In vivo imaging of lymphocytes in the CNS reveals different behaviour of naïve T cells in health and autoimmunity

<p>Abstract</p> <p>Background</p> <p>Two-photon laser scanning microscopy (TPLSM) has become a powerful tool in the visualization of immune cell dynamics and cellular communication within the complex biological networks of the inflamed central nervous system (CNS). Whereas many previous studies mainly focused on the role of effector or effector memory T cells, the role of naïve T cells as possible key players in immune regulation directly in the CNS is still highly debated.</p> <p>Methods</p> <p>We applied <it>ex vivo </it>and intravital TPLSM to investigate migratory pathways of naïve T cells in the inflamed and non-inflamed CNS. MACS-sorted naïve CD4+ T cells were either applied on healthy CNS slices or intravenously injected into RAG1 -/- mice, which were affected by experimental autoimmune encephalomyelitis (EAE). We further checked for the generation of second harmonic generation (SHG) signals produced by extracellular matrix (ECM) structures.</p> <p>Results</p> <p>By applying TPLSM on living brain slices we could show that the migratory capacity of activated CD4+ T cells is not strongly influenced by antigen specificity and is independent of regulatory or effector T cell phenotype. Naïve T cells, however, cannot find sufficient migratory signals in healthy, non-inflamed CNS parenchyma since they only showed stationary behaviour in this context. This is in contrast to the high motility of naïve CD4+ T cells in lymphoid organs. We observed a highly motile migration pattern for naïve T cells as compared to effector CD4+ T cells in inflamed brain tissue of living EAE-affected mice. Interestingly, in the inflamed CNS we could detect reticular structures by their SHG signal which partially co-localises with naïve CD4+ T cell tracks.</p> <p>Conclusions</p> <p>The activation status rather than antigen specificity or regulatory phenotype is the central requirement for CD4+ T cell migration within healthy CNS tissue. However, under inflammatory conditions naïve CD4+ T cells can get access to CNS parenchyma and partially migrate along inflammation-induced extracellular SHG structures, which are similar to those seen in lymphoid organs. These SHG structures apparently provide essential migratory signals for naïve CD4+ T cells within the diseased CNS.</p

Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation

Functional real-time analysis of the cellular and molecular mechanisms of neuronal dysfunction in chronic neuroinflammation

Für das Verständnis der Pathomechnismen in der Multiplen Sklerose (MS) ist die Darstellung funktioneller Zusammenhänge maßgeblich, mit bisher angewendeten Methoden jedoch nur bedingt möglich. In dieser Arbeit wurde die Zweiphotonenmikroskopie (TPLSM) auch in Kombination mit Fluoreszenzlebensdauermessung (FLIM) sowohl intravital im Tiermodell als auch in vitro an Patientenzellen genutzt, um die Reaktionen des Gewebes auf die Immunzellinvasion zu untersuchen. Hierbei wurde deutlich, dass das Gewebe des zentralen Nervensystems (ZNS) selbst entscheidende Änderungen in Form von Ausbildung eines retikulären Fasernetzwerkes vollzieht, die es erst ermöglichen, dass massenweise periphere Immunzellen einwandern. Die Immunzellen führen direkt und indirekt zur neuronalen Dysfunktion, welche mittels Messung der intrazellulären neuronalen Calciumkonzentration intravital analysiert wurde. Darüber hinaus konnte erstmals gezeigt werden, dass die Aktivität der NADPH-Oxidase (NOX) als Entstehungsort reaktiver Sauerstoffspezies (ROS) in direkter Korrelation mit der neuronalen Dysfunktion steht. Die Zellen, die für die Produktion der ROS im zentralen Nervensystem verantwortlich sind, waren neben peripheren eingewanderten Makrophagen und ortstständigen Mikroglia auch zu einem großen Teil Astrozyten. Die systemische Komponente der MS spiegelte sich in der Überaktivierung der NOX in peripheren CD11b+-Zellen mit signifikanten Unterschieden zwischen den Erkrankungsstadien bei MS-Patienten im Vergleich zu gesunden Probanden und Patienten wider. Eine Behandlung mit dem Antioxidans Epigallokatechingallat, einem Extrakt aus grünem Tee, senkte die NOX-Aktivierung auf gesunde Werte. Insgesamt zeigt diese Arbeit neue Zugänge auf, funktionelle Zusammenhänge neuroimmunologischer Erkrankungen im lebendigen Gewebe in Echtzeit zu erforschen.To fully understand the underlying pathological mechanisms of multiple sclerosis (MS), it is crucial to register and comprehend its functional dynamics. Yet until now, due to the lack of adequate detection methods, many interrelations in cell and metabolism dynamics remain elusive. In this work we introduced two-photon fluorescence microscopy (TPLSM) and its combination with NAD(P)H-fluorescence lifetime imaging (FLIM) both in an animal model for MS as well as human blood cells to investigate tissue and cell response to immune cell invasion. Thereby it became apparent how the central nervous system tissue itself undergoes changes in its extracellular matrix by developing a reticular meshwork that allows peripheral immune cells to infiltrate inflammatory lesions. These immune cells lead to neuronal dysfunction, which was analyzed through measuring intracellular neuronal calcium concentrations in vivo. Furthermore we showed for the first time how the activity of NADPH oxidase (NOX), the main source of reactive oxygen species (ROS), is directly correlated with neuronal dysfunction. The cellular source and the dynamics of ROS production were undetermined up to now, since freely diffusing ROS molecules cannot be localized and their production requires the assembly and not the mere expression of NOX subunits. Using intravital TPLSM and NAD(P)H-FLIM we identified infiltrating peripheral monocytes, activated resident microglia and astrocytes as the main cellular sources of ROS in EAE brainstem lesions. The systemic dimension of MS was mirrored in the over- activation of NOX enzymes in peripheral CD11b+ cells with significant differences between MS patients compared to healthy subjects. Administration of the anti-oxidant epigallocatechin-3-gallate, a green tea extract, counteracted this effect. Overall, this work establishes new intravital approaches to explore functional contexts in neuroinflammation and neurodegeneration in real time

Dominant-negative p53-overexpression in skeletal muscle induces cell death and fiber atrophy in rats.

The tumor suppressor p53 is thought to play a key role in the maintenance of cell size and homeostasis, but relatively little is known about its role in skeletal muscle. Based on its ability to suppress cell growth, we hypothesized that inhibiting the function of wild-type p53 through the overexpression of a dominant-negative p53 mutant (DDp53) could result in muscle fiber hypertrophy. To test this hypothesis, we electroporated adult rat tibialis anterior muscles with DDp53 and collected the tissue three weeks later. We confirmed successful overexpression of DDp53 on a histological and biochemical level and found pronounced changes to muscle architecture, metabolism, and molecular signaling. Muscle mass, fiber cross-sectional area, and fiber diameter significantly decreased with DDp53 overexpression. We found histopathological changes in DDp53 transfected muscle which were accompanied by increased levels of proteins that are associated with membrane damage and repair. In addition, DDp53 decreased oxidative phosphorylation complex I and V protein levels, and despite its negative effects on muscle mass and fiber size, caused an increase in muscle protein synthesis as assessed via the SUnSET technique. Interestingly, the increase in muscle protein synthesis was concomitant with a decrease in phospho-S6K1 (Thr389). Furthermore, the muscle wasting in the DDp53 electroporated leg was accompanied by a decrease in global protein ubiquitination and an increase in proteasome activity. In conclusion, overexpression of a dominant-negative p53 mutant in skeletal muscle results in decreased muscle mass, myofiber size, histological muscle damage, a metabolic phenotype, and perturbed homeostasis between muscle protein synthesis and degradation

Moving a mountain: Practical insights into mastering a major curriculum reform at a large European medical university

Aim: Undergraduate medical education is currently in a fundamental transition towards competency-based programs around the globe. A major curriculum reform implies a dual challenge: the change of the curriculum and the delivering organization. Both are closely interwoven. In this article, we provide practical insights into our approach of managing such a fundamental reform of the large undergraduate medical program at the Charité – Universitätsmedizin Berlin. Methods: Members of the project management team summarized the key features of the process with reference to the literature. Results: Starting point was a traditional, discipline-based curriculum that was reformed into a fully integrated, competency- based program. This change process went through three phases: initiation, curriculum development and implementation, and sustainability. We describe from a change management perspective, their main characteristics, and the approaches that were employed to manage them successfully. Conclusions: Our report is intended to provide practical insights and guidance for those institutions which are yet considering or have already started to undergo a major reform of their undergraduate programs towards competency medical education

A mutation in desmin makes skeletal muscle less vulnerable to acute muscle damage after eccentric loading in rats.

Desminopathy is the most common intermediate filament disease in humans. The most frequent mutation causing desminopathy in patients is a R350P DES missense mutation. We have developed a rat model with an analogous mutation in R349P Des. To investigate the role of R349P Des in mechanical loading, we stimulated the sciatic nerve of wild-type littermates (WT) (n&nbsp;=&nbsp;6) and animals carrying the mutation (MUT) (n&nbsp;=&nbsp;6) causing a lengthening contraction of the dorsi flexor muscles. MUT animals showed signs of ongoing regeneration at baseline as indicated by a higher number of central nuclei (genotype: P&nbsp;&lt;&nbsp;.0001). While stimulation did not impact central nuclei, we found an increased number of IgG positive fibers (membrane damage indicator) after eccentric contractions with both genotypes (stimulation: P&nbsp;&lt;&nbsp;.01). Interestingly, WT animals displayed a more pronounced increase in IgG positive fibers with stimulation compared to MUT (interaction: P&nbsp;&lt;&nbsp;.05). In addition to altered histology, molecular signaling on the protein level differed between WT and MUT. The membrane repair protein dysferlin decreased with eccentric loading in WT but increased in MUT (interaction: P&nbsp;&lt;&nbsp;.05). The autophagic substrate p62 was increased in both genotypes with loading (stimulation: P&nbsp;&lt;&nbsp;.05) but tended to be more elevated in WT (interaction: P&nbsp;=&nbsp;.05). Caspase 3 levels, a central regulator of apoptotic cell death, was increased with stimulation in both genotypes (stimulation: P&nbsp;&lt;&nbsp;.01) but more so in WT animals (interaction: P&nbsp;&lt;&nbsp;.0001). Overall, our data indicate that R349P Des rats have a lower susceptibility to structural muscle damage of the cytoskeleton and sarcolemma with acute eccentric loading

Generation of desminopathy in rats using CRISPR-Cas9.

BackgroundDesminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.MethodsUsing CRISPR-Cas9 technology, Des c.1045-1046 (AGG &gt; CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.ResultsWe found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P &lt; 0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P &lt; 0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P &lt; 0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P &lt; 0.05).ConclusionsWe created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression

Generation of desminopathy in rats using CRISPR-Cas9.

BackgroundDesminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.MethodsUsing CRISPR-Cas9 technology, Des c.1045-1046 (AGG&nbsp;&gt;&nbsp;CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n&nbsp;=&nbsp;6) between the age of 120-150&nbsp;days and an equal number of wild type littermates (n&nbsp;=&nbsp;6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.ResultsWe found a difference in the number of central nuclei between desmin mutants (1&nbsp;±&nbsp;0.4%) and wild type littermates (0.2&nbsp;±&nbsp;0.1%; P&nbsp;&lt;&nbsp;0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P&nbsp;&lt;&nbsp;0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P&nbsp;&lt;&nbsp;0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P&nbsp;&lt;&nbsp;0.05).ConclusionsWe created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression

Moving a mountain: Practical insights into mastering a major curriculum reform at a large European medical university

<p><b>Aim:</b> Undergraduate medical education is currently in a fundamental transition towards competency-based programs around the globe. A major curriculum reform implies a dual challenge: the change of the curriculum and the delivering organization. Both are closely interwoven. In this article, we provide practical insights into our approach of managing such a fundamental reform of the large undergraduate medical program at the Charité – Universitätsmedizin Berlin.</p> <p><b>Methods:</b> Members of the project management team summarized the key features of the process with reference to the literature.</p> <p><b>Results:</b> Starting point was a traditional, discipline-based curriculum that was reformed into a fully integrated, competency-based program. This change process went through three phases: initiation, curriculum development and implementation, and sustainability. We describe from a change management perspective, their main characteristics, and the approaches that were employed to manage them successfully.</p> <p><b>Conclusions:</b> Our report is intended to provide practical insights and guidance for those institutions which are yet considering or have already started to undergo a major reform of their undergraduate programs towards competency medical education.</p