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Noch immer ist die Behandlung von Neuropathien mit den gängigen therapeutischen Mitteln für viele Patienten sehr unbefriedigend. Als erfolgsversprechender therapeutischer Ansatz werden zur Zeit Wege erforscht, welche direkt in die molekularen Entstehungsmechanismen pathologischer Veränderungen und regenerationsfördernder Mechanismen eingreifen, um dadurch eine Heilung von Nervenschäden zu ermöglichen. Bisher sind die Erkenntnisse über diese Mechanismen nicht vollständig genug, um daraus eine sichere Behandlungsmöglichkeit abzuleiten. Wegweisende Erkenntnisse deuten sich allerdings durch Studien von unterschiedlichen Vertretern des Zytokinnetzwerkes an - darunter auch TNF-alpha - welche als molekulare Ursache neuropathischer Veränderungen diskutiert werden. In dieser Studie wurde an Knockoutmäusen der Einfluss des jeweiligen TNF-alpha-Rezeptors auf morphologische Veränderungen nach CCI (Chronic constriction injury) und Crush-Verletzung des N. ischiadicus untersucht. Nach 3,7,15 und 36 Tagen (CCI) bzw. 3,7 und 28 Tagen (Crush) wurden in Methylenblau gefärbten Semidünnschnitten intakte und degenerierte Nervenfasern, Makrophagen, Angioproliferation, Ödembildung udn Veränderung des Anteils nicht neuronaler Zellen lichtmikroskopisch beurteilt. Zusätzlich wurden Mac-1+ Makrophagen immunzytochemisch erfasst. Die Ergebnisse zeigten in beiden Modellen und bei beiden Knockouttypen eine starke axonale Schädigung, die von einer großen endoneuroalen Makrophagenansammlung begleitet war. Bei TNF-R1-/- Mäusen war eine stärkere und verlängerte Degeneration mit entsprechend höheren Makrophagenzahlen sichtbar. In den Immunzytochemischen Färbungen wiesen die TNF-R1-/- Mäuse hingegen den geringsten Makropahgenanteil auf.Trotz der starken Schädigung war die anschließende Regeneration im Gegensatz zu WT und TNF-R2-/- Mäusen besser. Die Ödembildung war bei den TNF-R2-/- nach CCI besonders stark ausgeprägt und von einer schlechten Regeneration gefolgt. Während die gefundenen Daten auf eine Beteiligung beider Rezeptoren während degenerativer Prozesse hindeuten, scheint insbesondere TNF-R2 regenerationsfördernde Effekte zu vermitteln.
Cellular responses to outer stimuli are the basis for all biological processes. Signal integration is achieved by protein cascades, recognizing and processing molecules from the environment. Factors released by pathogens or inflammation usually induce an inflammatory response, a signal often transduced by Tumour Necrosis Factor alpha (TNF). TNFα receptors TNF-R1 and TNF-R2 can in turn lead to apoptosis or proliferation via NF-B. These processes are closely regulated by membrane compartimentalization, protein interactions and trafficking. Fluorescence microscopy offers a reliable and non-invasive method to probe these cellular events. However, some processes on a native membrane are not resolvable, as they are well below the diffraction limit of microscopy. The recent development of super-resolution fluorescence microscopy methods enables the observation of these cellular players well below this limit: by localizing, tracking and counting molecules with high spatial and temporal resolution, these new fluorescence microscopy methods offer a previously unknown insight into protein interactions at the near-molecular level. Direct stochastic optical reconstruction microscopy (dSTORM) utilizes the reversible, stochastic blinking events of small commercially available fluorescent dyes, while photoactivated localization microscopy (PALM) utilizes phototransformation of genetically encoded fluorescent proteins. By photoactivating only a small fraction of the present fluorophores in each observation interval, single emitters can be localized with high precision and a super-resolved image can be reconstructed. Quantum Dot Triexciton imaging (QDTI) utilizes the three-photon absorption (triexcitonic) properties of quantum dots (QD) and to achieve a twofold resolution increase using conventional confocal microscopes. In this thesis, experimental approaches were implemented to achieve super-resolution microscopy in fixed and live-cells to study the spatial and temporal dynamics of TNF and other cellular signaling events. We introduce QDTI to study the three-dimensional cellular distribution of biological targets, offering an easy method to achieve resolution enhancement in combination with optical sectioning, allowing the preliminary quantification of labeled proteins. As QDs are electron dense, QDTI can be used for correlative fluorescence and transmission electron microscopy, proving the versatility of QD probes. Utilizing the phototransformation properties of fluorescent proteins, single-receptor tracking on live cells was achieved, applying the concept of single particle tracking PALM (sptPALM) to track the dynamics of a TNF-R1-tdEos chimera on the membrane. Lateral receptor dynamics can be tracked with high precision and the influences of ligand addition or lipid disruption on TNF-R1 mobility was observed. The results reveal complex receptor dynamics, implying internalization processes in response to TNFα stimulation and a role for membrane domains with reduced fluidity, so-called lipid raft domains, in TNF-R1 compartimentalization prior or post ligand induction. Comparisons with previously published FCS data show a good accordance, but stressing the increased data depth available in sptPALM experiments. Additionally, the active transport of NF-κB-tdEos fusions was observed in live neurons under chemical stimulation and/or inhibition. Contrary to phototransformable proteins that need no special buffers to exhibit photoconversion or photoactivation, dSTORM has previously been unsuitable for in vivo applications, as organic dyes relied on introducing the probes via immunostaining in concert with a reductive, oxygen-free medium for proper photoswitching behaviour. ATTO655 had been previously shown to be suitable for live-cell applications, as its switching behavior can be catalyzed by the reductive environment of the cytoplasm. By introducing the cell-permeant organic dye via a chemical tag system, a high specificity and low background was achieved. Here, the labeled histone H2B complex and thus single nucleosome movements in a live cell can be observed over long time periods and with ~20 nm resolution. Implementing these new approaches for imaging biological processes with high temporal and spatial resolution provides new insights into the dynamics and spatial heterogeneities of proteins, further elucidating their function in the organism and revealing properties that are usually only detectable in vitro.