@phdthesis{Griffoni2019,
  author    = {Griffoni, Chiara},
  title     = {Towards advanced immunocompetent skin wound models for in vitro drug evaluation},
  doi       = {10.25972/OPUS-19212},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192125},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models.},
  subject      = {Haut},
  language  = {en}
}
@phdthesis{Klein2015,
  author    = {Klein, Dennis},
  title     = {The pathogenic role of endogenous antibodies in a mouse model for Charcot-Marie-Tooth 1B neuropathy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121941},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Charcot-Marie-Tooth (CMT) type 1 neuropathies are a genetically heterogeneous group of non-treatable inherited disorders affecting the peripheral nervous system that lead to sensory and motor dysfunction. Secondary low grade inflammation, implicating the innate and adaptive immune system, could previously be identified as a substantial disease modifier in two mouse models for CMT1, CMT1B and 1X, respectively. However, the exact mechanism how the adaptive immune system contributes to disease pathogenesis is not completely understood. Based on observations that the accumulation of endogenous antibodies to myelin components is important for rapid myelin clearance after nerve injury during Wallerian degeneration, a possibly similar mechanism was considered for endogenous antibodies as disease amplifier in mice heterozygously deficient for P0 (P0het), mimicking some typical features of CMT1B. In this study an increased antibody deposition was detected in the affected peripheral nerves of P0het myelin mutant mice. By crossbreeding P0het mutants with mice specifically lacking B-lymphocytes, and therefore antibodies (JHD-/-), a decline of endoneurial macrophages together with a substantially ameliorated demyelination could be demonstrated in 6-month-old mutant mice. Moreover, reconstitution with murine IgGs reverted the neuropathic phenotype, substantiating that endogenous antibodies are potentially pathogenic at this early stage of disease. Unexpectedly, in 12-months-old P0het mutants, JHD deficiency resulted in disease aggravation accompanied by an increased inflammatory reaction and M2-polarized macrophage response. These observations suggest that in a mouse model for CMT1B, the lack of endogenous antibodies has a dichotomous effect: ameliorating early macrophage-mediated demyelination, as opposed to increasing inflammatory reactions leading to disease aggravation at older ages.},
  subject      = {Maus},
  language  = {en}
}
@phdthesis{Yuan2023,
  author    = {Yuan, Xidi},
  title     = {Aging and inflammation in the peripheral nervous system},
  doi       = {10.25972/OPUS-23737},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-237378},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Aging is known to be a risk factor for structural abnormalities and functional decline in the nervous system. Characterizing age-related changes is important to identify putative pathways to overcome deleterious effects and improve life quality for the elderly. In this study, the peripheral nervous system of 24-month-old aged C57BL/6 mice has been investigated and compared to 12-month-old adult mice. Aged mice showed pathological alterations in their peripheral nerves similar to nerve biopsies from elderly human individuals, with nerve fibers showing demyelination and axonal damage. Such changes were lacking in nerves of adult 12-month-old mice and adult, non-aged humans. Moreover, neuromuscular junctions of 24-month-old mice showed increased denervation compared to adult mice. These alterations were accompanied by elevated numbers of macrophages in the peripheral nerves of aged mice. The neuroinflammatory conditions were associated with impaired myelin integrity and with a decline of nerve conduction properties and muscle strength in aged mice. To determine the pathological impact of macrophages in the aging mice, macrophage depletion was performed in mice by oral administration of CSF-1R specific kinase (c-FMS) inhibitor PLX5622 (300 mg/kg body weight), which reduced the number of macrophages in the peripheral nerves by 70\%. The treated mice showed attenuated demyelination, less muscle denervation and preserved muscle strength. This indicates that macrophage-driven inflammation in the peripheral nerves is partially responsible for the age-related neuropathy in mice. Based on previous observations that systemic inflammation can accelerate disease progression in mouse models of neurodegenerative diseases, it was hypothesized that systemic inflammation can exacerbate the peripheral neuropathy found in aged mice. To investigate this hypothesis, aged C57BL/6 mice were intraperitoneally injected with a single dose of lipopolysaccharide (LPS; 500 μg/kg body weight) to induce systemic inflammation by mimicking bacterial infection, mostly via activation of Toll-like receptors (TLRs). Altered endoneurial macrophage activation, highlighted by Trem2 downregulation, was found in LPS injected aged mice one month after injection. This was accompanied by a so far rarely observed form of axonal perturbation, i.e., the occurrence of "dark axons" characterized by a damaged cytoskeleton and an increased overall electron density of the axoplasm. At the same time, however, LPS injection reduced demyelination and muscle denervation in aged mice. Interestingly, TREM2 deficiency in aged mice led to similar changes to LPS injection. This suggests that LPS injection likely mitigates aging-related demyelination and muscle denervation via Trem2 downregulation. Taken together, this study reveals the role of macrophage-driven inflammation as a pathogenic mediator in age-related peripheral neuropathy, and that targeting macrophages might be an option to mitigate peripheral neuropathies in aging individuals. Furthermore, this study shows that systemic inflammation may be an ambivalent modifier of age-related nerve damage, leading to a distinct type of axonal perturbation, but in addition to functionally counteracting, dampened demyelination and muscle denervation. Translationally, it is plausible to assume that tipping the balance of macrophage polarization to one direction or the other may determine the functional outcome in the aging peripheral nervous system of the elderly.},
  subject      = {Maus},
  language  = {en}
}