Universiẗat, Diss., 2004--Osnabrück.

No description available.

No description available.

Myelin is critical for the operation of a functional vertebrate nervous system, allowing for rapid saltatory conduction and providing trophic support to axons. In multiple sclerosis (MS), the immune system attacks myelin sheaths, leading to de-myelination of axons. De-myelinated axons not only lose their ability to conduct rapid nerve impulses, but are themselves susceptible to damage and loss. Long term demyelination leads to neuronal loss and the devastating symptoms of secondary stages of MS. One therapeutic approach which has been suggested is to improve the ability of oligodendrocyte precursor cells (OPCs) to differentiate into mature re-myelinating oligodendrocytes. This process is known to occur in vivo, however, the myelin produced appears reduced and the efficiency with which OPCs differentiate into myelinating oligodendrocytes (OLs) varies greatly. For example, the ability of OPCs from older mice to differentiate is reduced compared to those from young mice. This fact taken alongside the presence of many OPCs in some MS lesions which have failed to re-myelinate makes identifying compounds which can increase OPC differentiation into OLs a key goal for MS drug development. In this work, I use OPC to OL differentiation during zebrafish development as a model for differentiation of OLs more generally. Zebrafish are widely used for chemical screening, with recent developments in genetic manipulation, such as CRISPR/Cas9 gene editing and Tol2 transgenesis, allowing for production of targeted mutations and fluorescent reporter lines respectively. Retinoid X receptor-γ (RXRγ) has previously been identified as being transcriptionally upregulated during re-myelination. Moreover, treatment with 9-cis-retinoic acid, an agonist for the receptor, has been shown to improve remyelination in vivo in rats with toxin induced focal demyelination. I first present a manual chemical screen of a library of compounds designed to target RXRγ, from which I identify several compounds which reproducibly increase OLs in zebrafish. In order to assess whether any of the hit compounds could be acting as agonists for RXRγ, I have created a double knockout zebrafish lacking both genes coding for the zebrafish RXRγ homologues (rxrga and rxrgb). This line has been used to test the activity of hit compounds in a RXRγ loss of function background. Following this first chemical screen, it was clear that great improvements could be made to both throughput and robustness if the screen was automated. Using a commercially available fish handling robot which automates the imaging of plates of zebrafish embryos, known as the VAST BioImager, the throughput of our assay was increased from ~40 fish per day to up to around 300 to 400. We combined the VAST BioImager with a state of the art spinning disk confocal microscope, giving us (to the best of our knowledge) the world's fastest in vivo vertebrate screening system capable of orienting fish and imaging at sub-cellular resolution. This significant increase in rate of fish processing led to the need for an increase in the rate of image analysis. Much of the gains in throughput would be lost to time counting cells, and so I developed software to automate the image processing and analysis. The software developed is shown to closely match the abilities of a human to identify compounds which give significant increases in OLs, with very little human intervention required. In the final section of this work I present an example screen performed using the VAST BioImager in combination with the automated cell counting software, which I developed. The hits from this screen highlight our ability to automatically identify compounds that increase the number of OLs in the developing zebrafish. This method is broadly applicable to other central nervous system cell types and other methods of analysis can be integrated into the presented screening software.

acase@tulane.edu / Bringing a newly formulated drug used for neurological applications to the market is a highly time and labor-intensive process. The current pathway of bringing a drug to market requires extensive drug testing on animal models at the pre-clinical stage. Live animal models are expensive, low-throughput and increasingly recognized to be poor predictors of clinical outcomes in the process of drug development. An in vitro testing platform would address these above stated problems by providing a pre-screening process that could improve the high attrition rates of novel pharmaceutical compounds and also reduce the demand for the number of animals used for testing. This dissertation presents the progress of studies that were conducted to create a three-dimensional myelinated in vitro peripheral nerve-on-a-chip model that could be subject to electrophysiological and histological testing to be used as a tool for drug screening. In the first study, our model utilized an ultra-violet light cured methacrylated heparin hydrogel as the growth permissive substrate and a polyethylene glycol gel as a growth restrictive boundary that contained three-dimensional neural growth from an embryonic rat’s dorsal root ganglion explant. The model enabled electrophysiological field recording testing to measure metrics such as compound action potential amplitude and nerve conduction velocity. However, the heparin hydrogel presented issues with immunohistochemistry and histological studies leading us to recreate the model with a different growth permissive substrate. The second study utilized a methacrylated gelatin hydrogel in place of the heparin as the growth substrate. The dense neural growth was rapider than heparin while the gel allowed electrophysiological and histological testing to conclusively show the presence of myelin. Data from the histological testing was used to tabulate structural measurement such as percentage of myelinated axons and g-ratios which were then correlated with the electrophysiological data. This study paved way to use this model to simulate a demyelinating physiology and assess the effectiveness of a possible neuroprotective agent. The third and final study investigated the usage of the peripheral nerve-on-a-chip as model of demyelination by using forskolin and twitcher mouse serum, adapted from the established in vivo model of Krabbe’s disease. The effects of demyelination were observed using electrophysiological, immunohistochemistry, and histological studies. The corticosteroid dexamethasone was also included in the demyelination models to assess its extent of neuroprotection against the demyelinating agents. The results established a novel myelinated peripheral nerve-on-a-chip model which could be subject to electrophysiological, immunohistochemistry, and histological studies. The model has the potential to be used to simulate various pathologies and evaluate the efficacy of drugs before animal testing could be conducted. / 1 / Ashwin Sivakumar

Previous studies by our group revealed that chronic low grade inflammation implicating phagocytosing macrophages is a highly relevant mechanism in the pathogenesis of Charcot-Marie-Tooth disease. The lack of CSF-1, the primary regulator of macrophage function and survival, led to a robust and persistent amelioration of the phenotype in two authentic mouse models of CMT. Moreover, a close contact between CSF-1 producing fibroblasts and endoneurial macrophages carrying CSF-1R has been confirmed in nerve biopsies of CMT patients, further supporting the clinical significance of this pathway. In the current study we treated 3 distinct mouse models of CMT1: the PMP22tg mice as a model for CMT1A, the P0+/- mice as a model for CMT1B and the Cx32def mice as a model for CMT1X, with a CSF-1R specific kinase (c-FMS) inhibitor (800-1200 mg PLX5622/ kg chow) according to different treatment regimes mimicking an ideal early onset treatment, a late onset treatment and the withdrawal of the drug. Using the above mentioned doses of PLX5622, we documented a dramatic decrease in macrophage numbers in the PNS of all 3 myelin mutants, except for the quadriceps nerve of Cx32def mice. Fibroblast numbers remained unchanged in treated animals. Surprisingly, in spite of the decrease in the number of detrimental macrophages we could not detect an unequivocal phenotypic improvement. CMAP amplitudes were reduced in both wild type and myelin mutant mice treated with CSF-1R inhibitor in comparison to untreated littermates. Corresponding to the electrophysiological findings, the axon number and the percentage of large diameter axons were reduced in the quadriceps nerve of treated P0+/- and Cx32def mice. By contrast we observed a higher number of fully myelinated axons, in parallel with a decrease in the percentage of demyelinated (and hypermyelinated in PMP22tg mice) fibers in the ventral roots of P0+/- mice treated with CSF-1R inhibitor from 3 months up to 6 months of age and PMP22tg animals treated from 9 months up to 15 months of age. Our results indicate that CSF-1R inhibitor has the potential to improve the demyelinating phenotype of at least two models of CMT1. Nevertheless, further studies are necessary (for example with lower doses of the inhibitor) to minimize or even eliminate the putative neurotoxic effect we observed with high dose treatment conditions. / Vorhergehende Studien unserer Gruppe haben gezeigt, dass eine niedriggradige Entzündung, die von phagozytierenden Makrophagen ausgeht, von ausserordentlicher Bedeutung für die Pathogenese der Charcot-Marie-Tooth Krankheit ist. In Abwesenheit von CSF-1, des primären Regulators für Funktion und Überleben von Makrophagen, trat eine stabile und dauerhafte Verbesserung des Phänotyps in den zwei etablierten CMT Mausmodellen auf. Darüber hinaus konnte ein enger Kontakt zwischen CSF-1-produzierenden Fibroblasten und Makrophagen, die den zugehörigen Rezeptor CSF-1Rexprimieren, in Nervbiopsien von CMT Patienten bestätigt werden, was die klinische Relevanz dieses Mechanismus weiter verdeutlicht. In der aktuellen Studie wurden drei verschiedene CMT1 Mausmodelle, PMP22tg Mäuse als Modell für CMT1A, P0+/- Mäuse als Modell für CMT1B und Cx32-defiziente Mäuse als Modell für CMT1X, mit einem Inhibitor der CSF-1R spezifischen Kinase (c-FMS) (800-1200 mg PLX5622/kg Futter) behandelt. Der Inhibitor wurde gemäß den verschiedenen Behandlungsmethoden eingesetzt, um den Verlauf eineridealerweise frühzeitigen und einer spät-beginnenden Behandlung und des Entzug des Medikaments zu imitieren. Nach der Behandlung mit PLX5622 konnten wir im PNS aller drei Myelin-Mutanten, mit Ausnahme des N. quadriceps von Cx32-defizienten Mäusen, einen drastischen Rückgang der Makrophagenanzahl feststellen. Die Anzahl der Fibroblasten blieb in den behandelten Tieren unverändert. Überraschenderweise konnten wir, trotz des Rückgangs der Anzahl an schädlichen Makrophagen, keine einheitliche Verbesserung des Phänotyps dokumentieren. CMAP Amplituden waren nach CSF-1R Inhibitor Behandlung sowohl in Wild-Typ Mäusenals auch in den Myelin-Mutanten geringer als in unbehandelten Kontrolltieren. Passend zu den elektrophysiologischen Ergebnissen war die Anzahl an Axonen und die Prozentzahl an großkalibrigen Axonen im N. quadriceps von behandelten P0+/- und Cx32-defizienten Mäusen reduziert.Im Gegensatz dazu war eine erhöhte Menge an normalmyelinisierten Axonen, mit einer gleichzeitigen Reduktiondemyelinisierter Fasern (und hypermyelinisierten in PMP22tg Mäusen) in den ventralen Spinalwurzeln von P0+/- Mäusen zu beobachten, die mit dem CSF-1R Inhibitor im Alter von 3 bis 6 Monaten behandelt worden waren sowie bei PMP22tg Mäusen, die im Alter von 9 bis 15 Monaten den Inhibitor erhalten hatten. Unsere Ergebnisse deuten an, dass der CSF-1R Inhibitor das Potential besitzt, zumindest in zwei Modellen von CMT1 den demyelinisierenden Phänotyp zu verbessern. Dennoch müssen weitere Studien durchgeführt werden (z.B. die Verwendung einer niedrigeren Dosis des Inhibitors), um den möglichen neurotoxischen Effekt, der bei oben genannten Behandlungsbedingungen zu beobachten war, zu minimieren oder ganz zu beheben.

During aging, there is a decrease both in the stability of central nervous system (CNS) myelin once formed and in the efficiency of its repair by oligodendrocytes (OLs). To study CNS remyelination during aging, I used the cuprizone (a copper chelator) mouse model. Inclusion of cuprizone in the diet kills mature OLs and demyelinates axons in the rostral corpus callosum (RCC) of mice, which enabled me to characterize age-related changes (i.e., 2-16 months of age) in glial cell response during the recruitment (i.e., demyelination) and differentiation (i.e., remyelination) phases of myelin repair. I found that the time between 12 and 16 months of age is a critical period during which there is an age-related decrease in the number of OL lineage cells (Olig2Nuc+ve/GFAP-ve cells) in the RCC of both control mice and mice recovering from cuprizone-induced demyelination. My results also show there was an age-related impaired recruitment of progenitor cells to replace lost OLs even though there was no major age-related decrease in the size of the progenitor cell pool (PDGF á R+ve/GFAP-ve, and Olig2Nuc+ve/PDGFáR+ve cells). However, there were cuprizone-induced increased numbers of astrocyte progenitor cells (Olig2Cyto+ve/PDGFáR+ve) in these same mice; thus PDGFáR+ve progenitor cells in mice as old as 16 months of age retain the ability to differentiate into astrocytes, with this fate choice occurring following cytoplasmic translocation of Olig2. These data reveal for the first time age-related differences in the differentiation of PDGFáR+ve progenitor cells into OLs and astrocytes and lead me to suggest that during aging there must be a transcriptional switch mechanism in the progenitor cell fate choice in favour of astrocytes. This may at least partially explain the age-related decrease in efficiency of OL myelination and remyelination.

During aging, there is a decrease both in the stability of central nervous system (CNS) myelin once formed and in the efficiency of its repair by oligodendrocytes (OLs). To study CNS remyelination during aging, I used the cuprizone (a copper chelator) mouse model. Inclusion of cuprizone in the diet kills mature OLs and demyelinates axons in the rostral corpus callosum (RCC) of mice, which enabled me to characterize age-related changes (i.e., 2-16 months of age) in glial cell response during the recruitment (i.e., demyelination) and differentiation (i.e., remyelination) phases of myelin repair. I found that the time between 12 and 16 months of age is a critical period during which there is an age-related decrease in the number of OL lineage cells (Olig2Nuc+ve/GFAP-ve cells) in the RCC of both control mice and mice recovering from cuprizone-induced demyelination. My results also show there was an age-related impaired recruitment of progenitor cells to replace lost OLs even though there was no major age-related decrease in the size of the progenitor cell pool (PDGF á R+ve/GFAP-ve, and Olig2Nuc+ve/PDGFáR+ve cells). However, there were cuprizone-induced increased numbers of astrocyte progenitor cells (Olig2Cyto+ve/PDGFáR+ve) in these same mice; thus PDGFáR+ve progenitor cells in mice as old as 16 months of age retain the ability to differentiate into astrocytes, with this fate choice occurring following cytoplasmic translocation of Olig2. These data reveal for the first time age-related differences in the differentiation of PDGFáR+ve progenitor cells into OLs and astrocytes and lead me to suggest that during aging there must be a transcriptional switch mechanism in the progenitor cell fate choice in favour of astrocytes. This may at least partially explain the age-related decrease in efficiency of OL myelination and remyelination.

No description available.

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2002? / Includes bibliographical references (leaves 194-225).