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Imaging neuroinflammatory reactions in cerebral ischemia: role of thrombin

English title Imaging neuroinflammatory reactions in cerebral ischemia: role of thrombin
Applicant Hirt Lorenz
Number 135617
Funding scheme Project funding
Research institution Service de Neurologie Département des Neurosciences Cliniques CHUV
Institution of higher education University of Lausanne - LA
Main discipline Neurophysiology and Brain Research
Start/End 01.03.2012 - 31.08.2015
Approved amount 375'000.00
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All Disciplines (2)

Discipline
Neurophysiology and Brain Research
Neurology, Psychiatry

Keywords (6)

stroke; thrombin; glia; imaging; inflammation; cytokines

Lay Summary (English)

Lead
Lay summary
Imaging neuroinflammatory reactions in cerebral ischemia: role of thrombin
Stroke is an important public health issue. Most commonly stroke results from the occlusion of a cerebral artery leading to a lack of blood supply (ischemia) to part of the brain. For patients suffering an acute ischemic stroke, restoring blood flow by disruption of the blood clot (thrombolysis) within 4.5 h from symptom onset is the only available treatment. Thrombolysis can be applied to less than 10% of patients, therefore the development of novel effective therapies aimed at reducing post-ischemic brain damage is an urgent challenge. The evolution of tissue damage is influenced by neuroinflammatory events that involve the release of soluble pro-inflammatory mediators (cytokines/chemokines) and specialised cells activated locally (glial cells) or recruited from the circulating blood (leukocytes). Lorenz Hirt's Lab has been is studying mechanism of cell death and survival after ischemic stroke, focusing on a signalling pathway involving thrombin with activation of the c-Jun N-terminal kinase (JNK) via the thrombin receptor PAR-1. Thrombin is a coagulation factor, present in the brain and activated in response to ischemia. Interestingly, thrombin and the PAR-1 receptor are involved in the activation of glial cells. Identifying pathways of activation of microglia and astrocytes will provide new insights into microglia/astrocyte-mediated neuronal injury in stroke. The Hirt lab teamed up with Paola Bezzi’s lab, with expertise in dynamics of glial communication in the brain. Our aim is to combine our complementary scientific, medical and methodological skills to study neuroinflammation in stroke focusing on the role of the thrombin-JNK pathway.
Using models of cerebral ischemia in vivo (middle cerebral artery occlusion in mice) and in vitro (cell cultures, co-cultures or acute brain slices subjected to oxygen and glucose deprivation, OGD) we plan to study the activation of glial cells and the role of the thrombin pathway. The following recombinant mice are available: mice with fluorescent glial cells (CXCR1+/eGFP and GFAP/eGFP mice expressing GFP in microglia or astrocytes); mice deficient in the cerebral thrombin inhibitor (protease nexin-1, PN-1); mice expressing a reporter gene to study the expression of PN-1. Several pharmacological tools are also available to influence this signalling pathway.
Testing mice with fluorescent glia in our models will allow us to observe ischemia induced glial changes and the influence of pharmacologically targetting the thrombin-JNK pathway. Crossing these mice with PN-1 mutant mice will allow us to study further the effect of the thrombin pathway on glial activation. Using powerful imaging techniques (time-lapse confocal microscopy, Ca2+-imaging), we will study structural and functional analysis on in situ modifications of microglia and astrocytes and correlate these observations with immunohistochemical characterization of biological features (cyto-/chemokines, receptors, scaffold proteins, secretory organelles etc). The functional alteration of microglia/astrocyte-mediated neuro-inflammation will be studied at a cellular level (intracellular signalling) and at an integrated level (microglia-astrocyte-neuron interactions, assessment of neuronal activity) in cell culture.
Relevance: The central goal of this interdisciplinary project is to describe structural re-arrangements and functional modifications occurring in microglial and astroglial cells in stroke-induced neuroinflammation in reproducible ischemia models with powerful imaging methods in genetically engineered mice. Characterising the contribution of neuroinflammation to ischemic injury is very relevant to stroke research. The strength of this study relies on the clinical relevance of the experimental models and on the possibility of testing the effect of neuroprotective strategies already identified in the lab on neuroinflammation.




Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Spatio-temporal overview of neuroinflammation in an experimental mouse stroke model
Buscemi Lara, Price Melanie, Bezzi Paola, Hirt Lorenz (2019), Spatio-temporal overview of neuroinflammation in an experimental mouse stroke model, in Scientific Reports, 9(1), 507-507.
Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes
Buscemi Lara, Ginet Vanessa, Lopatar Jan, Montana Vedrana, Pucci Luca, Spagnuolo Paola, Zehnder Tamara, Grubišić Vladimir, Truttman Anita, Sala Carlo, Hirt Lorenz, Parpura Vladimir, Puyal Julien, Bezzi Paola (2017), Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes, in Cerebral Cortex, 27(3), 2365-2384.
Improved long-term outcome after transient cerebral ischemia in aquaporin-4 knockout mice
Hirt Lorenz, Fukuda Andrew M, Ambadipudi Kamalakar, Rashid Faisil, Binder Devin, Verkman Alan, Ashwal Stephen, Obenaus Andre, Badaut Jerome (2016), Improved long-term outcome after transient cerebral ischemia in aquaporin-4 knockout mice, in Journal of Cerebral Blood Flow & Metabolism, 37(1), 277-290.
Non-Invasive Diagnostic Biomarkers for Estimating the Onset Time of Permanent Cerebral Ischemia
Berthet Carole, Xin Lijing, Buscemi Lara, Benakis Corinne, Gruetter Rolf, Hirt Lorenz, Lei Hongxia (2014), Non-Invasive Diagnostic Biomarkers for Estimating the Onset Time of Permanent Cerebral Ischemia, in Journal of Cerebral Blood Flow & Metabolism, 34(11), 1848-1855.

Collaboration

Group / person Country
Types of collaboration
Department of Biotechnology, University of Rijeka Croatia (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Neurology and Physiology and Psychiatry, UCSF United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
UNIL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
CNR Institute of Neuroscience and Department of Biotechnology and Transl. Med, University of Milan Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Awards

Title Year
Paola Bezzi was awarded a professorship at University la Sapienza, in Rome, in the Department of Physiology and Pharmacology 2019

Associated projects

Number Title Start Funding scheme
112484 Investigation of the role of the serine protease thrombin in cerebral ischemia 01.01.2007 Project funding

Abstract

Imaging neuroinflammatory reactions in cerebral ischemia: role of thrombin1. Summary of the research planBackground: Stroke is an important public health issue. For patients suffering an acute ischemic stroke i.v thrombolysis administered within 4.5 h from symptom onset is the only available treatment and can be applied to less than 10% of patients. The development of novel effective therapies aimed at reducing post-ischemic brain damage is an urgent challenge. The evolution of tissue damage in the so-called penumbra is strongly affected by neuroinflammatory events that involve the release of soluble pro-inflammatory mediators (cytokines/chemokines) and specialised cells activated locally (glial cells) or recruited from the periphery (leukocytes). Lorenz Hirt's Lab has been involved in a very promising neuroprotective project using a TAT-coupled peptide inhibitor of the c-Jun N-terminal kinase (JNK) that plays a prominent role in ischemic neuronal death. The JNK cascade is part of the signalling pathway activated by thrombin (via activation of the PAR-1 receptor). Thrombin is a coagulation serine protease, present in the brain and activated in response to ischemia. Interestingly, thrombin and the PAR-1 receptor are involved in the activation of glial cells. Recent data from the lab indicate that thrombin induced tolerance also involves PAR-1 and JNK, pointing out the dual role of thrombin, which also influences neurite outgrowth. Identifying pathways of activation of microglia and astrocytes will provide new insights into microglia/astrocyte-mediated neuronal injury in stroke. The Hirt lab teamed up with Paola Bezzi’s lab (DBCM, UNIL, Lausanne), with expertise in the dynamics of glial communication in the brain. Our aim is to combine our complementary scientific, medical and methodological skills to study the brain neuroinflammation in stroke focusing on the role of the thrombin-JNK pathway.Projects: The ischemia models chosen for this project are: (i) a transient middle cerebral artery occlusion (MCAo) model in mice, modelling stroke and allowing the study of ischemic changes in the brain parenchyma in vivo and ex vivo; (ii) acute brain slices subjected to oxygen and glucose deprivation (OGD) in vitro; (iii) cell cultures or co-cultures subjected to OGD. The following recombinant mice are available: CXCR1+/eGFP and GFAP/eGFP mice expressing GFP in microglia or astrocytes, allowing monitoring of structural modifications of microglia and astrocytes ex vivo and in vivo; mice deficient in the thrombin inhibitor protease nexin-1 (PN-1-/-), with altered thrombin/PAR-1 signalling; mice expressing a reporter gene driven by the PN-1 promoter (PN-1 KI) to study the expression of PN-1.Project 1: Mice with fluorescent glia will be crossed with PN-1 mutants to generate: PN-1-/- X CXCR1+/eGFP or GFAP/eGFP; PN-1KI X CXCR1+/eGFP or GFAP/eGFP. The resulting mice will be subjected to MCAo to characterize the mechanisms of microglia and astroglia activation in both the early and late phases. Structural and functional analyses of microglia and astrocytes will be studied at different timepoints. The thrombin-JNK pathway will be targeted pharmacologically after ischemia in mice harbouring fluorescent glial cells.Project 2: Paola Bezzi’ and Lorenz Hirt’s laboratories will perform structural and functional analysis on in situ modifications of microglia and astrocytes in acute slices subjected to OGD and/or in acute slices derived from animals subjected to MCAo in vivo in the Hirt lab. Intrinsic microglial and astrocyte properties will be analysed morphologically in real-time with time-lapse confocal microscopy and with Ca2+-imaging, followed by immunohystochemical characterization of biological features (cyto-/chemokines, receptors, scaffold proteins, secretory organelles etc).Project 3: Paola Bezzi’s lab will study the functional alteration of microglia/astrocyte-mediated neuro-inflammation at a cellular level (intracellular signalling) and at an integrated level (microglia-astrocyte-neuron interactions, assessment of neuronal activity). This part of the project will be performed in cell cultures (pure cultures, co-cultures) subjected to OGD.Relevance: The central goal of this interdisciplinary project is to describe structural rearrangements and functional modifications occurring in microglial and astroglial cells in stroke-induced neuroinflammation in reproducible ischemia models with powerful imaging methods in genetically engineered mice. Characterising the contribution of neuroinflammation to ischemic injury is very relevant to stroke research. The strength of this study relies on the clinical relevance of the experimental models and on the possibility of testing the effect of neuroprotective strategies already identified in Dr. Hirt’s laboratory on the relevant structural/functional dynamics processes of neuroinflammation.
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