In adult mammals, including humans, neurons, and axons in the brain and spinal cord are inherently incapable of regenerating after injury. Studies of animals with innate capacity for regeneration are providing valuable insights into the mechanisms driving tissue healing. The aim of this review is to summarize recent data on regeneration mechanisms in the brain and spinal cord of zebrafish and neonatal mice. We infer that elucidating these mechanisms and understanding how and why they are lost in adult mammals will contribute to the development of strategies to promote central nervous system regeneration.

In mammals, spinal cord injury often results in permanent impairment of motor function owing to ineffective tissue regeneration. Unlike mammals, zebrafish have the remarkable ability to regenerate many tissues, including the spinal cord. Cross-species comparison is an attractive approach for revealing regeneration-specific mechanisms, but the large evolutionary distance between species sometimes hinders direct comparison. Recent studies have revealed that another model fish species, medaka, has a low regenerative ability in some tissues, making comparisons with them advantageous to revealing regeneration-specific mechanisms. However, their spinal cord regenerative ability has not been compared to other models. In this study, we functionally and histologically compared the spinal cord regeneration abilities of zebrafish and medaka. Swimming speed recovery was significantly lower in medaka than in zebrafish. Bridging of glia and neural tissue were thinner in medaka than in zebrafish. Axonal extension across the injured site was observed in zebrafish but not in medaka. Comparison of their gene expression profiles revealed genes involved in "Regeneration" were upregulated in zebrafish, whereas genes related to "Synaptic signaling" were downregulated in medaka. These results suggest that the ability to regenerate the spinal cord is lower in medaka than in zebrafish, making medaka an attractive model for revealing the mechanisms of spinal cord regeneration.

Wound healing after spinal cord injury involves highly coordinated interactions among multiple cell types, which are poorly understood. Astrocytes play a central role in creating a border against the non-neural lesion core. To do so, astrocytes undergo dramatic morphological changes by first thickening and elongating their processes and then overlapping them to form a physical barrier. We show here that the expression of a cell-surface receptor, Ryk, is induced in astrocytes after injury in both rodent and human spinal cords. Astrocyte-specific knockout of Ryk dramatically elongated the reactive astrocytes, accelerated the formation of the border, and reduced the size of the scar. Astrocyte-specific knockout of Ryk also accelerated the injury responses of multiple cell types. Single-cell transcriptomics analyses revealed a broad range of changes in cell signaling among astrocytes, microglia, fibroblasts, and endothelial cells after astrocyte-specific Ryk knockout, suggesting that Ryk not only regulates injury responses of astrocytes but may also regulate signals emanating from astrocytes and coordinate the responses of these cell types. The elongation of astrocyte processes is mediated by NrCAM, a cell adhesion molecule induced by astrocyte-specific conditional knockout of Ryk after spinal cord injury. Our findings suggest that Ryk is a promising therapeutic target to accelerate wound healing, promote neuronal survival, and enhance functional recovery.

Spinal cord injury in humans is a life-changing condition with no effective treatment. However, many non-mammalian vertebrates can fully regenerate their spinal cord after injury. Frogs such as Xenopus can regenerate the spinal cord at larval stages, but lose this capacity at metamorphosis. This makes them ideal models to elucidate molecular pathways underlying regenerative capacity by comparing responses to spinal cord injury in regenerative (R) and non-regenerative (NR) stages of the same species. Here we use quantitative proteomics with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) followed by Ingenuity Pathway Analysis (IPA) to identify functions and pathways that were differentially regulated after spinal cord injury between R and NR stages in Xenopus laevis. We find that many embryonic pathways of neuronal development are re-activated following SCI at the R but not at the NR stage. This is accompanied by the upregulation of regulatory proteins controlling transcription and translation at the R stage, but their downregulation at the NR stage. Conversely, lipid hydrolysis and uptake as well as mitochondrial oxidative phosphorylation is downregulated at the R, but upregulated at the NR stage. Taken together this suggests that dysregulation of lipid homeostasis and augmentation of oxidative stress play a key role in the loss of regenerative capacity of the spinal cord after metamorphosis. In identifying new factors regulating regenerative capacity in the vertebrate spinal cord, our findings suggest new potential therapeutic targets for promoting neural repair in the injured adult mammalian spinal cord.

Neuroinflammation-caused secondary injury is a key event after spinal cord injury (SCI). Dedicator of cytokinesis 2 (DOCK2) belonging to DOCK-A subfamily has a vital role in microglia polarization and neuroinflammation via mediating Rac activation. However, the role of DOCK2 in SCI is unclear. In the present study, SCI model in mice was established by an impactor at thoracic T10 level. DOCK2 expression was significantly increased in the spinal cord after SCI. After knocking down DOCK2 using a lentivirus-mediated method, SCI mice exhibited improved motor function recovery, as revealed by increased Basso Mouse Scale (BMS) score, angle of incline, and relatively coordinated footprint, and decreased damaged area in the spinal cord. DOCK2 deficiency reduced neuronal apoptosis in the spinal cord after injury. Besides, deficiency of DOCK2 suppressed neuroinflammation after SCI, demonstrated by the reduction in pro-inflammatory mediators including IFN-γ, IL-1β and IL-6 and the increase in IL-4, IL-10 and IL-13, anti-inflammatory factors. The CD86, iNOS and COX-2 were down-regulated in the spinal cord, whereas CD206, Arg-1 and TGF-β were up-regulated by DOCK2 deficiency. Rac activation was prevented by DOCK2 deficiency following SCI. In vitro experiments were conducted for further verification. Treatment of BV-2 microglia with lentivirus-mediated DOCK2 inhibited IFN-γ/LPS-induced pro-inflammatory microglia polarization but increased IL-4-induced anti-inflammatory microglia, through inhibiting Rac activation. In brief, our data reveal that DOCK2 deficiency improves functional recovery in mice after SCI, which is related to Rac activation.

Though spinal cord injury (SCI) causes irreversible sensory and motor impairments in human, adult zebrafish retain the potent regenerative capacity by injury-induced proliferation of central nervous system (CNS)-resident progenitor cells to develop new functional neurons at the lesion site. The hallmark of SCI in zebrafish lies in a series of changes in the epigenetic landscape, specifically DNA methylation and histone modifications. Decoding the post-SCI epigenetic modifications is therefore critical for the development of therapeutic remedies that boost SCI recovery process. Here, we have studied on Sirtuin1 (Sirt1), a non-classical histone deacetylase that potentially plays a critical role in neural progenitor cells (NPC) proliferation and axonal regrowth following SCI in zebrafish. We investigated the role of Sirt1 in NPC proliferation and axonal regrowth in response to injury in the regenerating spinal cord and found that Sirt1 is involved in the induction of NPC proliferation along with glial bridging during spinal cord regeneration. We also demonstrate that Sirt1 plays a pivotal role in regulating the HIPPO pathway through deacetylation-mediated inactivation of Dnmt1 and subsequent hypomethylation of yap1 promoter, leading to the induction of ctgfa expression, which drives the NPC proliferation and axonal regrowth to complete the regenerative process. In conclusion, our study reveals a novel cross-talk between two important epigenetic effectors, Sirt1 and Dnmt1, in the context of spinal cord regeneration, establishing a previously undisclosed relation between Sirt1 and Yap1 which provides a deeper understanding of the underlying mechanisms governing injury-induced NPC proliferation and axonal regrowth. Therefore, we have identified Sirt1 as a novel, major epigenetic regulator of spinal cord regeneration by modulating the HIPPO pathway in zebrafish.

Zebrafish are an increasingly popular model to study regeneration after spinal cord injury (SCI). The transparency of larval zebrafish makes them ideal to study cellular processes in real time. Standardized approaches, including age at the time of injury, are not readily available making comparisons of the results with other models challenging. In this study, we systematically examined the response to spinal cord transection of larval zebrafish at three different larval ages (3-, 5-, or 7-days post fertilization (dpf)) to determine whether the developmental complexity of the larvae affects the overall response to SCI. We then used imaging and behavioral analysis to evaluate whether differences existed based on the age of injury. Injury led to increased expression of cytokines associated with the immune response; however, we found that the timing of specific inflammatory markers changed with the age of the injury. We also observed changes in glial and axonal bridging with age. Young larvae (3 dpf) were better able to regenerate axons independent of the glial bridge, unlike older larvae (7 dpf), consistent with results seen in adult zebrafish. Finally, locomotor experiments demonstrated that some swimming behavior occurs independent of glial bridge formation, further highlighting the need for standardization of this model and functional recovery assays. Overall, we found differences based on the age of transection in larval zebrafish, underlining the importance of considering age when designing experiments aimed at understanding regeneration.

This chapter describes the pivotal role of animal models for unraveling the physiology of neuroglial cells in the central nervous system (CNS). The two rodent species Mus musculus (mice) and Rattus norvegicus (rats) have been indispensable in scientific research due to their remarkable resemblance to humans anatomically, physiologically, and genetically. Their ease of maintenance, short gestation times, and rapid development make them ideal candidates for studying the physiology of astrocytes, oligodendrocyte-lineage cells, and microglia. Moreover, their genetic similarity to humans facilitates the investigation of molecular mechanisms governing neural physiology. Mice are largely the predominant model of neuroglial research, owing to advanced genetic manipulation techniques, whereas rats remain invaluable for applications requiring larger CNS structures for surgical manipulations. Next to rodents, other animal models, namely, Danio rerio (zebrafish) and Drosophila melanogaster (fruit fly), will be discussed to emphasize their critical role in advancing our understanding of glial physiology. Each animal model provides distinct advantages and disadvantages. By combining the strengths of each of them, researchers can gain comprehensive insights into glial function across species, ultimately promoting the understanding of glial physiology in the human CNS and driving the development of novel therapeutic interventions for CNS disorders.

Degenerative diseases and injuries of central nervous system (CNS) often cause nerve cell apoptosis and neural dysfunction. Protection of surviving cells or inducing the differentiation of stem cells into functional cells is considered to be an important way of neurorepair. In addition, transdifferentiation technology emerged recently is expected to provide new solutions for nerve regeneration. Cell surface receptors are transmembrane proteins embedded in cytoplasmic membrane, and play crucial roles in maintaining communication between extracellular signals and intracellular signaling processes. The extracellular microenvironment changed dramatically upon neural lesion, exploring the biological function of signals mediated by cell surface receptors will help to develop molecular strategies for nerve regeneration. An increasing number of studies have reported that cell surface receptor-mediated signaling affects the survival, differentiation, and functioning of neural cells, and even regulate their trans-lineage reprogramming. Here, we provide a review on the roles of cell surface receptors in CNS regeneration, thus providing new cues for better treatment of neurodegenerative diseases or nerve injury.

Wound healing after spinal cord injury involves highly coordinated interactions among multiple cell types, which is poorly understood. Astrocytes play a central role in creating a border against the non-neural lesion core. To do so, astrocytes undergo dramatic morphological changes by first thickening the processes and then elongating and overlap them. We show here show that the expression of a cell-surface receptor, Ryk, is induced in astrocytes after injury in both rodent and human spinal cord. Astrocyte-specific knockout of Ryk dramatically elongated the reactive astrocytes and accelerated the formation of the border and reduced the size of the scar. Astrocyte-specific knockout of Ryk also accelerated the injury responses of multiple cell types, including the resolution of neuroinflammation. Single cell transcriptomics analyses revealed a broad range of changes cell signaling among astrocytes, microglia, fibroblasts, endothelial cell, etc, after astrocyte-specific Ryk knockout, suggesting that Ryk not only regulates the injury response of astrocytes but may also regulate signals which coordinate the responses of multiple cell types. The elongation is mediated by NrCAM, a cell adhesion molecule induced by astrocyte-specific conditional knockout of Ryk after spinal cord injury. Our findings suggest a promising therapeutic target to accelerate wound healing and promote neuronal survival and enhance functional recovery.

Stimulation of pancreatic beta cell regeneration could be a therapeutic lead to treat diabetes. Unlike humans, the zebrafish can efficiently regenerate beta cells, notably from ductal pancreatic progenitors. To gain insight into the molecular pathways involved in this process, we established the transcriptomic profile of the ductal cells after beta cell ablation in the adult zebrafish. These data highlighted the protein phosphatase calcineurin (CaN) as a new potential modulator of beta cell regeneration. We showed that CaN overexpression abolished the regenerative response, leading to glycemia dysregulation. On the opposite, CaN inhibition increased ductal cell proliferation and subsequent beta cell regeneration. Interestingly, the enhanced proliferation of the progenitors was paradoxically coupled with their exhaustion. This suggests that the proliferating progenitors are next entering in differentiation. CaN appears as a guardian which prevents an excessive progenitor proliferation to preserve the pool of progenitors. Altogether, our findings reveal CaN as a key player in the balance between proliferation and differentiation to enable a proper beta cell regeneration.

In goldfish, spinal cord injury triggers the formation of a fibrous scar at the injury site. Regenerating axons are able to penetrate the scar tissue, resulting in the recovery of motor function. Previous findings suggested that regenerating axons enter the scar through tubular structures surrounded by glial elements with laminin-positive basement membranes and that glial processes expressing glial fibrillary acidic protein (GFAP) are associated with axonal regeneration. How glia contribute to promoting axonal regeneration, however, is unknown. Here, we revealed that glial processes expressing vimentin or brain lipid-binding protein (BLBP) also enter the fibrous scar after spinal cord injury in goldfish. Vimentin-positive glial processes were more numerous than GFAP- or BLBP-positive glial processes in the scar tissue. Regenerating axons in the scar tissue were more closely associated with vimentin-positive glial processes than GFAP-positive glial processes. Vimentin-positive glial processes co-expressed matrix metalloproteinase (MMP)-14. Our findings suggest that vimentin-positive glial processes closely associate with regenerating axons through tubular structures entering the scar after spinal cord injury in goldfish. In intact spinal cord, ependymo-radial glial cell bodies express BLBP and their radial processes express vimentin, suggesting that vimentin-positive glial processes derive from migrating ependymo-radial glial cells. MMP-14 expressed in vimentin-positive glial cells and their processes might provide a beneficial environment for axonal regeneration.

The central and peripheral nervous systems (CNS and PNS, respectively) exhibit remarkable diversity in the capacity to regenerate following neuronal injury with PNS injuries being much more likely to regenerate than those that occur in the CNS. Glial responses to damage greatly influence the likelihood of regeneration by either promoting or inhibiting axonal regrowth over time. However, despite our understanding of how some glial lineages participate in nerve degeneration and regeneration, less is known about the contributions of peripheral satellite glial cells (SGC) to regeneration failure following central axon branch injury of dorsal root ganglia (DRG) sensory neurons. Here, using in vivo, time-lapse imaging in larval zebrafish coupled with laser axotomy, we investigate the role of SGCs in axonal regeneration. In our studies we show that SGCs respond to injury by relocating their nuclei to the injury site during the same period that DRG neurons produce new central branch neurites. Laser ablation of SGCs prior to axon injury results in more neurite growth attempts and ultimately a higher rate of successful central axon regrowth, implicating SGCs as inhibitors of regeneration. We also demonstrate that this SGC response is mediated in part by ErbB signaling, as chemical inhibition of this receptor results in reduced SGC motility and enhanced central axon regrowth. These findings provide new insights into SGC-neuron interactions under injury conditions and how these interactions influence nervous system repair.

Neuropathic pain (NP), a severe chronic pain condition, remains a substantial clinical challenge due to its complex pathophysiology and limited effective treatments. An association between the members of the Fibroblast Growth Factors (FGFs), particularly Fgf3, and the development of NP has become evident. In this study, utilizing a mouse model of NP, we observed a time-dependent increase in Fgf3 expression at both mRNA and protein levels within the dorsal root ganglia (DRG). Functional studies revealed that blocking Fgf3 expression mitigated nerve injury induced nociceptive hypersensitivity, suggesting its pivotal role in pain modulation. Moreover, our findings elucidate that Fgf3 contributes to pain hypersensitivity through the activation of the Akt/mTOR signaling in injured DRG neurons. These results not only shed light on the involvement of Fgf3 in nerve injury-induced NP but also highlight its potential as a promising therapeutic target for pain management. This study thereby advances our understanding of the molecular mechanisms underlying NP and opens new avenues for the development of effective treatment strategies.

Traumatic brain injury (TBI) and spinal cord injury (SCI) are neurological conditions that result from immediate mechanical injury, as well as delayed injury caused by local inflammation. Furthermore, TBI and SCI often lead to secondary complications, including pressure wounds of the skin, which can heal slowly and are prone to infection. Pressure wounds are localized areas of damaged tissue caused by prolonged pressure on the skin due to immobility and loss of neurological sensation. With the aim to ameliorate these symptoms, we investigated whether fibroblast growth factors 2 (FGF-2) could contribute to recovery. FGF-2 plays a significant role in both neurogenesis and skin wound healing. We developed a recombinant fusion protein containing FGF-2 linked to elastin-like polypeptides (FGF-ELP) that spontaneously self-assembles into nanoparticles at around 33 °C. The nanoparticle's size was ranging between 220 and 250 nm in diameter at 2 μM. We tested this construct for its ability to address neuronal and skin cell injuries. Hydrogen peroxide was used to induce oxidant-mediated injury on cultured neuronal cells to mimic the impact of reactive oxidants released during the inflammatory response in vivo. We found that FGF-ELP nanoparticles protected against hydrogen peroxide-mediated injury and promoted neurite outgrowth. In the skin cell models, cells were depleted from serum to mimic the reduced levels of nutrients and growth factors in chronic skin wounds. FGF-ELP increased the proliferation and migration of human keratinocytes, fibroblasts, and endothelial cells. FGF-ELP is, therefore, a potentially useful agent to provide both neuroprotection and promotion of cellular processes involved in skin wound healing.

Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, a transient population of injury-responsive neurons (iNeurons) show elevated plasticity 1 week post-injury. We found iNeurons are injury-surviving neurons that acquire a neuroblast-like gene expression signature after injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.

In humans and other adult mammals, axon regeneration is difficult in axotomized neurons. Therefore, spinal cord injury (SCI) is a devastating event that can lead to permanent loss of locomotor and sensory functions. Moreover, the molecular mechanisms of axon regeneration in vertebrates are not very well understood, and currently, no effective treatment is available for SCI. In striking contrast to adult mammals, many nonmammalian vertebrates such as reptiles, amphibians, bony fishes and lampreys can spontaneously resume locomotion even after complete SCI. In recent years, rapid progress in the development of next-generation sequencing technologies has offered valuable information on SCI. In this review, we aimed to provide a comparison of axon regeneration process across classical model organisms, focusing on crucial genes and signalling pathways that play significant roles in the regeneration of individually identifiable descending neurons after SCI. Considering the special evolutionary location and powerful regenerative ability of lamprey and zebrafish, they will be the key model organisms for ongoing studies on spinal cord regeneration. Detailed study of SCI in these model organisms will help in the elucidation of molecular mechanisms of neuron regeneration across species.

Spinal cord injury (SCI) affects between 250,000 to 500,000 individuals annually. After the initial injury, a delayed secondary cascade of cellular responses occurs causing progressive degeneration and permanent disability. One part of this secondary process is disturbance of ionic homeostasis. The K+ channel blocker, 4-aminopyridine (4-AP), is used clinically to alleviate symptoms of multiple sclerosis (MS). Several ongoing studies are being conducted to explore additional areas where 4-AP may have an effect, including stroke, traumatic brain injury, and nervous system recovery after SCI. The goal of our study was to determine whether 4-AP affects recovery from SCI in zebrafish (Danio rerio). Using the transgenic line Tg(gfap:EGFP), we created a spinal transection and tracked swim recovery. We found that constant treatment with 10 μM 4-AP increases swimming distance 40%. Live imaging demonstrated that treatment with 4-AP increases radial glial cells bridging at the site of injury in the presence of 4-AP. We conclude that 10 μM 4-AP is pro-regenerative after SCI.

Immune cells elicit a continuum of transcriptional and functional states after spinal cord injury (SCI). In mammals, inefficient debris clearance and chronic inflammation impede recovery and overshadow pro-regenerative immune functions. We found that, unlike mammals, zebrafish SCI elicits transient immune activation and efficient debris clearance, without causing chronic inflammation. Single-cell transcriptomics and inducible genetic ablation showed zebrafish macrophages are highly phagocytic and required for regeneration. Cross-species comparisons between zebrafish and mammalian macrophages identified transcription and immune response regulator ( tcim ) as a macrophage-enriched zebrafish gene. Genetic deletion of zebrafish tcim impairs phagocytosis and regeneration, causes aberrant and chronic immune activation, and can be rescued by transplanting wild-type immune precursors into tcim mutants. Conversely, genetic expression of human TCIM accelerates debris clearance and regeneration by reprogramming myeloid precursors into activated phagocytes. This study establishes a central requirement for elevated phagocytic capacity to achieve innate spinal cord repair.

The zebrafish is a powerful model organism for studying development and regeneration. However, there is a lack of a standardized reference diet for developmental and regeneration experiments. Most studies evaluate the rate of growth, survival, and fecundity. In this study, we compare three diets and their effects on growth and regeneration after a spinal cord injury (SCI). Fish were fed daily for 1 week with daily measurements of overall length and width of spinal injury. Fish fed a live rotifer diet grew 32%, whereas a commercially available diet only led to a 4% increase in body length. Similarly, differences in rate of regeneration were observed with over 80% of rotifer-fed larvae forming a glial bridge after injury compared to <10% of zebrafish fed with the commercial diet. Our data highlight the need for establishing a standardized diet for regeneration studies to improve research reproducibility.

Studying the properties of neural stem progenitor cells (NSPCs) in a fish model will provide new information about the organization of neurogenic niches containing embryonic and adult neural stem cells, reflecting their development, origin cell lines and proliferative dynamics. Currently, the molecular signatures of these populations in homeostasis and repair in the vertebrate forebrain are being intensively studied. Outside the telencephalon, the regenerative plasticity of NSPCs and their biological significance have not yet been practically studied. The impressive capacity of juvenile salmon to regenerate brain suggests that most NSPCs are likely multipotent, as they are capable of replacing virtually all cell lineages lost during injury, including neuroepithelial cells, radial glia, oligodendrocytes, and neurons. However, the unique regenerative profile of individual cell phenotypes in the diverse niches of brain stem cells remains unclear. Various types of neuronal precursors, as previously shown, are contained in sufficient numbers in different parts of the brain in juvenile Pacific salmon. This review article aims to provide an update on NSPCs in the brain of common models of zebrafish and other fish species, including Pacific salmon, and the involvement of these cells in homeostatic brain growth as well as reparative processes during the postraumatic period. Additionally, new data are presented on the participation of astrocytic glia in the functioning of neural circuits and animal behavior. Thus, from a molecular aspect, zebrafish radial glia cells are seen to be similar to mammalian astrocytes, and can therefore also be referred to as astroglia. However, a question exists as to if zebrafish astroglia cells interact functionally with neurons, in a similar way to their mammalian counterparts. Future studies of this fish will complement those on rodents and provide important information about the cellular and physiological processes underlying astroglial function that modulate neural activity and behavior in animals.

Spinal cord injuries cause irreversible loss of sensory and motor functions. In mammals, intrinsic and extrinsic inhibitions of neuronal regeneration obstruct neural repair after spinal cord injury. Although astrocytes have been involved in a growing list of vital homeostatic functions in the nervous system, their roles after injury have fascinated and puzzled scientists for decades. Astrocytes undergo long-lasting morphological and functional changes after injury, referred to as reactive astrogliosis. Although reactive astrogliosis is required to contain spinal cord lesions and restore the blood-spinal cord barrier, reactive astrocytes have detrimental effects that inhibit neuronal repair and remyelination. Intriguingly, elevated regenerative capacity is preserved in some non-mammalian vertebrates, where astrocyte-like glial cells display exclusively pro-regenerative effects after injury. A detailed molecular and phenotypic catalog of the continuum of astrocyte reactivity states is an essential first step toward the development of glial cell manipulations for spinal cord repair.

Unlike mammals, adult and larval zebrafish exhibit robust regeneration following traumatic spinal cord injury. This remarkable regenerative capacity, combined with exquisite imaging capabilities and an abundance of powerful genetic techniques, has established the zebrafish as an important vertebrate model for the study of neural regeneration. Here, we describe a protocol for the complete mechanical ablation of the larval zebrafish spinal cord. With practice, this protocol can be used to reproducibly injure upward of 100 samples per hour, facilitating the high-throughput screening of factors involved in spinal cord regeneration and repair.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection to their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain swim capacity within 8 weeks of injury. Here, we describe methods to perform complete spinal cord transections and to assess functional and cellular recovery during regeneration. For spinal cord injury, a complete transection is performed 4 mm caudal to the brainstem. Swim endurance is quantified as a central readout of functional spinal cord repair. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. To assess cellular regeneration, histological examination is performed to analyze the extents of glial and axonal bridging across the lesion.

Identification of signaling events that contribute to innate spinal cord regeneration in zebrafish can uncover new targets for modulating injury responses of the mammalian central nervous system. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, dynamic and regional regulation of JNK signaling help to direct glial cell behaviors during innate spinal cord regeneration.

Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, transient populations of injury-responsive neurons (iNeurons) show elevated plasticity between 1 and 3 weeks post-injury. Using cross-species transcriptomics and CRISPR/Cas9 mutagenesis, we found iNeurons are injury-surviving neurons that share transcriptional similarities with a rare population of spontaneously plastic mouse neurons. iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.

The fibroblast growth factor (FGF) family regulates various and important aspects of nervous system development, ranging from the well-established roles in neuronal patterning to more recent and exciting functions in axonal growth and synaptogenesis. In addition, FGFs play a critical role in axonal regeneration, particularly after spinal cord injury, confirming their versatile nature in the nervous system. Due to their widespread involvement in neural development, the FGF system also underlies several human neurological disorders. While particular attention has been given to FGFs in a whole-cell context, their effects at the axonal level are in most cases undervalued. Here we discuss the endeavor of the FGF system in axons, we delve into this neuronal subcompartment to provide an original view of this multipurpose family of growth factors in nervous system (dys)function. Video Abstract.

Glia play multifaceted roles in nervous systems in response to injury. Depending on the species, extent of injury and glial cell type in question, glia can help or hinder the regeneration of neurons. Studying glia in the context of successful regeneration could reveal features of pro-regenerative glia that could be exploited for new human therapies. Planarian flatworms completely regenerate their nervous systems after injury - including glia - and thus provide a strong model system for exploring glia in the context of regeneration. Here, we report that planarian glia regenerate after neurons, and that neurons are required for correct glial numbers and localization during regeneration. We also identify the planarian transcription factor-encoding gene ets-1 as a key regulator of glial cell maintenance and regeneration. Using ets-1 (RNAi) to perturb glia, we show that glial loss is associated with altered neuronal gene expression, impeded animal movement and impaired nervous system architecture - particularly within the neuropil. Importantly, our work reveals the inter-relationships of glia and neurons in the context of robust neural regeneration.

Spinal cord injury (SCI) is a devastating event that results in a wide range of physical impairments and disabilities. Despite the advances in our understanding of the biological response to injured tissue, no effective treatments are available for SCIs at present. Some studies have addressed this issue by exploring the potential of cell transplantation therapy. However, because of the abnormal microenvironment in injured tissue, the survival rate of transplanted cells is often low, thus limiting the efficacy of such treatments. Many studies have attempted to overcome these obstacles using a variety of cell types and animal models. Recent studies have shown the utility of zebrafish as a model of neural regeneration following SCIs, including the proliferation and migration of various cell types and the involvement of various progenitor cells. In this review, we discuss some of the current challenges in SCI research, including the accurate identification of cell types involved in neural regeneration, the adverse microenvironment created by SCIs, attenuated immune responses that inhibit nerve regeneration, and glial scar formation that prevents axonal regeneration. More in-depth studies are needed to fully understand the neural regeneration mechanisms, proteins, and signaling pathways involved in the complex interactions between the SCI microenvironment and transplanted cells in non-mammals, particularly in the zebrafish model, which could, in turn, lead to new therapeutic approaches to treat SCIs in humans and other mammals.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.

The vascular system is inefficiently repaired after spinal cord injury (SCI) in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord (SC) and restore motility, but the vascular response to injury has not been investigated. Here, we describe the zebrafish SC blood vasculature, starting in development with the initial vessel ingression in a body size-dependent manner, the acquisition of perivascular support and the establishment of ventral to dorsal blood circulation. The vascular organization grows in complexity and displays multiple barrier specializations in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and axons. Vascular repair involves an early burst of angiogenesis that creates dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-SC barrier is re-established. This study demonstrates that zebrafish can successfully re-vascularize the spinal tissue, reinforcing the value of this organism as a regenerative model for SCI.

Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Axon growth, glial bridging and adult neurogenesis are hallmarks of cellular regeneration during spinal cord repair. However, the correlation between these cellular regenerative processes and functional recovery remains to be elucidated. Whereas the majority of established functional regeneration metrics measure swim capacity, we hypothesize that gait quality is more directly related to neurological health. Here, we performed a longitudinal swim tracking study for 60 individual zebrafish spanning 8 weeks of spinal cord regeneration. Multiple swim parameters as well as axonal and glial bridging were integrated. We established rostral compensation as a new gait quality metric that highly correlates with functional recovery. Tensor component analysis of longitudinal data supports a correspondence between functional recovery trajectories and neurological outcomes. Moreover, our studies predicted and validated that a subset of functional regeneration parameters measured 1 to 2 weeks post-injury is sufficient to predict the regenerative outcomes of individual animals at 8 weeks post-injury. Our findings established new functional regeneration parameters and generated a comprehensive correlative database between various functional and cellular regeneration outputs.

One of the most common forms of the mammalian central nervous system (CNS) injuries is spinal cord injury (SCI), and any lesion to the CNS can result in a lifelong functional impairment since CNS axons cannot regenerate. The relative axon regenerating genes following spinal SCI were examined using the regenerative SN, pSN + DC, and non-regenerating DC lesion models. By using qRT-PCR, we discovered that fibroblast growth factor receptor-5 (FGFR5) was 4.2-fold more highly expressed in non-regeneration lesions compared to intact control and regenerating animals. Furthermore, in cultured dorsal root ganglion neurons (DRGN), short interfering RNA (siRNA)-mediated knockdown of FGFR5 had no effect on DRGN neurite outgrowth, indicating that the gene's suppression has no effect on axon regeneration and may play other roles in the CNS besides axon regeneration.

Neural stem cells (NSCs) derived from human pluripotent stem cells (hPSCs) are considered a major cell source for reconstructing damaged neural circuitry and enabling axonal regeneration. However, the microenvironment at the site of spinal cord injury (SCI) and inadequate intrinsic factors limit the therapeutic potential of transplanted NSCs. Here, it is shown that half dose of SOX9 in hPSCs-derived NSCs (hNSCs) results in robust neuronal differentiation bias toward motor neuron lineage. The enhanced neurogenic potency is partly attributed to the reduction of glycolysis. These neurogenic and metabolic properties retain after transplantation of hNSCs with reduced SOX9 expression in a contusive SCI rat model without the need for growth factor-enriched matrices. Importantly, the grafts exhibit excellent integration properties, predominantly differentiate into motor neurons, reduce glial scar matrix accumulation to facilitate long-distance axon growth and neuronal connectivity with the host as well as dramatically improve locomotor and somatosensory function in recipient animals. These results demonstrate that hNSCs with half SOX9 gene dosage can overcome extrinsic and intrinsic barriers, representing a powerful therapeutic potential for transplantation treatments for SCI.

Unlike mammals, adult zebrafish undergo spontaneous recovery after major spinal cord injury. Whereas reactive gliosis presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish elicit pro-regenerative bridging functions after injury. Here, we perform genetic lineage tracing, assessment of regulatory sequences and inducible cell ablation to define mechanisms that direct the molecular and cellular responses of glial cells after spinal cord injury in adult zebrafish. Using a newly generated CreERT2 transgenic line, we show that the cells directing expression of the bridging glial marker ctgfa give rise to regenerating glia after injury, with negligible contribution to either neuronal or oligodendrocyte lineages. A 1 kb sequence upstream of the ctgfa gene was sufficient to direct expression in early bridging glia after injury. Finally, ablation of ctgfa-expressing cells using a transgenic nitroreductase strategy impaired glial bridging and recovery of swim behavior after injury. This study identifies key regulatory features, cellular progeny, and requirements of glial cells during innate spinal cord regeneration.

Axolotls have been considered to be able to regenerate their skin completely. Our recent study updated this theory with the finding that the lattice structure of dermal collagen fibers was not fully regenerated after skin injury. We also discovered that nerves induce the regeneration of collagen fibers. The mechanism of collagen fiber regeneration remains unknown, however. In this study, we focused on the structure of collagen fibers with collagen braiding cells, and cell origin in axolotl skin regeneration. In the wounded dermis, cells involved in skin repair/regeneration were derived from both the surrounding dermis and the subcutaneous tissue. Regardless of cell origin, cells acquired the proper cell morphology to braid collagen fiber with nerve presence. We also found that FGF signaling could substitute for the nerve roles in the conversion of subcutaneous fibroblasts to lattice-shaped dermal fibroblasts. Our findings contribute to the elucidation of the fundamental mechanisms of true skin regeneration and provide useful insights for pioneering new skin treatments.

Adult zebrafish are capable of anatomical and functional recovery following severe spinal cord injury. Axon growth, glial bridging and adult neurogenesis are hallmarks of cellular regeneration during spinal cord repair. However, the correlation between these cellular regenerative processes and functional recovery remains to be elucidated. Whereas the majority of established functional regeneration metrics measure swim capacity, we hypothesize that gait quality is more directly related to neurological health. Here, we performed a longitudinal swim tracking study for sixty individual zebrafish spanning eight weeks of spinal cord regeneration. Multiple swim parameters as well as axonal and glial bridging were integrated. We established rostral compensation as a new gait quality metric that highly correlates with functional recovery. Tensor component analysis of longitudinal data supports a correspondence between functional recovery trajectories and neurological outcomes. Moreover, our studies predicted and validated that a subset of functional regeneration parameters measured 1 to 2 weeks post-injury is sufficient to predict the regenerative outcomes of individual animals at 8 weeks post-injury. Our findings established new functional regeneration parameters and generated a comprehensive correlative database between various functional and cellular regeneration outputs.

Spinal cord injury (SCI) can cause long-lasting disability in mammals due to the lack of axonal regrowth together with the inability to reinitiate spinal neurogenesis at the injury site. Deciphering the mechanisms that regulate the proliferation and differentiation of neural progenitor cells is critical for understanding spinal neurogenesis after injury. Compared with mammals, zebrafish show a remarkable capability of spinal cord regeneration. Here, we show that Rassf7a, a member of the Ras-association domain family, promotes spinal cord regeneration after injury. Zebrafish larvae harboring a rassf7a mutation show spinal cord regeneration and spinal neurogenesis defects. Live imaging shows abnormal asymmetric neurogenic divisions and spindle orientation defects in mutant neural progenitor cells. In line with this, the expression of rassf7a is enriched in neural progenitor cells. Subcellular analysis shows that Rassf7a localizes to the centrosome and is essential for cell cycle progression. Our data indicate a role for Rassf7a in modulating spindle orientation and the proliferation of neural progenitor cells after spinal cord injury.

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MAP2K signaling plays a key role in axon elongation during nervous system development. Here, we show that conditional expression of a constitutively kinase-activated BRAF in mature corticospinal neurons elicited the expression of a set of transcription factors previously implicated in the regeneration of zebrafish retinal ganglion cell axons and promoted regeneration and sprouting of corticospinal tract (CST) axons after spinal cord injury in mice. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, resulting in improved recovery of motor function. Noninvasive suprathreshold high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) activated the BRAF canonical downstream effectors MAP2K1/2 and modulated the expression of a set of regeneration-related transcription factors in a pattern consistent with that induced by BRAF activation. HF-rTMS enabled CST axon regeneration and sprouting, which was abolished in MAP2K1/2 conditional null mice. These data collectively demonstrate a central role of MAP2K signaling in augmenting the growth capacity of mature corticospinal neurons and suggest that HF-rTMS might have potential for treating spinal cord injury by modulating MAP2K signaling.

Zebrafish have a remarkable capacity for spontaneously regenerating their central nervous system. Larval zebrafish are optically transparent and therefore are widely used to dynamically visualize cellular processes in vivo, such as nerve regeneration. Regeneration of retinal ganglion cell (RGC) axons within the optic nerve has been previously studied in adult zebrafish. In contrast, assays of optic nerve regeneration have previously not been established in larval zebrafish. In order to take advantage of the imaging capabilities in the larval zebrafish model, we recently developed an assay to physically transect RGC axons and monitor optic nerve regeneration in larval zebrafish. We found that RGC axons rapidly and robustly regrow to the optic tectum. Here, we describe the methods for performing the optic nerve transections, as well as methods for visualizing RGC regeneration in larval zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

Human spinal cord injury (SCI) is characterized by permanent loss of damaged axons, resulting in chronic disability. In contrast, zebrafish can regenerate axonal projections following central nervous system injury and re-establish synaptic contacts with distant targets; elucidation of the underlying molecular events is an important goal with translational potential for improving outcomes in SCI patients. We generated transgenic zebrafish with GFP-labeled axons and transected their spinal cords at 10 days post-fertilization. Intravital confocal microscopy revealed robust axonal regeneration following the procedure, with abundant axons bridging the transection site by 48 h post-injury. In order to analyze neurological function in this model, we developed and validated new open-source software to measure zebrafish lateral trunk curvature during propulsive and turning movements at high temporal resolution. Immediately following spinal cord transection, axial movements were dramatically decreased caudal to the lesion site, but preserved rostral to the injury, suggesting the induction of motor paralysis below the transection level. Over the subsequent 96 h, the magnitude of movements caudal to the lesion recovered to baseline, but the rate of change of truncal curvature did not fully recover, suggesting incomplete restoration of caudal strength over this time course. Quantification of both morphological and functional recovery following SCI will be important for the analysis of axonal regeneration and downstream events necessary for restoration of motor function. An extensive array of genetic and pharmacological interventions can be deployed in the larval zebrafish model to investigate the underlying molecular mechanisms.

[This corrects the article DOI: 10.4103/1673-5374.338993].

In fishes and salamanders, but not mammals, neural stem cells switch back to neurogenesis after injury. The signalling environment of neural stem cells is strongly altered by the presence of damaged cells and an influx of immune, as well as other, cells. Here, we summarise our recently expanded knowledge of developmental, physiological and immune signals that act on neural stem cells in the zebrafish central nervous system to directly, or indirectly, influence their neurogenic state. These signals act on several intracellular pathways, which leads to changes in chromatin accessibility and gene expression, ultimately resulting in regenerative neurogenesis. Translational approaches in non-regenerating mammals indicate that central nervous system stem cells can be reprogrammed for neurogenesis. Understanding signalling mechanisms in naturally regenerating species show the path to experimentally promoting neurogenesis in mammals.

Many people around the world suffer from some form of paralysis caused by spinal cord injury (SCI), which has an impact on quality and life expectancy. The spinal cord is part of the central nervous system (CNS), which in mammals is unable to regenerate, and to date, there is a lack of full functional recovery therapies for SCI. These injuries start with a rapid and mechanical insult, followed by a secondary phase leading progressively to greater damage. This secondary phase can be potentially modifiable through targeted therapies. The growing literature, derived from mammalian and regenerative model studies, supports a leading role for mitochondria in every cellular response after SCI: mitochondrial dysfunction is the common event of different triggers leading to cell death, cellular metabolism regulates the immune response, mitochondrial number and localization correlate with axon regenerative capacity, while mitochondrial abundance and substrate utilization regulate neural stem progenitor cells self-renewal and differentiation. Herein, we present a comprehensive review of the cellular responses during the secondary phase of SCI, the mitochondrial contribution to each of them, as well as evidence of mitochondrial involvement in spinal cord regeneration, suggesting that a more in-depth study of mitochondrial function and regulation is needed to identify potential targets for SCI therapeutic intervention.