An isolated haemoperfused distal cow limb model for experimental studies on the pathogenesis of bovine laminitis

Titel & Inhaltsverzeichnis 1\. Einleitung 2\. Literaturübersicht 3\. Material & Methoden 4\. Ergebnisse Teil 1 5\. Ergebnisse Teil 2 6\. Ergebnisse Teil 3 7\. Ergebnisse Teil 4 8\. Diskussion 9\. Zusammenfassung 10\. Summary 11\. Literaturverzeichnis 12\. Anhang SOP 13\. Danksagung SelbstständigkeitserklärungDas Wissen über die Pathogenese der Klauenrehe ist bis heute trotz fortschreitender intensiver Forschungsarbeit unvollständig. Bei Untersuchungen zu diesem Thema sind isoliert perfundierte distale Gliedmaßen ein viel versprechendes Modell, um die biologischen Mechanismen zu ergründen, die hinter diesem Syndrom liegen. Dieses Modell stellt aus ethischer wie auch aus finanzieller Sicht eine Alternative zum Tierversuch dar. Ziel der vorliegenden Arbeit war die Entwicklung eines Modells der isoliert hämoperfundierten distalen Rindergliedmaße, basierend auf dem bereits vorhandenen Modell der isoliert hämoperfundierten Schweinegliedmaße, das für die Prüfung pharmakologischer Substanzen verwendet wird. Die isolierten distalen Gliedmaßen wurden von routinemäßig geschlachteten Rindern gewonnen und im Labor der Firma Vitro-tec Entwicklungsgesellschaft mbH, Berlin, perfundiert. Die Standardapparatur ermöglicht eine Perfusion für fünf Stunden unter nahezu physiologischen Verhältnissen. Die Ausgangswerte für den Perfusionsfluss und -druck wurden basierend auf Literaturangaben für Pferd und Schwein eingestellt. Der Vergleich verschiedener Versuchsgruppen ergab als optimalen Perfusionsfluss einen Wert von 190 ml/ min. Die Sauerstoffsättigung wurde bei 100 % und der pH-Wert zwischen 7,35 7,45 gehalten. Glukose wurde als Nährstoff zugesetzt. Die Gewichtzunahme der Gliedmaße sowie der Perfusionsdruck und der Organwiderstand wurden als Vitalitätsindikatoren verwendet. Die Gliedmaße durfte während der Perfusion nicht über 10 % an Gewicht zunehmen und der Perfusionsdruck durfte nicht über 150 mmHg steigen und nicht unter 30 mmHg absinken. Zusätzlich wurden systematische licht- und elektronenmikroskopische Untersuchungen an Gewebeproben aus allen Segmenten der Klauen durchgeführt, um die Vitalität des Gewebes anhand einer Standardliste morphologischer Kriterien zu überprüfen. Ausgewählte Perfusionen wurden thermographisch auf cold spots überprüft, die einen Indikator für ischämische Bezirke darstellen. Als Perfusionsmedium zeigte sich Vollelektrolytlösung, angereichert mit gewaschenen Erythrozyten (Hämatokrit 8 10 %) als am besten geeignet. Das Modell wurde in einer Serie von 23 Experimenten getestet. Dabei wurden für die Pathogenese der Klauenrehe relevante bioaktive Moleküle in ihrer Wirkung auf den Perfusionsdruck und die Gewebeveränderungen untersucht. Die modernen Hypothesen zur Pathogenese der Klauenrehe schreiben Störungen der Mikrozirkulation im dermalen Gefäßsystem der Klaue und direkten Schäden der Gefäßendothelien eine zentrale Rolle während des initialen Phase der Reheentwicklung zu. Diese Störungen und Schäden werden durch Toxine, vasoaktive Substanzen oder metobolische Abbauprodukte hervorgerufen. Daher wurden in den Perfusionsexperimenten dieser Arbeit die Effekte einiger der am häufigsten im Zusammenhang mit der Rehepathogenese diskutierten Substanzen getestet. Das Gewebe wurde unter Sauerstoffdefizit und verminderter Flussrate perfundiert und es wurde untersucht, ob Endotoxine, Laktat oder Histamin Änderungen der Perfusion, der Gefäße und Gewebveränderungen auslösen, die für die Reheentwicklung relevant sind. Es konnte gezeigt werden, dass eine Sauerstoffsättigung von 15 30 % über den Zeitraum von vier Stunden sowie die Histamin und Endotoxin von E. coli Veränderungen in den Perfusionsparametern und in der Gewebestruktur auslösen. Es konnten Druckanstieg, erhöhter Organwiderstand, Veränderungen der Gefäßpermeabilität, Ödembildung sowie epidermale Zellschäden nachgewiesen werden. Diese Veränderungen fügen sich nahtlos in die aktuellen Rehehypothesen ein. Der Zeitrahmen von fünf Stunden erlaubt jedoch keine endgültige Aussage darüber, ob dieser Zustand letztendlich zu einer Klauenrehe führt. Das Modell ist für verschiedene spezifische veterinärmedizinische Fragestellungen insbesondere zur weiteren Erforschung der Huf- und Klauenrehe interessant, aber auch für die Humanmedizin attraktiv. Vielfältige pharmakologische und toxikologische Einsatzgebiete sind denkbar. Durch eine Kombination mit modernen Analysen (z.B. PCR) von Gewebeproben aus den perfundierten Gliedmaßen lassen sich die Reaktionen der Zellen auf die eingesetzten Faktoren auf molekularer Ebene detektieren. Die Kombination des hier vorgestellten neuen ex vivo Modells mit molekularbiologischen Methoden insbesondere der real-time PCR zur quantitativen Analyse von Effekten auf das Expressionsprofil der Zellen wird für künftige Untersuchungen empfohlen.The actual knowledge on the pathomechanisms of bovine laminitis is still incomplete despite of progressively increasing research activities. Isolated perfused distal limbs are a promising model to study the biological mechanisms that lie behind the laminitis syndrome. For ethical and financial reasons experiments using this model are a preferable alternative to animal experiments. The aim of this work was to develop an isolated haemoperfused distal cow limb model basing on an already existing porcine limb model used already for studying the effects of pharmaceuticals. The isolated limbs were obtained from routinely slaughtered cows and subsequently perfused in the laboratory of a local biotech company (Vitro-tec Entwicklungsgesellschaft mbH, Berlin). A standard perfusion apparatus enables isolated limb perfusion for up to 5 hours under close to physiological conditions. Perfusion pressure and flow were calculated and adjusted basing on values available for horses and pigs. The comparison of the different initial experiments showed that 190 ml/min is an optimal value for the flow of perfusate. The oxygen saturation was adjusted to 100 % and the pH preset between 7.35 and 7.45. Glucose was added as nutrient. The weight increase of the limb and the perfusion pressure served as vitality indicators. The threshold for an acceptable weight gain of the distal limb was 10 % and for the upper and lower values of perfusion pressure 150 mmHg and 30 mmHg respectively. In addition, systematic light- and electron microscopic examination of tissue samples from all segments of the claws was carried out to evaluate tissue vitality according to a list of standardised morphological criteria. A thermographic camera was used to detect cold spots as an indicator for ischemic areas. A complete electrolyte solution added with washed erythrocytes (haematocrit 8 10 %) turned out to be optimal for perfusion and was used as standard perfusate. The model was challenged in a series of 23 experiments. Thereby the effects of candidate bioactive molecules relevant for the pathogenesis of laminitis on perfusion pressure and resulting tissue alterations were tested. The modern hypotheses on the pathogenesis of laminitis attribute a central role to disturbances in microcirculation and direct damage to the vascular endothelium in the initial phase of the development of laminitis. These disturbances and alterations are caused by toxins, vaso-active substances of metabolic by- products. Therefore, in the perfusion experiments of this work the effects of some of these substances most frequently discussed in relation to laminitis have been tested. The claw tissue was perfused under oxygen deficiency and reduced flow rates. Furthermore the model was challenged by endotoxin, lactate and histamine to detect whether these substances produce changes in perfusion or structure of the vascular system or cause tissue alterations relevant for the development of laminitis. It was possible to show that an oxygen deficiency between 15 30 % for 4 hours and agents like histamine and E. coli endotoxin provoke changes in perfusion parameters and alterations in tissue structure. The following changes were detected: An increase in pressure and organ resistance, alterations of vascular permeability, formation of oedema and cell damage. These alterations perfectly fit in the actual hypotheses on the pathogenesis of laminitis. The limited period of 5 hours however does not permit final conclusions whether this condition finally would lead to clinical laminitis. The model introduced here is interesting to investigations on a variety of specific questions in Veterinary Medicine. It is attractive in particular for further research into equine and bovine laminitis. Furthermore it is also attractive for research in human medicine with possible applications in Pharmacology and Toxicology. In combination with modern analytical methods such as PCR techniques the reactions of cells to the challenging factors could be detected on the molecular level. The combination of the described novel ex vivo model with real-time PCR for quantitative analysis of effects on the cellular protein expression is recommended for future investigations using this model

Nature and Validity of Complex Problem Solving

This thesis investigates the nature and validity of complex problem solving (CPS). The main focus lies on analyses of three research questions dealing with CPS’ (1) internal structure, its (2) structural stability combined with comparisons of performance differences across groups, and its (3) construct validity. In previous research, results on CPS’ (1) internal factor structure have been solely based on samples with high cognitive performance, (2) structural stability of CPS across groups has not been tested yet, and analyses of performance differences across groups have been rather scarce. Further, results on (3) construct validity dealing with the relation of CPS to other measures of cognitive performance are inconsistent. By applying a multiple task approach called MicroDYN to measure CPS, (1) internal factor structure is tested in groups of high school students, university students, and blue-collar workers, varying considerably in age and cognitive performance. Thereby, the interplay of theoretically derived CPS dimensions (use of strategies, knowledge acquisition, knowledge application) and their relation to characteristics of CPS tasks is analyzed in-depth. Further, for the first time in CPS research, (2) structural stability of CPS across groups is evaluated before performance differences are compared. Finally, (3) construct validity of CPS is investigated focusing on CPS’ incremental validity beyond reasoning in explaining school performance. In summary, the present work addresses several gaps in existing research. In chapter 1, the construct CPS and the measurement approach MicroDYN are introduced. Subsequently, previous results on internal structure, structural stability, performance differences, and construct validity are reported and followed by a brief description of the four empirical papers that are the main body of this thesis. The full papers are located in chapters 2 to 5. Whereas the first two papers are already in press after having successfully passed peer-review, the latter two papers are currently under revision. Furthermore, chapter 1 and chapter 6 refer to other papers including supplementary contributions of the author of this thesis on CPS research, which are listed as “additional papers” on page 10. The first empirical paper included in this thesis analysed internal structure and construct validity of CPS using data from a sample of university students. A 2-dimensional structure comprising the dimensions of knowledge acquisition and knowledge application fitted the data best and use of strategies as third dimension did not yield important information on CPS performance beyond knowledge acquisition. Further, CPS showed incremental validity beyond reasoning in explaining variance in school grades (cf. chapter 2). The second paper investigated structural stability and performance differences in CPS across high school students of different ages in a Hungarian sample and related performance in CPS to reasoning and parental education. Measurement invariance analyses based on structural equation models confirmed structural stability of CPS across groups and revealed that CPS performance increased with higher age. Further, CPS showed incremental validity beyond reasoning in analyses based on manifest variables. Moreover, parental education predicted performance in CPS (cf. chapter 3). The third contribution expands analyses of the second paper by comparing the structure of CPS across samples of high school students, university students, and blue-collar workers varying considerably in age and cognitive performance. As expected, CPS was measured invariant and participants in the academic track (university students, senior high school students) performed significantly better than participants in a non-academic track (blue-collar workers, junior high school students; cf. chapter 4). The fourth paper investigated structural stability across gender and nationality by comparing German and Hungarian high school student samples. CPS was measured invariant and analyses on performance differences showed that Germans outperformed Hungarians and males outperformed females. However, subsequent analyses revealed that performance differences could be partly explained by Hungarian females making less use of an efficient strategy (i.e., vary-one-thing-at-a-time) to generate knowledge. Further, influence of use of strategies as prerequisite for performance in CPS is discussed (cf. chapter 5). Chapter 6 provides a general discussion of this research. Papers consistently yielded a 2-dimensional internal structure of CPS comprising the dimensions knowledge acquisition and knowledge application. CPS was measured invariant across samples varying in age, gender, and nationality, which is a prerequisite for comparing competency across groups. Analyses of performance differences not only showed that participants with higher education performed better in CPS, but also deepened the understanding of the influence of use of strategies as prerequisite of CPS performance. Further, encouraging results on incremental validity of CPS beyond reasoning were found. After this summary of results, strengths of the papers are outlined and shortcomings combined with an outlook for future research are provided. In this respect, five issues are tackled: the operationalization of CPS, its convergent and criterion validity, relation of CPS to general mental ability, use of process data, and trainability of CPS. In summary, this thesis advances knowledge about CPS and emphasizes its usefulness as an indicator of cognitive performance in addition to traditional measures