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Microglia are dynamic immunosurveillance cells in the CNS. Whether microglia are protective or pathologic is context dependent; the outcome varies as a function of time relative to the stimulus, activation state of neighboring cells in the microenvironment or within progression of a particular disease. Although brain microglia can be “primed” using bacterial lipopolysaccharide (LPS)/endotoxin, it is unknown whether LPS delivered systemically can also induce neuroprotective microglia in the spinal cord. Here, we show that serial systemic injections of LPS (1 mg/kg, i.p., daily) for 4 consecutive days (LPSx4) consistently elicit a reactive spinal cord microglia response marked by dramatic morphologic changes, increased production of IL-1, and enhanced proliferation without triggering leukocyte recruitment or overt neuropathology. Following LPSx4, reactive microglia frequently contact spinal cord endothelial cells. Targeted ablation or selective expression of IL-1 and IL-1 receptor (IL-1R) in either microglia or endothelia reveal that IL-1-dependent signaling between these cells mediates microglia activation. Using a mouse model of ischemic spinal cord injury in male and female mice, we show that preoperative LPSx4 provides complete protection from ischemia-induced neuron loss and hindlimb paralysis. Neuroprotection is partly reversed by either pharmacological elimination of microglia or selective removal of IL-1R in microglia or endothelia. These data indicate that spinal cord microglia are amenable to therapeutic reprogramming via systemic manipulation and that this potential can be harnessed to protect the spinal cord from injury.SIGNIFICANCE STATEMENTData in this report indicate that a neuroprotective spinal cord microglia response can be triggered by daily systemic injections of LPS over a period of 4 d (LPSx4). The LPSx4 regimen induces morphologic transformation and enhances proliferation of spinal cord microglia without causing neuropathology. Using advanced transgenic mouse technology, we show that IL-1-dependent microglia–endothelia cross talk is necessary for eliciting this spinal cord microglia phenotype and also for conferring optimal protection to spinal motor neurons from ischemic spinal cord injury (ISCI). Collectively, these novel data show that it is possible to consistently elicit spinal cord microglia via systemic delivery of inflammogens to achieve a therapeutically effective neuroprotective response against ISCI.

Infections impair neurological outcome and increase mortality after spinal cord injury (SCI). Emerging data show that pathogens more easily infect individuals with SCI because SCI disrupts neural and humoral control of immune cells, culminating with the development of “SCI-Induced Immune Deficiency Syndrome” (SCI-IDS). Here, we review data that implicate autonomic dysfunction and impaired neuroendocrine signaling as key determinants of SCI-IDS. Although it is widely appreciated that mature leukocyte dysfunction is a canonical feature of SCI-IDS, new data indicate that SCI impairs the development and mobilization of immune cell precursors in bone marrow. Thus, this review will also explore how the post-injury acquisition of a “bone marrow failure syndrome” may be the earliest manifestation of SCI-IDS.

To our knowledge, this is the first article to apply metagenomics to characterize changes in gut microbial population dynamics caused by a clinically relevant model of central nervous system (CNS) trauma. It also utilizes the most current approaches in genome-resolved metagenomics and viromics to maximize the biological inferences that can be made from these data., Emerging data indicate that gut dysbiosis contributes to many human diseases, including several comorbidities that develop after traumatic spinal cord injury (SCI). To date, all analyses of SCI-induced gut dysbiosis have used 16S rRNA amplicon sequencing. This technique has several limitations, including being susceptible to taxonomic “blind spots,” primer bias, and an inability to profile microbiota functions or identify viruses. Here, SCI-induced gut dysbiosis was assessed by applying genome- and gene-resolved metagenomic analysis of murine stool samples collected 21 days after an experimental SCI at the 4th thoracic spine (T4) or 10th thoracic spine (T10) spinal level. These distinct injuries partially (T10) or completely (T4) abolish sympathetic tone in the gut. Among bacteria, 105 medium- to high-quality metagenome-assembled genomes (MAGs) were recovered, with most (n = 96) representing new bacterial species. Read mapping revealed that after SCI, the relative abundance of beneficial commensals (Lactobacillus johnsonii and CAG-1031 spp.) decreased, while potentially pathogenic bacteria (Weissella cibaria, Lactococcus lactis_A, Bacteroides thetaiotaomicron) increased. Functionally, microbial genes encoding proteins for tryptophan, vitamin B6, and folate biosynthesis, essential pathways for central nervous system function, were reduced after SCI. Among viruses, 1,028 mostly novel viral populations were recovered, expanding known murine gut viral species sequence space ∼3-fold compared to that of public databases. Phages of beneficial commensal hosts (CAG-1031, Lactobacillus, and Turicibacter) decreased, while phages of pathogenic hosts (Weissella, Lactococcus, and class Clostridia) increased after SCI. Although the microbiomes and viromes were changed in all SCI mice, some of these changes varied as a function of spinal injury level, implicating loss of sympathetic tone as a mechanism underlying gut dysbiosis. IMPORTANCE To our knowledge, this is the first article to apply metagenomics to characterize changes in gut microbial population dynamics caused by a clinically relevant model of central nervous system (CNS) trauma. It also utilizes the most current approaches in genome-resolved metagenomics and viromics to maximize the biological inferences that can be made from these data. Overall, this article highlights the importance of autonomic nervous system regulation of a distal organ (gut) and its microbiome inhabitants after traumatic spinal cord injury (SCI). By providing information on taxonomy, function, and viruses, metagenomic data may better predict how SCI-induced gut dysbiosis influences systemic and neurological outcomes after SCI.

Inflammation is a ubiquitous but poorly understood consequence of spinal cord injury (SCI). The mechanisms controlling this response are unclear but culminate in the sequential activation of resident and recruited immune cells. Collectively, these cells can exert divergent effects on cell survival and tissue repair. HMGB1 is a ubiquitously expressed DNA binding protein and also a potent inflammatory stimulus. Necrotic cells release HGMB1, but HMGB1 also is actively secreted by inflammatory macrophages. A goal of this study was to quantify spatio-temporal patterns of cellular HMGB1 expression in a controlled mouse model of experimental SCI then determine the effects of HMGB1 on post-SCI neuroinflammation and recovery of function. We documented SCI-induced changes in nuclear and cytoplasmic distribution of HMGB1 in various cell types after SCI. The data reveal a time-dependent increase in HMGB1 mRNA and protein with protein reaching maximal levels 24–72 h post-injury then declining toward baseline 14–28 days post-SCI. Although most cells expressed nuclear HMGB1, reduced nuclear labeling with increased cytoplasmic expression was found in a subset of CNS macrophages suggesting that those cells begin to secrete HMGB1 at the injury site. In vitro data indicate that extracelluar HMGB1 helps promote the development of macrophages with a neurotoxic phenotype. The ability of HMGB1 to elicit neurotoxic macrophage functions was confirmed in vivo; 72 h after injecting 500 ng of recombinant HMGB1 into intact spinal cord ventral horn, inflammatory CNS macrophages co-localized with focal areas of neuronal killing. However, attempts to confer neuroprotection after SCI by blocking HMGB1 with a neutralizing antibody were unsuccessful. Collectively, these data implicate HMGB1 as a novel regulator of post-SCI inflammation and suggest that inhibition of HMGB1 could be a novel therapeutic target after SCI. Future studies will need to identify better methods to deliver optimal concentrations of HMGB1 antagonists to the injured spinal cord.

Spinal cord injury (SCI) disrupts the autonomic nervous system (ANS), impairing its ability to coordinate organ function throughout the body. Emerging data indicate that the systemic pathology that manifests from ANS dysfunction exacerbates intraspinal pathology and neurological impairment. Precisely how this happens is unknown, although new data, in both humans and in rodent models, implicate changes in the composition of bacteria in the gut (i.e., the gut microbiota) as disease-modifying factors that are capable of affecting systemic physiology and pathophysiology. Recent data from rodents indicate that SCI causes gut dysbiosis, which exacerbates intraspinal inflammation and lesion pathology leading to impaired recovery of motor function. Postinjury delivery of probiotics containing various types of “good” bacteria can partially overcome the pathophysiologal effects of gut dysbiosis; immune function, locomotor recovery, and spinal cord integrity are partially restored by a sustained regimen of oral probiotics. More research is needed to determine whether gut dysbiosis varies across a range of clinically relevant variables, including sex, injury level, and injury severity, and whether changes in the gut microbiota can predict the onset or severity of common postinjury comorbidities, including infection, anemia, metabolic syndrome, and, perhaps, secondary neurological deterioration. Those microbial populations that dominate the gut could become “druggable” targets that could be manipulated via dietary interventions. For example, personalized nutraceuticals (e.g., pre- or probiotics) could be developed to treat the above comorbidities and improve health and quality of life after SCI.

Secondary manifestations of spinal cord injury beyond motor and sensory dysfunction can negatively affect a person's quality of life. Spinal cord injury is associated with an increased incidence of depression and anxiety; however, the mechanisms of this relationship are currently not well understood. Human and animal studies suggest that changes in the composition of the intestinal microbiota (dysbiosis) are associated with mood disorders. The objective of the current study is to establish a model of anxiety following a cervical contusion spinal cord injury in rats and to determine whether the microbiota play a role in the observed behavioural changes. We found that spinal cord injury caused dysbiosis and increased symptoms of anxiety-like behaviour. Treatment with a fecal transplant prevented both spinal cord injury-induced dysbiosis as well as the development of anxiety-like behaviour. These results indicate that an incomplete unilateral cervical spinal cord injury can cause affective disorders and intestinal dysbiosis, and that both can be prevented by treatment with fecal transplant therapy.

Spinal cord injury (SCI) disrupts the autonomic nervous system (ANS) and impairs communication with organ systems throughout the body, resulting in chronic multi-organ pathology and dysfunction. This dysautonomia contributes to the pronounced immunosuppression and gastrointestinal dysfunction seen after SCI. All of these factors likely contribute to the development of gut dysbiosis after SCI—an imbalance in the composition of the gut microbiota that can impact the development and progression of numerous pathological conditions, including SCI. The gut microbiota are the community of microbes (bacteria, viruses, fungi) that live in the GI tract and are critical for nutrient absorption, digestion, and immune system development. These microbes also communicate with the CNS through modulation of the immune system, production of neuroactive metabolites and neurotransmitters, and activation of the vagus nerve. After SCI, gut dysbiosis develops and persists for more than one year from the time of injury. In experimental models of SCI, gut dysbiosis is correlated with changes in inflammation and functional recovery. Moreover, probiotic treatment can improve locomotor recovery and immune function in the gut-associated lymphoid tissue (GALT). Since different types of bacteria produce different metabolites with unique physiological and pathological effects throughout the body, it may be possible to predict the prevalence or severity of post-injury immune dysfunction and other related comorbidities (e.g., metabolic disease, fatigue, anxiety) using microbiome sequencing data. As research identifies microbial-derived small molecules and the genes responsible for their production, it is likely that it will become feasible to manipulate these molecules to affect human biology and disease.

Acute oligodendrocyte (OL) death after traumatic spinal cord injury (SCI) is followed by robust neuron–glial antigen 2 (NG2)-positive OL progenitor proliferation and differentiation into new OLs. Inflammatory mediators are prevalent during both phases and can influence the fate of NG2 cells and OLs. Specifically, toll-like receptor (TLR) 4 signaling induces OL genesis in the naive spinal cord, and lack of TLR4 signaling impairs white matter sparing and functional recovery after SCI. Therefore, we hypothesized that TLR4 signaling may regulate oligodendrogenesis after SCI. C3H/HeJ (TLR4-deficient) and control (C3H/HeOuJ) mice received a moderate midthoracic spinal contusion. TLR4-deficient mice showed worse functional recovery and reduced OL numbers compared with controls at 24 h after injury through chronic time points. Acute OL loss was accompanied by reduced ferritin expression, which is regulated by TLR4 and needed for effective iron storage. TLR4-deficient injured spinal cords also displayed features consistent with reduced OL genesis, including reduced NG2 expression, fewer BrdU-positive OLs, altered BMP4 signaling and inhibitor of differentiation 4 (ID4) expression, and delayed myelin phagocytosis. Expression of several factors, including IGF-1, FGF2, IL-1β, and PDGF-A, was altered in TLR4-deficient injured spinal cords compared with wild types. Together, these data show that TLR4 signaling after SCI is important for OL lineage cell sparing and replacement, as well as in regulating cytokine and growth factor expression. These results highlight new roles for TLR4 in endogenous SCI repair and emphasize that altering the function of a single immune-related receptor can dramatically change the reparative responses of multiple cellular constituents in the injured CNS milieu. SIGNIFICANCE STATEMENT Myelinating cells of the CNS [oligodendrocytes (OLs)] are killed for several weeks after traumatic spinal cord injury (SCI), but they are replaced by resident progenitor cells. How the concurrent inflammatory signaling affects this endogenous reparative response is unclear. Here, we provide evidence that immune receptor toll-like receptor 4 (TLR4) supports OL lineage cell sparing, long-term OL and OL progenitor replacement, and chronic functional recovery. We show that TLR4 signaling is essential for acute iron storage, regulating cytokine and growth factor expression, and efficient myelin debris clearance, all of which influence OL replacement. Importantly, the current study reveals that a single immune receptor is essential for repair responses after SCI, and the potential mechanisms of this beneficial effect likely change over time after injury.

Kigerl et al. show that spinal cord injury causes profound changes in gut microbiota and that these changes in gut ecology are associated with activation of GALT immune cells. They show that feeding mice probiotics after SCI confers neuroprotection and improves functional recovery., The trillions of microbes that exist in the gastrointestinal tract have emerged as pivotal regulators of mammalian development and physiology. Disruption of this gut microbiome, a process known as dysbiosis, causes or exacerbates various diseases, but whether gut dysbiosis affects recovery of neurological function or lesion pathology after traumatic spinal cord injury (SCI) is unknown. Data in this study show that SCI increases intestinal permeability and bacterial translocation from the gut. These changes are associated with immune cell activation in gut-associated lymphoid tissues (GALTs) and significant changes in the composition of both major and minor gut bacterial taxa. Postinjury changes in gut microbiota persist for at least one month and predict the magnitude of locomotor impairment. Experimental induction of gut dysbiosis in naive mice before SCI (e.g., via oral delivery of broad-spectrum antibiotics) exacerbates neurological impairment and spinal cord pathology after SCI. Conversely, feeding SCI mice commercial probiotics (VSL#3) enriched with lactic acid–producing bacteria triggers a protective immune response in GALTs and confers neuroprotection with improved locomotor recovery. Our data reveal a previously unknown role for the gut microbiota in influencing recovery of neurological function and neuropathology after SCI.

Mouse models have provided key insight into the cellular and molecular control of human immune system function. However, recent data indicate that extrapolating the functional capabilities of the murine immune system into humans can be misleading. Since immune cells significantly affect neuron survival and axon growth and also are required to defend the body against infection, it is important to determine the pathophysiological significance of spinal cord injury (SCI)-induced changes in human immune system function. Research projects using monkeys or humans would be ideal; however, logistical and ethical barriers preclude detailed mechanistic studies in either species. Humanized mice, i.e., immunocompromised mice reconstituted with human immune cells, can help overcome these barriers and can be applied in various experimental conditions that are of interest to the SCI community. Specifically, newborn NOD-SCID-IL2rg(null) (NSG) mice engrafted with human CD34(+) hematopoietic stem cells develop normally without neurological impairment. In this report, new data show that when mice with human immune systems receive a clinically-relevant spinal contusion injury, spontaneous functional recovery is indistinguishable from that achieved after SCI using conventional inbred mouse strains. Moreover, using routine immunohistochemical and flow cytometry techniques, one can easily phenotype circulating human immune cells and document the composition and distribution of these cells in the injured spinal cord. Lesion pathology in humanized mice is typical of mouse contusion injuries, producing a centralized lesion epicenter that becomes occupied by phagocytic macrophages and lymphocytes and enclosed by a dense astrocytic scar. Specific human immune cell types, including three distinct subsets of human monocytes, were readily detected in the blood, spleen and liver. Future studies that aim to understand the functional consequences of manipulating the neuro-immune axis after SCI should consider using the humanized mouse model. Humanized mice represent a powerful tool for improving the translational value of pre-clinical SCI data.

Most spinal cord injury (SCI) research programs focus only on the injured spinal cord with the goal of restoring locomotor function by overcoming mechanisms of cell death or axon regeneration failure. Given the importance of the spinal cord as a locomotor control center and the public perception that paralysis is the defining feature of SCI, this “spinal-centric” focus is logical. Unfortunately, such a focus likely will not yield new discoveries that reverse other devastating consequences of SCI including cardiovascular and metabolic disease, bladder/bowel dysfunction and infection. The current review considers how SCI changes the physiological interplay between the spinal cord, the gut and the immune system. A suspected culprit in causing many of the pathological manifestations of impaired spinal cord-gut-immune axis homeostasis is the gut microbiota. After SCI, the composition of the gut microbiota changes, creating a chronic state of gut “dysbiosis”. To date, much of what we know about gut dysbiosis was learned from 16S-based taxonomic profiling studies that reveal changes in the composition and abundance of various bacteria. However, this approach has limitations and creates taxonomic “blindspots”. Notably, only bacteria can be analyzed. Thus, in this review we also discuss how the application of emerging sequencing technologies can improve our understanding of how the broader ecosystem in the gut is affected by SCI. Specifically, metagenomics will provide researchers with a more comprehensive look at post-injury changes in the gut virome (and mycome). Metagenomics also allows changes in microbe population dynamics to be linked to specific microbial functions that can affect the development and progression of metabolic disease, immune dysfunction and affective disorders after SCI. As these new tools become more readily available and used across the research community, the development of an “ecogenomic” toolbox will facilitate an Eco-Systems Biology approach to study the complex interplay along the spinal cord-gut-immune axis after SCI.

It is widely believed that microglia and monocyte-derived macrophages (collectively referred to as central nervous system (CNS) macrophages) cause excitotoxicity in the diseased or injured CNS. This view has evolved mostly from in vitro studies showing that neurotoxic concentrations of glutamate are released from CNS macrophages stimulated with lipopolysaccharide (LPS), a potent inflammogen. We hypothesized that excitotoxic killing by CNS macrophages is more rigorously controlled in vivo, requiring both the activation of the glutamate/cystine antiporter (system x(c)(-)) and an increase in extracellular cystine, the substrate that drives glutamate release. Here, we show that non-traumatic microinjection of low-dose LPS into spinal cord gray matter activates CNS macrophages but without causing overt neuropathology. In contrast, neurotoxic inflammation occurs when LPS and cystine are co-injected. Simultaneous injection of NBQX, an antagonist of AMPA glutamate receptors, reduces the neurotoxic effects of LPS+cystine, implicating glutamate as a mediator of neuronal cell death in this model. Surprisingly, neither LPS nor LPS+cystine adversely affects survival of oligodendrocytes or oligodendrocyte progenitor cells. Ex vivo analyses show that redox balance in microglia and macrophages is controlled by induction of system x(c)(-) and that high GSH:GSSG ratios predict the neurotoxic potential of these cells. Together, these data indicate that modulation of redox balance in CNS macrophages, perhaps through regulating system x(c)(-), could be a novel approach for attenuating injurious neuroinflammatory cascades.

Progranulin (proepithelin) is a pleiotropic growth-factor associated with inflammation and wound repair in peripheral tissues. It also has been implicated in the response to acute traumatic brain injury as well as to chronic neurodegenerative diseases. To determine whether changes in progranulin expression also accompany acute spinal cord injury, C57BL/6 mice were subjected to mid-thoracic (T9 level) contusion spinal cord injury and analyzed by immunohistochemical and biochemical methods. Whereas spinal cord sections prepared from non-injured laminectomy control animals contained low basal levels of progranulin immunoreactivity in gray matter, sections from injured animals contained intense immunoreactivity throughout the injury epicenter that peaked 7-14 days post injury. Progranulin immunoreactivity colocalized with myeloid cell markers CD11b and CD68, indicating that expression increased primarily in activated microglia and macrophages. Immunoblot analysis confirmed that progranulin protein levels rose after injury. On the basis of quantitative polymerase chain reaction analysis, increased protein levels resulted from a tenfold rise in progranulin transcripts. These data demonstrate that progranulin is dramatically induced in myeloid cells after experimental spinal cord injury and is positioned appropriately both spatially and temporally to influence recovery after injury.

Macrophages dominate sites of central nervous system (CNS) injury where they promote both injury and repair. These divergent effects may be caused by distinct macrophage subsets, i.e., “classically-activated” pro-inflammatory (M1) or “alternatively-activated” anti-inflammatory (M2) cells. Here, we show that an M1 macrophage response is rapidly induced then maintained at sites of traumatic spinal cord injury and that this response overwhelms a comparatively smaller and transient M2 macrophage response. The high M1:M2 macrophage ratio has significant implications for CNS repair. Indeed, we present novel data showing that only M1 macrophages are neurotoxic and M2 macrophages promote a regenerative growth response in adult sensory axons, even in the context of inhibitory substrates that dominate sites of CNS injury (e.g., proteoglycans and myelin). Together, these data suggest that polarizing the differentiation of resident microglia and infiltrating blood monocytes toward an M2 or “alternatively” activated macrophage phenotype could promote CNS repair while limiting secondary inflammatory-mediated injury.

There is growing recognition that psychological stress influences pain. Hormones that comprise the physiological response to stress (e.g., corticosterone; CORT) may interact with effectors of neuropathic pain. To test this hypothesis, mice received a spared nerve injury (SNI) after exposure to 60 min restraint stress. In stressed mice, allodynia was consistently increased. The mechanism(s) underlying the exacerbated pain response involves CORT acting via glucocorticoid receptors (GRs); RU486, a GR antagonist, prevented the stress-induced increase in allodynia whereas exogenous administration of CORT to non-stressed mice reproduced the allodynic response caused by stress. Since nerve injury-induced microglial activation has been implicated in the onset and propagation of neuropathic pain, we evaluated cellular and molecular indices of microglial activation in the context of stress. Activation of dorsal horn microglia was accelerated by stress; however, this effect was transient and was not associated with the onset or maintenance of a pro-inflammatory phenotype. Stress-enhanced allodynia was associated with increased dorsal horn extracellular signal-regulated kinase phosphorylation (pERK). ERK activation could indicate a stress-mediated increase in glutamatergic signaling, therefore mice were treated prior to SNI and stress with memantine, an N-methyl-D-aspartate receptor (NMDAR) antagonist. Memantine prevented stress-induced enhancement of allodynia after SNI. These data suggest that the hormonal responses elicited by stress exacerbate neuropathic pain through enhanced central sensitization. Moreover, drugs that inhibit glucocorticoids (GCs) and/or NMDAR signaling could ameliorate pain syndromes caused by stress.

Activation of macrophages via toll-like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non-pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFkappaB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2-/-) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild-type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin-1beta mRNA. In TLR2-/- mice, locomotor recovery also was impaired relative to SCI wild-type controls and novel patterns of myelin pathology existed within ventromedial white matter--an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post-injury sequelae and perhaps in regulating inflammation and gliosis after SCI.

Spinal contusion pathology in rats and mice is distinct. Cystic cavities form at the impact site in rats while a dense connective tissue matrix occupies the injury site in mice. Because inflammatory cells coordinate mechanisms of tissue injury and repair, we evaluated whether the unique anatomical presentation in spinally injured rats and mice is associated with a species-specific inflammatory response. Immunohistochemistry was used to compare the leukocytic infiltrate between rats and mice. Microglia/macrophage reactions were similar between species; however, the onset and magnitude of lymphocyte and dendritic cell (DC) infiltration were markedly different. In rats, T-cell numbers were highest between 3 and 7 days postinjury and declined by 50% over the next 3 weeks. In mice, significant T-cell entry was not evident until 14 days postinjury, with T-cell numbers doubling between 2 and 6 weeks. Dendritic cell influx paralleled T-cell infiltration in rats but was absent in mouse spinal cord. De novo expression of major histocompatability class II molecules was increased in both species but to a greater extent in mice. Unique to mice were cells that resembled lymphocytes but did not express lymphocyte-specific markers. These cells extended from blood vessels within the fibrotic tissue matrix and expressed fibronectin, collagen I, CD11b, CD34, CD13, and CD45. This phenotype is characteristic of fibrocytes, specialized blood-borne cells involved in wound healing and immunity. Thus, species-specific neuroinflammation may contribute to the formation of distinct tissue environments at the site of spinal cord injury in mice and rats. J. Comp. Neurol. 462:223–240, 2003. © 2003 Wiley-Liss, Inc.

Pattern recognition receptors (PRRs) are part of the innate immune response and were originally discovered for their role in recognizing pathogens by ligating specific pathogen associated molecular patterns (PAMPs) expressed by microbes. Now the role of PRRs in sterile inflammation is also appreciated, responding to endogenous stimuli referred to as "damage associated molecular patterns" (DAMPs) instead of PAMPs. The main families of PRRs include Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs), AIM2-like receptors (ALRs), and C-type lectin receptors. Broad expression of these PRRs in the CNS and the release of DAMPs in and around sites of injury suggest an important role for these receptor families in mediating post-injury inflammation. Considerable data now show that PRRs are among the first responders to CNS injury and activation of these receptors on microglia, neurons, and astrocytes triggers an innate immune response in the brain and spinal cord. Here we discuss how the various PRR families are activated and can influence injury and repair processes following CNS injury.

Stress and glucocorticoids exacerbate pain via undefined mechanisms. Macrophage migration inhibitory factor (MIF) is a constitutively expressed protein that is secreted to maintain immune function when glucocorticoids are elevated by trauma or stress. Here we show that MIF is essential for the development of neuropathic and inflammatory pain, and for stress-induced enhancement of neuropathic pain. Mif null mutant mice fail to develop pain-like behaviors in response to inflammatory stimuli or nerve injury. Pharmacological inhibition of MIF attenuates pain-like behaviors caused by nerve injury and prevents sensitization of these behaviors by stress. Conversely, injection of recombinant MIF into naive mice produces dose-dependent mechanical sensitivity that is exacerbated by stress. MIF elicits pro-inflammatory signaling in microglia and activates sensory neurons, mechanisms that underlie pain. These data implicate MIF as a key regulator of pain and provide a mechanism whereby stressors exacerbate pain. MIF inhibitors warrant clinical investigation for the treatment of chronic pain.

After central nervous system (CNS) trauma, axons have a low capacity for regeneration. Regeneration failure is associated with a muted regenerative response of the neuron itself, combined with a growth-inhibitory and cytotoxic post-injury environment. After spinal cord injury (SCI), resident and infiltrating immune cells (especially microglia/macrophages) contribute significantly to the growth-refractory milieu near the lesion. By targeting both the regenerative potential of the axon and the cytotoxic phenotype of microglia/macrophages, we may be able to improve CNS repair after SCI. In this review, we discuss molecules shown to impact CNS repair by affecting both immune cells and neurons. Specifically, we provide examples of pattern recognition receptors, integrins, cytokines/chemokines, nuclear receptors and galectins that could improve CNS repair. In many cases, signaling by these molecules is complex and may have contradictory effects on recovery depending on the cell types involved or the model studied. Despite this caveat, deciphering convergent signaling pathways on immune cells (which affect axon growth indirectly) and neurons (direct effects on axon growth) could improve repair and recovery after SCI. Future studies must continue to consider how regenerative therapies targeting neurons impact other cells in the pathological CNS. By identifying molecules that simultaneously improve axon regenerative capacity and drive the protective, growth-promoting phenotype of immune cells, we may discover SCI therapies that act synergistically to improve CNS repair and functional recovery.

Following spinal trauma, the limited physiological axonal sprouting that contributes to partial recovery of function is dependent upon the intrinsic properties of neurons as well as the inhibitory glial environment. The transcription factor p53 is involved in DNA repair, cell cycle, cell survival, and axonal outgrowth, suggesting p53 as key modifier of axonal and glial responses influencing functional recovery following spinal injury. Indeed, in a spinal cord dorsal hemisection injury model, we observed a significant impairment in locomotor recovery in p53−/−versus wild-type mice. p53−/−spinal cords showed an increased number of activated microglia/macrophages and a larger scar at the lesion site. Loss- and gain-of-function experiments suggested p53 as a direct regulator of microglia/macrophages proliferation. At the axonal level, p53−/−mice showed a more pronounced dieback of the corticospinal tract (CST) and a decreased sprouting capacity of both CST and spinal serotoninergic fibers.In vivoexpression of p53 in the sensorimotor cortex rescued and enhanced the sprouting potential of the CST in p53−/−mice, while, similarly, p53 expression in p53−/−cultured cortical neurons rescued a defect in neurite outgrowth, suggesting a direct role for p53 in regulating the intrinsic sprouting ability of CNS neurons. In conclusion, we show that p53 plays an important regulatory role at both extrinsic and intrinsic levels affecting the recovery of motor function following spinal cord injury. Therefore, we propose p53 as a novel potential multilevel therapeutic target for spinal cord injury.

Macrophages exert divergent effects in the injured CNS, causing either neurotoxicity or regeneration. The mechanisms regulating these divergent functions are not understood but can be attributed to the recruitment of distinct macrophage subsets and the activation of specific intracellular signaling pathways. Here, we show that impaired signaling via the chemokine receptor CX3CR1 promotes recovery after traumatic spinal cord injury (SCI) in mice. Deficient CX3CR1 signaling in intraspinal microglia and monocyte-derived macrophages (MDMs) attenuates their ability to synthesize and release inflammatory cytokines and oxidative metabolites. Also, impaired CX3CR1 signaling abrogates the recruitment or maturation of MDMs with presumed neurotoxic effects after SCI. Indeed, in wild-type mice, Ly6C(lo)/iNOS(+)/MHCII(+)/CD11c(-) MDMs dominate the lesion site, whereas CCR2(+)/Ly6C(hi)/MHCII(-)/CD11c(+) monocytes predominate in the injured spinal cord of CX3CR1-deficient mice. Replacement of wild-type MDMs with those unable to signal via CX3CR1 resulted in anatomical and functional improvements after SCI. Thus, blockade of CX3CR1 signaling represents a selective anti-inflammatory therapy that is able to promote neuroprotection, in part by reducing inflammatory signaling in microglia and MDMs and recruitment of a novel monocyte subset.

Following traumatic spinal cord injury (SCI), activated glia and inflammatory leukocytes contribute to both neurodegeneration and repair. The mechanisms that control these divergent functions are poorly understood. Toll-like receptors (TLRs) are a highly conserved family of receptors involved in pathogen recognition and host defense. However, recently it was shown that TLRs are expressed on a range of neuronal and non-neuronal cells (e.g., glia, stem/progenitor cells and leukocytes), and that nonpathogenic molecules released from sites of tissue injury, i.e., danger-associated molecular patterns (DAMPs), can activate cells via TLRs. This review will discuss how DAMPs acting at various TLRs may influence injury and repair processes of relevance to SCI, i.e., neurotoxicity, demyelination, growth cone collapse and stem/progenitor cell turnover.

Following traumatic spinal cord injury (SCI), activated glia and inflammatory leukocytes contribute to both neurodegeneration and repair. The mechanisms that control these divergent functions are poorly understood. Toll-like receptors (TLRs) are a highly conserved family of receptors involved in pathogen recognition and host defense. However, recently it was shown that TLRs are expressed on a range of neuronal and non-neuronal cells (e.g., glia, stem/progenitor cells and leukocytes), and that nonpathogenic molecules released from sites of tissue injury, i.e., danger-associated molecular patterns (DAMPs), can activate cells via TLRs. This review will discuss how DAMPs acting at various TLRs may influence injury and repair processes of relevance to SCI, i.e., neurotoxicity, demyelination, growth cone collapse and stem/progenitor cell turnover.

Susceptibility to neuroinflammatory disease is influenced in part by genetics. Recent data indicate that survival of traumatized neurons is strain dependent and influenced by polygenic loci that control resistance/susceptibility to experimental autoimmune encephalomyelitis (EAE), a model of CNS autoimmune disease. Here, we describe patterns of neurodegeneration and intraparenchymal inflammation after traumatic spinal cord injury (SCI) in mice known to exhibit varying degrees of EAE susceptibility [EAE-resistant (r) or EAE-susceptible (s) mice]. Spinal cords from C57BL/6 (EAE-s), C57BL/10 (EAE-r), BALB/c (EAE-r), and B10.PL (EAE-s) mice were prepared for stereological and immunohistochemical analysis at 6 hours or 3, 7, 14, 28, or 42 days following midthoracic (T9) spinal contusion injury. In general, genetic predisposition to EAE predicted the magnitude of intraparenchymal inflammation but not lesion size/length or locomotor recovery. Specifically, microglia/macrophage activation, recruitment of neutrophils and lymphocytes, and de novo synthesis of MHC class II were greatest in C57BL/6 mice and least in BALB/c mice at all times examined. However, lesion volume and axial spread of neurodegeneration were similar in C57BL/6 and BALB/c mice and were significantly greater than in C57BL/10 or B10.PL mice. Strains with marked intraspinal inflammation also developed the most intense lesion fibrosis. Thus, strain-dependent neuroinflammation was observed after SCI, but without a consistent relationship to EAE susceptibility or lesion progression. Only in C57BL/6 mice was the magnitude of intraspinal inflammation predictive of secondary neurodegeneration, functional recovery, or fibrosis.

To evaluate the direct effect of human cyclooxygenase-2 (hCox-2) on human breast tumor cell proliferation, invasion, and angiogenesis, hCox-2 cDNA was transfected into slow growing, non-metastatic MCF-7 human breast tumor cells that express low levels of Cox-2. Two stable transfectant clones, designated MCF-7/hCox-2 clones 8 and 10, had significantly decreased (P