Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.     

Endogenous neurogenesis following ischaemic brain injury: Insights for therapeutic strategies

Ischaemic stroke is among the most common yet most intractable types of central nervous system (CNS) injury in the adult human population. In the acute stages of disease, neurons in the ischaemic lesion rapidly die and other neuronal populations in the ischaemic penumbra are vulnerable to secondary injury. Multiple parallel approaches are being investigated to develop neuroprotective, reparative and regenerative strategies for the treatment of stroke. Accumulating evidence indicates that cerebral ischaemia initiates an endogenous regenerative response within the adult brain that potentiates adult neurogenesis from populations of neural stem and progenitor cells. A major research focus has been to understand the cellular and molecular mechanisms that underlie the potentiation of adult neurogenesis and to appreciate how interventions designed to modulate these processes could enhance neural regeneration in the post-ischaemic brain. In this review, we highlight recent advances over the last 5 years that help unravel the cellular and molecular mechanisms that potentiate endogenous neurogenesis following cerebral ischaemia and are dissecting the functional importance of this regenerative mechanism following brain injury.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.     

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Investigating regeneration and functional integration of CNS neurons: Lessons from zebrafish genetics and other fish species

Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

A dynamic spatiotemporal extracellular matrix facilitates epicardial-mediated vertebrate heart regeneration

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.     

Cell Therapy for CNS Trauma

Cell therapy plays an important role in multidisciplinary management of the two major forms of central nervous system (CNS) injury, traumatic brain injury and spinal cord injury, which are caused by external physical trauma. Cell therapy for CNS disorders involves the use of cells of neural or non-neural origin to replace, repair, or enhance the function of the damaged nervous system and is usually achieved by transplantation of the cells, which are isolated and may be modified, e.g., by genetic engineering, when it may be referred to as gene therapy. Because the adult brain cells have a limited capacity to migrate to and regenerate at sites of injury, the use of embryonic stem cells that can be differentiated into various cell types as well as the use of neural stem cells has been explored. Preclinical studies and clinical trials are reviewed. Advantages as well as limitations are discussed. Cell therapy is promising for the treatment of CNS injury because it targets multiple mechanisms in a sustained manner. It can provide repair and regeneration of damaged tissues as well as prolonged release of neuroprotective and other therapeutic substances.     

Progenitor cells as remote "bioreactors": neuroprotection via modulation of the systemic inflammatory response

Acute central nervous system(CNS)injuries such as spinal cord injury,traumatic brain injury,autoimmune encephalomyelitis,and ischemic stroke are associ- ated with significant morbidity,mortality,and health care costs worldwide.Preliminary research has shown potential neuroprotection associated with adult tissue derived stem/progenitor cell based therapies.While initial research indicated that engraftment and transdif- ferentiation into neural cells could explain the observed benefit,the exact mechanism remains controversial.A second hypothesis details localized stem/progenitor cell engraftment with alteration of the loco-regional milieu;however,the limited rate of cell engraftment makes this theory less likely.There is a growing amount of pre-clinical data supporting the idea that,after intravenous injection,stem/progenitor cells interact with immuno- logic cells located in organ systems distant to the CNS,thereby altering the systemic immunologic/inflammatory response.Such distant cell"bioreactors"could modulate the observed post-injury pro-inflammatory environment and lead to neuroprotection.In this review,we discuss the current literature detailing the above mechanisms of action for adult stem/progenitor cell based therapies in the CNS.     

The HeArt of regeneration

Fish and amphibian hearts are known to regenerate after partial resection, but the molecular mechanisms underlying this process remain unclear. In this issue of Cell, Lipilina et al. analyze regeneration in the zebrafish heart. Their work indicates that new cardiomyocytes originate from undifferentiated progenitor cells and reveals a critical role for the epicardium, the cellular layer that covers the heart.     

Regeneration of the central nervous system-principles from brain regeneration in adult zebrafish

Poor recovery of neuronal functions is one of the most common healthcare challenges for patients with different types of brain injuries and/or neurodegenerative diseases. Therapeutic interventions face two major challenges: (1) How to generate neurons de novo to replenish the neuronal loss caused by injuries or neurodegeneration (restorative neurogenesis) and (2) How to prevent or limit the secondary tissue damage caused by long-term accumulation of glial cells, including microglia, at injury site (glial scar). In contrast to mammals, zebrafish have extensive regenerative capacity in numerous vital organs, including the brain, thus making them a valuable model to improve the existing therapeutic approaches for human brain repair. In response to injuries to the central nervous system (CNS), zebrafish have developed specific mechanisms to promote the recovery of the lost tissue architecture and functionality of the damaged CNS. These mechanisms include the activation of a restorative neurogenic program in a specific set of glial cells (ependymoglia) and the resolution of both the glial scar and inflammation, thus enabling proper neuronal specification and survival. In this review, we discuss the cellular and molecular mechanisms underlying the regenerative ability in the adult zebrafish brain and conclude with the potential applicability of these mechanisms in repair of the mammalian CNS.     

Identification of Ca~(2+) signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells(NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3 r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor(IP3 R) type-1 and ryanodine receptor(RyR) type-2 are differentially expressed in cells with neuron-or radial glial-like properties. Furthermore, ATP but not caffeine(IP3 R and RyR agonists, respectively), induced the generation of Ca~(2+) transients in cells exhibiting neuron-or glial-like morphology. These results indicate the differential expression of components of the Ca~(2+) -signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca~(2+) signaling as a read-out.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Inflammation in Traumatic Brain Injury: Role of Cytokines and Chemokines

A traumatic injury to the adult mammalian central nervous system (CNS), such as a stab wound lesion, results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. The roles of cytokines and growth factors released locally by the damaged endogenous cells are recognized in controlling the cellular changes that occur following CNS injury. However, the role of chemokines, a novel class of chemoattractant cytokines, is only recently being studied in regulating inflammatory cell invasion in the injured/diseased CNS (1). The mRNAs for several chemokines have been shown to be upregulated in experimental allergic encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, but chemokine expression in traumatic brain injury has not been studied in detail. Astrocytes have been demonstrated to participate in numerous processes that occur following injury to the CNS. In particular, astrocytic expression of cytokines and growth factors in the injured CNS has been well reviewed (2). Recently a few studies have detected the presence of chemokines in astrocytes following traumatic brain injury (3,4). These studies have suggested that chemokines may represent a promising target for future therapy of inflammatory conditions. This review summarizes the events that occur in traumatic brain injury and discusses the roles of resident and non-resident cells in the expression of growth factors, cytokines and chemokines in the injured CNS.     

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.     

Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury

Studying regeneration in the central nervous system (CNS) is hampered by current histological and imaging techniques because they provide only partial information about axonal and glial reactions. Here we developed a tetrahydrofuran-based clearing procedure that renders fixed and unsectioned adult CNS tissue transparent and fully penetrable for optical imaging. In large spinal cord segments, we imaged fluorescently labeled cells by 'ultramicroscopy' and two-photon microscopy without the need for histological sectioning. We found that more than a year after injury growth-competent axons regenerated abundantly through the injury site. A few growth-incompetent axons could also regenerate when they bypassed the lesion. Moreover, we accurately determined quantitative changes of glial cells after spinal cord injury. Thus, clearing CNS tissue enables an unambiguous evaluation of axon regeneration and glial reactions. Our clearing procedure also renders other organs transparent, which makes this approach useful for a large number of preclinical paradigms.     

Contact inhibition in the failure of mammalian CNS axonal regeneration

Anamniote animals, such as fish and amphibians, are able to regenerate damaged CNS nerves following injury, but regeneration in the mammalian CNS tracts, such as the optic nerve, does not occur. However, severed adult mammalian retinal axons can regenerate into peripheral nerve segments grafted into the brain and this finding has emphasized the importance of the environment in explaining regenerative failure in the adult mammalian CNS. Following lesions, regenerating axons encounter the glial cells, oligodendrocytes and astro-cytes, and their derivatives, respectively myelin and the astrocytic scar. Experiments to investigate the influence of these components on axon growth in culture have revealed cell-surface and extracellular matrix molecules that inhibit axon extension and growth cone motility. Structural and functional characterization of these ligands and their receptors is underway, and may solve the interesting neurobiological conundrum posed by the failure of mammalian CNS regeneration. Simultaneously, this might allow new possibilities for treatment of the severe clinical disabilities resulting from injury to the brain and spinal cord.