The progression of this project, as well as the outcomes, are extremely relevant to the public and scientific researchers since imaging of the brain, integrating different imaging modalities and obtaining as much information as possible out of the images is essential to improve the treatment of a wide range of brain diseases. As reported previously, a pipeline for co-registration of anatomical MRI with histology and MSI images has been established.
The overall aims of this project were:
- Develop improved and novel multimodal methodologies and approaches for optical brain imaging which add functional and molecular value to the current state-of-art clinical imaging techniques of CT, PET and MRI.
- Establish an international multidisciplinary network of Early Stage Researchers (ESRs) and Experienced Researchers (ERs) that will (i) lay down the foundations by training a new generation of researchers familiar with working in this way, and (ii) enable the full exploitation of synergies between the involved partners.
WP1: AD and inflammation.
The goal of this WP was to study the interaction between soluble amyloid, inflammation and synaptic loss in AD. A number of innovative MRI methodologies for detection of early stage AD have been explored in small animal imaging µMRI studies (see WP 1.1). As reported previously, a pipeline for co-registration of anatomical MRI with histology and MSI images has been established (See WP 1.2). A co-registration workflow for MRI, IHC and MSI datasets has been developed, implemented and fully documented at both UA and Icometrix (see WP1.3). MSI imaging was not successful in visualizing amyloid related proteins which prevented to draw conclusion of combining MRI with MSI datasets. Nevertheless, a DCE-MRI study was performed to study the impact of cerebrovascular amyloid (and its removal by pharmacological treatment) on BBB-function (See WP 1.4).
WP2: Stroke – brain lesions and the role of stem cells and inflammation.
The goal of WP2 was 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. In WP1.1 in vivo monitoring of stem cell differentiation has been established. This allows in vivo cell fate imaging and its correlation with the mechanisms underlying stem cell mediated improvement. The multifactorial mechanisms of stroke treatment by mir-124 have been deciphered. This is a promising novel treatment strategy to ameliorate the detrimental effects of stroke induced inflammation. Proof of concept to visualize in vivo the activity of the inflammasome after stroke has been demonstrated. The role of endogenous neurogenesis as a homeostatic regulator of normal functional neuronal networks has been deciphered. Disturbances of the functional neuronal network after stroke are described in their time dependent development. The involved anatomic areas are identified and the stabilizing contribution of stem cell grafts has been described.
WP3: GBM and the immune response.
The goal of this WP was to test a potential new combination therapy for a type of aggressive and malignant brain tumour. We demonstrated in a mouse model of glioblastoma that the treatment with stem cells loaded with PLGA nanoparticles containing doxorubicin can slow tumour progression but only for a limited time. This treatment alone is not sufficient to stop tumour progression in nude mice where the median survival of animals is 3 weeks after tumour injection. We developed an optimized protocol for imaging stem cells in mouse brain for future experiments involving also optoacoustic imaging. Treatment with a combination of immunotherapeutic agents such as checkpoint blockers (Anti-CTLA-4 and Anti-PD-L1) was able to stop tumour progression in a syngeneic mouse model of glioblastoma. This is due to the fact that the chosen combination of immunotherapeutic agents is able to inhibit Tregs and allow the immune system to mount an effective anti-tumour response. Mice that responded to therapy were not only tumour progression free but showed signs of tumour regression.
WP4: TBI and repair.
The goal of this workpackage was 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 mechanisms of recovery. We successfully generated genetically modified stem cells for monitoring spatial and temporal dynamics in rodent brains after implantation.
WP5: Migraine – Stroke, Migraine-Related Hyperexcitability as a Risk Factor for Stroke Evolution.
The goal of this work package was 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. Significant results include the validated protocols for mouse MCAo and subsequent MRI analysis in relevant migraine-stroke mouse models and the automated stroke MRI segmentation tool and optimized immunohistochemical staining platform for mouse migraine-stroke brains using inflammatory (Iba1, M1, M2, GFAP) and neuronal (NeuN) markers.
WP6: Method development: MSI.
The goal of this work package was 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. Significant results include the development of the new imaging workflow for the detection and identification of different lipid species and sialylated gangliosides in murine mouse brain; the development of the high-throughput dual-polarity MALDI imaging for the detection of different lipid species from the same tissue section and new molecular insight of the song control nuclei and the interconnected circuits in the song learning system in Zebra Finches.
WP7: Method development: optoacoustic imaging.
The goal of this work package was 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. It was implemented for sO2 measurements in healthy tissue and hypoxic tumors. High resolution images of blood oxygenation were obtained and a good correspondence with laboratory gold-standard was shown. Assumingly, eMSOT points to a technique that has potential to become the gold-standard in in vivo imaging of blood oxygenation, offering accurate label-free insights into assessing physiology, hemodynamics and metabolism in vivo. We further evaluated the sensitivity of MSOT combined with appropriate spectral processing methods for the application of cell imaging (7.2). In addition, we established a 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 4 μm axial and 18 μm lateral resolution, maintainable through several millimeters of depth. The raster-scanning was performed in a continuous-discrete manner to expedite the measurements. The feasibility of imaging of brain slices and visualization of different brain structures and patterns was shown with this experimental setup. For WP7.4 the applicability of RSOM for raster scanning of Ca2+ sensitive reporter proteins has been demonstrated in reduced acute brain slice preparation (currently, these results are in the process of preparation for publication).