Traumatic brain injury (TBI) is associated with cerebral edema, blood brain barrier breakdown, and neuroinflammation that contribute to the degree of injury severity and functional recovery. Unfortunately, there are no effective proactive treatments for limiting immediate or long-term consequences of TBI. Therefore, the objective of this study was to determine the efficacy of methylene blue (MB), an antioxidant agent, in reducing inflammation and behavioral complications associated with a diffuse brain injury. Here we show that immediate MB infusion (intravenous; 15–30 minutes after TBI) reduced cerebral edema, attenuated microglial activation and reduced neuroinflammation, and improved behavioral recovery after midline fluid percussion injury in mice. Specifically, TBI-associated edema and inflammatory gene expression in the hippocampus were significantly reduced by MB at 1 d post injury. Moreover, MB intervention attenuated TBI-induced inflammatory gene expression (interleukin [IL]-1β, tumor necrosis factor α) in enriched microglia/macrophages 1 d post injury. Cell culture experiments with lipopolysaccharide-activated BV2 microglia confirmed that MB treatment directly reduced IL-1β and increased IL-10 messenger ribonucleic acid in microglia. Last, functional recovery and depressive-like behavior were assessed up to one week after TBI. MB intervention did not prevent TBI-induced reductions in body weight or motor coordination 1–7 d post injury. Nonetheless, MB attenuated the development of acute depressive-like behavior at 7 d post injury. Taken together, immediate intervention with MB was effective in reducing neuroinflammation and improving behavioral recovery after diffuse brain injury. Thus, MB intervention may reduce life-threatening complications of TBI, including edema and neuroinflammation, and protect against the development of neuropsychiatric complications.
cytokines; fluid percussion injury; intervention; microglia; recovery
Alternative activation of microglia/macrophages (M2a) by interleukin (IL)-4 is purported to support intrinsic growth and repair processes after CNS injury. Nonetheless, alternative activation of microglia is poorly understood in vivo, particularly in the context of inflammation, injury, and aging. Here, we show that aged mice (18–19 months) had reduced functional recovery after spinal cord injury (SCI) associated with impaired induction of IL-4 receptor α (IL-4Rα) on microglia. The failure to successfully promote an IL-4/IL-4Rα response in aged mice resulted in attenuated arginase (M2a associated), IL-1β, and chemokine ligand 2 (CCL2) expression, and diminished recruitment of IL-4Rα+ macrophages to the injured spinal cord. Furthermore, the link between reduced IL-4Rα expression and reduced arginase, IL-1β, and CCL2 expression was confirmed using adult IL-4Rα knock-out (IL-4RαKO) mice. To better understand IL-4Rα-mediated regulation of active microglia, a series of studies was completed in mice that were peripherally injected with lipopolysaccharide and later provided IL-4 by intracerebroventricular infusion. These immune-based studies demonstrate that inflammatory-induced IL-4Rα upregulation on microglia was required for the induction of arginase by IL-4. In addition, IL-4-mediated reprogramming of active microglia enhanced neurite growth ex vivo and increased inflammatory gene expression (i.e., IL-1β and CCL2) and the corresponding recruitment of CCR2+/IL-4Rα+/arginase+ myeloid cells in vivo. IL-4 reprogrammed active microglia to a unique and previously unreported phenotype (arginase+/IL-1β+) that augmented neurite growth and enhanced recruitment of peripheral IL-4Rα+ myeloid cells to the CNS. Moreover, this key signaling cascade was impaired with age corresponding with reduced functional recovery after SCI.
advanced age; IL-4; inflammation; M2a; microglia; spinal cord injury
Autonomic dysreflexia (AD), a potentially dangerous complication of high-level spinal cord injury (SCI) characterized by exaggerated activation of spinal autonomic (sympathetic) reflexes, can cause pulmonary embolism, stroke, and, in severe cases, death. People with high-level SCI also are immune compromised, rendering them more susceptible to infectious morbidity and mortality. The mechanisms underlying postinjury immune suppression are not known. Data presented herein indicate that AD causes immune suppression. Using in vivo telemetry, we show that AD develops spontaneously in SCI mice with the frequency of dysreflexic episodes increasing as a function of time postinjury. As the frequency of AD increases, there is a corresponding increase in splenic leucopenia and immune suppression. Experimental activation of spinal sympathetic reflexes in SCI mice (e.g., via colorectal distension) elicits AD and exacerbates immune suppression via a mechanism that involves aberrant accumulation of norepinephrine and glucocorticoids. Reversal of postinjury immune suppression in SCI mice can be achieved by pharmacological inhibition of receptors for norepinephrine and glucocorticoids during the onset and progression of AD. In a human subject with C5 SCI, stimulating the micturition reflex caused AD with exaggerated catecholamine release and impaired immune function, thus confirming the relevance of the mouse data. These data implicate AD as a cause of secondary immune deficiency after SCI and reveal novel therapeutic targets for overcoming infectious complications that arise due to deficits in immune function.
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 naïve 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.
MIF; Pain; Stress; Glucocorticoids; Axon; Neuroplasticity; Microglia; Macrophage; Cytokine; Inflammation
The lack of reproducibility in many areas of experimental science has a number of causes, including a lack of transparency and precision in the description of experimental approaches. This has far-reaching consequences, including wasted resources and slowing of progress. Additionally, the large number of laboratories around the world publishing articles on a given topic make it difficult, if not impossible, for individual researchers to read all of the relevant literature. Consequently, centralized databases are needed to facilitate the generation of new hypotheses for testing. One strategy to improve transparency in experimental description, and to allow the development of frameworks for computer-readable knowledge repositories, is the adoption of uniform reporting standards, such as common data elements (data elements used in multiple clinical studies) and minimum information standards. This article describes a minimum information standard for spinal cord injury (SCI) experiments, its major elements, and the approaches used to develop it. Transparent reporting standards for experiments using animal models of human SCI aim to reduce inherent bias and increase experimental value.
axonal injury; axonal regeneration; MIASCI; spinal cord injury
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 xc-) 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 xc- 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 xc-, could be a novel approach for attenuating injurious neuroinflammatory cascades.
system xc-; redox; neuroinflammation; spinal cord injury; glutamate
Spinal cord ischemia and paralysis are devastating perioperative complications that can accompany open or endovascular repair surgery for aortic aneurysms. Here, we report on the development of a new mouse model of spinal cord ischemia with delayed paralysis induced by cross-clamping the descending aorta.
Transient aortic occlusion was produced in mice by cross clamping the descending aorta through a lateral thoracotomy. To establish an optimal surgical procedure with limited mortality, variable cross-clamp times and core temperatures were tested between experiments.
The onset of paresis or paralysis and postsurgical mortality varied as a function of cross-clamp time and core temperature that was maintained during the period of cross-clamp. Using optimal surgical parameters (7.5 min cross-clamp duration @ 33°C core temperature), the onset of paralysis is delayed 24–36 h postreperfusion and > 95% of mice survive through 9 weeks postsurgery. These mice are further stratified into two groups, with 70% (n = 19/27) of mice developing severe hindlimb paralysis and the remaining mice showing mild, though still permanent, behavioral deficits.
This new model should prove useful as a preclinical tool for screening neuroprotective therapeutics and for defining the basic biological mechanisms that cause delayed paralysis and neurodegeneration after transient spinal cord ischemia.
Emerging data indicate that traumatic injury to the brain or spinal cord activates B lymphocytes, culminating in the production of antibodies specific for antigens found within and outside the central nervous system (CNS). In this article, we summarize what is known about the effects of CNS injury on B cells. We outline the potential mechanisms for CNS trauma-induced B cell activation and discuss the potential consequences of these injury-induced B cell responses. Based on recent data, we hypothesize that a subset of autoimmune B cell responses initiated by CNS injury are pathogenic and that targeted inhibition of B cells could improve recovery in brain and spinal cord injured patients.
Traumatic injury or disease of the spinal cord and brain elicits multiple cellular and biochemical reactions that together cause or are associated with neuropathology. Specifically, injury or disease elicits acute infiltration and activation of immune cells, death of neurons and glia, mitochondrial dysfunction, and the secretion of substrates that inhibit axon regeneration. In some diseases, inflammation is chronic or non-resolving. Ligands that target PPARs (peroxisome proliferator-activated receptors), a group of ligand-activated transcription factors, are promising therapeutics for neurologic disease and CNS injury because their activation affects many, if not all, of these interrelated pathologic mechanisms. PPAR activation can simultaneously weaken or reprogram the immune response, stimulate metabolic and mitochondrial function, promote axon growth and induce progenitor cells to differentiate into myelinating oligodendrocytes. PPAR activation has beneficial effects in many pre-clinical models of neurodegenerative diseases and CNS injury; however, the mechanisms through which PPARs exert these effects have yet to be fully elucidated. In this review we discuss current literature supporting the role of PPAR activation as a therapeutic target for treating traumatic injury and degenerative diseases of the CNS.
Alzheimer’s disease; astrocyte; experimental autoimmune encephalomyelitis (EAE); macrophage; multiple sclerosis; spinal cord injury; ALS, amyotrophic lateral sclerosis; Arg1, Arginase 1; BMP, bone morphogenetic protein; 15d-PGJ2, 15-deoxy-Δ-12,14-prostaglandin J-2; EAE, experimental autoimmune encephalomyelitis; GR, glucocorticoid receptor; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MS, multiple sclerosis; NF-κB, nuclear factor κB; NGF, nerve growth factor; OPC, oligodendrocyte precursor cell; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; SCI, spinal cord injury; SHP-2, Src homology region 2-containing protein tyrosine phosphatase-2; TBI, traumatic brain injury; Th1, T helper type 1; TNFα, tumour necrosis factor α; UCP, uncoupling protein
Post-traumatic immune suppression renders individuals with spinal cord injury (SCI) susceptible to infection. Normally, proper immune function is regulated by collaboration between the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis and involves the controlled release of glucocorticoids (GCs) and norepinephrine (NE). Recently, we showed that after high thoracic (T3) SCI, aberrant levels of GCs and NE accumulate in the blood and spleen, respectively. These changes are associated with splenic atrophy, splenic leucopenia, increased intrasplenic caspase-3 levels and suppressed B lymphocyte function. Since GCs boost SNS function, in part by increasing the expression and affinity of beta-2 adrenergic receptors (β2ARs) while simultaneously preventing β2AR down-regulation, we predicted that surges in stress hormones (i.e., GCs and NE) in the blood and spleen of mice with high-level SCI would act concurrently to adversely affect lymphocyte function and survival. Here, we show that post-SCI concentrations of GCs enhance the sensitivity of lymphocytes to β2AR stimulation causing an increase in intracellular Bim (Bcl2-Interacting Mediator of Cell Death) and subsequent apoptosis. In vivo, the combined antagonism of GC receptors and β2ARs significantly diminished lymphocyte Bim levels and SCI-induced splenic lymphopenia. Together, these data suggest that pharmacological antagonists of the HPA/SNS axes should be considered as adjunct therapies for ameliorating post-traumatic immune suppression in quadriplegics and high paraplegics.
CNS injury; sympathetic nervous system; hypothalamic-pituitary-adrenal axis; Bim
Historically, microglia/macrophages are quantified in the pathological central nervous system (CNS) by counting cell profiles then expressing the data as cells/mm2. However, because it is difficult to visualize individual cells in dense clusters and in most cases it is unimportant to know the absolute number of macrophages within lesioned tissue, alternative methods may be more efficient for quantifying the magnitude of the macrophage response in the context of different experimental variables (e.g., therapeutic intervention or time post-injury/infection). The present study provides the first in-depth comparison of different techniques commonly used to quantify microglial/macrophage reactions in the pathological spinal cord. Individuals from the same and different laboratories applied techniques of digital image analysis (DIA), standard cell profile counting and a computer-assisted cell counting method with unbiased sampling to quantify macrophages in focal inflammatory lesions, disseminated lesions caused by autoimmune inflammation or at sites of spinal trauma. Our goal was to find a simple, rapid and sensitive method with minimal variability between trials and users. DIA was consistently the least variable and most time-efficient method for assessing the magnitude of macrophage responses across lesions and between users. When used to evaluate the efficacy of an anti-inflammatory treatment, DIA was 5–35x faster than cell counting and was sensitive enough to detect group differences while eliminating inter-user variability. Since lesions are clearly defined and single profiles of microglia/macrophages are difficult to discern in most pathological specimens of brain or spinal cord, DIA offers significant advantages over other techniques for quantifying activated macrophages.
image analysis; macrophages; microglia; inflammation; CNS; immunohistochemistry
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.
interleukin-4; interferon-γ; dorsal root ganglion; regeneration; neurotoxicity; spinal cord injury
Traumatic spinal cord injury (SCI) in mammals causes widespread glial activation and recruitment to the CNS of innate (e.g., neutrophils, monocytes) and adaptive (e.g., T and B lymphocytes) immune cells. To date, most studies have sought to understand or manipulate the post-traumatic functions of astrocytes, microglia, neutrophils or monocytes. Significantly less is known about the consequences of SCI-induced lymphocyte activation. Yet, emerging data suggest that T and B cells are activated by SCI and play significant roles in shaping post-traumatic inflammation and downstream cascades of neurodegeneration and repair. Here, we provide neurobiologists with a timely review of the mechanisms and implications of SCI-induced lymphocyte activation, including a discussion of different experimental strategies that have been designed to manipulate lymphocyte function for therapeutic gain.
autoimmune; lymphocyte; autoantibody; T cell; B cell; CNS injury
Traumatic injury to the mammalian spinal cord activates B cells, which culminates in the synthesis of autoantibodies. The functional significance of this immune response is unclear. Here, we show that locomotor recovery was improved and lesion pathology was reduced after spinal cord injury (SCI) in mice lacking B cells. After SCI, antibody-secreting B cells and Igs were present in the cerebrospinal fluid and/or injured spinal cord of WT mice but not mice lacking B cells. In mice with normal B cell function, large deposits of antibody and complement component 1q (C1q) accumulated at sites of axon pathology and demyelination. Antibodies produced after SCI caused pathology, in part by activating intraspinal complement and cells bearing Fc receptors. These data indicate that B cells, through the production of antibodies, affect pathology in SCI. One or more components of this pathologic immune response could be considered as novel therapeutic targets for minimizing tissue injury and/or promoting repair after SCI.
Trauma to the central nervous system (CNS) triggers intraparenchymal inflammation and activation of systemic immunity with the capacity to exacerbate neuropathology and stimulate mechanisms of tissue repair. Despite our incomplete understanding of the mechanisms that control these divergent functions, immune-based therapies are becoming a therapeutic focus. This review will address the complexities and controversies of post-traumatic neuroinflammation, particularly in spinal cord. In addition, current therapies designed to target neuroinflammatory cascades will be discussed.
macrophages; lymphocytes; neuroinflammation; spinal cord injury; traumatic brain injury; blood-brain barrier
Individuals with spinal cord injury (SCI) are highly susceptible to infection. This post-traumatic immune suppression is thought to occur via alterations in sympathetic nervous system (SNS) or hypothalamic-pituitary-adrenal (HPA) axis function. Normally, the HPA axis and SNS help coordinate proper immune function. After SCI, the HPA axis becomes activated and descending input to sympathetic preganglionic neurons (SPNs) is impaired. Because lymphoid organs are innervated by SPNs distributed throughout the thoracolumbar spinal cord, we predicted level-dependent immune suppression after SCI due to activation of the HPA axis and loss of descending input to SPNs. We tested this hypothesis by measuring indices of HPA (circulating corticosterone; CORT) and SNS function (norepinephrine (NE) in spleen) as well as antigen-specific antibody synthesis against an exogenous non-self protein following high or low level SCI. Using a mid-thoracic (T9) spinal contusion injury model, we found that CORT was elevated after SCI with aberrant patterns of diurnal CORT synthesis evident through at least the first 24 hours post-injury. However, splenic NE and antibody synthesis were similar to uninjured controls. Injury severity did not change these parameters. Indeed, CORT, NE and antibody synthesis were similar after T9 contusion or transection SCI. In contrast, high level SCI (T3) caused sustained increases in CORT and splenic NE along with impaired antibody synthesis and elevated splenocyte apoptosis. The immunosuppressive effects of T3 SCI were caused by NE acting at β2-adrenergic receptors (β2AR) and could be reversed using β2AR blockers. Interestingly, impaired antibody after T3 SCI could be mimicked after T9 SCI with a β2AR agonist. These data illustrate the immunosuppressive effects of the SNS after high-level SCI and indicate that immune deficits may be overcome using β-blockers.
CNS injury; sympathetic nervous system; hypothalamic-pituitary-adrenal axis; antibodies; apoptosis; vaccines
These experiments were completed as part of an NIH-NINDS contract entitled “Facilities of Research Excellence – Spinal Cord Injury (FORE-SCI) – Replication”. Our goal was to replicate pre-clinical data from Simard et al. (2007) showing that glibenclamide, an FDA approved anti-diabetic drug that targets sulfonylurea receptor 1 (SUR1)-regulated Ca2+ activated, [ATP]i-sensitive nonspecific cation channels, attenuates secondary intraspinal hemorrhage and secondary neurodegeneration caused by hemicontusion injury in rat cervical spinal cord. In an initial replication attempt, the Infinite Horizons impactor was used to deliver a standard unilateral contusion injury near the spinal cord midline. Glibenclamide was administered continuously via osmotic pump beginning immediately post-SCI. The ability of glibenclamide to limit intraspinal hemorrhage was analyzed at 6, 12 and 24 hours using a colorimetric assay. Acute recovery (24 hours) of forelimb function also was assessed. Analysis of data from these initial studies revealed no difference between glibenclamide and vehicle-treated SCI rats. Later, it was determined that differences in primary trauma affect the efficacy of glibenclamide. Indeed, the magnitude and distribution of primary intraspinal hemorrhage was greater when the impact was directed to the dorsomedial region of the cervical hemicord (as in our initial replication experiment), as compared to the dorsolateral spinal cord (as in the Simard et al. experiment). In three subsequent experiments, injury was directed to the dorsolateral spinal cord. In each case, glibenclamide reduced post-traumatic hemorrhage 24-48 hours post-injury. In the third experiment, we also assessed function and found that acute reduction of hemorrhage led to improved functional recovery. Thus, independent replication of the Simard et al. data was achieved. These data illustrate that the injury model and type of trauma can determine the efficacy of pre-clinical pharmacological treatments after SCI.
cervical spinal cord injury; hemorrhage; secondary injury; sulfonylurea receptor; glibenclamide; injury models
These experiments were completed as part of an NIH-NINDS contract entitled “Facilities of Research Excellence-Spinal Cord Injury (FORE-SCI)—Replication”. Our goal was to replicate data from a paper published by Dr. Lloyd Guth and colleagues in which combined injections of lipopolysaccharide, indomethacin and pregnenolone (referred to herein as LIP therapy) conferred marked neuroprotection in a pre-clinical model of spinal cord injury (SCI). Specifically, post-injury injection of the combination LIP therapy was found to significantly reduce tissue damage at/nearby the site of injury and significantly improve recovery of locomotor function. In this report, we confirm the primary observations made by Guth et al., however, the effects of LIP treatment were modest. Specifically, LIP treatment improved myelin and axon sparing, axonal sprouting while reducing lesion cavitation. However, spontaneous recovery of locomotion, as assessed using historical (Tarlov scoring) and more current rating scales (i.e., BBB scoring), was not affected by LIP treatment. Instead, more refined parameters of functional recovery (paw placement accuracy during grid walk) revealed a significant effect of treatment. Possible explanations for the neuroprotective effects of LIP therapy are described along with reasons why the magnitude of neuroprotection may have differed between this study and that of Guth and colleagues.
Neuroinflammation; LPS; Steroids; Spinal cord injury; Replication
Injured CNS tissue often contains elevated iron and its storage protein ferritin, which may exacerbate tissue damage through pro-oxidative mechanisms. Therefore, therapeutic studies often target iron reduction as a neuroprotective strategy. However, iron may be crucial for oligodendrocyte replacement and remyelination. For instance, we previously showed that intraspinal TLR4 macrophage activation induced the generation of new ferritin+ oligodendrocytes, and that iron chelation significantly reduced this oligodendrogenic response. Since macrophages can secrete ferritin, we hypothesize that ferritin is a macrophage-derived signal that promotes oligodendrogenesis. To test this, we microinjected ferritin into the intact adult rat spinal cords. Within 6h, NG2+ progenitor cells proliferated and accumulated ferritin. By 3d, many of these cells had differentiated into new oligodendrocytes. However, acute neuron and oligodendrocyte toxicity occurred in gray matter. Interestingly, ferritin+ NG2 cells and macrophages accumulated in the area of cell loss, revealing that NG2+ cells thrive in an environment that is toxic to other CNS cells. To test if ferritin can be transferred from macrophages to NG2 cells in vivo, we loaded macrophages with fluorescent ferritin then transplanted them into intact spinal white matter. Within 3–6d, proliferating NG2 cells migrated into the macrophage transplants and accumulated fluorescently-labeled ferritin. These results show that activated macrophages can be an in vivo source of ferritin for NG2 cells, which induces their proliferation and differentiation into new oligodendrocytes. This work has relevance for conditions in which iron-mediated injury and/or repair likely occur, such as hemorrhage, stroke, spinal cord injury, aging, Parkinson’s disease and Alzheimer’s disease.
iron; spinal cord injury; myelin; macrophage; progenitor; inflammation
We recently reported that the neuropathic pain medication, gabapentin (GBP; Neurontin), significantly attenuated both noxious colorectal distension (CRD)-induced autonomic dysreflexia (AD) and tail pinch-induced spasticity compared to saline-treated cohorts 2–3 weeks after complete high thoracic (T4) spinal cord injury (SCI). Here we employed long-term blood pressure telemetry to test, firstly, the efficacy of daily versus acute GBP treatment in modulating AD and tail spasticity in response to noxious stimuli at 2 and 3 weeks post-injury. Secondly, we determined whether daily GBP alters baseline cardiovascular parameters, as well as spontaneous AD events detected using a novel algorithm based on blood pressure telemetry data. At both 14 and 21 days after SCI, irrespective of daily treatment, acute GBP given 1 h prior to stimulus significantly attenuated CRD-induced AD and pinch-evoked tail spasticity; conversely, acute saline had no such effects. Moreover, daily GBP did not alter 24 h mean arterial pressure (MAP) or heart rate (HR) values compared to saline treatment, nor did it reduce the incidence of spontaneous AD events compared to saline over the three week assessment period. Power spectral density (PSD) analysis of the MAP signals demonstrated relative power losses in mid frequency ranges (0.2–0.8 Hz) for all injured animals relative to low frequency MAP power (0.02–0.08 Hz). However, there was no significant difference between groups over time post-injury; hence, GBP had no effect on the persistent loss of MAP fluctuations in the mid frequency range after injury. In summary, the mechanism(s) by which acute GBP treatment mitigate aberrant somatosensory and cardiophysiological responses to noxious stimuli after SCI remain unclear. Nevertheless, with further refinements in defining the dynamics associated with AD events, such as eliminating requisite concomitant bradycardia, the objective repeatability of automatic detection of hypertensive crises provides a potentially useful tool for assessing autonomic function pre- and post-SCI, in conjunction with experimental pharmacotherapeutics for neuropathic pain, such as GBP.
neuropathic pain; colorectal distension; power spectral density; telemetry; blood pressure; heart rate
Traumatic spinal cord injury (SCI) affects the activation, migration, and function of microglia, neutrophils and monocyte/macrophages. Because these myeloid cells can positively and negatively affect survival of neurons and glia, they are among the most commonly studied immune cells. However, the mechanisms that regulate myeloid cell activation and recruitment after SCI have not been adequately defined. In general, the dynamics and composition of myeloid cell recruitment to the injured spinal cord are consistent between mammalian species; only the onset, duration, and magnitude of the response vary. Emerging data, mostly from rat and mouse SCI models, indicate that resident and recruited myeloid cells are derived from multiple sources, including the yolk sac during development and the bone marrow and spleen in adulthood. After SCI, a complex array of chemokines and cytokines regulate myelopoiesis and intraspinal trafficking of myeloid cells. As these cells accumulate in the injured spinal cord, the collective actions of diverse cues in the lesion environment help to create an inflammatory response marked by tremendous phenotypic and functional heterogeneity. Indeed, it is difficult to attribute specific reparative or injurious functions to one or more myeloid cells because of convergence of cell function and difficulties in using specific molecular markers to distinguish between subsets of myeloid cell populations. Here we review each of these concepts and include a discussion of future challenges that will need to be overcome to develop newer and improved immune modulatory therapies for the injured brain or spinal cord.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0032-6) contains supplementary material, which is available to authorized users.
Monocytes; macrophages; neutrophils; microglia; cytokine; chemokine
In spinal cord injury (SCI), block of Sur1-regulated NCCa-ATP channels by glibenclamide protects penumbral capillaries from delayed fragmentation, resulting in reduced secondary hemorrhage, smaller lesions and better neurological function. All published experiments demonstrating a beneficial effect of glibenclamide in rat models of SCI have used a cervical hemicord impact calibrated to produce primary hemorrhage located exclusively ipsilateral to the site of impact. Here, we tested the hypothesis that glibenclamide also would be protective in a model with more extensive, bilateral primary hemorrhage. We studied the effect of glibenclamide in 2 rat cervical hemicord contusion models with identical impact force (10 g, 25 mm), one with the impactor positioned laterally to yield unilateral primary hemorrhage (UPH), and the other with the impactor positioned more medially, yielding larger, bilateral primary hemorrhages (BPH) and 6-week lesion volumes that were 45% larger. Functional outcome measures included: modified (unilateral) Basso, Beattie, and Bresnahan scores, angled plane performance, and rearing times. In the UPH model, the effects of glibenclamide were similar to previous observations, including a functional benefit as early as 24 h after injury and 6-week lesion volumes that were 57% smaller than controls. In the BPH model, glibenclamide exerted a significant benefit over controls, but the functional benefit was smaller than in the UPH model and 6-week lesion volumes were 33% smaller than controls. We conclude that glibenclamide is beneficial in different models of cervical SCI, with the magnitude of the benefit depending on the magnitude and extent of primary hemorrhage.
Spinal cord injury; Hemorrhage; Glibenclamide
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, Ly6Clo/iNOS+/MHCII+/CD11c− MDMs dominate the lesion site whereas CCR2+/Ly6Chi/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.
fractalkine; CX3CL1; CX3CR1; microglia; macrophage; neuroprotection; chimera; spinal cord injury
In this review, we first provide a brief historical perspective, discussing how peripheral nerve injury (PNI) may have caused World War I. We then consider the initiation, progression, and resolution of the cellular inflammatory response after PNI, before comparing the PNI inflammatory response with that induced by spinal cord injury (SCI).
In contrast with central nervous system (CNS) axons, those in the periphery have the remarkable ability to regenerate after injury. Nevertheless, peripheral nervous system (PNS) axon regrowth is hampered by nerve gaps created by injury. In addition, the growth-supportive milieu of PNS axons is not sustained over time, precluding long-distance regeneration. Therefore, studying PNI could be instructive for both improving PNS regeneration and recovery after CNS injury. In addition to requiring a robust regenerative response from the injured neuron itself, successful axon regeneration is dependent on the coordinated efforts of non-neuronal cells which release extracellular matrix molecules, cytokines, and growth factors that support axon regrowth. The inflammatory response is initiated by axonal disintegration in the distal nerve stump: this causes blood-nerve barrier permeabilization and activates nearby Schwann cells and resident macrophages via receptors sensitive to tissue damage. Denervated Schwann cells respond to injury by shedding myelin, proliferating, phagocytosing debris, and releasing cytokines that recruit blood-borne monocytes/macrophages. Macrophages take over the bulk of phagocytosis within days of PNI, before exiting the nerve by the circulation once remyelination has occurred. The efficacy of the PNS inflammatory response (although transient) stands in stark contrast with that of the CNS, where the response of nearby cells is associated with inhibitory scar formation, quiescence, and degeneration/apoptosis. Rather than efficiently removing debris before resolving the inflammatory response as in other tissues, macrophages infiltrating the CNS exacerbate cell death and damage by releasing toxic pro-inflammatory mediators over an extended period of time. Future research will help determine how to manipulate PNS and CNS inflammatory responses in order to improve tissue repair and functional recovery.
Macrophage; microglia; axotomy; Wallerian degeneration; phagocytosis; neuroinflammation; inflammation; spinal cord injury; galectin-1
The past three decades have seen an explosion of research interest in spinal cord injury (SCI) and the development of hundreds of potential therapies that have demonstrated some promise in pre-clinical experimental animal models. A growing number of these treatments are seeking to be translated into human clinical trials. Conducting such a clinical trial, however, is extremely costly, not only for the time and money required to execute it, but also for the limited resources that will then no longer be available to evaluate other promising therapies. The decision about what therapies have sufficient pre-clinical evidence of efficacy to justify testing in humans is therefore of utmost importance. Here, we have developed a scoring system for objectively grading the body of pre-clinical literature on neuroprotective treatments for acute SCI. The components of the system include an evaluation of a number of factors that are thought to be important in considering the “robustness” of a therapy's efficacy, including the animal species and injury models that have been used to test it, the time window of efficacy, the types of functional improvements effected by it, and whether efficacy has been independently replicated. The selection of these factors was based on the results of a questionnaire that was performed within the SCI research community. A modified Delphi consensus-building exercise was then conducted with experts in pre-clinical SCI research to refine the criteria and decide upon how to score them. Finally, the grading system was applied to a series of potential neuroprotective treatments for acute SCI. This represents a systematic approach to developing an objective method of evaluating the extent to which the pre-clinical literature supports the translation of a particular experimental treatment into human trials.
Delphi; grading system; neuroprotection; spinal cord injury