With the advent of transgenic mouse models expressing cortical amyloid pathology, the potential to study its progression in an intact brain has been realized. Multiphoton microscopy provides a non-destructive means of imaging with micron resolution up to 500 microm deep into the cortex. We detail a surgical procedure and discuss a multiphoton imaging approach that allows for labeling and chronic visualization of amyloid-beta deposits through a cranial window. The ability to monitor these hallmarks of Alzheimer's disease enables studies aimed at evaluating the efficacy of treatment and prevention strategies.
"MPFM has been combined with in vivo fluorescence probes for cellualr imaging in diverse organs such as skin , kidney , heart , and brain . It has also been applied as a tool to study the development, progression and potential treatment of pathological conditions such as cancer  and Alzheimer's disease . In tumor biology for instance, MPFM of transgenic mice with GFP-fluorescent cells has been used to track single cell behavior within different tumor microenvironments  . "
[Show abstract][Hide abstract] ABSTRACT: Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell-matrix mechanical interactions in 3-D culture models, tissue explants and living animals.
Experimental Cell Research 06/2013; 319(16). DOI:10.1016/j.yexcr.2013.06.018 · 3.25 Impact Factor
"The uniquely designed PCA presented here has the advantage to detect and visualize α-syn oligomerization in post mortem tissue with minimal tissue processing, but could potentially be utilized to monitor the formation of α-syn oligomers over time in the brain of a living animal using two-photon microscopy. Recent advances in two-photon microscopy have enabled in vivo visualization of protein aggregation and neurodegeneration in the brain of an Alzheimer’s disease mouse model [22,33,34] and protein degradation in the brain of a PD animal model [35-37]. Here, using two-photon microscopy, we demonstrate the ability to image and detect α-syn oligomers in vivo. "
[Show abstract][Hide abstract] ABSTRACT: Rat models of Parkinson's disease are widely used to elucidate the mechanisms underlying disease etiology or to investigate therapeutic approaches. Models were developed using toxins such as MPTP or 6-OHDA to specifically target dopaminergic neurons resulting in acute neuronal loss in the substantia nigra or by using viral vectors to induce the specific and gradual expression of alpha synuclein in the substantia nigra. The detection of alpha- synuclein oligomers, the presumed toxic species, in these models and others has been possible using only indirect biochemical approaches to date. Here we coinjected AAVs encoding alpha-synuclein fused to the N- or C-terminal half of VenusYFP in rat substantia nigra pars compacta and describe for the first time a novel viral vector rodent model with the unique ability to directly detect and track alpha synuclein oligomers ex vivo and in vivo.
Viral coinjection resulted in widespread VenusYFP signal within the nigrostriatal pathway, including cell bodies in the substantia nigra and synaptic accumulation in striatal terminals, suggestive of in vivo alpha-synuclein oligomers formation. Transduced rats showed alpha-synuclein induced dopaminergic neuron loss in the substantia nigra, the appearance of dystrophic neurites, and gliosis in the striatum. Moreover, we have applied in vivo imaging techniques in the living mouse to directly image alpha-synuclein oligomers in the cortex.
We have developed a unique animal model that provides a tool for the Parkinson's disease research community with which to directly detect alpha- synuclein oligomers in vivo and screen therapeutic approaches targeting alpha-synuclein oligomers.
"The general techniques for cranial window preparation and in vivo multiphoton imaging have been published previously (Skoch et al., 2005; Holtmaat et al., 2009). In this study, we have used a modified approach, developed by our group, to optimize the application of blood-brain-barrier impermeant agents to cortical areas (Unni et al.). "
[Show abstract][Hide abstract] ABSTRACT: Increased intracellular levels of α-synuclein are implicated in Parkinson's disease and related disorders and may be caused by alterations in the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal pathway (ALP). A critical question remains how α-synuclein is degraded by neurons in vivo. To address this, our study uses α-synuclein transgenic mice, expressing human α-synuclein or α-synuclein-eGFP under the (h)PDGF-β promoter, in combination with in vivo pharmacologic and multiphoton imaging strategies to systematically test degradation pathways in the living mouse brain. We demonstrate that the UPS is the main degradation pathway for α-synuclein under normal conditions in vivo while with increased α-synuclein burden the ALP is recruited. Moreover, we report alterations of the UPS in α-synuclein transgenic mice and age dependence to the role of the UPS in α-synuclein degradation. In addition, we provide evidence that the UPS and ALP might be functionally connected such that impairment of one can upregulate the other. These results provide a novel link between the UPS, the ALP, and α-synuclein pathology and may have important implications for future therapeutics targeting degradation pathways.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2011; 31(41):14508-20. DOI:10.1523/JNEUROSCI.1560-11.2011 · 6.34 Impact Factor
Michael Rullmann, Juergen Dukart, Karl-Titus Hoffmann, Julia Luthardt, Solveig Tiepolt, Marianne Patt, Hermann-Josef Gertz, Matthias L Schroeter, John Seibyl, Walter J Schulz-Schaeffer, Osama Sabri, Henryk Barthel
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