Optimierte Verfahren zur morphologischen und histochemischen Auswertung der Niere

Immersionsfixierungen von Mäuse- und Rattennieren ermöglichen nur eine ungenügende Strukturerhaltung für licht- und elektronenmikroskopische Auswertungsansätze, als Goldstandard gilt daher die Perfusionsfixierung. Etablierte Protokolle hierfür wurden in früheren Arbeiten für Glutaraldehyd-haltige Fixierlösungen erstellt, welche sich jedoch allgemein nicht für immunhistochemische Präparationen eignen. Hierfür müssen üblicherweise separate Versuchstiergruppen mit Formaldehyd-haltigen Fixierlösungen per Immersion oder Perfusion fixiert werden, was in beiden Fällen häufig mit starken qualitativen Einschränkungen der Strukturerhaltung verbunden ist. Aufgrund der verbesserten Generierung von immer neuen experimentellen Tiermodellen und neuen mikroskopischen Auswertungsansätzen wie Konfokalmikroskopie und Super-Resolution-Lichtmikroskopie besteht daher ein immer größer werdender Bedarf an hochwertig fixiertem Nierengewebe für immunhistochemische sowie ultrastrukturelle Auswertungen. Um sämtliche konventionellen licht- und elektronenmikroskopischen Methoden sowie auch immunhistochemischen und biochemischen Methoden anhand von nur einem Versuchstier durchführen zu können, optimierte ich im Rahmen meiner Dissertation die Zusammensetzung von Formaldehyd-haltigen Fixierlösungen für Perfusionsfixierungen von Mäusen und Ratten durch Anpassung der Vehikelosmolarität, des kolloidosmotischen Drucks, des Puffersystems und des Fixans. Durch diese optimierte Fixierung erreichte ich eine hochwertige Strukturerhaltung von Rinde und äußerem Mark durch zwei Standardprotokolle (Protokoll 1 mit Zusatz von Hydroxyethylstärke, Protokoll 2 ohne Zusatz von Hydroxyethylstärke), welche mit einem klassischen Protokoll zur Perfusionsfixierung mit Glutaraldehyd verglichen wurden (Protokoll 3 mit Zusatz von Hydroxyethylstärke). Im Rahmen von Variationen dieser Protokolle konnte durch verringerte Vehikelosmolarität die ultrastrukturelle Darstellung von Podozyten und kortikalen Tubuli optimiert werden, während sich eine hohe Vehikelosmolarität und ein hoher kolloidosmotischer Druck allgemein für gute Strukturerhaltung des äußeren Marks eigneten. Zusätzlich optimierte ich die Auswertung von Nierengewebe durch Anpassung der Gewebe- und Schnittpräparation sowie der verwendeten mikroskopischen Techniken. Hierfür stellte ich durch neue Präparationsansätze qualitativ hochwertige Ultradünnschnitte für großflächige Digitalisierungen mit dem Transmissionselektronenmikroskop und neuartigen Techniken per Rasterelektronenmikroskop her. Dies erhielt den räumlichen Zusammenhang komplexer Nierenstrukturen wie Glomeruli von der Übersicht bis zum hochauflösenden Detail und ermöglichte damit eine stufenlose Vergrößerung. Die Nutzung von Siliziumplättchen als Trägermaterial für Ultradünnschnitte im Rasterelektronenmikroskop zeigte viele Vorteile im Vergleich zu klassischen Grids bei der Verwendung im Transmissionselektronenmikroskop; sie vereinfachten die Probenpräparation, erhöhten drastisch die Stabilität der Präparate und ermöglichten eine großflächige Abbildung barrierefreier Ultradünnschnittareale. Die Digitalisierung von Ultradünnschnittarealen per Rasterelektronenmikroskop konnte durch Verwendung eines neuartigen Mehrstrahl-Rasterelektronenmikroskops darüber hinaus um das ca. 100-fache beschleunigt werden, wodurch komplette Ultradünnschnitte innerhalb von ca. 30 min. in guter Qualität digitalisiert wurden.Immersion fixation of rodent kidneys leads to poor structural preservation for light- and electron microscopical analysis. Thus, perfusion fixation represents the goldstandard. Therefore, protocols for glutaraldehyde containing fixative solutions were established by previous authors for optimized structural preservation of rodent kidneys. Glutaraldehyde-fixed tissue, however, is generally not suitable for immunohistochemical approaches. Therefore, separate animal groups are usually used to perform formaldehyde fixation via immersion or perfusion, both associated with severe drawbacks in structural preservation. Based on today’s versatile experimental animal models and innovative microscopy techniques, such as confocal microscopy and superresolution microscopy, there is an increasing demand on high quality fixed tissue for combined immunohistochemical and ultrastructural analysis. My aim was to provide high quality fixed tissue as a basis for all conventional light- and electron microscopical techniques as well as immunohistochemical and biochemical techniques using one single animal. Therefore, I optimized fixation solutions for perfusion fixation of mice and rat kidneys by adapting vehicle osmolarity, colloidosmotic pressure, buffer solution and fixative type. Based on this improved fixation, renal cortex and outer medulla were preserved with superior quality using two standard protocols (protocol 1 containing hydroxy ethyl starch and protocol 2 without addition of hydroxy ethyl starch). Morphology was compared to a standard protocol, containing glutaraldehyde and hydroxy ethyl starch (protocol 3). Variations of these protocols with reduced vehicle osmolarity improved ultrastructural preservation especially of podocytes and cortical proximal tubular epithelium cells, whereas raised vehicle osmolarity and colloid osmotic pressure improved ultrastructural preservation of medullary components. In addition, improvements of tissue processing and section preparation further increased overall quality. By combining new approaches in transmission- and scanning electron microscopy with my adapted tissue processing protocols, I was able to digitize large thin section areas with superior quality as compared to conventional approaches. As a result, complex microanatomical architecture was preserved in the digitized datasets, facilitating smooth zooming. Compared to traditional transmission electron microscopy, improvements of thin section processing were mainly based on a more stable preparation using silicon substrate and imaging using a scanning electron microscope. Thin section digitization was further sped up using a new and powerful multi-beam scanning electron microscope by approximately 100-fold, enabling whole thin section digitization in about 30 min. with satisfactory quality

Successful plasmapheresis and immunoglobulin treatment for severe lipid storage myopathy: Doing the right thing for the wrong reason

Three consecutive skeletal muscle biopsies during a several months time-frame, showing different degrees of neutral lipid storage. This is highlighted by Oil-red-O stains (D, E, F) and electron microscopy (G, H, I). Note the impact on mitochondrial morphology with so called 'parking lots (K, L). Zooming 'in and out' into the ultrastructure, using the nanotomy platform provides interesting detailled information (http://nanotomy.org). ​

Systemic sclerosis-associated myositis features minimal inflammation and characteristic capillary pathology

Systemic sclerosis represents a chronic connective tissue disease featuring fibrosis, vasculopathy and autoimmunity, affecting skin, multiple internal organs, and skeletal muscles. The vasculopathy is considered obliterative, but its pathogenesis is still poorly understood. This may partially be due to limitations of conventional transmission electron microscopy previously being conducted only in single patients. The aim of our study was therefore to precisely characterize immune inflammatory features and capillary morphology of systemic sclerosis patients suffering from muscle weakness. In this study, we identified 18 individuals who underwent muscle biopsy because of muscle weakness and myalgia in a cohort of 367 systemic sclerosis patients. We performed detailed conventional and immunohistochemical analysis and large-scale electron microscopy by digitizing entire sections for in-depth ultrastructural analysis. Muscle biopsies of 12 of these 18 patients (67%) presented minimal features of myositis but clear capillary alteration, which we termed minimal myositis with capillary pathology (MMCP). Our study provides novel findings in systemic sclerosis-associated myositis. First, we identified a characteristic and specific morphological pattern termed MMCP in 67% of the cases, while the other 33% feature alterations characteristic of other overlap syndromes. This is also reflected by a relatively homogeneous clinical picture among MMCP patients. They have milder disease with little muscle weakness and a low prevalence of interstitial lung disease (20%) and diffuse skin involvement (10%) and no cases of either pulmonary arterial hypertension or renal crisis. Second, large-scale electron microscopy, introducing a new level of precision in ultrastructural analysis, revealed a characteristic capillary morphology with basement membrane thickening and reduplications, endothelial activation and pericyte proliferation. We provide open-access pan-and-zoom analysis to our datasets, enabling critical discussion and data mining. We clearly highlight characteristic capillary pathology in skeletal muscles of systemic sclerosis patients

Genetic and epigenetic characterization of posterior pituitary tumors

Pituicytoma (PITUI), granular cell tumor (GCT), and spindle cell oncocytoma (SCO) are rare tumors of the posterior pituitary. Histologically, they may be challenging to distinguish and have been proposed to represent a histological spectrum of a single entity. We performed targeted next-generation sequencing, DNA methylation profiling, and copy number analysis on 47 tumors (14 PITUI; 12 GCT; 21 SCO) to investigate molecular features and explore possibilities of clinically meaningful tumor subclassification. We detected two main epigenomic subgroups by unsupervised clustering of DNA methylation data, though the overall methylation differences were subtle. The largest group (n = 23) contained most PITUIs and a subset of SCOs and was enriched for pathogenic mutations within genes in the MAPK/PI3K pathways (12/17 [71%] of sequenced tumors: FGFR1 (3), HRAS (3), BRAF (2), NF1 (2), CBL (1), MAP2K2 (1), PTEN (1)) and two with accompanying TERT promoter mutation. The second group (n = 16) contained most GCTs and a subset of SCOs, all of which mostly lacked identifiable genetic drivers. Outcome analysis demonstrated that the presence of chromosomal imbalances was significantly associated with reduced progression-free survival especially within the combined PITUI and SCO group (p = 0.031). In summary, we observed only subtle DNA methylation differences between posterior pituitary tumors, indicating that these tumors may be best classified as subtypes of a single entity. Nevertheless, our data indicate differences in mutation patterns and clinical outcome. For a clinically meaningful subclassification, we propose a combined histo-molecular approach into three subtypes: one subtype is defined by granular cell histology, scarcity of identifiable oncogenic mutations, and favorable outcome. The other two subtypes have either SCO or PITUI histology but are segregated by chromosomal copy number profile into a favorable group (no copy number changes) and a less favorable group (copy number imbalances present). Both of the latter groups have recurrent MAPK/PI3K genetic alterations that represent potential therapeutic targets

Microglia regulate myelin growth and integrity in the central nervous system

Myelin is required for the function of neuronal axons in the central nervous system, but the mechanisms that support myelin health are unclear. Although macrophages in the central nervous system have been implicated in myelin health(1), it is unknown which macrophage populations are involved and which aspects they influence. Here we show that resident microglia are crucial for the maintenance of myelin health in adulthood in both mice and humans. We demonstrate that microglia are dispensable for developmental myelin ensheathment. However, they are required for subsequent regulation of myelin growth and associated cognitive function, and for preservation of myelin integrity by preventing its degeneration. We show that loss of myelin health due to the absence of microglia is associated with the appearance of a myelinating oligodendrocyte state with altered lipid metabolism. Moreover, this mechanism is regulated through disruption of the TGFβ1–TGFβR1 axis. Our findings highlight microglia as promising therapeutic targets for conditions in which myelin growth and integrity are dysregulated, such as in ageing and neurodegenerative disease(2,3)

DNA methylation-based classification of sinonasal tumors

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs

Morphological Characteristics of Idiopathic Inflammatory Myopathies in Juvenile Patients

Background: In juvenile idiopathic inflammatory myopathies (IIMs), morphological characteristic features of distinct subgroups are not well defined. New treatment strategies require a precise diagnosis of the subgroups in IIM, and, therefore, knowledge about the pathomorphology of juvenile IIMs is warranted. Methods: Muscle biopsies from 15 patients (median age 8 (range 3–17) years, 73% female) with IIM and seven controls were analyzed by standard methods, immunohistochemistry, and transmission electron microscopy (TEM). Detailed clinical and laboratory data were accessed retrospectively. Results: Proximal muscle weakness and skin symptoms were the main clinical symptoms. Dermatomyositis (DM) was diagnosed in 9/15, antisynthetase syndrome (ASyS) in 4/15, and overlap myositis (OM) in 2/15. Analysis of skeletal muscle tissues showed inflammatory cells and diffuse upregulation of MHC class I in all subtypes. Morphological key findings were COX-deficient fibers as a striking pathology in DM and perimysial alkaline phosphatase positivity in anti-Jo-1-ASyS. Vascular staining of the type 1 IFN-surrogate marker, MxA, correlated with endothelial tubuloreticular inclusions in both groups. None of these specific morphological findings were present in anti-PL7-ASyS or OM patients. Conclusions: Morphological characteristics discriminate IIM subtypes in juvenile patients, emphasizing differences in aetiopathogenesis and supporting the notion of individual and targeted therapeutic strategies