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Periodic Report Summary 1 - BRAINPATH (Molecular Imaging of Brain Pathophysiology)

WP1: AD and inflammation: The goal of this WP is to study the interaction between soluble amyloid, inflammation and synaptic loss in AD. We were able to create a 3D stack from sequential 2D histological images which was then co-registered to a 3D anatomical MRI scan. Currently we are working on optimizing the co-registration of different histological stainings to the 3D stack of MBP stained images, as well as the co-registration of different MRI modalities to the 3D anatomical MRI scan. (Terzopoulos et al 2015). Resting State fMRI (rsfMRI) has been proposed in both preclinical model of AD and in clinical investigations as potential biomarker of early-stage neurodegeneration as it probes for synaptic connectivity which is thought to be affected early-on by the neurotoxic soluble amyloid. the data obtained support the further research of rsfMRI in mice models of AD to detect and monitor early stage AD pathology. Combined, the results suggest rsfMRI may possibly be used as a non-invasive read-out tool to detect alterations of neurotransmitter systems induced by pathology or treatment.

WP2: Stroke – brain lesions and the role of stem cells and inflammation. The goal of WP2 is to understand better the interrelationship between stem cell grafts and inflammatory activity following cerebral lesions for the purpose of developing a preclinical, robust regenerative therapy for stroke. Cell number-dependent survival of engrafted radial glia-like NSCs could not be verified. Because analysis of the data revealed that photon emission was decreasing for all cell numbers over time and, based on the slope of the graph, that engrafting high numbers of NSC into the brain of mice does not result in a better survival than engrafting smaller numbers. Based on histology afterwards is concluded that stem cell engraftment results in a local inflammatory response. We are currently establishing non-invasive imaging methods to further investigate this post engraftment inflammation. Successful in vitro labelling of murine monocytes/macrophages and their in vivo detectability in the murine brain. Establishment of a protocol to selectively observe macrophages and analyze their distinct contribution. We could detect accumulating microglia/macrophages surrounding and inside the stem cell graft. Theses microglia/macrophages appear as amoeboid or intermediate cells with round cell bodies and short processes indicating an activated state. In comparison, microglia/macrophages at the contralateral hemisphere show ramified morphology with small cell bodies and long branching processes. Hence, stem cell engraftment results in a local inflammatory response. We are currently establishing non-invasive imaging methods to further investigate this post engraftment inflammation. The development and building of the multimodality animal bed for MRI and optical imaging (OI) with Medres GmbH, a new animal cradle was designed from material compatible for use in a high-field MRI system and optimised for use in the optical imaging system from Perkin-Elmer, the IVIS Spectrum. This multimodal animal cradle type has successfully been tested and validated together with Medres GmbH. It is now ready to be entered into longitudinal in vivo imaging protocols.

WP3: GBM and the immune response. The goal of this WP is to test a potential new combination therapy for a type of aggressive and malignant brain tumour. After loading the MSCs with nanoparticles their tumour-tropic properties are still intact and the loading did not affect the tumour-tropic migratory attributes of the MSCs. Demonstration of a significant cytotoxic effect of doxorubicin-loaded MSCs when co-cultered with glioma cells in vitro. However, it is also revealed that the cytotoxic effect of Doxo-loaded mMSCs is temporary and has to be calibrated in vivo to achieve a good synergy with the combined immunotherapy proposed. New reporter proteins for enhanced optical and opto-acoustic imaging in the brain were developed and evaluated. For this a Luc2 gene has been fused to near infrared proteins iRFP670 and iRFP720 that have optoacustic potential. We generated lentiviral vector carrying the fusion proteins and successfully transduced mouse MSCs and also human MSCs.

WP4: TBI and repair. The goal of this work package is to combine imaging technologies, such as MRI together with optical imaging, to ensure that the cell implantation is accurate, to determine that no ill effects follow grafting and to understand the mechanism of recovery. Two NIRF cyanine dyes, HQ5 and IRDye 800CW, respectively, were identified that possess necrosis avidity both in vitro and in vivo in a mouse model. Transduction with the Luc2-copGFP reporter gene construct did not influence senescence, proliferation and doubling time of the HFBSCs. Green fluorescence of copGFP was immediately visible after transduction and persisted for at least 15 passages. Luc2 expression was concomitant with copGFP expression. Additional loading with red-fluorescent TMSR50 nanoparticles did not affect cellular viability, as compared to non-loaded cells.

WP5: Migraine – Stroke, Migraine-Related Hyperexcitability as a Risk Factor for Stroke Evolution. The goal of this work package is to identify, and intervene with, the underlying molecular and neurobiological mechanisms by which migraine influences stroke susceptibility and the evolution of brain damage after stroke. Validated protocols for mouse MCAo and subsequent MRI analysis in relevant migraine-stroke mouse models. Optimized immunohistochemical staining and analysis platform for mouse migraine-stroke brains using inflammatory (Iba1, M1, M2, GFAP) and neuronal (NeuN) markers. Successful development prototype K+ biosensor and implementation in intracortical K+ measurements in relation to spreading depression in mice (Odijk et al., 2014). Successful development of MUA/EEG setup to detect neuronal excitability and CSD susceptibility parameters for studies in relevant migraine-stroke mouse models (Tolner et al., 2015).

WP6: Method development: MSI. The goal of this work package is to utilize high structural resolving power offered by ion mobility mass spectrometry and high resolution mass spectrometry to elucidate local molecular signalling pathways on complex biological surfaces. We developed a pipeline for MSI of transgenic murine brains. The pipeline includes data acquisition, data pre-processing, multivariate data analysis, and co-registration of the MSI data with the Allen Brain Atlas (ABA). The pipeline was applied to examine molecular changes within the brains of transgenic mouse models. Biological validation of previous co-registration SIMS and ABA results. MS imaging datasets were acquired using DESI (Desorption electrospray ionization) and MALDI coupled to ion mobility. Experiments were carried out on transgenic mouse models and compared with control mice. Lipid classes imaged with either of the methods were compared. Moreover differences in lipid occurrence between KO and control mice were investigated. The main outcome of the project is databasing lipid classes accessible through DESI vs. MALDI MS imaging.

WP7: Method development: optoacoustic imaging. The goal of this work package is to provide project partners and the wider scientific community with a new tool for in vivo optical visualization of brain pathophysiology at high resolution. The novel inversion algorithm accounting for wavelength dependent optical fluence was tested to accurately quantify measures of blood sO2 in deep tissue. The algorithm implements the eigenspectra MSOT (eMSOT) method, a novel methodology that efficiently accounts for the wavelength dependent light attenuation in tissue. Characterization of improvements of sO2 quantification in deep tissue. In-vivo sO2 quantification in superficial layers of mouse brain and the monitoring of it during alternating breathing conditions. Development of method of brain slice preparation and high-resolution opto-acoustic imaging, in real-time. By using raster-scan optoacoustic mesoscopy (RSOM) via a newly developed cutting protocol on a vibratome of freshly excised brain (without any fixation or embedding) in slices of 200 – 1000 µm, which is optimal for imaging purposes. RSOM imaging of brain slices was performed in epi-illumination mode. This imaging modality has yielded 4μm axial and 18μm lateral resolution, maintanable through several millimeters of depth. The raster-scanning was performed in a continuous-discrete manner to expedite the measurements.

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Life Sciences
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