Alexander Rodriguez Guerrero

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

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-α 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.

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

BackgroundRecent 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.MethodsMR16-1 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)-γ and tumor necrosis factor-α) 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-γ and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia.ResultsLFB-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-γ-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.ConclusionThe 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.

Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

IntroductionMesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).MethodsCommercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.ResultsAt 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.ConclusionThis scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.

Tumor Necrosis Factor-α Antagonist Reduces Apoptosis of Neurons and Oligodendroglia in Rat Spinal Cord Injury

Study Design. To examine the effects of a tumor necrosis factor (TNF)-&agr; antagonist (etanercept) on rat spinal cord injury and identify a possible mechanism for its action. Objective. To elucidate the contribution of etanercept to the pathologic cascade in spinal cord injury and its possible suppression of neuronal and oligodendroglial apoptosis. Summary of Background Data. Etanercept has been recently used successfully for treatment of inflammatory disorders. However, only a few studies have examined its role in suppressing neuronal and oligodendroglial apoptosis in spinal cord injury. Methods. Etanercept or saline (control) was administered by intraperitoneal injection 1 hour after thoracic spinal cord injury in rats. The expressions and localizations of TNF-&agr;, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2) were examined by immunoblot and immunohistochemical analyses. Spinal cord tissue damage between saline- and etanercept-treated groups was also compared after hematoxylin-eosin and luxol fast blue (LFB) staining. The Basso-Beattie-Bresnahan (BBB) scale was used to evaluate rat locomotor function after etanercept administration. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were counted and the immunoreactivity to active caspase-3 and caspase-8 was examined after etanercept administration. Results. Immunoblot and double immunofluorescence staining revealed suppression of TNF-&agr;, TNFR1, and TNFR2 expression after administration of etanercept in the acute phase of spinal cord injury. LFB staining demonstrated potential myelination in the etanercept-treated group from 2 week after spinal cord injury, together with an increased BBB locomotor score. Double immunofluorescence staining showed a significant decrease in TUNEL-positive neurons and oligodendroglia from 12 hour to 1 week in the gray and white matters after etanercept administration. Immunoblot analysis demonstrated overexpression of activated caspase-3 and caspase-8 after spinal cord injury, which was markedly inhibited by etanercept. Conclusion. Our results indicated that etanercept reduces the associated tissue damage of spinal cord injury, improves hindlimb locomotor function, and facilitates myelin regeneration. This positive effect of etanercept on spinal cord injury is probably attributable to the suppression of TNF-&agr;, TNFR1, TNFR2, and activated caspase-3 and caspase-8 overexpressions, and the inhibition of neuronal and oligodendroglial apoptosis.

Targeted retrograde gene delivery of brain-derived neurotrophic factor suppresses apoptosis of neurons and oligodendroglia after spinal cord injury in rats.

Study Design. Histologic and immunohistochemical studies after targeted retrograde adenovirus (AdV)-mediated brain-derived neurotrophic factor (BDNF) gene delivery via intramuscular injection in rats with injured spinal cord. Objective. To investigate the neuroprotective effect of targeted retrograde AdV-BDNF gene transfection in the traumatically injured spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. Summary of Background Data. Several studies investigated the neuroprotective effects of neurotrophins including BDNF on spinal cord injury, with respect to prevention of neural cell apoptosis in injured spinal cord. However, no report has described the potential effect of targeted retrograde neurotrophic factor gene delivery in injured spinal cord on prevention of neural cell apoptosis. Methods. AdV-BDNF or AdV-LacZ was used for retrograde delivery via bilateral sternomastoid muscles to the spinal accessory motoneurons immediately after spinal cord injury in rats. Localization of β-galactosidase expression produced by LacZ gene or AdV-BDNF gene transfection was examined by immunofluorescence staining and double staining of cell markers (NeuN, RIP, GFAP, OX-42, and NG2) in the injured spinal cord. TUNEL-positive cells were counted and immunoreactivity to active caspase-3 and NG2 was examined after gene injection. Results. Retrograde delivery of LacZ marker gene was identified in cervical spinal neurons and glial cells including oligodendrocytes in the white matter. AdV-BDNF transfection resulted in a significant decrease in the number of TUNEL-positive apoptotic cells by downregulating the caspase apoptotic pathway, with significant promotion of NG2 expression in injured spinal cord, compared with AdV-LacZ injection. Conclusion. Our results suggest that targeted retrograde BDNF gene delivery suppresses apoptosis of neurons and oligodendrocytes in the injured rat spinal cord.

Tumor necrosis factor-alpha and its receptors contribute to apoptosis of oligodendrocytes in the spinal cord of spinal hyperostotic mouse (twy/twy) sustaining chronic mechanical compression.

Study Design. To examine the distribution of apoptotic cells and expression of tumor necrosis factor (TNF)-α and its receptors in the spinal hyperostotic mouse (twy/twy) with chronic cord compression using immunohistochemical methods. Objective. To study the mechanisms of apoptosis, particularly in oligodendrocytes, which could contribute to degenerative change and demyelination in chronic mechanical cord compression. Summary of Background Data. TNF-α acts as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury. Chronic spinal cord compression caused neuronal loss, myelin destruction, and axonal degeneration. However, the biologic mechanisms of apoptosis in chronically compressed spinal cord remain unclear. Methods. The cervical spinal cord of 34 twy mice aged 20 to 24 weeks and 11 control animals were examined. The apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining. The expression and the localization of TNF-α, TNF receptor 1 (TNFR1), and TNF receptor 2 (TNFR2) were examined using immunoblot and immnohistochemical analysis. Results. The number of TUNEL-positive cells in the white matter increased with the severity of compression, which was further increased bilaterally in the white matter of twy/twy mice. Double immunofluorescence staining showed that the number of cells positive for TUNEL and RIP, a marker of oligodendrocytes, increased in the white matter with increased severity of cord compression. Immunoblot analysis demonstrated overexpression of TNF-α, TNFR1, and TNFR2 in severe compression. The expression of TNF-α appeared in local cells including microglia while that of TNFR1 and TNFR2 was noted in apoptotic oligodendrocytes. Conclusion. Our results suggested that the proportion of apoptotic oligodendrocytes, causing spongy axonal degeneration and demyelination, correlated with the magnitude of cord compression and that overexpression of TNF-α, TNFR1, and TNFR2 seems to participate in apoptosis of such cells in the chronically compressed spinal cord.

High-mobility group box-1 and its receptors contribute to proinflammatory response in the acute phase of spinal cord injury in rats.

Study Design. To examine the localization and expression of high-mobility group box-1 (HMGB-1) protein and its receptors after rat spinal cord injury. Objective. To elucidate the contribution of HMGB-1 and its receptors as potential candidates in a specific upstream pathway to the proinflammatory response leading to a cascade of secondary tissue damage after spinal cord injury. Summary of Background Data. HMGB-1 was recently characterized as a key cytokine with a potential role in nucleosome formation and regulation of gene transcription. No studies have investigated the role of HMGB-1 in spinal cord injury. Methods. Injured thoracic spinal cord from 62 rats aged 8 to 12 weeks and spinal cord from 20 control rats were examined. HMGB-1 was localized by immunofluorescence staining, costaining with cell markers, and by immunoelectron microscopy. The expression of HMGB-1 and its receptors, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)2, and TLR4 were also examined by immunohistochemistry. Results. HMGB-1 expression appeared earlier than that of tumor necrosis factor-&agr;, interleukin (IL)-1&bgr;, and IL-6 in the spinal cord injury rats, with the HMGB-1 produced by both macrophages and neurons. HMGB-1 translocated from nucleus to cytoplasm in some neurons at an early stage after neural injury. Increased expression of HMGB-1, RAGE, and TLRs was observed after injury, and interaction of HMGB-1 with RAGE or TLRs, particularly in macrophage, was confirmed at 3 days after injury. Conclusion. Our results demonstrated an earlier onset in the expression of HMGB-1 than in tumor necrosis factor-&agr;, IL-1&bgr;, and IL-6 after spinal cord injury. The release of HMGB-1 from neurons and macrophages is mediated through the HMGB-1/RAGE or TLR pathways. HMGB-1 seems to play at least some roles in the proinflammatory cascade originating the secondary damage after the initial spinal cord injury.

Vertebroplasty-augmented short-segment posterior fixation of osteoporotic vertebral collapse with neurological deficit in the thoracolumbar spine: comparisons with posterior surgery without vertebroplasty and anterior surgery

OBJECT The surgical approach and treatment of thoracolumbar osteoporotic vertebral collapse with neurological deficit have not been documented in detail. Anterior surgery provides good decompression and solid fusion, but the surgery-related risk is relatively higher than that associated with the posterior approach. In posterior surgery, the major problem after posterior correction and instrumentation is failure to support the anterior spinal column, leading to loss of correction of kyphosis. The aim of this study was to evaluate the efficacy of reinforcing short-segment posterior fixation with vertebroplasty and to compare the outcome with those of posterior surgery without vertebroplasty and anterior surgery, retrospectively. METHODS The authors studied 83 patients who underwent surgical treatment for a single thoracolumbar osteoporotic vertebral collapse with neurological deficit. Twenty-eight patients treated by posterior surgery combined with vertebroplasty (Group A), 25 patients treated by posterior surgery without vertebroplasty (Group B), and 30 patients treated by anterior surgery (Group C) were followed up for a mean postoperative period of 4.4 years. Neurological outcome, visual analog scale pain score, and radiographic results were compared in the 3 groups. RESULTS Postoperative (4-6 weeks) and follow-up neurological outcome and visual analog scale scores were not significantly different among the 3 groups. Postoperative kyphotic angle was significantly reduced in Group B compared with Group C (p = 0.007), whereas the kyphotic angle was not significantly different among the 3 groups at follow-up. The mean ± SD loss of correction at follow-up was 4.6° ± 4.5°, 8.6° ± 6.2°, and 4.5° ± 5.9° in Groups A, B, and C, respectively. The correction loss at follow-up in Group B was significantly higher compared with Groups A and C (p = 0.0171 and p = 0.0180, respectively). CONCLUSIONS The results suggest that additional reinforcement with vertebroplasty reduces the kyphotic loss and instrumentation failure, compared with patients without the reinforcement of vertebroplasty. Vertebroplasty-augmented short-segment fixation seems to offer immediate spinal stability in patients with thoracolumbar osteoporotic vertebral collapse; the effect seems equivalent to that of anterior reconstruction.

The Prevalence and Phenotype of Activated Microglia/Macrophages within the Spinal Cord of the Hyperostotic Mouse (twy/twy) Changes in Response to Chronic Progressive Spinal Cord 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.

Early transcutaneous electrical nerve stimulation reduces hyperalgesia and decreases activation of spinal glial cells in mice with neuropathic pain.

Summary Application of early transcutaneous electrical nerve stimulation in a mouse model of neuropathic pain suppressed hyperalgesia and attenuated spinal microglial activation. ABSTRACT Although transcutaneous electrical nerve stimulation (TENS) is widely used for the treatment of neuropathic pain, its effectiveness and mechanism of action in reducing neuropathic pain remain uncertain. We investigated the effects of early TENS (starting from the day after surgery) in mice with neuropathic pain, on hyperalgesia, glial cell activation, pain transmission neuron sensitization, expression of proinflammatory cytokines, and opioid receptors in the spinal dorsal horn. Following nerve injury, TENS and behavioral tests were performed every day. Immunohistochemical, immunoblot, and flow cytometric analysis of the lumbar spinal cord were performed after 8 days. Early TENS reduced mechanical and thermal hyperalgesia and decreased the activation of microglia and astrocytes (P < 0.05). In contrast, the application of TENS at 1 week (TENS‐1w) or 2 weeks (TENS‐2w) after injury was ineffective in reducing hyperalgesia (mechanical and thermal) or activation of microglia and astrocytes. Early TENS decreased p‐p38 within microglia (P < 0.05), the expression levels of protein kinase C (PKC‐&ggr;), and phosphorylated anti‐phospho‐cyclic AMP response element‐binding protein (p‐CREB) in the superficial spinal dorsal horn neurons (P < 0.05), mitogen‐activated protein (MAP) kinases, and proinflammatory cytokines, and increased the expression levels of opioid receptors (P < 0.05). The results suggested that the application of early TENS relieved hyperalgesia in our mouse model of neuropathic pain by inhibiting glial activation, MAP kinase activation, PKC‐&ggr;, and p‐CREB expression, and proinflammatory cytokines expression, as well as maintenance of spinal opioid receptors. The findings indicate that TENS treatment is more effective when applied as early after nerve injury as possible.