Stroke research in the European Union

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1 Stroke research in the European Union

2 Interested in European research? RTD info is our quarterly magazine keeping you in touch with main developments (results, programmes, events, etc.). It is available in English, French and German. A free sample copy or free subscription can be obtained from: European Commission Directorate-General for Research Information and Communication Unit BE-1049 Brussels Fax Stroke research in the European Union EUROPEAN COMMISSION Directorate-General for Research Medical and Public Health Research - Unit F2 Contact: Virginija Dambrauskaite Helpdesk: Internet: Directorate-General for Research EUR 22707

3 Table of Contents Introduction...5 LEGAL NOTICE Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information. The views expressed in this publication are the sole responsibility of the author and do not necessarily reflect the views of the European Commission. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server (http://ec.europa.eu). Cataloguing data can be found at the end of this publication. Luxembourg: Office for Official Publications of the European Communities, 2007 ISBN ISSN European Communities, 2007 Reproduction is authorised provided the source is acknowledged. Printed in Belgium Printed on white chlorine-free paper Copyright pictures: Shutterstock, istock photos Europe Direct is a service to help you find answers to your questions about the European Union Freephone number (*): (*) Certain mobile telephone operators do not allow access to numbers or these calls may be billed. Summary of Stroke Research Workshop...7 Achievements, Opportunities and Future Goals in Stroke Research... 8 Cerebral Reperfusion... 9 Optimizing the Delivery of Stroke Care Brain Protection Recovery after Stroke Cerebrovascular Biology Stroke imaging Stroke Prevention Conclusion Acknowledgements References Support for Stroke Research through EU Research Programmes Connectivity in language rehabilitation in stroke and dementia Robotic assistance in neuro and motor Rehabilitation Gene search towards the identification of novel therapeutic strategies against brain ischemia Collaborative evaluation of rehabilitation in stroke across Europe The use of stroke registers to assess the quality of stroke management across Europe Ultrasonographic monitoring and early diagnosis of stroke European stroke prevention in reversible ischaemia trial European carotid stenting network Molecular basis of vascular events leading to thrombotic stroke Genetic regulation of the end-stage clotting process that leads to thrombotic stroke Glutamate Receptor Interacting Proteins as Novel Neuroprotective Targets Multipotent adult progenitor cells to treat stroke Pre-clinical evaluation of stem cell therapy in stroke Towards a stem cell therapy for stroke... 32

4 Introduction Stroke is a major cause of disability and death in Europe. In recent years large scale clinical trials have provided the evidence base for clinical approaches to primary and secondary prevention of stroke. New imaging techniques now enable to diagnose stroke more rapidly and precisely. The progress in treatment was made through the availability of lytic therapy for ischemic stroke, the most common type of brain attack. With the currently available treatment options further improvements in this area to a large extent depend on improved delivery of health care. Today, the results achieved and remaining challenges in preventing and treating stroke create opportunities for biomedical research. For many years the European Community has contributed to funding stroke research through its Research Framework Programmes. 4 On October 2005, the Research Directorate General of the European Commission invited a group of leading European experts in the field to provide an overview of the results of European stroke research and to discuss the way forward. A number of participants were coordinators of EU-funded projects. The programme for the workshop reflected the objectives: A keynote lecture by Professor Hennerici gave an overview of European stroke research. Subsequently, results from existing European stroke research initiatives were presented, including several EU-funded projects. These presentations illustrated the potential of multidisciplinary research activities that involve cooperation between European industry, basic science research institutes and university hospitals. The central part of the workshop was a round table discussion during which priorities for stroke research in Europe for the next few years were explored. This booklet summarises the discussion held at the workshop on stroke research priorities. The discussion was centred on the following topics: Cerebral reperfusion Optimising the delivery of stroke care Brain protection Recovery after stroke Cerebrovascular biology Stroke imaging Stroke prevention Some of the identified priorities in these areas will possibly be funded through the upcoming 7 th EU-Research Framework Programme. It will be important to align European research activities in this field more broadly, bringing in also the national and regional research programmes. In this context it is helpful that the outcome report of this workshop was already presented to a large audience of experts in the field at the 2006 European Stroke Conference and was disseminated through the journal «Cerebrovascular Diseases». 5 This brochure now brings together the analysis of future research needs with a summary of the results of the stroke research projects funded under the European research programme FP5 and FP6. It is hoped that knowledge gained from coordinated research will be translated into further improvements of stroke prevention and care to the benefit of European citizens.

5 6 Summary of Stroke Research Workshop Brussels - October 25, 2005 Stephen Meairs, Nils Wahlgren, Ulrich Dirnagl, Olle Lindvall, Peter Rothwell, Jean-Claude Baron, Konstantin Hossmann, Britta Engelhardt, José Ferro, James McCulloch, Markku Kaste, Matthias Endres, Jari Koistinaho, Anna Planas, Denis Vivien, Rick Dijkuizen, Anna Czlonkowska, Arne Hagen, Alan Evans, Gennaro De Libero, Zoltan Nagy, Daiva Rastenyte, Jürgen Reess, Antoni Davalos, Gian Luigi Lenzi, Pierre Amarenco, Michael Hennerici 7 About one million strokes occur each year in the European Union 1. Indeed, about 25% of men and 20% of women can expect to suffer a stroke if they live to be 85 years old. As a cause of death worldwide, stroke is second only to coronary heart disease 2. Although stroke is a major cause of death, mortality data underestimate the true burden of stroke. This is chronic disability. Since stroke causes disability more often than death, stroke patients frequently require long hospital stays followed by ongoing support in the community, or nursing-home care. Stroke is consequently a major drain on health-care funding. The number one cause of disability in the European Union is stroke. The total incidence of stroke is projected to increase considerably over the next two decades. This is because of the rapid increase in the elderly population. It is predicted that stroke will account for 6.2% of the total burden of illness in Thus, without more effective strategies for the prevention, treatment, and rehabilitation of stroke, the cost of this disease will increase dramatically. The European Commission hosted a European Stroke Workshop in Brussels on October 25, 2005, gathering top European experts in clinical stroke management, basic science, stroke research and industry. The primary objective of the workshop was to exchange views on the topic of stroke and to identify research activities that could potentially result in major advances in the areas of stroke prevention, treatment and recovery. These research priorities should address the most pressing scientific, clinical and industrial needs in the stroke field. Participants agreed that answers to the questions posed by these research priorities must significantly help in reducing the stroke burden to the European Union. The following is a summary of the ideas that were presented, discussed and agreed upon during the Stroke Workshop. Several of the key topics, particularly those in basic science stroke research and imaging, span prevention, therapy and recovery, thus conceivably making contributions to multiple areas of stroke management.

6 Achievements, Opportunities and Future Goals in Stroke Research 8 The last 25 years have seen remarkable progress in the understanding of the pathophysiology of ischemic stroke. European research centres have been and remain at the forefront of basic and clinical stroke research, having made seminal contributions to stroke pathophysiology and clinical management of stroke. This first wave of stroke research produced relevant animals models of cerebral ischemia that allowed the identification of mechanisms that contribute to tissue damage. It is now recognized that the critical reduction of blood supply to an area of the brain leads to a complex cascade of events, which evolves over time and space. This knowledge has opened the possibility to intercept this cascade therapeutically. In animal models of stroke, brain tissue can be successfully protected from damage. However, attempts to translate these findings into clinical practice have not yet been successful, although recently positive results have been obtained in clinical trials with the free-radical scavenger NXY-059. Only recently it has been understood that the capacity of the nervous system for regeneration, including the formation of neurons in adulthood, is greater than previously believed. This has opened new horizons for the treatment of stroke utilizing strategies to induce the brain s own mechanisms of protection and regeneration. Moreover, it has led to treatment with stem cells of various sources, which have successfully restored brain function after experimental stroke. Impressive developments have occurred in stroke trial methodology and in new treatment approaches. Aspirin and other antiplatelet agents are now used in secondary stroke prevention, as well as anticoagulants for the subgroup of patients who experience a stroke from a cardiac source of emboli. Blood pressure lowering after stroke and transitory ischemic attack (TIA) helps to prevent recurrent stroke and cardiac events. Accumulating evidence also suggests that statins have a prominent role in secondary prevention of stroke. Endarterectomy for symptomatic high-grade stenosis of the carotid artery has proven to protect against new ipsilateral stroke. Experiences from research on interventions to combat acute stroke are mixed. We now know that only a short therapeutic window of a few hours is available for reopening the blood supply to the ischemic brain. This has resulted in calls upon programmes to increase public awareness and to improve the prompt availability of stroke care through well-organised services. Tissue plasminogen activator (t-pa) is the only drug approved by the EMEA for intravenous thrombolysis of acute ischaemic stroke. The establishment of thrombolysis as the first successful therapeutic strategy in acute stroke, the development of novel non-invasive brain imaging strategies, as well as advances in trial design and hyperacute patient recruitment have raised the hope that we will be able to protect the brains of patients with stroke in the near future. Current trials take advantage of second generation thrombolytics with lesser side effects on the vasculature and brain parenchyma. Moreover, imaging technologies like perfusion- and diffusion-weighted MRI allow the identification of tissue at risk and can be used to identify patients in which brain protective therapy is likely to be successful. A second wave of combined efforts Cerebral Reperfusion T-PA is the only drug that is able to improve the outcome of patients with ischemic stroke. By enhancing the endogenous formation of plasmin from plasminogen, the blood clot occluding a cerebral vessel can be dissolved by disruption of fibrin. Although the reperfusion strategy has proven to be a successful concept for treating acute stroke, there are still considerable scientific opportunities to further develop this approach and to achieve improved benefit for patients. One experience from the use of t-pa in acute ischaemic stroke is that a substantial proportion of the patients do not improve, or only mildly improve, after administration of the drug. Identification of this lack of treatment response and development of an alternative strategy to overcome this problem is a scientific priority. Accumulating evidence is already available about such approaches, but these need to be refined and evaluated in an extended clinical setting. One possible explanation for an insufficient treatment response is that the dose of t-pa, restricted by safety concerns, is insufficient to dissolve particularly dense thromboembolic clots. Increased knowledge of the significance of variations in the size, composition and location of the blood clot would be useful for advancing this approach. Fibrinolytic gene polymorphism may be another factor explaining individual variability in treatment response. Mapping of such variability and development of new pharmacological agents which may enhance the fibrinolytic response of t-pa are interesting challenges. It has also been suggested that observations of neurotoxic effects in in basic and clinical stroke research can now capitalize on these promising developments. European research is in an excellent position to make significant advances in the field of stroke. Core skills of established centres of excellence in basic research provide ample opportunities when combined with the EU ethos for collaboration and interdisciplinarity. Collaborative efforts of European stroke researchers can draw on already existing networks, established in large part by EU-funding. The Stroke Workshop participants identified the following research fields in which a European research offensive can be highly successful on an immediate to intermediate time scale. animal stroke models of t-pa may be relevant for the clinical response in human stroke patients and restrict the beneficial effects of reperfusion. There may be additional ways to enhance the reperfusion concept. Combinations of intravenous thrombolysis with intraarterial approaches are promising and need clinical large-scale evaluation. Ultrasound may facilitate fibrinolysis. The effect of thrombolysis in combination with potential neuroprotective drugs needs further exploration. Further research into vascular biology mechanisms may provide enhanced understanding of the importance of the blood brain barrier during reperfusion and identify markers that can predict hemorrhage induced by thrombolytics. Not only do relatively few patients benefit from t-pa because of the narrow time window, its neurotoxic activities may also limit its clinical potential. Novel Research Priorities Cerebral Reperfusion Identify and test non-neurotoxic thrombolytics, understand mechanisms by which current thrombolytics damage vessels and neurons. Determine the most optimal approaches of ultrasound facilitation of clot lysis. Evaluate methods for delivering thrombolytic agents using microbubble targeting. Obtain knowledge on the bioeffects of therapeutic ultrasound applications for treatment of stroke to optimize therapeutic goals and limit adverse side-effects. Organise clinical trials of intraarterial approaches integrated with intravenous thrombolysis. Develop methods to identify individual variability in response to thromboembolic occlusion in cerebral arteries. 9

7 Brain Protection thrombolytic agents need to be developed and their effectiveness tested to overcome this hurdle. Intraarterial interventions used for locally applied pharmacological agents or mechanical impact on the occlusion may be used in case of treatment failure or in patients for which predictions may indicate that intravenous thrombolysis will be less successful. Ultrasound can induce microstreams within the clot resulting in in-depth distribution of t-pa to facilitate lysis. Ultrasound in combination with novel micro- and nanoparticles offers further therapeutic potentials 10 Prompt therapy of stroke is of fundamental importance for limiting the acute lesion ( time is brain ). Early identification of stroke patients, optimization of initial medical management, and techniques for early evaluation of symptoms, have a high likelihood of being successful and need systematic evaluation. General treatment of stroke including blood pressure, blood glucose and fever is based on consensus of experts, not on scientific evidence. We need reliable evidence to guide acute stroke care starting from pre-hospital emergency medical services continuing through emergency wards to stroke unit care. The positive outcomes of clinical trials documenting the effect of stroke units, thrombolysis and recently haemostasis have not been followed by a consistent implementation into clinical practice. An extensive network of more than 350 acute stroke centres with focus in the European Union provides a strong support for implementation services with accumulation of data from around 7000 treatments in the Safe Implementation of Thrombolysis in Stroke (SITS) database. Updated information on the use of stroke units are also required, since the data bank of the Stroke Unit Trialists Collaboration is based upon studies carried out years ago. Modern stroke unit care is something totally different and its efficacy needs to be scientifically evaluated. Success is unlikely unless scientific methods are used to explore factors which delay and which facilitate implementation of evidence-based treatments. State-of-the art stroke unit care must also be evaluated and information of its efficacy distributed and implemented widely to decrease human and economic burden of stroke. for stroke treatment, i.e. thrombolysis, targeted drug delivery and increased collateral flow to the ischemic penumbra. Rapid technological progress is being made in several European centers in both ultrasound imaging and therapeutic applications of ultrasound. Cerebral bioeffects of these new technologies, however, are not well understood, and appropriate in vitro and in vivo models for investigating these bioeffects are lacking. There is considerable fragmentation in European efforts and standardization in this area is lacking. Optimizing the Delivery of Stroke Care Broad scale evaluation of different levels of stroke care in academic settings, rural areas, prehospital care in EU member states, of factors which may delay or facilitate safe broad implementation of thrombolysis and other stroke interventions. This may create a basis for bringing effective stroke interventions to patients. By developing new approaches for prehospital management, the time between onset of symptoms and initiation of therapy can be shortened and patients may be better selected for therapy. Early biomarkers may also help in patient selection. The development of novel ultrasound imaging equipment or other promising technologies for stroke detection may allow mobile stroke imaging. When linked to advanced telemedicine systems, such diagnostic equipment could provide information to guide prehospital thrombolysis and neuroprotection. Research Priorities Optimizing the Delivery of Stroke Care Produce new data on optimal care of acute stroke patients and identify factors which may delay and which may facilitate broad implementation of evidence-based stroke interventions Identify methods for optimal hyperacute, prehospital management of stroke and subject them to clinical evaluation. Link new mobile brain imaging equipment for prehospital diagnosis of stroke with advanced telemedicine to provide new strategies for prehospital therapy of stroke. The treatment window for thrombolysis is so narrow that only a minority of patients arrive early enough in hospitals. Neuroprotecting agents could have a longer time window for treatment and less severe side effects. They could not only add to the efficacy of thrombolysis and also reduce the risk involved in it, but could also be effective as a stand alone treatment or be combined with other brainprotective treatments including hypothermia in patients not eligible for thrombolysis. Recent experimental research has identified many basic mechanisms involved in progress of ischemic penumbra in brain infarction. This opens new possibilities for more effective therapies and also for combination therapies. The same holds true for agents enhancing neuronal regeneration after experimental stroke. These observations may open totally new treatment strategies for enhanced neuronal recovery, reorganisation and improved neuroplasticity. Success in the field of enhanced neurorecovery in basic research will translate to patient treatment and also could widen the time window of acute stroke therapy to allow many more acute stroke patients benefit from these breakthroughs. Recent advances in understanding the signalling mechanisms of ischemic damage and endogenous neuroprotection set the stage for a focussed effort to close gaps in our knowledge, and to develop therapeutic strategies from this knowledge. We need to protect not only neurons, but the whole network of brain cells (astrocytes, oligodendrocytes, microglia, etc.) affected by focal cerebral ischemia We therefore will need to focus more on white matter injury. Surprisingly, white matter injury is rarely addressed in current stroke research, despite its relevance for the potential of the neurons to recover, and for a particular type of cerebral ischemic syndrome, vascular dementia. It is becoming clear that a successful treatment of stroke will need to block various mechanisms of damage simultaneously, hence the need for combination therapy. The preclinical establishment of combination therapy is demanding, and only feasible as a joint effort of many laboratories (large multicenter preclinical studies). EU scientists also need to establish common criteria for performing such research and to assure quality. Establishing SOPs and rules for conducting and reporting experimental stroke studies would also facilitate systematic reviews, and help to close the gap between clinical and experimental stroke research. An important message of recent stroke research is that to harness a full therapeutic potential, brain protective strategies should not only focus on the brain. Specifically, brain-cardiovascular, and neuralimmune interactions have been identified as highly relevant for tissue damage as well as repair after a 11

8 12 stroke. For example, infection is the most important complication after a stroke, as well as the number one cause of death after the first 24 hours after symptom onset. There is a great need for clinical breakthroughs for neuroprotecting and neurorecovery enhancing treatments for acute stroke treatment. Based on progress in basic research we now understand much more about ischemic cell death. This information opens a gate for successful translation to stroke patient treatment and now is the time to take the challenge. Recovery after Stroke Based on advances in stem cell biology there is potential for therapy of chronic and acute brain disorders such as stroke. However, a number of important issues need to be explored before clinical trials of stem cell therapy in stroke patients can be considered. Transplanted cells of different sources have induced incomplete recovery in animals and humans affected with stroke, probably through trophic support. In a future clinical application, the grafted stem cells Research Priorities Brain Protection Identify strategies to protect not only neurons, but also vascular and glial cells after focal cerebral ischemia. Focus more on white matter injury, small vessel disease and address vascular dementia. Conduct multinational preclinical trials on combination therapy. Establish SOPs, good laboratory practice, and reporting standards for experimental stroke research. Identify and understand cardiovascular - brain and neural immune interactions after stroke, develop therapeutic strategies. Evaluate neuroprotecting and neurorecovery enhancing treatments as stand alone and combined therapies in clinical trials. most probably have to be of human origin. Research should now focus on testing various types of stem cells (embryonic stem cells, stem cells from fetal and adult brain, and from other tissues) in animal models of stroke for their ability to generate and replace those particular neurons which have died, to remyelinate the demyelinated axons, and to repair the damaged neural circuitries. Only cells which can act through either some or all of these mechanisms, directly or after predifferentiation in culture, are likely to induce improvement of major therapeutic value in a clinical setting. It is also necessary to learn how to control the proliferation and differentiation of the stem cells into the specific cellular phenotypes. Behavioral recovery after transplantation of the most promising cell types has to be optimized in animal models of stroke. The grafted cells should be able to survive, integrate and differentiate in large numbers. The time window after the stroke when transplantation will lead to maximum restitution of neural circuitries and functional recovery should be determined. It will be important to be able to influence the pathological tissue environment after stroke including inflammatory and immune reactions to allow for efficient repair. One valuable strategy to promote recovery will probably be to combine stem cell and gene therapy. The stem cells could be genetically modified to improve their survival and differentiation, or genes could be delivered directly into the stroke-damaged brain to improve the migration and functional integration of the grafted cells. Stroke-damaged adult rodent brain has some capacity for neuronal replacement from its own neural stem cells. During several months after stroke, neural stem cells in the subventricular zone, lining the lateral ventricles, generate new striatal neurons which migrate to the damaged area. Stroke-induced neurogenesis is maintained in the aged brain. It is now important to establish whether endogenous neurogenesis contributes to functional recovery after stroke, and if it occurs in humans. Also, it needs to be determined whether endogenous neural stem cells can be triggered to produce cortical neurons, which are needed for improvement in most strokedamaged brains. Strategies to increase survival of new neurons and stimulate their incorporation into reorganizing neural circuitries should be developed. Research Priorities Recovery after Stroke Test various types of stem cells in animal models of stroke for their ability to generate and replace neurons which have died, to remyelinate the demyelinated axons, and to repair damaged neural circuitries. Explore contribution of endogenous neurogenesis to functional recovery after stroke. Learn how to trigger production of cortical neurons and stimulate integration into neural circuitries. Combine stem cell and gene therapy for stroke recovery. Stem cells could be genetically modified to improve their survival and differentiation, or genes could be delivered directly into the stroke-damaged brain to improve the migration and functional integration of the grafted cells. Further develop imaging techniques for assessment of grafted and endogenous stem cell survival, migration, and function after stroke. The development of imaging techniques for assessment of grafted and endogenous stem cell survival, migration, and function after stroke is necessary for the advancement of this research field. Some promising data are already available using magnetic resonance imaging. Safety issues are of major importance. Strategies to eliminate the risk for tumor formation, in particular, from embryonic stem cells have to be developed. Prior to clinical application, other adverse effects of stem cell therapy in stroke, such as involuntary and inappropriate movements and sensory dysfunction, should be carefully screened for in animals. There is a need to define which subset of stroke patients will be suitable for stem cell therapy. This will depend both on stroke pathology, location, and symptomatology in patients, as well as on which specific neuron types can be generated from stem cells. 13 Cerebrovascular Biology Stroke is a vascular disease. Clinically successful stroke treatment and prevention modalities are thrombolysis and platelet inhibition, i.e. vascular approaches. The physiology and pathophysiology of the neurovascular unit, the complex network of endothelial, smooth muscle, glial, and neuronal interactions, needs to be understood to devise new preventive as well as protective strategies. An interdisciplinary effort of neuroscientists, vascular biologists and neurologists is clearly required. Our basic understanding of vascular development and vascular interfaces, has greatly improved over the last decade. In contrast, our knowledge on the regulation of blood-brain barrier (BBB) function and the neurovascular unit is still behind. This knowledge needs to be expanded and to be integrated, extended and applied to stroke pathology. Barrier characteristics of brain endothelial cells are rapidly lost in stro-

9 Stroke imaging 14 ke. We know that maintenance of the BBB endothelium depends on continued crosstalk with the CNS microenvironment, but the cellular and molecular mechanisms involved in this maintenance are unknown. There is evidence that improvement in clinical outcome after stroke could be achieved by maintaining BBB integrity and/or accelerated reestablishment of BBB function. Only recently we have come to understand that vessel formation and brain cell development are closely linked. Neoangiogenesis is tightly linked to neuroneogenesis, and the vasculature can provide scaffolds for neuronal migration. It is likely that these physiological processes are also highly relevant after brain damage, e.g. after a stroke. Interdisciplinary work of neuroscientists, stem cell biologists, and vascular biologists can clarify this issue. Recruitment of cells of the innate and adaptive immune system contributes to stroke pathogenesis. However, the sequence of molecular steps involved in the recruitment of different leukocyte subpopulations across the BBB is not completely understood Over the last 20 years an impressive repertoire of small animal stroke models has been developed. However, these efforts have almost exclusively focused on large vessel occlusion. Models to study Research Priorities Cerebrovascular Biology Perform research on the molecular and cellular biology of the BBB to understand the regulation of BBB characteristics in CNS endothelium as a prerequisite to understand the pathophysiology of the CNS microcirculation during stroke. Understand the cellular and molecular mechanisms with which endothelial cells of the BBB react to focal cerebral ischemia, identify targets to protect these cells, the BBB, and the neurovascular unit against damage. Define markers of BBB disruption, and markers that can predict hemorrhage induced by thrombolytics. Understand the sequence of molecular traffic signals involved in leukocyte recruitment across the BBB in order to specifically prevent the immigration of pathogenic leukocytes into the CNS, while at the same time maintaining the recruitment of potential repair cells. Understand and target angiogenesis, vasculogenesis, arteriogenesis after (or before) a stroke. Study the interaction of angiogenesis and neurogenesis, in particular after stroke. Develop small animal models for lacunar stroke. Investigate mechanisms of damage as they relate to non-large vessel occlusion. small vessel disease are needed to expand our understanding of these important clinical entities. The recent advances in morphological, functional, and molecular brain imaging have greatly contributed to the progress in understanding of stroke pathophysiology, as well as in establishing successful treatment strategies. Approaches have been developed to stratify stroke patients for treatment (e.g. diffusion/perfusion mismatch), to non-invasively assess the brain circuitry involved in functional recovery (e.g. fmri), and to monitor novel treatment strategies, such as cell therapy (e.g. MION label MR). However, the potential of these techniques has to be further developed, and the underlying concepts need to be validated before patients can benefit from these advances on a broader scale. The penumbra concept is an important breakthrough in stroke research. Novel imaging techniques now allow characterization of a penumbra in individual patients. However, each approach has a different underlying concept of penumbra. Not only relevant to stroke, but also to other acute and chronic brain disorders is the urgent need to develop MR, CT, PET, ultrasound, and optical imaging techniques to study: Stroke Prevention Specific research on stroke prevention is clearly warranted. High quality studies in this field have not been undertaken due to the complex nature of heterogeneous stroke syndromes with different underlying pathophysiologies. Likewise, there has been a notable lack of translation of important developments in imaging and vascular biology into stroke prevention trials, particularly in studies of the elderly. As atherosclerosis, hypertension and diabetes are important risk factors for stroke, continued support of research in these areas is also needed. The identification of rare, Mendelian forms of stroke has greatly benefited our understanding of the common, multifactorial forms of stroke, because they allow us to identify key pathways that lead to a stroke, which are likely to be involved also in the development of other forms of stroke. Large scale European interdisciplinary efforts of molecular geneticists, cellular biologists, physiologists, and clinicians are needed to further our understanding of genetic susceptibility to stroke, as well as to the genetic influence on acute stroke pathology as well as recovery. Such knowledge should significantly contribute to further development Research Priorities Stroke Imaging Understand what the underlying pathophysiology is that produces a penumbra when PET, MR, ultrasound or CT measure it. Validate the underlying concept with invasive methods in experimental models. European stroke researchers need to be integrated and team up with networks of scientists in which new imaging methodologies are being developed and validate, and contribute stroke specific aspects and models. Develop new approaches for molecular imaging. Use imaging to better understand lesion maturation and progression and clinical deterioration, and to monitor the effect of treatments. Gene expression at the transcriptional level Protein expression Protein-protein interaction Cell homing, invasion, and migration, as well as differentiation Functional activity, neural pathways and networks, plasticity and reorganisation Research Priorities Stroke Prevention Novel risk factors for stroke should be identified. Moreover, we must learn how existing risk factors differ by subtype. Large networks should be developed to achieve the sample sizes necessary to study this highly heterogeneous clinical syndrome. Understand genetic susceptibility to stroke, as well as to the genetic influence on acute stroke pathology and recovery. of stroke prevention strategies. Prevention research in this area should be targeted to: determine the most effective preventive strategy during the high risk acute phase after a TIA or minor stroke. determine overall prognosis of particular clinical syndromes, such as TIA and stroke associated with large artery atherosclerosis in the posterior circulation, so that the need for treatment can be properly determined and treatment trials planned. predict the risk of stroke in individuals with syndromes, such as asymptomatic carotid stenosis or recent TIA, so that potentially risky or costly treatments can be 15

10 targeted at those individuals who are most likely to benefit. investigate the potential of screening the eldery «primary prevention» population to identify individuals who are at high risk of stroke. Conclusion Support for Stroke Research through EU Research Programmes Research Framework- Programme 5 ( ) Research Framework Programme 6 ( ) 16 The workshop highlighted a number of research priorities for the area of stroke research in Europe. Implicit in some of these priorities identified is the need for reinforced collaboration within the stroke research community. In addition to identifying research priorities, examples of successful European collaborations in stroke research were presented at the workshop. It will be important to build on these experiences in the future. An option could be to create a more formal stroke research network. Acknowledgements We wish to thank the European Union for hosting this workshop. We are indebted to Dr. Virginija Dambrauskaite, both for her excellent organization of the workshop and for her helpful contributions to the workshop summary. We also thank Dr. Elmar Nimmesgern and Dr. Alain Vanvossel for their valuable guidance in this endeavor. References 1. Sudlow CL, Warlow CP. Comparable studies of the incidence of stroke and its pathological types: results from an international collaboration. International Stroke Incidence Collaboration. Stroke. 1997;28(3): Murray CJL, Lopez AD. The Global Burden of Disease: a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1990 and projected to Boston: Harvard University Press, Menken M, Munsat TL, Toole JF. The global burden of disease study: implications for neurology. Arch Neurol. 2000;57(3): Quality of life and management of living resources Shared-cost research projects: 1. QLK6-CT Connectivity in language rehabilitation in stroke and dementia (LSDE) 2. QLK6-CT Robotic assistance in neuro and motor rehabilitation (GENTLE/S) 3. QLG3-CT Gene search towards the identification of novel therapeutic strategies against brain ischemia (StrokeGene) 4. QLK6-CT Collaborative evaluation of rehabilitation in stroke across Europe (CERISE) 5. QLG4-CT The use of stroke registers to assess the quality of stroke management across Europe (EROS) 6. QLG1-CT Ultrasonographic monitoring and early diagnosis of stroke (UMEDS) Concerted action projects: 7. QLK6-CT European stroke prevention in reversible ischaemia trial (ESPRIT) 8. QLK6-CT European carotid stenting network (ECSN) Theme 1 Life sciences: genomics and biotechnology for health Specific Targeted Research projects: 9. LSHM-CT Molecular basis of vascular events leading to thrombotic stroke (MOLSTROKE) 10. LSHM-CT Genetic regulation of the end-stage clotting process that leads to thrombotic stroke (EuroClot) 11. LSHM-CT Glutamate receptor interacting proteins as novel neuroprotective targets (GRIPANNT) 12. LSHB-CT Multipotent adult progenitor cells to treat stroke (STROKEMAP) 13. LSHB-CT Pre-clinical evaluation of stem cell therapy in stroke (STEMS) 14. LSHB-CT Towards a stem cell therapy for stroke (STEMSTROKE) S. Karger AG, Basel Meairs S, Wahlgren N, Dirnagl U, Lindvall O, et al.: Cerebrovascular Diseases 2006;22:75-82 This article is reproduced with kind permission of S. Karger AG, Basel.

11 1. Connectivity in language rehabilitation in stroke and dementia 2. Robotic assistance in neuro and motor Rehabilitation Project Acronym: LSDE Project number: QLK6-CT EC contribution: Dates: 01/03/ /02/2003 Duration: 36 months Project Acronym: GENTLE/S Project number: QLK6-CT EC contribution: Dates: 01/02/ /04/2003 Duration: 39 months 18 Overall objectives of the project The underlying goal of this project is to research the neurophysiological basis for the restitution of language function in stroke. Language, like other complex cognitive functions, is represented in the brain in an extensive network of specialised areas. It is now known that the brain is a learning and problem-solving organ, whose neural organisation can change in response to experience and practice. This reorganisation can occur within the specialised areas, but it can also involve a change in the interactions between those areas (i.e., a change in connectivity). This inherent capacity of the brain may also contribute to a functional restoration after stroke. Thus, this project aims to examine the connectivity in normal and disordered language processing, to investigate the neurobiological determinants of a favourable recovery, and to examine changes in functional activation and connectivity in response to language therapy as well as drug intervention. Experimental approach and working method We examine the functional specialisation of different brain areas with the help of modern imaging techniques such as EEG, MEG, and functional magnetic resonance imaging. These techniques can provide converging fine-grained spatial and temporal information on language processes in the brain. We are further preparing the ground to investigate the above questions by developing and applying new techniques to assess the interplay between different languagerelevant brain regions (i.e., techniques assessing effective connectivity). Empirically, we investigate language processes in adults with regular schooling as well as in illiterate adults, to get a better picture of the effects of experience and learning in healthy brain function. This will enable a better understanding of the neurobiological markers of functional restitution and rehabilitation after stroke, a knowledge which can be directly applied to the planning of therapeutical intervention programmes for persons who have suffered a stroke with resulting language disorders. Achievements and results We have developed an algorithm to estimate the connectivity between brain areas, and validated it with data from earlier reports of interactions between specific brain regions. Further, we refined EEG and MEG signal analysis tools used to indicate brain connectivity. We also improved existing techniques to characterize the integrity of main fibre pathways between cortical areas. We have already applied some of these new techniques to examine connectivity in language learning paradigms in healthy adults. Preliminary results from imaging studies with individuals who have suffered a stroke have provided data on brain reorganisation. Paradigms to examine language function in illiterate individuals have been validated. Overall objectives of the project The GENTLE/S project is seeking to develop and demonstrate machine-mediated physiotherapies in the treatment of stroke rehabilitation for older patients. Experimental approach and working method GENTLE/S has taken a rapid prototyping approach to developing the technology for machine-mediated stroke rehabilitation. This allowed the technical partners to produce a viable clinical prototype within fourteen months of the project start date and also allowed clinical partners to begin a pilot clinical study within the first half of the allotted project time. Results from this pilot study along with ergonomic information on the use of the machine, was then available for analysis and to refine the design approach. After a revision of the design a more substantial clinical study was undertaken. A parallel activity was begun on the kinematics and biomechanics of the person using the mechanism, which has allowed both a good understanding of the forces and range of movement involved. It proved possible to use robotic techniques to back estimate joint torques for a greater clinical understanding. An important aspect of the research was to assess not only the clinical impact but the readiness of the technology for a market that could be at a hospital, a patient s home, a rural health-centre or a medical practice. Achievements and results The project has identified the user needs via consultation with the user community and via a workshop with clinicians and people who have had a stroke to discuss the important aspects in the design of the system. Two clinical prototypes produced and two clinical evaluation sites in two countries selected, the first at the Tallaghn Hospital, Dublin, and the second at the RBH-Battle Hospital in Reading. Partners from these two organizations have developed a detailed experimental protocol and used this as the basis of a submission to the respective ethics committees. Ethical approval has been granted for the study to proceed at one site, with the committees decision pending for the second site. Work was completed on a second phase prototype based on a modified HapticMaster haptic interface from Fokker Control. This prototype was fully validated both from a technical and a clinical aspect. Dissemination activities included a poster, brochures and publications in both academic and general literature as well as two international television broadcasts on the project. 19 Cornelius Weiller Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Neurologie Martinistr. 52 D Hamburg, Germany Tel: , Fax: William Harwin University of Reading (UK), Department of Cybernetics Whiteknights, Reading, Berks RG6 6AY, UK Tel: +44 (0) ; Fax: +44 (0)

12 3. Gene search towards the identification of novel therapeutic strategies against brain ischemia 4. Collaborative evaluation of rehabilitation in stroke across Europe Project Acronym: StrokeGene Project number: QLG3-CT EC contribution: Dates: 01/09/ /08/2003 Duration: 36 months Project Acronym: CERISE Project number: QLK6-CT EC contribution: Dates: 01/01/ /04/2006 Duration: 52 months 20 Overall objectives of the project Current therapies for brain damage after stroke or heart failure are inefficient and, therefore, require urgent improvement. The main objective of the proposal is the development of novel therapeutic strategies based on the analysis of gene-related responses that lead to either reversible or irreversible brain injury. To search for such genes, high throughput gene expression profiling is carried out in clinically relevant brain ischemia models, with precise characterization of the pathophysiology. The second objective is the identification of genes that regulate life and death decisions in ischemically damaged neurons, thus providing a better insight into the molecular mechanisms of ischemic brain injury. Finally, the proposal aims to promote interdisciplinary research in laboratories from different European countries, and to foster interactions between research laboratories and industry. Experimental approach and working method Clinically relevant experimental models of stroke and heart failure induced brain damage are produced and characterized by electrophysiological, morphological and pictorial biochemical methods. Brain regions with reversible and irreversible brain injury are localized and mrna is isolated from these regions under control conditions, and at various times after the onset of the ischemic injury. Ischemia-regulated genes are identified by automated DNA chip based technology, by the recently developed Digital Expression Pattern Display *DEPD* and by Multi-Western blot analysis, all of which provide powerful tools for high throughput gene expression profiling under complex pathophysiological conditions. Comparisons of the gene expression patterns in the various samples have identified those genes that are possible key regulators of cellular death and survival. Konstantin - A. Hossmann Max Planck Institut für neurologische Forschung Gleueler Str. 50, D Köln, Germany Tel: +49 (0) ; Fax: +49 (0) Achievements and results Establishment of clinically relevant brain ischemia models and identification of brain regions undergoing reversible and irreversible injury Isolation of mrna from reversibly and irreversibly damaged brain tissue, and from preconditioned cortex (i.e. with increased tolerance to ischaemia). Establishment of microarray gene expression profiling and Multi-Western blot analysis methods. Identification of more than 300 transcripts and more than 180 proteins that are up- or down-regulated during focal ischemia. Identification of time-related changes in gene expression associated with brain preconditioning in mice. Depth-resolved two-photon in vivo microscopy for the study of neuronal calcium dynamics and blood flow during sensory stimulation. Development of a new mitochondria-targeted fluorescent probe for the study of mitochondrial dysfunction in ischemia. Analysis of nestin expression as a marker of ischemic stress and nestin promoter/enhancer requirements in response to CNS injury. Discovery that brain preconditioning (i.e. increased tolerance to ischaemia) is associated with a fold increase in the expression of the a7-nicotinic acetylcholine receptors. Gene expression analysis by DEPD after 1 hour middle cerebral artery occlusion and cardiac arrest. Analysis of expression data: generation of a list of temporary co-regulated genes after cardiac arrest and of gene clusters that are differentially regulated after CA and MCAO. Establishment of nestin promoter/enhancer sequences that lead to elevated gene expression in response to injury in transgenic mice. New data on the molecular biology of the stroke and dementia syndrome CADASIL on a cellular basis. The observation that knock out of the Notch 3 gene gives no obvious phenotype. The discovery of a novel, astroglia-associated mechanism for the coupling between neuronal function and blood flow. Overall objectives of the project The aim of this study is to compare the outcome of stroke rehabilitation in different centres across Europe and additionally to examine differences in the amount and the content of therapy, organisational characteristics and input of man-hours of the staff. Four principal components will be studied in an integrated way: 1. Comparison of the pattern of recovery and outcome after stroke in four rehabilitation centres in four European countries; 2. Identification of the difference in the provision of stroke services in these centres, which might account for differences in outcome. This includes consideration of the amount of time spent in therapy and the content of the physical and occupational therapy; 3. Analysis of the organisational characteristics of the different units providing rehabilitation. The analysis focuses on three classes of variables: management and characteristics of labour division; staff characteristics and characteristics of the physical environment. Furthermore, qualitative research methods will allow description of cultural differences and interaction processes; 4.The final stage of the study will bring together these four elements, to identify the best clinical practice in stroke rehabilitation and to acquire evidence for the best disease management and decision-making. Experimental approach and working method A prospective longitudinal study to examine the impact of rehabilitation on the pattern of recovery after stroke (4 centres across Europe). Variations in case-mix will be corrected using multiple regression analysis. Several aspects of recovery will be assessed using an agreed protocol on admission to the rehabilitation centres, at discharge and at 2, 4 and 6 months after stroke. The nature and amount of therapy offered will be documented, on matched patients. Treatment sessions will be videotaped. The amount, content and context of therapy will be documented with time sampling and related to recovery. Finally the analysis of management, staff characteristics and physical environment will allow identification of services that offer optimal therapy at what input of man-hours. This study will identify ways of improving the rehabilitation services for stroke patients and as result enable people to cope better with disability. Achievements and results At this time, the data collection on the time sampling and content of therapy is completed. The data collection on the recovery pattern and organisation of the rehabilitation centres is completed. Intermediate analyses have shown large discrepancies in the intake profiles of the stroke patients between the four different centres. These discrepancies will have to be taken into account when comparing the recovery patterns. The analysis of the time sampling data shows that there are great differences between centres in the average therapy input per day. In the next year, the analyses will be completed and the link between recovery pattern, content and amount of therapy and organisational aspects will be explored. The results will be disseminated at national and international level. Willy De Weerdt K.U.Leuven, Faculty of Physical Education and Physiotherapy, Department of Rehabilitation Sciences, Tervuursevest 101, B-3001 Leuven (Heverlee), Belgium Tel: , Fax:

13 5. The use of stroke registers to assess the quality of stroke management across Europe 6. Ultrasonographic monitoring and early diagnosis of stroke Project Acronym: EROS Project number: QLG4-CT EC contribution: Dates: 01/10/ /09/2006 Duration: 48 months Project Acronym: UMEDS Project number: QLG1-CT EC contribution: dates: 01/10/ /03/2006 Duration: 42 months 22 Overall objectives of the project The European Register Of Stroke (EROS) programme is a 4 years study assessing the quality of stroke care provision on outcome in the area of the public health call of the Framework V programme. The programme has 4 main objectives: 1. To develop methods to estimate: the range of models of care for stroke, the case mix of stroke patients, the appropriateness of stroke care and outcome after stroke across Europe. 2. To assess the appropriateness of acute stroke care, secondary prevention and rehabilitation after stroke against National and European guidelines. 3. To understand the factors (socio-demographic, organisational and clinical) that contribute to the variations in 1) quality (adherence to guidelines) and 2) outcome after stroke. 4. To develop and use methods to quantify the resource consequences of the different models of stroke care and study the determinant of cost, in particular the optimal size of stroke unit facilities and their cost efficiency. Overall the main aim of the programme is to estimate the quality of care an impact of stroke care across Europe using a variety of different methods. Many of the objectives will be achieved by setting up population registers in the different centres. Charles Wolfe Kings College London, Dept. Of Public Health 5 th Floor Capital House 42 Weston Street London SE1 3QD, UK Tel: , Fax: Experimental approach and working method This is both an epidemiological (population) and health services research programme. Population based registers have been set up in 8 countries within the programme. The reviewing of the evidence and site visits have involved developing a quality of stroke care tool. Achievements and results The EROS project has a website which outlines what the project is about and has information and links to other centres. Website: The tools for estimating the impact of stroke and for assessing the quality and outcomes of care are developed. Data Collection in 7 population based stroke register is ongoing to address the study deliverables. Overall objectives of the project The main objective of the UMEDS project was to establish ultrasound (US) techniques for early diagnosis and bedside monitoring of stroke. This involved expansion of recent developments in US contrast agent (microbubble) technology and US imaging techniques to enable both qualitative and quantitative evaluation of brain tissue perfusion. Development of new technologies to meet these goals included an automated system for detection of microemboli circulating in the bloodstream, a novel device for imaging the brain directly through the skull bone, a dual transducer system for microemboli detection and enhanced features for commercial ultrasound perfusion imaging of the brain. The project also addressed the potential of novel microbubbles specially targeted to blood platelets for characterizing microemboli, for visualization of acute vessel occlusion and for thrombolysis of blood clots. Experimental approach and working method Microbubbles were synthesized that contain antibodies to activated platelets for both targeted imaging and thrombolysis of human thrombus. The binding of these microbubbles was demonstrated by specific, custommade in vitro assays on both human and rat platelet substrates as well as on human clots. The targeted microbubbles were then tested both in vitro and in an in vivo animal model. Studies were also undertaken to ascertain optimal modes of microbubble delivery and their application to brain perfusion measurements. Different types of approaches for characterizing brain perfusion with ultrasound and microbubbles were investigated. The results were integrated into special image analysis software. Animal models of ischemic and hemorrhagic stroke were used to study possible effects of microbubbles used for stroke monitoring. Results arising from experimental data on brain imaging and perfusion studies provided specifications for design of enhanced features to improve commercial brain imaging applications. Construction of new ultrasound equipment followed detailed specification and design. System prototypes were then subjected to rigorous testing and clinical evaluation. Achievements and results Great progress was made in attaching antibodies to microbubbles for better imaging of human thrombus and for targeted clot lysis with ultrasound. Importantly, the safety of ultrasound monitoring of acute cerebral infarction and hemorrhage with microbubbles was demonstrated in animal models. Unexpectedly, results of these studies showed a significant reduction in infarct volume in rats sonicated with microbubbles, thus suggesting a protective role in stroke. Imaging of microbubbles in humans showed a good correlation to magnetic resonance perfusion studies for detecting acute cerebral perfusion deficits. Using image analysis software developed in the project, time-intensity curves were established for different areas in the brain parenchyma. In a multi-center clinical study it was shown that ultrasound imaging with contrast agent is highly sensitive and specific for diagnosis of ischemic vs. hemorrhagic stroke. Enhancements in ultrasound brain imaging were implemented on new commercial scanners. These included a novel technique for real time ultrasound imaging of microbubbles at very low energies through the transtemporal bone window. Moreover, a totally new ultrasound brain scanner was developed in this project. It is based upon so-called time-reversal mirrors and allows direct imaging through the human skull. Automatic embolus detection equipment was developed. Moreover, a multi-gate/multi-channel system for acquisition and processing of intra- and extracranial Doppler signals with integrated automatic embolic signal detection was developed. These novel systems were extensively tested in a clinical environment with excellent results. 23 Stephen Meairs Neurologische Klinik, Universitätsklinikum Mannheim der Universität Heidelberg Theodor-Kutzer-Ufer, D Mannheim, Germany Tel: ; Fax:

14 7. European stroke prevention in reversible ischaemia trial 8. European carotid stenting network Project Acronym: ESPRIT Project number: QLK6-CT EC contribution: dates: 01/01/ /12/2006 Duration: 48 months Project Acronym: ECSN Project number: QLK6-CT EC contribution: dates: 01/01/ /12/2006 Duration: 48 months 24 Objectives The aim of ESPRIT is to compare the efficacy and safety of two new treatment modalities (A and B): A) oral anticoagulation (INR ) and B) the combination of mg acetylsalicylic acid and 400 mg dipyridamole daily with that of the standard treatment C) mg acetylsalicylic acid daily to find a more effective treatment in the secondary prevention for patients with cerebral ischaemia of arterial origin. Experimental approach and working method Patients who have had a transient ischaemic attack or non-disabling ischaemic stroke of arterial origin have an annual risk of 7 to 12% for subsequent nonfatal stroke, nonfatal myocardial infarction or death (from all vascular causes) without treatment. The standard treatment acetylsalicylic acid (ASA) prevents only 13% of these events; this is far from ideal. ESPRIT is a randomised, international, multicentre trial in which three treatments are compared: a) AC (INR 2-3), b) ASA combined with dipyridamole c) ASA. The primary measure of outcome is the composite event nonfatal stroke, nonfatal myocardial infarction, death from all vascular causes or major bleeding complication, whichever occurs first. A total of 4500 patients will be enrolled. Since cerebrovascular disease is one of the major European health problems, especially in the elderly, it is of utmost importance to look for more effective prevention than that by ASA. Data from secondary prevention trials in other cerebrovascular diseases suggest that oral anticoagulation (AC) or ASA combined with dipyridamole are more effective in the secondary prevention. A more effective treatment will reduce disability, handicap and mortality and prevent loss of quality of life in many (especially elderly) patients. Overall objectives of the project Stroke is a major cause of disability in the ageing population. Atherosclerotic carotid artery stenosis is an important cause of stroke, which can be prevented by carotid endarterectomy. However, endarterectomy requires an incision in the neck and carries a small risk of operative stroke. Stenting, a new endovascular method of treating carotid stenosis, avoids some of the discomforts and complications of surgery. However, stenting also risks procedural stroke and may have an unacceptable incidence of restenosis. Evidence is required to inform patients, doctors and providers in the EU when choosing between these treatments. We have therefore started an international randomised trial to compare the risks and benefits of stenting with endarterectomy. A European network of centres will harmonise stenting techniques, safeguard the safety of EU patients thus treated and ensure the trial results are generalisable throughout the EU. Thus the overall objectives of this project are to: 1. Set up a European network of collaborating centres performing carotid stenting and surgery for the prevention of stroke in patients with carotid stenosis. 2. Encourage harmonised clinical practice and standards for the investigation and treatment of carotid stenosis to improve the quality of stroke prevention within the EC. 3. Carry out a randomised clinical trial within the network to compare the risks, benefits and economic value of carotid stenting and surgery. 4. Facilitate dissemination of knowledge and experience of the procedures involved and subsequent results throughout the network. Experimental approach and working method The experimental approach is that of a randomised controlled clinical trial to compare two treatment policies. The working method is the recruitment of collaborating centres with appropriately qualified clinicians to randomise patients to one or other treatment in the trial, and assist in the dissemination of results. Achievements and results An active network has been set up to disseminate information about the treatment of carotid stenosis. An ongoing clinical trial has also been set up that is successfully continuing to recruit both patients and centres. On 31 December 2003 there were 24 active centres and 244 patients had been recruited. 25 J. van Gijn University Medical Center Utrecht, Department of Neurology PO Box Heidelberglaan 100 NL-3508 GA Utrecht, the Netherlands Tel: (31-30) , Fax: (31-30) Martin M. Brown Institute of Neurology, University College London National Hospital for Neurology & Neurosurgery Queen Square, London WC1N 3BG, UK Tel: , Fax:

15 9. Molecular basis of vascular events leading to thrombotic stroke Project Acronym: MOLSTROKE Project number: LSHM-CT EC contribution: Starting date: 1/01/2005 Duration: 36 months 26 Summary Stroke kills about 5 million people annually and is also the leading cause of disability and dementia in adults. Early recognition of individuals at risk for stroke would significantly alleviate the heavy social and economical burdens due to stroke. Advances in identification of vulnerable individuals require development of entirely new strategies. Stroke is triggered by thrombosis occurring after rupture of atherosclerotic plaques, and MOLSTROKE focuses on identifying pathogenetic molecular mechanisms and vascular protagonists defining vulnerable plaques and contributing to plaque rupture. MOLSTROKE assembles 7 partners with multidisciplinary backgrounds and exploits innovative technological approaches together with testing of novel pathogenetic hypotheses. The priority research areas of MOLSTROKE comprise 1) concomitant wide genomic and histoproteomic screening of lesional vascular tissue to identify novel pathogenetic markers, 2) Early inflammatory events, which are the key to atherosclerosis progression and hence primary prevention, and 3) atherosclerotic plaque instability, which leads to the acute clinical thrombotic events. The proposed investigations will implement novel technologies of differential display of unknown genes and vascular tissue arrays. Thus, weighted identification of stroke denominators can be accomplished and thereby lead to improved diagnostic and treatment modalities. The research armoury spans genomics, tissue arrays, molecular biology, cell biology, immunology, biochemistry, gene transfer, animal models and integrative bioinformatic software tools. MOLSTROKE will maximize the scientific, educational and commercial potential of the proposal by electronic data-sharing, communication networks, shared research tools, and training programmes. s Gennaro De Libero Therese Resink Experimental Immunology Signal Transduction Department of Research, University Hospital Basel CH-4030 Basel, Switzerland Problem Stroke is a major killer since about 5 million people die each year of this disease. Stroke is also the major cause of disability in adults, and the second most important cause of dementia in Western countries. Among those who survive a stroke, the risk of a second stroke is very high. Currently known risk factors for stroke (age, cardiovascular diseases, atrial fibrillation, arterial hypertension, diabetes mellitus, carotid stenosis) are of low sensitivity and specificity. It is important to identify new markers to more appropriately predict the possible development of stroke and to identify those subjects with potential benefit from preventive therapy. Ischemic strokes account for 83% of all strokes and thrombotic strokes represent 52% of all ischemic strokes. Thrombotic stroke is a consequence of atherosclerotic disease and is caused by destabilization of atheromatous plaque with ensuing thrombosis and vessel occlusion locally or distally due to embolism. A large body of data supports that inflammation plays a major role in the pathophysiology of atherosclerosis and stroke. Understanding the mechanisms responsible for the initiation, establishment, maturation and persistence of atherosclerotic lesions is far from being fully accomplished. The current research focus is definition of new criteria to recognize vulnerable plaques and vulnerable patients. These criteria should be based on better definition of the pathogenetic mechanisms of plaque rupture. They should utilize inexpensive and relatively non-invasive screening methods that are capable of adding predictive value to measurements of established risk factors. Moreover, they should be readily applicable to an asymptomatic population. Aim The main scientific and technological goals of MOL- STROKE are to identify mechanisms and molecular protagonists that participate in the vascular pathological events leading to thrombotic stroke. MOL- STROKE addresses objectives using two concomitant strategies. The first strategy is non-hypothesis driven and will utilize wide-screening technologies together with novel bioinformatics tools to identify genes and proteins preferentially expressed in atheromatous plaques as well as molecular, biochemical, and cellular patterns potentially involved in plaque rupture. The second strategy is based on hypothesis-driven studies, taking into consideration current knowledge of the pathogenesis of vascular lesions at the level of molecular protagonists and mediators of inflammation and vascular cell responses to inflammation. Expected results: Identification of genes of potential pathogenetic importance Generation and use of novel bioinformatics tools Localize and quantify differentially expressed proteins in symptomatic atherosclerosis Identify molecular targets of novel therapeutic approaches to prevent symptomatic atherosclerosis and stroke Strategy for immunotherapy of atherosclerosis and stroke prevention Strategy for lipid-based immunomodulation of atherosclerosis Development of new therapeutic strategies (antiinflammatory and/or anti-angiogenic) for prevention of thrombotic stroke Translational impact to other diseases in which inflammation and excessive angiogenesis occur (e.g. cancer, diabetic retinopathy, arthritis, psoriasis) Results to date MOLSTROKE project began in January During this first year a tissue bank has been initiated, arterial and venous tissue arrays from atherosclerotic patients and vessel transplant donors have been constructed. Initial array investigations have been performed. Protocols for RNA extraction from plaque tissues have been established and preliminary microarray studies have been performed. Genes potentially involved in plaque rupture have been analyzed and immune-response in mouse models of atherosclerosis is under investigation. Identification of plaque lipids involved in stimulation of acquired immunity has been initiated. A panel of T cell clones specific for lipids present in plaques has been established. The influence of lipids on the innate immunity (DC differentiation, investigated with gene arrays, proteomics and lipidomics) have been initiated. Relevant lipids have been synthesiszed. Lipid effects on angiogenesis have been studied using microarray analyses, in vivo and in vitro angiogenenic assays, and cellular signalling. Angiogenic properties of cell adhesion molecules have been investigated. In vitro studies on the pro-angiogenic interactions between T cells, macrophages and endothelial cells have been initiated. These initial studies have already confirmed that plaque lipids have multiple biological effects on different cell types. In particular, the initial results obtained in MOLSTROKE have shown the unique capacity of lipids to stimulate acquired immunity, innate immunity and to modulate neo-angiogenesis. Expected end results are the exact species identification of individual lipids that, by stimulating chronic inflammatory response and neo-angiogenesis within plaques, directly participate in plaque rupture and eventual stroke. This project may lead to understanding of the molecular mechanisms of plaque rupture and thereby facilitate therapeutic targeting of individuals at risk. 27

16 10. Genetic regulation of the end-stage clotting process that leads to thrombotic stroke 11. Glutamate Receptor Interacting Proteins as Novel Neuroprotective Targets Project Acronym: EuroClot Project number: LSHM-CT EC contribution: Starting date: 1/01/2005 Duration: 36 months Project Acronym: GRIPANNT Project number: LSHM-CT EC contribution: Starting date: 1/08/2005 Duration: 36 months 28 Summary Thrombotic stroke is a disabling condition - affecting an estimated 650,000 Europeans annually, with considerable mortality and costing over 30 billion/yr. This project aims to unravel the genetic basis of thrombotic stroke leading to new diagnostics and drug targets. Genetic factors account for a substantial component of the incidence & mortality of stroke. There is little effective therapy. EuroClot aims to identify and validate potentially therapeutically useful genes associated with thrombotic stroke using a novel approach. Stroke is a complex end-point disease involving the interaction of many pathologic processes, such as vessel wall atheroma, hypertension, platelet function & coagulation. EuroClot focuses on uncovering the genes that control the end-stage of the coagulation process that leads directly to the production of the thrombus (clot) that causes vascular obstruction and tissue death. Clinical studies indicate that alterations in fibrin structure and/ or function create a prothrombotic phenotype, which increases vascular risk. Twin studies have shown a substantial genetic component to levels of activation peptides and the final common pathway of thrombus (fibrin structure/function). Aim To identify the major genes involved in variations of the end-stage clotting process and investigate the role of these novel genes (and existing candidate genes) in the pathogenesis of stroke across Europe. EuroClot will study stroke intermediate phenotypes in over 3000 twins from GenomEUtwin project involving 8 countries and 700 subjects from extended families. Genes will be validated in 1000 stroke cases including those from the large European prospective MORGAM study. Cross-European differences in allelic frequencies will be examined along with their relative impacts. Phenotyping will be standardised and harmonised and a European database established. Close links with European SMEs will ensure that all findings from EuroClot are maximally exploited to develop future novel diagnostics and therapeutic targets. Summary Excitotoxicity contributes significantly to neuronal cell death in a number of neurological conditions including stroke, head trauma, and Huntington s disease. The recent discovery of the proteins that anchor and interact with glutamate receptors opens for a new strategical approach to cytoprotective therapy. The present project aims at exploiting this conceptual advance to provide a platform for cytoprotective therapies that do not interfere unduly with synaptic transmission. Glutamate receptor interacting proteins ( interactors ) serve dual purpose. They determine the level and site of glutamate receptor expression within the cells and connect the receptors to specific intracellular signalling pathways. Both roles are interesting from a therapeutical perspective. Thus, excitotoxicity might be alleviated by modulation of the surface expression of glutamate receptors, as well as by interfering with their downstream signalling. The first part of the project aims at providing a more complete picture of the functional roles of interactors (WP1-3). It is envisaged that we will be able to identify novel interactors and that we will be in a position to understand, at a molecular level, how the different interactors connect with glutamate receptors and with each other. This part of the project will also elucidate the principles that govern the turnover and surface expression of glutamate receptors and the mechanisms that couple the individual receptors to specific downstream effectors of excitotoxicity. The second part (WP4 and 5) aims at exploiting the increased insight obtained through the first part of the project to design ways to alleviate excitotoxicity in different model systems. In designing these experiments the complex of glutamate receptor interacting proteins will be viewed as a nodal point in orchestrating the surface expression of receptors and in activating appropriate and inappropriate (excitotoxic) signalling pathways. The present project will be based on a unique combination of methods that draws full advantage of the technological advances made over the last few years. 29 Tim Spector Twin Research Unit, St Thomas Hospital Lambeth Palace Road London SE1 7EH, United Kingdom Tel: +44 (0) ; Fax: +44 (0) Ole Petter Ottersen University of Oslo, CMBN Postboks 1105 Blindern N-0317, Norway Tel: , Fax

17 12. Multipotent adult progenitor cells to treat stroke 13. Pre-clinical evaluation of stem cell therapy in stroke Project Acronym: STROKEMAP Project number: LSHB-CT EC contribution: Starting date: 1/10/2006 Duration: 36 months Project Acronym: STEMS Project number: LSHB-CT EC contribution: Starting date: 1/12/2006 Duration: 36 months 30 Summary Successful therapy for stroke will be achieved using cells that can limit ischemic injury and differentiate into the multiple cell types needed for restoring blood flow and neural circuits, and would be available for therapy off the shelf. As Multipotent Adult Progenitor Cells (MAPCs) differentiate into vascular and neural cells, and reconstitute damaged tissues in vivo, we hypothesize that MAPCs is an ideal allogeneic cell product to treat stroke. In WP1, WP2 and WP3, we will develop approaches to generate committed vascular cells and neuroprogenitors, and identify key molecular events that guide differentiation. WPs 4-7 will rigorously evaluate the pre-clinical efficacy of allogeneic MAPCs or their progeny in stroke. This will include development of noninvasive imaging techniques to follow the fate of grafted cells and evaluate their impact on CNS function (WP4). We will compare the efficacy of MAPCs with that of till now goldstandard stem cell populations in stroke, and determine mechanisms underlying observed effects (WP5). We will examine the immunogenicity of MAPCs and their differentiated progeny in vitro (WP6) and in vivo using mice with a human immune system (WP7). Studies in WP9 will develop clinical grade culture systems to generate human MAPCs and, if needed, lineage committed progeny. Studies in WP8 will develop a framework in which to develop clinical grade stem cell products in an ethically responsible manner. The management, exploitation and dissemination of the project will be ensured through WP10. These studies will lay the foundation for clinical trials of MAPCs in stroke in Europe in subsequent years. The highly innovative methods, tools and technologies that will be developed will not only be applicable in the area of stem cell based therapies for stroke, but may significantly advance use of human stem cells in regenerative medicine. Summary Using stem cells (SC) multipotent properties has become a challenging research field for most clinical areas, especially in disciplines lacking treatment options such as brain disorders and lesions. In particular, stroke or ischemic cerebrovascular disease accounts for roughly half of the patients hospitalised for neurological diseases and is associated with a large proportion of the health care costs in Europe. Until now, all neuroprotective approaches having yielded positive results in animal models have proven ineffective in clinical trials. SC clonal populations expected capacity to self-renew and differentiate efficiently into the desired cell type promise to produce beneficial effects in many diseases. Several studies indicate the therapeutic potential of SC in stroke after transplantation of various SC types. However, standardisation of conditions to regulate SC proliferation and differentiation to produce the «ideal» graft need to be better defined; changes induced by their transplantation into lesioned brain structures are unknown, as is the full extent of functional improvement at long-term post-stroke delays. STEMS deals with the therapeutic aim of using SC to reduce stroke-induced brain dysfunctions. Its main objective is to determine the extent and limits of SC therapy in stroke to pave the way for clinical trials. It will be achieved through the completion of six successive and complementary goals: - Definition of standard experimental conditions for proliferation, guided differentiation and mass-production - Identification of the best transplantation protocol regarding poststroke delays in a rat model of focal ischemia - Control of safety and compatibility aspects - Quantification of the effects of SC transplantation on functional impairments - Transposition of the optimal experimental conditions to non-human primates - Definition of the relevant human cell therapy product and operating procedures to be applied to stroke patient. 31 Catherine Verfaillie Katholieke Universiteit Leuven Department Oncology - Hematology Transplantation Oude Markt 13 B-3000 Leuven, Belgium Tel: , Fax Brigitte Onteniente Institut National de la Recherche Médicale INSERM UMR rue du général Sarrail F Créteil, France Tel: , Fax

18 14. Towards a stem cell therapy for stroke European Commission EUR Luxembourg: Office for Official Publications of the European Communities pp x 29.7 cm ISBN ISSN Project Acronym: STEMSTROKE Project number: LSHB-CT EC contribution: Starting date: 1/1/2007 Duration: 36 months Summary Stroke is a major cause of long-term disability in humans, but effective treatments are lacking. The StemStroke consortium comprises 5 highly qualified academic research teams and one SME which, together with excellent clinicians in the stroke field, will perform innovative research leading to the first preclinical protocol for application of stem cell therapy in stroke patients. Human neural stem cell (NSC) lines will be isolated from the foetal and adult brain, and from embryonic stem cells. Cellular and molecular mechanisms regulating the proliferation, migration, survival, and differentiation of the NSC lines after transplantation into the stroke-damaged rodent brain will be determined. In parallel, Stem- Stroke will unravel mechanisms regulating self-repair after stroke through formation of new neurons from the adult brain s own NSCs. The StemStroke will explore the morphological and functional integration of grafted and endogenously generated NSCs and their progeny in the stroke-damaged brain, and develop new in vivo imaging and behavioural tests, relevant for the human situation, for assessment of stem cell function and recovery of sensory, motor and cognitive deficits. Finally, StemStroke will optimize transplantation- and endogenous neurogenesis-based strategies and create an important preclinical protocol which can be rapidly translated into human trials. The teams contain top-level expertise in animal models of stroke and MRI-based in vivo imaging, stem cell, molecular, and cellular biology, molecular genetics, animal behaviour and psychology, translational research, and clinical cell therapy. Through the participating SME, commercial exploitation of the knowledge emerging from the project is ensured. The complementarity of expertise within the consortium, together with the intellectual and technological resources available from each partner, will ensure efficient and high-quality performance and feasibility of achieving its ambitious S&T goals. SALES AND SUBSCRIPTIONS Publications for sale produced by the Office for Official Publications of the European Communities are available from our sales agents throughout the world. You can find the list of sales agents on the Publications Office website (http://publications.europa.eu) or you can apply for it by fax (352) Contact the sales agent of your choice and place your order. Zaal Kokaia Lund University, Faculty of Medicine BMC B-10 Klinikagatan 26 S Lund, Sweden Tel: , Fax:

19 KI-NA EN-C

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