Astrocyte-Neuron Interactions in Spinal Cord Injury

Abstract

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.

1. Introduction

This chapter provides an overview of the complex and fascinating roles of astrocytes after spinal cord injury. We first review the disease burden and multicellular complications associated with spinal cord injuries. With astrocytes displaying a range of reactivity states in response to different insults, we discuss the advantages and limitations of implementing different models and injury paradigms, highlighting the value of performing comparative studies to generate a comprehensive catalog of astrocyte reactivity states and functions. We then delve into the mechanisms that direct reactive astrogliosis and the dual consequences of reactive astrogliosis as a defense mechanism and anti-regenerative effector after spinal cord injury.

1.1. Spinal Cord Injury: Disease Burden and Multicellular Complications

1.1.1. Spinal Cord Injury Is a Devastating Condition and a Challenging Medical Priority

Spinal cord injuries (SCIs) comprise traumatic and non-traumatic damages that alter the anatomy and function of the spinal cord. Common causes of traumatic SCI include motor vehicle accidents, falls, sports-related injuries, or violence, whereas non-traumatic SCI results from acute or chronic diseases such as cancer, infection, or degenerative disk disease [1, 2]. SCI has devastating, long-term consequences for the physical, psychological, financial, and social well-being of patients and their caregivers. Over the past 30 years, the number of SCI cases has shown a 52.7% increase with over nine million cases worldwide and North America reporting the highest number of cases among developed regions [3]. With less than 1% of SCI patients experiencing complete neurological recovery by hospital discharge, over one million people are affected in North America alone with direct lifetime costs ranging between $1.1 and 4.6 million per patient [2, 4]. With the increased lifespan of the human population, the global burden of living with disability increased by 65.4% from 1990 to 2019 [3]. SCI is commonly viewed as a problem of motor control and sensory loss, resulting in at least partial paralysis. However, SCI drives systemic immune dysregulation across the body, negatively impacting major organ systems and general homeostatic mechanisms [5, 6]. Major concurrent SCI complications, such as neuropathic pain, increased risk of infection, bowel and bladder dysfunction, cardiovascular problems, and predisposition to obesity, diabetes, and liver disease, are challenging medical priorities [5, 7]. Despite advanced medical care, even SCI patients in developed regions have significantly reduced lifespan. SCI is therefore a medical priority that requires concerted research efforts to extend the lifespan and improve the health span of SCI patients.

1.1.2. Spinal Cord Injury Triggers Multicellular and Systemic Injury Complications

SCI leads to loss of motor, sensory, and autonomic functions. Understanding the role of astrocyte-neuron interactions in SCI requires a thorough understanding of various SCI complications, which cooperatively disrupt astrocyte functions after injury. SCI elicits complex multicellular responses that cause permanent neurological deficits and impede spontaneous regeneration in mammals [8–13]. Injury responses following SCI vary depending on the location, severity, and time of injury. SCI has been divided into four temporally distinct phases: acute (0 to 2 days), subacute (2 to 14 days), intermediate (14 days to 6 months), and chronic (>6 months) phases in humans. Acute SCI is marked by the primary physical damage that causes compression or transection to the spinal cord, disrupts spinal vascular flow, and compromises the blood-spinal cord barrier. Primary SCI damages trigger spontaneous cascades of inflammatory, glial, neuronal, and systemic complications that result in neurotoxic inflammation, overt glial cell reactivity, and cyclical cell loss [8, 9, 12, 13]. Even in the acute-to-subacute stages, multiple cellular complications result in secondary damages that are often more detrimental than the primary lesion. Shortly after injury, disrupted vascular flow and exaggerated inflammation cause swelling and additional compression that exacerbate the primary injury. Rapid disruption to calcium homeostasis and ischemic toxicity mediates neuronal and glial cell death, releasing high levels of glutamate into the extracellular milieu. Glutamate, which is cleared by astrocytes under homeostatic conditions, is poorly reabsorbed by surviving astrocytes [14, 15]. Glutamate cytotoxicity and necroptosis lead to cyclical propagation of secondary cell death. Excess glutamate activates NMDA and AMPA receptors, leading to cyclical propagation of glutamate cytotoxicity and necroptosis [14]. Intermediate-to-chronic SCI is marked by scarring, cystic cavitation, neural circuit remodeling, and vascular reorganization, which form additional barriers to regeneration [16–22]. At these stages, the extracellular milieu of the lesioned cord is flooded by reactive cell-derived or debris-derived molecules that are potent inhibitors of axon regeneration. Alterations in the composition of the extracellular matrix result in the accumulation of inhibitory proteoglycans in and around the lesion [20, 23–26]. Inhibitory molecules released by degenerating oligodendrocytes include neurite outgrowth inhibitor A (Nogo-A), oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG). Myelin-associated glycoproteins, upon binding to Nogo and TNF receptors, cause growth cone collapse and neurite retraction in a Rho/ROCK-dependent mechanism [25, 27–30]. Together, these injury complications exacerbate the inherently limited ability of the mammalian spinal cord to replenish lost neurons via adult neurogenesis or to regrow lesioned axon tracts. As primary SCI damages are irreversible, the major goals of SCI therapies have been to target and contain secondary damages. Considering the multicellular complications associated with secondary SCI damages, individual therapeutic approaches are unlikely to yield major effects. Thus, combinatorial strategies have been pursued as a more promising therapeutic avenue [31–34]. Comprehensive and simultaneous examination of neuronal and non-neuronal cells after SCI is therefore fundamental to understanding and manipulating the multicellular complexities of SCI injuries.

1.2. Models of Spinal Cord Injury

Animal models are indispensable for understanding the pathophysiology of SCI, investigating mechanisms of innate spinal cord repair, and testing regenerative therapies. An ideal SCI model should (a) mimic the anatomical and pathophysiological complications of human SCI, (b) possess an endogenous regenerative capacity, (c) be amenable to genetic and pharmacological manipulations, and (d) allow for high-powered and inexpensive studies and therapeutic testing. In the absence of a single animal model that fulfills all these criteria, multiple animal models and injury paradigms have been deployed.

1.2.1. Injury Paradigms

Animal models of SCI include contusion, compression, or transection injuries. These SCI models could be tweaked to target specific injury levels along the rosto-caudal axis of the injured animal and a range of injury severity from mild SCI to severe SCI [35]. Contusion models, which involve a weight-induced impact on the spinal cord, are thought to generate injuries that closely mimic the etiology and outcome of human SCI [36–38]. Compression or crush injuries are created by constricting the exposed cord with forceps or weight application [39–41]. Transection models can be complete transections or partial hemisections and are commonly used to study axon regrowth mechanisms.

1.2.2. Mammalian Models Mimic SCI Complications

Large animal models, such as non-human primate or swine models, are ideal to mimic the pathophysiology of SCI patients, including lesion size and the formation of cystic cavitation post-injury. However, substantial ethical, economical, and technical concerns are associated with large animal models including sample size, cost, and housing requirements. Therefore, large animal models can form an important intermediate model to validate preclinical data prior to initiating clinical studies. In contrast to large animal models, rat and mouse models are less expensive and are prominently used for SCI research and therapeutic testing. While the injury response in rats is anatomically similar to human lesions, including the formation of cystic cavities in the lesion core, mouse SCI lesions do not cavitate [42, 43]. Nonetheless, inherent differences in molecular signaling, regenerative capacity, and decreased amenability to genetic studies have challenged the throughput and translatability of rat SCI studies.

1.2.3. Regenerative Vertebrates Present Role Models for Innate Spinal Cord Repair

Unlike mammals, vertebrate species including teleost fish and amphibians have an innate ability to spontaneously recover from severe SCI. Following complete transection of spinal cord tissues, adult zebrafish are capable of reversing paralysis and regaining swim function within 6 to 8 weeks of injury [44, 45]. Pro-regenerative injury responses involving neurons, glia, and immune cells cooperate to achieve spontaneous and efficient repair in zebrafish [46–52]. Early after SCI, potent populations of progenitor cells, including central canal-surrounding ependymo-radial glial cells, are activated to replenish lost neurons and glia [46, 48, 50, 51]. Newly differentiated motor neurons and interneurons populate the regenerate tissue, as pre-existing neurons regrow axons across lesioned tissues [49–51]. Though less studied, astrocyte-like glial cells enact instrumental pro-regenerative responses throughout the course of regeneration [45, 48, 53]. However, a holistic understanding of the cellular interactions that coordinate the pro-regenerative responses to direct SC regeneration in zebrafish is to be acquired. Thus, how and why injury responses differ between zebrafish and mammalian astrocytes requires comprehensive molecular investigation and cross-species comparisons.

1.3. Astrocyte Responses after Mammalian Spinal Cord Injury

Astrocytes are the largest and most abundant glial cells in mammals (Freeman and Rowitch, 2013). They play developmental and physiological roles that are critical for the development, survival, and function of the central nervous system. Under physiological conditions, astrocytes provide trophic and metabolic support for neurons and play central roles in synapse development, pruning, and maturation [54, 55]. Astrocytes control the blood-spinal cord barrier and modulate spinal blood flow, fluid availability, and ion homeostasis [56, 57]. Spinal astrocytes are highly heterogeneous. Morphological, molecular, and functional differences between white and gray matter astrocytes have emerged in recent years [58, 59]. Accumulating evidence suggests that Gfap is predominantly expressed in white matter astrocytes, while S100β tends to be expressed in the astrocytes located in the gray matter [58, 59]. Even within the gray matter, astrocytes are regionally specialized into anatomical regions that correlate with the respective neurotransmitter properties of each region [60]. As astrocytes are shown to be involved in a growing list of vital homeostatic functions in the nervous system, their roles after injury have fascinated and puzzled scientists for decades.

1.3.1. The Glial Scar

Mammalian SCI is marked by scarring. The “glial scar,” a historically used umbrella term that refers to SCI scarring, was thought to be detrimental for regeneration and blamed on astrocytes [12, 61, 62]. However, emerging technology and cell-specific manipulations have enabled a finer view and deeper understanding of SCI scarring. In response to SCI, multiple cell types rapidly migrate into the lesion site. Lesion-induced cells include immune cells (macrophages, neutrophils, and lymphocytes), fibroblasts, and pericytes in addition to glial cells (microglia, OPCs, and astrocytes). Microglia and macrophages are the first responders, with microglia proliferating and migrating toward the lesion, while monocyte-derived macrophages infiltrate from the periphery via the broken blood-spinal cord barrier. Lesion surrounding astrocytes proliferate, elongate, and form a cellular border that separates the necrotic lesion core from the spared neural tissue that surrounds the lesion [63–65]. With renewed appreciation for the anatomy and complexity of injury-induced scarring, the SCI scar has been more recently categorized into distinct fibrotic and glial compartments. While the fibrotic core is comprised of infiltrating immune cells, fibroblasts, and pericytes, the glial scar is comprised of microglia, NG2 glia, and astrocytes. Concomitantly, the concept that the glial scar is both detrimental and beneficial for functional recovery following SCI has emerged [12, 13, 21, 66]. Thus, the glial scar plays dual roles in pathophysiology of SCI.

In the acute stages of SCI, astrocytes proliferate and migrate toward the lesion site [53, 67, 68]. The border formed by reactive astrocytes isolates the fibrotic compartment of the scar from adjacent preserved neural tissues. Thus, scar-bordering astrocytes play a dual role by (a) limiting the expansion of the inflammatory core and further expansion of the fibrotic lesion core and (b) protecting spared neural tissues from exposure to scar-induced insults that trigger secondary damage [12, 69]. This initial astrocyte response is also necessary to reestablish the blood-spinal cord barrier [39, 70–72]. Subsequently, changes in the gene expression profiles of reactive astrocytes alter the microenvironmental landscape of the lesioned spinal cord, resulting in neurotoxicity and extrinsic inhibition of axon sprouting and functional recovery [22, 73–77]. Astrocytes increase the expression of intermediate filaments (Gfap, nestin, and vimentin) [71, 78–80] and release a variety of cytokines (TNF-α, IL-6, IL-10, and IL-1β) [81–83], chemokines (CCL2 and CCL3) [84, 85], growth factors (BDNF and GDNF) [86, 87], toxic amino acids (GABA and glutamate) [88, 89], and extracellular matrix molecules and proteoglycans (CSPGs, collagen I, fibronectin, and MMP-9) [78, 79, 90]. These molecules stimulate a variety of cellular responses by neurons, OPCs, and microglia, in addition to triggering cyclical reactivity in surrounding astrocytes [91]. In summary, mammalian astrocytes display multifarious injury responses that are overshadowed by anti-regenerative scar-forming cells and inhibitory extracellular molecules [12, 92, 93]. Thus, in light of our increased ability to examine the regional and functional heterogeneity of astrocyte subpopulations in response to SCI, it is important to move away from the umbrella term of the glial scar and to understand the concept and complexity of astrocyte reactivity in response to SCI.

1.3.2. Astrocyte Reactivity

In response to SCI, astrocytes undergo long-lasting morphological and functional changes referred to as “reactive astrogliosis” or “astrocyte reactivity” [94]. Various intrinsic and extrinsic factors stimulate reactive astrogliosis and play critical roles in the pathology and outcomes of SCI. Reactive astrogliosis results in a range of changes in the transcriptional profiles of reactive astrocytes and the adoption of newly acquired biochemical, morphological, metabolic, and physiological states in response to injury [95]. By causing reactive astrocytes to gain new functions and/or lose their ability to maintain homeostatic functions in the nervous system, reactive astrogliosis is an essential predictor and modulator of SCI outcome.

Cellular hypertrophy, thickening of astrocytic processes, and elevated expression of intermediate filament proteins such as Gfap are the minimum criteria used to define reactive astrocytes. Additional markers of astrocyte reactivity include Plaur, Mmp2, Mmp13, Axin2, Nes, and Ctnnb1, complement 3, type I collagen, Cdh2, Sox9, and chondroitin sulfate proteoglycan (CSPG)-related genes including Xylt1, Csgalnact1, Chst11, Pcan, Acan, and Slit2. While some of these genes have been associated with specific phenotypes such as A1/A2, neurotoxic/neuroprotective, and reactive/scar-forming [75, 96], multiple gene combinations are typically concomitantly expressed and inferring the functional phenotype of a reactive astrocyte based on a handful of markers proved to be misleading. Notably, astrocyte reactivity is a broad terminology that encompasses a continuum of transcriptional and functional states that astrocytes can adopt in response to diverse pathologies. Even within the pathology of SCI, astrocyte reactivity can vastly differ between injuries depending on the location of the lesion, time, and severity of the injury [91]. As the loss of some homeostatic functions can happen simultaneously with gains in protective and/or detrimental functions, computing the overall phenotype of a single reactive astrocyte into beneficial or detrimental is virtually impossible. Adding to the complexity of profiling a single astrocyte, the heterogeneity in astrocyte population prior to and after SCI adds to the challenges of determining astrocyte-induced phenotypes based on their specific reactivity states. In the absence of a prototypical reactive astrocyte and the wrong assumption of categorizing astrocytes into binary phenotypes, astrocyte reactivity has been a challenging research topic in SCI. An updated consensus among astrocyte biologists is to move beyond binary definitions to reflect the increasing complexity and functional heterogeneity of astrocytes [95].

1.3.3. Mechanisms of Astrocyte Activation

Following SCI, mechanical disruption of astrocyte membranes and processes activates mechanosensitive ion channels, resulting in rapid influx of extracellular sodium and calcium ions into astrocytes [97–100]. Further studies showed that membrane stretching is sufficient to release ions and signaling molecules including calcium, ATP, endothelin 1, and matrix metalloproteinases 9 [101–104]. In addition to mechanically induced reactivity, an array of chemokines, cytokines, transcription factors, and growth factors mediate astrocyte reactivity. These pathways include NF-κB, Stat3, Tgfβ, and MAPK. Proinflammatory cytokines such as TNF-α, interleukin-6, and interleukin-1β trigger the reactivity of astrocytes during the acute phase after SCI [81, 105, 106]. Early injury also triggers microglial activation, which induces the activation of astrocytes by secreting interleukin-1α, TNF, and C1q [107]. Reactive astrocytes release TNF-α, interleukin-6, and MMP9, which in turn activate more astrocytes in a feedback loop [108]. Stat3 signaling is a central pathway that directs astrocyte reactivity. Mice with astrocyte-specific deletion of Stat3 fail to upregulate Gfap, eliciting increased cell hypertrophy and exacerbated scar formation after SCI [109]. Stat3 deletion was also shown to limit the migration of astrocytes, resulting in widespread infiltration of inflammatory cells, degeneration of neurons, demyelination of axons, and severe motor deficits [71]. Conversely, Stat3 activation enhances the migration of reactive astrocytes toward the lesion, reduces the size of the fibrotic scar, and improves functional recovery [71]. These results provided a potential intervention strategy that aims to target Stat3 signaling pathway for SCI treatment. Additional astrocyte-derived signaling pathways that impact SCI include TGF-β signaling, which greatly contributes to the formation of reactive astrocytes and is a key upstream pathway in astrocyte reactivity. TGF-β could increase the expression of anti-regenerative molecules such as CSPGs, laminin, and fibronectin in reactive astrocytes [110]. These examples highlight multimodal modes by which SCI triggers astrocyte reactivity.

1.4. Reactive Astrocytes Play Dual Roles after Spinal Cord Injury

1.4.1. Reactive Astrocytes Have Detrimental Effects

Extrinsic Inhibition of Neuronal Repair

Reactive astrocytes form a physical barrier that obstructs axonal growth. Reactive astrocytes secrete inhibitory proteins, such as CSPGs, which inhibit axon growth in vitro and in vivo. While CSPG-rich regions show inhibited axon regrowth, chondroitinase ABC, which removes CSPG glycosaminoglycan chains, was shown to counteract the inhibitory activity of CSPGs and facilitate axonal regeneration and functional recovery [16, 111, 112]. Besides, reactive astrocytes have a role in inhibiting the differentiation of neuronal progenitor cells by expressing insulin-like growth factor-binding proteins and CSPGs [113].

Inhibitory Roles on OPCs

Oligodendrocyte precursor cells (OPCs) have extremely powerful remyelination ability as they can proliferate and differentiate to replenish oligodendrocytes that are lost after SCI. CSPGs play major inhibitory roles in neurons and OPCs. Astrocyte-derived CSPGs inhibit the migration and differentiation of OPCs in vitro, and the number of OPCs surrounding the lesion significantly increased when treated with the enzyme chondroitinase ABC [114, 115]. In addition to CSPGs, other astrocyte-derived molecules, such as BMP and ET1, can also inhibit the differentiation of OPCs and influence remyelination [116, 117].

Astrocytes Induce Edema and Secondary Injuries After SCI

In the acute-to-subacute stages of SCI, edema develops rapidly at the lesion site and extends rostrocaudally beyond the lesion, leading to spinal fluid accumulation and increasing the overall volume of the spinal cord [118]. In contrast to vasogenic edema, which refers to the accumulation of interstitial fluid in the extracellular space following blood-spinal cord barrier disruption, cytotoxic edema refers to intracellular fluid accumulation in astrocytes [119]. Cytotoxic edema, a common pathology in compression injuries, causes secondary compression damages that exacerbate the primary injury and impact neurological outcome [118, 120–122]. As surgical decompressive interventions do not fully relieve edema or local pressure in the spinal cord, biological interventions are in great need. Water channels play central roles in the initiation and resolution of distinct forms of injury-induced edema. Aquaporin 4 (AQP4) is ubiquitously expressed in astrocytes, with prominent localization to the astrocytic end feet and glia limitans, which serve as direct contact points between the spinal cord and its surrounding cerebrospinal fluid [123]. Following thoracic compression injury, AQP4 mutant mice show reduced water content, enhanced neuronal survival, myelin sparing, and improved functional recovery [124, 125]. In contrast, in a model of thoracic contusion injury that causes vasogenic edema rather than cytotoxic edema, AQP4 deletion exacerbates injury outcomes, revealing a protective role for AQP4 in the clearance of vasogenic edema [126]. Thus, AQP4 water channels play a protective role in contusion or transection SCI (vasogenic edema), but a deleterious role in compression SCI (cytotoxic edema).

Astrocyte Reactivity and Neuropathic Pain

Chronic pain affects >80% of SCI patients [127]. The pain may be nociceptive, neuropathic, or a combination of nociceptive and neuropathic. While musculoskeletal pain is the predominant source of nociceptive pain, neuropathic pain is the most common type of pain in SCI patients [127]. Reactive astrogliosis is associated with the development of neuropathic pain [128]. Multiple signaling pathways activated in reactive astrocytes have been shown to contribute to neuropathic pain. Glutamate transporters are downregulated in astrocytes in pain. Indeed, overexpression of GLT1 and GLAST in spinal astrocytes or reduction of extracellular glutamate levels is sufficient to prevent injury-induced allodynia [129–131]. These findings are consistent with a model in which decreased glutamate transporter in astrocytes leads to excessive activation of excitatory glutamatergic pathway, resulting in the development of neuropathic pain. JNK and ERK are major signaling pathways that have been implicated in the development of neuropathic pain [132]. In addition to JNK and glutamate signaling, various astrocytic molecules have been implicated in reactive astrogliosis-induced neuropathic pain, including the gap junction protein connexin 43 and the nuclear receptor protein PPARˠ [133, 134]. Connexin 43 is upregulated after SCI, suggesting increased connectivity between adjacent astrocytes [135]. Despite limited knowledge that implicates few molecules in neuropathic pain development after SCI, our understanding of the signaling pathways that trigger or limit pain remains limited.

1.4.2. Reactive Astrocytes Are a Defense Mechanism

Containing the Lesion and Impacting the Immune System

At the acute stages of SCI, reactive astrocytes migrate rapidly around the lesion to seclude infiltrating immune cells and limit the extent of inflammation, which has markedly positive effects on functional recovery [71]. Supporting this beneficial role of astrocyte reactivity, selective and conditional ablation of reactive astrocytes in mouse SCI models results in significantly increased and prolonged infiltration of inflammatory cells around the lesion [39]. Various endogenous and exogenous factors trigger the release and accumulation of cellular debris and neurotoxic factors in the extracellular spaces after SCI. Moreover, reactive astrocytes can influence immune cell activation and mobilization by releasing various molecules such as TNF-α, TGF-β, and proteoglycans [136–140]. CSPGs have a close relationship with immune activity as they can recruit chemokines and growth factors that enhance the connection of immune cells [91].

Additional Beneficial Outputs of Astrocyte Reactivity in SCI

SCI-induced disruption to the blood-spinal cord barrier exposes the lesioned cord to endogenous and exogenous blood-borne molecules that can result in detrimental effects on functional recovery. In response to these insults, reactive astrocytes upregulate Sonic hedgehog (Shh) as part of a reactive signaling cascade that restores tight junctions and directs the repair of the blood-spinal cord barrier [141]. Consequently, loss of reactive astrocytes leads to failure in repairing the damaged barrier. Reactive astrocytes can also reduce the impact of glutamate cytotoxicity on neurons and OPCs by clearing excess glutamate from blood or necrotic cells [142]. Reactive astrocytes were recently reported to play a crucial role in clearing cellular debris and neurotoxic factors from the lesion environment. Intriguingly, reactive astrocytes have the ability to phagocytose dead cells in vitro and in vivo via the upregulation of ABCA1 [143, 144]. Early astrocyte reactivity is therefore an ostensibly conserved response that evolved to protect the central nervous system against external injuries.

1.4.3. Species Differences in Astrocyte Reactivity

It is important to note that the timeline and outcomes of astrocyte reactivity differ among different species and SCI models. In rodents, microglia become activated before astrocytes, and both glial cell populations proliferate and migrate toward the injury site. In primates, glial cell activation is delayed relative to rodents. The response of astrocytes eventually leads to the formation of a dense astroglial border surrounding the fibrotic lesion core.

Though the complexities of glial cell responses to injury have been extensively studied in mammals, our understanding of how astrocytes respond to SCI in highly regenerative vertebrates such as zebrafish is relatively limited. In fact, a detailed description of zebrafish astrocytes was only reported in 2020 [145]. Following SCI in zebrafish, specialized glial cells form a bridge that is thought to provide a physical and signaling scaffold for cellular regrowth across the lesion [45, 53]. Previous studies implicated Fgf and Ctgf signaling in glial bridge formation in adult zebrafish. The glial bridging functions of Fgf signaling were established using transgenic dominant-negative Fgf receptor manipulations, sprouty mutants, and Fgf8 injection [53]. ctgfa genetic loss of function was shown to impair glial bridging, axon regrowth, and functional SC repair [45]. Genetic evidence indicates that ctgfa functions within a multi-nodal gene regulatory network that directs epithelial-to-mesenchymal transition and is necessary and sufficient to reprogram pro-regenerative glia after injury [48]. However, since Fgf and Ctgf are secreted extracellular proteins, the glial requirements of these molecules during bridging and of glial bridging during SC regeneration remained unclear. Previous studies suggested axon regrowth proceeds independently of the projection of glial processes across the lesion [46, 146, 147]. However, ablation of ctgfa⁺ cells was shown to be sufficient to impair glial bridging, axon regrowth, and functional recovery after SCI, indicating ctgfa⁺ cells are required during SC repair [148]. Regenerating axons often associate with elongated bipolar astrocytes across vertebrates, including zebrafish and mammals [45, 53, 149–151]. Bridging glia from adult zebrafish retain an elongated morphology [45, 53] and possess a mesenchymal signature that correlates with increased plasticity and immature astrocytic cell identity [48]. Similarly, in adult mice, a small proportion of elongated astrocytes, termed “astroglial bridges,” correlate with increased axon regrowth under genetic manipulations such as PTEN deletion [151]. We thus propose that future comparative studies will shed light on the molecular similarities and differences between murine elongated astrocytes and zebrafish bridging glia. Indeed, zebrafish glia possess an astrocyte-like cell identity as transcriptomes of zebrafish bridging glia are thought to be most similar to scar-bordering astrocytes in mammals. These findings shed light on glial bridging as an effective, natural mechanism of spinal cord regeneration and established astrocyte reactivity as an indispensable component to achieve spinal cord repair.

2. Conclusion

In summary, spinal cord injuries are devastating conditions that require concerted efforts and combinatorial approaches. Astrocytes undergo a range of long-lasting morphological and functional changes in response to neural damages. It is likely that reactive astrogliosis evolved as an innate protective response to spinal cord injury. Yet, the anti-regenerative effects of reactive astrocytes present a major burden to spinal cord repair. Unraveling the molecular complexities of reactive astrogliosis using state-of-the-art technologies will offer unprecedented opportunities to modulate the phenotypic outputs of reactive astrocytes to promote spinal cord repair.

Abbreviations

AQP

Aquaporin

CCL

CC Chemokine ligand

CSPG

Chondroitin sulfate proteoglycan

CTGF

Connective tissue growth factor

FGF

Fibroblast growth factor

Gfap

Glial fibrillary acidic protein

IL

Interleukin

MAG

Myelin-associated glycoprotein

OMgp

Oligodendrocyte-myelin glycoprotein

OPC

Oligodendrocyte precursor cell

SCI

Spinal cord injury

Shh

Sonic hedgehog

TNF

Tumor necrosis factor