Identification and Characterization of Reactive Astrocytes Following Optic Nerve Injury in Zebrafish




Neve, Luis Daniel

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Following nerve injury, teleost fish are capable of initiating robust signaling cascades leading to the repair of damaged tissue and a functional recovery of nerves: a phenomenon absent in amniotes. When nerves of the CNS are injured, amniotic organisms initiate an inflammatory-like response that results in a permanent morphological change of the injured tissue, creating a collagen-rich scar around the injury site referred to as the glial scar. Reactive astrocytes are the major cellular components of the glial scar, the formation of which seems to have both a protective and inhibitory function following nerve damage in mammals. The transition of astrocytes to a reactive state, a condition referred to as reactive gliosis, is characterized by hypertrophy and increased intermediate filament production, detected immunocytochemically by increased expression of glial fibrillary acidic protein (GFAP). However, the astrocytes in the optic nerve of zebrafish express cytokeratin, not GFAP, as their intermediate filament. Unlike GFAP expressing astrocytes, cytokeratin expression by astrocytes of the optic nerve of fish does not seem to increase in response to injury. The absence of differential intermediate filament expression in response to injury, in addition to an absence of GFAP, poses a problem when trying to identify reactive astrocytes in zebrafish. In mammals, recent studies of the protein bystin have shown it to be up-regulated during the astrocyte transition from a quiescent to a reactive state, making it a suitable marker for reactivity. A bystin-like gene (bysl) has been characterized in zebrafish; however, little has been shown as far as its involvement in neural tissue injury. The goal of the study presented in this thesis was to determine if astrocytes in the optic nerve of zebrafish become reactive following nerve lesion and if reactivity could be detected via immuno-labeling of Bysl. Immunohistochemistry was performed using an anti-bystin antibody (clone S20) which recognizes an epitope with 90% homology to the predicted amino acid sequence of Bysl. Labeling with the anti-bystin antibody was observed in the cytoplasm of cytokeratin-expressing astrocytes in the optic nerve of zebrafish within 12 hours after nerve lesion. Measurements of the average nuclear size show a significant enlargement of nuclei in Bysl positive astrocytes of the injured optic nerve in comparison to astrocytes of the contralateral nerve, further suggesting anti-bystin is labeling hypertrophic reactive astrocytes. We also observed colocalization of Bysl and ATF3, a transcription factor shown to be upregulated by various stressors in the CNS, in cells showing morphology expected for hypertrophic astrocytes, suggesting Atf3 may have a function in the reactive transition. This study is the first to show the presence of reactive astrocytes following injury in the zebrafish optic nerve, further supporting the use of zebrafish as a model organism with relevance to human health.



Astrocyte, Zebrafish, Reactive astrogliosis, Reactive astrocytes, Atf3, Optic nerve injury


Neve, L. D. (2011). <i>Identification and characterization of reactive astrocytes following optic nerve injury in zebrafish</i> (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.


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