Visually Driven Behavior Assays to Study Functionality and Recovery after Damage of the Aged African Turquoise Killifish Visual System.

Loss of vision is a prominent feature of aging and vision is considered by many to be the most valuable sense to be lost. In our graying society, we are increasingly challenged by age-related deterioration of the central nervous system (CNS), as well as by age-associated neurodegenerative diseases and brain injuries, all often affecting the visual system and thus its performance. Here, we describe two visually driven behavior assays to evaluate visual performance upon aging or CNS damage in the fast-aging killifish. The first test, the optokinetic response (OKR), measures the reflexive eye movement triggered by motion in the visual field and allows assessment of visual acuity. The second assay, the dorsal light reflex (DLR), evaluates the swimming angle based on input of light coming from above. The OKR can be used to study the effect of aging on visual acuity as well as visual improvement and recovery after rejuvenation therapy or visual system injury or disease, whereas the DLR is best used to assess functional repair after a unilateral optic nerve crush.

Morphological Analysis of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As the number of elderly individuals is increasing in modern society, the need for a relevant gerontology model is higher than ever before. Aging can be defined by specific cellular hallmarks, described by López-Otín and colleagues, who provided a map which can be used to scavenge the aging tissue environment. As revealing the presence of individual hallmarks does not necessarily indicate aging, here we provide different (immuno)histochemical approaches that can be used to investigate several aging hallmarks-namely, genomic damage, mitochondrial dysfunction/oxidative stress, cellular senescence, stem cell exhaustion, and altered intercellular communication-in the killifish retina, optic tectum, and/or telencephalon at a morphological level. In combination with molecular and biochemical analysis of these aging hallmarks, this protocol offers the opportunity to fully characterize the aged killifish central nervous system.

The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment.

In our graying world population, we are increasingly facing brain injuries and age-associated neurodegenerative diseases, which are often characterized by axonal pathology. Here, we propose the killifish visual/retinotectal system as a model for investigating central nervous system repair, more specifically axonal regeneration, in an aging context. We first describe an optic nerve crush (ONC) injury paradigm in killifish to induce and study both de- and regeneration of retinal ganglion cells (RGCs) and their axons. Subsequently, we summarize several methods for mapping different steps of the regenerative process-namely, axonal regrowth and synapse reformation-using retro- and anterograde tracing methods, (immuno)histochemistry, and morphometrical analyses.

Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish.

As modern society is graying, aging research and biogerontology models, in which the aging process can be studied, are becoming increasingly important. A proper aging model can be defined as one that displays many of the aging hallmarks. Here, we provide two different practical approaches-namely, real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting-that can be used to investigate cellular senescence (RT-qPCR for p21 and p27), altered intercellular communication/inflammaging (RT-qPCR for il-10, sirt-1, il-6, il-1b, il-8, and tnf), and oxidative stress (western blotting for 4-HNE) in the killifish central nervous system, and, more specifically, in the retina, optic tectum, and telencephalon. These molecular and biochemical analyses are a first step in confirming the aging characteristics but should preferably be combined with morphological analyses.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics.

Axonal regeneration in the central nervous system is an energy-intensive process. In contrast to mammals, adult zebrafish can functionally recover from neuronal injury. This raises the question of how zebrafish can cope with this high energy demand. We previously showed that in adult zebrafish, subjected to an optic nerve crush, an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair. We postulate a 'dendrites for regeneration' paradigm that might be linked to intraneuronal mitochondrial reshuffling, as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously. Here, we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments (dendrites, somas, and axons) during the regenerative process. Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction, whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth. Upon dendritic regrowth in the retina, mitochondrial density inside the retinal dendrites returned to baseline levels. Moreover, a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage. Taken together, these findings suggest that during optic nerve injury-induced regeneration, mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

The age factor in optic nerve regeneration: Intrinsic and extrinsic barriers hinder successful recovery in the short-living killifish.

As the mammalian central nervous system matures, its regenerative ability decreases, leading to incomplete or non-recovery from the neurodegenerative diseases and central nervous system insults that we are increasingly facing in our aging world population. Current neuroregenerative research is largely directed toward identifying the molecular and cellular players that underlie central nervous system repair, yet it repeatedly ignores the aging context in which many of these diseases appear. Using an optic nerve crush model in a novel biogerontology model, that is, the short-living African turquoise killifish, the impact of aging on injury-induced optic nerve repair was investigated. This work reveals an age-related decline in axonal regeneration in female killifish, with different phases of the repair process being affected depending on the age. Interestingly, as in mammals, both a reduced intrinsic growth potential and a non-supportive cellular environment seem to lie at the basis of this impairment. Overall, we introduce the killifish visual system and its age-dependent regenerative ability as a model to identify new targets for neurorepair in non-regenerating individuals, thereby also considering the effects of aging on neurorepair.

The killifish visual system as an in vivo model to study brain aging and rejuvenation.

Worldwide, people are getting older, and this prolonged lifespan unfortunately also results in an increased prevalence of age-related neurodegenerative diseases, contributing to a diminished life quality of elderly. Age-associated neuropathies typically include diseases leading to dementia (Alzheimer's and Parkinson's disease), as well as eye diseases such as glaucoma and age-related macular degeneration. Despite many research attempts aiming to unravel aging processes and their involvement in neurodegeneration and functional decline, achieving healthy brain aging remains a challenge. The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived reported vertebrate that can be bred in captivity and displays many of the aging hallmarks that have been described for human aging, which makes it a very promising biogerontology model. As vision decline is an important hallmark of aging as well as a manifestation of many neurodegenerative diseases, we performed a comprehensive characterization of this fish's aging visual system. Our work reveals several aging hallmarks in the killifish retina and brain that eventually result in a diminished visual performance. Moreover, we found evidence for the occurrence of neurodegenerative events in the old killifish retina. Altogether, we introduce the visual system of the fast-aging killifish as a valuable model to understand the cellular and molecular mechanisms underlying aging in the vertebrate central nervous system. These findings put forward the killifish for target validation as well as drug discovery for rejuvenating or neuroprotective therapies ensuring healthy aging.

Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri.

The aging central nervous system (CNS) of mammals displays progressive limited regenerative abilities. Recovery after loss of neurons is extremely restricted in the aged brain. Many research models fall short in recapitulating mammalian aging hallmarks or have an impractically long lifespan. We established a traumatic brain injury model in the African turquoise killifish (Nothobranchius furzeri), a regeneration-competent vertebrate that evolved to naturally age extremely fast. Stab-wound injury of the aged killifish dorsal telencephalon unveils an impaired and incomplete regeneration response when compared to young individuals. In the young adult killifish, brain regeneration is mainly supported by atypical non-glial progenitors, yet their proliferation capacity clearly declines with age. We identified a high inflammatory response and glial scarring to also underlie the hampered generation of new neurons in aged fish. These primary results will pave the way to unravel the factor age in relation to neurorepair, and to improve therapeutic strategies to restore the injured and/or diseased aged mammalian CNS.

Injury-induced Autophagy Delays Axonal Regeneration after Optic Nerve Damage in Adult Zebrafish.

Optic neuropathies comprise a group of disorders in which the axons of retinal ganglion cells (RGCs), the retinal projection neurons conveying visual information to the brain, are damaged. This results in visual impairment or even blindness, which is irreversible as adult mammals lack the capacity to repair or replace injured or lost neurons. Despite intensive research, no efficient treatment to induce axonal regeneration in the central nervous system (CNS) is available yet. Autophagy, the cellular recycling response, was shown repeatedly to be elevated in animal models of optic nerve injury, and both beneficial and detrimental effects have been reported. In this study, we subjected spontaneously regenerating adult zebrafish to optic nerve damage (ONC) and revealed that autophagy is enhanced after optic nerve damage in zebrafish, both in RGC axons and somas, as well as in macroglial cells of the retina, the optic nerve and the visual target areas in the brain. Interestingly, the pattern of the autophagic response in the axons followed the spatiotemporal window of axonal regrowth, which suggests that autophagy is ongoing at the growth cones. Pharmacological inhibition of the recycling pathway resulted in accelerated RGC target reinnervation, possibly linked to increased mechanistic target of rapamycin (mTOR) activity, known to stimulate axonal regrowth. Taken together, these intriguing findings underline that further research is warranted to decipher if modulation of autophagy could be an effective therapeutic method to induce CNS regeneration.

Müller glia-myeloid cell crosstalk accelerates optic nerve regeneration in the adult zebrafish.

Neurodegenerative disorders, characterized by progressive neuronal loss, eventually lead to functional impairment in the adult mammalian central nervous system (CNS). Importantly, these deteriorations are irreversible, due to the very limited regenerative potential of these CNS neurons. Stimulating and redirecting neuroinflammation was recently put forward as an important approach to induce axonal regeneration, but it remains elusive how inflammatory processes and CNS repair are intertwined. To gain more insight into these interactions, we investigated how immunomodulation affects the regenerative outcome after optic nerve crush (ONC) in the spontaneously regenerating zebrafish. First, inducing intraocular inflammation using zymosan resulted in an acute inflammatory response, characterized by an increased infiltration and proliferation of innate blood-borne immune cells, reactivation of Müller glia, and altered retinal cytokine expression. Strikingly, inflammatory stimulation also accelerated axonal regrowth after optic nerve injury. Second, we demonstrated that acute depletion of both microglia and macrophages in the retina, using pharmacological treatments with both the CSF1R inhibitor PLX3397 and clodronate liposomes, compromised optic nerve regeneration. Moreover, we observed that csf1ra/b double mutant fish, lacking microglia in both retina and brain, displayed accelerated RGC axonal regrowth after ONC, which was accompanied with unusual Müller glia proliferative gliosis. Altogether, our results highlight the importance of altered glial cell interactions in the axonal regeneration process after ONC in adult zebrafish. Unraveling the relative contribution of the different cell types, as well as the signaling pathways involved, may pinpoint new targets to stimulate repair in the vertebrate CNS.

Designing neuroreparative strategies using aged regenerating animal models.

In our ever-aging world population, the risk of age-related neuropathies has been increasing, representing both a social and economic burden to society. Since the ability to regenerate in the adult mammalian central nervous system is very limited, brain trauma and neurodegeneration are often permanent. As a consequence, novel scientific challenges have emerged and many research efforts currently focus on triggering repair in the damaged or diseased brain. Nevertheless, stimulating neuroregeneration remains ambitious. Even though important discoveries have been made over the past decades, they did not translate into a therapy yet. Actually, this is not surprising; while these disorders mainly manifest in aged individuals, most of the research is being performed in young animal models. Aging of neurons and their environment, however, greatly affects the central nervous system and its capacity to repair. This review provides a detailed overview of the impact of aging on central nervous system functioning and regeneration potential, both in non-regenerating and spontaneously regenerating animal models. Additionally, we highlight the need for aging animal models with regenerative capacities in the search for neuroreparative strategies.

Prior Exposure to Immunosuppressors Sensitizes Retinal Microglia and Accelerates Optic Nerve Regeneration in Zebrafish.

As adult mammals lack the capacity to replace or repair damaged neurons, degeneration and trauma (and subsequent dysfunction) of the central nervous system (CNS) seriously constrains the patient's life quality. Recent work has shown that appropriate modulation of acute neuroinflammation upon CNS injury can trigger a regenerative response; yet, the underlying cellular and molecular mechanisms remain largely elusive. In contrast to mammals, zebrafish retain high regenerative capacities into adulthood and thus form a powerful model to study the contribution of neuroinflammation to successful regeneration. Here, we used pharmacological immunosuppression methods to study the role of microglia/macrophages during optic nerve regeneration in adult zebrafish. We first demonstrated that systemic immunosuppression with dexamethasone (dex) impedes regeneration after optic nerve injury. Secondly, and strikingly, local intravitreal application of dex or clodronate liposomes prior to injury was found to sensitize retinal microglia. Consequently, we observed an exaggerated inflammatory response to subsequent optic nerve damage, along with enhanced tectal reinnervation. In conclusion, we found a strong positive correlation between the acute inflammatory response in the retina and the regenerative capacity of the optic nerve in adult zebrafish subjected to nerve injury.

Extensive growth is followed by neurodegenerative pathology in the continuously expanding adult zebrafish retina.

The development of effective treatments for age-related neurodegenerative diseases remains one of the biggest medical challenges today, underscoring the high need for suitable animal model systems to improve our understanding of aging and age-associated neuropathology. Zebrafish have become an indispensable complementary model organism in gerontology research, yet their growth-control properties significantly differ from those in mammals. Here, we took advantage of the clearly defined and highly conserved structure of the fish retina to study the relationship between the processes of growth and aging in the adult zebrafish central nervous system (CNS). Detailed morphological measurements reveal an early phase of extensive retinal growth, where both the addition of new cells and stretching of existent tissue drive the increase in retinal surface. Thereafter, and coinciding with a significant decline in retinal growth rate, a neurodegenerative phenotype becomes apparent,-characterized by a loss of synaptic integrity, an age-related decrease in cell density and the onset of cellular senescence. Altogether, these findings support the adult zebrafish retina as a valuable model for gerontology research and CNS disease modeling and will hopefully stimulate further research into the mechanisms of aging and age-related pathology.