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Enhancement of research competitiveness in molecular imaging<br/>at the Ernst Moritz Arndt University of Greifswald

Final Report Summary - ENVISION (Enhancement of research competitiveness in molecular imaging<br/>at the Ernst Moritz Arndt University of Greifswald)

Executive Summary:
The EnVision project was designed to enhance the competitiveness in molecular imaging of the University Medicine Greifswald. Eight research groups with a longstanding interest in developmental and pathophysiological molecular processes joined in a collaborative approach to enhance their competitiveness in molecular imaging. A central aim was to establish multi-photon imaging at the University Medicine Greifswald. Therefore the main objectives were to acquire a suitable multi-photon microscope that would allow live molecular imaging on viable tissues and direct in vivo observations, and to acquire the knowledge to apply methods of molecular imaging, especially multi-photon microscopy, to the different research projects of the eight groups.

At the beginning of the project, scientists for the different sub-projects on molecular imaging were hired. A multi-photon microscope and additional equipment were purchased. The microscope was set up in a new research building at the heart of the new medical campus. Scientists received specific training on the multi-photon microscope by the manufacturer and by experienced external senior scientists. Junior as well as senior scientists of the eight scientific groups visited the European partner labs, and scientists of the European partner labs visited Greifswald. Thus, there was a lively exchange of knowledge and a considerable training on molecular imaging. Furthermore, scientists participated in a workshop on bioethics in animal research. Two international workshops on "Molecular Imaging in Medical Research" were held in the building of the Krupp Kolleg in Greifswald – the first in May 2012 and the second in September 2013. Thanks to the overall improved capabilities, progress was achieved in all eight groups and their different scientific goals were reached, as documented by 16 publications in peer-reviewed international journals arising from the EnVision project.

All groups have greatly benefitted from the EnVision project and gained international reputation. This will support the international recognition of Greifswald as a scientific center of excellence. The opportunity to invite leading scientists to Greifswald for the workshop and to send scientists abroad to work with the strategic partners has helped to form new co-operations. As expected both major goals were reached at the end of the project: Scientific advances were made and the competitiveness of Greifswald as a scientific center was improved in the expanding field of molecular imaging. The success of EnVision will also contribute to the regional development, from which the population of Pomerania will benefit. Furthermore, the EnVision project will promote scientific progress in medical research for improving European health care.

Project Context and Objectives:
Molecular imaging is one of the fastest growing areas in basic and applied scientific research. It allows completely novel insights into basic cellular processes and thus creates the potential to better understand crucial pathophysiological mechanisms of a wide variety of diseases and to delineate effective diagnostic and therapeutic methods. In particular, two-photon imaging is an innovative optical technique that has quickly become state of the art for monitoring fluorescent signals in a variety of organisms. With many advantages over conventional confocal microscopy, such as greater image resolution, deeper penetration (up to 400 μm), and much less photo damage, two-photon microscopy has already proven to be an extremely useful tool for imaging live cells or tissues. Due to its tremendous versatility, recent efforts have adapted this technique to allow visualization of cells directly in living animals. Therefore the main objective of this project is to achieve a higher research capacity in molecular imaging at the University Medicine Greifswald, Germany,
• by systematically establishing a central imaging infrastructure,
• by greatly enhancing the methodological know-how through recruitment of experienced researchers,
• by close hands-on interaction with high-level European strategic partners, and
• by targeted dissemination activities.

To this end eight groups from departments, institutes and clinics of the University Medicine Greifswald joined to form an interdisciplinary Molecular Imaging Group, covering several thematic foci:
1) Organismal aging and regeneration (Molecular Neurobiology Group - Dressel group, Clinic of Neurology)
2) Cellular imaging to clarify neuromuscular diseases (Molecular Neurophysiology Group - Brinkmeier group, Institute of Pathophysiology)
3) Infection biology and immune defense (Bröker group, Dept. of Immunology)
4) Vascular malformations (Felbor group, Institute of Human Genetics)
5) Chronic kidney disease (Renal Cell Biology Group - Endlich group, Dept. of Anatomy and Cell Biology)
6) Pharmacology of membrane transporter (Center of Drug Absorption and Transport - Jedlitschky group, Dept. of Pharmacology)
7) Pancreas in health and disease (Gastroenterology Group - Lerch/Mayerle group, Dept. of Internal Medicine)
8) Vascular imaging in cardiovascular diseases and myocardial regeneration (Cardiovascular Diseases and Vascular Biology Group - Felix group, Dept. of Cardiology)
Project Results:
A detailed description of the S&T results/foregrounds for improvement of infrastructure and for the eight research groups is given in the following according to the work package (WP) structure of the EnVision project.


WP1. Infrastructure

1) Two-Photon Microscope; 2) Equipment for tissue conservation and processing: freezer for cryopreservation of biological material, zebrafish aquarium module, automatic tissue processor for immunofluorescence, small animal anesthesia unit.

The organizing principle was to have one central core facility for molecular imaging, which could be utilized for a wide variety of individual research projects within the Molecular Imaging Group and with collaborating institutions. Technical specifications were prepared for the necessary equipment and national and international supplier companies were contacted for offers. Offers from the supplier companies were collected, and the supplier company was selected based on performance/price optimum. The suppliers set up the equipment on site and completed user trainings.

The two-photon microscope was purchased on 20.07.2011 and timely delivered. On the basis of the offers it was decided to obtain a Zeiss Laser Scanning Microscope Axio Imager with a Ti-S-laser from Coherent. The microscope was initially located in the Ellernholzstrasse in immediate proximity to the confocal laser-scanning microscope purchased within the previous EU supported project ImpactG project (Grant no. 229750). When the new research building at the central campus of the university medicine was completed in spring 2012 both microscopes were relocated to the new facility. Thus we have now a modern molecular imaging facility with a central location for all research groups.

Equipment for tissue conservation and processing:
Freezer for cryopreservation of biological material was ordered and timely delivered. The freezer works well. Purchase date: 07.03.2011
Automatic tissue processor for immunofluorescence purchase date: 11.05.2011
Small animal anesthesia unit was purchased together with the two-photon microscope on 20.07.2011 and timely delivered. It was installed next to the two-photon microscope to keep studied animals for in vivo imaging in anesthesia and it works well.
Zebrafish aquarium module for zebrafish husbandry. Purchase date: 26.09.2011. The aquarium module contains 36 separate aquarium boxes. In each box up to 20 fish are kept together. The purchased aquarium module is the third module in the Renal Cell Biology Group, making it possible to breed additional transgenic zebrafish strains. To visualize podocytes in the pronephric glomerulus of zebrafish larvae by two-photon microscopy it was necessary to generate a completely transparent fish without any absorbing or reflecting pigments and with fluorescently labeled podocytes. This goal could be achieved thanks to the additional aquarium module.


WP2. Improvement of S&T experience and knowledge of researchers in application of two-photon microscopy to aging and neural repair studies

Actions during the first period:
The PI of WP2 visited the cooperation partner Prof. Hilary Carswell for one week in Glasgow, in addition this partner was visited by Antje Vogelgesang for 3 weeks who is employed in WP4. The PI of WP4 Barbara Bröker and Antje Vogelgesang are long-standing close cooperation partners of the PI of WP2. They also closely cooperate in the current EnVision projects. In addition to the experienced scientist Romy Baguhl, who is employed in WP2, additional members of the research group of the PI participate in the two-photon imaging research. Christy Joseph, PhD, is an experienced scientist in investigating neuroprotection in experimental stroke. To further enhance the knowledge of the entire research team of WP2 an experienced scientist Jennifer Shearer from the group of our cooperation partner Prof. Hilary Carswell will join us for a two month period in our laboratory in 2013. This will provide us with the unique opportunity to learn hands on at our own facility from an experienced scientist in the field.

Actions during the second period:
Training on two-photon-microscopy for animal studies: scientific exchange with our partner Hilary Carswell, Glasgow UK. In this project we had the unique opportunity to have experienced post-doctoral level scientists join our laboratory. Dr. Jennifer Shearer is a post-doctoral level scientist who has long worked in the group of Dr. Carswell. She joined us in Greifswald from 1st of April to the 30th of June. This visit allowed us to receive hands on training in our own facilities, thus avoiding the loss of knowledge often encountered in transferring methods between facilities. Furthermore since the methods were relevant not only to WP2 but also to WP4 postdocs we had the opportunity for members of both groups to benefit from Dr. Shearer. Dr. Romy Baguhl (WP2) and Dr. Antje Vogelgesang (WP4) worked very closely with Dr. Shearer. Additionally, Dr. Christy Joseph and Donna Sunny received training. Similarly the members of the WP2 benefitted when Dr. Jonathan Coles, an outstanding scientist who pioneered many developments in imaging was hosted by Barbara Bröker and Antje Vogelgesang (both WP4) from 11.8.-18.8.2013.

Expert scientist exchange from Greifswald to Glasgow. The close cooperation with our partner in Glasgow has resulted in a common ERA-Net proposal including two additional partners from Poland and Germany that is currently under evaluation. Alexander Dressel will travel to Glasgow if we get selected to submit the full proposal for this project.

Expert scientist exchange from Glasgow to Greifswald. Our cooperation partner and member of the international advisory board joined us for the second workshop on molecular imaging. We used the opportunity to learn from her in an additional seminar hosted by the WP2 leader Alexander Dressel and the WP4 scientist Antje Vogelgesang immediately before the start of the workshop.


WP3. Improvement of S&T experience and knowledge of researchers in two-photon microscopy for the study of neuromuscular diseases

Actions during the first period:
Close collaboration with the strategic partners on the use of advanced technological equipment has been initiated and extended. An expert scientist, Dr. Christian Cognard, from the University of Poitiers visited the University of Greifswald from September 12-14, 2011. Dr. Cognard is member of the scientific advisory board of Envision and head of the imaging center at the University of Poitiers. In Greifswald Dr. Cognard gave a talk on visualization of cell surface structures: “Surface and deeper networks structural alterations in mdx cardiomyocytes: a "plant staking" role for dystrophin?" The presentation was a highlight on an international workshop, which took place in Greifswald on Transport Processes in Neurodegenerative and Neuromuscular Diseases.

The work package leader of WP3, Prof. Heinrich Brinkmeier, visited the laboratories of both strategic partners Christian Cognard and Bruno Constantin at the University of Poitiers from 5.-7. July 2012. During the visit further steps of knowledge transfer and training activities for the hired researcher, Y. Zhang, MSc, were discussed. The two strategic partners provided complementary opportunities for WP3. Dr. Cognard is an expert of cellular imaging including the newly developed method of scanning ion-conductance microscope (SICM). The work presented in Greifswald Dr. Cognard was partly based on the SCIM technology. Dr. Constantin is highly experienced in cellular imaging as well, but his work is additionally focusing on the evaluation of macromolecular complexes in cells and signal transduction pathways. The latter investigations are mainly based on biochemical techniques and provide complementary opportunities for the research on cellular mechanisms of neuromuscular diseases studied in the Institute of Pathophysiology. Dr. Constantin has been invited to the next workshop organized by the Envision project. The workshop will take place in September 2013 and Bruno Constantin has already assured his participation.

Actions during the second period:
Close collaboration with the strategic partners on the use of advanced technological equipment for molecular imaging has been extended in the second period of the project. Two expert scientists, Dr. Bruno Constantin and Dr. Elisabeth Aguettaz, from the University of Poitiers visited the University of Greifswald from 19.-21. Sept. 2013.
They gave two talks on “Ultrastructural and functional alterations of E-C coupling” and of “Calcium imaging in migrating cells”. The presentations were given on the second workshop on “Molecular Imaging in Medical Research” in Greifswald in Sept. 2013.

Training on two-photon-microscopy: scientific exchange with our partners Prof. Christian Cognard and Prof. Bruno Constantin, Poitiers France. After the stay of our co-worker and employee Yaxin Zhang in Poitiers in July 2012, we were not able to arrange another long-term exchange of a scientist. The major reason for this was the new situation at the Institute de Physiologie et Biologie Cellulaire in Poitiers. Both of our strategic partners belong to the French national center for sciences, CNRS (Centre national de la recherche scientifique). From the beginning of 2012 CNRS aimed to reorganize groups and main research topics in our partner institute. The former focus “neuromuscular diseases” was replaced by research on tumor cell biology and migration of tumor cells. Both topics are related to intracellular calcium and this was reflected in the talk given by Bruno Constantin on “Calcium imaging in migrating cells” in Sept. 2013.

Expert scientific exchange from Greifswald to Poitiers. The work package leader of WP3, Prof. Heinrich Brinkmeier, visited the laboratories of both strategic partners Christian Cognard and Bruno Constantin at the University of Poitiers from 5.-7. July 2012. Scientific discussions have been held around imaging techniques for studying muscle fibers and cardiomyocytes. Techniques included confocal microscopy and newly developed technique of scanning ion-conductance microscope (SICM).

Expert scientific exchange from Poitiers to Greifswald. Two expert scientists, Dr. Bruno Constantin and Dr. Elisabeth Aguettaz, from the University of Poitiers visited the University of Greifswald from 19.-21. Sept. 2013. Elisabeth Aguettaz is member of the group of Christian Cognard and visited our university for discussions on common projects. Ms Aguettaz came as a substitute for the strategic partner Christian Cognard and gave a talk on the second workshop on “Molecular Imaging in Medical Research” in Greifswald, 20. Sept. 2013. In her presentation she showed an application of the newly developed SCIM technique and gave an important contribution to the session on “Imaging in neuromuscular diseases”. Bruno Constantin gave an impressive introduction into his new field of research “cellular imaging and cell migration”.


WP4. Rodent models for molecular imaging of infection

Actions during the first period:
Our cooperation partner Rikard Holmdahl, Stockholm, is an expert in animal breeding and is running one of the most modern animal facilities. Antje Vogelgesang visited his lab for one week to learn about the quality standards of housing, breeding and bioethics applied in Karolinska University. Moreover she was taught how to induce and score experimental autoimmune encephalomyelitis (EAE), a demyelinating disease of the central nervous system in rats and mice that reflects the pathogenesis of Multiple Sclerosis in humans. This neurological disorder can be compared to stroke since both diseases feature autoimmune brain tissue destruction.

In addition Antje Vogelgesang visited (in cooperation with WP2) Hilary Carswell lab in Glasgow, twice. Overall she spent three weeks in the Carswell lab, which is an expert cooperation partner for two-photon imaging. Antje Vogelgesang was taught how to set up experiments for brain imaging and was supplied with protocols and measures for individual lab operating systems. Antje Vogelgesang also presented our current research and preliminary results to our cooperation partners.

Actions during the second period:
Training on animal models at Glasgow lab. Due to pregnancy of Antje Vogelgesang additional exchange was taken by the master student Julia Ickler. She spend two month (February - April 2014) in Hilary Carswells lab to establish an immunocyte staining in murine brain slices. The invasion of different immune cells (granulocytes, T cells) as well as their activation status can be characterized post stroke and their localization in respect to the infarction area can be monitored.

Expert scientific exchange from Greifswald. The PI of the WP4 Prof Bröker had the opportunity to join the laboratory of another of her close cooperation partner from April 1st to September 30th, 2014 as a guest scientist. This exchange is building on the close cooperation that has been established during the EnVision project focusing on modern imaging of immune responses.

Expert scientific exchange from MIR to Greifswald. As described above Antje Vogelgesang was taught how to set up experiments for brain imaging and was supplied with protocols and measures for individual lab operating systems in 2012 which were set up accordingly in Greifswald. In 2013 Jennifer Shearer from Hilary Carswell's lab visited and introduced Antje Vogelgesang into the technique to create a window into the skull of mice for live imaging purposes with the newly set upped system. Jonathan Coles, senior scientist from Glasgow, visited Greifswald for one week in august 2013. He has a longstanding experience in two-photon imaging. With his help we were able to further improve the generated images. Moreover he presented his current research.


WP5. Improvement of S&T experience and knowledge of researchers in zebrafish live imaging using two-photon microscopy

Actions during the first period:
We succeeded in hiring a well-suited scientist, Katharina Schmidt, in February 2011. She started to work on the project on March 15, 2011. However, since she had just become pregnant, she left for maternity leave on August 31, 2011. After the birth of her child on October 4, 2011, she remained on maternity leave until September 30, 2012. Given her pregnancy and her small baby, it was impossible to send her to the Heisenberg lab in Austria.

In December 2011, we succeeded to hire a second well-suited scientist, Dr. Ahmed Kotb, as a substitute of Katharina Schmidt. He started to work on the project on January 21, 2012. Since he had to be trained on working with zebrafish first (handling, breeding, microinjection, confocal imaging etc.), there was no scientific exchange until December 2012. Nevertheless, he has already started to work with the two-photon microscope in Greifswald, and he is now ready to be sent to the Heisenberg lab in Austria.

Facing the problems with sending scientists to the Heisenberg lab, we enhanced the communication with Prof. Heisenberg by phone and E-mail to get support in setting up two-photon microscopy for the zebrafish in our group. In addition, Prof. Heisenberg visited Greifswald in May 2012 during the week of our first international workshop. Thanks to the close communication, Prof. Heisenberg's visit, and the commitment of a senior scientist (Prof. Nicole Endlich), we succeeded in getting the first images of the zebrafish pronephros in August 2011. Dr. Kotb was then involved in the experiments, in which we imaged GFP-labeled podocytes in the pronephros of zebrafish over a period of up to 18 h. We were able to show for the first time, that podocyte processes are stationary structures over many hours. This is a spectacular finding, since many people in the kidney field believe that podocytes and their processes are motile structures showing considerable movement on the time scale of minutes. The finding has been submitted as a manuscript in December 2012, and we are currently working on the revision.

Actions during the second period:
Training on live imaging in zebrafish at IST lab, Vienna. Dr. Kotb, who was hired in December 2011, was trained in the Heisenberg group at the IST (Klosterneuburg, Austria) in July 2013.

Expert scientific exchange from Greifswald to IST. Two senior scientists from Greifswald (Prof. K. Endlich, Prof. N. Endlich) visited Prof. Heisenberg and his group at the IST (Klosterneuburg, Austria) in February 2013.


WP6. Improvement of S&T experience and knowledge of researchers in longitudinal two-photon microscopy of stem cells

Actions during the first period:
During the first year, next generation sequencing analyses were performed on DNA of six patients that are affected by CCMs using a SOLiD 5500XL sequencer from Life Technologies to find new genes which might be involved in maintaining vascular integrity. The genes identified will be analyzed in zebrafish and cell cultures to characterize possible functions. For this purpose, Stefanie Spiegler received expert introduction into confocal, spinning-disc, and two-photon fluorescence laser-scanning microscopy by our strategic partners Dr. Bettina Kirchmaier and Prof. Stefan Schulte-Merker at the Developmental and Stem Cell Biology Department (Schulte-Merker group), Hubrecht Institute-KNAW & University Medical Centre Utrecht, the Netherlands. These techniques enable imaging of the vascular network of living zebrafish under different conditions. The goal for Stefanie Spiegler`s first short-term training course (1 month) was to get an introduction into the zebrafish animal model and the above-mentioned imaging techniques. Emphasis was placed on novel technologies and laboratory applications of two-photon microscopy.

On the occasion of her first visit in the Netherlands, Stefanie Spiegler also received expert guidance in next generation sequencing data processing by Drs. Kornelia Neveling, Alexander Hoischen, and Christian Gilissen, Department of Human Genetics, Radboud University Nijmegen Medical Centre. Custom-made tools for data analysis gave the chance to search for overlapping mutations in the same genes while performing several filter steps in parallel. Back at the Institute of Human Genetics in Greifswald, manual validation of the remaining genes through appropriate parameters led to further reduction to a couple of candidate genes that were validated by Sanger sequencing.

During Stefanie Spiegler’s second stay in Utrecht (long-term training / 2 months), one promising candidate gene was evaluated in zebrafish using a Morpholino-knockdown approach. Again, special emphasis was placed on the investigation of vascular morphogenesis by longitudinal two-photon microscopy. Prior to this stay, suitable morpholinos had been designed and in preparation of specificity tests all of the cloning work had been done. Together with Dr. Bettina Kirchmaier, knockdown experiments in zebrafish were initiated. The Morpholino specificity and efficiency was tested and in situ hybridizations for the localization of candidate gene expression were started. To study the effect of the candidate gene in a stable mutational background, targeted mutagenesis using the newly reported TALEN approach was also initiated during this long-term training in Utrecht, i.e. double-strand DNA breaks at the zebrafish locus of our candidate were induced by synthetic nucleases (TALEN). Injection of TALEN mRNA into fertilized one cell stage transgenic zebrafish embryos will now lead to adult knockout fish that will give the newly induced specific mutation to their progeny. This recently described approach will spare the Morpholino-induced knockdown which is more expensive, time consuming and variable and will be the prime selection for future studies. The intention is to spend three further months in the Netherlands to analyze adult knockout zebrafish in April/May/June 2013.

Actions during the second period:
Stefanie Spiegler received instruction in the use of the two-photon microscope situated in Greifswald and the ZEN software at the hands of Dr. Stefan Groß (Zeiss). With assistance of Dr. Romy Baguhl, the microscope was used to image vascular structures of living transgenic zebrafish expressing vasculature-linked fluorescent protein.

Expert scientific exchange from Greifswald to Utrecht. Stefanie Spiegler visited our strategic partner Prof. Stefan Schulte-Merker at the Developmental and Stem Cell Biology Department, Hubrecht Institute-KNAW & University Medical Centre Utrecht, the Netherlands.

Expert scientific exchange from Utrecht to Greifswald. Representing Prof. Schulte-Merker, Dr. Bettina Kirchmaier visited Greifswald in September 2013 and participated in the 2nd EnVision workshop with a talk.


WP7. Improvement of S&T experience and knowledge of researchers in molecular imaging of drug absorption and transport

Actions during the first period:
Our strategic partner is Prof. Michael Freissmuth, head of the Department of Pharmacology at the Medical University in Vienna. A major focus of the recent research of his group is the molecular machinery underlying the trafficking of neurotransmitter transporters from the endoplasmic reticulum to the target membrane domain. Here, his group did pioneering work using state-of-the-art molecular imaging techniques including fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS) as well as single particle tracking and imaging. Prof. Freissmuth is also deputy speaker of the Special Research Program SFB35 of the Austrian Science Fund/FWF "Transmembrane Transporters in Health and Disease". In this excellence network several Austrian research groups work together to unravel the molecular mechanisms of transport activity of transmitter transporters and efflux pumps. We (Dr. Igor Mosyagin, the employed researcher, and Dr. Gabriele Jedlitschky, the WP leader) attended the annual workshops of this SFB in September 2011 and September 2012 and presented our studies on “Protein interactions and regulation of the ABC transporter MRP4 (ABCC4)” and “Characterization of expression and localization of the transporter SLC35F1” at the respective poster sessions. We intensively discussed the imaging aspects of the projects with Prof. Freissmuth and other participants (see also detailed reports). During the stay in September 2012 also a technical training was given to us in confocal microscopy and FRET as described in the detailed exchange reports. Furthermore, special tools were provided by the cooperation partner that will enable us to establish the FRET technic in Greifswald. Prof. Freissmuth visited us in Greifswald at the beginning of the project in 2010 as well as in May 2012 during the first international workshop of the EnVision program (May 10-12, 2012). Here, he gave a talk on “ER-export and targeting of neurotransmitter transporters”. We also discussed common projects and technical aspects of molecular imaging.

Actions during the second period:
Training on fluorescent microscopy at Prof. Freissmuth's lab, Vienna, and expert scientific exchange. A major aim of this project was to study transporter localization as well as their regulation especially through protein-protein interactions. These can be studied on the one hand in living cells by means of molecular imaging technics such as fluorescence resonance energy transfer (FRET). Therefore, we (Dr. Igor Mosyagin, the employed researcher, and Dr. Gabriele Jedlitschky, the WP leader) continued our training in FRET measurement and analysis during our stay in Vienna in September 2013. This time we were able to use vector constructs for CFP-tagged MRP4 and an YFP-tagged adaptor protein, which we had prepared before in Greifswald, as described in more detail in the exchange reports. On the other hand transgenic mice can provide new insights in transporter function as well as in the regulation by protein interactions. In a recent study of our partners it could be demonstrated that dopamine transporter (DAT) function is impaired in mice with targeted calmodulin kinase IIα (αCaMKII) mutations and in a mouse model of the Angelman syndrome, which is characterized by severe developmental delay, including impaired motor coordination and language deficits. By use of fluorescence microscopy (also demonstrated to us) co-localization of αCaMKII and DAT in primary dopaminergic neurons could be studied. The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. We discussed visualization of the dopaminergic network in murine brain by means of two-photon microscopy also in the context of the two visits of Prof. Freissmuth in Greifswald during the project period. In addition, we had again the opportunity to attend the annual workshops of the SFB "Transmembrane Transporters in Health and Disease" organized by our partner institute in September 2013 and present our study on: “The SLC35F1 transporter: expression and localization in brain”. This presentation of Dr. Mosyagin was selected for a poster award.

Application of the learned fluorescence microscopy technics in Greifswald. The staff training in bioethics in animal research which was given by Dr. Olaf Grisk in Greifswald as well as the training in imaging techniques in Vienna enabled us also to study transporter localization and function in mice including transgenic animals. In cooperation with the Department of Neuropathology in Greifswald we studied the role of P-gp in Aβ accumulation in a double transgenic Alzheimer's disease mouse model. These animals, which expressed the human amyloid precursor protein (APP) and were deficient in presenilin 1, were treated for 60 and 120 days with an extract of Hypericum perforatum (St. John’s Wort; SWJ) and P-gp as well as Aβ were visualized in brain. This study showed that long-term administration of SJW resulted in a potent decrease of Aβ accumulation in the brains of the double transgenic mice, and in a significant upregulation of cerebrovascular P gp expression. The induction of transporters such as P-gp may be a novel therapeutic strategy to protect the brain from Aβ accumulation. Furthermore, Dr. Mosyagin was also involved in studies on the tissue distribution of atorvastatin in transporter-deficient mice, in particular in P-gp and MRP4 knock-out mice (unpublished work; part of MD thesis of Matthias Müller, University Medicine Greifswald). In P-gp knock-out mice the brain/blood ratio of atorvastatin was significantly increased indicating that P-gp limits the brain penetration of this drug (unpublished results). A major focus of the work of Dr. Mosyagin, however, was the characterization of the transporter SLC35F1. This protein belongs to the human SLC35 family of transporters some of that (subfamilies A-D) have been proposed to transport nucleotide-activated sugars from cytosol into the lumen of Golgi complex or endoplasmic reticulum and may thus be involved in the formation of proteoglycans and glycoproteins. Since high SLC35F1 mRNA levels have been detected especially in brain, we have investigated the expression of SLC35F1 as well as its cellular localization in murine brain. Confocal fluorescence microscopy studies revealed expression of SLC35F1 in some neurons of cerebrum und in Purkinje cells of the cerebellum. The localization of SLC35F1 in specific neurons in cerebrum and cerebellum indicates a role of this transporter in neuronal function or differentiation.


WP8. Improvement of S&T experience and knowledge of researchers in molecular imaging of organ inflammation

Actions during the first period:
CFTRtm1HGU mice develop no spontaneous phenotype and have no exocrine pancreatic insufficiency, clinically a precondition to develop pancreatitis. Pancreatitis in CFTR-knock-out animals was more severe as indicated by higher serum amylase and lipase activities as well as greater intrapancreatic trypsinogen activation. Myeloperoxidase activity in the pancreas was significantly increased in CFTRtm1HGU animals as early as 1h after onset. In isolated acini upon supramaximal CCK stimulation, however, intracellular trypsin and elastase activities were identical between wild type and CFTRtm1HGU mice. Co-incubation of activated leukocytes from CFTRtm1HGU mice with wild type animals and vice versa did not result in any changes in premature intracellular zymogen activation. Within this project Simon Trapp (year one period 2 month) investigated the role of ductal cells in the laboratory of Professor Hegyi.

Prof. Hegyi visited Greifswald from the 19th to 21st of November 2013. He gave a seminar on the topic of ion exchange in ductal pancreatic cells, which was well attended and talked to the people in the project. A publication strategy was decided on. Prof. Lerch visited Szeged twice in the last 12 month and a lively collaboration is ongoing.

Actions during the second period:
Training on ductal cell isolation in Szeged. To transfer the in vivo imaging using dual-photon microscopy to ductal cells. We have refreshed our experience in preparing ductal cells and Professor Lerch visited Szeged in November 2013.

Expert scientific exchange from Greifswald to Szeged. Istvan Hriz, Zoltan Rakonczay and Peter Hegyi all visited Greifswald from the 16th until the 22nd of September in 2013. Several projects were discussed:
1) The University of Szeged has become a recruiting center for the multicenter, multinational randomized, placebo-controlled trial using intravenous Magnesium for the prevention of post-ERCP pancreatitis (MagPEP).
2) In line with our project proposal we discussed and outlined a work schedule to delineate the role of pancreatic ducts for the in induction of pancreatitis. To test this hypothesis, we will determine the specific role of the duct in the induction of acute pancreatitis using well-established disease models and mice with deletion of the Na/H exchanger regulatory factor-1 that have selectively impaired ductal function. Acute necrotizing pancreatitis will be induced by i.p. administration of cerulein or by intraductal administration of sodium taurocholate. The pancreatic expression of Na/H exchanger regulatory factor-1 and cystic fibrosis transmembrane conductance regulator will be analyzed by immunohistochemistry. In vivo pancreatic ductal secretion was studied in anesthetized mice. Functions of pancreatic acinar and ductal cells as well as inflammatory cells will be analyzed in vitro. Greifswald will contribute to this study by studying the systemic inflammatory response of this model.
3) Acute pancreatitis recently became the most common cause for hospitalization among gastrointestinal diseases. There is no specific therapy against the disease due to insufficient understanding of pathogenesis. Animal and in vitro models indicated that reduced pancreatic ductal bicarbonate secretion and consequent intraductal acidosis is an early event in acute pancreatitis, but human data are lacking. Therefore, our aim was to determine the luminal pH in the main pancreatic duct during acute pancreatitis in humans. Intraductal pH was measured in patients with acute biliary pancreatitis (n = 6) during endoscopic retrograde cholangio-pancreatography. Patients with common bile stones without pancreatitis (n = 5) and a patient with an adenoma of the papilla of Vater served as controls. The luminal pH was significantly lower in the group of acute biliary pancreatitis vs. controls (6.97 ± 0.13 vs. 7.79 ± 0.20). Greifswald agreed to study this finding in mice with the technique outline above in the two-photon microscopy.
All three projects are ongoing at current but the consortium grant has fostered our collaboration.

Expert scientific exchange from Szeged to Greifswald. To transfer the in vivo imaging using dual-photon microscopy to ductal cells. We have refreshed our experience in preparing ductal cells and Professor Lerch visited Szeged in November 2013.


WP9. Improvement of S&T experience and knowledge of researchers in intravital cardiovascular imaging

Actions during the first period:
In previous investigations we were able to show that intravenously injected cardiac stem cells home to diseased hearts. In our current work we identified different proteins which are expressed or secreted by cardiac stem cells under different conditions and might be responsible for cardiac regeneration (secretome analysis and in vitro immunohistochemistry, fluorescence microscopy). We were able to show secretion of these markers also by injected stem cells (two-photon microscopy). In the future we would like to detect these markers in vivo and characterize their expression using a heart failure mouse model. At the same time we will attempt to trace the differentiation process of the injected cardiac stem cells by two-photon microscopy.

Actions during the second period:
Study of regenerative capacity of cardiac stem cells in diseased heart tissue. Many studies showed that the differentiation rate of transplanted stem cells is too low to sufficiently explain the observed cardiac improvement after cell transplantation. Therefore it is assumed that the improvement of cardiac function is at least partly caused by the paracrine activity of injected stem cells in the diseased heart. Our group was able to show that cardiac stem cells respond to pathophysiological conditions in vitro with an increased secretion of soluble factors which can participate in cardiac regeneration via paracrine actions.

Firstly, MRI was used to determine the cardiac function of heart failure animals after injection of stem cells and compared to the cardiac parameters of animals which had received saline only. Afterwards immunohistochemical staining of the explanted organs served to elucidate the secretory activity of the cardiac stem cells two weeks after transplantation using two-photon microscopy. We observed an improvement of cardiac function after cell injection and were able to show engraftment of transplanted stem cells. In addition we detected co-expression of stem cell markers (GFP+, green) and paracrine factors like galectin-1, which suggests that the secretion of soluble factors is induced in hearts under pathophysiological conditions and hints to a paracrine action of these cells. In the future this possible paracrine action could be tested in an in vivo study, where conditioned media of cardiac stem cells is injected into diseased mice hearts and the functional improvement is detected via MRI. Histological studies determining the level of fibrosis and neoangiogenesis as well as immunohistological stainings could enlighten the mode of action of the factors.

Study of interaction between biomaterials and cells after stent implantation. To study the interaction of different cell types with various kinds of stent materials including migration and adhesion silicon tubes were colonized with human umbilical vein endothelial cells (HUVEC) under different shear stress conditions. After 7-9 days an endothelial cell monolayer formed and different kinds of stent material, like PLLA, P(4HB) where inserted into the silicon tube while bare metal stent served as a control. Afterwards, the silicon tubes were perfused with human plasma for 24 h in order to analyze the interaction of e.g. thrombocytes with diverse stent material and to determine the degree of activation and adhesion of these cells caused by the respective stent materials. Flow cytometry and immunohistochemical analyses (confocal microscopy and electron microscopy) revealed that the degree of thrombosis depends on the stent material being used. This finding will be important for future clinical application of stent materials.

Following this experiment the interaction of blood cells and diverse stent materials was investigated under different shear stress conditions in ex vivo pig arteries using a tissue chamber system. Following stenting the perfusion of arteries with human plasma was carried out. After 24 h the vessel was fixed and sliced for histological and immunohistological analysis.
Potential Impact:
All groups have greatly benefitted from the EnVision project and gained international reputation. This will support the international recognition of Greifswald as a scientific center of excellence. The opportunity to invite leading scientists to Greifswald for the workshop and to send scientists abroad to work with the strategic partners has helped to form new co-operations. As expected both major goals were reached at the end of the project: Scientific advances were made and the competitiveness of Greifswald as a scientific center was improved in the expanding field of molecular imaging. The success of EnVision will also contribute to the regional development, from which the population of Pomerania will benefit. Furthermore, the EnVision project will promote scientific progress in medical research for improving European health care.

The EnVision project included several dissemination activities: scientific publications, publications in the lay press, a project web page (www.envision-project.de) and two international workshops. The international workshops are described in following.

First International Workshop on Molecular Imaging in Medical Research (May 10-12, 2012).
The First International Workshop on Molecular Imaging in Medical Research was organized by four of the eight groups participating in the EnVision project. Initially it was planned in the grant application that the project leaders of WP2, 3, 5 and 6 would organize the workshop. Due to a greater overlap with regard to contents, the Steering Committee decided that the project leaders of WP2, 4, 5 and 7 would organize the workshop. The title of the workshop was changed accordingly.

Outstanding scientists from leading laboratories throughout the world with extensive experience in Molecular Imaging gathered for this conference in the Krupp Kolleg in Greifswald. The conference was announced both in the scientific community and the lay press. More than 130 participants attended the conference.

The following external speakers and chairs contributed to the First International Workshop on Molecular Imaging:
Carl-Philipp Heisenberg (Klosterneuburg, Austria)
Andreas Schedl (Nice, France)
Christoph Englert (Jena, Germany)
Arindam Majumdar (Lund, Sweden)
Alan Davidson (Auckland, New Zealand)
Mario Schiffer (Hannover, Germany)
Albrecht Kramer-Zucker (Freiburg, Germany)
Ivica Grgic (Boston, USA/ Marburg, Germany)
Matthias Gunzer (Essen, Germany)
Hilary Carswell (Glasgow, UK)
Denis Bragin (Albuquerque, USA)
Alexander Flügel (Goettingen, Germany)
Simon Foster (Sheffield, UK)
Helena Radbruch (Berlin, Germany)
Erin Schuetz (Memphis, USA)
Anne Nies (Stuttgart, Germany)
Stefan Engelhardt (Munich, Germany)
Michael Freissmuth (Vienna, Austria)
Mattias Rickhag (Copenhagen, Denmark)

On Saturday afternoon after the international workshop, the scientists of the EnVision project received a seminar on “Technical aspects of multi-photon imaging”. This “by invitation only” seminar was designed to give technical advice on the “nuts and bolts” of two-photon imaging to the scientists directly involved in the EnVision project. Dr. Denis Bragin gave the seminar and Dr. Hilary Carswell joined him. So the participants were able to address highly specific issues of experimental procedures and obtained valuable information that should greatly facilitate the experimental design of multi-photon imaging in Greifswald. 15 participants attended the seminar.

Second International Workshop on Molecular Imaging in Medical Research (September 19-21, 2013).
The Second International Workshop on Molecular Imaging in Medical Research was organized by four of the eight groups participating in the EnVision project. The first international workshop was organized by the project leaders of WP2, 4, 5 and 7. Thus, the Steering Committee decided that the project leaders of WP3, 6, 8 and 9 would organize the second workshop.

Outstanding scientists from leading laboratories throughout the world with extensive experience in Molecular Imaging gathered for this conference in the Krupp Kolleg in Greifswald. The conference was announced both in the scientific community and the lay press. More than 130 participants attended the conference.

The following external speakers and chairs contributed to the Second International Workshop on Molecular Imaging:
Volker Straub (Newcastle upon Tyne, UK)
Matias Mosqueira (Heidelberg
Jens Kockskämper (Marburg, Germany)
Elizabeth Aguettaz (Poitiers, France)
Martin Vielreicher (Erlangen, Germany)
Bruno Constantin (Poitiers, France)
Urs Rüegg (Geneva, Switzerland)
Bettina Kirchmaier (Utrecht, The Netherlands)
Andreas Fischer (Heidelberg, Germany)
Ulrich Sure (Duisburg-Essen, Germany)
Marc van Zandvoort (Maastricht, The Netherlands)
Gabor Foldes (London, UK)
Remco Megens (Munich, Germany)
David Criddle (Liverpool, UK)
Peter Thorn (Queensland, Australia)
Peter Hegyi (Szeged, Hungary)
Zoltan Rakonczay, Jr (Szeged, Hungary)
Ashok K. Saluja (Minneapolis, USA)
Alexei Tepikin (Liverpool, UK)
List of Websites:
Website:
www.project-envision.de

Coordinator:
Prof. Dr. Karlhans Endlich
Dept. of Anatomy and Cell Biology
University Medicine Greifswald
Friedrich-Loeffler-Str. 23c
17487 Greifswald, Germany