Daisuke Sugita

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.

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment

Bone marrow‐derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC‐based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)‐positive bone marrow‐chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post‐SCI improved motor function and relieved SCI‐induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C‐γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p‐p38 mitogen‐activated protein kinase and extracellular signal‐regulated kinase (p‐ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double‐positive hematogenous macrophages without decreasing the CD11b‐positive and GFP‐negative activated spinal‐microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood‐spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor‐α, interleukin‐6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte–macrophage colony‐stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal‐resident microglia and the activity and recruitment of hematogenous macrophages. Stem Cells 2015;33:1902–1914

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.

Ossification process involving the human thoracic ligamentum flavum: role of transcription factors.

IntroductionOssification of the ligamentum flavum (OLF) of the spine is associated with serious neurologic compromise, but the pathomechanism of this process remains unclear. The objective of this study was to investigate the pathomechanism of the ossification process, including the roles of various transcriptional factors in the ossification of human thoracic ligamentum flavum.MethodsSections of the thoracic ligamentum flavum were obtained from 31 patients with OLF who underwent posterior thoracic decompression, and from six control patients free of OLF. Cultured ligamentum flavum cells (n = 6, each) were examined with real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis for Sry-type high-mobility group box 9 (Sox9), runt-related transcription factor 2 (Runx2), muscle segment homeobox 2 (Msx2), Osterix, distal-less homeobox 5 (Dlx5), and AP-1. The harvested sections were examined with hematoxylin-eosin, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method, and immunohistochemistry for the transcriptional factors.ResultsCompared with the control, the OLF showed disorganization of the elastic fiber bundles and abundant hypertrophic chondrocytes in the ossification front. TUNEL-positive chondrocytes were found near the ossified plaques. The mRNA expression levels of Sox9, Runx2, Msx2, and AP-1 in cultured cells from the ligamentum flavum of OLF patients were significantly different from those of the control. OLF samples were strongly immunoreactive to Sox9, Runx2, and Msx2 at proliferating chondrocytes in the fibrocartilage area. Hypertrophic chondrocytes were positive for Runx2, Osterix, Dlx5, and AP-1.ConclusionsThe ossification process in OLF seems to involve chondrocyte differentiation under the unique expression of transcriptional factors. Accumulation of hypertrophic chondrocytes was evident around the calcified area at the ossification front, and we suggest that the differentiation of these cells seems to be concerned with the ossification process.

Cyclic tensile strain facilitates the ossification of ligamentum flavum through β-catenin signaling pathway: in vitro analysis.

Study Design. Histological, immunohistochemical, and real-time reverse transcription-polymerase chain reaction analyses of the expression of cell signaling and transcriptional factors in human ossification of ligamentum flavum (OLF). Objective. To test the hypothesis that &bgr;-catenin plays a role in the ossification of OLF cells in response to cyclic tensile strain. Summary of Background Data. Several studies have investigated the roles of biomechanical and metabolic factors in the development and progression of OLF, based on the importance of genetic and biological factors. The process of ossification includes enchondral ossification, although such pathology remains poorly defined. Methods. Using real-time reverse transcription-polymerase chain reaction, we analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process (&bgr;-catenin, Runx2, Sox9, and osteopontin) in cultured OLF cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexercell FX-3000 (Flexercell International, Hillsborough, NC), applied for 0, 6, 12, or 24 hours. The localization of these factors was examined in decalcified paraffin OLF sections by immunohistochemistry. Controlled samples were harvested from nonossified ligamentum flavum of patients who underwent thoracic posterior surgical procedures. Results. Under resting conditions (no tensile strain), the mRNA levels of &bgr;-catenin, Runx2, Sox9, and osteopontin in cultured OLF cells were significantly higher than in the control non-OLF cells. Application of cyclic tensile strain to OLF cells resulted in significant increases in mRNA expression levels of &bgr;-catenin, Runx2, Sox9, and osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and osteopontin. Immunoreactivity of &bgr;-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area. Conclusion. Our results indicated that cyclic tensile strain applied to OLF cells activated their ossification through a process mediated by the &bgr;-catenin signaling pathway.

(18)F-FDG PET/CT for Diagnosis of Osteosclerotic and Osteolytic Vertebral Metastatic Lesions: Comparison with Bone Scintigraphy.

Study Design A retrospective study. Purpose The aims of this study were to investigate the diagnostic value of 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) in PET/computed tomography (CT) in the evaluation of spinal metastatic lesions. Overview of Literature Recent studies described limitations regarding how many lesions with abnormal 18F-FDG PET findings in the bone show corresponding morphologic abnormalities. Methods The subjects for this retrospective study were 227 patients with primary malignant tumors, who were suspected of having spinal metastases. They underwent combined whole-body 18F-FDG PET/CT scanning for evaluation of known neoplasms in the whole spine. 99mTc-methylene diphosphonate bone scan was performed within 2 weeks following PET/CT examinations. The final diagnosis of spinal metastasis was established by histopathological examination regarding bone biopsy or magnetic resonance imaging (MRI) findings, and follow-up MRI, CT and 18F-FDG PET for extensively wide lesions with subsequent progression. Results From a total of 504 spinal lesions in 227 patients, 224 lesions showed discordant image findings. For 122 metastatic lesions with confirmed diagnosis, the sensitivity/specificity of bone scan and FDG PET were 84%/21% and 89%/76%, respectively. In 102 true-positive metastatic lesions, the bone scan depicted predominantly osteosclerotic changes in 36% and osteolytic changes in 19%. In 109 true-positive lesions of FDG PET, osteolytic changes were depicted predominantly in 38% while osteosclerotic changes were portrayed in 15%. Conclusions 18F-FDG PET in PET/CT could be used as a substitute for bone scan in the evaluation of spinal metastasis, especially for patients with spinal osteolytic lesions on CT.

Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.

Study Design. Histological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL). Objective. To examine the hypothesis that Ihh signaling in correlation with Sox9 and parathyroid-related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL. Summary of Background Data. In enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy. Methods. Samples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from patients with cervical spondylotic myelopathy/radiculopathy without OPLL. The harvested sections were stained with hematoxylin-eosin and toluidine blue, examined by transmission electron microscopy, and immunohistochemically stained for Ihh, PTHrP, Sox9, type X, XI collagen, and alkaline phosphatase. Immunoblot analysis was performed in cultured cells derived from the posterior longitudinal ligaments in the vicinity of the ossified plaque and examined for the expression of these factors. Results. The ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes and was strongly positive for PTHrP in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 than those in non-OPLL cells. Conclusion. Our results indicated that overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification and enhances bone formation in OPLL.

Modified Metaphyseal‐Loading Anterolaterally Flared Anatomic Femoral Stem: Five‐ to Nine‐Year Prospective Follow‐Up Evaluation and Results of Three‐Dimensional Finite Element Analysis

We have designed a proximal-fitting, anterolaterally flared, arc-deposit hydroxyapatite-coated anatomical femoral stem (FMS-anatomic stem; KYOCERA Medical, Osaka, Japan) for cementless total hip arthroplasty (THA) for Japanese patients with dysplastic hip osteoarthritis, using a nonlinear three-dimensional finite element analysis simulating loading conditions. The Anatomic Fit stem was modified in the region of the arc-sprayed surface, to allow more proximal appearance of spot welds. The aim of the present study was to analyze the clinical and radiographic outcomes of patients who underwent THA using this stem. We reviewed 73 consecutive patients (79 hips; 13 men 16 hips; 60 women 63 hips; age at surgery, 57.6 years, range, 35-78) who underwent cementless THA using the Anatomic Fit stem, at a follow-up period of 7.1 years (range, 5.1-9.4). Harris Hip score improved from 40.7 ± 17.1 before surgery to 91.0 ± 5.2 points at follow-up. The 7.1-year stem survival rate was 100%. Radiographs at follow-up confirmed the stability of the femoral stems within the femoral canal in all cases, with sufficient bone ingrowth. None of the patients had subsidence of the stem exceeding 2.0 mm within the femoral canal or changes in varus or valgus position of more than 2.0°. The Anatomic Fit stem provided excellent results. The nonlinear three-dimensional finite element analysis demonstrated that the stem-bone relative motion was 10 µm at the proximal end of the stem and proximal load transfer. Our analysis confirmed reduced radiolucency around the stem, minimal subsidence, appropriate stress shielding, and promising medium-term stability within the femoral canal.

Postoperative gait analysis and hip muscle strength in patients with pelvic ring fracture

The aims of present study were (1) to determine changes in kinematic and kinetic variables at 3 and 12 months after open reduction and internal fixation (ORIF) of pelvic ring fracture and (2) to determine the factor(s) associated with gait disorders that correlate with gait parameters measured at 12 months after surgery. Nineteen patients with pelvic ring fractures underwent ORIF and examined at 3 and 12 months postoperatively. The study also included a similar number of age-matched control subjects. Peak hip abduction angle, peak hip extension moment in the stance, peak hip abduction moment, and peak ankle plantarflexion moment at 3 months after ORIF were significantly lower than the respective control values. At 12 months, complete recovery was noted in peak hip abduction moment and peak ankle plantarflexion moment, whereas the recovery in peak hip abduction angle and peak hip extension moment in the stance was partial. The existence of neurological lesions and strength asymmetry of hip abductor and adductor at 3 months post-ORIF correlated with decreased peak hip abduction moment after ORIF. Our results highlighted characteristic gait patterns up to 12 months after ORIF for pelvic fracture, and these patterns correlated with neurological lesion and weakness of hip abductor and adductor muscles.