Etablierung von standardisierten Probennahmeplänen für Organe und Gewebe porziner Tiermodelle in der biomedizinischen Forschung

Porzine Tiermodelle haben in der biomedizinischen Forschung in den letzten Jahren aufgrund der dem Menschen sehr ähnlichen Physiologie und Anatomie des Schweines und Fortschritten auf dem Gebiet der Gentechnik, erheblich an Bedeutung gewonnen. Schweinemodelle werden in verschiedenen Bereichen der translationalen Medizin genutzt. Hierzu zählen die Grundlagenforschung, die Erfassung von Erbkrankheiten des Menschen anhand gentechnisch veränderter „maßgeschneiderter“ porziner Modelle, die Erforschung von Pathogenitätsmechanismen, die toxikopathologische Evaluation neuer Wirkstoffe und die Entwicklung und Erprobung neuer therapeutischer Ansätze. Für diverse Nagetierspezies existieren etablierte Protokolle zur standardisierten Gewinnung von Organ- und Gewebeproben für toxikopathologische Untersuchungen. Für Schweinemodelle hingegen waren derartige Probennahmepläne bislang nicht verfügbar. In der vorliegenden Arbeit wurden standardisierte Probennahmeprotokolle für mehr als fünfzig verschiedene porzine Organe und Gewebe entwickelt und praktisch erprobt. Die Protokolle ermöglichen die reproduzierbare, standardisierte Generierung von repräsentativem Probenmaterial. Die Probennahmepläne beinhalten detaillierte Angaben zur Entnahmestelle, der Anzahl der zu entnehmenden Proben, ihrer Orientierung und weiteren Prozessierung für unterschiedliche qualitative und quantitative morphologische Analyseverfahren sowie molekularbiologische und biochemische Untersuchungen. Neben relevanten anatomischen Besonderheiten des Schweines und Hinweisen zur Sektionstechnik werden die Probennahmeprotokolle für sämtliche Organe und Gewebe durch detaillierte Schemazeichnungen sowie makroskopische und histologische Abbildungen veranschaulicht und durch den zu veranschlagenden Zeit-, Kosten- und Personalaufwand ergänzt. In Anpassung an unterschiedliche Studienziele werden für die einzelnen Organe und Gewebe unterschiedliche Probennahmeprotokolle (Typ-I – Typ-III) vorgestellt. Diese können frei kombiniert und an das experimentelle Design einer jeweiligen Studie angepasst werden. Typ-I Probennahmeprotokolle sind für Übersichtsuntersuchungen morphologischer Alterationen und molekulare Analysen, beispielweise in toxikopathologischen Studien, vorgesehen. Hierbei erfolgt die Entnahme von Proben für histopathologische Routineuntersuchungen und molekularbiologische Analysen aus definierten Lokalisationen mit festgelegter Orientierung. Typ-II und Typ-III Probennahmeprotokolle wurden zur Generierung von Proben für detaillierte Untersuchungen einzelner Organe und Gewebe mit unterschiedlichen Analyseverfahren (Typ-II), beziehungsweise für die Erstellung von Gewebe-Biobanken (Typ-III) entwickelt. Zur reproduzierbaren Generierung repräsentativer Proben für ein breites Spektrum an quantitativen und qualitativen Untersuchungen werden die Entnahmelokalisationen der Proben in Typ-II und Typ-III Studien durch systematisch zufällige Verfahren bestimmt. Die Gewinnung von für verschiedene Analyseverfahren geeigneten Proben ermöglicht die Durchführung initial nicht geplanter Untersuchungen zur Beantwortung von sich erst im Verlauf einer Studie ergebenden Fragestellungen. Dies kann zur Verringerung der in einer Studie benötigten Tierzahl beitragen. Die im Februar 2016 im Fachjournal Toxicologic Pathology veröffentlichten Probennahmeprotokolle leisten einen Beitrag zur standardisierten Gewinnung von qualitativ hochwertigen, repräsentativen Probenmaterials als Voraussetzung für die Vergleichbarkeit von Ergebnissen unterschiedlicher Studien porziner Tiermodelle in der biomedizinischen Forschung.During the past decade(s), porcine animal models have gained a steadily growing popularity in biomedical research. This is due to the similar physiology and anatomy of pigs and humans, and the technical advances in genetic modification of the porcine genome. Porcine models are used in diverse areas of translational medicine, including basic research, generation of “tailored”, genetically modified pig models of human diseases, investigations of pathophysiological processes, surgery, transplantation research, toxicity testing of pharmacological substances, as well as development of new therapeutic strategies. For various rodent species used as experimental animal models, standardized sampling guidelines for reproducible collection of organ and tissue specimen exist. For porcine models, however, such guidelines have not been published so far. In the present work, standardized sampling guidelines for more than 50 porcine organs and tissues were developed to facilitate the reproducible generation of representative specimen. For each organ and tissue, the sampling guidelines indicate the relevant anatomic features, and provide precise advices on the appropriate necropsy techniques and the estimated time and personnel expenses for sampling. Illustrated by detailed schematic drawings, macroscopic pictures and microscopic images of histological slides, the protocols specify the sampling locations and give recommendations on the adequate number of samples, their orientation, and the subsequent processing of the specimen for different qualitative and quantitative morphological investigations, as well as for molecular and biochemical analyses. According to the aims and purposes of different studies, different types of sampling protocols are provided (type I-III), which can be individually combined and adapted to the experimental design of a specific study. Type-I sampling protocols are designed for studies in which a broad set of organs/tissues is examined in an overview fashion, using routine histopathological techniques and/or standard molecular analyses, as e.g., in general toxicity studies. Here, the samples are collected from deliberately defined locations and in predefined orientations. Type-II and type-III sampling protocols were developed for advanced, detailed studies of distinct organs and tissues, using a wide range of different analytical methods (type-II), respectively for biobank projects (type-III), where particularly large numbers of different types of samples from a wide range of different organs and tissues are required. To enable the reproducible generation of representative specimen, the sampling locations in type-II and type-III studies are determined by efficient, systematic random sampling strategies. From each of the sampled locations, sub-samples are taken and processed for a broad spectrum of different analysis methods, including analyses not necessarily specified or planned at the time point of sampling. A suchlike ‘‘forward-looking’’ sampling strategy allows to perform additional experiments without a repeated generation of new samples, and might thus contribute to reduce the number of animals in a study. The sampling protocols presented in this study were published in “Toxicologic Pathology” in February 2016. Their broad application will ensure the efficient generation of representative, high-quality samples of porcine organs and tissues, and contribute to the reproducibility of results and the intra-/interstudy comparability of research projects involving pigs as animal models

Tissue Sampling Guides for Porcine Biomedical Models

This article provides guidelines for organ and tissue sampling adapted to porcine animal models in translational medical research. Detailed protocols for the determination of sampling locations and numbers as well as recommendations on the orientation, size, and trimming direction of samples from approximate to 50 different porcine organs and tissues are provided in the Supplementary Material. The proposed sampling protocols include the generation of samples suitable for subsequent qualitative and quantitative analyses, including cryohistology, paraffin, and plastic histology;immunohistochemistry;in situ hybridization;electron microscopy;and quantitative stereology as well as molecular analyses of DNA, RNA, proteins, metabolites, and electrolytes. With regard to the planned extent of sampling efforts, time, and personnel expenses, and dependent upon the scheduled analyses, different protocols are provided. These protocols are adjusted for (I) routine screenings, as used in general toxicity studies or in analyses of gene expression patterns or histopathological organ alterations, (II) advanced analyses of single organs/tissues, and (III) large-scale sampling procedures to be applied in biobank projects. Providing a robust reference for studies of porcine models, the described protocols will ensure the efficiency of sampling, the systematic recovery of high-quality samples representing the entire organ or tissue as well as the intra-/interstudy comparability and reproducibility of results

The Munich MIDY Pig Biobank - A unique resource for studying organ crosstalk in diabetes

OBJECTIVE: The prevalence of diabetes mellitus and associated complications is steadily increasing. As a resource for studying systemic consequences of chronic insulin insufficiency and hyperglycemia, we established a comprehensive biobank of long-term diabetic INSC94Y transgenic pigs, a model of mutant INS gene-induced diabetes of youth (MIDY), and of wild-type (WT) littermates. METHODS: Female MIDY pigs (n = 4) were maintained with suboptimal insulin treatment for 2 years, together with female WT littermates (n = 5). Plasma insulin, C-peptide and glucagon levels were regularly determined using specific immunoassays. In addition, clinical chemical, targeted metabolomics, and lipidomics analyses were performed. At age 2 years, all pigs were euthanized, necropsied, and a broad spectrum of tissues was taken by systematic uniform random sampling procedures. Total beta cell volume was determined by stereological methods. A pilot proteome analysis of pancreas, liver, and kidney cortex was performed by label free proteomics. RESULTS: MIDY pigs had elevated fasting plasma glucose and fructosamine concentrations, C-peptide levels that decreased with age and were undetectable at 2 years, and an 82% reduced total beta cell volume compared to WT. Plasma glucagon and beta hydroxybutyrate levels of MIDY pigs were chronically elevated, reflecting hallmarks of poorly controlled diabetes in humans. In total, ∼1900 samples of different body fluids (blood, serum, plasma, urine, cerebrospinal fluid, and synovial fluid) as well as ∼17,000 samples from ∼50 different tissues and organs were preserved to facilitate a plethora of morphological and molecular analyses. Principal component analyses of plasma targeted metabolomics and lipidomics data and of proteome profiles from pancreas, liver, and kidney cortex clearly separated MIDY and WT samples. CONCLUSIONS: The broad spectrum of well-defined biosamples in the Munich MIDY Pig Biobank that will be available to the scientific community provides a unique resource for systematic studies of organ crosstalk in diabetes in a multi-organ, multi-omics dimension