High-density speckle contrast optical tomography (SCOT) for three dimensional tomographic imaging of the small animal brain

High-density speckle contrast optical tomography (SCOT) utilizing tens of thousands of source-detector pairs, was developed for in vivo imaging of blood flow in small animals. The reduction in cerebral blood flow (CBF) due to local ischemic stroke in a mouse brain was transcanially imaged and reconstructed in three dimensions. The reconstructed volume was then compared with corresponding magnetic resonance images demonstrating that the volume of reduced CBF agrees with the infarct zone at twenty-four hours.Peer ReviewedPostprint (author's final draft

High-density speckle contrast optical tomography of cerebral blood flow response to functional stimuli in the rodent brain

Noninvasive, three-dimensional, and longitudinal imaging of cerebral blood flow (CBF) in small animal models and ultimately in humans has implications for fundamental research and clinical applications. It enables the study of phenomena such as brain development and learning and the effects of pathologies, with a clear vision for translation to humans. Speckle contrast optical tomography (SCOT) is an emerging optical method that aims to achieve this goal by directly measuring three-dimensional blood flow maps in deep tissue with a relatively inexpensive and simple system. High-density SCOT is developed to follow CBF changes in response to somatosensory cortex stimulation. Measurements are carried out through the intact skull on the rat brain. SCOT is able to follow individual trials in each brain hemisphere, where signal averaging resulted in comparable, cortical images to those of functional magnetic resonance images in spatial extent, location, and depth. Sham stimuli are utilized to demonstrate that the observed response is indeed due to local changes in the brain induced by forepaw stimulation. In developing and demonstrating the method, algorithms and analysis methods are developed. The results pave the way for longitudinal, nondestructive imaging in preclinical rodent models that can readily be translated to the human brain.This project was funded by Fundació CELLEX Barcelona, Ministerio de Economía y Competitividad/FEDER (PHOTODEMENTIA, DPI2015-64358-C2-1-R), Instituto de Salud Carlos III/FEDER (MEDPHOTAGE, DTS16/00087), the “Severo Ochoa” Program for Centers of Excellence in R\&D (SEV-2015-0522), the Obra Social “la Caixa” Foundation (LlumMedBcn), AGAUR-Generalitat (2017 SGR 1380), LASERLAB-EUROPE IV, and “Fundació La Marató TV3.

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding

Objective Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. Approach and Results We used wild-type mice and Selplg(-/-) and Fut7(-/-) mice defective in P-selectin binding and lymphopenic Rag2(-/-) mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4(+) memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. Conclusions The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke

Tracking Asian tiger mosquito introductions in the Netherlands using Nextstrain

The Asian tiger mosquito Aedes albopictus is an undesirable invasive mosquito species that causes considerable nuisance through its biting behaviour, and has been proven to transmit more than 22 different viruses under laboratory conditions. Human-aided transportation, the capacity of winter diapause, and possibly global warming have contributed to the global invasion of Ae. albopictus. The species was found for the first time in the Netherlands in 2005, and since 2010 has been found introduced at many locations throughout the country. Elucidating the origin of these introduced mosquitoes could help the authorities on the planning and evaluation of the risk-based surveillance of Aedes invasive mosquitoes. This study aims to determine the genomic diversity of Ae. albopictus that is represented within and between collection sites with a database consisting of Ae. albopictus specimens from past introductions in the Netherlands, specimens from populations from other regions in the world, and data from specimens present in databases. In this study, complete mitochondrial genomes were sequenced, a recommended marker for phylogeography analysis of Ae. albopictus. Metadata is presented in a Nextstrain build containing 254 Ae. albopictus genomes up to October 2020. Overall, the phylogeny results of the Nextstrain build reveals a low mitogenomic diversity within Ae. albopictus. Genomic diversity of Ae. albopictus specimens found in the Netherlands fall within one main cluster which is hypothesised to represent the globally invasive strain of the species. Other organisations are stimulated to share data or materials for inclusion and improvement of the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/Aedes/20210728.info:eu-repo/semantics/publishedVersio

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke

Stem Cell Mediation of Functional Recovery after Stroke in the Rat

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Regenerative strategies of stem cell grafting have been demonstrated to be effective in animal models of stroke. In those studies, the effectiveness of stem cells promoting functional recovery was assessed by behavioral testing. These behavioral studies do, however, not provide access to the understanding of the mechanisms underlying the observed functional outcome improvement. [Methodology/Principal Findings]: In order to address the underlying mechanisms of stem cell mediated functional improvement, this functional improvement after stroke in the rat was investigated for six months after stroke by use of fMRI, somatosensory evoked potentials by electrophysiology, and sensorimotor behavior testing. Stem cells were grafted ipsilateral to the ischemic lesion. Rigorous exclusion of spontaneous recovery as confounding factor permitted to observe graft-related functional improvement beginning after 7 weeks and continuously increasing during the 6-month observation period. The major findings were i) functional improvement causally related to the stem cells grafting; ii) tissue replacement can be excluded as dominant factor for stem cell mediated functional improvement; iii) functional improvement occurs by exclusive restitution of the function in the original representation field, without clear contributions from reorganization processes, and iv) stem cells were not detectable any longer after six months. [Conclusions/Significance]: A delayed functional improvement due to stem cell implantation has been documented by electrophysiology, fMRI and behavioral testing. This functional improvement occurred without cells acting as a tissue replacement for the necrotic tissue after the ischemic event. Combination of disappearance of grafted cells after six months on histological sections with persistent functional recovery was interpreted as paracrine effects by the grafted stem cells being the dominant mechanism of cell activity underlying the observed functional restitution of the original activation sites. Future studies will have to investigate whether the stem cell mediated improvement reactivates the original representation target field by using original connectivity pathways or by generating/activating new ones for the stimulus.Financial support from the Hertie Foundation (Germany), and EU grants of the FP-6: DiMI (LSHB-CT-2005-512146), EMIL (LSHC-CT-2004-503569) and Stem Stroke (LSHB-CT-2006-037526) are gratefully acknowledged.Peer Reviewe

Poststroke Lung Infection by Opportunistic Commensal Bacteria Is Not Mediated by Their Expansion in the Gut Microbiota

Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection.Funded by Fundació la Marató de TV3 (ref. 201723-30-31-32) to Drs Planas, Urra, and Sancho; the Ministerio de Ciencia e Innovación (MICINN)/AEI/10.13039/501100011033 and European Regional Development Fund (ERDF) A way of making Europe by the European Union (PID2020-113202RB-I00 to Dr Planas); CSIC Interdisciplinary Thematic Platform Plataforma Temática Interdisciplinar (PTI)+Neuro-Aging of the Consejo Superior de Investigaciones Científicas (Dr Planas) funded M. Gallizioli. The work of Instituto de Productos Lácteos (IPLA)-Consejo Superior de Investigaciones Científicas (CSIC) group was partly financed by grant AYUD/2021/ 50981 from Principality of Asturias. The work of Institut de Química Avançada de Catalunya (IQAC)-CSIC was financed by Ministerio de Economía y Competitividad (AGL2017-83599-R) and MICINN (PID2020-117009RB-I00). Dr Díaz-Marugan was funded by La Caixa Foundation (ID 100010434; code LCF/BQ/DE16/11570021). Dr Arboleya received a postdoctoral Juan de la Cierva contract (MICINN, Ref. IJCI-2017-32156). Work in Dr Sancho laboratory was funded by Centro de Investigaciones Cardiovasculares (CNIC), European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091, and MICINN (PID2019-108157RB/AEI/10.13039/501100011033).Peer reviewe

MRI detection of secondary damage after stroke Chronic iron accumulation in the thalamus of the rat brain

[Background and purpose]: Iron plays a central role in many metabolic processes. Under certain pathological situations it accumulates, producing negative effects such as increasing damage by oxidative stress. The present study examined long-term iron accumulation in a stroke model with secondary degeneration, using MRI and histological techniques.[Methods]: Male Wistar rats (n=22) were subjected to 60 minutes MCA occlusion. MR images (T2- and T2*-weighted) were obtained weekly between weeks 1 and 7 after reperfusion, and at weeks 10, 14, 20, and 24. Histological iron detection and immunohistochemical examination for different markers (NeuN, GFAP, OX-42, HO-1, and APP) were performed at the 3 survival time points (3, 7, and 24 weeks).[Results]: Infarcts affecting MCA territory were evident on T2-weighted imaging, and all animals showed deficits on behavioral tests. In the thalamus, T2 hyperintensity was detected 3 weeks after stroke, and disappeared around week 7 when T2*-weighted images showed a marked hypointensity in that area. Histology revealed neuronal loss in the thalamus, accompanied by strong microglial reactivity and microglial HO-1 expression. APP deposits were detected in the thalamus from week 3 on and persisted until week 24. Iron storage was detected in microglia at week 3, in the parenchyma at week 7, and around APP deposits at week 24.[Conclusions]: T2*-weighted MRI allows the detection of secondary damage in the thalamus after MCAO. Iron accumulation in the thalamus is mediated by HO-1 expression in reactive microglia.The investigation was supported by grants from the EU-FP6 program: DiMI (LSHB-CT-2005-512146), EMIL (LSHC-CT-2004-503569) and StemStroke (LSHB-CT-2006-037526).Peer reviewe

Validation of in vivo imaging by invasive histological and immunohistochemcial methods

Capítulo en: Himmelreich, Uwe; Hoehn, Mathias; Planas, Anna M. (eds.). In vivo Imaging of Animal Models: Stroke. Dordrecht: Springer, 2013, p.93-99. ISBN 978-94-007-1548-6Peer Reviewe