The Neuroscience Group at the Ernst-Moritz-Arndt ...



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4.1 Final publishable summary report

4.1.1 Executive summary

Neuroscience is the fastest growing area in basic scientific research. Neuroscience is an interdisciplinary field designed to improve our understanding of neural communication mechanisms in health and disease. Treatments, cures, and, above all, knowledge are the possibilities that continue to be discovered through neuroscientific research. The Neuroscience Group at the Medical Faculty of the University of Greifswald promotes interdisciplinary investigations from the level of gene expression in single neurons to imaging of localized regions of the human brain and neurorehabilitation. The Neuroscience Group at the Ernst-Moritz-Arndt University is located in a convergence region, and the paucity of high-tech, expensive equipment for research, the lack of a critical mass of experienced researchers, a reduced level of experience and knowledge of researchers as well as fewer international collaborations with highly competitive groups put severe limitations on the impact of our publications and our capacity to attract research funds from EU programs. Therefore the major aim of this proposal was to redress this balance by (i) stimulating the realization of the full research potential of our Neuroscience Group by acquisition of cutting-edge technology and equipment; (ii) increasing the quality and international impact of our research by cooperation with high profile institutions and recruitment of experienced researchers; (iii) improving S&T experience and knowledge of researchers by short term and long term trainings with our Strategic Partners and exchange of know-how and experience with the scientific experts; (iv) organization of workshops to facilitate knowledge transfer at international level; (v) plenary meetings; (vi) dissemination and promotional activities. The equipment was timely delivered. Since then it has been in use and allowed studies on genetic studies in aging and brain diseases. The confocal laser microscope is used to study cellular structures at 3D level. The MagPro X100 Magnetic Stimulator is a high-performance, non-invasive magnetic stimulator for use in both the clinic and in medical research. By the use of MagPro X100 patterned transcranial stimulation, e.g. TBS could be introduced in the neurorehabilitation research unit. To improve S&T experience and knowledge of researchers in molecular and cellular neuroscience by training studies six experienced researchers have been sent to our cooperating partners. At the same time the project leaders and the corresponding project partners did exchange know-how in specific areas of neuroscience. Three international open workshops on (i) brain regeneration after injuries, (ii) on brain stimulation and brain repair mechanisms, (iii) transport processes in neurodegenerative and neuromuscular diseases, have been organized. On these occasions three booklets for the presentation and publicity of the project achievements have been published along with five publications with our cooperating partners. The workshops have been preceded by press conferences.

These measures allowed the Neuroscience Group in Greifswald to (i) increase our international visibility by organizing 3 workshops; (ii) acquire a highly competitive research infrastructure; (iii) publish a number of good publications. Unfortunately, until to date, it did not help to successfully contribute to other EU Framework Program projects.

4.1.2. Summary description of project context and objectives (max 4 pages)

Project context

Neuroscience is a systemic approach helping us to understand the molecular basis but also physiology of brain disorders. Knowledge, treatments and cures continue to be discovered through brain research. The combination of structural and functional studies allows the integrative investigation of neural development, cellular responses to injury, diseases and genetic modification. Further, the mechanisms that underlie communication between nerve cells and the processing of information through brain and spinal cord circuits can be elucidated. The recent development of probes and microscopic techniques for localizing critical molecules in neurons as well as new noninvasive imaging methods that can directly link brain function to behavior, have provided breakthroughs in the way we view the brain, revealing the diversity and complexity of the constituent neurons and circuits.

The Neuroscience Group at the Medical Faculty of the University of Greifswald promotes interdisciplinary investigations from the level of gene expression in single neurons to imaging of localized regions of the human brain and neurorehabilitation. Ongoing studies encompass: behavioral neuroscience; neurorehabilitation; functional neuroimaging and sensorimotor integration; molecular neurophysiology; molecular and cellular mechanisms underlying recovery of brain tissue and function after stroke; adult brain neurogenesis and tissue regeneration. Although there are quite a number of neuroscience institutions in Europe conducting high quality research, there is a growing need for research and development in this area. The Neuroscience Group at the Ernst Moritz Arndt University (EMAU) Greifswald is located in a convergence region, and the paucity of high-tech, expensive equipment for research, the lack of a critical mass of experienced researchers, a reduced level of experience and knowledge of researchers as well as fewer international collaborations with highly competitive groups put severe limitations on the impact of our publications and our capacity to attract research funds from EU programs.

Progress beyond the state of the art

Laboratory of Molecular Neurobiology

The goal of regenerative medicine is to restore cells, tissues and structures that are lost or damaged after disease, injury or ageing in humans. The Laboratory of Molecular Neurobiology is an entity devoted to research and development that is located in the Clinic of Neurology of the Ernst Moritz Arndt University in Greifswald. The overall objectives of the studies in this work program are to study the molecular and cellular reactions to injury in those (mammalian) systems which do not classically regenerate and to ask whether the early events following injury evoke inhibitory responses and/or characteristic responses in stem cells which can be traced by monitoring the transcriptome and proteome profiles. The specific aims of our group are to (i) uncover cellular and molecular mechanisms underlying neurorehabilitation after stroke in aged rats, (ii) examine the role of adult brain neurogenesis for tissue repair after injuries, (iii) investigate possibilities to positively influence/augment adult neurogenesis from stem cells and therefore improve neurorehabilitation after tissue damage in the CNS; (iv) to monitor the recovery of function after injuries to the central nervous system. To this end we will use a battery of functional tests including: (1) sensorimotor tests like the Rota-rod; Cylinder Test; Adhesive Tape Removal Test; (2) learning and memory tests including the Morris water maze; Elevated Plus Maze; T-Maze; (3) fear conditioning, including the startle box test.

Although we are addressing highly relevant areas of neuroscience research, we are aware that our investigative approach is not causal. In order to identify specific genetic mechanisms underlying recovery of tissue and function after stroke we have to turn to transgenic mice and use functional genomics and proteomics in conjunction with in vitro models to identify specific genes and proteins that are involved in the recovery process after stroke. By this project we shall significantly improve the research infrastructure in neuroscience. Using new technology acquired by this measure we aim at discovering regeneration-relevant genes. Further, by exchange of know-how we hope to establish new methods of investigation in brain repair. Gene expression will be modulated using double-stranded small interfering RNA (siRNA). Further, the acquired experience will allow us to study axonal outgrowth of embryonic CNS axons on organotypic slice cultures. Such studies require the acquisition of the following equipment: (1) a modern confocal laser scanning microscope for the analyses and phenotyping of cells in the nervous system and 3D reconstructions of biological structures, and (2) a quantitative real-time PCR system.

These ambitious projects require adequate equipment and the expertise of experienced strategic partners like the Institute of Brain Research (BRI) in Zurich represented by Dr. Oliver Raineteau and the Laboratory of Neural Stem Cell Biology, University of Lund, represented by Dr. Zaal Kokaia. The Institute of Brain Research is one of the outstanding and experienced laboratories in Europe devoted to studying molecular and cellular mechanisms underlying tissue and functional recovery after injuries to the central nervous system.

The Laboratory of Neural Stem Cell Biology has pioneered the clinical application of cell transplantation in the human brain and provided the proof of principle that neuronal replacement can restore motor function in patients with Parkinson’s disease. This partner has also discovered a novel mechanism for self-repair after stroke based on neuronal replacement from the adult brain’s own neural stem cells (NSCs). Strengthening the research and technological development capacities will be based on advanced demonstrative training studies concerning the acquired equipment and the transfer of knowledge from Zürich and Lund to Greifswald by short- and long-term term trainings. The newly recruited personnel of the molecular neurobiology group will visit the Brain Research Institute in Zurich to learn and acquire new techniques related to (i) conditional expression of genes both in vivo and in vitro (ii) an overlay assay using postnatal slices from various brain regions as a substrate to study influence of secreted cues onto cell migration and differentiation in the brain; (iii) organotypic slice cultures from cortex and hippocampus with the aim to investigate gene function, functional imaging and behavior of different cell types in situ; (iv) creation of artificial structures which support the migration of neuronal precursor cells in the injured brain (v) learn various paradigms of behavioural recuperation after injuries to the central nervous system.

The newly recruited personnel of the molecular neurobiology and the experimental neurology groups will be trained in Lund how to dissect the embryonic and adult rodent brains to remove the neurogenic zones such as ventricular and sub-ventricular zones. They will also learn how to dissociate dissected tissue into single cell suspension and plate to expand as monolayer culture or neurospheres. The researchers will be trained to follow the growth of the culture and after sufficient expansion prepare the cell suspension for the intracerebral transplantation. They will also get familiarized with rodent stereotaxic frame for intracebral injection of NSCs in the defined brain regions. The training of the researchers will also include transcardial perfusion of the rodents with implanted NSCs, sectioning of the brains for immunocytochemical staining as free-floating sections and fluorescent microscopy for identification and characterization of implanted NSCs. The researchers will also be trained in behavioural methods aimed at assessing recovery after injuries to the central nervous system.

In addition, the researcher capacity in Greifswald will benefit from the exchange of know-how and experience on specific topics related to regenerative medicine through trans-national two-way secondments of experts between the molecular neurobiology and experimental neurology groups and the Institute of Brain Research in Zurich and the Laboratory of Neural Stem Cell Biology, University of Lund.

Laboratory of Neurorehabilitation

Previous research of the neurorehabilitation research group focused on (a) the detailed analysis of behavioural deficits after stroke, i.e. “behavioural plasticity”, in conditions such as somatosensory deficits, paresis, and apraxia, (b) clinical scale development to monitor impairment and activity limitations, (c) related alterations of brain network activities and prediction of recovery based on cognitive performance and brain activity [EEG analysis], (d) development and clinical evaluation (RCTs) of neuroscience-based training techniques for motor recovery after stroke, and (e) a first concomitant investigation of lesion- and training-induced cortical plasticity [TMS].

The objective of this support action is to further exploit the group’s research potential, thus contributing to the regional development with stroke (and its rehabilitation) being one major health and disability challenge of a European regional population with demographic changes indicating higher proportions of elderly patients. This exploitation will be fostered by taking advantage of the knowledge and experience existing in other regions of Europe.

So far, rehabilitation techniques developed by the group were based on therapist-centred interventions. The research group’s results are competitive and achieved international attention. The group does, however, not hold expertise in potential future rehabilitation interventions based on brain stimulation. This will be sought during this support action. The action plan as indicated below will enable the research group to participate in this research that could have a major impact on stroke rehabilitation in the near future. Another specific research development strategy of the neurorehabilitation research group will be to establish the technical equipment as well as knowledge and training of researchers, to engage in research using neuro-navigation based brain diagnostics and stimulation (rTMS) in stroke patients and focusing on brain recovery and plasticity by innovative interventions. Equipment for brain stimulation (rTMS) will be purchased. Research staff of the neurorehabilitation research group will visit the Physiology and Pathophysiology of Human Motor Control Research Group at the Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, Queen Square, London, U.K., and the Department of Cognitive Neuroscience, Faculty of Psychology, Maastricht University, the Netherlands, to be educated in brain stimulation and neuro-navigation techniques. Experts from these centres will be invited to visit the neurorehabilitation research group in Greifswald.

Laboratory of Molecular Neurophysiology

The combination of molecular biological methods, cellular imaging and cell physiological techniques allows the functional analysis of ion channels in the neuromuscular system. Dysfunction of ion channels, altered gene expression or altered regulation of channel proteins can lead to disturbances of cellular excitability, to cell damage and even to cell death and these conditions can cause severe neuromuscular diseases. The Laboratory of Molecular Neurophysiology is part of the Institute of Pathophysiology at the Ernst Moritz Arndt University of Greifswald. Our overall objectives in this program are to characterize gene expression, cellular localization and function of cation channels that are responsible for the background Ca2+ influx into muscle fibers. A disturbed cellular Ca2+ homeostasis can lead to muscle fiber necrosis and cell death and seems to play a key role in muscular dystrophies. Antagonists of ion channels that inhibit the background Ca2+ influx may be promising tools or at least lead structures to develop a pharmacotherapy for muscular dystrophy.

Our strategic partners are Drs. Hanns Lochmüller and Volker Straub from the Institute of Human Genetics in Newcastle, UK. Dr. Lochmüller is an expert for construction and application of reporter systems in vivo and in vitro. Transfection of reporter molecules into cultured muscle cells and isolated fibres will be established in collaboration with our partner. Distribution of TRPC3 channels in normal and dystrophin-deficient muscle fibres will be studied by confocal laser microscopy.

Carrying out the suggested studies requires the acquisition of a modern confocal laser scanning microscope. Using this microscope will make it possible to analyze the cellular localization and trafficking of TRP channels in living cells. The combination of confocal microscopy and electrophysiology will give new insights in the functional activation of TRP cation channels. Confocal microscopy relies on the use of fluorescent reporter molecules, a technique that is well developed in the laboratory of the strategic partner at the Institute of Human Genetics in Newcastle. Exchange of researchers, collaboration and visits will improve the research potential and competitive position of the Laboratory of Molecular Neurophysiology in Greifswald.

Laboratory of Molecular Neuropathology

Deposition of the beta-amyloid peptide (Abeta) in the brain occurs during normal aging and is augmented in Alzheimer’s disease (AD). Since cells of the brain continuously produce Abeta, it has been suggested that decreased clearance of the peptide from the brain at the blood brain barrier (BBB) could contribute to the buildup of Abeta. Mechanistic studies on Alzheimer’s disease require clearly the use of transgenic animals and the expertise of an experienced strategic partner like the Institute of Neuropathology of the University of Zurich represented by Prof. Dr. Adriano Aguzzi. This institute is one of the outstanding and experienced laboratories in Europe devoted to studying the molecular and cellular mechanisms underlying neurodegenerative diseases like Alzheimer’s disease or Creutzfeldt-Jakob-Disease. Strengthening the research and technological development capacities will be based on advanced demonstrative training studies and the transfer of knowledge from Zurich to Greifswald by long-term trainings. The scientific staff members of our group will visit the Institute of Neuropathology to learn and acquire new techniques including (1) preparation of Abeta using brain extracts from humans with AD or APP/PS1 transgenic mice; (2) injection of Abeta preparations intracerebrally into FVBAPP/PS1-MDR1a/b-/-- mice and FVBAPP/PS1-MDR1a/b+/+ mice, respectively. In addition, the researcher capacity in Greifswald will benefit from exchange of know-how and experiences.

Objectives

The major objectives of this project were:

1. Acquisition, development or upgrading of research equipment of the Neuroscience Group at the Medical Faculty, Ernst Moritz Arndt University of Greifswald Including a confocal microscope devoted to live imaging and molecular physiology.

2. Recruitment of incoming experienced researchers. The Neuroscience Group in Greifswald does not have a critical mass of skilled researchers. Therefore a team of 6 researchers will be recruited. Each of them will be involved in collaborative research involving at least two network laboratories.

3. Improve S&T experience and knowledge of researchers by exchange of know-how and experience with scientific experts from several Strategic Partners.

4. The network will have a strong focus on training of the integrative researchers and other young scientists in the participating laboratories. The training includes demonstrative applications with acquired equipment and collaborative sessions with several strategic partners

5. Organization of three workshops with international participation to facilitate knowledge transfer at international level.

6. Plenary meetings. Each year there will be a plenary meeting involving all the members of the Neuroscience Group in Greifswald, the integrative researchers and members of our Strategic Partners who will be invited as plenary speakers. At the plenary meetings there will be a press office and access for patient groups.

7. Dissemination and promotional activities like a Neuroscience Group’s Webpage and Booklets on Regenerative Medicine and Stroke.

4.1.3. Description of the main S&T results/foreground (max 25 pages)

WP1: Improvement of Research Capacity for Neuroscience at Ernst Moritz Arndt University of Greifswald

D1.1 Employment of Researchers Report

By this project we were able to increase our research capacity by hiring:

1) Four postdoctoral fellows: Drs. Ana-Maria Buga, Senthil Sivanesan, Anja Brenn and Marianne Christel

2) We further recruited a physician, Mr. Schüttauf and a PhD student Shadap Syed Raza. Two other experienced PhD students were recruited and earned their doctoral degree during the project period, Stephanie Hübner and Mirjam Krautwald. One medical student working for the project in the Institute of Pathophysiology submitted his thesis (Thom Lange).

D1.2 Equipment Purchasing Reports

Acquisition and upgrading of research equipment for the molecular neurobiology group.

The equipment was timely delivered. Since then it has been in use and allowed studies on genetic studies in aging and brain diseases.

a) The confocal laser microscope can be used to study cellular structures at 3D level. To this end many tissue sections have to be stained using the second acquired device.

b) Semi-automat for immunostaining of tissue sections

c) Finally, the Real Time PCR System allows the study of individual gene expression in health and mental diseases. By this technological upgrade the “Laboratory of Molecular Neurobiology” has significantly increased its competitiveness at national and international level.

The Use of Resources

A combined application of automatic tissue staining (b) followed by Confocal Laser Scanning Microscope (a) for brain regeneration after injuries is shown in Fig. 1 below:

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Figure 1. Visualization of the vascular nische (red) and neuronal progenitor cells (doublecortin, grün) in the brain after stroke.

The Real Time PCR System (c) has been extensively used to study changes in genetic events underlying brain regeneration after stroke. The results are summarized for genes of the oxidative stress in Table 1 and Fig. 2.

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Figure 2. Typical amplification curves for several stroke-genes using the RT-PCR machine

Acquisition and upgrading of research equipment for the molecular neurophysiology group.

The equipment was designed to improve capacity and quality of physiological measurements in the molecular neurophysiology group. All equipment was timely delivered, i.e. in May 2009, is functioning well and included:

a) Capillary electrode Puller: Sutter P-2000

b) Patch clamp amplifier EPC10

c) Amplifier SEC-05x

d) motorized microscope stage for xy-scanning

e) Micromanipulator systems

The Use of Resources

The capillary electrode puller (a) was used to draw glass pipettes with small diameters (1- few µm) to allow recordings from single cells and for solution application to individual cells. The amplifiers (b, c) were used for intracellular recordings of electrical currents from cells and/or voltage changes. The new equipment made the collection and evaluation of such cell physiological data more standardized and much easier to handle.

Of great help for the investigations was the motorized microscope stage (d), since it allowed to store positions of single cells in a petri dish and re-inspect these cells later. Depending on the scientific question between 10 and 100 cells could be investigated in stained form (Figure 3). Even signals from living cells have been recorded in that way and the results have been already published (publication No. 3 of this report).

Figure 3. Staining of muscle fibers with fluorescence indicators using a microscope with motorized scanning table. Nuclei (green, left), cation channel TRPC3 (red, middle) and overlay of both stainings (right).

Acquisition, development and upgrading of research equipment for the neurorehabilitation research group.

MagPro X100 Magnetic StimulatorThe MagPro X100 Magnetic Stimulator is a high-performance, non-invasive magnetic stimulator for use in both the clinic and in medical research has been purchased on 21.04.2009.

The Use of Resources

The magnetic stimulator is used for both, diagnostic and therapeutic purposes (Fig. 4). In a combined mode with a neuronavigation system, the magnetic brain stimulation using a coil with a relatively focal magnetic field can be navigated to specific brain areas where the excitability can be measured by single magnetic impulses or altered by repetitive impulses. With patterned repetitive transcranial magnetic stimulation (rTMS) the local excitability of certain brain areas can either be diminished or enhanced. This gives a potential opportunity for research of brain dynamics both in healthy subjects (see publications No. 6 and 7 of this report) and subjects with brain disease such as stroke.

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Figure 4. TMS lab of the neurorehabilitation group showing the purchased magnetic stimulator in combination with the neuronavigation system for its use for focal brain stimulation.

WP2: Improvement of S&T experience and knowledge of researchers in

molecular neurobiology

D 2.2 Report on exchange of know-how and experience on molecular neurobiology (36th month)

D 2.3 Report on long term training in regenerative neuroscience (36th month)

To this end Dr. Ana-Maria Buga, affiliated to the Clinic of Neurology, Greifswald has joined the Laboratory of Neural Stem Cell Biology (UL) for one month, between 01.01.2011-31.07.2011. Dr. Buga learned (i) to stain brain sections by immunofluorescence; (ii) to quantify inflammation after stroke using stereological methods. This knowledge has been transferred to Greifswald. In the following we give a short account of the transferred know-how to Greifswald.

Protocol for immunhistochemie by fluorescence staining

Microglia: Mitotically active microglia were phenotyped by using a combination of mouse anti-rat antibody recognizing a cytoplasmic determinant of activated brain macrophages ED1 (clone MCA341), conjugated with FITC in conjunction with the rat anti-BrdU antibodies (Table 1).

Circulating blood cells and hematopoietic progenitor cells: The following antibodies were used to identify circulating blood cells with BrdU-positive nuclei: mouse anti-rat CD4, a marker for T helper cells; mouse anti-rat CD8, a marker for cytotoxic T cells; mouse anti-rat CD11b, which reacts with the CR3 complement receptor (C3bi) expressed on rat monocytes, granulocytes, macrophages, dendritic cells, NK cells, and a subset of lymphocytes; goat anti-rat CD34, a marker for hematopoietic progenitor cells and vascular endothelial cells; mouse anti-CD45, a marker for rat leukocyte common antigen; rabbit anti-myeloperoxidase, a marker for immature myeloid cells; and rabbit anti-rat polymorphonuclear leukocytes (PMN).

Oligodendrocyte progenitors and astrocytes: Oligodendroglia were double-immunolabeled with rabbit anti-NG2-specific antibodies (1:3000, Chemicon, Temecula, CA) and rat anti-BrdU-specific antibodies. Astrocytes were double-immunolabeled with rabbit anti-GFAP-specific antibodies, and rat anti-BrdU-specific antibodies. The antigen-antibody complexes were visualized with donkey anti-rabbit rhodamine-conjugated antibodies (1:4000) and donkey anti-rat FITC-conjugated antibodies (1:2000), respectively (Table 1).

Basal lamina and BrdU-positive cells: To determine the relationship between proliferating cells and the vascular basal lamina, sections were double-immunolabeled with rabbit anti-laminin-specific antibodies and rat anti-BrdU-specific antibodies. The antigen-antibody complexes were visualized with a mixture of donkey anti-rabbit Cy5-conjugated antibodies (1:2000) and donkey anti-rat rhodamine-conjugated antibodies (1:5000).

Brain capillaries and BrdU-positive cells: To determine the relationship between proliferating cells and the vascular wall, sections were double-immunolabeled with mouse anti-rat endothelial cell antigen (RECA)-specific antibodies and rat anti-BrdU-specific antibodies. The antigen-antibody complexes were visualized with a mixture of donkey anti-mouse FITC-conjugated antibodies (1:3000) and donkey anti-rat rhodamine-conjugated antibodies (1:5000).

Protocol for counting of cells after chemical staining: The number of labelled cells at the reperfusion times of 3, 7, 14 and 28 days was determined by counting cells on every tenth section in systematic random series across the entire infarcted volume. To this end, a sequence of confocal counting images of 161x242x25 µm, spaced 0.1 µm apart across a 25 µm-thick section and covering 30% of the infarcted area, was taken for fluorescently labelled cells. The resulting images were loaded into the 3-D analysis software “Volocity” (IMPROVISION, Coventry, UK) and computed using a Macintosh computer. The obtained images clearly indicated that most BrdU-labeled cells occurred, depending on the post-stroke stage, in dense, clonal-like clusters such that individual cells could not readily be distinguished, and hence an accurate count based on stereological methods could not be obtained. However, in many instances, the 3-D reconstruction method allows to differentiate closely spaced cells and thus to count all BrdU-labeled cells within the 3-D spaces with reasonable accuracy and reliability. Nevertheless, at time points where proliferation was at its maximum, such as 7 days post-stroke, counting was less precise because of the many mitotic cells with a clonal-like appearance. The relative mean number of BrdU-positive cells was then calculated per group, time point, and age by multiplying the number of cells per section times 3.3 (the counting boxes that were quantitated covered one third of the area of each section) times the section interval of 10.

WP3: Collaboration with strategic partners and training for the improvement of research capacity in cellular and molecular neuroscience at EMAU Greifswald

D3.2 Report on training and collaboration on neurorestoration with strategic partners (36th month)

Dr. Senthil Sivanesan could not be sent to Lund, Schweden. Instead we sent Dr. Buga for one month, between 01.08.2011-31.08.2011. Dr. Buga learned (i) to visualize apoptotic (dying) cells and to (ii) calculate the volume of the damage inflicted by stroke using stereological approaches. In the following we give a short account of the transferred know-how to Greifswald.

Terminal deoxynucleotidyltransferase-mediated UTP nick end labeling.

One of the cellular events associated with cerebral ischemia is cell death by apoptosis. In order to estimate the number and phenotype of cells undergoing apoptosis, we employed terminal deoxynucleotidyltransferase-mediated UTP nick end labeling (TUNEL) on free-floating sections. Briefly, sections were incubated with equilibration buffer for 30 min at room temperature followed by TdT-reaction solution diluted 1:1 with TUNEL dilution buffer (Roche Diagnostics, Mannheim, Germany). After 1 hr at 37°C, the reaction was stopped by the addition of Stop Buffer. After washing in PBS, sections were blocked in 3% donkey serum and 0.3% Tween in PBS overnight at 4°C. For peroxidase detection, TUNEL-treated sections were incubated with a sheep anti-digoxigenin-FITC antibody (1:1000; Roche Diagnostics) in 3% donkey serum, 1xPBS, 0,3% Tween for 2 days at 4°C. TUNEL labelling was visualized by incubation with biotinylated anti-sheep IgG (1:400; Jackson ImmunoResearch, West Grove, PA) followed by the avidin–biotin peroxidase complex (Vector Laboratories). The horseradish peroxidase reaction was detected with 0.05% diaminobenzidine (DAB) and 0.03% H2O2. To determine non-specific labelling, some sections were incubated without the Klenow fragment enzyme.

Determination of Infarct Volume

To assess the size of the infarct induced by transient focal ischemia, every twentieth section was stained with an immunological marker of neuronal viability (NeuN). Low power images of the stained sections were taken with a video camera and the images were printed. Then the infarcted areas were demarcated with a pencil and the images were scanned. Finally, the area and the partial volume of Every twentieth section were obtained using the NIH image analysis program. Integration of the resulting partial volumes gave the volume of the ipsilateral hemisphere along with the volume of the cortical infarct, which was then expressed as percent of the total volume of the hemisphere.

WP4: Collaboration with strategic partners and training for the improvement of research capacity in the treatment of neurodegenerative diseases at EMAU Greifswald

D4.2 Report on training and collaboration on neurodegenerative diseases with strategic partners (36th month)

To achieve this objective we sent Stephanie Hübner to Lund for 3 months (01.09.2010-31.12.2010)

During her stay, Stephanie Huebner had the opportunity to work with a rat model of stroke for cell therapy purposes. We delivered aged rats to the group of Dr. Kokaia and our researcher was trained to make transplantations for therapeutical purposes using human and rat stem cells. The visit has increased her experience in this field of stroke models and offered us the possibility to implement cell therapy for stroke in our research laboratory in Greifswald.

Techniques to prepare, cultivate and differentiate rat fetal mesencephalic neural stem and progenitor cells were learned. These cells are the progenitors for the dopaminergic system and in adults the loss of them leads to Parkinson disease.

Several treatments of these neural progenitor cells were tested in order to increase the dopaminergic differentiation. Treatment included growth factors, mechanical stress, hyperbaric oxygen, interleukins and others to enhance/inhibit certain cellular pathways. Some of these treatments were combined to see if there exists co-activation. To check if the treatment was toxic for the cells every treatment was tested with PI/Hoechst double staining to count the amount of dead cells after 24 and 48 hours post-treatment.

It is known that MAP-kinases (p38, JNK, ERK1/2) activate cellular processes, which can increase dopaminergic differentiation, e.g. interleukin-1beta. To detect activation of those kinase pathways FACE (fast activated cell-based enzyme-linked immunosorbent assay), a modified ELISA technique, was used. Therefore the cells were fixed 1, 2 and 3 hours after treatment and the ELISA’s were performed for p38, JNK and ERK1/2 to calculate the ratio between phosphorylated (activated) and total protein concentrations.

As the final step promising treatments/ combination of treatments were used for differentiation experiments. After treatment cells were allowed to differentiate for seven days, whereas in some cases treatment need to be renewed with the change of medium, e.g. growth factors. After fixations the cells where stained for several markers to determine and calculate the ratio of the different cell types. GalC, GFAP, Map2ab and TH where used to stain oligodendrocytes, astrocytes, mature neurons and pre-dopaminergic neurons.

Figure 4: Representative microphotographs showing mesencephalic neural stem cells during differentiation. a) GalC staining demonstrating oligodendroglial differentiation. b) Phase contrast images demonstrating astoglial and oligodendroglial differentiation. Living cells with blue soma. c) Typical "neurosphere" during neuronal differentiation (Map2 staining). Cell nuclei are counter-stained with DAPI. d) Bright-field images during neural differentiation; cells counter-stained with Hoechst (cell nuclei) and propidiumiodide (dead cells).

WP5: Improvement S&T experience and knowledge of researchers in molecular neuropathology

D5.2 Long term training report on the pathomechanisms of the Alzheimer’s disease (36th month)

To achieve this objective we sent Anja Brenn to the Institute of Neuropathology, University Hospital Zürich, Switzerland for 3 months (7. April 2010 to 30th July 2010). She worked in the group of Dr. Jeppe Falsig Pedersen in the Institute of Prof. Dr. Adriano Aguzzi. The following is a part of the report by Dr. Anja Brenn (Introduction and aim of the project) The complete training report is attached to this report as a pdf file:

During the research stay, I had the chance to work – under the supervision of Dr. Jeppe Falsig Pedersen - on a project with the following title: Role of Microglial Phagocytosis in Prion Disease

Introduction: Prion diseases, or transmissible spongiform encephalopathies, are fatal neurodegenerative disorders, affecting humans and a variety of animals. Microgliosis is besides spongiosis, neuronal cell loss, astrogliosis and prion plaque deposition, one of the most known hallmarks of prion infection and can be observed very early on the course of disease. Microglial activation even precedes the deposition of prion plaques and neurodegeneration. In the later stages of prion disease, microglia is shown to accumulate abundantly around prion plaques.

Microglia are resident immune cells of the brain and are of myeloid origin. Their role is in the first line of defence against injury and infection. Any disturbances of the central nervous system (CNS) homeostasis lead to the activation of microglia resulting in a rapid change in their phenotype. Depending on the mechanism of activation, microglia may release pro-inflammatory or neuroprotective factors and can also exert phagocytosis. Whether their activation in prion disease aggravates or allays the progression of disease remains to be elucidated. CD11b-HSVTK-mice (TK) express the herpes simplex virus thymidine kinase (HSVTK) under the CD-11b-promotor of myeloid cells. HSVTK transforms the pro-drug Ganciclovir (GCV) into a cytotoxic metabolite, leading to a selective ablation of proliferating myeloid cells. Preliminary data showed that the depletion of microglia in prion infected organotypic cerebellar slice cultures by GCV administration resultes in a markedly increased deposition of proteinase K resistant PrPSc and higher prion titers. Microglial ablation is also associated with the significant increase of neurodegeneration (unpublished data). Reconstitution of cerebellar slice cultures with wild type peritoneal macrophages leads to a decrease in PrPSc deposition and a reduction of cerebellar granule cell death. These findings indicate a role of microglia in prion containment, potentially by phagocytosis and degradation of prions.

Aim of the project: The aim of the proposed project was to investigate the role of microglial phagocytosis in prion disease and its possible role in prion containment. Organotypic cerebellar slice cultures prepared from Tga20-TK-mice (Tga20 mice overexpress PrPC), represent a suitable ex vivo model to study prion-induced neurodegeneration (Falsig 2008). These cultures will be prion inoculated and microglia will be depleted. Organotypic slices are incubated with infectious inoculum as free-floating sections, washed and cultured for up to 8 weeks. To modify microglial phagocytosis, microglia deficient slice cultures will be reconstituted with murine embryonic stem cell derived microglia (ESdM) precursors overexpressing GFP.

(see results including figures in a pdf file attached to this report: D5.2.Long-term-training-report_ABrenn_2010.pdf)

WP6: Collaboration with a strategic partner and training for the improvement of research capacity for molecular neurophysiology at the EMAU Greifswald

D6.2 Report on training and collaboration on molecular neurophysiology (36th month)

To achieve this objective we sent Mirjam Krautwald to Newcastle, UK, for one month (June, 2011). There she learned advanced techniques to improve her skills in confocal microscopy and in addition she got an introduction in magnetic resonance imaging of murine organs.

The following report was written by Mirjam Krautwald, PhD, about her stay in the Institute of Human Genetics in Newcastle:

During my visit at the partner institute in Newcastle (Institute for Genetic Medicine, Newcastle University, United Kingdom, Head: Prof. Hanns Lochmüller) I had the opportunity to learn several techniques and methods for the functional characterization of cation conducting membrane proteins in muscle tissue and of mice. In the first project the analysis of skeletal muscle function of mice in vivo by Magnetic Resonance Imaging (MRI) was demonstrated. In another project I was able to test the reactivity and specificity of two polyclonal rabbit-antisera against the two cation conduction membrane proteins TRPV2 and TRPV4 (TRP, transient receptor potential). Experiments included Western Blot Analysis, immunohistochemistry and confocal laser scanning microscopy of isolated muscle cells and tissue sections.

1. Analysis of skeletal muscles function in vivo by Magnetic Resonance Imaging (MRI)

The divalent cation Ca2+ is a common signalling molecule in living cells. Changes in the cytosolic concentration of free Ca2+ play a central role in many cellular processes. For example, Ca2+ can act as a messenger during signal transduction, muscle contraction, transmitter release, gene expression as well as cell proliferation and apoptosis. Because of its important physiological role the intracellular or resting concentration of Ca2+ underlies a precise regulation by Ca2+-sensing or Ca2+-binding proteins and cation conducting channels of cellular membranes. To maintain the normal intracellular concentration Ca2+ is actively pumped to the extracellular space or into intracellular Ca2+ stores, such as the endoplasmic reticulum and mitochondria. After specific signals the cytoplasmic free Ca2+ concentration can increase up to 100-fold of normal level by opening channels in the endoplasmic reticulum or the plasma membrane.

The pathomechanism of muscular dystrophy is very complex and involves muscle degeneration and dysregulation of calcium homeostasis of muscle fibres. Whereas the enhanced mechanical sensitivity of muscle fibres is caused by membrane fragility depending on the absence of the cytoskeletal membrane protein dystrophin, the molecular mechanisms of the high calcium uptake of dystrophic muscle fibres are still unknown. Nevertheless, calcium overload in skeletal muscles is proposed to force fibre degeneration by activation of calcium-dependent proteolysis followed by necrosis and detrimental impact on mitochondrial structure and function.

Magnetic Resonance Imaging (MRI) of muscles allows a sensitive identification of muscle degeneration and replacement by fat or connective tissue and provides great anatomical details.

To study the function of calcium channels in muscular dystrophy, cardiac muscles of dystrophic mdx mice were analysed by Magnetic Resonance Imaging. Therefore, mice were treated with the aminoglycoside antibiotic streptomycin from onset of disease to chronically block calcium channel function. At the age of twenty weeks the development of cardiac muscle were imaged after infusion of the common MRI contrast agent manganese. Mice were anesthetized with 5% isoflurane and a cannula was placed in the tail vein. The anaesthesia was maintained at 1,5% in oxygen and the mouse was placed on a sled. Body temperature, respiration and heart function were monitored by cutaneous ECG-electrodes. After calibration and shimming of the scanning area, the manganese infusion (190 nm Mn2+ per g bodyweight) via the tail vein by an automatic pump was started and series of 10 images were acquired every 5 min for an hour. The progress of the disease and the development of cardiac muscles of treated and untreated animals then could be analysed by computer based software programs and evaluation of captured images.

2. Analysis of polyclonal rabbit antisera against TRPV2 and TRPV4

The proteins TRPV2 and TRPV4 are members of the conserved superfamily of non-selective cation conducting membrane proteins called TRP (transient receptor potential) channels. Although the physiological function of TRPV2 and TRPV4 in skeletal muscles is not known in detail, these proteins seem to be involved not only in processes like muscle development and Ca2+ homeostasis, but also in disease progression of certain muscular disorders.

To understand the molecular and physiological function in more detail the expression, subcellular localization and activity of TRPV2 and TRPV4 in healthy and dystrophic skeletal muscle should be analyzed by immunohistochemistry and confocal laser scanning microscopy. Commercial antibodies against these proteins were normally peptide-generated resulting in a more or less unspecific reactivity. To obtain more specific antibodies against TRPV2 and TRPV4 we cloned and expressed the large cytoplasmic N-terminal region of both proteins in Escherichia coli. The proteins were purified by electroelution from polyacrylamid gels and were used for immunization of rabbits in 2010/2011. The specificity of these TRPV2 and TRPV4 antisera were then analysed in cooperation with our partner.

Because of the low expression level of TRP channels I learned a new method for the specific preparation of membrane fractions from different tissues, in order to concentrate larger amounts of these proteins. The skeletal muscles Tibialis anterior and Musculus soleus as well as samples from kidney and brain of C57BL/10 mice were homogenized with a tissue ruptor in tissue lysis buffer (10 mM Tris-HCl, pH 7,4) supplemented with specific protease inhibitors (PMSF and protease inhibitor complete – Roche). After addition of 1% Triton X100, the samples were incubated on ice for 30 min under gentle agitation. For pre-clearing samples were centrifuged at 700xg for 10 min at 4 °C. Cellular membranes were harvested from the homogenate by centrifugation at 10.000xg for 30 min. The membrane pellets were resuspended in tissue lysis buffer and the protein concentration was measured using BCA. 20 µg from each membrane preparation were separated by SDS-PAGE (SDS-polyacrylamid gel electrophoresis) and proteins were transferred onto special PVDF membranes. For Western blot analyses, membranes were blocked in 5% dry milk for one hour and incubated with the monospecific polyclonal antisera against TRPV2 and TRPV4 as well as respective preimmunsera at dilutions of 1:5000 and 1:20.000. Binding of peroxidase-conjugated anti-rabbit-IgG antibodies was detected by chemiluminescence.

The reactivity of both antisera was also tested by immunofluorescence analysis of cryo-sections of skeletal muscles and kidney from mice. 10 µm sections on poly-Lysin coated microscope slides were blocked with 4% bovine serum albumin and then incubated with the monospecific polyclonal antibodies against TRPV2 and TRPV4 at dilutions of 1:1000. Serial sections were incubated with a commercial polyclonal antibody against dystrophin (abcam) at a dilution of 1:1000 as a control. Bound antibodies were detected with a fluorescence-conjugated rabbit antibody (Alexa Fluor 594, Invitrogen). Samples were mounted with a glycerol-based fluorescence-medium (Vectashield, Vector Labs) supplemented with Dapi for chromatin counterstaining. Fluorescence was imaged by confocal laser scanning microscopy at a NikonTiE microscope.

D6.3 Report on exchange of know-how and experience on neuromuscular disorders (36th month)

The collaboration with Drs. Volker Straub and Hanns Lochmüller, Institute of Human Genetics, Newcastle, UK, has been very intensive during 2010 and 2011 and also very fruitful. I met Dr. Lochmüller on a scientific meeting in Ulm, Germany in March 2011 personally. Hanns Lochmüller an I myself gave talks on an International Symposium on Muscle Degeneration and Regeneration (We., 30. March, 2011). There we discussed the progress of our common projects and future perspectives of these projects. Meanwhile a first common publication of our groups in a peer- reviewed journal has appeared (see publication No. 4 of this report).

There was no chance to invite Dr. Lochmüller to the International Workshop, organized by Silke Vogelgesang and Heinrich Brinkmeier, in Sep. 2011 in Greifswald, since he was himself involved in a grant application to the EC exactly during the time of our workshop. Thus, he sent his coworker Michaela Guglieri, Newcastle, UK, who gave an interesting talk on our workshop, visited the laboratories and discussed with us current and future projects.

WP7. Initiation of collaboration with strategic partners and training for the improvement of research capacity for the neurorehabilitation research group

D7.3 Advanced training activities report on neuronavigation (36th month)

Close collaboration with the strategic partners on the use of advanced technological equipment has been carried out with

1. Prof. John Rothwell from Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, Queen Square, London (INL) (advanced technology for brain stimulation: Transcranial Magnetic Stimulation, TMS).

2. Dr. Alexander T. Sack, Assistant Professor, Department of Cognitive Neuroscience, Faculty of Psychology, Maastricht University, the Netherlands (MUN) (advanced technology for brain stimulation and neuronavigation).

Prof. Thomas Platz, the leader of the neurorehabilitation research group visited the labs of both strategic partners at the beginning of the project period to discuss specific steps of knowledge transfer and training activities. Professor John Rothwell from the Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, Queen Square, London (INL) and Dr. Alexander T. Sack, Assistant Professor, Department of Cognitive Neuroscience, Faculty of Psychology, Maastricht University, the Netherlands (MUN), had in reverse been visiting Greifswald to help to establish these technologies. The two strategic partners’ laboratories provided complementary opportunities for the neurorehabilitation research group that in combination facilitated the above mentioned group’s development. While Professor Rothwell is a specialist in TMS and neurophysiology of the motor system, Dr. Sack is a specialist in neuronavigation-based TMS. During the project period from 2009 to 2012 the technology of neuronavigation-based TMS with up-to-date stimulation protocols for brain research (e.g. theta burst stimulation, TBS) was successfully established in the neurorehabilitation research group’s lab in Greifswald.

Advanced training activities

The focus of this task was on having on site expertise for the acquired equipment. Advanced training activities concerning the acquired equipment included:

(i) Neuronavigation- and transcranial magnetic stimulation-based assessment of the motor system after stroke. Expertise on the use of high-resolution structural Magnetic Resonance-based individual head and brain models in combination with Transcranial Magnetic Stimulation for neurorehabilitation purposes has been transferred to the Neurorehabilitation Center in Greifswald. To this end Prof. Platz has visited the Institute of Neurology, Queen Square, London (INL) and the Department of Cognitive Neuroscience, Faculty of Psychology, Maastricht University (MUN) at the beginning of the project. The use of the technology has then been started in Greifswald. In addition, the experienced researcher hired for the project, Dr. Christel was visiting the lab in Maastricht for 1 month in 2010. During that period she had been trained in the application of the above mentioned methods that could then successfully be applied in Greifswald. Since then neuronavigated TMS has been established as a method in Greifswald.

(ii) Neuronavigation- and TMS-based (therapeutic) stimulation of the motor system after stroke

A therapeutic approach to enhance motor recovery after stroke could be brain stimulation combined with training strategies. Professor John Rothwell from the Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, Queen Square, London (INL) is a leading scientist in the field. In close collaboration with him specific protocols including new types of repetitive stimulation (TBS) had been implemented in Greifswald.

Further, specific protocols to use brain stimulation with TBS in a neuronavigated approach to different brain sites and in combination with training (motor learning) have been developed in close collaboration with both strategic partners. Such a newly designed protocol had then first been implemented during Dr. Christels visit in Dr. Sack’s in 2010, was testes with healthy volunteers, and was then transferred to Greifswald. A further step was to develop protocols that could specifically be used in future research of neuronavigation- and TMS-based therapeutic stimulation of the motor and other systems after stroke. These issues had been developed together with Professor John Rothwell. In addition, advanced scientific applications as presented by world leading groups in the field at the 2nd workshop of Impact G on brain stimulation and brain repair in 09.2010 had been discussed with invited researchers and were consequently implemented in Greifswald. Protocols include potential future interventions for diverse impairments such as paresis, aphasia, and neglect after stroke. The second hired researcher Mr. Schüttauf participated in an educational initiative of the Department of Cognitive Neuroscience, Faculty of Psychology, Maastricht University (MUN) in 03.2012 to receive the latest knowledge on the technology.

At the end of the project, WP7 has fully achieved its objectives: Neuronavigated TMS with modern stimulation techniques has been established for both diagnostic and especially for potentially therapeutic purposes in stroke victims. The group is now well prepared to initiate cutting edge clinical research in the field. Two clinical studies are currently in preparation. In addition, the group’s visibility in the regional public and the relevant international scientific community has well been enhanced.

4.1.4. The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).

Introduction

The impact of the recently completed project for the University of Greifswald and beyond that cannot be overestimated. The impact has become visible and obvious on several levels: (i) for integration and collaboration between the local research groups in the field of neuroscience and neuromuscular research, (ii) for neuroscience research at the Faculty of Medicine (now called University Medicine Greifswald), (iii) for the University of Greifswald and its development and growing relevance in biomedical sciences, (iv) for the visibility of Greifswald as an important place of teaching an research in Germany, preferentially in the field of biomedical sciences and medicine and (v) for the town itself, the region Vorpommern in the North-East of Germany and its relevance, visibility and economical development. Further, the project strengthened the collaboration between researchers in Greifswald and partners at important places in Europe and linked local research groups to strategic partners all over Europe. The latter point will have far-reaching consequences for further collaboration and upcoming projects.

Strategic impact

With this project, the Ernst Moritz Arndt University of Greifswald aimed to improve its research capacity in Molecular Neurobiology, Behavioural Neuroscience, Experimental Neurology, Molecular Neurophysiology, Molecular Neuropathology and Neurorehabilitation by

• improvement of equipment capacity

• achievement of a researcher capacity

• improvement of S&T experience by collaboration with strategic partners and training

The improvement of research capacities in terms of advanced analytical equipment and trained neuroscientists has been achieved. However, we cold not hire all scientists on a long term. Close cooperation with the strategic partners has been achieved. Some will continue after the project ended. Three international dissemination workshops on research and training in neuroscience were realized, an International Workshop on "Brain Regeneration after Injuries" in March 2010, an International Workshop on "Brain Stimulation and Brain Repair Mechanisms" in Sept. 2010 and an International Workshop on “Transport Processes in Neurodegenerative and Neuromuscular Diseases” in Sept. 2011. All workshops were of great success, were well recognized by the University and the local and national press. Further, the workshops brought together well-respected experts from all over Europe in three specific fields of neuroscience research. These contacts and up-to-date information will be very valuable for future plans.

We also aimed to make our Neuroscience Group more competitive in attracting research grants both at national and international levels and to attract more researchers in the field of neuroscience. This has partly been achieved. In 2009 Oliver von Bohlen und Halbach (Prof. of Anatomy and Cell Biology), a neuroscientist, joined the Medical Faculty in Greifswald. This has further strengthened our capacity in molecular and cellular neuroscience. We are confident in rising further funds for neuroscience in the next years in two disciplines: Molecular and cellular neuroscience on the one hand and clinical brain imaging on the other hand. These two disciplines had been combined in the current project, but it turned out that their methodological and scientific approaches are too different to successfully attract common grants. Thus, Greifswald has to attain the critical mass of researchers in both disciplines molecular neuroscience and brain imaging.

The Neuroscience Group’s greatest impact is in the field of regenerative medicine. Regenerative medicine is essentially a collaborative effort. The combination of structural and functional studies represent an integrative focus for experimental investigation of neural development; cellular responses to injury, disease, or genetic modification; and the mechanisms that underlie communication between nerve cells and the processing of information through brain and spinal cord circuits. The chosen strategic partners from different European countries, all with excellent research achievements in their respective field, constituted an ideal complementary mentoring group to back our progress towards strengthening the research profile of neuroscience in Greifswald. Such an integrative composition of strategic partners was only possible at the European level.

Socio-economic impact

The EU funds enabled us to create the intellectual and physical infrastructure that will nurture and sustain physician-neuroscientists in the region.

After the 3 year funding period provided by the EU, we anticipated that the researchers hired will continue to strengthen and expand the neurosciences at the Medical Faculty of Greifswald in the following ways:

1. The EU funds will enable the training and long-term integration of young scientists into the nascent neuroscience community in Greifswald. These young scientists will be in an excellent position to fill sorely needed openings at the University. For example, two permanent positions in Neuropathology at the Institute of Pathology will need to be filled in 2012.

2. The Institute of Pathophysiology and the Rehabilitation Hospital intended to employ two of the hired researchers for at least another 3 years after the funding period ended in April 2012. This has been achieved and the sustained our capacity in neuroscience research. The researcher in the Institute of Pathophysiology obtained her Ph.D. within the funding period and has qualified for a post-doc position in the institute. The researcher hired for the neurorehabilitation group, an MD, has been offered a position in the Rehabilitation Hospital. He is now mainly doing clinical work, but with his background in basic research he is very valuable for the initiation of basic and/or clinical research in the field of neurorehabilitation.

3. In addition, the Clinic of Neurology has permanent openings for young physicians who wish to pursue a career in translational neuroscience, with an emphasis on stroke, Parkinson’s disease, Alzheimer’s disease and epilepsy.

4. Last but not least, through the support of the EU we expect to greatly increase our competitiveness in neuroscience at the University of Greifswald by enhancing the ability of the new researchers to establish independent, extramurally funded research programs at the University. We also anticipate that the sustainability of the initiative will be strengthened by enhanced collaborative interactions among scientists both within the University and throughout the EU.

For the training in partner laboratories an obligatory return mechanism has been implemented in their individual contracts. All scientists returned to Greifswald but eventually we could not give contracts to all to continue the projects initiated by this measure.

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