@phdthesis{Schulte2023,
  author    = {Schulte, Annemarie},
  title     = {\(In\) \(vitro\) reprogramming of glial cells from adult dorsal root ganglia into nociceptor-like neurons},
  doi       = {10.25972/OPUS-30311},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303110},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Plexus injury often occurs after motor vehicle accidents and results in lifelong disability with severe neuropathic pain. Surgical treatment can partially restore motor functions, but sensory loss and neuropathic pain persist. Regenerative medicine concepts, such as cell replacement therapies for restoring dorsal root ganglia (DRG) function, set high expectations. However, up to now, it is unclear which DRG cell types are affected by nerve injury and can be targeted in regenerative medicine approaches. This study followed the hypothesis that satellite glial cells (SGCs) might be a suitable endogenous cell source for regenerative medicine concepts in the DRG. SGCs originate from the same neural crest-derived cell lineage as sensory neurons, making them attractive for neural repair strategies in the peripheral nervous system. Our hypothesis was investigated on three levels of experimentation. First, we asked whether adult SGCs have the potential of sensory neuron precursors and can be reprogrammed into sensory neurons in vitro. We found that adult mouse DRG harbor SGC-like cells that can still dedifferentiate into progenitor-like cells. Surprisingly, expression of the early developmental transcription factors Neurog1 and Neurog2 was sufficient to induce neuronal and glial cell phenotypes. In the presence of nerve growth factor, induced neurons developed a nociceptor-like phenotype expressing functional nociceptor markers, such as the ion channels TrpA1, TrpV1 and NaV1.9. In a second set of experiments, we used a rat model for peripheral nerve injury to look for changes in the DRG cell composition. Using an unbiased deep learning-based approach for cell analysis, we found that cellular plasticity responses after nerve injury activate SGCs in the whole DRG. However, neither injury-induced neuronal death nor gliosis was observed. Finally, we asked whether a severe nerve injury changed the cell composition in the human DRG. For this, a cohort of 13 patients with brachial plexus injury was investigated. Surprisingly, in about half of all patients, the injury-affected DRG showed no characteristic DRG tissue. The complete entity of neurons, satellite cells, and axons was lost and fully replaced by mesodermal/connective tissue. In the other half of the patients, the basic cellular entity of the DRG was well preserved. Objective deep learning-based analysis of large-scale bioimages of the "intact" DRG showed no loss of neurons and no signs of gliosis. This study suggests that concepts for regenerative medicine for restoring DRG function need at least two translational research directions: reafferentation of existing DRG units or full replacement of the entire multicellular DRG structure. For DRG replacement, SGCs of the adult DRG are an attractive endogenous cell source, as the multicellular DRG units could possibly be rebuilt by transdifferentiating neural crest-derived sensory progenitor cells into peripheral sensory neurons and glial cells using Neurog1 and Neurog2.},
  subject      = {Spinalganglion},
  language  = {en}
}
@phdthesis{Mayer2019,
  author    = {Mayer, Rafaela},
  title     = {OxPAPC as an endogenous agonist of TRPA1 channels on nociceptors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175890},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Non-steroidal antiinflammatory drugs are most commonly used for inflammatory and postoperative pain. But they lack effectiveness and specificity, leading to severe side effects, like gastric ulcers, asthma and severe bleeding. Oxidized 1-palmitoyl-2-arachinidonoyl-sn-glycero-3-phosphocholine (OxPAPC) plays an important role in inflammatory pain. PAPC is a common phosphatidylcholine of membranes, which can be oxidized by reactive oxygen species. In preliminary experiments, our group found that local injection of OxPAPC in rat paws induces hyperalgesia. In this study we examined the effect of OxPAPC on transient receptor potential A1 (TRPA1), an ion channel expressed in C-fiber neurons. Furthermore, we investigated if intracellular cysteine residues of TRPA1 were necessary for agonist-channel-interactions and if a subsequent TRPA1 activation could be prevented by OxPAPC scavengers. To answer these questions, we performed calcium imaging using HEK-293 cells stably expressing hTRPA1, or transiently expressing the triple mutant channel hTRPA1-3C and na{\"i}ve DRG neurons. Cells were incubated with the ratiometric, fluorescent dye Fura-2/AM and stimulated with OxPAPC. The change of light emission after excitation with 340 and 380 nm wavelengths allowed conclusions regarding changes of intracellular calcium concentrations after TRPA1 activation. In our investigation we proved evidence that OxPAPC activates TRPA1, which caused a flow of calcium ions into the cytoplasm. The TRPA1-specific channel blocker HC-030031 eliminated this agonist-induced response. TRPA1-3C was not completely sensitive to OxPAPC. The peptide D-4F and the monoclonal antibody E06 neutralized OxPAPC-induced TRPA1 activation. In this work, the importance of OxPAPC as a key mediator of inflammatory pain and as a promising target for drug design is highlighted. Our results indicate that TRPA1 activation by OxPAPC involves cysteine-dependent mechanisms, but there are other, cysteine-independent activation mechanisms as well. Potential pharmaceuticals for the treatment of inflammatory pain are D-4F and E06, whose efficiency has recently been confirmed in the animal model by our research group.},
  subject      = {Schmerzforschung},
  language  = {en}
}
@phdthesis{Leffler2003,
  author    = {Leffler, Andreas},
  title     = {TRPV1 ist ein polymodaler Rezeptor von nozizeptiven Spinalganglienzellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-10748},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2003},
  abstract  = {In der vorliegenden Arbeit wurde mittels der Whole-Cell Patch-Clamp Methode sensible Neurone von transgenen M{\"a}usen untersucht, bei denen das Gen f{\"u}r TRPV1 (transient receptor potential V1) deletiert wurde. Das Ergebniss wurde mit den Daten von Wildtyp M{\"a}usen verglichen. TRPV1 (fr{\"u}her VR1; vanilloid receptor 1) wird nahezu selektiv in sensiblen Neuronen exprimiert und wird im heterologen Expressionssystem durch Vanilloide, Hitze (> 43°C) und Protonen aktiviert. Durch diese Eigenschaften scheint TRPV1 f{\"u}r die rezeptiven Eigenschaften polymodaler Nozizeptoren von großer Bedeutung zu sein. Als ein Model des peripheren afferenten Neurons wurde die Aktivierbarkeit kultivierter Spinalganglienzellen durch Vanilloide, Protonen und Hitze elektrophysiologisch untersucht. W{\"a}hrend etwa 35\% der Wildtyp-Zellen Vanilloid-sensibel waren, fehlte in Zellen der TRPV1-knockout Maus jegliche Vanilloid-Sensibilit{\"a}t. Auch bei der Protonen-Sensibilit{\"a}t wurde eine signifikante Reduktion in TRPV1-knockout Zellen beobachtet. In Wildtyp-Zellen wurde eine hohe Protonen-Sensibilit{\"a}t fast ausschliesslich in Vanilloid-sensiblen Zellen beobachtet. Hitze-induzierte Einw{\"a}rtsstr{\"o}me mit einer Aktivierungsschwelle bei 43°C wurden ausschliesslich in Vanilloid-sensiblen Zellen der Wildtyp-Maus beobachtet. Dagegen wurden Hitze-induzierte Einw{\"a}rtsstr{\"o}me mit einer Aktivierungsschwelle {\"u}ber 53°C in sowohl Wildtyp- als auch in TRPV1-knockout Zellen beobachtet. Im Bezug auf die Bedetung von TRPV1, wurde die Funktionalit{\"a}t zwei distinkter Populationen von Spinalganglienzellen, NGF- bzw. GDNF-abh{\"a}ngigen Neuronen, durch eine Lebendf{\"a}rbung mit IB4-FITC untersucht. Hinsichtlich Vanilloid-, Protonen-, Hitze-Sensibilit{\"o}t wurden jedoch keine Unterschiede zwischen IB4-negative und IB4-positive Neuronen beobachtet. Die vorliegende Studie zeigt damit, dass TRPV1 f{\"u}r Vanilliod-Sensibilit{\"a}t sensibler Neurone essentiell ist. Weiterhin konnte gezeigt werden, dass TRPV1 ein wichtiges Transduktionselement f{\"u}r sowohl die Protonen-Sensibilit{\"a}t als auch f{\"u}r die Hitze-Sensibilit{\"a}t in Spinalganglienzellen darstellt. Die Daten dieser zellul{\"a}ren Untersuchungen konnten in weiteren in vitro und in vivo Untersuchungen best{\"a}tigt werden (Caterina et al., 2000).},
  language  = {de}
}