked distribution of angles with maximum frequency at θe = 34.54°, with Δθe=11.44°. This angle value is very close to the inclination of the tubulin heterodimmer with respect to the long axis of the microtubule. © 2010 Copyright SPIE - The International Society for Optical Engineering.

Amaro S.,Hospital Clinic of Barcelona | Amaro S.,Institute dInvestigacions Biomediques August Pi Sunyer IDIBAPS | Laredo C.,Hospital Clinic of Barcelona | Laredo C.,Institute dInvestigacions Biomediques August Pi Sunyer IDIBAPS | And 14 more authors.
Stroke | Year: 2016

Background and Purpose - Identification of neuroprotective therapies in acute ischemic stroke is imperative. We report a predefined analysis of the URICO-ICTUS trial (Efficacy Study of Combined Treatment With Uric Acid and r-tPA in Acute Ischemic Stroke) assessing the efficacy of uric acid (UA) compared with placebo to prevent early ischemic worsening (EIW) and the relevance of collateral circulation. Methods - URICO-ICTUS was a double-blind, placebo-controlled, phase 2b trial where a total of 411 patients treated with alteplase within 4.5 hours of stroke onset were randomized (1:1) to receive UA 1000 mg (n=211) or placebo (n=200) before the end of alteplase infusion. EIW defined an increment ≥4 points in the National Institutes of Health Stroke Scale score within 72 hours of treatment in the absence of hemorrhage or recurrent stroke. Logistic regression models assessed the interaction between therapy and the collateral circulation in 112 patients who had a pretreatment computed tomographic angiography. Results - EIW occurred in 2 of 149 (1%) patients with good outcome and 23 of 262 (9%) patients with poor outcome (χ2; P=0.002). EIW occurred in 7 of 204 (3%) patients treated with UA and in 18 of 200 (9%) patients treated with placebo (χ2; P=0.01). There was a significant interaction between the efficacy of UA to prevent EIW and collaterals (P=0.029), with lower incidence in patients with good collaterals treated with UA compared with placebo (2% versus 15%, respectively; P=0.048). Conclusions - UA therapy may prevent EIW after acute stroke in thrombolysed patients. Optimal access of UA to its molecular targets through appropriate collaterals may modify the magnitude of the neuroprotective effect. © 2016 American Heart Association, Inc.

Penas C.,Cell Medica | Font-Nieves M.,Institute for Biomedical Research of Barcelona IIBB | Fores J.,University of Barcelona | Petegnief V.,Institute for Biomedical Research of Barcelona IIBB | And 3 more authors.
Cell Death and Differentiation | Year: 2011

Disconnection of the axon from the soma of spinal motoneurons (MNs) leads either to a retrograde degenerative process or to a regenerative reaction, depending on the severity and the proximity to the soma of the axonal lesion. The endoplasmic reticulum (ER) is a continuous membranous network that extends from the nucleus to the entire cytoplasm of the neuronal soma, axon and dendrites. We investigated whether axonal injury is sensed by the ER and triggers the activation of protective mechanisms, such as the unfolded protein response (UPR) and autophagy. We found early (at 3 days) accumulation of beclin1, LC3II and Lamp-1, hallmarks of autophagy, in both degenerating MNs after spinal root avulsion and in non-degenerating MNs after distal nerve section, although Lamp-1 disappeared by 5 days only in the former. In contrast, only degenerating MNs presented early activation of IRE1α, revealed by an increase of the spliced isoform of Xbp1 and accumulation of ATF4 in their nucleus, two branches of the UPR, and late BiP downregulation in association with cytoskeletal and organelle disorganization. We conclude that BiP decrease is a signature of the degenerating process, as its overexpression led to an increase in MN survival after root avulsion. Besides, Bcl2 is strongly implicated in the survival pathway activated by BiP overexpression. © 2011 Macmillan Publishers Limited All rights reserved.

De La Rosa X.,Institute for Biomedical Research of Barcelona IIBB | Santalucia T.,Institute for Biomedical Research of Barcelona IIBB | Fortin P.-Y.,University of Bordeaux Segalen | Purroy J.,Institute for Biomedical Research of Barcelona IIBB | And 5 more authors.
European Journal of Nuclear Medicine and Molecular Imaging | Year: 2013

Purpose: Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. Methods: A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Results: Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. Conclusion: This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core. © 2012 Springer-Verlag Berlin Heidelberg.

PubMed | University of Bordeaux Segalen and Institute for Biomedical Research of Barcelona IIBB
Type: Journal Article | Journal: Biomedical optics express | Year: 2014

Gene promoter activity can be studied in vivo by molecular imaging methods using reporter gene technology. Transcription of the reporter and the reported genes occurs simultaneously. However, imaging depends on reporter protein translation, stability, and cellular fate that may differ among the various proteins. A double transgenic mouse strain expressing the firefly luciferase (lucF) and fluorescent mPlum protein under the transcriptional control of the thermo-inducible heat-shock protein (Hspa1b) promoter was generated allowing to follow up the reporter proteins by different and complementary in vivo imaging technologies. These mice were used for in vivo imaging by bioluminescence and epi fluorescence reflectance imaging (BLI & FRI) and as a source of embryonic fibroblast (MEF) for in vitro approaches. LucF, mPlum and endogenous Hsp70 mRNAs were transcribed simultaneously. The increase in mRNA was transient, peaking at 3 h and then returning to the basal level about 6 h after the thermal stimulations. The bioluminescent signal was transient and initiated with a 3 h delay versus mRNA expression. The onset of mPlum fluorescence was more delayed, increasing slowly up to 30 h after heat-shock and remaining for several days. This mouse allows for both bioluminescence imaging (BLI) and fluorescence reflectance imaging (FRI) of Hsp70 promoter activation showing an early and transient lucF activity and a retrospective and persistent mPlum fluorescence. This transgenic mouse will allow following the transient local induction of Hsp-70 promoter beyond its induction time-frame and relate into subsequent dynamic biological effects of the heat-shock response.

Doucerain C.,Institute for Biomedical Research of Barcelona IIBB | Doucerain C.,August Pi i Sunyer Biomedical Research Institute IDIBAPS | Urra X.,August Pi i Sunyer Biomedical Research Institute IDIBAPS | Planas A.M.,Institute for Biomedical Research of Barcelona IIBB | And 3 more authors.
Journal of Neuroimmunology | Year: 2014

Heat shock protein 70 (Hsp-70) can act as a danger signal and activate immune responses. We studied the presence of Hsp-70 in lymphoid tissue and plasma of acute stroke patients and asymptomatic controls free of neurological disease. Immunofluorescence, Western blotting, qRT-PCR and flow cytometry studies were performed. Plasma Hsp-70 concentration at day 7 was similar in patients and controls, whereas patients disclosed stronger immunoreactivity to Hsp-70 in lymphoid tissue than controls. Most Hsp-70. + cells were antigen presenting cells located in T cell zones. Stronger immunoreactivity to Hsp-70 was associated with smaller infarctions and better functional outcome. © 2014 Elsevier B.V.

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