Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Transplantation of mesenchymal stem cells promotes an alternative pathway of macrophage activation and functional recovery after spinal cord injury

Abstract Mesenchymal stem cells (MSC) derived from bone marrow can potentially reduce the acute inflammatory response in spinal cord injury (SCI) and thus promote functional recovery. However, the precise mechanisms through which transplanted MSC attenuate inflammation after SCI are still unclear. The present study was designed to investigate the effects of MSC transplantation with a special focus on their effect on macrophage activation after SCI. Rats were subjected to T9-T10 SCI by contusion, then treated 3 days later with transplantation of 1.0×10(6) PKH26-labeled MSC into the contusion epicenter. The transplanted MSC migrated within the injured spinal cord without differentiating into glial or neuronal elements. MSC transplantation was associated with marked changes in the SCI environment, with significant increases in IL-4 and IL-13 levels, and reductions in TNF-a and IL-6 levels. This was associated simultaneously with increased numbers of alternatively activated macrophages (M2 phenotype: arginase-1- or CD206-positive), and decreased numbers of classically activated macrophages (M1 phenotype: iNOS- or CD16/32-positive). These changes were associated with functional locomotion recovery in the MSC-transplanted group, which correlated with preserved axons, less scar tissue formation, and increased myelin sparing. Our results suggested that acute transplantation of MSC after SCI modified the inflammatory environment by shifting the macrophage phenotype from M1 to M2, and that this may reduce the effects of the inhibitory scar tissue in the subacute/chronic phase after injury to provide a permissive environment for axonal extension and functional recovery.

Concise review:bone marrow for the treatment of spinal cord injury: mechanisms and clinical applications

Transplantation of bone marrow stem cells into spinal cord lesions enhances axonal regeneration and promotes functional recovery in animal studies. There are two types of adult bone marrow stem cell; hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). The mechanisms by which HSCs and MSCs might promote spinal cord repair following transplantation have been extensively investigated. The objective of this review is to discuss these mechanisms; we briefly consider the controversial topic of HSC and MSC transdifferentiation into central nervous system cells but focus on the neurotrophic, tissue sparing, and reparative action of MSC grafts in the context of the spinal cord injury (SCI) milieu. We then discuss some of the specific issues related to the translation of HSC and MSC therapies for patients with SCI and present a comprehensive critique of the current bone marrow cell clinical trials for the treatment of SCI to date.

The haematopoietic stem cell niche:new insights into the mechanisms regulating haematopoietic stem cell behaviour

The concept of the haematopoietic stem cell (HSC) niche was formulated by Schofield in the 1970s, as a region within the bone marrow containing functional cell types that can maintain HSC potency throughout life. Since then, ongoing research has identified numerous cell types and a plethora of signals that not only maintain HSCs, but also dictate their behaviour with respect to homeostatic requirements and exogenous stresses. It has been proposed that there are endosteal and vascular niches within the bone marrow, which are thought to regulate different HSC populations. However, recent data depicts a more complicated picture, with functional crosstalk between cells in these two regions. In this review, recent research into the endosteal/vascular cell types and signals regulating HSC behaviour are considered, together with the possibility of a single subcompartmentalised niche.

A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro

The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p <0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p <0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.

Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells

The delicately orchestrated process of bone fracture healing is not always successful and long term non union of fractured bone occurs in 5-20% of all cases. Atrophic fracture non unions have been described as the most difficult to treat and this is thought to arise through a cellular and local failure of osteogenesis. However, little is known about the presence and osteogenic proficiency of cells in the local area of non union tissue. We have examined the growth and differentiation potential of cells isolated from human non union tissues compared with normal human bone marrow mesenchymal stromal cells (BMSC). We report the isolation and culture expansion of a population of non union stromal cells (NUSC) which have a CD profile similar to that of BMSC, i.e. CD34-ve, CD45-ve and CD105+ve. The NUSC demonstrated multipotentiality and differentiated to some extent along chondrogenic, adipogenic and osteogenic lineages. However, and importantly, the NUSC showed significantly reduced osteogenic differentiation and mineralization in vitro compared to BMSC. We also found increased levels of cell senescence in NUSC compared to BMSC based on culture growth kinetics and cell positivity for senescence associated beta galactosidase (SA-beta-Gal) activity. The reduced capacity of NUSC to form osteoblasts was associated with significantly elevated secretion of Dickkopf-1 (Dkk-1) which is an important inhibitor of Wnt signalling during osteogenesis, compared to BMSC. Conversely, treating BMSC with levels of rhDkk-1 that were equivalent to those levels secreted by NUSC inhibited the capacity of BMSC to undergo osteogenesis. Treating BMSC with NUSC conditioned medium also inhibited the capacity of the BMSC to undergo osteogenic differentiation when compared to their treatment with BMSC conditioned medium. Our results suggest that the development of fracture non union is linked with a localised reduced capacity of cells to undergo osteogenesis, which in turn is associated with increased cell senescence and Dkk-1 secretion.

The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro:implications for degenerative disc disease

The avascular nature of the human intervertebral disc (IVD) is thought to play a major role in disc pathophysiology by limiting nutrient supply to resident IVD cells. In the human IVD, the central IVD cells at maturity are normally chondrocytic in phenotype. However, abnormal cell phenotypes have been associated with degenerative disc diseases, including cell proliferation and cluster formation, cell death, stellate morphologies, and cell senescence. Therefore, we have examined the relative influence of possible blood-borne factors on the growth characteristics of IVD cells in vitro.

Initiation and progression of ossification of the posterior longitudinal ligament of the cervical spine in the hereditary spinal hyperostotic mouse (twy/twy)

Ossification of the posterior longitudinal ligament (OPLL) is a significantly critical pathology that can eventually cause serious myelopathy. Ossification commences in the vertebral posterior longitudinal ligaments, and intensifies and spreads with the progression of the disease, resulting in osseous projections and compression of the spinal cord. However, the paucity of histological studies the underlying mechanisms of calcification and ossification processes remain obscure. The pathological process could be simulated in the ossifying process of the ligament in mutant spinal hyperostotic mouse (twy/twy). The aim of this study is to observe that enlargement of the nucleus pulposus followed by herniation, disruption and regenerative proliferation of annulus fibrosus cartilaginous tissues participated in the initiation of ossification of the posterior longitudinal ligament of twy/twy mice.

Apoptosis of neurons and oligodendrocytes in the spinal cord of spinal hyperostotic mouse (twy/twy):possible pathomechanism of human cervical compressive myelopathy

Cervical compressive myelopathy is the most serious complication of cervical spondylosis or ossification of the posterior longitudinal ligament (OPLL) and the most frequent cause of spinal cord dysfunction. There is little information on the exact pathophysiological mechanism responsible for the progressive loss of neural tissue in the spinal cord of such patients. In this study, we used the spinal hyperostotic mouse (twy/twy) as a suitable model of human spondylosis, and OPLL to investigate the cellular and molecular changes in the spinal cord. Mutant twy/twy mouse developed ossification of the ligamentum flavum at C2-C3 and exhibited progressive paralysis.

Blockade of interleukin 6 signaling improves the survival rate of transplanted bone marrow stromal cells and increases locomotor function in mice with spinal cord injury

Bone marrow stromal cells (BMSCs) have the potential to improve functional recovery in patients with spinal cord injury (SCI); however, they are limited by low survival rates after transplantation in the injured tissue. Our objective was to clarify the effects of a temporal blockade of interleukin 6 (IL-6)/IL-6 receptor (IL-6R) engagement using an anti-mouse IL-6R monoclonal antibody (MR16-1) on the survival rate of BMSCs after their transplantation in a mouse model of contusion SCI. MR16-1 cotreatment improved the survival rate of transplanted BMSCs, allowing some BMSCs to differentiate into neurons and astrocytes, and improved locomotor function recovery compared with BMSC transplantation or MR16-1 treatment alone. The death of transplanted BMSCs could be mainly related to apoptosis rather than necrosis. Transplantation of BMSC with cotreatment of MR16-1 was associated with a decrease of some proinflammatory cytokines, an increase of neurotrophic factors, decreased apoptosis rates of transplanted BMSCs, and enhanced expression of survival factors Akt and extracellular signal-regulated protein kinases 1/2. We conclude that MR16-1 treatment combined with BMSC transplants helped rescue neuronal cells and axons after contusion SCI better than BMSCs alone by modulating the inflammatory/immune responses and decreasing apoptosis. © 2013 by the American Association of Neuropathologists, Inc.

The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinalcord compression:implications for human cervical compressive myelopathy

Background:Cervical compressive myelopathy, e.g. due to spondylosis or ossification of the posterior longitudinal ligament is a common cause of spinal cord dysfunction. Although human pathological studies have reported neuronal loss and demyelination in the chronically compressed spinal cord, little is known about the mechanisms involved. In particular, the neuroinflammatory processes that are thought to underlie the condition are poorly understood. The present study assessed the localized prevalence of activated M1 and M2 microglia/macrophages in twy/twy mice that develop spontaneous cervical spinal cord compression, as a model of human disease.Methods:Inflammatory cells and cytokines were assessed in compressed lesions of the spinal cords in 12-, 18- and 24-weeks old twy/twy mice by immunohistochemical, immunoblot and flow cytometric analysis. Computed tomography and standard histology confirmed a progressive spinal cord compression through the spontaneously development of an impinging calcified mass.Results:The prevalence of CD11b-positive cells, in the compressed spinal cord increased over time with a concurrent decrease in neurons. The CD11b-positive cell population was initially formed of arginase-1- and CD206-positive M2 microglia/macrophages, which later shifted towards iNOS- and CD16/32-positive M1 microglia/macrophages. There was a transient increase in levels of T helper 2 (Th2) cytokines at 18 weeks, whereas levels of Th1 cytokines as well as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and macrophage antigen (Mac) -2 progressively increased.Conclusions:Spinal cord compression was associated with a temporal M2 microglia/macrophage response, which may act as a possible repair or neuroprotective mechanism. However, the persistence of the neural insult also associated with persistent expression of Th1 cytokines and increased prevalence of activated M1 microglia/macrophages, which may lead to neuronal loss and demyelination despite the presence of neurotrophic factors. This understanding of the aetiopathology of chronic spinal cord compression is of importance in the development of new treatment targets in human disease. © 2013 Hirai et al.

Bone marrow-derived mesenchymal stem cells become anti-angiogenic when chondrogenically or osteogenically differentiated:implications for bone and cartilage tissue engineering

Osteochondral tissue repair requires formation of vascularized bone and avascular cartilage. Mesenchymal stem cells stimulate angiogenesis both in vitro and in vivo but it is not known if these proangiogenic properties change as a result of chondrogenic or osteogenic differentiation. We investigated the angiogenic/antiangiogenic properties of equine bone marrow-derived mesenchymal stem cells (eBMSCs) before and after differentiation in vitro. Conditioned media from chondrogenic and osteogenic cell pellets and undifferentiated cells was applied to endothelial tube formation assays using Matrigel™. Additionally, the cell secretome was analysed using LC-MS/MS mass spectrometry and screened for angiogenesis and neurogenesis-related factors using protein arrays. Endothelial tube-like formation was supported by conditioned media from undifferentiated eBMSCs. Conversely, chondrogenic and osteogenic conditioned media was antiangiogenic as shown by significantly decreased length of endothelial tube-like structures and degree of branching compared to controls. Undifferentiated cells produced higher levels of angiogenesis-related proteins compared to chondrogenic and osteogenic pellets. In summary, eBMSCs produce an array of angiogenesis-related proteins and support angiogenesis in vitro via a paracrine mechanism. However, when these cells are differentiated chondrogenically or osteogenically, they produce a soluble factor(s) that inhibits angiogenesis. With respect to osteochondral tissue engineering, this may be beneficial for avascular articular cartilage formation but unfavourable for bone formation where a vascularized tissue is desired. © Copyright 2014, Mary Ann Liebert, Inc.

The development of Cannabidiol as a psychiatric therapeutic:a review of its antipsychotic efficacy and possible underlying pharmacodynamic mechanisms

Cannabidiol (CBD), a once-considered inert cannabis constituent, is one of two primary constituents of cannabis, alongside delta-9-tetrahydrocannabinol (?9-THC/THC). In the last 30 years, CBD has become implicated with a range of pharmaceutical mechanisms of great therapeutic interest and utility. This review details the literature speculating CBD’s attenuation of psychotic symptoms, particularly in light of a marked elevation in mean THC concentrations, and a concomitant decline in CBD concentrations in the prevalent U.K street market cannabis derivatives since c. 2000. CBD is purported to exhibit pharmacology akin to established atypical antipsychotics, whilst THC has been implicated with the precipitation of psychosis, and the induction of associated symptoms. The aim of the review was to clarify the conjecture surrounding CBD’s antipsychotic efficacy, before going on to detail prominent theories about its associated pharmacodynamics. Were CBD’s antipsychotic efficacy established, then there is potential for major latent anthropological repercussions to manifest, such as significant elevations in psychosis manifestations in the U.K. The review found a largely affirmative body of evidence asserting CBD’s antipsychotic efficacy. CBD exhibited capacity to attenuate natural and artificially induced psychoses in both animal and human cohorts, the latter of which included individuals considered resistant to conventional treatment. CBD also shows promising potential for use as an antipsychotic drug for Parkinson’s disease (PD) patients with psychosis, owing to its low rate of extra-pyramidal side-effect induction. A range of potential pharmacological mechanisms behind CBD’s neuroleptic pharmacology are outlined, with particular emphasis on its prevention of the hydrolysis and reuptake of the endogenous cannabinoid, anandamide. However, given the nebular aetiological basis for psychoses, explicit conclusions on how CBD attenuates psychotic symptoms remains to be determined.