Limitaties op oogzenuw regeneratie in killivissen: de rol van littekenweefsel en de veroudering van niet-neuronale cellen

Aging is associated with a progressive decline in the regenerative capacity of the central nervous system (CNS), contributing to the increasing burden of neurodegenerative diseases. In mammals, CNS injury results in limited repair, characterized by neuronal loss, chronic inflammation, and inhibitory scar formation. In contrast, regeneration-competent species such as zebrafish exhibit efficient neuronal survival, axonal regrowth, and functional recovery following CNS damage. The African turquoise killifish represents a unique model system, exhibiting robust regenerative capacity in young adulthood followed by a rapid, age-dependent decline in regenerative competence. This thesis aimed to investigate how neuronal resilience and the extrinsic injury environment contribute to regenerative outcomes, and how these processes are altered by aging. To this end, the retinotectal system—comprising the retina, optic nerve and optic tectum—of killifish was used as a model with optic nerve crush (ONC) serving as the injury paradigm. In a first research chapter, retinal ganglion cell (RGC) survival following ONC was compared across mice, zebrafish, and killifish using standardized retinal whole-mount analyses. Significant interspecies differences in RGC density and survival were identified. Zebrafish showed high neuronal resilience with minimal RGC loss, whereas mice exhibited rapid degeneration with low long-term survival (~10-20%). Killifish displayed an intermediate phenotype that worsened with age, with approximately 70% RGC survival in young adults and temporally distinct primary and secondary phases of RGC degeneration. These findings establish the killifish as a distinctive model that allows the dissection of primary and secondary mechanisms driving neuronal degeneration. In a second research chapter, a more severe injury paradigm—complete optic nerve transection (ONT)—was applied in killifish to further assess regenerative capacity. In contrast to zebrafish, which maintain robust regeneration following ONT, killifish showed a marked reduction in RGC survival (~25%) and failed to reinnervate their target neurons in the optic tectum. Despite this, surviving RGCs retained intrinsic axonal growth capacity, as evidenced by aberrant axonal projections within the retina. At the lesion site, a dense collagen-rich gliofibrotic scar formed, acting as a major barrier to regeneration. Reducing scar formation through partial transection (pONT) improved neuronal survival, potentially supporting axonal regrowth and restoring target reinnervation. These results demonstrate that extrinsic environmental factors, particularly scar formation, critically limit regeneration, even when intrinsic growth capacity is preserved. Together, these findings position the killifish as a model to study how age-related changes in the non-neuronal environment contribute to declining regenerative capacity. A final research chapter focused on the aging optic nerve as a glia-enriched system to investigate changes in the extrinsic environment. Bulk RNA sequencing revealed age-dependent transcriptional alterations associated with hallmarks of human aging, including chronic inflammation, gliosis, metabolic and energetic dysfunction, and cellular senescence. As the optic nerve is largely composed of non-neuronal cells, these findings reflect a strong impact of aging on its most abundant populations, namely glial and immune cells. This supports the notion that changes in the non-neuronal environment play a key role in limiting regeneration. Future integration with single-cell approaches will enable further characterization of cell type-specific contributions, including the potential roles of other optic nerve populations such as fibroblast-like cells. Overall, this thesis demonstrates that regenerative outcomes in the CNS are determined by the interaction between intrinsic neuronal properties and the extrinsic injury environment. While killifish largely retain intrinsic growth capacity, regeneration is constrained by injury-induced and age-related changes in the surrounding tissue. By positioning the killifish as an a unique model within the spectrum of regeneration, this work provides a conceptual framework to further study the mechanisms underlying the transition from successful regeneration to regenerative failure during adulthood and aging.