Reorganization of the intermediate filament (IF) network during axonal regeneration is accompanied by changes in the expression of various IF proteins. An increase in expression of the neuronal IF subunit gefiltin in goldfish retinal ganglion cells (RGCs) has been linked to the unique ability of the goldfish optic nerve to regenerate following injury. Evidence suggests that the optic tectum may regulate the expression of gefiltin during regeneration. The goal of this thesis was to determine the function of the tectum in the regulation of gefiltin mRNA expression during optic fiber regeneration in the goldfish. It was found that gefiltin mRNA levels in the RGCs of animals that received an optic nerve crush (ONC group) increased by 10 days, peaked from 20 to 38 days at around 5.5-fold over normal, and declined to near normal by 115 days. In animals that had the entire tectum removed and an optic nerve crush (ETR group), gefiltin mRNA levels increased by 10 days, peaked at 20 days at around 5.5- to 6.5-fold over normal, and although they dropped slightly thereafter, they remained elevated at around 5-fold over normal for at least 115 days. When axons regenerated to the ipsilateral tectal lobe as a result of a left tectal lobe removal and left eye removal surgery the expression pattern of gefiltin mRNA paralleled that of the ONC group. It was also found that the abundance of gefiltin subunits in the retina was elevated at 30 days of regeneration in ONC and ETR animals, and that levels in the nerve were reconstituted to 80% of normal by 30 days. These results demonstrate that increases in gefiltin mRNA and protein levels during optic nerve regeneration are independent of the tectum, whereas the downregulation of gefiltin mRNA levels is entirely dependent upon the tectum. (Abstract shortened by UMI.)

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This study aimed to better understand the clearance of myelin debris during Wallerian degeneration in the goldfish visual paths. Myelin debris was first examined immunohistochemically in the presence or absence of regenerating axons. From these preliminary experiments it was apparent that the clearance of myelin debris was not affected by regenerating axons and that the debris was removed in a differential pattern along the visual pathway. Specifically, in the distal stump of the nerve as well as in the optic tract, myelin debris had been effectively cleared by one-month postoperative, while in the cranial segment of the nerve debris persisted for at least 6 weeks after injury. The differential pattern of myelin debris in the optic nerve and tract was then analyzed qualitatively and quantitatively using thick and thin plastic sections at various time points during regeneration. The results suggested that highly activated peripheral macrophages were responsible for the effective clearance of myelin in the distal nerve stump. In the optic tract a number of cellular properties, including their unique population of astrocytes may have enhanced the rate of debris clearance. By contrast, in the cranial segment of the nerve persistent debris was found both intracellularly in phagosomes and extracellularly, suggesting that the resident phagocytes were deficient in effecting both phagocytosis and emigration. Deficient phagocytosis may be a result of the production of anti-inflammatory cytokines in this region, while the failure to emigrate is most likely due to the rigid network of astrocytes in the nerve.

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Patterns of proliferation and changes in total cell number in the optic nerve, tract and tectum of goldfish have been examined following optic nerve crush or optic enucleation, using bromodeoxyuridine to label the proliferating cells. In general, an increase in proliferation and total cell number in all parts of the visual system was observed peaking between 7 and 14 days and resolving itself to normal or near normal levels by 32 days postoperative. Enucleation resulted in elevated proliferation values as compared to animals with an optic nerve crush, at least one early timepoint in each part of the visual system, but overall, there is little to suggest that axons are exerting a major effect on the cellular response. Finally, a seasonal effect on the proliferative response of non-neuronal cells and axonal regrowth has been demonstrated. Fish acclimatized under autumn-like conditions showed a faster initiation of the non-neuronal cell response and an enhanced rate of axonal regrowth when compared to fish acclimatized under spring-like conditions. It is believed that photoperiod plays a major role in the seasonal effects observed with temperature playing only a minor one.

This study aims at providing a new technique for the isolation and culture of goldfish microglial cells. So far no protocol has been designed for the growth of these cells in vitro, despite the growing interest in the remarkable capacity of goldfish central nervous system (CNS) for regenerating severed axons. This newly developed technique has little or no similarity to those used in the isolation of mammalian microglia, and is distinguished by its simple setup and its fast yield for microglial cells. In addition, a virtually pure population of microglia was generated when plated on untreated plastic dishes, eliminating further need for purification. This technique may thus provide a starting point for future characterization of the microglial cells in vitro, which may eventually help toward building a better understanding of the function and biology of these cells. A preliminary morphological characterization of the cells has also been conducted, in addition to groundwork experiments on the phagocytic activity of these cells in vitro, using myelin to stimulate phagocytosis. These assays were oriented toward providing a comparison to the mammalian cultures of microglia, and so far, displayed several similarities in morphologies and phagocytosis.

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No description available.