Final Report Summary - GLOWBRAIN (Combining Stem Cells and Biomaterials for Brain Repair - Unlocking the Potential of the Existing Brain Research through Innovative In Vivo Molecular Imaging)
Executive Summary:
The main objective of the proposed project GlowBrain was to upgrade and unlock the existing excellence in brain research with specific twist in activities based on development of stem cell and biomaterials applications.
The core idea of this project was based on combined biomaterials and stem cell applications in the brain monitored by combined bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). This allowed to enhance the stem cell delivery and integration in the brain and to monitor both the stem cells and their effects in the living animals. The institutional capacities were upgraded and a GlowBrain platform was assembled in order to achieve the top-notch research organized in a unique way, not present elsewhere in Europe. Exchange of know-how with partnering institutions helped to collect and implement the knowledge to make the platform functional.
Our main S&T objective was to introduce a novel approach for stem cell based therapy of nervous system. This was achieved by using the biomaterials to enhance and control stem cells, and by in vivo monitoring of stem cell behavior after transplantation through complementary combination of two imaging modalities - bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). In order to decrease the most common obstacle in stem cell applications, low survival and integration rate of cells faced with hostile environment, we used biomaterials which supported formation of 3D cellular networks.
BLI was used to in vivo monitor molecular events in the brain with the help of the specific transgenic animals carrying luciferase as bioluminescence reporter under promoter of genes relevant to the brain repair and regeneration. The appropriate mouse lines were collected in our facility.
MRI was used to in vivo precisely localize transplanted stem cells in the brain. Apart from precise anatomical location, MRI was used to distinguish brain pathology (in particular after ischemic lesion through middle cerebral artery occlusion) and repair processes in the mouse brain. In order to be visible the stem cells were labeled by ultra-small superparamagnetic iron oxide (USPIO) visible by MRI. This allowed to track the transplanted stem cell population and to assess their distribution within the brain.
The advantage of combination of BLI and MRI is that the brain can be visualized both by luciferase markers and superparamagnetic particles. This enabled visualization of the brain by MRI and characterization of its reaction by luciferase as gene expression marker by BLI. The setup of such a complete capacities upgrade was very complex and it was based on our previous knowledge and abilities, combined with twinning and exchange of know-how with our partners.
The ability to visualize the brain in the living animals represents a leading edge asset, which together with all other already existing expertise establishes the highly versatile preclinical platform for brain research.
Project Context and Objectives:
There is a high and urgent need to attack brain diseases, which represent one of the main burdens of the modern society. Using parameters of the World Health Organization and the European Brain Council it became clear that brain diseases account for 35% of the burden of all diseases in Europe with a strong tendency to reach almost 50% as the population ages. On the other hand, only 15% of the research budget is allocated to this field, which corresponds to the urgent need to direct substantial financial support to neuroscience research in the next decades. Moreover, 95% of therapeutic efforts are based only on symptomatic relief of brain diseases (i.e. stroke, Alzheimer’s disease, amyotrophic lateral sclerosis, brain trauma), while direct action on pathogenetic mechanisms are vastly limited and significantly lower than in other organ systems.
The ongoing efforts of scientific community to improve this situation include not only immediate action through testing available therapies, but as well relates to completely new concepts of therapy, one of which is referred as cell therapy, in which applied cells would support and/or replace the brain cells. The application of this innovative concepts relies on increase of the basic knowledge about brain development, organization and function together with the development of the new complex technologies related to stem cell preparation, transplantation and monitoring.
University of Zagreb School of Medicine (UZSM) is the central medical institution in Croatia, and its part Croatian Institute for Brain Research (CIBR) is the central institution for research in neuroscience which coordinates basic, clinical and translational neuroscience. There is neither similar organization nor equivalent excellence in neuroscience in Croatia nor in the nearby convergency regions. This excellence was recognized recently by awarding The Center of Excellence in Basic, Clinical and Translational Neuroscience.
The main objective of the proposed project GlowBrain was to upgrade and unlock the existing excellence in brain research with specific twist in activities based on development of stem cell and biomaterials applications. This will be achieved using the already existing contacts with ERA partners enabling: 1) significant exchange of know-how, 2) recruitment of experienced researchers, 3) introduction of the novel infrastructure and 4) development of intellectual property competencies.
The mouse brain research represents a central point of basic research at CIBR. The research infrastructure for mouse brain research is already highly developed and includes several important premises, i.e. platforms. They include: (1) mouse facility, (2) neuroanatomy platform, (3) neurodevelopmental platform, (4) microscopy platform, (5) molecular biology platform, and (6) behavioral platform, plus two platforms already under construction (7) stem cell platform, and (8) biomaterials platform. Actually, GlowBrain project aims to complete two platforms under construction (i.e. 7 and 8) and to add in vivo molecular imaging as the platform number 9.
The in vivo molecular imaging changes the use of animals in experiments, in particular transgenic animals. Previously, the analyzes of the molecular mechanisms were based on the single end point data only, i.e. as a result of analysis after sacrificing the animal. The molecular markers were visualized with enzymatic or fluorescent reporters in the obtained tissues under the microscope. On the contrary, the in vivo molecular imaging strategy is based on employing reporter molecules developed to study biological processes in living animals in real time. This enabled novel approach of time-dependent studies of molecular processes in living animals.
Imaging modalities frequently utilized in in vivo research are mostly adaptations from familiar, clinical imaging instrumentation such as X-ray, ultrasound, CT, SPECT, PET and MRI. From this rapidly evolving field we have selected two flagship technologies: Bioluminescent Imaging (BLI) and Magnetic Resonance Imaging (MRI). Exciting possibility employed in the this project is to combine these two techniques, which offers a chance for giant progression in this highly innovative field.
The core idea of this project was based on combined biomaterials and stem cell applications in the brain monitored by combined bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). This allowed to enhance the stem cell delivery and integration in the brain and to monitor both the stem cells and their effects in the living animals. The institutional capacities were upgraded and a highly needed added value was obtained in order to achieve the top-notch research organized in a unique way, not present elsewhere in Europe. Exchange of know-how with partnering institutions with proved excellence in applicable science and patenting opened a new dimension enabling the progress in development of innovations and protection of intellectual property.
Our main S&T objective was to introduce a novel approach for stem cell based therapy of nervous system. This was achieved by using the biomaterials to enhance and control stem cells, and by in vivo monitoring of stem cell behavior after transplantation through complementary combination of two imaging modalities - bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). In order to decrease the most common obstacle in stem cell applications, low survival and integration rate of cells faced with hostile environment, we used biomaterials which supported formation of 3D cellular networks.
BLI was used to in vivo monitor gene activity of transplanted stem cells. To visualize through BLI the mouse brain the transgenic animals were needed, already carrying luciferase as bioluminescence reporter under promoter of genes involved in differentiation of stem cells toward neurons. Many of these mouse lines were collected in our facility or in the facilities of our Partnering Organizations. BLI enables visualization of the subset of brain cells which would express the bioluminescence reporter at the corresponding molecular event. This enabled to monitor the onset, location and duration of the particular brain event. The markers could range from those that are specific for neurons, glia or non-neural phenotypes; for those specific for established cell types, and for those describing various types of neuronal activity, i.e. axonal sprouting, or neuroinflammation. The high sensitivity of BLI is indeed the major asset enabling the monitoring all necessary subtle steps in this complex process.
MRI was used to in vivo precisely localize transplanted stem cells in the brain. Apart from precise anatomical location, MRI was used to distinguish brain pathology (in particular after ischemic lesion through MCAO) and repair processes in the mouse brain. In order to be visible the stem cells should be marked by appropriate particles adhered to the cell surface and visible by MRI, like ultra-small superparamagnetic iron oxide (USPIO). This allowed to track the complete transplanted stem cell population and to assess their distribution within the brain.
The advantage of combination of BLI and MRI is that the brain could carry both luciferase markers and superparamagnetic particles. This should enable visualization of the one cellular process in the brain by MRI and characterization of another by luciferase as gene expression marker by BLI. Through this double labeling strategy, the precise localization of molecular and cellular events viewed by MRI would be enhanced with ability to detect sensitive signal viewed by MRI. This actually points to the superb added value of the combined facility and introduced multimodal imaging.
The proof of concept was the establishing of the whole procedure of in vivo molecular imaging of transplanted biomaterials-supported stem cells in the mouse brain after ischemic injury induced by middle cerebral artery occlusion (MCAO). The setup of such a complete capacities upgrade was very complex and it was based on our previous knowledge and abilities, combined with twinning and exchange of know-how with our partners.
Specific objectives necessary to establish the integrated platform were (1) establishing the differentiation, labeling and transplantation of stem cells, (2) development of advanced techniques in application of biomaterials in the brain, (3) visualizing brain and stem cells with BLI, (4) visualizing brain and stem cells with MRI, (5) multimodal imaging BLI/MRI of brain and stem cells, and (6) assessing the brain repair in mouse model of the stroke (MCAO).
Therefore, the ability to visualize the stem cells and the brain in the living animals represents a leading edge asset, which together with all other already existing expertise establishes the highly versatile platform for stem cell research in the brain.
Project Results:
Our main S&T objective was to introduce a novel approach in preclinical research on stem cell based therapies of the nervous system. This was achieved by establishment three main pillars of the upgraded research activities: A) stem cells, B) biomaterials and C) in vivo animal imaging system. The biomaterials are expected to enhance and control stem cells, and in vivo monitoring of stem cell behavior after transplantation needs to be evaluated through complementary combination of two imaging modalities - bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). The research organised around the combination of these three pillars was referred as the GlowBrain platform.
The major result and major foreground of the GlowBrain project is the newly established GlowBrain platform. The major part of platform is located in the new laboratory referred as Laboratory for Regenerative Neuroscience, and nicknamed GlowLab. The capital equipment acquired by GlowBrain project is located in the GlowLab, but the GlowLab is wider than just hosting the instruments. Similarly, the GlowBrain platform is not only instruments and architectural space, but as well a functional combination of knowledge. In the sense of the procedures and laboratory space, the GlowBrain platform includes together with the GlowLab the previously existing laboratories and facilities of Croatian Institute for Brain Research, which as well got upgraded by the new procedures and new functionalities.
The major topic, connecting knowledge, space and equipment, is understanding of brain pathologies and preclinical validation of potential cures. The animal experimental model used is mouse, and the central disease model is the stroke. In order to get insight in the stroke pathology and subsequent brain repair, the preclinical mouse model of the brain ischemic injury was used. The standardized approach applied in the GlowBrain platform to create this injury in the mouse brain is medial cerebral artery occlusion (MCAO). Moreover, during the GlowBrain course we added unique model of human disease using intermittent hypoxia to model in mice a human disease - obstructive sleep apnea.
When the GlowBrain was conceived the equipment to be purchased was intended to be located in the main building of the Croatian Institute for Brain Research. Major equipment needed a specially organized dedicated space was MRI, and in the first period of our negotiations on the instrument acquisition with the MRI producers, the planning was based on this location. As MRI needed 3 rooms, one for the instrument, one for the supporting machinery (i.e. technical room), and one for the computers and researchers running the system (i.e. control room), plus many connections, this prompted the discussion about the equipment final location. The building of Croatian Institute for Brain Research was the newest on the campus, completed in 1990, and it has the best conditions for research at our institution, University of Zagreb School of Medicine (UZSM, Croatian Institute for Brain Research is an integral part of the University of Zagreb School of Medicine). The modifications necessary to host the machines implied that new building would require some substantial renovations. Subsequently, the alternative space was considered and finally decision was made to provide the GlowBrain platform an additional space in another building of Department of Anatomy. The advantages of this solution was that the GlowBrain could organize its space in a best possible way and provide to new equipment all necessary conditions designed and prepared for GlowBrain platform. The disadvantages were that new space was in basement of an old building (completed in 1908), and this basement was abandoned 20 years ago, and at decision moment humid and in a very bad shape. The challenging task of basement renovation was successfully accomplished, and resulting laboratory space is an exquisite example of a new laboratory. When considering the final result, the new laboratory represents a real gem for brain research in Europe, and it is the excellent space for the acquired equipment. To reach this level was not easy, but it made us more proud that we could achieve this. We can state that we have the best preclinical in vivo imaging facility between Vienna and Athens.
The new laboratory represents a synergy between financial resources of GlowBrain and financial resources of the institution. The construction works were fully covered by institutional money and in this sense it showed how important for the institution was the GlowBrain.
The new Laboratory for Regenerative Neuroscience integrated in cca 200 sqm main functionalities of the GlowBrain platform. The core facility is indeed in vivo imaging, but together with it, there is dedicated space for the animals being involved in the experiments, operation room for the animals where the mouse stroke model takes place, and cell culture, where stem cells and biomaterials are prepared.
The workflow in GlowLab corresponds to the main idea of GlowBrain platform, combining biomaterials and stem cell research to treat ischemic brain injury (stroke) in mouse and validating effects by in vivo imaging. The laboratory consists from 3 areas, each area has its own cleanness and security levels. The first area is dedicated to data analysis and represents the area where the researchers and eventual guests can freely enter. In this area computers for image and data processing are located and researchers can work on their desks. The other two areas are restricted and the entrance is allowed only to selected trained personnel. The lower level of restricted access is allowed to the area where in vivo imaging and cell culture is situated. The in vivo imaging consists of 3 rooms: technical room housing supporting equipment for MRI, the MRI room with the scanner itself, and the control room. In the control room there are the computers running MRI, animal table to prepare animals for imaging, and as well the preclinical optical imager for bioluminescence (BLI) and fluorescene (FLI) imaging.
In the same restricted area, nearby the control room, is a cell culture dedicated primarily to cultivation of the stem cells. These cells could come from the outside (e.g. human stem cells) or could be harvested from the animals in the nearby animal area (e.g. as it is the current use of neural stem cells obtained from mouse fetal brains).
The third area being the most restricted is dedicated to the animals held in individual ventilated cages in two animal rooms, one for rats and another for mice. The space was organized as an experimental mouse facility. The breeding of animals and cleaning and disinfection of cages is located in the main building of Croatian Institute for Brain Research. Together with rooms for animals, in GlowLab there is one room as a clean space (e.g. for the clean cages), and another room as a dirty space (e.g. for the dirty cages). The dirty cages exit through the technical exit/entrance to be transported to the main building and subsequently return cleaned through the same exit/entrance.
The animal area has two additional rooms important for the GlowBrain platform. One is Operations, where the animals are operated and treated. This includes the mouse models for the brain pathology either as MCAO, being a stroke model, or another types of operations, e.g. spinal cord injury or traumatic brain injury. Another aspect of Operations is treatment of animals, where stem cells need to be introduced in animal through stereotaxic injection. The next door room is a multipurpose room, which can serve as a space for animal recovery (e.g. the temperature can be set higher than in the rest of the space), or for simple behavioral analysis, or as a quarantine for the specific guest animals.
The GlowLab allows specific workflow related to GlowBrain project through specifically designed communicating windows. The stem cells from the cell culture are transferred to the animal area through specially designed window, animals are treated with stem cells, they are housed in animal rooms and then carried to in vivo imaging area (again through specially designed window). The windows enable transfer of materials from one restricted area to another without a need of researchers to change clothes or use specific paths to maintain a clean facility.
The important feature of the facility is that it is BSL2 ready. The air pressure is differential from the area to area, enabling the air flow from the cleaner spaces (animal area) to the dirtier spaces (data analysis area). The GlowLab has all necessary connections which enable electricity, heating/cooling, air flow, air pressures, information network, medical gasses, and exhausts for the MRI equipment (helium) or animal preparations (anesthetics). Together with cca 200 sqm of floor, there is a labyrinth of 200 sqm of connections in the ceiling, and cca 200 sqm of equipment outside the building to provide all necessary conditions in the laboratory. We affirm that the GlowLab is organized and constructed as a top notch facility to give the maximum versatility and possibility to the equipment purchased from the GlowBrain project.
Although the major asset of the GlowBrain, the purchased instruments for in vivo imaging are located in GlowLab, the GlowBrain platform exceeds the GlowLab and it includes laboratories and spaces in main building of Croatian Institute for Brain Research, at Dpt. of Histology and Embryology, and Dpt. of Pathology.
The GlowBrain procedures are based on standardized and well treated animals, which are hosted in the Section for Laboratory Animals of Croatian Institute for Brain Research (e.g. animal house). The animal house was upgraded by GlowBrain in order to host more animals in better conditions. As Croatia joined European Union during the course of GlowBrain, we adapted European standards. The animal house was reviewed by local authorities and the license for animal work issued (one of the first at University of Zagreb). Moreover, the groups using animals together with ethical approval of Institutional Review Board, received as well approval from the Ministry of Agriculture to work with animals. Again, due to the acquired EU regulations, new internationally and nationally licensed courses for animal work were attended by all users of the animal house and all group leaders using animals in their projects and they acquired the personal licenses for the animal work. To sum up – we actively followed the extensive upgrade of animal standards and we are completely up to date having all permissions and documentation for the animal work, but as well following actively in daily practice the recommendations and practices of animal welfare.
The major modification in animals used for GlowBrain purposes is that we needed to change the background of the animals. Our mouse lines were all kept on C57Bl6 black background. After we acquired optical imager and started to image animals, the first step was to shave animals in order to remove black hair and allow light to pass through. This was feasible for short experiments, but for long term imaging of the same animal (week and above) the reaction on hair removal was noticeable – the small hairs growing faster, and the skin getting pigmented. As we imaged neuroinflammation, we could not distinguish whether the inflammation was due to the hair removal and if the hair removal could influence our measurements. Therefore, the C57Bl6 albino strain was acquired and all mouse strains we had (cca 500 mice as an average number of animals present in the animal house in the same time) needed to be backcrossed to this new albino strain. Actually, we could not do partial backcross only to the lines we would use for imaging, as we would have two groups of animals, one on black and another on albino background, subsequently not comparable in our experiments. Therefore, we decided to have all animals on the albino C57Bl6 background. Currently, this process is completed and all lines were backcrossed to albino strain. Theoretical threshold to be considered isogenic is 5 generations, and with most mouse lines we are already on this step. The rederivation of animals represented a bold necessary step, which is a very important time consuming result of the GlowBrain, allowing us to proceed with the experiments.
The core GlowBrain procedures in the mouse house include the ischemic lesion induction and we currently use two procedures: (1) transient ischemic lesion by middle cerebral artery occlusion (MCAO), and (2) intermittent hypoxia.
Transient ischemic lesion by MCAO was significantly upgraded by GlowBrain. We discussed extensively how to perform procedure with our partners and they had trained our personnel at their site and in Zagreb how to do it. Moreover, we acquired laser color doppler in order to measure the blood flow through the brain at the location of middle cerebral artery. The major advantage of the MCAO procedure is that it is the best clinically relevant model of the human ischemic stroke, as the same artery is affected in majority of human stroke cases. The major disadvantage of this mouse model is that it is highly variable. The variability in our setting is reduced by measuring the blood flow during the procedure, and having trained personnel to perform lesion in a standardized fashion. The introduced ischemia is temporary and it lasts for 1 hour and after that reperfusion occurs. The animals are routinely tested subsequently by measuring the neurological score, and now with the functionality of MRI we added the necessary step of verifying the lesion by MRI. This provided us an important prerequisite to be able to perform transient MCAO in a standardized and validated form.
Intermittent hypoxia is a unique approach developed through GlowBrain activities and represents the model for the human obstructive sleep apnea. The development of this unique model represents an important added value of the GlowBrain. Obstructive sleep apnea is a huge unrecognized human health problem where during the night the breathing completely stops due to snoring and person remains hypoxic for the short periods of time. The brain consequences of this intermittent hypoxia are still unknown and it is hypothesized that they hugely contribute to the raise of dementia in Europe and globally. The setting included development of the hypoxic chamber for mice dedicated to mimic exactly obstructive sleep apnea and this is now patent pending (patent submission number is GB1602071.1). Through this GlowBrain platform added unique in house developed mouse model of major human disease, obstructive sleep apnea.
The animal house is as well location for stem cell and biomaterials treatment platform were we organized a comprehensive stereotaxic injection setup to inject cells, nanoparticles and biomaterials to the mouse brain in the standardized fashion.
The next developed foregrounds are related to the stem cells and biomaterials. Protocols and procedures were organized and performed in the Laboratory for Stem Cells of Croatian Institute for Brain Research, where the important advances of the stem cell culture were achieved. The stem cell culture was needed to be standardized and evaluated. The major goal was to choose the best way to grow mouse neural stem cells obtained from the fetal mouse brain. In addition, we introduced mesenchymal stem cell culture from mouse. Finally, the human stem cells were introduced in a separate cell culture in the Dpt. of Histology and Embryology, where human stem cells were isolated from oral mucosa, an easy way to get patient specific stem cells.
The part of the GlowBrain platform dedicated to the biomaterials and nanoparticles was the Section for Neurogenetics, Cytogenetics and Developmental Genetics of Croatian institute for Brain Research. Before GlowBrain this Section was place for molecular genetics analyses of the transgenic animals, therefore it was recently renamed in the Section for Regenerative Neuroscience, Medical Genetics and Developmental Genetics. Together with previous activities, activities spectrum was extended to nanoparticles and biomaterials. Naively, at the very beginning we assumed that we could concentrate only on in vivo experiments with marked stem cells. In a nutshell the first proposed concept was that the various partners will synthesize nanoparticles and biomaterials and we would test them in the animal disease model. As soon as we started to create the experiments we realized that before testing the nanoparticles and biomaterials in the mouse model of stroke we need to perform 2 important, necessary and inevitable steps. The first was testing of biocompatibility of the compounds in vitro on the stem cells, to verify whether the treated stem cells are comparable to non-treated. This as well tests in general the suitability of any compound to be injected in the brain. The second step included in vivo testing of the biocompatibility of the compound when injected in the brain. Interestingly, we suggest that these two steps represent the missing link between the material scientists (e.g. chemists) and biomedical scientists, and when provided would allow many of the compounds to be tested in preclinical settings. Therefore, our effort was necessary, but as well essential for the cross-talk between the disciplines.
The in vitro testing included (1) general toxicity testing, this tested for the cell survival and proliferation, (2) differentiation testing by immunohistochemistry and RT-PCR, (3) labelling efficiency testing, (4) labelling mechanism testing, and (5) oxidative stress testing. The innovative step in these tests was the use of flow cytometry, which allowed the more precise measurements of the toxicity and cell labeling. The settings as well included a revival of institutional Centre for Electron Microscopy, which is located at Dpt. of Pathology. It was needed for labelling evaluation, but as well it was highly praised by our partners and it would be the base for continuous collaboration with our Partnering Organization Paracelsus Medical University.
The biomaterial combined with stem cells to be transplanted to mouse brain and to enhance stem cell capabilities was alginate. Alginate was chosen as a polysaccharide biomaterial tested already in vitro by our collaborator Simona Casarosa. Therefore, in this particular case we could skip in vitro tests and went directly for in vivo biocompatibility. For in vivo testing of biocompatibility we used the advantages of the introduced preclinical in vivo imaging and of a unique model of Tlr-2/luc mice, we had in our mouse collection. Tlr-2/luc mice are improtant, as by bioluminescent imaging they allow to measure neuroinflammation and to verify if alginate injection in the brain elicits neuroinflammation. The results showed that it was comparable to injection of the vehicle (PBS), therefore represented an inevitable reaction to the stereotaxic injection not to be attributed to specific effect of the alginate. These results are part of defended PhD thesis of Angela Bozza, and of the manuscript in preparation.
In setting up the procedures vital for the GlowBrain platform, we used in vivo imaging to validate as well nanoparticle applications in the brain, stem cells visualization labeled with nanoparticles, bioluminescent cell visualization, and stem cell and biomaterial visualization.
These performed experiments showed platform abilities. Still it should be noted that standardization and full functionality will require time, and that the GlowLab started to be fully functional right at the very end of GlowBrain project. Moreover, one of the consequence of the GlowBrain project is increased perceptual value of the standardization of the procedures and this will be continued in the GlowLab, in particular as this would be the base for planned contract research, which is important for exploitation plan.
The following settings specific for the GlowBrain platform are elements of the achieved foreground:
1. Collection of transgenic animals bearing luciferase and fluorescent reporters and knock-out animals of genes relevant to brain damage repair
The mouse lines in our collection are rather numerous and they were selected to cover diverse aspects of the GlowBrain platform. They include the following mouse lines:
a. The mice carrying luciferase (luc) and green fluorescent protein (GFP) reporters (the luc reporter is aimed to be used for bioluminescent imaging, and the GFP for the immunohistochemistry analysis on the histological slides):
- Tlr2-luc/GFP
Luc and GFP reporters are driven by Tlr2 promoter. TLR2 (Toll-like receptor 2) is a receptor located mainly on the microglia cells and it binds various ligands important for innate immunity response. For stroke pathology of the major importance are particles of necrotic cells, which died due to the ischemia. They belong to Damage-Associated Molecular Pattern molecules (DAMPs) also known as Danger-Associated Molecular Pattern molecules, and they represent host molecules that can initiate and perpetuate an innate inflammatory response. Upon binding to TLR2 they initiate a signaling cascade leading to activation of microglia and neuroinflammatory response. These mice were used to image the inflammatory response after stroke, but as well after newly introduced mouse model of intermittent hypoxia. They are the major asset to verify the in vivo biocompatibility of intended therapeutic or diagnostic approach. Therefore there were used to evaluate brain response to stem cell therapies, their combination with biomaterials (alginate in particular), and application of nanoparticles alone or as a cell label.
- Gap43-luc/GFP
Luc and GFP reporters are driven by Gap43 promoter. GAP43 (Growth Associated Protein 43) is expressed at high levels in neuronal growth cones during development and during axonal regeneration, and subsequently being associated with growth and plasticity. It is used to image the brain repair after postischemic lesion. During GlowBrain it was in particular used to develop a novel way of imaging apoptosis by the use of caged luciferin, luciferin bound to DEVD peptide to be cleaved by Caspases 3 and 7, and subsequently released for imaging.
- Nestin-luc/GFP
Luc and GFP reporters are driven by nestin promoter. Nestin is an intermediary filament protein characteristic for neural progenitors. It is expressed during neuronal differentiation, and it serves as a marker for neural stem cells either after transplantation or if endogenous stem cells being activated in brain diseases, e.g. after ischemic lesion.
- Dcx-luc/GFP
Luc and GFP reporters are driven by Dcx promoter. DCX (Doublecortin) is a microtubule-associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures. Its use for imaging is similar as of nestin-luc/GFP mice, as it is as well marker of transplanted or endogenous neural stem cells.
- Tg(CAG-luc,-GFP)L2G85Chco/J
This is ubiquitously expressed luciferase and GFP reporter. The reporter genes are under the control of the chicken beta actin promoter coupled with the cytomegalovirus (CMV) immediate early enhancer (CAG). This mouse line is in particular useful to be a source for stem cells, which can be visualized after transplantation to the brain.
b. The mice carrying fluorescent reporters. The acquired instrument for preclinical optical imaging together with bioluminescence imaging (BLI) is capable as well for fluorescence imaging (FLI). The markers could be visible on histological slides directly or via immunohistochemistry.
- Tg(Thy1-YFP16)Jrs
The yellow fluorescent protein as a reporter is under control of Thy-1 promotor. In this particular line the neurons are exclusively marked, but only some enabling to see the whole neuron with all processes including the dendritic spines. This particular mouse line is currently used as a source for stem cell transplantation in the brain in order to visualize the differentiated neurons in the brain.
- Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J
This is ubiquitous mouse line with Tomato reporter under control of gene trap ROSA promoter. It is used to track stem cells with FLI and histology after transplantation to the brain.
c. Knock-out animals
- Tlr2
This is the knock-out of Tlr2 gene, showed to have different post-stroke development of the events. It is used in the GlowBrain platform as a major model for testing of influences of neuroinflammation after ischemic lesion.
2. Mouse models for human stroke and obstructive sleep apnea
As the mouse model for stroke we use the medial cerebral artery occlusion (MCAO). The model was enhanced by monitoring of the blood flow by laser color doppler, and subsequently the determination of the ischemic lesion by MRI. This enables to exclude animals were the procedure was not correct, or variability is out of proportions. The standardization of MCAO is a major prerequisite for all corresponding experiments.
The mouse model of obstructive sleep apnea was introduced as a specific result of GlowBrain together with our partner from Budapest. The protocol lasts for 21 days, 8 hours per day during the daylight period, in the interval of 90 seconds the mice are in normal (21%) oxygen and in hypoxic (5.7% O2) conditions. To enable this protocol we have designed a patent pending intermittent hypoxia system (GB1602071.1) which is attached to the cage used routinely for the animals, providing the needed fast exchange of gases which are monitored by appropriate sensors.
3. Stem cell culture and brain transplantations
We currently use 3 types of stem cells, 2 from mice: neural stem cells and mesenchymal stem cells, and 1 from the humans: oral mucosa stem cells. The stem cell culture and differentiation procedures are developed in details. The brain transplantations are performed by stereotaxic apparatus. In addition, we are able to transplant cells intravenously and intraarterialy, still these procedures are rarely used.
4. In vitro and in vivo biocompatibility validation of nanoparticles and biomaterials
The setup includes a row of analysis, which includes the physical-chemical characterization, in vitro tests, and in vivo validation. The physico-chemical characterization includes transmission electron microscopy, dynamic light scattering, and electrophoretic light scattering. In vitro tests were made on stem cells and on permanent cell lines (e.g. HEK-23), and they include: cell labeling efficiency testing by flow cytometry, cell viability tests, cell proliferation tests, determination of entry mechanism by transmission electron microscopy, measurements of intracellular reactive oxygen species, measurements of intracellular glutathione levels, measurements of glutathione peroxidase and superoxide dismutase activity, measurements of mitochondrial and cellular membrane potential and COMET essay. In vivo biocompatibility test is based on brain imaging of Tlr2-luc activity, which shows the neuroinflammation after nanoparticle application.
5. In vivo preclinical optical imaging of fluorescent nanoparticles
The distribution of gold nanoparticles labeled by fluoresecent dye was established by in vivo FLI in the mouse brain.
6. In vivo preclinical bioluminescence imaging of the unique mouse models bearing luciferase reporters
The imaging of Tlr2-luc and Gap43-luc were used mainly in the evaluation of the post-ischemic brain consequences. As a unique approach developed through GlowBrain, we introduced imaging of caged luciferin, DEVD luciferin, which was our innovation, being the first to introduce this type of imaging. DEVD luciferin cannot be used by luciferase, and therefore it is referred as caged. After cleavage of DEVD by caspase 3 or 7, the luciferin is released being available for luciferase and imaging. As caspases 3 and 7 are involved in apoptosis, the obtained image represents a subset of luciferase expressing region (e.g. Gap43 expression in our case), which is in the same time caspase positive. Moreover, we have shown that the cells expressing both Gap43 and caspase are not apoptotic, but the co-expression defines the neurons in stress after stroke, which will subsequently either undergo apoptosis or axonal sprouting and recovery.
7. In vivo magnetic resonance imaging of the mouse brain
The magnetic resonance imaging consists of the various modalities and the acquired machine enables the maximum flexibility to apply any imaging protocol to be imagined. This refers to the potential of the machine, which we would use for the next decades. Currently, we use the MRI modalities mostly used in human MRI, T1, T2, and diffusion weighted imaging. To image stroke in mouse, the ischemic lesion is imaged by T2-weighted and T2-maps.
8. Ex vivo magnetic resonance imaging of the anatomical and pathological samples
The ex vivo MRI enables to collect the samples not only from the brain, but as well from other parts of the body and it allows as well for the imaging of human samples. We have currently imaged many different samples as a result of our training. The research-oriented samples were mouse and rat brains, postnatal mouse brain, sheep tissue-engineered cartilage and human breast tissue. The contrast can be enhanced by the use of gadolinium salts.
9. In vivo magnetic resonance imaging of the mouse and rat body
In vivo imaging was used for mouse stroke model using the specific brain coil. The whole body imaging is perforemed by acquired specific coils for mice and for rats.
10. Evaluation of the mouse ischemic lesion consequences and repair using multimodal in vivo imaging.
The multimodal imaging was enabled by specific construction of the holder to be used in both instruments. Although at the very beginning we were told that such a holder exists as commercially available, what we got was not a close to what was needed. Therefore, we engaged in the constructions of our custom made holder, which would enable the heating and monitoring of animals during imaging, being compatible with the acquired mouse monitoring MRI setup. Mouse stroke and labeled stem cells were visualized in both instruments. After imaging in both imaging systems the overlay of images was achieved by general software ImageJ. The multimodal imaging is new emerging research topic, and we expect to develop the various modes and postprocessing approaches of multimodal imaging in our future research projects.
11. Stem cells tracking using in vivo imaging
The labeled stem cells were imaged by bioluminescence, MRI, and X-ray imaging. The stem cells were labeled by magnetic nanoparticles and those cells were imaged on MRI and synchrotron radiation micro computed tomography (Elletra synchrotron in Trieste, Italy). The Gap43-labeled stem cells were imaged by bioluminescence imaging. The response of the brain to stem cells and biomaterials combination was imaged by Tlr2-luc mice.
The proof of concept of GlowBrain was the establishing of the whole procedure of in vivo molecular imaging of transplanted biomaterials-supported stem cells in mouse brain after ischemic injury induced by middle cerebral artery occlusion (MCAO). We have assembled all the parts of this puzzle: we introduced ischemic injury in the brain, we transplanted stem cells and biomaterials, and we imaged living animals both by bioluminescence imaging on Preclinical optical system Perkin Elmer IVIS Spectrum, and by magnetic resonance imaging on Bruker 7 Tesla Biospec 20/70 USR.
Specific objectives necessary to establish the integrated platform were (1) establishing the differentiation, labeling and transplantation of stem cells, (2) development of advanced techniques in application of biomaterials in the brain, (3) visualizing brain and stem cells with BLI, (4) visualizing brain and stem cells with MRI, (5) multimodal imaging BLI/MRI of brain and stem cells, and (6) assessing the brain repair in mouse model of the stroke (MCAO). All these specific objectives were reached. Although reached, the objectives 5 and 6 remain our goals beyond the duration of the GlowBrain project. Multimodal imaging is a novel approach which would be further developed by our team and by other groups, and correlation analysis of the imaging with the brain repair represents one of our major scientific goals to be used by the GlowBrain platform. Therefore we can say that although GlowBrain was completed, GlowBrain platform is just starting by attacking the major research question in the field.
When compared to the background, what was achieved with GlowBrain project is without a doubt a profound change. The GlowBrain had 7 Work Packages. The first one (WP1) was related to the management of the project entitled “Coordination and management”. Although this is not a classical Working Package expected to produce a project results, it actually brought us tangible advancement in how the project should be taken care of and how to manage it. Therefore, corresponding to the WP1 we would discuss foreground category “Organization and management”. The WP2 was “Exchange of know-how and experience” and its major outcome is foreground category “Knowledge”, the WP3 Recruitment of the experienced researchers resulted in the foreground category “Personnel”, and WP4 “Acquisition and upgrading of research equipment” relates to the category “Equipment”. The fifth Work Package (WP5) “Intellectual property and innovation capacity building” relates to foreground category “IP skills”, and WP6 “Dissemination and promotional activities” to newly developed structure related to the foreground category “Responsible research and innovation”. Finally, the Work Package 7 was untypical package “Ex-post Evaluation of UniZAG research potential”, but it generated a document containing future looking elements related to GlowBrain planning, and ideas presented there were incorporated in current planning. It provided us as well the insights toward external evaluation of our future activities. This aspect of the foreground would be included in already presented category “Organization and management”. The categories will not be presented in the order of Work Packages but will follow different order: Equipment, Personnel, IP skills, Responsible research and innovation, Organization and management, and Knowledge.
Equipment:
During GlowBrain project we have bought two preclinical imaging instruments, magnetic resonance imager for in vivo imaging of small laboratory animals, and optical imager for mouse bioluminescence and fluorescence imaging. Moreover, we have upgraded the mouse facility. In synergy of the GlowBrain and its hosting institution University of Zagreb School of Medicine, the institution invested in reconstruction of an old basement where the new laboratory space was prepared. Through this a new laboratory was created, Laboratory for Regenerative Neuroscience or GlowLab. This laboratory consists of GlowBrain Imaging Facility, small animal facility, space for animal experiments, and cell culture. At the end of the project, GlowBrain Imaging Facility is functional, the experiments which include animal imaging are running and animals are imaged on the routine basis.
Personnel:
By carefully organizing GlowBrain financial resources it was possible to employ more people than initially planned. These employees represent the first part of the foreground related to the personnel, which was employed using funds directly provided by GlowBrain. Objective was to employ experienced researchers, if possible, returnees, which would develop the independent stem cells and biomaterials research for brain repair based on enhanced research potential, maintain and run the BLI and MRI equipment and ensure sustainability of introduced research.
All planned personnel were employed. Moreover, after obtaining permission from the Project Officer we employed two additional Postdoctoral scientists and additional Experienced Engineer. This in total meant that we employed 9 researchers: 2 senior scientists, 1 research manager, 4 postdoctoral scientists, and 2 experienced engineers. Together with 9 researchers we employed 1 administrative officer and 1 innovation manager, making together 11 direct GlowBrain employees.
Instead of originally planning 6 researchers, by careful use of resources we could employ 9 researchers (50% more). All of them stayed employed till the end of the second reporting period, and subsequently moved to other positions during the final three months of the GlowBrain. From these researchers 1 senior scientist, 1 research manager and 2 postdoctoral scientists were coming from abroad (4 of total 9; 44%) and 3 of them are Croatian returnees from their positions abroad. As Innovation Manger is as well Croatian returnee, this brings in total 5 scientists from abroad (of 11 total; 45%) employed by the project. From total of 11 employees, 6 (55%) were females.
In relation to sustainability of their positions beyond GlowBrain duration, we were challenged by the fact that it is not at all easy to get the position at our institution. The reasons are related by general cuts in Croatian public sector, specific cuts in relation to the financing of science and cumbersome procedural rules, which prohibit new employments. Despite this negative circumstances we succeeded to assure further employement for 4 employees and we plan to do it for 2 more, Igor Pongrac and Marin Radmilović, which will bring us to 6/6 planned, ie. 100%. It is important to notice that 2 more were not in need to be employed by us, Roland Pochet, which got retired, and Josip Pavičić, who continued with his own business using experiences he got through GlowBrain. In sense of unemployed status after GlowBrain, all of them found new positions, only one postdoctoral scientist from private reasons left to Pakistan, and now searches for job there.
GlowBrain activities involved many more scientists than those employed by the GlowBrain financial resources. Some of these UZSM employees have their status of independent researcher (Assistant Professor status and up) and are in position to guide their own projects (Principal Investigator status). 18 of them were involved in the GlowBrain project, and through their activities they already use GlowBrain platform and they will be first to contribute to sustainability of GlowBrain platform by their results. Moreover, these scientists involved in the GlowBrain activities are as well involved in the management of the institution and they are positioned at various leading positions in the organizational hierarchy of UZSM. This includes the very leading positions of Dean, Vice Deans, Vice Rectors, and Director of Croatian Institute for Brain Research.
Together with those leading scientists of UZSM, GlowBrain influenced as well a row of early stage researchers, besides of those being directly employed by GlowBrain. Some of them were involved in GlowBrain secondments, others were involved in application of gathered knowledge in GlowBrain platform. They were developed within GLowBrain environment and represent a future resource of new GlowBrain-associated researchers. In total, more than 30 scientists builds their careers around GlowBrain platform. They make power and future of GlowBrain foreground.
IP skills:
The foreground resulting from GlowBrain in relation to the management of intellectual property is already recognized at the institutional level and incorporated in its policies. This included setting up of institutional Committee for intellectual property managment, which then made institutional Regulations on intellectual property management. The Regulation was accepted by the Faculty Council (major governing body of institution), and it represents framework to operate in relation to institutional IP. The Committee approved all GlowBrain generated IP-related documents for institutional use. Moreover, there are 8 international IP experts with whom we established a permanent collaboration and we organized 4 IP Workshops.
These advancements already paid off as our institution was recognized at University level to be one of the rare with regulated IP policies, which enabled us to compete for national grants in relation to IP protection and development, and to be awarded 2 of them, being the best institution in this respect.
The upgrade of institutional IP policies includes as well:
- Raising awareness of researchers on generated IP,
- Processing all IP issues efficiently according to set policies in coordination of Technology Transfer Office, Committee for managing the intellectual property, and Faculty authorities,
- Active standpoint toward IP education, where IP related subjects are integral part of all UZSM PhD courses
- Applying for the national and international resources to improve IP management,
- Contributing to the University Master study on IP management,
- Regularly sending UZSM personnel for education at the University Master study at our University and similar workshops abroad.
The major challenge of the IP policies is the collaboration and participation in the activities of industry and SMEs, which could prosper on IP generated in our institution. This would be a next challenge to the institutional Committee to clear the path for joint activities with industry which would bring benefits to both sides.
In relation to IP, GlowBran generated IP which was submitted as a Patent application AN AUTOMATED SYSTEM FOR INDUCING HYPOXIA, GB1602071.1. We are currently exploring licensing possibilities for patent expecting to have a final decision on it in a year.
Responsible research and innovation:
The dissemination activities of GlowBrain were an important part of its activity and we consider them very successful. The following foreground was generated through the dissemination activities:
- GlowBrain brand - GlowBrain as a project acronym was well received and it caught attention. Therefore, and according to the advice of the Ex-Post Evaluators, GlowBrain name will remain beyond project duration. Name will be mainly used in relation to the GlowBrain platform located mainly in the GlowLab. Subsequently GlowBrain web page, Facebook profile and YouTube channel will remain functional in relation to ongoing activities of the GlowBrain platform.
- Regional Network for Regenerative Medicine and Stem Cell Therapies (RegMed) - At the end of GlowBrain activities RegMed was transformed in the professional association Croatian Association for Regenerative Medicine and Stem Cell Therapies (RegMedHr). This assured the sustainability of this professional interdisciplinary network. The plan of RegMedHr activities includes organization of international conferences, which will keep GlowBrain brand in the title.
- The Night when Brains are Glowing - This public activity enabled us to communicate directly with general public about achievements of GlowBrain project and to highlight importance of science for society. This dialogue was highly appreciated and it would be continued. Major issues in continuing this activity are the financial resources. Therefore, new GlowBrain night, scheduled for the end of 2016 / beginning 2017, will be based on public lectures with entrance fees. The lectures will be accompanied with the different media installations and entertainment part in similar way to the previous Nights. In the same way as already organised GlowBrain night was connected to 25 years of Croatian Institute for Brain Research, the next event will be connected to 100 years of UZSM.
Organization and management:
The activities related to organization and management of GlowBrain helped us to grasp management skills necessary for such a complex endeavor. It was very important to synchronize management of project with the institutional management as GlowBrain was dedicated to whole institution. This was successfully achieved and, even more, the institution realized the importance of GlowBrain and invested substantial amount of money in reconstruction and organization of space to host GlowBrain equipment, which is now GlowLab. Although GlowBrain needed to have its own management structure it was always in concordance with the management of Croatian Institute for Brain Research and UZSM. Subsequently at the end of GlowBrain, project management structure was integrated with management structure of the Croatian Institute for Brain Research, and it will function within its organizational bodies. The achieved structure is as follows.
The major organizational units of Croatian Institute for Brain Research (CIBR) are Sections. GlowBrain is represented in 3 Sections of CIBR. Two of these 3 were upgraded by GlowBrain still did not have a major change, therefore they stayed the same:
- Section for Laboratory Animals and
- Section for Electron Microscopy.
The third section was significantly changed. Previously it was
- Section for Neurogenetics, Cytogenetics and Developmental Genetics,
and now it was renamed in the
- Section for Neurogenetics, Medical Genetics and Regenerative Neuroscience.
This Section previously consisted from 2 Laboratories: Laboratory for Neurogenetics and Developmental Genetics, and Laboratory for Cytogenetics. Number and names of laboratories evolved through time and now in order to encompass recent changes it was reorganized to consist of 4 laboratories:
- Laboratory for Neurogenetics and Developmental Genetics,
- Laboratory for Stem Cells,
- Laboratory for Medical Genetics and
- Laboratory for Regenerative Neuroscience – GlowLab.
The management of the GlowBrain was integrated as well within the management structure of the Center of Excellence for Basic, Clinical and Translational Neuroscience. As the experience of Ex-Post Evaluation was positive, we plane to engage external experts in a form of different types of advisory boards. Croatian Institute for Brain Research already has Advisory Board and its membership was renewed at the end of 2015. Two representatives of GlowBrain Special Partnering Organisation, Laval University in Quebec, Canada are now members of the CIBR Advisory Board, prof. Jasna Križ and prof. Jean Pierre Julien. GlowBrain gathered through WP5 acitivities the 8 IP experts who become our permanent experts in the IP field, and finally GlowBrain platform will form External Advisory Board beyond duration of GlowBrain project.
Knowledge:
Knowledge is the most important foreground category resulting from the GlowBrain. Here some aspects of this foreground in relation of the GlowBrain results will be described.
The Exchange of Know-How with our partners
The major tool for knowledge exchange were secondments to and from our partners. This was very active part of GlowBrain and we organized 60 secondements (38.16 person months) to our partners and 35 secondements (9.01 person months) to our institution.
Development and exchange of knowledge in stem cell cultures took place during 32 secondments performed by following researchers: Lada Brkić Ahmed (6 visits to Lund), Marcell Bago (6 visits from Budapest to Zagreb), Dora Polšek (8 visits to Budapest), Marija Renić (11 visits to Salzburg) and Julila Marschallinger (one visit from Salzburg to Zagreb based on that topic). Lada Brkić Ahmed mastered isolation and analyses of cell fates in Lund partner laboratory, which is one of the most recognized European centers for stem cells and cell engineering. She was responsible for transfer of several methods of cell isolation and manipulation with different cell types, in particular stem cells and microglia. Dora Polšek and Marija Renić worked on stem cells in ischemic conditions and other cell subtypes, including microglia and stem cells from adult animals. Complementary approaches from three different laboratories (combined with already existing experience in Zagreb) yielded 10 standard operating procedures (SOPs) covering range from isolation of stem cells to different types of procedures based on stem cells. Budapest partner was regularly sending one scientist (Marcell Bago) to learn hypoxia in vivo methods from Zagreb coordinator and to work on further development of this technique. In addition, Julila Marschallinger from Salzburg was working on exchange of experience in isolation of neural stem cells. As an added value, secondments between Zagreb and Budapest resulted in patent application for chamber with controlled set of ischemic conditions while secondments between Zagreb and Salzburg resulted in a common article published in Nature Communication. Another added value is visible in parallel development of cultures originating from human oral mucosa in collaboration with the University of Zagreb School of Dentistry, which was in last year awarded two grants by Zagreb University.
Stem cell labeling and genetic modifications have been mastered during 11 secondments performed by following researchers: Marija Lovrić (2 visits to Prague), Lada Brkić Ahmed (6 visits to Lund), Uliana Kostiv (2 visits from Prague to Zagreb) and Somayyeh Hamzei (one visit from Cologne to Zagreb). During her first secondment (6 weeks in Prague) Marija Lovrić learned several techniques of stem cell labeling based on iron particles and their analyses based on spectroscopy, light scattering, atomic force microscopy and thermo-gravimetric analyses. During the second secondment she learned techniques of labeling stem cells with magnetic nanoparticles specifically prepared for MRI. Prague partner has sent dr. Uliana Kostiv two times to develop further methods of stem cell labeling in Zagreb environment which yielded several improvements of the procedure. This method is becoming recognized as one of the most important specific expertises based on stem cell labeling for magnetic resonance imaging. Cologne partner institution has sent Somayyeh Hamzei to help to develop new stem cell labeling techniques based on immunohistochemistry. During her 6 months stay in Lund, dr. Lada Brkić Ahmed mastered several methods of genetic manipulation with stem cells, which will be further utilized in combination with already existing protocols for cell transfection. One of the main outcomes of these secondments was newly established platform for stem cells labeling by nanoparticles and manipulation by cells using genetic modifications. This platform is now utilized by several groups in Zagreb. Thus one of the most prominent added values is ability of GlowBrain platform to test newly synthetized particles. Moreover, experience gathered in stem cell labeling has been successfully used during research for two PhD thesis which were finished in the second half of 2015 (Nina Kosi used labeled stem cells to trace them in the brain affected by stroke and Ivan Alić used labeled and genetically modified stem cells to analyze their differentiation after stroke).
Synthesis of innovative materials and their application have been mastered during 7 secondments performed by Marija Lovrić (2 visits to Minho), Lejla Ferhatović Hamzić (2 visits to Minho) and Marta Skelin (3 visits to Minho). Each of researchers has spent 6-12 weeks at University of Minho (and 3Bs research institution associated with University of Minho), which are one of the most recognized European centers for synthesis and application of biomaterials. During these secondments they learnt techniques of synthesis of various materials: methacrylated gums, hydrogels, modified hydrogels, polymers of various types, hydrogels in moulds, cell encapsulation by hydrogels and some others. In addition, Marta Skelin specifically learnt methods of production of extracellular matrix scaffolds and placental scaffolds. Some researchers have participated in advanced workshops dedicated to application of materials for stem cell cultivation (e.g. Lada Brkić Ahmed and Igor Pongrac participated in Cost NAMABIO action organized workshop in Cyprus).
The main outcome of these activities were experiments which combined biomaterials and stem cells in Zagreb (performed by Igor Pongrac and group of Simona Casarosa, University of Trento).
Visualizing brain and stem cells with BLI have been mastered by following researchers: Marija Renić (3 secondments performed in Salzburg partner institution) and Lada Brkić Ahmed (advanced level workshop organized by Perkin Elmer in Bruxelles). Since the instrument has arrived in the middle of the project, many trainings have been performed in Zagreb, including partner institution: Siniša Škokić was hosting both basic and advanced training level Perkin Elmer workshops for all interested researchers in Zagreb. Mariana Carvalho from partner institution Minho spent one month in Zagreb testing various concentrations of nanoparticles and dyes for tracing stem cells in brain using BLI. Adrian Manescu from Ancona partner institution visited Zagreb 11 times during project duration: majority of these secondments have been based on discussion and further development of imaging techniques for visualization of brain and stem cells. One of the outcomes of these trainings were SOPs for various procedures based on BLI. As a clear added value it can be listed that expertise in micro CT and other modalities coming from dr. Manescu was combined with BLI and MRI in Zagreb which resulted in the project proposal: “Marked stem cells tracking in mice ischemic brains by synchrotron radiation micro computed tomography”.
Visualizing brain and stem cells with MRI was covered by 13 secondments performed by the experienced engineer dr. Siniša Škokić who was hired to focus on installation and running of MRI. They included several levels of introductory trainings, safety and security trainings, operation with Bruker software, scan enhancements in different imaging modalities, animal handling, anesthesia control, monitoring of physiological parameters and image post-processing. Partners were as well involved in this exchange of know-how, so they visited Zagreb several times and helped in fine tuning of the instrument. Marina Dobrivojević spent two secondments in Cologne learning to work with animal models for MRI and basics of running of MR instrument. Exchanging of this knowledge also included other groups: dr. Mazzoni from University of Trieste used two visits to learn method of MRI from Zagreb group. Dr. Manescu from Ancona has visited Zagreb facility 11 times. Some of these visits have been based on learning MRI.
Use of small animals as disease models in neuroscience was covered in many secondments performed by both coordinator and partners. Specific requirements for animal facility and MRI was mastered by dr. Škokić, who collected experience from Cologne partner and used it for design of the new Zagreb facility linked to MRI. Dora Polšek and Lada Brkić Ahmed exchanged knowledge and know how in manipulation with different transgenic mouse lines used in neuroscience research.
One of the most notable results of these secondments was established collaboration between Zagreb and Budapest (performed by Dora Polšek from Zagreb and Marcell Bago from Budapest side) which resulted in development of a special chamber for intermittent hypoxia. This chamber is now in process of patent pending. Dunja Gorup has spent one month in Milan participating Young Investigator Training Program where she mastered some new advances in imaging methods and behavior studies. Several visits of partners to Zagreb were used to work with animal models present in Zagreb. Budapest (6 secondments), Salzburg (10 secondments of 6 people) and Cologne (Luam Hammelrath) partners were sending PhD students to exchange experience with personel involved in work with our models in Zagreb. Marcell Bago from Budapest was involved in developing intermittent ischemia model, while Salzburg group had 6 researchers involved in learning various histology methods, including those ones for electron microscopy and work with human tissue. In addition, two partner groups have been involved in learning MCAO stroke model from Zagreb coordinator group (dr. Casarosa group from Trento and Joanna Adamczak from Cologne). An added value in this section was obtained by 9 secondments of Marina Dobrivojević (visit to partner in Ancona and Trieste) who was working on application of X ray microtomography on imaging of mouse brain and tracing of transplanted stem cells in the brain.
Standardized procedures:
During GlowBrain we have organized and documented many standardized procedures (e.g. standardized operating procedures, SOPs). Although before GlowBrain we had the habit and prepared SOPs, GlowBrain increased complexity of our research procedures. New equipment and new approaches brought us to the frontier where we should search for our own approaches. Subsequently we can use many different approaches to address our question, and one of our research questions is to find out which is the most appropriate way. For example, if we want to use MRI to characterize the stroke, at first it seems very easy, there is a stroke, and we should measure the volume of the stroke area in the affected brain. Still there are many variables which can influence our measurement, at least 3 ways how to perform major stroke mouse model, middle cerebral artery occlusion, then the time of ischemia, the time of imaging, modality of imaging, etc. Consequently, which SOP one would use for MRI to evaluate the stroke requires much of evaluation to prove which approach will be the most relevant to our scientific question.
Although the SOPs can be considered as a basic step, easily learned from our partners or from the other laboratories with more experience, we are considering this as a major challenge for future, which if solved appropriately will answer research question or if research question could not be solved, it will at least be replied with relevant answer. In conclusion, SOPs represent a very important foreground of GlowBrain and their further development will be the major aspect of our future activities.
The knowledge was gathered and exchanged, the collaborations were developed and fostered at 5 scientific events organized by GlowBrain. These are:
- Final conference “Stem cell and biomaterial applications for brain repair" at on 27.-29.5.2015.
- GlowBrain 1st Workshop "Stem Cell Techniques" on 23-25.05.2013.
- GlowBrain 2nd Workshop "Application of biomaterials and in vivo imaging in stem cell research" on 27-29.03.2014.
- GlowBrain 3rd Workshop "Visualization of molecular markers in the brain" on 29.-31.1.2015.
- GlowBrain 4th Workshop "Preclinical In Vivo Optical Imaging Regional Meeting" on 5.11.2015.
- GlowBrain 5th Workshop "Biomedical Imaging Meeting" on 18.12.2015.
These scientific events served as well educational purposes as they included the hands-on demonstrations of the new procedures. GlowBrain is as well actively involved in teaching activities at our institution, which includes the MD and PhD studies, in particular the PhD studies in Neuroscience. In sense of GlowBrain collaborations efforts a comprehensive table of research topics and developed collaborations was provided in deliverable D6.5 Exploitation plan.
In conclusion, GlowBrain platform is versatile and would be used for different purposes adapted to serve the research topics of scientists from and outside of our institution. Still the major purpose for GlowBrain platform will be core GlowBrain activity, testing the combination of stem cells and biomaterials for stroke, developing further the major research line of the GlowBrain. The introduced GlowBrain platform will enable the enhanced level of research within European research area, opening paths in Croatia in the novel scientific fields:
- Regenerative Medicine (due to introduction of therapeutic strategies involving brain repair and regeneration),
- Stem Cell Therapies (stem cells being core activity of GlowBrain research),
- Nanomedicine (through integration of nanoparticle applications in the brain),
- Tissue Engineering (due to combination of biomaterials and stem cell research),
- Molecular Imaging (through visualization of disease molecular markers in tissues and animals).
Potential Impact:
GlowBrain impact
Whenever GlowBrain was presented to public, one of topics was to explain that investment in science is important in Europe and in particular in Croatia. Taking considerable attention due to its dissemination activities, and being one of the biggest FP7 project granted to Croatia, GlowBrain was in position to exert some influences on general public opinion. Therefore, the major argument raised was that the money received for GlowBrain was for the benefit of European and Croatian society. Investing in science is vital for society, and specific explaining why particular research financed through GlowBrain was important. Current economy is knowledge-driven economy and relies on science producing new knowledge. Subsequently, society lacking its own knowledge-producing activity has actually no chance to develop its economy, as it has no fuel that economy needs – the knowledge.
GlowBrain conveyed the following general messages:
- Science is vital for knowledge-based economy,
- In our effort to converge to the best we need to complement the traditional research approaches with the new technologies (GlowBrain contributed by new molecular imaging technologies),
- In order to be needed at the European level we have to find our own complementary niche, which would attract the partnerships in producing knowledge, but as well applying the knowledge (GlowBrain contributed by niche of in vivo multimodal imaging of stem cells after stroke),
- As Croatia is very small country only by joining our efforts together we could use our resources (knowledge, equipment, finances),
- Showing its success GlowBrain shared good news that there is no limit to achieve given goals. We made specifically desinged T-shirts to convey the good news. T-shirts were given away during our public events organized during Workshops, Final Conference and 2 public events organized, 2 Nights when brains are glowing. The message on the T-shirt was “Yes, we scan!” due to the fact we installed two scanners, MRI and optical scanner, and Yes, we scan! indeed with them,
- The fact that GlowBrain employed young scientists, some of them returning from abroad, was always highlighted. Science gives jobs, and it reverses brain drain into brain gain. The young scientists who went abroad are among best educated people of Croatia, therefore having them back home is a huge benefit for Croatia.
As stated above we always provoked debate about justification and a need to spend for science, and subsequently about GlowBrain imapct. As a specific challenge we used the most expensive instrument we acquired, magnetic resonance imager, which costed roughly 1.5 million Euros. Magnetic resonance imager is indeed expensive and most of these instruments are used for human diagnostic. Instead to buy a human scanner which would be used to diagnose sick people in need, we acquired scanner to put mice and rats in it and perform research activity.
The reason is a need for new therapeutic approaches to help sick people. Our major target was stroke, which is a major health problem, being first cause of disabilities and second cause of death. The stroke has no efficient therapy right now, hence there is an important need for advancement in this field. In order to create new therapies, experimental animals and preclinical experiments are essential tools, which subsequently lead to the clinical trials on humans.
As its major result, GlowBrain organized a preclinical platform to test new therapies for brain, in particular for the stroke. Moreover, preclinical platform was organized similarly to clinical setting. After we induce stroke in mice, animals are monitored alive to evaluate effects of given therapies. The MRI machine is critical for this step, providing the fine morphology of the brain, extent of stroke affected area, and post-stroke recovery. Moreover, the labeled stem cells when transplanted to the brain can be followed by MRI in living animal and consequences of introduced stem cell therapies can be evaluated. By helping mice after experimental stroke, we are able to contribute to future stroke therapies. While human scanner helps those people which are diagnosed with its help, animal MRI has potential to help many more humans in the future. GlowBrain platform is a comprehensive and necessary preclinical setup designed to contribute to the new therapies, for the brain diseases, and in particular for the stroke.
GlowBrain contributed to Croatian science to provide a necessary link of preclinical research on the brain. Neuroscience is recognized as one of the strongest research areas in Croatia, exemplifying by Center of Excellence in Basic, Clinical and Translational Neuroscience, and the current platform enables translational and applied biomedical research on brain diseases. It contributes as well to major priority of the biomedical research, which we refer as a path of drug development. The drug development path starts in basic science research, continues to preclinical research, translates to clinic, and finally after clinical trials it ends by approved use in patients. This path is long and cumbersome, but if a country and its research community can connect it together, it represents an enormous asset, and GlowBrain platform serves in Croatia to be a necessary node on this path.
The argument important for GlowBrain societal implications is specific economic power of the preclinical biomedical research. The health care industry increases its GDP share, being estimated to be 19.6% of US GDP in 2016. Therefore, preclinical research of GlowBrain contributes to increasingly important field of economic activity. It is unique as well by the fact that not only it enables economic benefits, but in the same time it gives health to the people, so compared to other economic activities it has a double effect on society.
GlowBrain specific societal impact for Europe is:
- Introducing a complementary niche of multimodal imaging of the mouse brain necessary for European Research Area,
- Counteracting 2-speed Europe,
- Contributing to preclinical stroke research, providing a platform which uses unique mouse models not present in other laboratories,
- It developed a medically relelvant innovation, a patent-pending mouse model for human disease - obstructive sleep apnea.
GlowBrain specific societal impact for Croatia is:
- Changing the perspective that high-technological science is necessary and possible in Croatia,
- Highlighting the need of preclinical research for medical applications,
- Contributing to drug development path in Croatia,
- Contributing to necessary upgrade of IP capabilities in the biomedical field
- Reversing brain drain in brain gain,
- Integrating research activities in institution, in country, and in European Research Area,
- Raising awareness of vital role of science in society.
Dissemination activities
The dissemination activities of GlowBrain were numerous and they were performed on professional and general public level throughout the duration of GlowBrain.
We would just mention some highlights:
- Srecko Gajovic, GlowBrain coordinator was for 2012 chosen among 100 people that changed Croatia to better by one of major daily Croatian newspaper, Jutarnji list,
- Facebook profile of GlowBrain, https://www.facebook.com/GlowBrainCroatia/ follows 1403 people, the profile is in Croatian (Croatia has a bit less than 4.3 million inhabitants, the total number of Croats in the world is estimated to 8 million),
- At inauguration of Horizon2020 programme in Croatia, GlowBrain was the only project invited for presentation,
- In 2015, Srecko Gajovic gave TEDx talk with topic “Why our brains are glowing?”, https://www.youtube.com/watch?v=4jfHBuEbPZc
GlowBrain was recognized in Croatia as EU scientific project receiving the most amount of money, which was only temporary as later other REGPOT project was awarded higher amount. Still the perception did not change as the other project did not make the awareness on this fact (actually, we made their job, answering that we are not the first anymore).
As a fact sheet on March 30, 2016:
- 74 congress abstracts were made
- 13 scientific publications
- 7 PhD thesis
- 16 public lectures
- 32 published text in newspapers and journals
- 8 radio appearances
- 17 TV appearances
- 13514 web page visits
- 1384 Facebook likes
- 1190 YouTube channel views
- 3 workshops organized
- 2 public events hosted by GlowBrain
The GlowBrain organized two public events “The night when brains are glowing” for policy makers and political establishment with participation of selected celebrities, general public and extensive media coverage.
The first Night was organized on September 20, 2013. at Lauba Gallery in Zagreb. It showed a special movie about the GlowBrain, the participants were invited to make their smartphones to “glow” through special android application, and a Millenium Photograph with “glowing” mobile phones was made. The event was attended and greeted with Vice president of Croatian Parliament and Croatian member of European Parliament among others. Croatian singer Massimo Savic and actor Goran Grgic participated in program. GlowBrain activites were presented on so called SmartSquare by GlowBrain members holding tablets with special presentations. The timing of meeting was when two national congresses were held, Croatian Neuroscience Congress and Croatian Pharmacology Congress. This enabled Congress participants, general public and colleagues researchers to attend and mix together.
The second Public Event “The night when brains are glowing” was organized on 14th September 2015 at the premises of University of Zagreb School of Medicine, in front of the Laboratory for Regenerative Neuroscience (GlowLab), Šalata 11, Zagreb. Public Event was organized as a part of the celebration of 25 years of Croatian Institute for Brain research. It was as well a grand opening of GLowBrain acquired instrument for pre-clinical imaging of small animals by magnetic resonance, Bruker BioSpec 70/20 USR, as a GlowBrain major acquisition.
Guests were welcomed at Smart Square in front of GlowLab by GlowBrain team and students, who presented GlowBrain project. The visitors registered at the entrance, and were offered promotional materials prepared for them: abstract books, brochure, pencils, foders, t-shirts, puzzles, all with EU logo. Plaster brain models and brain puzzles were prepared in order to explain to the visitors the purpose of GlowBrain project. Together with Prof. Gajović, GlowBrain coordinator, the speakers were: on behalf of the President of Republic of Croatia, academician Željko Reiner, Vice-President of Croatian Parliament; academician Zvonko Kusić, President of Croatian Academy of Sciences and Arts; professor Miloš Judaš, Vice-Rector for science, inter-institutional and international cooperation of University of Zagreb; academician Davor Miličić, Dean of the UZSM; academician Ivica Kostović, Honorary Director of CIBR; and Lovorka Grgurević, Head of the Department of Anatomy and Clinical Anatomy. A tour around a newly renovated Laboratory for Regenerative Neuroscience (GlowLab) and MRI Bruker BioSpec 70/20 USR, first such device in this part of Europe, funded by GlowBrain was presented to the visitors.
After GlowLab tour visitors enjoyed entertainment and program of second public event „Night when brains are glowing“, including Croatian singer Dražen Žanko, jazz quartet The Brainiacs and electronic music DJ. Finally, documentary movie about reconstruction of the Laboratory for regenerative neuroscience was projected, and common millennium photograph, with specific GlowBrain android application to light up smartphones and create illusion of glowing brains, was made.
In conclusion, by diverse and comprehensive dissemination activities, GlowBrain awareness was achieved in both professional community and general public.
Exploitation of results
The GlowBrain platform represents a flagship of new interdisciplinary technologies aimed to upgrade the medicine. These interdisciplinary technologies include:
- Regenerative Medicine (due to introduction of therapeutic strategies involving brain repair and regeneration)
- Stem Cell Therapies (stem cells being the core activity of the GlowBrain research)
- Nanomedicine (through integration of nanoparticle applications in the brain)
- Tissue Engineering (due to the combination of biomaterials and stem cell research)
- Molecular Imaging (through visualization of disease molecular markers in the animals and tissues)
The GlowBrain and its activities were highly transdisciplinary. The newly created platform is not only dedicated to research in neuroscience, but it represents a versatile tool opened to various research approaches. Its exploitation not only fosters the connection of different disciplines, which can benefit from the platform, but as well depends on their mutual collaboration:
- Mathematicians (providing mathematical models for image analysis)
- Physicists (imaging and postprocessing of images)
- Chemists (synthesis and characterization of nanoparticles and macromolecular biomaterials)
- Biologists (biological characterization of the in vitro and in vivo disease models)
- Medical doctors (providing animal models for disease and translational component)
- Electrical Engineers (being involved in complex imaging and experimental technologies, e.g. image collection and interpretation)
- Mechanical Engineers (providing interfaces between animals and imaging equipment)
- IT specialists (collecting and analyzing plethora of data generated by the platform).
If we consider only GlowBrain employees, they included electrical and mechanical engineers, chemists, biologists, biotechnologists, and medical doctors. All the above disciplines have already been gathered around the platform and attracted to dedicate at least part of their activities to it. This was as well psychologically connected to positive attitude of GlowBrain message of opened possibilities and collaborative success.
The platform exploitation efforts and its sustainability depend mainly on its financial sustainability. This part represents the most difficult challenge for the platform as we are developing not only interdependence of research interests, but as well the interdependence of financial resources, enabling the resources flow through the GlowBrain platform to create financial gain across the network, which would be used for platform sustainability, but as well its further development.
GlowBrain scientific results and research potential was already realized in the row of new projects awarded to the UZSM and related to the GlowBrain, the most of them ongoing beyond the end of the GlowBrain.
GlowBrain related grants are:
1. H2020 “BIOengineered grafts for Cartilage Healing In Patients – BioChip” (H2020-PHC-2015-single-stage_RTD Personalising health and care; 2015-2018, Partner – Srećko Gajović)
2. ERC = ScienceSquared (ERC-SUPPORT-2014: ERC Support for novel ways to highlight the work funded by the ERC and reach out a wider public; 2015-2018, Partner – Srećko Gajović)
3. H2020 Coordination Action in support of the sustainability and globalisation of the Joint Programming Initiative on Neurodegenerative Diseases — JPsustaiND (H2020-HCO-2015; 2015-2019, Partner – Dinko Mitrečić)
4. Structural Fund – European Social Fund project – Upgrade of young investigator competencies in the technologies of regenerative medicine in the brain diseases - Young Brain (HR.3.2.01; 2015-2016, leader Dinko Mitrečić)
5. Bilateral Grant Croatia – Serbia, financed by Croatian government – Stem cell application in the experimental treatments of amyothrophic lateral sclerosis (leader Dinko Mitrečić)
6. University of Zagreb Project – Gap43 gene function in stem cell differentiation and their response to stroke (leader Dinko Mitrečić)
7. University of Zagreb Project – Isolation, characterisation and differentiation potential of oral mucosa stem cells (partner Dinko Mitrečić)
8. COST Action CM1403 - The European upconversion network - from the design of photon-upconverting nanomaterials to biomedical applications (MC member Srećko Gajović)
9. COST Action CA15214 - An integrative action for multidisciplinary studies on cellular structural networks. (MC member Srećko Gajović)
10. COST Action CA15124 - A new Network of European BioImage Analysts to advance life science imaging (NEUBIAS) (MC member Dinko Mitrečić)
11. Italy Elettra grant 20135435 - Ischemic brain analysis by phase-contrast micro computed tomography (partner M. Dobrivojevic)
12. Italy Elettra grant 20140286 - Marked stem cells tracking in mice ischemic brains by microCT (partner M. Dobrivojevic)
13. Italy Elettra grant 20155224 - Marked stem cells tracking in mice ischemic brains by synchrotron radiation micro computed tomography (partner M. Dobrivojevic)
14. Center for Excellence in Basic, Clinical and Translational Neuroscience (whole GlowBrain platform included, awarded to UZSM)
15. Center for Excellence in Reproductive and Regenerative Medicine (awarded to UZSM)
16. Center for Excellence in Bioethics (awarded to University of Zagreb School of Philosophy, Partner Srećko Gajović)
17. Croatian Sceince Foundation Fellowship for Ph.D. student Marta Skelin (mentor Marija Ćurlin)
18. Croatian Science Foundation Fellowship for Ph.D. student Nikola Habek (mentor Aleksandra Sinđić)
The major financial contribution important for sustainability of the GlowBrain platform is assured already from first 4 projects on the list.
The above described projects, although rather diverse, have a clear connection to GlowBrain. They are a major result of perception by GlowBrain professional environment that GlowBrain platform is bringing something new to community and that equipment, knowledge and skills acquired are important. BioChip shares with GlowBrain topic of regenerative medicine applied to cartilage, and GlowBrain with experiences in developing new technologies and management of EU project was an excellent base for this proposal. Science Squared is a result of extensive public promotion of the project, and experiences of these public promotions have been used to approach this collaborative project and win the position in successful consortium. JPsustaiND represents recognition of GlowBrain at European Neuroscience level. Croatia as a partner in this project was given a task to motivate and engage Central East European neuroscientists to be active in JPND (Joint Programme for Neurodegenerative Diseases). Finally, structural project Young Brain was currently only structural call offered for individual scientists to apply. This does not mean no structural funds were awarded, but they have been decided at the level of universities or research institutes. GlowBrain was involved as well in institutional applications to structural funds and in this way GlowBrain platform is directly included in one Center of Excellence, and his participants in 2 other Centers of Excellence. According to information from Ministry of Science roughly 30 million of Euros will be available for Centers of Excellence in 2016.
GlowBrain platform has unique features indicating its commercial potential. These unique features are:
1. Patent pending system for intermittent hypoxia in mouse,
2. Unique transgenic mouse models with molecular markers for brain inflammation and repair,
3. Unique combination of instruments and organization dedicated for brain research – GlowBrain platform located in the GlowLab,
These features can be organized to offer unique services, which could be competitive at European and global level.
Recently, many tested compounds, candidates for drugs for neurological diseases, were shown to be beneficial in preclinical settings, but they subsequently failed in clinical trials. Not only there was no cure found, but as well this was connected to the financial losses as both preclinical and clinical trials were very expensive. Finally, search for cure did not get more intensive, but pharmaceutical companies started to avoid neuroscience in order to prevent losses. This could be improved by investing more in basic research, and by better understanding of brain diseases enabling the design of better therapies. Crucial moment to develop efficient therapy is validation whether the promising preclinical study would be beneficial in clinical trials. Our GlowBrain services should contribute to this by in vivo tracking of molecular markers and events in the brain, which would allow testing of promising compounds in the living brain on unique mouse models using patented disease models.
The above described rationale is base for contract research to be offered to pharma companies. Moreover, we as well have mastered many techniques, which are not unique but valuable, which could be included as a part of portfolio. In order to achieve this goal of contract research services, there is still a long way to go, mainly in organizing the controllable, standardized and validated procedures to be offered on the market.
The use of GlowBrain platform for contract research will rely on new innovation and IP policies introduced at institutional level by improving innovation management of GlowBrain. Still there is uncertainty in perception of a commercial activities on university as public institution. Therefore, we would suggest a model or privately owned spin off, which would license technology, rent equipment and space of GlowLab in order to offer contract research. Still other possibilities are as well opened.
The exploitation of results oriented toward professional community is directed to open possibilities and to support innovations.
GlowBrain platform is an ideal platform to boost innovations. It installed the new technologies and new directions in order to reach a new way to attack major health issues. Moreover, it trained young people to be open minded and innovation prone. GlowBrain orientation to early stage researchers was already confirmed by first Structural Fund we obtained, which is dedicated to the young people “Young Brain”. The continuous activity of GlowBrain beyond the project duration is based on support to innovation and freedom of ideas. This is a message we share among the wide scientific community – if one has a research idea, GlowBrain is a versatile platform to welcome it and make it real, either within the platform or as an example to help others to go their own way. In conclusion, GlowBrain serves as an innovation platform supporting innovation of others, which in return will support the sustainability of the GlowBrain.
To exploit the full potential of platform, we intend to further develop the mechanisms to measure and to predict impact of activities The impact measurement methodology is still to be defined at the European level, and we would by our activities contribute to this effort.
The societal GlowBrain potential toward general public is currently formalized by our activities within international Navigating Knowledge Landscape Network (NKLN). NKLN was founded in Zagreb at the meeting of RegMed network and the founders are Anna Lydia Svalastog (Norway), Joachim Allgaier (Austria), Lucia Martinelli (Italy) and Srećko Gajović (Coratia). The Navigating Knowledge Landscapes Network is an international and interdisciplinary group with an explorative interest in area of online and offline communication concerning health and biomedical knowledge. The aim of the Navigating Knowledge Landscapes Network is to explore and describe how individuals navigate through landscapes of health-related knowledge in digital society. The reason for NKLN, i.e. to explore navigation in an increasing amount of digital information, is an urgent need to empower the patients and other stakeholders to make better informed health-related decisions.
Moroever, within the spectrum of GlowBrain activities Regional Network for Stem Cell Therapies was concieved as a body covering diverse activities in professional community and in society. Already at the beginning, at the first meeting of the Network, the name was modified to this comprehensive task and the new name was Regional Network for Regenerative Medicine and Stem Cell Therapies (RegMed). We succeeded to include in network basic scientists, clinicians, representatives of the industry, business angels, lawyers, patent attorneys, phylosophers, bioethicians and theologists. Approach diversity indeed corresponded to complexity of GlowBrain approach in establishing the new medical technology. In December 2015 the network was formalized as an association referred as Croatian Association for Regenerative Medicine and Stem Cell Therapy (RegMedHr). The association is not only an important result of GlowBrain activities, but as well it serves as support for further sustainability of GlowBrain and wide base of Exploitation Plan for future use of the GlowBrain platform.
In conclusion, GlowBrain has already made significant advances in sustaining its activities beyond the project duration. These activities will assure the exploitation of newly created GlowBrain platform. This is achieved by already ongoing new international (H2020) projects, increasing the regional potential by being actively engaged in synergy with structural funds, and through network of collaboration attracting potential users of GlowBrain platform. Economic potential of GlowBrain should be realized by the unique services to be offered to industry partners, and its societal potential accomplished by supporting innovation and clarifying its impact for society benefits.
List of Websites:
The project website is http://glowbrain.hiim.hr/.
The Facebook profile is https://www.facebook.com/GlowBrainCroatia.
The YouTube movies are http://www.youtube.com/results?search_query=glowbrain.
The official project email address is glowbrain@hiim.hr.
The main objective of the proposed project GlowBrain was to upgrade and unlock the existing excellence in brain research with specific twist in activities based on development of stem cell and biomaterials applications.
The core idea of this project was based on combined biomaterials and stem cell applications in the brain monitored by combined bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). This allowed to enhance the stem cell delivery and integration in the brain and to monitor both the stem cells and their effects in the living animals. The institutional capacities were upgraded and a GlowBrain platform was assembled in order to achieve the top-notch research organized in a unique way, not present elsewhere in Europe. Exchange of know-how with partnering institutions helped to collect and implement the knowledge to make the platform functional.
Our main S&T objective was to introduce a novel approach for stem cell based therapy of nervous system. This was achieved by using the biomaterials to enhance and control stem cells, and by in vivo monitoring of stem cell behavior after transplantation through complementary combination of two imaging modalities - bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). In order to decrease the most common obstacle in stem cell applications, low survival and integration rate of cells faced with hostile environment, we used biomaterials which supported formation of 3D cellular networks.
BLI was used to in vivo monitor molecular events in the brain with the help of the specific transgenic animals carrying luciferase as bioluminescence reporter under promoter of genes relevant to the brain repair and regeneration. The appropriate mouse lines were collected in our facility.
MRI was used to in vivo precisely localize transplanted stem cells in the brain. Apart from precise anatomical location, MRI was used to distinguish brain pathology (in particular after ischemic lesion through middle cerebral artery occlusion) and repair processes in the mouse brain. In order to be visible the stem cells were labeled by ultra-small superparamagnetic iron oxide (USPIO) visible by MRI. This allowed to track the transplanted stem cell population and to assess their distribution within the brain.
The advantage of combination of BLI and MRI is that the brain can be visualized both by luciferase markers and superparamagnetic particles. This enabled visualization of the brain by MRI and characterization of its reaction by luciferase as gene expression marker by BLI. The setup of such a complete capacities upgrade was very complex and it was based on our previous knowledge and abilities, combined with twinning and exchange of know-how with our partners.
The ability to visualize the brain in the living animals represents a leading edge asset, which together with all other already existing expertise establishes the highly versatile preclinical platform for brain research.
Project Context and Objectives:
There is a high and urgent need to attack brain diseases, which represent one of the main burdens of the modern society. Using parameters of the World Health Organization and the European Brain Council it became clear that brain diseases account for 35% of the burden of all diseases in Europe with a strong tendency to reach almost 50% as the population ages. On the other hand, only 15% of the research budget is allocated to this field, which corresponds to the urgent need to direct substantial financial support to neuroscience research in the next decades. Moreover, 95% of therapeutic efforts are based only on symptomatic relief of brain diseases (i.e. stroke, Alzheimer’s disease, amyotrophic lateral sclerosis, brain trauma), while direct action on pathogenetic mechanisms are vastly limited and significantly lower than in other organ systems.
The ongoing efforts of scientific community to improve this situation include not only immediate action through testing available therapies, but as well relates to completely new concepts of therapy, one of which is referred as cell therapy, in which applied cells would support and/or replace the brain cells. The application of this innovative concepts relies on increase of the basic knowledge about brain development, organization and function together with the development of the new complex technologies related to stem cell preparation, transplantation and monitoring.
University of Zagreb School of Medicine (UZSM) is the central medical institution in Croatia, and its part Croatian Institute for Brain Research (CIBR) is the central institution for research in neuroscience which coordinates basic, clinical and translational neuroscience. There is neither similar organization nor equivalent excellence in neuroscience in Croatia nor in the nearby convergency regions. This excellence was recognized recently by awarding The Center of Excellence in Basic, Clinical and Translational Neuroscience.
The main objective of the proposed project GlowBrain was to upgrade and unlock the existing excellence in brain research with specific twist in activities based on development of stem cell and biomaterials applications. This will be achieved using the already existing contacts with ERA partners enabling: 1) significant exchange of know-how, 2) recruitment of experienced researchers, 3) introduction of the novel infrastructure and 4) development of intellectual property competencies.
The mouse brain research represents a central point of basic research at CIBR. The research infrastructure for mouse brain research is already highly developed and includes several important premises, i.e. platforms. They include: (1) mouse facility, (2) neuroanatomy platform, (3) neurodevelopmental platform, (4) microscopy platform, (5) molecular biology platform, and (6) behavioral platform, plus two platforms already under construction (7) stem cell platform, and (8) biomaterials platform. Actually, GlowBrain project aims to complete two platforms under construction (i.e. 7 and 8) and to add in vivo molecular imaging as the platform number 9.
The in vivo molecular imaging changes the use of animals in experiments, in particular transgenic animals. Previously, the analyzes of the molecular mechanisms were based on the single end point data only, i.e. as a result of analysis after sacrificing the animal. The molecular markers were visualized with enzymatic or fluorescent reporters in the obtained tissues under the microscope. On the contrary, the in vivo molecular imaging strategy is based on employing reporter molecules developed to study biological processes in living animals in real time. This enabled novel approach of time-dependent studies of molecular processes in living animals.
Imaging modalities frequently utilized in in vivo research are mostly adaptations from familiar, clinical imaging instrumentation such as X-ray, ultrasound, CT, SPECT, PET and MRI. From this rapidly evolving field we have selected two flagship technologies: Bioluminescent Imaging (BLI) and Magnetic Resonance Imaging (MRI). Exciting possibility employed in the this project is to combine these two techniques, which offers a chance for giant progression in this highly innovative field.
The core idea of this project was based on combined biomaterials and stem cell applications in the brain monitored by combined bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). This allowed to enhance the stem cell delivery and integration in the brain and to monitor both the stem cells and their effects in the living animals. The institutional capacities were upgraded and a highly needed added value was obtained in order to achieve the top-notch research organized in a unique way, not present elsewhere in Europe. Exchange of know-how with partnering institutions with proved excellence in applicable science and patenting opened a new dimension enabling the progress in development of innovations and protection of intellectual property.
Our main S&T objective was to introduce a novel approach for stem cell based therapy of nervous system. This was achieved by using the biomaterials to enhance and control stem cells, and by in vivo monitoring of stem cell behavior after transplantation through complementary combination of two imaging modalities - bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). In order to decrease the most common obstacle in stem cell applications, low survival and integration rate of cells faced with hostile environment, we used biomaterials which supported formation of 3D cellular networks.
BLI was used to in vivo monitor gene activity of transplanted stem cells. To visualize through BLI the mouse brain the transgenic animals were needed, already carrying luciferase as bioluminescence reporter under promoter of genes involved in differentiation of stem cells toward neurons. Many of these mouse lines were collected in our facility or in the facilities of our Partnering Organizations. BLI enables visualization of the subset of brain cells which would express the bioluminescence reporter at the corresponding molecular event. This enabled to monitor the onset, location and duration of the particular brain event. The markers could range from those that are specific for neurons, glia or non-neural phenotypes; for those specific for established cell types, and for those describing various types of neuronal activity, i.e. axonal sprouting, or neuroinflammation. The high sensitivity of BLI is indeed the major asset enabling the monitoring all necessary subtle steps in this complex process.
MRI was used to in vivo precisely localize transplanted stem cells in the brain. Apart from precise anatomical location, MRI was used to distinguish brain pathology (in particular after ischemic lesion through MCAO) and repair processes in the mouse brain. In order to be visible the stem cells should be marked by appropriate particles adhered to the cell surface and visible by MRI, like ultra-small superparamagnetic iron oxide (USPIO). This allowed to track the complete transplanted stem cell population and to assess their distribution within the brain.
The advantage of combination of BLI and MRI is that the brain could carry both luciferase markers and superparamagnetic particles. This should enable visualization of the one cellular process in the brain by MRI and characterization of another by luciferase as gene expression marker by BLI. Through this double labeling strategy, the precise localization of molecular and cellular events viewed by MRI would be enhanced with ability to detect sensitive signal viewed by MRI. This actually points to the superb added value of the combined facility and introduced multimodal imaging.
The proof of concept was the establishing of the whole procedure of in vivo molecular imaging of transplanted biomaterials-supported stem cells in the mouse brain after ischemic injury induced by middle cerebral artery occlusion (MCAO). The setup of such a complete capacities upgrade was very complex and it was based on our previous knowledge and abilities, combined with twinning and exchange of know-how with our partners.
Specific objectives necessary to establish the integrated platform were (1) establishing the differentiation, labeling and transplantation of stem cells, (2) development of advanced techniques in application of biomaterials in the brain, (3) visualizing brain and stem cells with BLI, (4) visualizing brain and stem cells with MRI, (5) multimodal imaging BLI/MRI of brain and stem cells, and (6) assessing the brain repair in mouse model of the stroke (MCAO).
Therefore, the ability to visualize the stem cells and the brain in the living animals represents a leading edge asset, which together with all other already existing expertise establishes the highly versatile platform for stem cell research in the brain.
Project Results:
Our main S&T objective was to introduce a novel approach in preclinical research on stem cell based therapies of the nervous system. This was achieved by establishment three main pillars of the upgraded research activities: A) stem cells, B) biomaterials and C) in vivo animal imaging system. The biomaterials are expected to enhance and control stem cells, and in vivo monitoring of stem cell behavior after transplantation needs to be evaluated through complementary combination of two imaging modalities - bioluminescent imaging (BLI) and magnetic resonance imaging (MRI). The research organised around the combination of these three pillars was referred as the GlowBrain platform.
The major result and major foreground of the GlowBrain project is the newly established GlowBrain platform. The major part of platform is located in the new laboratory referred as Laboratory for Regenerative Neuroscience, and nicknamed GlowLab. The capital equipment acquired by GlowBrain project is located in the GlowLab, but the GlowLab is wider than just hosting the instruments. Similarly, the GlowBrain platform is not only instruments and architectural space, but as well a functional combination of knowledge. In the sense of the procedures and laboratory space, the GlowBrain platform includes together with the GlowLab the previously existing laboratories and facilities of Croatian Institute for Brain Research, which as well got upgraded by the new procedures and new functionalities.
The major topic, connecting knowledge, space and equipment, is understanding of brain pathologies and preclinical validation of potential cures. The animal experimental model used is mouse, and the central disease model is the stroke. In order to get insight in the stroke pathology and subsequent brain repair, the preclinical mouse model of the brain ischemic injury was used. The standardized approach applied in the GlowBrain platform to create this injury in the mouse brain is medial cerebral artery occlusion (MCAO). Moreover, during the GlowBrain course we added unique model of human disease using intermittent hypoxia to model in mice a human disease - obstructive sleep apnea.
When the GlowBrain was conceived the equipment to be purchased was intended to be located in the main building of the Croatian Institute for Brain Research. Major equipment needed a specially organized dedicated space was MRI, and in the first period of our negotiations on the instrument acquisition with the MRI producers, the planning was based on this location. As MRI needed 3 rooms, one for the instrument, one for the supporting machinery (i.e. technical room), and one for the computers and researchers running the system (i.e. control room), plus many connections, this prompted the discussion about the equipment final location. The building of Croatian Institute for Brain Research was the newest on the campus, completed in 1990, and it has the best conditions for research at our institution, University of Zagreb School of Medicine (UZSM, Croatian Institute for Brain Research is an integral part of the University of Zagreb School of Medicine). The modifications necessary to host the machines implied that new building would require some substantial renovations. Subsequently, the alternative space was considered and finally decision was made to provide the GlowBrain platform an additional space in another building of Department of Anatomy. The advantages of this solution was that the GlowBrain could organize its space in a best possible way and provide to new equipment all necessary conditions designed and prepared for GlowBrain platform. The disadvantages were that new space was in basement of an old building (completed in 1908), and this basement was abandoned 20 years ago, and at decision moment humid and in a very bad shape. The challenging task of basement renovation was successfully accomplished, and resulting laboratory space is an exquisite example of a new laboratory. When considering the final result, the new laboratory represents a real gem for brain research in Europe, and it is the excellent space for the acquired equipment. To reach this level was not easy, but it made us more proud that we could achieve this. We can state that we have the best preclinical in vivo imaging facility between Vienna and Athens.
The new laboratory represents a synergy between financial resources of GlowBrain and financial resources of the institution. The construction works were fully covered by institutional money and in this sense it showed how important for the institution was the GlowBrain.
The new Laboratory for Regenerative Neuroscience integrated in cca 200 sqm main functionalities of the GlowBrain platform. The core facility is indeed in vivo imaging, but together with it, there is dedicated space for the animals being involved in the experiments, operation room for the animals where the mouse stroke model takes place, and cell culture, where stem cells and biomaterials are prepared.
The workflow in GlowLab corresponds to the main idea of GlowBrain platform, combining biomaterials and stem cell research to treat ischemic brain injury (stroke) in mouse and validating effects by in vivo imaging. The laboratory consists from 3 areas, each area has its own cleanness and security levels. The first area is dedicated to data analysis and represents the area where the researchers and eventual guests can freely enter. In this area computers for image and data processing are located and researchers can work on their desks. The other two areas are restricted and the entrance is allowed only to selected trained personnel. The lower level of restricted access is allowed to the area where in vivo imaging and cell culture is situated. The in vivo imaging consists of 3 rooms: technical room housing supporting equipment for MRI, the MRI room with the scanner itself, and the control room. In the control room there are the computers running MRI, animal table to prepare animals for imaging, and as well the preclinical optical imager for bioluminescence (BLI) and fluorescene (FLI) imaging.
In the same restricted area, nearby the control room, is a cell culture dedicated primarily to cultivation of the stem cells. These cells could come from the outside (e.g. human stem cells) or could be harvested from the animals in the nearby animal area (e.g. as it is the current use of neural stem cells obtained from mouse fetal brains).
The third area being the most restricted is dedicated to the animals held in individual ventilated cages in two animal rooms, one for rats and another for mice. The space was organized as an experimental mouse facility. The breeding of animals and cleaning and disinfection of cages is located in the main building of Croatian Institute for Brain Research. Together with rooms for animals, in GlowLab there is one room as a clean space (e.g. for the clean cages), and another room as a dirty space (e.g. for the dirty cages). The dirty cages exit through the technical exit/entrance to be transported to the main building and subsequently return cleaned through the same exit/entrance.
The animal area has two additional rooms important for the GlowBrain platform. One is Operations, where the animals are operated and treated. This includes the mouse models for the brain pathology either as MCAO, being a stroke model, or another types of operations, e.g. spinal cord injury or traumatic brain injury. Another aspect of Operations is treatment of animals, where stem cells need to be introduced in animal through stereotaxic injection. The next door room is a multipurpose room, which can serve as a space for animal recovery (e.g. the temperature can be set higher than in the rest of the space), or for simple behavioral analysis, or as a quarantine for the specific guest animals.
The GlowLab allows specific workflow related to GlowBrain project through specifically designed communicating windows. The stem cells from the cell culture are transferred to the animal area through specially designed window, animals are treated with stem cells, they are housed in animal rooms and then carried to in vivo imaging area (again through specially designed window). The windows enable transfer of materials from one restricted area to another without a need of researchers to change clothes or use specific paths to maintain a clean facility.
The important feature of the facility is that it is BSL2 ready. The air pressure is differential from the area to area, enabling the air flow from the cleaner spaces (animal area) to the dirtier spaces (data analysis area). The GlowLab has all necessary connections which enable electricity, heating/cooling, air flow, air pressures, information network, medical gasses, and exhausts for the MRI equipment (helium) or animal preparations (anesthetics). Together with cca 200 sqm of floor, there is a labyrinth of 200 sqm of connections in the ceiling, and cca 200 sqm of equipment outside the building to provide all necessary conditions in the laboratory. We affirm that the GlowLab is organized and constructed as a top notch facility to give the maximum versatility and possibility to the equipment purchased from the GlowBrain project.
Although the major asset of the GlowBrain, the purchased instruments for in vivo imaging are located in GlowLab, the GlowBrain platform exceeds the GlowLab and it includes laboratories and spaces in main building of Croatian Institute for Brain Research, at Dpt. of Histology and Embryology, and Dpt. of Pathology.
The GlowBrain procedures are based on standardized and well treated animals, which are hosted in the Section for Laboratory Animals of Croatian Institute for Brain Research (e.g. animal house). The animal house was upgraded by GlowBrain in order to host more animals in better conditions. As Croatia joined European Union during the course of GlowBrain, we adapted European standards. The animal house was reviewed by local authorities and the license for animal work issued (one of the first at University of Zagreb). Moreover, the groups using animals together with ethical approval of Institutional Review Board, received as well approval from the Ministry of Agriculture to work with animals. Again, due to the acquired EU regulations, new internationally and nationally licensed courses for animal work were attended by all users of the animal house and all group leaders using animals in their projects and they acquired the personal licenses for the animal work. To sum up – we actively followed the extensive upgrade of animal standards and we are completely up to date having all permissions and documentation for the animal work, but as well following actively in daily practice the recommendations and practices of animal welfare.
The major modification in animals used for GlowBrain purposes is that we needed to change the background of the animals. Our mouse lines were all kept on C57Bl6 black background. After we acquired optical imager and started to image animals, the first step was to shave animals in order to remove black hair and allow light to pass through. This was feasible for short experiments, but for long term imaging of the same animal (week and above) the reaction on hair removal was noticeable – the small hairs growing faster, and the skin getting pigmented. As we imaged neuroinflammation, we could not distinguish whether the inflammation was due to the hair removal and if the hair removal could influence our measurements. Therefore, the C57Bl6 albino strain was acquired and all mouse strains we had (cca 500 mice as an average number of animals present in the animal house in the same time) needed to be backcrossed to this new albino strain. Actually, we could not do partial backcross only to the lines we would use for imaging, as we would have two groups of animals, one on black and another on albino background, subsequently not comparable in our experiments. Therefore, we decided to have all animals on the albino C57Bl6 background. Currently, this process is completed and all lines were backcrossed to albino strain. Theoretical threshold to be considered isogenic is 5 generations, and with most mouse lines we are already on this step. The rederivation of animals represented a bold necessary step, which is a very important time consuming result of the GlowBrain, allowing us to proceed with the experiments.
The core GlowBrain procedures in the mouse house include the ischemic lesion induction and we currently use two procedures: (1) transient ischemic lesion by middle cerebral artery occlusion (MCAO), and (2) intermittent hypoxia.
Transient ischemic lesion by MCAO was significantly upgraded by GlowBrain. We discussed extensively how to perform procedure with our partners and they had trained our personnel at their site and in Zagreb how to do it. Moreover, we acquired laser color doppler in order to measure the blood flow through the brain at the location of middle cerebral artery. The major advantage of the MCAO procedure is that it is the best clinically relevant model of the human ischemic stroke, as the same artery is affected in majority of human stroke cases. The major disadvantage of this mouse model is that it is highly variable. The variability in our setting is reduced by measuring the blood flow during the procedure, and having trained personnel to perform lesion in a standardized fashion. The introduced ischemia is temporary and it lasts for 1 hour and after that reperfusion occurs. The animals are routinely tested subsequently by measuring the neurological score, and now with the functionality of MRI we added the necessary step of verifying the lesion by MRI. This provided us an important prerequisite to be able to perform transient MCAO in a standardized and validated form.
Intermittent hypoxia is a unique approach developed through GlowBrain activities and represents the model for the human obstructive sleep apnea. The development of this unique model represents an important added value of the GlowBrain. Obstructive sleep apnea is a huge unrecognized human health problem where during the night the breathing completely stops due to snoring and person remains hypoxic for the short periods of time. The brain consequences of this intermittent hypoxia are still unknown and it is hypothesized that they hugely contribute to the raise of dementia in Europe and globally. The setting included development of the hypoxic chamber for mice dedicated to mimic exactly obstructive sleep apnea and this is now patent pending (patent submission number is GB1602071.1). Through this GlowBrain platform added unique in house developed mouse model of major human disease, obstructive sleep apnea.
The animal house is as well location for stem cell and biomaterials treatment platform were we organized a comprehensive stereotaxic injection setup to inject cells, nanoparticles and biomaterials to the mouse brain in the standardized fashion.
The next developed foregrounds are related to the stem cells and biomaterials. Protocols and procedures were organized and performed in the Laboratory for Stem Cells of Croatian Institute for Brain Research, where the important advances of the stem cell culture were achieved. The stem cell culture was needed to be standardized and evaluated. The major goal was to choose the best way to grow mouse neural stem cells obtained from the fetal mouse brain. In addition, we introduced mesenchymal stem cell culture from mouse. Finally, the human stem cells were introduced in a separate cell culture in the Dpt. of Histology and Embryology, where human stem cells were isolated from oral mucosa, an easy way to get patient specific stem cells.
The part of the GlowBrain platform dedicated to the biomaterials and nanoparticles was the Section for Neurogenetics, Cytogenetics and Developmental Genetics of Croatian institute for Brain Research. Before GlowBrain this Section was place for molecular genetics analyses of the transgenic animals, therefore it was recently renamed in the Section for Regenerative Neuroscience, Medical Genetics and Developmental Genetics. Together with previous activities, activities spectrum was extended to nanoparticles and biomaterials. Naively, at the very beginning we assumed that we could concentrate only on in vivo experiments with marked stem cells. In a nutshell the first proposed concept was that the various partners will synthesize nanoparticles and biomaterials and we would test them in the animal disease model. As soon as we started to create the experiments we realized that before testing the nanoparticles and biomaterials in the mouse model of stroke we need to perform 2 important, necessary and inevitable steps. The first was testing of biocompatibility of the compounds in vitro on the stem cells, to verify whether the treated stem cells are comparable to non-treated. This as well tests in general the suitability of any compound to be injected in the brain. The second step included in vivo testing of the biocompatibility of the compound when injected in the brain. Interestingly, we suggest that these two steps represent the missing link between the material scientists (e.g. chemists) and biomedical scientists, and when provided would allow many of the compounds to be tested in preclinical settings. Therefore, our effort was necessary, but as well essential for the cross-talk between the disciplines.
The in vitro testing included (1) general toxicity testing, this tested for the cell survival and proliferation, (2) differentiation testing by immunohistochemistry and RT-PCR, (3) labelling efficiency testing, (4) labelling mechanism testing, and (5) oxidative stress testing. The innovative step in these tests was the use of flow cytometry, which allowed the more precise measurements of the toxicity and cell labeling. The settings as well included a revival of institutional Centre for Electron Microscopy, which is located at Dpt. of Pathology. It was needed for labelling evaluation, but as well it was highly praised by our partners and it would be the base for continuous collaboration with our Partnering Organization Paracelsus Medical University.
The biomaterial combined with stem cells to be transplanted to mouse brain and to enhance stem cell capabilities was alginate. Alginate was chosen as a polysaccharide biomaterial tested already in vitro by our collaborator Simona Casarosa. Therefore, in this particular case we could skip in vitro tests and went directly for in vivo biocompatibility. For in vivo testing of biocompatibility we used the advantages of the introduced preclinical in vivo imaging and of a unique model of Tlr-2/luc mice, we had in our mouse collection. Tlr-2/luc mice are improtant, as by bioluminescent imaging they allow to measure neuroinflammation and to verify if alginate injection in the brain elicits neuroinflammation. The results showed that it was comparable to injection of the vehicle (PBS), therefore represented an inevitable reaction to the stereotaxic injection not to be attributed to specific effect of the alginate. These results are part of defended PhD thesis of Angela Bozza, and of the manuscript in preparation.
In setting up the procedures vital for the GlowBrain platform, we used in vivo imaging to validate as well nanoparticle applications in the brain, stem cells visualization labeled with nanoparticles, bioluminescent cell visualization, and stem cell and biomaterial visualization.
These performed experiments showed platform abilities. Still it should be noted that standardization and full functionality will require time, and that the GlowLab started to be fully functional right at the very end of GlowBrain project. Moreover, one of the consequence of the GlowBrain project is increased perceptual value of the standardization of the procedures and this will be continued in the GlowLab, in particular as this would be the base for planned contract research, which is important for exploitation plan.
The following settings specific for the GlowBrain platform are elements of the achieved foreground:
1. Collection of transgenic animals bearing luciferase and fluorescent reporters and knock-out animals of genes relevant to brain damage repair
The mouse lines in our collection are rather numerous and they were selected to cover diverse aspects of the GlowBrain platform. They include the following mouse lines:
a. The mice carrying luciferase (luc) and green fluorescent protein (GFP) reporters (the luc reporter is aimed to be used for bioluminescent imaging, and the GFP for the immunohistochemistry analysis on the histological slides):
- Tlr2-luc/GFP
Luc and GFP reporters are driven by Tlr2 promoter. TLR2 (Toll-like receptor 2) is a receptor located mainly on the microglia cells and it binds various ligands important for innate immunity response. For stroke pathology of the major importance are particles of necrotic cells, which died due to the ischemia. They belong to Damage-Associated Molecular Pattern molecules (DAMPs) also known as Danger-Associated Molecular Pattern molecules, and they represent host molecules that can initiate and perpetuate an innate inflammatory response. Upon binding to TLR2 they initiate a signaling cascade leading to activation of microglia and neuroinflammatory response. These mice were used to image the inflammatory response after stroke, but as well after newly introduced mouse model of intermittent hypoxia. They are the major asset to verify the in vivo biocompatibility of intended therapeutic or diagnostic approach. Therefore there were used to evaluate brain response to stem cell therapies, their combination with biomaterials (alginate in particular), and application of nanoparticles alone or as a cell label.
- Gap43-luc/GFP
Luc and GFP reporters are driven by Gap43 promoter. GAP43 (Growth Associated Protein 43) is expressed at high levels in neuronal growth cones during development and during axonal regeneration, and subsequently being associated with growth and plasticity. It is used to image the brain repair after postischemic lesion. During GlowBrain it was in particular used to develop a novel way of imaging apoptosis by the use of caged luciferin, luciferin bound to DEVD peptide to be cleaved by Caspases 3 and 7, and subsequently released for imaging.
- Nestin-luc/GFP
Luc and GFP reporters are driven by nestin promoter. Nestin is an intermediary filament protein characteristic for neural progenitors. It is expressed during neuronal differentiation, and it serves as a marker for neural stem cells either after transplantation or if endogenous stem cells being activated in brain diseases, e.g. after ischemic lesion.
- Dcx-luc/GFP
Luc and GFP reporters are driven by Dcx promoter. DCX (Doublecortin) is a microtubule-associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures. Its use for imaging is similar as of nestin-luc/GFP mice, as it is as well marker of transplanted or endogenous neural stem cells.
- Tg(CAG-luc,-GFP)L2G85Chco/J
This is ubiquitously expressed luciferase and GFP reporter. The reporter genes are under the control of the chicken beta actin promoter coupled with the cytomegalovirus (CMV) immediate early enhancer (CAG). This mouse line is in particular useful to be a source for stem cells, which can be visualized after transplantation to the brain.
b. The mice carrying fluorescent reporters. The acquired instrument for preclinical optical imaging together with bioluminescence imaging (BLI) is capable as well for fluorescence imaging (FLI). The markers could be visible on histological slides directly or via immunohistochemistry.
- Tg(Thy1-YFP16)Jrs
The yellow fluorescent protein as a reporter is under control of Thy-1 promotor. In this particular line the neurons are exclusively marked, but only some enabling to see the whole neuron with all processes including the dendritic spines. This particular mouse line is currently used as a source for stem cell transplantation in the brain in order to visualize the differentiated neurons in the brain.
- Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J
This is ubiquitous mouse line with Tomato reporter under control of gene trap ROSA promoter. It is used to track stem cells with FLI and histology after transplantation to the brain.
c. Knock-out animals
- Tlr2
This is the knock-out of Tlr2 gene, showed to have different post-stroke development of the events. It is used in the GlowBrain platform as a major model for testing of influences of neuroinflammation after ischemic lesion.
2. Mouse models for human stroke and obstructive sleep apnea
As the mouse model for stroke we use the medial cerebral artery occlusion (MCAO). The model was enhanced by monitoring of the blood flow by laser color doppler, and subsequently the determination of the ischemic lesion by MRI. This enables to exclude animals were the procedure was not correct, or variability is out of proportions. The standardization of MCAO is a major prerequisite for all corresponding experiments.
The mouse model of obstructive sleep apnea was introduced as a specific result of GlowBrain together with our partner from Budapest. The protocol lasts for 21 days, 8 hours per day during the daylight period, in the interval of 90 seconds the mice are in normal (21%) oxygen and in hypoxic (5.7% O2) conditions. To enable this protocol we have designed a patent pending intermittent hypoxia system (GB1602071.1) which is attached to the cage used routinely for the animals, providing the needed fast exchange of gases which are monitored by appropriate sensors.
3. Stem cell culture and brain transplantations
We currently use 3 types of stem cells, 2 from mice: neural stem cells and mesenchymal stem cells, and 1 from the humans: oral mucosa stem cells. The stem cell culture and differentiation procedures are developed in details. The brain transplantations are performed by stereotaxic apparatus. In addition, we are able to transplant cells intravenously and intraarterialy, still these procedures are rarely used.
4. In vitro and in vivo biocompatibility validation of nanoparticles and biomaterials
The setup includes a row of analysis, which includes the physical-chemical characterization, in vitro tests, and in vivo validation. The physico-chemical characterization includes transmission electron microscopy, dynamic light scattering, and electrophoretic light scattering. In vitro tests were made on stem cells and on permanent cell lines (e.g. HEK-23), and they include: cell labeling efficiency testing by flow cytometry, cell viability tests, cell proliferation tests, determination of entry mechanism by transmission electron microscopy, measurements of intracellular reactive oxygen species, measurements of intracellular glutathione levels, measurements of glutathione peroxidase and superoxide dismutase activity, measurements of mitochondrial and cellular membrane potential and COMET essay. In vivo biocompatibility test is based on brain imaging of Tlr2-luc activity, which shows the neuroinflammation after nanoparticle application.
5. In vivo preclinical optical imaging of fluorescent nanoparticles
The distribution of gold nanoparticles labeled by fluoresecent dye was established by in vivo FLI in the mouse brain.
6. In vivo preclinical bioluminescence imaging of the unique mouse models bearing luciferase reporters
The imaging of Tlr2-luc and Gap43-luc were used mainly in the evaluation of the post-ischemic brain consequences. As a unique approach developed through GlowBrain, we introduced imaging of caged luciferin, DEVD luciferin, which was our innovation, being the first to introduce this type of imaging. DEVD luciferin cannot be used by luciferase, and therefore it is referred as caged. After cleavage of DEVD by caspase 3 or 7, the luciferin is released being available for luciferase and imaging. As caspases 3 and 7 are involved in apoptosis, the obtained image represents a subset of luciferase expressing region (e.g. Gap43 expression in our case), which is in the same time caspase positive. Moreover, we have shown that the cells expressing both Gap43 and caspase are not apoptotic, but the co-expression defines the neurons in stress after stroke, which will subsequently either undergo apoptosis or axonal sprouting and recovery.
7. In vivo magnetic resonance imaging of the mouse brain
The magnetic resonance imaging consists of the various modalities and the acquired machine enables the maximum flexibility to apply any imaging protocol to be imagined. This refers to the potential of the machine, which we would use for the next decades. Currently, we use the MRI modalities mostly used in human MRI, T1, T2, and diffusion weighted imaging. To image stroke in mouse, the ischemic lesion is imaged by T2-weighted and T2-maps.
8. Ex vivo magnetic resonance imaging of the anatomical and pathological samples
The ex vivo MRI enables to collect the samples not only from the brain, but as well from other parts of the body and it allows as well for the imaging of human samples. We have currently imaged many different samples as a result of our training. The research-oriented samples were mouse and rat brains, postnatal mouse brain, sheep tissue-engineered cartilage and human breast tissue. The contrast can be enhanced by the use of gadolinium salts.
9. In vivo magnetic resonance imaging of the mouse and rat body
In vivo imaging was used for mouse stroke model using the specific brain coil. The whole body imaging is perforemed by acquired specific coils for mice and for rats.
10. Evaluation of the mouse ischemic lesion consequences and repair using multimodal in vivo imaging.
The multimodal imaging was enabled by specific construction of the holder to be used in both instruments. Although at the very beginning we were told that such a holder exists as commercially available, what we got was not a close to what was needed. Therefore, we engaged in the constructions of our custom made holder, which would enable the heating and monitoring of animals during imaging, being compatible with the acquired mouse monitoring MRI setup. Mouse stroke and labeled stem cells were visualized in both instruments. After imaging in both imaging systems the overlay of images was achieved by general software ImageJ. The multimodal imaging is new emerging research topic, and we expect to develop the various modes and postprocessing approaches of multimodal imaging in our future research projects.
11. Stem cells tracking using in vivo imaging
The labeled stem cells were imaged by bioluminescence, MRI, and X-ray imaging. The stem cells were labeled by magnetic nanoparticles and those cells were imaged on MRI and synchrotron radiation micro computed tomography (Elletra synchrotron in Trieste, Italy). The Gap43-labeled stem cells were imaged by bioluminescence imaging. The response of the brain to stem cells and biomaterials combination was imaged by Tlr2-luc mice.
The proof of concept of GlowBrain was the establishing of the whole procedure of in vivo molecular imaging of transplanted biomaterials-supported stem cells in mouse brain after ischemic injury induced by middle cerebral artery occlusion (MCAO). We have assembled all the parts of this puzzle: we introduced ischemic injury in the brain, we transplanted stem cells and biomaterials, and we imaged living animals both by bioluminescence imaging on Preclinical optical system Perkin Elmer IVIS Spectrum, and by magnetic resonance imaging on Bruker 7 Tesla Biospec 20/70 USR.
Specific objectives necessary to establish the integrated platform were (1) establishing the differentiation, labeling and transplantation of stem cells, (2) development of advanced techniques in application of biomaterials in the brain, (3) visualizing brain and stem cells with BLI, (4) visualizing brain and stem cells with MRI, (5) multimodal imaging BLI/MRI of brain and stem cells, and (6) assessing the brain repair in mouse model of the stroke (MCAO). All these specific objectives were reached. Although reached, the objectives 5 and 6 remain our goals beyond the duration of the GlowBrain project. Multimodal imaging is a novel approach which would be further developed by our team and by other groups, and correlation analysis of the imaging with the brain repair represents one of our major scientific goals to be used by the GlowBrain platform. Therefore we can say that although GlowBrain was completed, GlowBrain platform is just starting by attacking the major research question in the field.
When compared to the background, what was achieved with GlowBrain project is without a doubt a profound change. The GlowBrain had 7 Work Packages. The first one (WP1) was related to the management of the project entitled “Coordination and management”. Although this is not a classical Working Package expected to produce a project results, it actually brought us tangible advancement in how the project should be taken care of and how to manage it. Therefore, corresponding to the WP1 we would discuss foreground category “Organization and management”. The WP2 was “Exchange of know-how and experience” and its major outcome is foreground category “Knowledge”, the WP3 Recruitment of the experienced researchers resulted in the foreground category “Personnel”, and WP4 “Acquisition and upgrading of research equipment” relates to the category “Equipment”. The fifth Work Package (WP5) “Intellectual property and innovation capacity building” relates to foreground category “IP skills”, and WP6 “Dissemination and promotional activities” to newly developed structure related to the foreground category “Responsible research and innovation”. Finally, the Work Package 7 was untypical package “Ex-post Evaluation of UniZAG research potential”, but it generated a document containing future looking elements related to GlowBrain planning, and ideas presented there were incorporated in current planning. It provided us as well the insights toward external evaluation of our future activities. This aspect of the foreground would be included in already presented category “Organization and management”. The categories will not be presented in the order of Work Packages but will follow different order: Equipment, Personnel, IP skills, Responsible research and innovation, Organization and management, and Knowledge.
Equipment:
During GlowBrain project we have bought two preclinical imaging instruments, magnetic resonance imager for in vivo imaging of small laboratory animals, and optical imager for mouse bioluminescence and fluorescence imaging. Moreover, we have upgraded the mouse facility. In synergy of the GlowBrain and its hosting institution University of Zagreb School of Medicine, the institution invested in reconstruction of an old basement where the new laboratory space was prepared. Through this a new laboratory was created, Laboratory for Regenerative Neuroscience or GlowLab. This laboratory consists of GlowBrain Imaging Facility, small animal facility, space for animal experiments, and cell culture. At the end of the project, GlowBrain Imaging Facility is functional, the experiments which include animal imaging are running and animals are imaged on the routine basis.
Personnel:
By carefully organizing GlowBrain financial resources it was possible to employ more people than initially planned. These employees represent the first part of the foreground related to the personnel, which was employed using funds directly provided by GlowBrain. Objective was to employ experienced researchers, if possible, returnees, which would develop the independent stem cells and biomaterials research for brain repair based on enhanced research potential, maintain and run the BLI and MRI equipment and ensure sustainability of introduced research.
All planned personnel were employed. Moreover, after obtaining permission from the Project Officer we employed two additional Postdoctoral scientists and additional Experienced Engineer. This in total meant that we employed 9 researchers: 2 senior scientists, 1 research manager, 4 postdoctoral scientists, and 2 experienced engineers. Together with 9 researchers we employed 1 administrative officer and 1 innovation manager, making together 11 direct GlowBrain employees.
Instead of originally planning 6 researchers, by careful use of resources we could employ 9 researchers (50% more). All of them stayed employed till the end of the second reporting period, and subsequently moved to other positions during the final three months of the GlowBrain. From these researchers 1 senior scientist, 1 research manager and 2 postdoctoral scientists were coming from abroad (4 of total 9; 44%) and 3 of them are Croatian returnees from their positions abroad. As Innovation Manger is as well Croatian returnee, this brings in total 5 scientists from abroad (of 11 total; 45%) employed by the project. From total of 11 employees, 6 (55%) were females.
In relation to sustainability of their positions beyond GlowBrain duration, we were challenged by the fact that it is not at all easy to get the position at our institution. The reasons are related by general cuts in Croatian public sector, specific cuts in relation to the financing of science and cumbersome procedural rules, which prohibit new employments. Despite this negative circumstances we succeeded to assure further employement for 4 employees and we plan to do it for 2 more, Igor Pongrac and Marin Radmilović, which will bring us to 6/6 planned, ie. 100%. It is important to notice that 2 more were not in need to be employed by us, Roland Pochet, which got retired, and Josip Pavičić, who continued with his own business using experiences he got through GlowBrain. In sense of unemployed status after GlowBrain, all of them found new positions, only one postdoctoral scientist from private reasons left to Pakistan, and now searches for job there.
GlowBrain activities involved many more scientists than those employed by the GlowBrain financial resources. Some of these UZSM employees have their status of independent researcher (Assistant Professor status and up) and are in position to guide their own projects (Principal Investigator status). 18 of them were involved in the GlowBrain project, and through their activities they already use GlowBrain platform and they will be first to contribute to sustainability of GlowBrain platform by their results. Moreover, these scientists involved in the GlowBrain activities are as well involved in the management of the institution and they are positioned at various leading positions in the organizational hierarchy of UZSM. This includes the very leading positions of Dean, Vice Deans, Vice Rectors, and Director of Croatian Institute for Brain Research.
Together with those leading scientists of UZSM, GlowBrain influenced as well a row of early stage researchers, besides of those being directly employed by GlowBrain. Some of them were involved in GlowBrain secondments, others were involved in application of gathered knowledge in GlowBrain platform. They were developed within GLowBrain environment and represent a future resource of new GlowBrain-associated researchers. In total, more than 30 scientists builds their careers around GlowBrain platform. They make power and future of GlowBrain foreground.
IP skills:
The foreground resulting from GlowBrain in relation to the management of intellectual property is already recognized at the institutional level and incorporated in its policies. This included setting up of institutional Committee for intellectual property managment, which then made institutional Regulations on intellectual property management. The Regulation was accepted by the Faculty Council (major governing body of institution), and it represents framework to operate in relation to institutional IP. The Committee approved all GlowBrain generated IP-related documents for institutional use. Moreover, there are 8 international IP experts with whom we established a permanent collaboration and we organized 4 IP Workshops.
These advancements already paid off as our institution was recognized at University level to be one of the rare with regulated IP policies, which enabled us to compete for national grants in relation to IP protection and development, and to be awarded 2 of them, being the best institution in this respect.
The upgrade of institutional IP policies includes as well:
- Raising awareness of researchers on generated IP,
- Processing all IP issues efficiently according to set policies in coordination of Technology Transfer Office, Committee for managing the intellectual property, and Faculty authorities,
- Active standpoint toward IP education, where IP related subjects are integral part of all UZSM PhD courses
- Applying for the national and international resources to improve IP management,
- Contributing to the University Master study on IP management,
- Regularly sending UZSM personnel for education at the University Master study at our University and similar workshops abroad.
The major challenge of the IP policies is the collaboration and participation in the activities of industry and SMEs, which could prosper on IP generated in our institution. This would be a next challenge to the institutional Committee to clear the path for joint activities with industry which would bring benefits to both sides.
In relation to IP, GlowBran generated IP which was submitted as a Patent application AN AUTOMATED SYSTEM FOR INDUCING HYPOXIA, GB1602071.1. We are currently exploring licensing possibilities for patent expecting to have a final decision on it in a year.
Responsible research and innovation:
The dissemination activities of GlowBrain were an important part of its activity and we consider them very successful. The following foreground was generated through the dissemination activities:
- GlowBrain brand - GlowBrain as a project acronym was well received and it caught attention. Therefore, and according to the advice of the Ex-Post Evaluators, GlowBrain name will remain beyond project duration. Name will be mainly used in relation to the GlowBrain platform located mainly in the GlowLab. Subsequently GlowBrain web page, Facebook profile and YouTube channel will remain functional in relation to ongoing activities of the GlowBrain platform.
- Regional Network for Regenerative Medicine and Stem Cell Therapies (RegMed) - At the end of GlowBrain activities RegMed was transformed in the professional association Croatian Association for Regenerative Medicine and Stem Cell Therapies (RegMedHr). This assured the sustainability of this professional interdisciplinary network. The plan of RegMedHr activities includes organization of international conferences, which will keep GlowBrain brand in the title.
- The Night when Brains are Glowing - This public activity enabled us to communicate directly with general public about achievements of GlowBrain project and to highlight importance of science for society. This dialogue was highly appreciated and it would be continued. Major issues in continuing this activity are the financial resources. Therefore, new GlowBrain night, scheduled for the end of 2016 / beginning 2017, will be based on public lectures with entrance fees. The lectures will be accompanied with the different media installations and entertainment part in similar way to the previous Nights. In the same way as already organised GlowBrain night was connected to 25 years of Croatian Institute for Brain Research, the next event will be connected to 100 years of UZSM.
Organization and management:
The activities related to organization and management of GlowBrain helped us to grasp management skills necessary for such a complex endeavor. It was very important to synchronize management of project with the institutional management as GlowBrain was dedicated to whole institution. This was successfully achieved and, even more, the institution realized the importance of GlowBrain and invested substantial amount of money in reconstruction and organization of space to host GlowBrain equipment, which is now GlowLab. Although GlowBrain needed to have its own management structure it was always in concordance with the management of Croatian Institute for Brain Research and UZSM. Subsequently at the end of GlowBrain, project management structure was integrated with management structure of the Croatian Institute for Brain Research, and it will function within its organizational bodies. The achieved structure is as follows.
The major organizational units of Croatian Institute for Brain Research (CIBR) are Sections. GlowBrain is represented in 3 Sections of CIBR. Two of these 3 were upgraded by GlowBrain still did not have a major change, therefore they stayed the same:
- Section for Laboratory Animals and
- Section for Electron Microscopy.
The third section was significantly changed. Previously it was
- Section for Neurogenetics, Cytogenetics and Developmental Genetics,
and now it was renamed in the
- Section for Neurogenetics, Medical Genetics and Regenerative Neuroscience.
This Section previously consisted from 2 Laboratories: Laboratory for Neurogenetics and Developmental Genetics, and Laboratory for Cytogenetics. Number and names of laboratories evolved through time and now in order to encompass recent changes it was reorganized to consist of 4 laboratories:
- Laboratory for Neurogenetics and Developmental Genetics,
- Laboratory for Stem Cells,
- Laboratory for Medical Genetics and
- Laboratory for Regenerative Neuroscience – GlowLab.
The management of the GlowBrain was integrated as well within the management structure of the Center of Excellence for Basic, Clinical and Translational Neuroscience. As the experience of Ex-Post Evaluation was positive, we plane to engage external experts in a form of different types of advisory boards. Croatian Institute for Brain Research already has Advisory Board and its membership was renewed at the end of 2015. Two representatives of GlowBrain Special Partnering Organisation, Laval University in Quebec, Canada are now members of the CIBR Advisory Board, prof. Jasna Križ and prof. Jean Pierre Julien. GlowBrain gathered through WP5 acitivities the 8 IP experts who become our permanent experts in the IP field, and finally GlowBrain platform will form External Advisory Board beyond duration of GlowBrain project.
Knowledge:
Knowledge is the most important foreground category resulting from the GlowBrain. Here some aspects of this foreground in relation of the GlowBrain results will be described.
The Exchange of Know-How with our partners
The major tool for knowledge exchange were secondments to and from our partners. This was very active part of GlowBrain and we organized 60 secondements (38.16 person months) to our partners and 35 secondements (9.01 person months) to our institution.
Development and exchange of knowledge in stem cell cultures took place during 32 secondments performed by following researchers: Lada Brkić Ahmed (6 visits to Lund), Marcell Bago (6 visits from Budapest to Zagreb), Dora Polšek (8 visits to Budapest), Marija Renić (11 visits to Salzburg) and Julila Marschallinger (one visit from Salzburg to Zagreb based on that topic). Lada Brkić Ahmed mastered isolation and analyses of cell fates in Lund partner laboratory, which is one of the most recognized European centers for stem cells and cell engineering. She was responsible for transfer of several methods of cell isolation and manipulation with different cell types, in particular stem cells and microglia. Dora Polšek and Marija Renić worked on stem cells in ischemic conditions and other cell subtypes, including microglia and stem cells from adult animals. Complementary approaches from three different laboratories (combined with already existing experience in Zagreb) yielded 10 standard operating procedures (SOPs) covering range from isolation of stem cells to different types of procedures based on stem cells. Budapest partner was regularly sending one scientist (Marcell Bago) to learn hypoxia in vivo methods from Zagreb coordinator and to work on further development of this technique. In addition, Julila Marschallinger from Salzburg was working on exchange of experience in isolation of neural stem cells. As an added value, secondments between Zagreb and Budapest resulted in patent application for chamber with controlled set of ischemic conditions while secondments between Zagreb and Salzburg resulted in a common article published in Nature Communication. Another added value is visible in parallel development of cultures originating from human oral mucosa in collaboration with the University of Zagreb School of Dentistry, which was in last year awarded two grants by Zagreb University.
Stem cell labeling and genetic modifications have been mastered during 11 secondments performed by following researchers: Marija Lovrić (2 visits to Prague), Lada Brkić Ahmed (6 visits to Lund), Uliana Kostiv (2 visits from Prague to Zagreb) and Somayyeh Hamzei (one visit from Cologne to Zagreb). During her first secondment (6 weeks in Prague) Marija Lovrić learned several techniques of stem cell labeling based on iron particles and their analyses based on spectroscopy, light scattering, atomic force microscopy and thermo-gravimetric analyses. During the second secondment she learned techniques of labeling stem cells with magnetic nanoparticles specifically prepared for MRI. Prague partner has sent dr. Uliana Kostiv two times to develop further methods of stem cell labeling in Zagreb environment which yielded several improvements of the procedure. This method is becoming recognized as one of the most important specific expertises based on stem cell labeling for magnetic resonance imaging. Cologne partner institution has sent Somayyeh Hamzei to help to develop new stem cell labeling techniques based on immunohistochemistry. During her 6 months stay in Lund, dr. Lada Brkić Ahmed mastered several methods of genetic manipulation with stem cells, which will be further utilized in combination with already existing protocols for cell transfection. One of the main outcomes of these secondments was newly established platform for stem cells labeling by nanoparticles and manipulation by cells using genetic modifications. This platform is now utilized by several groups in Zagreb. Thus one of the most prominent added values is ability of GlowBrain platform to test newly synthetized particles. Moreover, experience gathered in stem cell labeling has been successfully used during research for two PhD thesis which were finished in the second half of 2015 (Nina Kosi used labeled stem cells to trace them in the brain affected by stroke and Ivan Alić used labeled and genetically modified stem cells to analyze their differentiation after stroke).
Synthesis of innovative materials and their application have been mastered during 7 secondments performed by Marija Lovrić (2 visits to Minho), Lejla Ferhatović Hamzić (2 visits to Minho) and Marta Skelin (3 visits to Minho). Each of researchers has spent 6-12 weeks at University of Minho (and 3Bs research institution associated with University of Minho), which are one of the most recognized European centers for synthesis and application of biomaterials. During these secondments they learnt techniques of synthesis of various materials: methacrylated gums, hydrogels, modified hydrogels, polymers of various types, hydrogels in moulds, cell encapsulation by hydrogels and some others. In addition, Marta Skelin specifically learnt methods of production of extracellular matrix scaffolds and placental scaffolds. Some researchers have participated in advanced workshops dedicated to application of materials for stem cell cultivation (e.g. Lada Brkić Ahmed and Igor Pongrac participated in Cost NAMABIO action organized workshop in Cyprus).
The main outcome of these activities were experiments which combined biomaterials and stem cells in Zagreb (performed by Igor Pongrac and group of Simona Casarosa, University of Trento).
Visualizing brain and stem cells with BLI have been mastered by following researchers: Marija Renić (3 secondments performed in Salzburg partner institution) and Lada Brkić Ahmed (advanced level workshop organized by Perkin Elmer in Bruxelles). Since the instrument has arrived in the middle of the project, many trainings have been performed in Zagreb, including partner institution: Siniša Škokić was hosting both basic and advanced training level Perkin Elmer workshops for all interested researchers in Zagreb. Mariana Carvalho from partner institution Minho spent one month in Zagreb testing various concentrations of nanoparticles and dyes for tracing stem cells in brain using BLI. Adrian Manescu from Ancona partner institution visited Zagreb 11 times during project duration: majority of these secondments have been based on discussion and further development of imaging techniques for visualization of brain and stem cells. One of the outcomes of these trainings were SOPs for various procedures based on BLI. As a clear added value it can be listed that expertise in micro CT and other modalities coming from dr. Manescu was combined with BLI and MRI in Zagreb which resulted in the project proposal: “Marked stem cells tracking in mice ischemic brains by synchrotron radiation micro computed tomography”.
Visualizing brain and stem cells with MRI was covered by 13 secondments performed by the experienced engineer dr. Siniša Škokić who was hired to focus on installation and running of MRI. They included several levels of introductory trainings, safety and security trainings, operation with Bruker software, scan enhancements in different imaging modalities, animal handling, anesthesia control, monitoring of physiological parameters and image post-processing. Partners were as well involved in this exchange of know-how, so they visited Zagreb several times and helped in fine tuning of the instrument. Marina Dobrivojević spent two secondments in Cologne learning to work with animal models for MRI and basics of running of MR instrument. Exchanging of this knowledge also included other groups: dr. Mazzoni from University of Trieste used two visits to learn method of MRI from Zagreb group. Dr. Manescu from Ancona has visited Zagreb facility 11 times. Some of these visits have been based on learning MRI.
Use of small animals as disease models in neuroscience was covered in many secondments performed by both coordinator and partners. Specific requirements for animal facility and MRI was mastered by dr. Škokić, who collected experience from Cologne partner and used it for design of the new Zagreb facility linked to MRI. Dora Polšek and Lada Brkić Ahmed exchanged knowledge and know how in manipulation with different transgenic mouse lines used in neuroscience research.
One of the most notable results of these secondments was established collaboration between Zagreb and Budapest (performed by Dora Polšek from Zagreb and Marcell Bago from Budapest side) which resulted in development of a special chamber for intermittent hypoxia. This chamber is now in process of patent pending. Dunja Gorup has spent one month in Milan participating Young Investigator Training Program where she mastered some new advances in imaging methods and behavior studies. Several visits of partners to Zagreb were used to work with animal models present in Zagreb. Budapest (6 secondments), Salzburg (10 secondments of 6 people) and Cologne (Luam Hammelrath) partners were sending PhD students to exchange experience with personel involved in work with our models in Zagreb. Marcell Bago from Budapest was involved in developing intermittent ischemia model, while Salzburg group had 6 researchers involved in learning various histology methods, including those ones for electron microscopy and work with human tissue. In addition, two partner groups have been involved in learning MCAO stroke model from Zagreb coordinator group (dr. Casarosa group from Trento and Joanna Adamczak from Cologne). An added value in this section was obtained by 9 secondments of Marina Dobrivojević (visit to partner in Ancona and Trieste) who was working on application of X ray microtomography on imaging of mouse brain and tracing of transplanted stem cells in the brain.
Standardized procedures:
During GlowBrain we have organized and documented many standardized procedures (e.g. standardized operating procedures, SOPs). Although before GlowBrain we had the habit and prepared SOPs, GlowBrain increased complexity of our research procedures. New equipment and new approaches brought us to the frontier where we should search for our own approaches. Subsequently we can use many different approaches to address our question, and one of our research questions is to find out which is the most appropriate way. For example, if we want to use MRI to characterize the stroke, at first it seems very easy, there is a stroke, and we should measure the volume of the stroke area in the affected brain. Still there are many variables which can influence our measurement, at least 3 ways how to perform major stroke mouse model, middle cerebral artery occlusion, then the time of ischemia, the time of imaging, modality of imaging, etc. Consequently, which SOP one would use for MRI to evaluate the stroke requires much of evaluation to prove which approach will be the most relevant to our scientific question.
Although the SOPs can be considered as a basic step, easily learned from our partners or from the other laboratories with more experience, we are considering this as a major challenge for future, which if solved appropriately will answer research question or if research question could not be solved, it will at least be replied with relevant answer. In conclusion, SOPs represent a very important foreground of GlowBrain and their further development will be the major aspect of our future activities.
The knowledge was gathered and exchanged, the collaborations were developed and fostered at 5 scientific events organized by GlowBrain. These are:
- Final conference “Stem cell and biomaterial applications for brain repair" at on 27.-29.5.2015.
- GlowBrain 1st Workshop "Stem Cell Techniques" on 23-25.05.2013.
- GlowBrain 2nd Workshop "Application of biomaterials and in vivo imaging in stem cell research" on 27-29.03.2014.
- GlowBrain 3rd Workshop "Visualization of molecular markers in the brain" on 29.-31.1.2015.
- GlowBrain 4th Workshop "Preclinical In Vivo Optical Imaging Regional Meeting" on 5.11.2015.
- GlowBrain 5th Workshop "Biomedical Imaging Meeting" on 18.12.2015.
These scientific events served as well educational purposes as they included the hands-on demonstrations of the new procedures. GlowBrain is as well actively involved in teaching activities at our institution, which includes the MD and PhD studies, in particular the PhD studies in Neuroscience. In sense of GlowBrain collaborations efforts a comprehensive table of research topics and developed collaborations was provided in deliverable D6.5 Exploitation plan.
In conclusion, GlowBrain platform is versatile and would be used for different purposes adapted to serve the research topics of scientists from and outside of our institution. Still the major purpose for GlowBrain platform will be core GlowBrain activity, testing the combination of stem cells and biomaterials for stroke, developing further the major research line of the GlowBrain. The introduced GlowBrain platform will enable the enhanced level of research within European research area, opening paths in Croatia in the novel scientific fields:
- Regenerative Medicine (due to introduction of therapeutic strategies involving brain repair and regeneration),
- Stem Cell Therapies (stem cells being core activity of GlowBrain research),
- Nanomedicine (through integration of nanoparticle applications in the brain),
- Tissue Engineering (due to combination of biomaterials and stem cell research),
- Molecular Imaging (through visualization of disease molecular markers in tissues and animals).
Potential Impact:
GlowBrain impact
Whenever GlowBrain was presented to public, one of topics was to explain that investment in science is important in Europe and in particular in Croatia. Taking considerable attention due to its dissemination activities, and being one of the biggest FP7 project granted to Croatia, GlowBrain was in position to exert some influences on general public opinion. Therefore, the major argument raised was that the money received for GlowBrain was for the benefit of European and Croatian society. Investing in science is vital for society, and specific explaining why particular research financed through GlowBrain was important. Current economy is knowledge-driven economy and relies on science producing new knowledge. Subsequently, society lacking its own knowledge-producing activity has actually no chance to develop its economy, as it has no fuel that economy needs – the knowledge.
GlowBrain conveyed the following general messages:
- Science is vital for knowledge-based economy,
- In our effort to converge to the best we need to complement the traditional research approaches with the new technologies (GlowBrain contributed by new molecular imaging technologies),
- In order to be needed at the European level we have to find our own complementary niche, which would attract the partnerships in producing knowledge, but as well applying the knowledge (GlowBrain contributed by niche of in vivo multimodal imaging of stem cells after stroke),
- As Croatia is very small country only by joining our efforts together we could use our resources (knowledge, equipment, finances),
- Showing its success GlowBrain shared good news that there is no limit to achieve given goals. We made specifically desinged T-shirts to convey the good news. T-shirts were given away during our public events organized during Workshops, Final Conference and 2 public events organized, 2 Nights when brains are glowing. The message on the T-shirt was “Yes, we scan!” due to the fact we installed two scanners, MRI and optical scanner, and Yes, we scan! indeed with them,
- The fact that GlowBrain employed young scientists, some of them returning from abroad, was always highlighted. Science gives jobs, and it reverses brain drain into brain gain. The young scientists who went abroad are among best educated people of Croatia, therefore having them back home is a huge benefit for Croatia.
As stated above we always provoked debate about justification and a need to spend for science, and subsequently about GlowBrain imapct. As a specific challenge we used the most expensive instrument we acquired, magnetic resonance imager, which costed roughly 1.5 million Euros. Magnetic resonance imager is indeed expensive and most of these instruments are used for human diagnostic. Instead to buy a human scanner which would be used to diagnose sick people in need, we acquired scanner to put mice and rats in it and perform research activity.
The reason is a need for new therapeutic approaches to help sick people. Our major target was stroke, which is a major health problem, being first cause of disabilities and second cause of death. The stroke has no efficient therapy right now, hence there is an important need for advancement in this field. In order to create new therapies, experimental animals and preclinical experiments are essential tools, which subsequently lead to the clinical trials on humans.
As its major result, GlowBrain organized a preclinical platform to test new therapies for brain, in particular for the stroke. Moreover, preclinical platform was organized similarly to clinical setting. After we induce stroke in mice, animals are monitored alive to evaluate effects of given therapies. The MRI machine is critical for this step, providing the fine morphology of the brain, extent of stroke affected area, and post-stroke recovery. Moreover, the labeled stem cells when transplanted to the brain can be followed by MRI in living animal and consequences of introduced stem cell therapies can be evaluated. By helping mice after experimental stroke, we are able to contribute to future stroke therapies. While human scanner helps those people which are diagnosed with its help, animal MRI has potential to help many more humans in the future. GlowBrain platform is a comprehensive and necessary preclinical setup designed to contribute to the new therapies, for the brain diseases, and in particular for the stroke.
GlowBrain contributed to Croatian science to provide a necessary link of preclinical research on the brain. Neuroscience is recognized as one of the strongest research areas in Croatia, exemplifying by Center of Excellence in Basic, Clinical and Translational Neuroscience, and the current platform enables translational and applied biomedical research on brain diseases. It contributes as well to major priority of the biomedical research, which we refer as a path of drug development. The drug development path starts in basic science research, continues to preclinical research, translates to clinic, and finally after clinical trials it ends by approved use in patients. This path is long and cumbersome, but if a country and its research community can connect it together, it represents an enormous asset, and GlowBrain platform serves in Croatia to be a necessary node on this path.
The argument important for GlowBrain societal implications is specific economic power of the preclinical biomedical research. The health care industry increases its GDP share, being estimated to be 19.6% of US GDP in 2016. Therefore, preclinical research of GlowBrain contributes to increasingly important field of economic activity. It is unique as well by the fact that not only it enables economic benefits, but in the same time it gives health to the people, so compared to other economic activities it has a double effect on society.
GlowBrain specific societal impact for Europe is:
- Introducing a complementary niche of multimodal imaging of the mouse brain necessary for European Research Area,
- Counteracting 2-speed Europe,
- Contributing to preclinical stroke research, providing a platform which uses unique mouse models not present in other laboratories,
- It developed a medically relelvant innovation, a patent-pending mouse model for human disease - obstructive sleep apnea.
GlowBrain specific societal impact for Croatia is:
- Changing the perspective that high-technological science is necessary and possible in Croatia,
- Highlighting the need of preclinical research for medical applications,
- Contributing to drug development path in Croatia,
- Contributing to necessary upgrade of IP capabilities in the biomedical field
- Reversing brain drain in brain gain,
- Integrating research activities in institution, in country, and in European Research Area,
- Raising awareness of vital role of science in society.
Dissemination activities
The dissemination activities of GlowBrain were numerous and they were performed on professional and general public level throughout the duration of GlowBrain.
We would just mention some highlights:
- Srecko Gajovic, GlowBrain coordinator was for 2012 chosen among 100 people that changed Croatia to better by one of major daily Croatian newspaper, Jutarnji list,
- Facebook profile of GlowBrain, https://www.facebook.com/GlowBrainCroatia/ follows 1403 people, the profile is in Croatian (Croatia has a bit less than 4.3 million inhabitants, the total number of Croats in the world is estimated to 8 million),
- At inauguration of Horizon2020 programme in Croatia, GlowBrain was the only project invited for presentation,
- In 2015, Srecko Gajovic gave TEDx talk with topic “Why our brains are glowing?”, https://www.youtube.com/watch?v=4jfHBuEbPZc
GlowBrain was recognized in Croatia as EU scientific project receiving the most amount of money, which was only temporary as later other REGPOT project was awarded higher amount. Still the perception did not change as the other project did not make the awareness on this fact (actually, we made their job, answering that we are not the first anymore).
As a fact sheet on March 30, 2016:
- 74 congress abstracts were made
- 13 scientific publications
- 7 PhD thesis
- 16 public lectures
- 32 published text in newspapers and journals
- 8 radio appearances
- 17 TV appearances
- 13514 web page visits
- 1384 Facebook likes
- 1190 YouTube channel views
- 3 workshops organized
- 2 public events hosted by GlowBrain
The GlowBrain organized two public events “The night when brains are glowing” for policy makers and political establishment with participation of selected celebrities, general public and extensive media coverage.
The first Night was organized on September 20, 2013. at Lauba Gallery in Zagreb. It showed a special movie about the GlowBrain, the participants were invited to make their smartphones to “glow” through special android application, and a Millenium Photograph with “glowing” mobile phones was made. The event was attended and greeted with Vice president of Croatian Parliament and Croatian member of European Parliament among others. Croatian singer Massimo Savic and actor Goran Grgic participated in program. GlowBrain activites were presented on so called SmartSquare by GlowBrain members holding tablets with special presentations. The timing of meeting was when two national congresses were held, Croatian Neuroscience Congress and Croatian Pharmacology Congress. This enabled Congress participants, general public and colleagues researchers to attend and mix together.
The second Public Event “The night when brains are glowing” was organized on 14th September 2015 at the premises of University of Zagreb School of Medicine, in front of the Laboratory for Regenerative Neuroscience (GlowLab), Šalata 11, Zagreb. Public Event was organized as a part of the celebration of 25 years of Croatian Institute for Brain research. It was as well a grand opening of GLowBrain acquired instrument for pre-clinical imaging of small animals by magnetic resonance, Bruker BioSpec 70/20 USR, as a GlowBrain major acquisition.
Guests were welcomed at Smart Square in front of GlowLab by GlowBrain team and students, who presented GlowBrain project. The visitors registered at the entrance, and were offered promotional materials prepared for them: abstract books, brochure, pencils, foders, t-shirts, puzzles, all with EU logo. Plaster brain models and brain puzzles were prepared in order to explain to the visitors the purpose of GlowBrain project. Together with Prof. Gajović, GlowBrain coordinator, the speakers were: on behalf of the President of Republic of Croatia, academician Željko Reiner, Vice-President of Croatian Parliament; academician Zvonko Kusić, President of Croatian Academy of Sciences and Arts; professor Miloš Judaš, Vice-Rector for science, inter-institutional and international cooperation of University of Zagreb; academician Davor Miličić, Dean of the UZSM; academician Ivica Kostović, Honorary Director of CIBR; and Lovorka Grgurević, Head of the Department of Anatomy and Clinical Anatomy. A tour around a newly renovated Laboratory for Regenerative Neuroscience (GlowLab) and MRI Bruker BioSpec 70/20 USR, first such device in this part of Europe, funded by GlowBrain was presented to the visitors.
After GlowLab tour visitors enjoyed entertainment and program of second public event „Night when brains are glowing“, including Croatian singer Dražen Žanko, jazz quartet The Brainiacs and electronic music DJ. Finally, documentary movie about reconstruction of the Laboratory for regenerative neuroscience was projected, and common millennium photograph, with specific GlowBrain android application to light up smartphones and create illusion of glowing brains, was made.
In conclusion, by diverse and comprehensive dissemination activities, GlowBrain awareness was achieved in both professional community and general public.
Exploitation of results
The GlowBrain platform represents a flagship of new interdisciplinary technologies aimed to upgrade the medicine. These interdisciplinary technologies include:
- Regenerative Medicine (due to introduction of therapeutic strategies involving brain repair and regeneration)
- Stem Cell Therapies (stem cells being the core activity of the GlowBrain research)
- Nanomedicine (through integration of nanoparticle applications in the brain)
- Tissue Engineering (due to the combination of biomaterials and stem cell research)
- Molecular Imaging (through visualization of disease molecular markers in the animals and tissues)
The GlowBrain and its activities were highly transdisciplinary. The newly created platform is not only dedicated to research in neuroscience, but it represents a versatile tool opened to various research approaches. Its exploitation not only fosters the connection of different disciplines, which can benefit from the platform, but as well depends on their mutual collaboration:
- Mathematicians (providing mathematical models for image analysis)
- Physicists (imaging and postprocessing of images)
- Chemists (synthesis and characterization of nanoparticles and macromolecular biomaterials)
- Biologists (biological characterization of the in vitro and in vivo disease models)
- Medical doctors (providing animal models for disease and translational component)
- Electrical Engineers (being involved in complex imaging and experimental technologies, e.g. image collection and interpretation)
- Mechanical Engineers (providing interfaces between animals and imaging equipment)
- IT specialists (collecting and analyzing plethora of data generated by the platform).
If we consider only GlowBrain employees, they included electrical and mechanical engineers, chemists, biologists, biotechnologists, and medical doctors. All the above disciplines have already been gathered around the platform and attracted to dedicate at least part of their activities to it. This was as well psychologically connected to positive attitude of GlowBrain message of opened possibilities and collaborative success.
The platform exploitation efforts and its sustainability depend mainly on its financial sustainability. This part represents the most difficult challenge for the platform as we are developing not only interdependence of research interests, but as well the interdependence of financial resources, enabling the resources flow through the GlowBrain platform to create financial gain across the network, which would be used for platform sustainability, but as well its further development.
GlowBrain scientific results and research potential was already realized in the row of new projects awarded to the UZSM and related to the GlowBrain, the most of them ongoing beyond the end of the GlowBrain.
GlowBrain related grants are:
1. H2020 “BIOengineered grafts for Cartilage Healing In Patients – BioChip” (H2020-PHC-2015-single-stage_RTD Personalising health and care; 2015-2018, Partner – Srećko Gajović)
2. ERC = ScienceSquared (ERC-SUPPORT-2014: ERC Support for novel ways to highlight the work funded by the ERC and reach out a wider public; 2015-2018, Partner – Srećko Gajović)
3. H2020 Coordination Action in support of the sustainability and globalisation of the Joint Programming Initiative on Neurodegenerative Diseases — JPsustaiND (H2020-HCO-2015; 2015-2019, Partner – Dinko Mitrečić)
4. Structural Fund – European Social Fund project – Upgrade of young investigator competencies in the technologies of regenerative medicine in the brain diseases - Young Brain (HR.3.2.01; 2015-2016, leader Dinko Mitrečić)
5. Bilateral Grant Croatia – Serbia, financed by Croatian government – Stem cell application in the experimental treatments of amyothrophic lateral sclerosis (leader Dinko Mitrečić)
6. University of Zagreb Project – Gap43 gene function in stem cell differentiation and their response to stroke (leader Dinko Mitrečić)
7. University of Zagreb Project – Isolation, characterisation and differentiation potential of oral mucosa stem cells (partner Dinko Mitrečić)
8. COST Action CM1403 - The European upconversion network - from the design of photon-upconverting nanomaterials to biomedical applications (MC member Srećko Gajović)
9. COST Action CA15214 - An integrative action for multidisciplinary studies on cellular structural networks. (MC member Srećko Gajović)
10. COST Action CA15124 - A new Network of European BioImage Analysts to advance life science imaging (NEUBIAS) (MC member Dinko Mitrečić)
11. Italy Elettra grant 20135435 - Ischemic brain analysis by phase-contrast micro computed tomography (partner M. Dobrivojevic)
12. Italy Elettra grant 20140286 - Marked stem cells tracking in mice ischemic brains by microCT (partner M. Dobrivojevic)
13. Italy Elettra grant 20155224 - Marked stem cells tracking in mice ischemic brains by synchrotron radiation micro computed tomography (partner M. Dobrivojevic)
14. Center for Excellence in Basic, Clinical and Translational Neuroscience (whole GlowBrain platform included, awarded to UZSM)
15. Center for Excellence in Reproductive and Regenerative Medicine (awarded to UZSM)
16. Center for Excellence in Bioethics (awarded to University of Zagreb School of Philosophy, Partner Srećko Gajović)
17. Croatian Sceince Foundation Fellowship for Ph.D. student Marta Skelin (mentor Marija Ćurlin)
18. Croatian Science Foundation Fellowship for Ph.D. student Nikola Habek (mentor Aleksandra Sinđić)
The major financial contribution important for sustainability of the GlowBrain platform is assured already from first 4 projects on the list.
The above described projects, although rather diverse, have a clear connection to GlowBrain. They are a major result of perception by GlowBrain professional environment that GlowBrain platform is bringing something new to community and that equipment, knowledge and skills acquired are important. BioChip shares with GlowBrain topic of regenerative medicine applied to cartilage, and GlowBrain with experiences in developing new technologies and management of EU project was an excellent base for this proposal. Science Squared is a result of extensive public promotion of the project, and experiences of these public promotions have been used to approach this collaborative project and win the position in successful consortium. JPsustaiND represents recognition of GlowBrain at European Neuroscience level. Croatia as a partner in this project was given a task to motivate and engage Central East European neuroscientists to be active in JPND (Joint Programme for Neurodegenerative Diseases). Finally, structural project Young Brain was currently only structural call offered for individual scientists to apply. This does not mean no structural funds were awarded, but they have been decided at the level of universities or research institutes. GlowBrain was involved as well in institutional applications to structural funds and in this way GlowBrain platform is directly included in one Center of Excellence, and his participants in 2 other Centers of Excellence. According to information from Ministry of Science roughly 30 million of Euros will be available for Centers of Excellence in 2016.
GlowBrain platform has unique features indicating its commercial potential. These unique features are:
1. Patent pending system for intermittent hypoxia in mouse,
2. Unique transgenic mouse models with molecular markers for brain inflammation and repair,
3. Unique combination of instruments and organization dedicated for brain research – GlowBrain platform located in the GlowLab,
These features can be organized to offer unique services, which could be competitive at European and global level.
Recently, many tested compounds, candidates for drugs for neurological diseases, were shown to be beneficial in preclinical settings, but they subsequently failed in clinical trials. Not only there was no cure found, but as well this was connected to the financial losses as both preclinical and clinical trials were very expensive. Finally, search for cure did not get more intensive, but pharmaceutical companies started to avoid neuroscience in order to prevent losses. This could be improved by investing more in basic research, and by better understanding of brain diseases enabling the design of better therapies. Crucial moment to develop efficient therapy is validation whether the promising preclinical study would be beneficial in clinical trials. Our GlowBrain services should contribute to this by in vivo tracking of molecular markers and events in the brain, which would allow testing of promising compounds in the living brain on unique mouse models using patented disease models.
The above described rationale is base for contract research to be offered to pharma companies. Moreover, we as well have mastered many techniques, which are not unique but valuable, which could be included as a part of portfolio. In order to achieve this goal of contract research services, there is still a long way to go, mainly in organizing the controllable, standardized and validated procedures to be offered on the market.
The use of GlowBrain platform for contract research will rely on new innovation and IP policies introduced at institutional level by improving innovation management of GlowBrain. Still there is uncertainty in perception of a commercial activities on university as public institution. Therefore, we would suggest a model or privately owned spin off, which would license technology, rent equipment and space of GlowLab in order to offer contract research. Still other possibilities are as well opened.
The exploitation of results oriented toward professional community is directed to open possibilities and to support innovations.
GlowBrain platform is an ideal platform to boost innovations. It installed the new technologies and new directions in order to reach a new way to attack major health issues. Moreover, it trained young people to be open minded and innovation prone. GlowBrain orientation to early stage researchers was already confirmed by first Structural Fund we obtained, which is dedicated to the young people “Young Brain”. The continuous activity of GlowBrain beyond the project duration is based on support to innovation and freedom of ideas. This is a message we share among the wide scientific community – if one has a research idea, GlowBrain is a versatile platform to welcome it and make it real, either within the platform or as an example to help others to go their own way. In conclusion, GlowBrain serves as an innovation platform supporting innovation of others, which in return will support the sustainability of the GlowBrain.
To exploit the full potential of platform, we intend to further develop the mechanisms to measure and to predict impact of activities The impact measurement methodology is still to be defined at the European level, and we would by our activities contribute to this effort.
The societal GlowBrain potential toward general public is currently formalized by our activities within international Navigating Knowledge Landscape Network (NKLN). NKLN was founded in Zagreb at the meeting of RegMed network and the founders are Anna Lydia Svalastog (Norway), Joachim Allgaier (Austria), Lucia Martinelli (Italy) and Srećko Gajović (Coratia). The Navigating Knowledge Landscapes Network is an international and interdisciplinary group with an explorative interest in area of online and offline communication concerning health and biomedical knowledge. The aim of the Navigating Knowledge Landscapes Network is to explore and describe how individuals navigate through landscapes of health-related knowledge in digital society. The reason for NKLN, i.e. to explore navigation in an increasing amount of digital information, is an urgent need to empower the patients and other stakeholders to make better informed health-related decisions.
Moroever, within the spectrum of GlowBrain activities Regional Network for Stem Cell Therapies was concieved as a body covering diverse activities in professional community and in society. Already at the beginning, at the first meeting of the Network, the name was modified to this comprehensive task and the new name was Regional Network for Regenerative Medicine and Stem Cell Therapies (RegMed). We succeeded to include in network basic scientists, clinicians, representatives of the industry, business angels, lawyers, patent attorneys, phylosophers, bioethicians and theologists. Approach diversity indeed corresponded to complexity of GlowBrain approach in establishing the new medical technology. In December 2015 the network was formalized as an association referred as Croatian Association for Regenerative Medicine and Stem Cell Therapy (RegMedHr). The association is not only an important result of GlowBrain activities, but as well it serves as support for further sustainability of GlowBrain and wide base of Exploitation Plan for future use of the GlowBrain platform.
In conclusion, GlowBrain has already made significant advances in sustaining its activities beyond the project duration. These activities will assure the exploitation of newly created GlowBrain platform. This is achieved by already ongoing new international (H2020) projects, increasing the regional potential by being actively engaged in synergy with structural funds, and through network of collaboration attracting potential users of GlowBrain platform. Economic potential of GlowBrain should be realized by the unique services to be offered to industry partners, and its societal potential accomplished by supporting innovation and clarifying its impact for society benefits.
List of Websites:
The project website is http://glowbrain.hiim.hr/.
The Facebook profile is https://www.facebook.com/GlowBrainCroatia.
The YouTube movies are http://www.youtube.com/results?search_query=glowbrain.
The official project email address is glowbrain@hiim.hr.
