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2.
J Mol Neurosci ; 71(3): 527-544, 2021 Mar.
Article in English | MEDLINE | ID: mdl-32833183

ABSTRACT

Mechanisms and progression of ischemic injuries in the retina are still incompletely clarified. Therefore, the time course of microglia activation as well as resulting cytokine expression and downstream signaling were investigated. Ischemia was induced in one eye by transiently elevated intraocular pressure (60 min) followed by reperfusion; the other eye served as a control. Eyes were processed for RT-qPCR and immunohistochemistry analyses at 2, 6, 12, and 24 h as well as at 3 and 7 days. Already 2 h after ischemia, more microglia/macrophages were in an active state in the ischemia group. This was accompanied by an upregulation of pro-inflammatory cytokines, like IL-1ß, IL-6, TNFα, and TGFß. Activation of TLR3, TLR2, and the adaptor molecule Myd88 was also observed after 2 h. NFκB revealed a wave-like activation pattern. In addition, an extrinsic caspase pathway activation was noted at early time points, while enhanced numbers of cleaved caspase 3+ cells could be observed in ischemic retinae throughout the study. Retinal ischemia induced an early and strong microglia/macrophage response as well as cytokine and apoptotic activation processes. Moreover, in early and late ischemic damaging processes, TLR expression and downstream signaling were involved, suggesting an involvement in neuronal death in ischemic retinae. Graphical Abstract.


Subject(s)
Cytokines/metabolism , Glaucoma/metabolism , Microglia/metabolism , Retina/metabolism , Toll-Like Receptors/metabolism , Animals , Cytokines/genetics , Male , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Rats , Retina/cytology , Signal Transduction , Toll-Like Receptors/genetics
3.
Front Cell Neurosci ; 13: 174, 2019.
Article in English | MEDLINE | ID: mdl-31133806

ABSTRACT

Neuronal damage and impaired vision in different retinal disorders are induced, among other factors, by ischemia/reperfusion (I/R). Since the mechanisms and the progression of ischemic injury are still not completely clarified, a timeline of this retinal degeneration is needed. In this study, we investigated protein and mRNA alterations at 2, 6, 12, and 24 h as well as 3 and 7 days after ischemia to determine the course of an ischemic insult through the whole retina. Moreover, functional analyses were performed at later stages. We detected a significant functional loss of cells in the inner nuclear layer and photoreceptors at 3 and 7 days. Additionally, the thickness of the whole retina was decreased at these points in time, indicating a severe degradation of all retinal layers. Immunohistological and qRT-PCR analyses of retinal ganglion cells (RGCs), glial cells, AII amacrine, cone and rod bipolar as well as cone and rod photoreceptor cells confirmed this first assumption. Our results show that all investigated cell types were damaged by ischemia induction. Especially RGCs, cone bipolar cells, and photoreceptor cones are very sensitive to I/R. These cells were lost shortly after ischemia induction with a progressive course up to 7 days. In addition, Müller cell gliosis was observed over the entire period of time. These results provide evidence, that I/R induces damage of the whole retina at early stages and increases over time. In conclusion, our study could demonstrate the intense impact of an ischemic injury. The ischemic defect spreads across the whole retina right up to the outer layers in the long-term and thus seems to impair the visual perception already during the stimulus processing. In addition, our findings indicate that the cone pathway seems to be particularly affected by this damage.

4.
Sci Rep ; 7(1): 14081, 2017 10 26.
Article in English | MEDLINE | ID: mdl-29074975

ABSTRACT

Lensless, coherent X-ray diffraction microscopy has been drawing considerable attentions for tomographic imaging of whole human cells. In this study, we performed cryogenic coherent X-ray diffraction imaging of human erythrocytes with and without malaria infection. To shed light on structural features near the surface, "ghost cells" were prepared by the removal of cytoplasm. From two-dimensional images, we found that the surface of erythrocytes after 32 h of infection became much rougher compared to that of healthy, uninfected erythrocytes. The Gaussian roughness of an infected erythrocyte surface (69 nm) is about two times larger than that of an uninfected one (31 nm), reflecting the formation of protein knobs on infected erythrocyte surfaces. Three-dimensional tomography further enables to obtain images of the whole cells with no remarkable radiation damage, whose accuracy was estimated using phase retrieval transfer functions to be as good as 64 nm for uninfected and 80 nm for infected erythrocytes, respectively. Future improvements in phase retrieval algorithm, increase in degree of coherence, and higher flux in combination with complementary X-ray fluorescence are necessary to gain both structural and chemical details of mesoscopic architectures, such as cytoskeletons, membraneous structures, and protein complexes, in frozen hydrated human cells, especially under diseased states.


Subject(s)
Erythrocytes/pathology , Malaria/diagnostic imaging , Malaria/pathology , Microscopy/methods , Tomography, X-Ray/methods , X-Ray Diffraction/methods , Cells, Cultured , Cryopreservation , Erythrocytes/metabolism , Humans , Imaging, Three-Dimensional/methods , Malaria/metabolism , Proteins/metabolism , Tomography, X-Ray/instrumentation
5.
Sci Rep ; 6: 24264, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-27080570

ABSTRACT

Mounting evidence indicated that human mesenchymal stem cells (hMSCs) are responsive not only to biochemical but also to physical cues, such as substrate topography and stiffness. To simulate the dynamic structures of extracellular environments of the marrow in vivo, we designed a novel surrogate substrate for marrow derived hMSCs based on physically cross-linked hydrogels whose elasticity can be adopted dynamically by chemical stimuli. Under frequent mechanical stress, hMSCs grown on our hydrogel substrates maintain the expression of STRO-1 over 20 d, irrespective of the substrate elasticity. On exposure to the corresponding induction media, these cultured hMSCs can undergo adipogenesis and osteogenesis without requiring cell transfer onto other substrates. Moreover, we demonstrated that our surrogate substrate suppresses the proliferation of hMSCs by up to 90% without any loss of multiple lineage potential by changing the substrate elasticity every 2nd days. Such "dynamic in vitro niche" can be used not only for a better understanding of the role of dynamic mechanical stresses on the fate of hMSCs but also for the synchronized differentiation of adult stem cells to a specific lineage.


Subject(s)
Cell Differentiation , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Stress, Mechanical , Antigens, Surface/genetics , Antigens, Surface/metabolism , Biomarkers , Cell Culture Techniques , Cell Proliferation , Cell Self Renewal , Cells, Cultured , Elasticity , Gene Expression , Humans , Models, Biological , Phenotype
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