A Study of the Pathomorphology of Non-Neoplastic Changes in Canine Mammary Glands

Deckblatt \- Impressum Dank Inhaltsverzeichnis 1 Einleitung und Zielstellung 1 2 Literaturübersicht 2 2.1 Anatomie der Milchdrüse 2 2.2 Nicht neoplastische pathologische Veränderungen der Milchdrüse 10 3 Eigene Untersuchungen 25 3.1 Material 25 3.3 Methoden 26 3.4 Mikroskopische Untersuchung 29 4 Ergebnisse 30 4.1 Milchdrüsengewebe der Hündin im Anöstrus 30 4.2 Nicht laktierendes Milchdrüsengewebe der Hündin während des Pro- und Metöstrus 36 4.3 Milchdrüsengewebe der Hündin während der Laktation 38 4.4 Zum Auftreten von physiologischen und pathologischen Hyperplasien und ihre Differenzierung 41 4.5 Altersbedingte Befunde im Milchdrüsengewebe der Hündin 42 4.6 Veränderungen nach Ovarektomie 45 4.7 Mastitis 47 4.8 Zysten 53 4.9 Akzessorische Zitzen 54 4.10 Sonstige pathologische Befunde 55 4.11 Gesamtpalette und Häufigkeit der nicht tumorösen Veränderungen in der Milchdrüse der Hündin 56 4.12 Kombiniertes Auftreten von nicht tumorösen Veränderungen 59 4.13 Lokalisation der nicht tumorösen Veränderungen im Drüsenkomplex und in der Milchleiste 62 4.14 Epithelzellzählung 67 4.15 Altersverteilung nicht tumoröser Veränderungen 70 5 Diskussion und Schlussfolgerungen 73 6 Zusammenfassung 94 7 Summary 96 8 Literaturverzeichnis 98 9 Anhang 113 Abkürzungsverzeichnis 119 Danksagung 120 Selbständigkeitserklärung 195Im Gegensatz zu den stark bearbeiteten Neoplasien der Mamma des Hundes bestehen nur wenige Kenntnisse über nicht neoplastische Veränderungen der Mamma. Zielstellung der vorliegenden Arbeit war es daher, durch pathologisch- anatomische und histopathologische Untersuchungen der Mamma einen Überblick über Art und Häufigkeit nicht tumoröser Veränderungen des Milchdrüsengewebes zu gewinnen. Als Untersuchungsmaterial dienten 60 Hündinnen verschiedener Rassen und Altersgruppen, die zur Sektion an das Institut für Veterinär- Pathologie der Freien Universität Berlin und in das Staatliche Veterinär- und Lebensmitteluntersuchungsamt Cottbus überwiesen worden waren. Die Mammaleisten der Hündinnen wurden zunächst makroskopisch untersucht. Gleichzeitig wurden die Eierstöcke auf ihren Funktionszustand geprüft. Die in fünfprozentigem Formalin fixierten einzelnen Mammarkomplexe mit Zitze von jeder Hündin wurden in Paraffin eingebettet und in vier verschiedenen Drüsenebenen (Zitzenspitze, Zitzenteil der Zisterne, Drüsenteil der Zisterne und Drüsenkörper) geschnitten. Die Schnitte wurden mit Hämatoxylin und Eosin gefärbt. Weiterhin wurden zur Darstellung der kollagenen Fasern die van Gieson-Färbung und zur Darstellung des Myoepithels immunhistologische Methoden angewendet. Insgesamt wurden 471 Mammarkomplexe (60 Hündinnen)untersucht. Bei 66,2 % der untersuchten Mammarkomplexe wurden histologische Veränderungen der Milchdrüse (Abweichungen vom anöstrischen Normzustand)festgestellt. Dabei ist grundsätzlich zwischen physiologischen und pathologischen nicht neoplastischen Veränderungen zu unterscheiden. Es traten unter den 51 Probanden (Abweichungen vom anöstrischen Normzustand) auf:Physiologische Befunde (n=24) \- Physiologische Hyperplasien während des Pro- und Metöstrus und der Laktation (n=15) \- Involutionen der Milchdrüse im Alter (n=9) Pathologische nicht tumoröse Veränderungen (n=45) \- Pathologische Hyperplasien im Anöstrus (n=9)und nach Entfernung der Eierstöcke (n=4) \- Atrophien nach Entfernung der Eierstöcke (n=10) \- Akute und chronische Mastitiden (n=9) - Zysten (n=4) \- Akzessorische Zitzen (n=9) Bei 20 Tieren traten mikroskopisch feststellbare Neoplasien auf. Die nicht neoplastischen Veränderungen der Mamma waren bevorzugt an den beiden kaudalen Mammarkomplexen sowie im zisternalen und alveolären Drüsenteil zu finden. Aus der Untersuchung geht weiter hervor, dass nicht nur ein Befund bei einer Hündin gefunden werden konnte, sondern es wurden auch 2 oder 3 Befunde am Gesäuge gleichzeitig bei einer Hündin gesehen. ie häufigsten Kombinationen waren pathologische Hyperplasien mit Geschwulst (n=7)oder physiologische Hyperplasien mit Mastitis (n=8). Hinweise auf die Möglichkeit, dass pathologische Hyperplasien Vorläufer neoplastischer Prozesse sein können, lassen sich aus den Untersuchungen nicht ableiten.Whilst neoplasia in canine mammae has been the subject of a considerable number of studies, little is known about non-neoplastic changes of the mammae. The aim of the present study was therefore to conduct pathological anatomical and histopathological examinations of mammary glands in order to draw up a survey of the type and frequency of non-tumorous changes in the lactiferous tissues. The material under examination consisted of 60 bitches of various breeds and ages that had been sent for dissection to the Institute of Veterinary Pathology at the Free University Berlin and to the State Veterinary and Food Inspection Office in Cottbus. First, the bitches' mammary regions were macroscopically examined. At the same time, the functional condition of the ovaries was checked. The individual mammary complexes, including teat, from each bitch were fixed in 5% formalin and embedded in paraffin before being cut at four different levels of the gland (tip of teat, middle of teat, glandular section of the cisterns and glandular section of the alveoli). The cuts were dyed using haematoxylin and eosin. In addition, van Gieson colouring was applied in order to show up the collagenous fibres, and an immunohistological method was used to show up the myoepithelial tissues. A total of 471 mammary complexes (60 bitches) were examined. In 66.2% of the mammary complexes tested, histological changes (deviations from the normal state during anoestrus) were evident in the mammary gland. It is important to distinguish between physiological and non-neoplastic changes. The following findings were made: Physiological findings (n=24) \- physiological hyperplasia during prooestrus, metoestrus and lactation (n=15) \- involution of the mammary glands in old age (n=9) Pathological non-tumorous changes (n=45) \- pathological hyperplasia during anoestrus (n=9) and after removal of the ovaries (n=4) \- atrophy after removal of the ovaries (n=10) \- acute and chronic mastitis (n=9) \- cysts (n=4) \- accessory teats (n=9) In 20 animals microscopic neoplasia was found. The non-neoplastic changes in the mammae were most frequently found in the two caudal mammary complexes, as well as in the cisternal and alveolar sections of the gland. The study also revealed that more than one abnormality might be found in a single bitch, two or three different findings sometimes being discovered simultaneously in the mammary regions of one animal. The most common combinations were hyperplasia with growth (n=7) and hyperplasia with mastitis (n=8). No evidence was found in the study for the possibility that pathological hyperplasia can be the precursor of neoplastic processes

Embryonic Development in Arabidopsis Thaliana: From the Zygote Division to the Shoot Meristem

Postembryonic organ formation of plants is fueled with cells from the stem cell niches in the shoot and root meristems. During the last two decades many players that regulate stem cell maintenance have been identified. With these factors in hand, the mechanisms establishing stem cell niches during embryo development can be addressed. Here we discuss current models of how the shoot meristem stem cell niche arises during Arabidopsis embryo pattern formation

Efficacy and safety of acupuncture for chronic pain caused by gonarthrosis: A study protocol of an ongoing multi-centre randomised controlled clinical trial [ISRCTN27450856]

BACKGROUND: Controlled clinical trials produced contradictory results with respect to a specific analgesic effect of acupuncture. There is a lack of large multi-centre acupuncture trials. The German Acupuncture Trial represents the largest multi-centre study of acupuncture in the treatment of chronic pain caused by gonarthrosis up to now. METHODS: 900 patients will be randomised to three treatment arms. One group receives verum acupuncture, the second sham acupuncture, and the third conservative standard therapy. The trial protocol is described with eligibility criteria, detailed information on the treatment definition, blinding, endpoints, safety evaluation, statistical methods, sample size determination, monitoring, legal aspects, and the current status of the trial. DISCUSSION: A critical discussion is given regarding the considerations about standardisation of the acupuncture treatment, the choice of the control group, and the blinding of patients and observers

Design and planning of a transdisciplinary investigation into farmland pollinators: rationale, co-design, and lessons learned

To provide a complete portrayal of the multiple factors negatively impacting insects in agricultural landscapes it is necessary to assess the concurrent incidence, magnitude, and interactions among multiple stressors over substantial biogeographical scales. Trans-national ecological field investigations with wide-ranging stakeholders typically encounter numerous challenges during the design planning stages, not least that the scientific soundness of a spatially replicated study design must account for the substantial geographic and climatic variation among distant sites. ‘PoshBee’ (Pan-European assessment, monitoring, and mitigation of Stressors on the Health of Bees) is a multi-partner transdisciplinary agroecological project established to investigate the suite of stressors typically encountered by pollinating insects in European agricultural landscapes. To do this, PoshBee established a network of 128 study sites across eight European countries and collected over 50 measurements and samples relating to the nutritional, toxicological, pathogenic, and landscape components of the bees’ environment. This paper describes the development process, rationale, and end-result of each aspect of the of the PoshBee field investigation. We describe the main issues and challenges encountered during the design stages and highlight a number of actions or processes that may benefit other multi-partner research consortia planning similar large-scale studies. It was soon identified that in a multi-component study design process, the development of interaction and communication networks involving all collaborators and stakeholders requires considerable time and resources. It was also necessary at each planning stage to be mindful of the needs and objectives of all stakeholders and partners, and further challenges inevitably arose when practical limitations, such as time restrictions and labour constraints, were superimposed upon prototype study designs. To promote clarity for all stakeholders, for each sub-component of the study, there should be a clear record of the rationale and reasoning that outlines how the final design transpired, what compromises were made, and how the requirements of different stakeholders were accomplished. Ultimately, multi-national agroecological field studies such as PoshBee benefit greatly from the involvement of diverse stakeholders and partners, ranging from field ecologists, project managers, policy legislators, mathematical modelers, and farmer organisations. While the execution of the study highlighted the advantages and benefits of large-scale transdisciplinary projects, the long planning period emphasized the need to formally describe a design framework that could facilitate the design process of future multi-partner collaborations

Systematics of the Neotropical Genus Leptodactylus Fitzinger, 1826 (Anura: Leptodactylidae): Phylogeny, the Relevance of Non-molecular Evidence, and Species Accounts

A phylogeny of the species-rich clade of the Neotropical frog genus Leptodactylus sensu stricto is presented on the basis of a total evidence analysis of molecular (mitochondrial and nuclear markers) and non-molecular (adult and larval morphological and behavioral characters) sampled from > 80% of the 75 currently recognized species. Our results support the monophyly of Leptodactylus sensu stricto, with Hydrolaetare placed as its sister group. The reciprocal monophyly of Hydrolaetare and Leptodactylus sensu stricto does not require that we consider Hydrolaetare as either a subgenus or synonym of Leptodactylus sensu lato. We recognize Leptodactylus sensu stricto, Hydrolaetare, Adenomera, and Lithodytes as valid monophyletic genera. Our results generally support the traditionally recognized Leptodactylus species groups, with exceptions involving only a few species that are easily accommodated without proposing new groups or significantly altering contents. The four groups form a pectinate tree, with the Leptodactylus fuscus group diverging first, followed by the L. pentadactylus group, which is sister to the L. latrans and L. melanonotus groups. To evaluate the impact of non-molecular evidence on our results, we compared our total evidence results with results obtained from analyses using only molecular data. Although non-molecular evidence comprised only 3.5% of the total evidence matrix, it had a strong impact on our total evidence results. Only one species group was monophyletic in the molecular-only analysis, and support differed in 86% of the 54 Leptodactylus clades that are shared by the results of the two analyses. Even though no non-molecular evidence was included for Hydrolaetare, exclusion of that data partition resulted in that genus being nested within Leptodactylus, demonstrating that the inclusion of a small amount of non-molecular evidence for a subset of species can alter not only the placement of those species, but also species that were not scored for those data. The evolution of several natural history and reproductive traits is considered in the light of our phylogenic framework. Invasion of rocky outcrops, larval oophagy, and use of underground reproductive chambers are restricted to species of the Leptodactylus fuscus and L. pentadactylus groups. In contrast, larval schooling, larval attendance, and more complex parental care are restricted to the L. latrans and L. melanonotus groups. Construction of foam nests is plesiomorphic in Leptodactylus but their placement varies extensively (e.g., underground chambers, surface of waterbodies, natural or excavated basins). Information on species synonymy, etymology, adult and larval morphology, advertisement call, and geographic distribution is summarized in species accounts for the 30 species of the Leptodactylus fuscus group, 17 species of the L. pentadactylus group, eight species of the L. latrans group, and 17 species of the L. melanonotus group, as well as the three species that are currently unassigned to any species group.Se presenta una filogenia del género Leptodactylus, un ciado neotropical rico en especies, basada en análises combinados de datos moleculares (marcadores nuclear y mitocondriales) y no moleculares (caracteres de la morfología de adultos y larvas así como de comportamiento) se muestrearon > 80% de las 75 especies reconocidas. Los resultados apoyan la monofília de Leptodactylus sensu stricto, con Hydrolaetare como su grupo hermano. La monofília recíproca de Hydrolaetare y Leptodactylus no requiere considerar a Hydrolaetare como un subgénero o sinónimo de Leptodactylus sensu lato. Se reconocen Leptodactylus sensu stricto, Hydrolaetare, Adenomera y Lithodytes como géneros monofiléticos válidos. Los resultados en general resuelven los grupos tradicionalmente reconocidos de Leptodactylus, con excepciones de algunas especies que son reasignadas sin la necesidad de proponer nuevos grupos o alterar significativamente el contenido de los grupos tradicionales. Los cuatro grupos de especies forman una topología pectinada donde el grupo de L. fuscus tiene una posición basal, seguido por el grupo de L. pentadactylus que es el grupo hermano al clado formado por los grupo de L. latrans y L. melanonotus. Se estimó el impacto de los datos no moleculares en los resultados, comparándose los resultados de evidencia total con los de los análises de datos moleculares solamente. Los datos no moleculares representan un 3.5% de la matriz de evidencia total, pero estos datos tuvieron un impacto significativo en los resultados del análisis de evidencia total. En el análisis estrictamente molecular solamente un grupo de especies resultó monofilético, y el apoyo difirió en 86% de los 54 ciados de Leptodactylus compartidos entre los dos análises. A pesar que datos no moleculares no fueron incluidos para Hydrolaetare, la exclusión de evidencia no molecular resultó en el género estar dentro de Leptodactylus, demostrando que la inclusión de evidencia no molecular pequeña para un subgrupo de especies altera no solamente la posición topológica de esas especies, sino tambien de las especies para las cuales dichos datos no fueron codificados. La evolución de patrones de historia natural y reprodución se evalúan en el contexto filogenético. La invasión de afloramientos rocosos y la construción de cámaras de reprodución subterraneas está limitada a los grupos de Leptodactylus fuscus y L. pentadactylus, mientras que la oofagia larval está restringida al grupo de L. pentadactylus. Por otro lado, los cárdumenes larvales, la proteción del cárdumen, y otros comportamientos parentales complejos carecterizan al clado formado por los grupos de especies de L. latrans y L. melanonotus. Los resúmenes de especies incluyen información de sinonimias, etimología, morfología de adultos y larvas, cantos, y distribución geográfica para las 30 especies del grupo de Leptodactylus fuscus, 17 especies del grupo L. pentadactylus, ocho especies del grupo de L. latrans, 17 especies del grupo de L. melanonotus, así como para las tres especies que actualmente no se encuentran asociadas a ninguno de los grupos de especies.Taran Grant was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico Proc. 307001/2011-3 and Fundação de Amparo à Pesquisa do Estado de São Paulo Proc. 2012/10000-5

Label-free detection of neuronal differentiation in cell populations using high-throughput live-cell imaging of PC12 cells.

<p>Detection of neuronal cell differentiation is essential to study cell fate decisions under various stimuli and/or environmental conditions. Many tools exist that quantify differentiation by neurite length measurements of single cells. However, quantification of differentiation in whole cell populations remains elusive so far. Because such populations can consist of both proliferating and differentiating cells, the task to assess the overall differentiation status is not trivial and requires a high-throughput, fully automated approach to analyze sufficient data for a statistically significant discrimination to determine cell differentiation. We address the problem of detecting differentiation in a mixed population of proliferating and differentiating cells over time by supervised classification. Using nerve growth factor induced differentiation of PC12 cells, we monitor the changes in cell morphology over 6 days by phase-contrast live-cell imaging. For general applicability, the classification procedure starts out with many features to identify those that maximize discrimination of differentiated and undifferentiated cells and to eliminate features sensitive to systematic measurement artifacts. The resulting image analysis determines the optimal post treatment day for training and achieves a near perfect classification of differentiation, which we confirmed in technically and biologically independent as well as differently designed experiments. Our approach allows to monitor neuronal cell populations repeatedly over days without any interference. It requires only an initial calibration and training step and is thereafter capable to discriminate further experiments. In conclusion, this enables long-term, large-scale studies of cell populations with minimized costs and efforts for detecting effects of external manipulation of neuronal cell differentiation.</p

Comparison of Gini indices for the hold-out data, the technically independent data set, the biologically independent, and the experimentally distinct data set.

<p>(A) stimulation without mitomycin (B) stimulation under mitomycin treatment. The hold-out data and technically independent data sets are marked by the solid lines with blue triangles or red circles, respectively. Additionally, the dashed/dotted lines in panel (A) depict the biologically independent/experimentally distinct test sample, wherein the triangles and circles mark the respective technically independent replicates within this setup. Each point represents the median Gini index of the five independent FDA classifiers and the error bars mark the % c.i. A Gini index of corresponds to perfect separation of differentiated and undifferentiated images. Since in the training data set and in the biologically independent data set the final cell density was high, the detection performance degrades such that the Gini index declines at later days. This is due to increasing build up of cell clumps rendering the differentiated cell morphology much harder to detect, even to the human eye. The experimentally distinct data set started from a lower cell density such that fewer cell clumps occurred, making the detection of differentiation feasible until day .</p