Vergleichende fluoreszenzoptische und liquorcytologische Untersuchungen an Versuchstieren nach intracerebraler Mumpsvirusapplikation

Eine der häufigsten viralen Infektionskrankheiten im Kindesalter ist gegenwärtig die Parotitis epidemica. Mit dem Auftreten einer abakteriellen Meningitis bei Kindern als Folge einer Mumpsvirusinfektion ist in über 80% der Fälle zu rechnen. 1. In serologisch bestätigten Mumpsfällen wurden Liquorproben von Kindern mit einer Meningitis immunfluoreszenzoptisch untersucht. In den lympho-monozytären Liquorzellen konnte das Mumpsantigen in 64% der Fälle nachgewiesen werden. 2. Der immunfluoreszenzoptisch Nachweis des Mumpsantigens in Liquorzellen stellt eine Erweiterung der diagnostischen Möglichkeiten bei serösen Meningitiden des Kindes unklarer Äthiologie dar. 3. Während der Prüfung von Mumpsviren reagieren intracerebral infizierte Affen mit einer statistisch gesicherten Erhöhung der Liquorzellzahl. Bei Affen, die mit einer neuropathogenen Variante infiziert waren, sind die Liquorzellzahlen auch 28 Tage p.i. signifikant höher im Vergleich zu mit Impfstoff infizierten Tieren. Der Nachweis des Mumpsantigens in den Liquorzellen beweist die Spezifität der experimentellen Mumpsmeningitis bei Affen. Das virale Antigen wird nur innerhalb der ersten Versuchswoche in den Liquorzellen detektiert, nach 3 – 4wöchiger Versuchsdauer ist das Mumpsvirus im Liquor der Affen nicht mehr nachweisbar. Diese Befunde sind mit den Befunden bei natürlichen Mumpsmeningitiden der Kinder vergleichbar. 4. Minischweine und Katzen reagieren auf intracerebral appliziertes Mumpsvirus mit charakteristischen liquorzytologischen Veränderungen, die mit den Befunden beim Affen korrelieren. Sie reagieren auf intracerebral inokuliertes Mumpsvirus bereits in der ersten Woche mit einer deutlichen Liquorpleozytose. Die Höhe der Zellzahl im Liquor ist bei Mumpswildviren (α = 0,05) bedeutend größer als bei den Impfviren. Die Zellzahlerhöhung ist nach 8 Wochen nicht auf die Normalwerte zurückgegangen. 5. Der immunfluoreszenzoptische Nachweis des Mumpsantigens in den Liquorzellen der Minischweine und Katzen ist zeitlich begrenzt. Es ergaben sich Unterschiede zwischen Mumpsviren vom Wild- und Impftyp. Der Nachweis des viralen Antigens in den Liquorzellen war 4 Wochen p.i. nicht mehr möglich. 6. Das Differentialzellbild des Liquors ist bei allen Tiermodellen durch lympho-monozytäre Zellen gekennzeichnet, wobei monozytäre Zellformen zu jedem Zeitpunkt der Infektion überwiegen. 7. Vor der Anwendung von Lebendimpfstoffen beim Menschen ist eine tierexperimentelle Sicherheitsprüfung notwendig. Die Einbeziehung der Liquordiagnostik (Verlauf der Pleozytose und der fluoreszenzoptische Antigennachweis in den Liquorzellen) ist eine wesentliche Ergänzung zur Charakterisierung der neurotropen Eigenschaften des viralen Mumpsantigens.One of the most common viral infectious diseases in childhood is currently parotitis epidemica. The occurrence of abacterial meningitis in children as a result of mumps virus infection can be expected in more than 80% of cases. 1. In serologically confirmed mumps cases, cerebrospinal fluid samples from children with meningitis were examined by immunofluorescence. Mumps antigen was detected in the lympho-monocytic cerebrospinal fluid (CSF) cells in 64% of cases. 2. Immunofluorescence detection of mumps antigen in CSF cells represents an extension of diagnostic possibilities in serous meningitis of the child of unclear etiology. 3. During mumps virus testing, intracerebrally infected monkeys respond with a statistically confirmed increase in CSF cell count. In monkeys infected with a neuropathogenic variant, CSF cell counts are significantly higher even 28 days p.i. compared with vaccine-infected animals. Detection of mumps antigen in CSF cells demonstrates the specificity of experimental mumps meningitis in monkeys. The viral antigen is detected in the CSF cells only within the first week of the experiment; after 3 - 4 weeks of the experiment, the mumps virus is no longer detectable in the CSF of the monkeys. These findings are comparable to the findings in natural mumps meningitis of children. 4. Minipigs and cats respond to intracerebrally applied mumps virus with characteristic liquor cytologic changes that correlate with the findings in monkeys. They respond to intracerebrally inoculated mumps virus with marked CSF pleocytosis as early as the first week. The level of cell number in CSF is significantly greater in wild mumps virus (α = 0.05) than in vaccine virus. The cell count increase did not return to normal values after 8 weeks. 5. Immunofluorescence detection of mumps antigen in CSF cells of minipigs and cats is temporal. Differences were found between wild-type and vaccine-type mumps viruses. Detection of viral antigen in CSF cells was no longer possible 4 weeks p.i.. 6. The differential cell pattern of CSF is characterized by lympho-monocytic cells in all animal models, with monocytic cell forms predominating at each time point of infection. 7. Animal safety testing is required prior the use of live vaccines in humans. The inclusion of CSF diagnostics (course of pleocytosis and fluorescence antigen detection in CSF cells) is an essential addition to characterize the neurotropic properties of the viral mumps antigen

Combining Optogenetics and fMRI to Study Cerebral Networks in Animal Models

Functional magnetic resonance imaging is a well-established technique to examine brain activity and networks in animal models. In contrast to electrophysiology, optogenetics enables the control of specific cell types. A second advantage is that optogenetic stimulation can trigger excitatory as well as inhibitory effects. This study investigated optogenetic manipulation of neuronal activity and connectivity changes in mice and rats, using fMRI as an analytical method. The work addresses (1) glutamate release in mice and its use as a neurotransmitter and (2) oxytocin release in rats and its impact on neuronal networks. In fMRI, changes of metabolism and blood flow are described by the hemodynamic response function. The influence of the optogenetic stimulation on the hemodynamic response function was examined and characterized for both species. In the first part, glutamatergic neurons in the left hippocampus of mice were optogenetically stimulated via channelrhodopsin-2. Sham animals, without virus injection, were used as a control group to study unspecific effects induced by the laser stimulation. In transgenic (α-CamKII-Cre) mice, the direct hippocampal activation was investigated as well as its projections to other regions. Additionally, the impact of the dorsal-ventral position of the optical fiber on the hippocampal-prefrontal projections was investigated. We could demonstrate that the hemodynamic response measured in the hippocampus of mice reached its maximum earlier compared to the hemodynamic response function in humans. An explanation for this observation may be the smaller body size and the faster metabolism of mice. A highly significant increase of the BOLD signal was found for the optogenetic stimulation of glutamatergic neurons in the left hippocampus and its projecting areas, like the contralateral hippocampus and prefrontal regions. Furthermore, a negative correlation of prefrontal activation and the fiber depth was measured, which may be explained by the larger amount of stimulated neurons. In the second part, we optogenetically stimulated oxytocin-releasing neurons either in the amygdala or in the paraventricular nucleus of rats. We hypothesize that changes in the oxytocin associated networks of the basal ganglia and the olfactory system should result from the optogenetic stimulation. Long-term changes in connectivity were investigated. Therefore, changes in correlations between different brain regions were calculated using the resting-state measurements before and after the optogenetic stimulation. Moreover, seed regions of defined functional networks were determined and voxel-based changes in resting-state correlation to the seed region were investigated. Additionally, short-term connectivity changes were examined in a psychophysiological interaction analysis as well as the direct activation induced by the laser stimulation. We found that both channelrhodopsin-2 groups showed increased connectivity in the olfactory and basal ganglia networks compared to the control group. However, no short-term network changes were observed comparing the laser on and laser off condition. This might be explained by the hormonal characteristics of oxytocin, leading to a more global and prolonged response. To summarize, this thesis demonstrates the successful combination of optogenetics and fMRI as a tool for basic neuropsychiatric research. It proves the successful manipulation, not only of single neurons, but also of neuronal networks in vivo by optogenetic stimulation

Development of a farm-specific real-time quantitative RT-PCR assay for the detection and discrimination of wild-type porcine reproductive respiratory syndrome virus and the vaccine strain in a farm under eradication

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases of swine causing severe economic losses worldwide, therefore intensive efforts are taken to eliminate PRRS virus (PRRSV) from infected herds for complete eradication. The most efficient, fastest but at the same time the most expensive eradication method is depopulation-repopulation. In order to reduce costs, a number of farms prefer to perform their eradication process with continuous production using modified live vaccine (MLV) immunisation. However, the commercial PRRSV RT-PCR kits do not have the capacity to discriminate infected from vaccinated animals. In this paper, we describe a simple discriminatory duplex TaqMan RT-PCR assay based on common forward and reverse primers, as well as two differently labelled MLV- and wild-type PRRSV-specific probes. The discriminatory PCR test we designed is a fast and efficacious method for processing large quantities of samples. The assay is cheap, flexible, easy to apply in different herds using different MLVs, but should be checked, and can be modified based on the sequence data obtained during the permanent monitoring examinations. Owing to its simplicity the test can serve as a significant complementary assay for PRRS control and elimination/eradication

The desert ant's celestial compass system

Die Wüstenameise, Cataglyphis, orientiert sich vor allem mittels Wegintegration. Über einen Heimvektor, den sie aus Distanz und Richtung einzelner Wegabschnitte berechnet, kann sie auf dem kürzesten Weg zu ihrem Ausgangspunkt zurückkehren. Zur Bestimmung der zurückgelegten Strecken verwendet sie einen Schrittintegrator. Die Laufrichtung wird hauptsächlich über Himmelsinformationen (Polarisationsmuster, Sonnenstand und Spektral- und Intensitätsgradienten) definiert. In dieser Arbeit über die Orientierungsfähigkeit von Cataglyphis fortis wird die Rolle des Polarisationskompasses bei der Bestimmung der Laufrichtung untersucht. In verschiedenen Versuchen wurden der Polarisationskompass der Ameise mit Hilfe eines Polarisationsfilters gezielt manipuliert und künstliche Konfliktsituationen erzeugt. Die Richtungsbestimmung wurde vom Polarisationskompass dominiert, wenn allein die Information des Polarisationskompass und idiothetische Richtungsinformation zur Verfügung standen. Erfuhren die Ameise widersprüchliche Informationen von Sonnen- und Polarisationskompass, berechneten sie eine mittlere Heimlaufrichtung, was eine gemeinsame neuronale Verarbeitung der beiden Signale voraussetzt. Diese These wurde durch Transferexperimente gestützt. In einer weiteren Versuchsreihe wurde die Wahrnehmung des Polarisationsmusters durch direkte Manipulation der entsprechenden Region (DRA) im Ameisenauge untersucht. Standen der Ameise in beiden Augen die frontale oder caudale DRA zur Verfügung führte dies zu einem deutlichen Orientierungsverlust. Die intakte DRA eines Auges erlaubte eine zielgerichtete Fortbewegung, die jedoch von der Erwartungsrichtung abwich. Zusammenfassend zeigen die Ergebnisse der vorliegenden Studie, dass der Polarisationskompass die präziseste Richtungsinformation liefert und den Himmelskompass der Wüstenameise dominiert.The desert ant, Cataglyphis, navigates predominantly by means of path integration. The information about the distance and direction of individual path segments is integrated into a home vector, which allows the ant to return to the starting point on the shortest way. The distances covered are determined by a stride integrator. The heading direction is inferred mainly via celestial cues: the sky’s polarization pattern, the position of the sun, and the spectral and intensity gradient. This thesis focuses generally on the orientation abilities of Cataglyphis fortis and particularly on the role of the polarization compass to determine the heading direction. In the experiments, the ant’s polarization compass was selectively manipulated using a polarization filter and artificial cue conflict situations were created. The ants relied exclusively on the polarization compass to determine their heading direction if only idiothetic information and information from the polarization compass were available. When the ants experienced contradicting information detected via the sun and the polarization compass systems, an intermediate homing direction was calculated, suggesting a combined neural processing of both signals. This statement was supported by transfer experiments. In a further series of experiments, the input part of the polarization compass was manipulated. Particular regions of the ant’s eye (DRA) that detect polarized light were occluded. Ants with only the frontal or caudal parts of the DRA became disoriented, whereas ants with the entire DRA of one eye were able to perform more precise paths, although deviated from the expected direction. Overall, the results from this thesis suggest that the polarization compass provides the most accurate directional information and dominates the celestial compass system of the desert ant

Biodegradable nano-carriers as pulmonary delivery systems for steroids

The aim of this dissertation is to investigate and optimize the preparation of steroid loaded particles, to increase drug loading by enhanced polymer-drug interactions, and the surfactant free stabilization of nanoparticles in the dry state. Different particle preparation methods are described and advantages and drawbacks are discussed. As sample molecules for drug loading, budesonide and estradiol are explored. In chapter II, the versatile properties of nano- and microparticles are introduced and the advantages of nanoparticulate drug delivery to the lungs are elucidated. Especially, efficient and preservative delivery of nanoobjects to specific regions of the lungs is focused. Local pulmonary and systemic diseases are depicted and potential treatment by polymeric nanoparticles is presented. Nanoparticle preparations for small molecular drug delivery are discussed as well as for protein and peptides or nucleonic acids. In chapter III, loading of a corticosteroid hormone, budesonide, into particles is investigated. Drawbacks and opportunities of different preparation methods on drug loading of steroids in poly(lactide-co-glycolide) particles are illustrated. Nanoparticles dispersed in water are prepared by two fundamental different methods: “solvent displacement” and “solvent emulsion”. Drug loading and release are determined as key characteristics of the particles gained. Microparticles are prepared for comparison by spray-drying and a more sophisticated method, electro spraying. The aim of this chapter is to select preparation parameters enhancing drug loading and prolonged release for a combination of drug and polymer. In contrast, the chapter IV deals with the optimization of polymer properties to increase drug loading for estradiol, another steroid hormone. Cyclodextrins can form inclusion complexes with small molecules and increase drug solubility. Introduction of β-cyclodextrins in the polymer matrix are expected to increased drug-polymer interactions. Different core molecules are grafted with PLGA. The synthesized polymers are analyzed by size exclusion chromatography, differential scanning calorimetry, nuclear magnetic resonance spectroscopy and tested for hemolytic activity. Nanoparticles are prepared by “solvent displacement” and characterized regarding to size, drug loading and drug release in comparison to particles of cyclodextrin-polymer blends. The scope of this study is to investigate if drug loading efficacy for PLGA-nanoparticles can be increased by optimization of the polymer matrix by introduction of cyclodextrin binding sites. Finally, prepared nanoparticles are stabilized in chapter V. Stabilization in dry state is difficult for nanoobjects; high surface energy forces the particles to aggregation. The selection of appropriate spacer molecules is intended to reduce aggregation forces. Spray-drying is selected as the method of choice, since it is well established in drug manufacturing, and continuous fabrication is possible. Additionally, the application of suitable preparation conditions enables the microparticles to be used directly as transport vehicles for pulmonary delivery. Surface active substances can irritate the sensitive endothelia in the lungs, so that surfactants should be avoided. The aim of this investigation was to establish surfactant free microcarriers for the transport of redispersible nanoparticles. Chapter VI presents a short summary and gives some perspectives of this work