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Abstract Background There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. Methods The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation. Results The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia. Conclusions Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.

Study Design: Retrospective study of prospectively collected data., Objective: To investigate the influence of cartilaginous endplates (CEs) in herniated discs on clinical symptoms and postoperative outcomes in patients with lumbar disc herniation (LDH) in different age groups., Summary of Background Data: LDH involving CEs, which are hard and less resorptive, is frequently observed with increasing age and appears to affect the natural course and clinical outcomes following discectomy., Methods: Overall, 239 patients who underwent microscopic discectomy were included. Main outcomes were evaluated using motor strength, visual analog scale (VAS) for back and leg pain, and Rolland-Morris Disability Questionnaire. The effects of CEs on clinical variables and postoperative outcomes were compared between two groups (<50 y and ≥50 y). Furthermore, we investigated the characteristics of CE avulsions in each group and examined the association between CE occupancy rate and clinical symptoms., Results: CEs were predominantly observed with increasing age and were more frequently detected in patients with Modic changes in both groups (P<0.001). A higher proportion of LDH with a ≥20% occupancy rate was found in patients aged <50 years (P=0.009) and was associated with a decrease in motor strength preoperatively (P=0.007). Postoperative VAS score for low back pain (LBP) was higher in patients with CEs than in those without CEs in the ≥50-year-old group (P<0.001). In multiple regression analysis, the presence of CEs was independently associated with residual LBP at 1 year postoperatively in older patients (β=0.46, P<0.001)., Conclusion: Avulsion-type herniations in patients aged <50 years had a higher CE occupancy rate, which is a potential cause of preoperative motor weakness. Clinical outcomes following discectomy improved regardless of the presence of CEs, however, cartilaginous herniation in patients aged ≥50 years may affect residual LBP at 1 year., Competing Interests: Declaration of Conflicting Interests: The authors (s) declare no potential conflicts of interest regarding the research, authorship, or publication of this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Surgery could regain the ability to walk even in non-ambulatory patients with spinal cord compression due to metastatic spine disease. However, many patients cannot reach the stage of independent ambulation because most are at an advanced disease stage. This study investigated the regained independent ambulation rate after surgery and prognostic factors for independent ambulation after metastatic spinal cord compression surgery., Methods: In a retrospective cohort study, 38 non-ambulatory patients with spinal metastases at the cervical or thoracic lesions, who underwent surgery, were included. All surgeries were performed using laminectomy and posterior fixation. Recovery rates of independent ambulation and its prognostic factors were examined. Independent ambulation was defined as the use of a walking aid without wheelchair requirement. Factors, including age, tumor type, visceral organ metastasis, past systematic cancer therapy, neurological grade, the time from leg-symptom onset to non-ambulatory stage, and the time from non-ambulatory stage to surgery, were investigated., Results: The regained independent ambulation rate was 18% (7/38). Compared to non-ambulatory patients, those who regained independent ambulation were more likely to have less past systematic therapy (14% [1/7] vs. 74% [23/31], P=0.003) and slow paralysis progression (over seven days from leg-symptom onset to non-ambulatory stage) (86% [6/7] vs. 23% [7/31], P=0.002)., Conclusions: Recovery to independent ambulation in non-ambulatory patients with metastatic spinal cord compression was poor, even if surgery was performed. Absence of past systematic therapy and slow paralysis progression were favorable factors for regaining independent ambulation., Competing Interests: Human subjects: Consent was obtained or waived by all participants in this study. Kyushu University Institutional Review Board issued approval 22098-00. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Iida et al.)

Objectives: This study aimed to determine the risk factors for vertebral fractures requiring surgery in patients with ankylosing spondylitis (AS)., Methods: We included 60 patients with AS diagnosed by using the modified New York criteria and who were treated in our department from April 2004 to March 2019. We evaluated age, sex, disease duration, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), ankylosed sacroiliac joint, bamboo spine, number of ankylosed vertebrae, and treatment (nonsteroidal antiinflammatory drugs (NSAIDs)), prednisolone (PSL), conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), biological disease-modifying antirheumatic drugs (bDMARDs), spine surgery for vertebral fracture) at the final follow-up of the nonsurgical group and the preoperative follow-up of the surgical group., Results: At the final follow-up, the mean age was 49 years, 46 patients (75%) were male, and the mean disease duration was 27 years. Additionally, 8 (13.3%) and 43 patients (71%) underwent surgical and medical treatments, respectively. The group of surgery for vertebral fracture had significantly higher CRP levels, which was also significantly associated with vertebral fracture surgery by multivariate analysis., Conclusions: CRP was identified as a risk factor for vertebral fractures requiring surgery. Control of systemic inflammation in patients with AS may reduce the risk of vertebral fractures requiring surgery., (© Japan College of Rheumatology 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Background: Spinal metastases can impair mobility, worsening the Karnofsky Performance Status (KPS). Surgery for spinal metastases has the potential to improve KPS and extend prognosis, but it is crucial to recognize the elevated risk of perioperative complications. Therefore, the development of a new scoring system to accurately predict perioperative complications in spinal metastatic surgery is essential., Methods: We conducted a retrospective observational study with 86 patients who underwent surgical intervention for spinal metastases. Patients were divided into two groups based on the presence or absence of perioperative complications within 14 days after surgery. Various factors related to perioperative complications were assessed through univariate and multivariate analyses. We established a clinical prognostic scoring system called the Perioperative Complications following Metastatic Spinal Surgery (PERCOM) score and evaluated its precision using receiver operating characteristic (ROC) analysis., Results: Five variables (age, KPS, primary prostate cancer, Albumin, and Hemoglobin) identified in the univariate analysis were assigned binary values of 0 or 1. The PERCOM score was then calculated for each patient by summing the individual points, ranging from 0 to 5. The optimal threshold determined by ROC curve analysis for the PERCOM score was 2 points, with a sensitivity of 86 % and a specificity of 56 %., Conclusions: The composite PERCOM score effectively predicted perioperative complications in spinal metastasis surgery. To further validate its precision, a prospective multicenter study is needed., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

BACKGROUND Postoperative acute liver failure, a complication following spine surgery, can cause delayed emergence from total intravenous anesthesia. Here, we report a case of acute severe postoperative liver failure following posterior spinal correction and fusion in a patient with congenital scoliosis. CASE REPORT A girl's congenital scoliosis worsened, and posterior spinal correction and fusion was scheduled. General anesthesia was induced with sevoflurane, fentanyl, target-controlled-infusion with propofol, and rocuronium. General anesthesia was maintained using target-controlled-infusion with propofol and remifentanil. The operation was completed with no remarkable complications. The operative time was 516 min and the anesthesia time was 641 min in the prone position. Emergence from anesthesia was poor, and it took 68 min to remove the tracheal tube after discontinuation of the anesthetic agents. The patient was drowsy and was transferred to her room in a general ward without reporting any pain, nausea, or dyspnea. On postoperative day 1, the results of laboratory investigations were suggestive of acute liver failure; contrast-enhanced computed tomography revealed a poorly enhanced area in the umbilical portion of the left liver lobe portal vein, indicating ischemic liver damage. Although no additional treatment was administered for acute liver failure, the patient recovered over time, and laboratory values normalized. No other postoperative complications were observed, and the patient was discharged on postoperative day 1. CONCLUSIONS Delayed emergence from general anesthesia may be due to acute liver failure following posterior spinal correction and fusion. There are several possible causes of postoperative liver failure, including anesthetics, prone position, and spinal surgery.

Background: In patients with cervical spinal cord injury (SCI), we need to make accurate prognostic predictions in the acute phase for more effective rehabilitation. We hypothesized that a multivariate prognosis would be useful for patients with cervical SCI., Methods: We made two predictive models using Multiple Linear Regression (MLR) and Artificial Neural Networks (ANNs). We adopted MLR as a conventional predictive model. Both models were created using the same 20 clinical parameters of the acute phase data at the time of admission. The prediction results were classified by the ASIA Impairment Scale. The training data consisted of 60 cases, and prognosis prediction was performed for 20 future cases (test cohort). All patients were treated in the Spinal Injuries Center (SIC) in Fukuoka, Japan., Results: A total of 16 out of 20 cases were predictable. The correct answer rate of MLR was 31.3%, while the rate of ANNs was 75.0% (number of correct answers: 12)., Conclusion: We were able to predict the prognosis of patients with cervical SCI from acute clinical data using ANNs. Performing effective rehabilitation based on this prediction will improve the patient's quality of life after discharge. Although there is room for improvement, ANNs are useful as a prognostic tool for patients with cervical SCI.

Study Design: Retrospective study., Objective: To investigate the characteristics of newly developing Modic changes following discectomy and their impact on residual low back pain (LBP) in the early postoperative stage of lumbar disc herniation., Methods: We included 96 patients who underwent microscopic discectomy. Through MRI, we assessed new developments of Modic changes and the progression of disc degeneration at the surgical level. The presence of cartilaginous endplates was evaluated using resected specimens, and the main outcome was assessed using the visual analog scale (VAS). Further, the prevalence and time course of Modic changes, and their effects on clinical outcomes in the early postoperative period were examined., Results: A new development of Modic changes was detected in 28% of cartilaginous herniations at 6 months. Modic changes were observed more frequently in patients with cartilaginous herniation than in those without cartilaginous herniation postoperatively ( P < .001). The VAS scores for LBP up to 6 months were greater in patients with Modic changes ( P < .001) than those without; however, no significant differences were identified in the presence or absence of Modic changes over the year follow-up. The development of Modic changes was closely associated with residual LBP at 6 months (β:0.511, P < .001)., Conclusions: Modic changes develop predominantly in patients with avulsion-type herniation than in those with annular rupture at an earlier phase after discectomy. Furthermore, disc herniation with cartilaginous endplates may be associated with a slower decrease in LBP for up to 6 months, supporting the notion that newly developing endplate changes may cause residual LBP., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Neonatal spinal cord injury (SCI) shows better functional outcomes than adult SCI. Although the regenerative capability in the neonatal spinal cord may have cues in the treatment of adult SCI, the mechanism underlying neonatal spinal cord regeneration after SCI is unclear. We previously reported age-dependent variation in the pathogenesis of inflammation after SCI. Therefore, we explored differences in the pathogenesis of inflammation after SCI between neonatal and adult mice and their effect on axon regeneration and functional outcome. We established two-day-old spinal cord crush mice as a model of neonatal SCI. Immunohistochemistry of the spinal cord revealed that the nuclear translocation of NF-κB, which promotes the expression of chemokines, was significantly lower in the astrocytes of neonates than in those of adults. Flow cytometry revealed that neonatal astrocytes secrete low levels of chemokines to recruit circulating neutrophils (e.g., Cxcl1 and Cxcl2) after SCI in comparison with adults. We also found that the expression of a chemokine receptor (CXCR2) and an adhesion molecule (β2 integrin) quantified by flow cytometry was lower in neonatal circulating neutrophils than in adult neutrophils. Strikingly, these neonate-specific cellular properties seemed to be associated with no neutrophil infiltration into the injured spinal cord, followed by significantly lower expression of inflammatory cytokines (Il-1β, Il-6 and TNF-α) after SCI in the spinal cords of neonates than in those of adults. At the same time, significantly fewer apoptotic neurons and greater axonal regeneration were observed in neonates in comparison with adults, which led to a marked recovery of locomotor function. This neonate-specific mechanism of inflammation regulation may have potential therapeutic applications in controlling inflammation after adult SCI.

In osteoclastogenesis, the metabolism of metal ions plays an essential role in controlling reactive oxygen species (ROS) production, mitochondrial biogenesis, and survival, and differentiation. However, the mechanism regulating metal ions during osteoclast differentiation remains unclear. The metal-binding protein metallothionein (MT) detoxifies heavy metals, maintains metal ion homeostasis, especially zinc, and manages cellular redox levels. We carried out tests using murine osteoclast precursors to examine the function of MT in osteoclastogenesis and evaluated their potential as targets for future osteoporosis treatments. MT genes were significantly upregulated upon differentiation from osteoclast precursors to mature osteoclasts in response to receptor activators of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation, and MT3 expression was particularly pronounced in mature osteoclasts among MT genes. The knockdown of MT3 in osteoclast precursors demonstrated a remarkable inhibition of differentiation into mature osteoclasts. In preosteoclasts, MT3 knockdown suppressed the activity of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways upon RANKL stimulation, leading to affect cell survival through elevated cleaved Caspase 3 and poly (ADP-ribose) polymerase (PARP) levels. Additionally, ROS levels were decreased, and nuclear factor erythroid 2-related factor 2 (NRF2) (a suppressor of ROS) and the downstream antioxidant proteins, such as catalase (CAT) and heme oxygenase 1 (HO-1), were more highly expressed in the MT3 preosteoclast knockdowns. mitochondrial ROS, which is involved in mitochondrial biogenesis and the production of reactive oxygen species, were similarly decreased because cAMP response element-binding (CREB) and peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) were less activated due to MT3 depletion. Thus, by modulating ROS through the NRF2 pathway, MT3 plays a crucial role in regulating osteoclast differentiation and survival, acting as a metabolic modulator of intracellular zinc ions., (© 2023. The Author(s).)

Background: Spinal cord injury (SCI) is a devastating disease that results in permanent paralysis. Currently, there is no effective treatment for SCI, and it is important to identify factors that can provide therapeutic intervention during the course of the disease. Zinc, an essential trace element, has attracted attention as a regulator of inflammatory responses. In this study, we investigated the effect of zinc status on the SCI pathology and whether or not zinc could be a potential therapeutic target., Methods: We created experimental mouse models with three different serum zinc concentration by changing the zinc content of the diet. After inducing contusion injury to the spinal cord of three mouse models, we assessed inflammation, apoptosis, demyelination, axonal regeneration, and the number of nuclear translocations of NF-κB in macrophages by using qPCR and immunostaining. In addition, macrophages in the injured spinal cord of these mouse models were isolated by flow cytometry, and their intracellular zinc concentration level and gene expression were examined. Functional recovery was assessed using the open field motor score, a foot print analysis, and a grid walk test. Statistical analysis was performed using Wilcoxon rank-sum test and ANOVA with the Tukey-Kramer test., Results: In macrophages after SCI, zinc deficiency promoted nuclear translocation of NF-κB, polarization to pro-inflammatory like phenotype and expression of pro-inflammatory cytokines. The inflammatory response exacerbated by zinc deficiency led to worsening motor function by inducing more apoptosis of oligodendrocytes and demyelination and inhibiting axonal regeneration in the lesion site compared to the normal zinc condition. Furthermore, zinc supplementation after SCI attenuated these zinc-deficiency-induced series of responses and improved motor function., Conclusion: We demonstrated that zinc affected axonal regeneration and motor functional recovery after SCI by negatively regulating NF-κB activity and the subsequent inflammatory response in macrophages. Our findings suggest that zinc supplementation after SCI may be a novel therapeutic strategy for SCI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kijima, Ono, Kobayakawa, Saiwai, Hara, Yoshizaki, Yokota, Saito, Tamaru, Iura, Haruta, Kitade, Utsunomiya, Konno, Edgerton, Liu, Sakai, Maeda, Kawaguchi, Matsumoto, Okada and Nakashima.)

We proposed a bimodal artificial intelligence that integrates patient information with images to diagnose spinal cord tumors. Our model combines TabNet, a state-of-the-art deep learning model for tabular data for patient information, and a convolutional neural network for images. As training data, we collected 259 spinal tumor patients (158 for schwannoma and 101 for meningioma). We compared the performance of the image-only unimodal model, table-only unimodal model, bimodal model using a gradient-boosting decision tree, and bimodal model using TabNet. Our proposed bimodal model using TabNet performed best (area under the receiver-operating characteristic curve [AUROC]: 0.91) in the training data and significantly outperformed the physicians' performance. In the external validation using 62 cases from the other two facilities, our bimodal model showed an AUROC of 0.92, proving the robustness of the model. The bimodal analysis using TabNet was effective for differentiating spinal tumors., Competing Interests: Authors declare that they have no competing interests., (© 2023 The Authors.)

Background: Progression of kyphosis after laminoplasty sometimes results in the recurrence of myelopathy with lamina closure. However, only a few case reports have been published on the reoperation of double-door laminoplasty using the suture method. This study investigated the incidence and clinical features of reoperation cases caused by the recurrence of myelopathy with lamina closure after double-door laminoplasty using a modified Kirita-Miyazaki suture method., Methods: A total of 169 patients who underwent double-door laminoplasty were included in this study, with a mean follow-up duration of 6.6 years (range: 2-16). All surgeries were double-door laminoplasties in which the open lamina was sutured to the paravertebral muscle. The reoperation rate for myelopathy recurrence due to lamina closure and the associated risk factors were investigated. The risk factors included age, history, cervical alignment, C2-7 lordosis, the cervical sagittal vertical axis, and C7 slope., Results: The reoperation rate for recurrence of myelopathy by lamina closure was 3.0% (5/169). All patients showed kyphosis progression after surgery; the spinal cord was more compressed by closed lamina than before the initial surgery. The reoperation group had more patients with neuromuscular or psychiatric disorders (60% [3/5] vs. 2% [4/164]; p  < 0.001), kyphotic alignments (60% [3/5] vs. 10% [16/164]; p  < 0.001), and cases with less than -10° of C2-7 lordosis (60% [3/5] vs. 7% [11/164]; p  < 0.001)., Conclusions: Double-door laminoplasty with the suture method may not be suitable for patients with a neuromuscular or psychiatric disease or those with preoperative C2-7 lordosis less than -10°., Competing Interests: None., (© 2023 Published by Elsevier B.V. on behalf of Professor P K Surendran Memorial Education Foundation.)

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism through which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in the injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3 -/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8 -/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8 -/- bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism through which migrating macrophages attract astrocytes and affect the pathophysiology and outcome after SCI., (© 2023. The Author(s).)

Joint contracture causes distressing permanent mobility disorder due to trauma, arthritis, and aging, with no effective treatment available. A principal and irreversible cause of joint contracture has been regarded as the development of joint capsule fibrosis. However, the molecular mechanisms underlying contracture remain unclear. We established a mouse model of knee joint contracture, revealing that fibrosis in joint capsules causes irreversible contracture. RNA-sequencing of contracture capsules demonstrated a marked enrichment of the genes involved in the extracellular region, particularly periostin (Postn). Three-dimensional magnetic resonance imaging and immunohistological analysis of contracture patients revealed posterior joint capsule thickening with abundant type I collagen (Col1a2) and POSTN in humans. Col1a2-GFP TG ; Postn -/- mice and chimeric mice with Col1a2-GFP TG ; tdTomato TG bone marrow showed fibrosis in joint capsules caused by bone marrow-derived fibroblasts, and POSTN promoted the migration of bone marrow-derived fibroblasts, contributing to fibrosis and contracture. Conversely, POSTN-neutralizing antibody attenuated contracture exacerbation. Our findings identified POSTN as a key inducer of fibroblast migration that exacerbates capsule fibrosis, providing a potential therapeutic strategy for joint contracture., (© 2023 Federation of American Societies for Experimental Biology.)

Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is a spectrum of heterogeneous diseases commonly recognised by skin and osteoarticular lesions. There have been reports of some surgical cases of the progressive, destructive spondylitis associated with SAPHO syndrome, wherein the destructive spondylitis was considered to have developed due to the progression of spondylitis with SAPHO syndrome as the pathogenic bacteria were not isolated. We herein report a surgical case of destructive cervical spondylitis associated with SAPHO syndrome. A 54-year-old woman with a history of palmoplantar pustulosis suffered severe neck pain for 6 months. Radiography and computeed tomography showed sclerosed and collapsed cervical vertebrae, and the patient was referred to our hospital for further evaluation and management upon suspicion of infection or spondylitis with SAPHO syndrome. For the severe neck pain and progressive destruction of cervical vertebrae, we performed posterior fusion surgery with subsequent anterior fusion. Cutibacterium acnes (C. acnes) was isolated by enrichment culture with thioglycolate broth from both the anterior and the posterior tissue samples. We diagnosed pyogenic spondylitis secondary to C. acnes infection and administered doxycycline for 6 weeks after the first surgery. The neck pain was resolved and cervical fusion was achieved one year postoperatively. C. acnes infection could elicit destructive spondylitis. An enrichment culture should be performed to isolate the pathogenic bacteria in cases of destructive spondylitis with SAPHO syndrome., (© Japan College of Rheumatology 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Spinal cord injury (SCI) causes reactive astrogliosis, the sequential phenotypic change of astrocytes in which naïve astrocytes (NAs) transform into reactive astrocytes (RAs) and subsequently become scar-forming astrocytes (SAs), resulting in glial scar formation around the lesion site and thereby limiting axonal regeneration and motor/sensory functional recovery. Inhibiting the transformation of RAs into SAs in the acute phase attenuates the reactive astrogliosis and promotes regeneration. However, whether or not SAs once formed can revert to RAs or SAs is unclear. We performed selective isolation of astrocytes from glial scars at different time points for a gene expression analysis and found that the expression of Sox9, an important transcriptional factor for glial cell differentiation, was significantly increased in chronic phase astrocytes (CAs) compared to SAs in the sub-acute phase. Furthermore, CAs showed a significantly lower expression of chondroitin sulfate proteoglycan (CSPG)-related genes than SAs. These results indicated that SAs changed their phenotypes according to the surrounding environment of the injured spinal cord over time. Even though the integrin-N-cadherin pathway is critical for glial scar formation, collagen-I-grown scar-forming astrocytes (Col-I-SAs) did not change their phenotype after depleting the effect of integrin or N-cadherin. In addition, we found that Col-I-SAs transplanted into a naïve spinal cord formed glial scar again by maintaining a high expression of genes involved in the integrin-N-cadherin pathway and a low expression of CSPG-related genes. Interestingly, the transplanted Col-I-SAs changed NAs into SAs, and anti-β 1 -integrin antibody blocked the recruitment of SAs while reducing the volume of glial scar in the chronic phase. Our findings indicate that while the characteristics of glial scars change over time after SCI, SAs have a cell-autonomous function to form and maintain a glial scar, highlighting the basic mechanism underlying the persistence of glial scars after central nervous system injury until the chronic phase, which may be a therapeutic target., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In crush syndrome, massive muscle breakdown resulting from ischemia-reperfusion muscle injury can be a life-threatening condition that requires urgent treatment. Blood reperfusion into the ischemic muscle triggers an immediate inflammatory response, and neutrophils are the first to infiltrate and exacerbate the muscle damage. Since free zinc ion play a critical role in the immune system and the function of neutrophils is impaired by zinc depletion, we hypothesized that the administration of a zinc chelator would be effective for suppressing the inflammatory reaction at the site of ischemia-reperfusion injury and for improving of the pathology of crush syndrome. A crush syndrome model was created by using a rubber tourniquet to compress the bilateral hind limbs of mice at 8 weeks. A zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) was administered immediately after reperfusion in order to assess the anti-inflammatory effect of the chelator for neutrophils. Histopathological evaluation showed significantly less muscle breakdown and fewer neutrophil infiltration in TPEN administration group compared with control group. In addition, the expression levels of inflammatory cytokine and chemokine such as IL-6, TNFα, CXCL1, CXCL2, CXCR2, CCL2 in ischemia-reperfusion injured muscle were significantly suppressed with TPEN treatment. Less dilatation of renal tubules in histological evaluation in renal tissue and significantly better survival rate were demonstrated in TPEN treatment for ischemia-reperfusion injury in crush syndrome. The findings of our study suggest that zinc chelators contributed to the resolution of exacerbation of the inflammatory response and attenuation of muscle breakdown in the acute phase after crush syndrome. In addition, our strategy of attenuation of the acute inflammatory reaction by zinc chelators may provide a promising therapeutic strategy not only for crush syndrome, but also for other diseases driven by inflammatory reactions., (© 2022. The Author(s).)

Study Design: Retrospective diagnostic analysis., Objectives: To establish a new predictor of surgical outcome after surgery for intradural extramedullary spinal tumor (IDEMT) in the thoracic spine, we introduced shape factor (SF), a mathematical description of the morphology of the spinal cord. SF was calculated by dividing object area by the square of perimeter., Materials and Methods: Forty-three consecutive patients with IDEMT, detected by magnetic resonance imaging at the thoracic level with myelopathic signs, were included. Preoperative transverse cross-sectional area (CSA) and perimeter of the spinal cord (perimeter) at the level of maximal compression were measured. SF was calculated as 4π × CSA/(perimeter) 2 . The association between clinicoradiological factors and surgical outcome of IDEMT was statistically analyzed., Results: Mean CSA, perimeter, and SF were 27.8 ± 15.8 mm 2 , 28.8 ± 6.1 mm, and 0.385 ± 0.14, respectively. A histogram distribution revealed that perimeter and SF, but not CSA, fit the normal distribution. The patients were subdivided into 2 groups according to postoperative modified Japanese Orthopedic Association Score (mJOA). [group F (favorable): n = 32, mJOA ≥ 9; group UF (unfavorable): n = 11, mJOA < 9). Group UF had significantly lower mean CSA and SF. In univariate analysis of possible predictive factors for IDEMT surgery, greater age, lower preoperative mJOA, and lower SF were significantly associated with unfavorable outcome. In multivariate analysis, lower SF was the only significant predictor of postoperative outcome (odds ratio = 2.66, 95% CI 1.10-6.39, p = 0.0115)., Conclusion: Measurements of CSA and perimeter, followed by calculation of SF, may provide valuable quantitative information for the outcome of surgery for IDEMT.

Background: Malignant tumors occurring around both the spinal column and posterior chest wall are uncommon. Surgical resection of chest wall tumors adjacent to the spinal column is still challenging due to the surrounding anatomical structures. The purpose of the present study was to evaluate the long-term outcomes of surgical management in malignant tumors involving the spinal column and posterior chest wall., Methods: Between 1999 and 2007, 10 consecutive patients underwent en bloc resection combined with the posterior chest wall in the treatment of malignant tumors around the spinal column. There were 6 males and 4 females with a mean age at the surgery of 40.9 years old (range, 14-62 years old). The mean postoperative follow-up period was 159.7 months (range, 84-245 months). The clinical history, physical examination, laboratory data, radiological findings, and operative findings for each patient were retrospectively reviewed., Results: All surgeries were performed via a combined anterior and posterior approach. The mean numbers of partially resected vertebrae and ribs were 3.1 and 4.1, respectively. Lower or upper lobectomy was performed in four patients, and the diaphragm was partially resected in two patients. The surgical margin was wide in seven patients and marginal in two patients. Although five patients had postoperative respiratory problem, all patients improved immediately without life-threatening complications. There were no patients with respiratory insufficiency after surgery. One patient with osteosarcoma died of lung metastases 99 months after surgery. At the final follow-up, only one patient had local recurrence, five had been continuously disease-free, and three were alive with no evidence of disease., Conclusions: En bloc resection and reconstruction in selected patients with malignant tumors involving both the spinal column and posterior chest wall demonstrated good long-term results for local control and the respiratory function., Competing Interests: Declaration of competing interest None., (Copyright © 2021 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Study Design: Basic science study., Objective: The aim of this study was to examine whether epidural fat tissue (EFT) transplantation can prevent epidural adhesion after laminectomy more efficiently than subcutaneous fat tissue (SFT) transplantation., Summary of Background Data: Epidural adhesion is almost inevitable after laminectomy. Although many materials have been used to prevent adhesion, none has been widely accepted. As EFT is an ectopic fat tissue located on the dura mater and there is no adhesion between EFT and the dura mater, we focused on the efficacy of EFT for adhesion prevention., Methods: We examined the differences in histology and gene expression between EFT and SFT of mice. We performed laminectomy at the 10th thoracic level and immediately transplanted EFT or SFT to the dura mater in mice. At 6 weeks after transplantation, we performed histological and gene expression analyses and evaluated the adhesion tenacity. In addition, we examined the characteristic differences between human EFT and SFT., Results: The adipocytes of EFT were significantly smaller than those of SFT in mice and humans. The gene expression of inflammatory cytokine and fibrosis-related factors was significantly higher in SFT than in EFT. At 6 weeks after transplantation, the percentage of the remaining fat area over the dura mater was significantly greater in the EFT group than in SFT group, and the adhesion tenacity score was significantly lower in the EFT group than that in the SFT group. An RNA sequencing analysis revealed 1921 differentially expressed genes (DEGs) between human EFT and SFT, and a Gene Ontology term associated with the inflammatory response was most highly enriched in SFT., Conclusion: EFT has different molecular and histological profiles from SFT and EFT grafting is more effective for epidural adhesion prevention than conventional SFT transplantation after laminectomy in a mouse model.Level of Evidence: N/A., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Multiple spinal cord tumors in a single patient are very rare and most often seen in cases of neurofibromatosis and associated disorders. Schwannomatosis, which is characterized by the development of multiple schwannomas without vestibular schwannomas, has been newly defined as a distinct form of neurofibromatosis. The purpose of the present study was to describe and review the clinical and radiological features and the management of patients with multiple spinal schwannomas without vestibular schwannomas., Methods: Between 1986 and 2016, 19 patients with multiple spinal schwannomas without vestibular schwannoma were diagnosed and treated. Of the 19 patients, 13 were males, and 6 were females. The mean age at the first surgery for spinal schwannoma was 45.2 years old. The mean follow-up period was 123.4 months. The clinical features and radiological findings of the patients with multiple spinal schwannomas were retrospectively reviewed., Results: Among the 19 patients, there were more than 140 spinal schwannomas. The most common area of spinal schwannoma was the thoracolumbar-lumbar region. Initial symptoms and chief complaints caused by spinal schwannomas were primarily pain in the trunk or extremities in 17 (89.5%) of 19 patients. More than 60 spinal schwannomas were surgically resected. Multiple spinal surgeries were required in six patients. In all 19 patients, surgical treatment has provided successful relief of symptoms and neurological recovery., Conclusions: Surgical treatment was safe and effective in patients with multiple spinal schwannomas without vestibular schwannomas. After surgery, we recommend that all patients be followed with magnetic resonance imaging to monitor for asymptomatic tumors or detect new tumors early., Competing Interests: Declaration of competing interest None., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Spinal meningioma is a common benign intradural spinal tumor. It has been reported that the local recurrence rate after surgical resection increases with longer follow-up duration. Simpson grade 1 resection could reduce the risk of recurrence, but this procedure needs dural reconstruction, which would cause cerebrospinal fluid (CSF) leakage or iatrogenic spinal cord injury. Saito et al. reported dura preservation technique to reduce the risk of CSF leakage, in which the meningioma together with the inner layer of the dura is removed and the outer layer is preserved for simple dural closure. The long-term outcomes with this technique have never been investigated. In this study, we retrospectively analyzed the data of 38 surgically treated patients (dura preservation technique, 12 patients; Simpson grade 2 resection, 26 patients) to assess the long-term recurrence rate (mean, 121.5 months; range, 60-228 months). The local recurrence rate in the dura preservation group was 8.3% (1 of 12 cases), which was similar to that in Simpson grade 2 resection group (2 of 26 cases [7.7%]). Although this case series did not indicate the significant difference in the recurrence rates between the dura preservation group and Simpson grade 2 group, we consider that this technique still has advantages for surgically less invasiveness in terms of dural reconstruction which is necessary for Simpson grade 1 and higher possibility of complete resection of tumors compared with Simpson grade 2 resection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: Compression of the spinal cord by thoracic ossification of the posterior longitudinal ligament (T-OPLL) often causes severe thoracic myelopathy. Although surgery is the most effective treatment for T-OPLL, problems associated with surgical intervention require resolution because surgical outcomes are not always favorable, and a small number of patients experience deterioration of their neurological status after surgery. The aim of the present study was to examine the surgery-related risk factors contributing to poor clinical outcomes for myelopathy caused by T-OPLL., Methods: Data were extracted from the records of 55 patients with thoracic myelopathy due to T-OPLL at institutions in the Fukuoka Spine Group. The mean follow-up period was 5.3 years. Surgical outcomes were assessed using the Japanese Orthopaedic Association (JOA) scale. To investigate the definitive factors associated with surgical outcomes, univariate and multivariate regression analyses were performed with several patient-related and surgery-related factors, including preoperative comorbidities, radiological findings, JOA score, surgical methods, surgical outcomes, and complications., Results: Neurological status improved in 33 patients (60.0%) and deteriorated in 10 patients (18.2%) after surgery. The use of instrumentation was significantly associated with an improved outcome. In the comparison of surgical approaches, posterior decompression and fusion resulted in a significantly higher neurological recovery rate than did anterior decompression via a posterior approach and fusion or decompression alone. It was also found that postoperative neurological status was significantly poorer when there were fewer instrumented spinal levels than decompression levels. CSF leakage was a predictable risk factor for deterioration following surgery., Conclusions: It is important to identify preventable risk factors for poor surgical outcomes for T-OPLL. The findings of the present study suggest that intraoperative CSF leakage and a lower number of instrumented spinal fusion levels than decompression levels were exacerbating factors for the neurological improvement in T-OPLL surgery.

Purpose: To describe the characteristic features of post-carbon-ion radiotherapy (CIRT) vertebral pathological fractures (VPFs) in upper cervical primary malignant spinal tumors (PMSTs) treated by occipito-cervical (OC) fusion., Methods: OC fusion was performed for three consecutive patients with post-CIRT VPFs. The clinical results and imaging findings, including bone single-photon emission computed tomography (SPECT)/CT were prospectively collected., Results: No surgery-related wound complication and surgical site infection were noted. One patient experienced re-fracture and displacement of dens with the loosening of occipital screws and was treated by posterior revision surgery. At the final follow-up, all patients were alive without evidence of disease, and the solid OC fusion was confirmed. Bone SPECT/CT clearly revealed the effect of CIRT on bone turnover in the irradiated field., Conclusion: The OC fusion with autologous bone grafts was a reliable option for the treatment of post-CIRT VPCs in the patients with upper cervical PMSTs. In addition, evaluation of the bone turnover at the irradiated field by bone SPECT/CT would help surgeons select an effective plan of care, such as fusion level and postoperative care.

Purpose: To evaluate the clinical outcome of combination of carbon-ion radiotherapy with separation surgery (CIRT-SS) in patients with primary spinal/paraspinal sarcoma (PSPS) and epidural spinal cord compression (ESCC)., Methods: CIRT-SS was performed in 11 consecutive patients. Patients treated in the primary and salvage settings were categorized into Group A (n = 8) and Group B (n  = 3), respectively. Clinical results and imaging findings were collected, with a particular focus on ESCC grade, treatment-associated adverse events (AEs), and the locoregional control (LRC) rate and overall survival (OS)., Results: The median follow-up period from the start of CIRT-SS was 25 months (7-57 months). ESCC was improved by SS in all cases. No patients exhibited radiation-induced myelopathy (RIM), but three developed Grade 3 vertebral compression fracture (VCF) during follow-up. Locoregional recurrences were observed in four patients [Group A: 1 (12.5%), Group B: 3 (100%)]. Over the entire follow-up period, three patients developed distant metastases and two patients died. The 2-year LRC rate and OS were 70% and 80%, respectively., Conclusion: CIRT-SS in the primary setting achieved acceptable LRC and OS without RIM in patients with PSPS and with ESCC. VCF was the most frequent AE associated with CIRT-SS.

Surgery in the prone position is associated with a variety of complications due to the positioning, including the widely recognized peripheral nerve compression injuries and brachial plexus neuropathy. Previous studies have reported that thin body habitus is a predisposing risk factor for the compressive peripheral nerve injuries due to the prone position surgery. However, prone-position-related brachial plexus injury in patients who are overweight due to hypertrophic muscles have never been reported. Here we report a case of a professional sumo wrestler with severe thoracic ossification of the posterior longitudinal ligament (OPLL). Thoracic OPLL was successfully treated by posterior spinal fusion and decompression surgery. Despite a preoperative simulation and intraoperative inspection of the patient's surgical positioning, he suffered from bilateral upper extremity paralysis immediately after the surgery. Postoperative axillary MRI image revealed a high-intensity area on both sides of his pectoral muscles and axillary fossa, which implied that the pectoral muscles between the ribs and chest pad were pushed out toward the axillary fossa, resulting in compressive brachial plexus injury. His upper extremity motor paralysis was fully recovered in 6 months, but he still has mild tingling sensation even after 12 months of his surgery. In conclusion, overweight patients with hypertrophic muscles pose a risk for brachial plexus entrapment injury by pectoral muscles during prone-position surgery, and therefore it would be more effective to use a wide chest pad to reduce the pressure on the pectoral muscles to prevent it from being pushed out toward the axillary fossa., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: To investigate the clinical outcome and prognostic factors of malignant spinal dumbbell tumors (m-SDTs)., Methods: We retrospectively reviewed the clinical outcome of 22 consecutive cases of m-SDTs and analyzed the prognostic factors associated with worse outcome., Results: Nineteen of the 22 cases were managed with surgery (86%), and gross total resection (GTR) was achieved in four cases (21%). The duration of overall survival (OS) ranged from 3 to 140 months, with a median survival time of 15.3 months. The 5 year OS rate was 55.6%. In multivariate analysis, histological subtype (high-grade malignant peripheral nerve sheath tumor) (hazard ratio [HR] 14.9, p = 0.0191), GTR (HR 0.07, p = 0.0343), and presence of local recurrences (HR 11.2, p = 0.0479) were significant and independent predictors of OS., Conclusions: On the basis of clinical data, we propose that GTR and prevention of local recurrence may improve the clinical outcome of m-SDTs., Competing Interests: Conflicts of Interest: The authors declare that there are no relevant conflict of interest.

Introduction: Several reports have demonstrated the surgical treatment strategy for patients with dialysis-associated spondylosis in the cervical spine (CDAS) with destructive spondyloarthropathy (DSA). However, studies focusing on the clinical outcome of patients with CDAS without DSA remain scarce. We aimed to review the treatment strategy of patients with CDAS but without DSA., Methods: The clinical data and surgical records of consecutive patients with CDAS without DSA (n = 9; D-group) and cervical spondylotic myelopathy (CSM) (n = 30; C-group) who underwent modified double-door laminoplasty(DDL) were reviewed retrospectively. We investigated four radiologic factors in the pre-and postoperative periods that have been reported to be the risk factors for worsening of clinical symptoms in various studies and examined statistical comparison between the D and C groups., Results: In the D group, the pre- versus postoperative C2-C7 sagittal angles were not significantly different, and only two patients (22%) had kyphosis postoperatively. There was a significant difference in the pre- and postoperative C2-C7 angles in the two groups (P = 0.031). Regarding the change in segmental alignment, the local open angle increased at the C4/C5 level in the D group. Also there was a significant difference in the local angles between the two groups at C4/5 and C5/6 (P = 0.00038, and 0.037), suggesting that postoperative segmental mobility at C4/5 and C5/6 was higher in the D group than in the C group., Conclusions: In the present study, DDL in patients with CDAS without DSA did not adversely affect the postoperative alignment and stability compared with CSM patients with CSM. However, patients in the D group may have a chance to develop DSA change at the C4/5 level in the future, and careful long-term follow-up is warranted., Competing Interests: Conflicts of Interest: The authors declare that there are no conflicts of interest.

The interaction of transplanted stem cells with local cellular and molecular cues in the host CNS microenvironment may affect the potential for repair by therapeutic cell populations. In this regard, spinal cord injury (SCI), Alzheimer's disease, and other neurological injuries and diseases all exhibit dramatic and dynamic changes to the host microenvironment over time. Previously, we reported that delayed transplantation of human CNS-derived neural stem cells (hCNS-SCns) at 9 or 30 d post-SCI (dpi) resulted in extensive donor cell migration, predominantly neuronal and oligodendrocytic donor cell differentiation, and functional locomotor improvements. Here, we report that acute transplantation of hCNS-SCns at 0 dpi resulted in localized astroglial differentiation of donor cells near the lesion epicenter and failure to produce functional improvement in an all-female immunodeficient mouse model. Critically, specific immunodepletion of neutrophils (polymorphonuclear leukocytes) blocked hCNS-SCns astroglial differentiation near the lesion epicenter and rescued the capacity of these cells to restore function. These data represent novel evidence that a host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population, and support targeting the inflammatory microenvironment in combination with cell transplantation after SCI. SIGNIFICANCE STATEMENT The interaction of transplanted cells with local cellular and molecular cues in the host microenvironment is a key variable that may shape the translation of neurotransplantation research to the clinical spinal cord injury (SCI) human population, and few studies have investigated these events. We show that the specific immunodepletion of polymorphonuclear leukocyte neutrophils using anti-Ly6G inhibits donor cell astrogliosis and rescues the capacity of a donor cell population to promote locomotor improvement after SCI. Critically, our data demonstrate novel evidence that a specific host immune cell population can block the potential for functional repair derived from a therapeutic donor cell population., (Copyright © 2017 the authors 0270-6474/17/379269-19$15.00/0.)

Spinal cord injury (SCI) is a devastating disorder for which the identification of exacerbating factors is urgently needed. We demonstrate that transient hyperglycemia during acute SCI is a detrimental factor that impairs functional improvement in mice and human patients after acute SCI. Under hyperglycemic conditions, both in vivo and in vitro, inflammation was enhanced through promotion of the nuclear translocation of the nuclear factor κB (NF-κB) transcription factor in microglial cells. During acute SCI, hyperglycemic mice exhibited progressive neural damage, with more severe motor deficits than those observed in normoglycemic mice. Consistent with the animal study findings, a Pearson χ(2) analysis of data for 528 patients with SCI indicated that hyperglycemia on admission (glucose concentration ≥126 mg/dl) was a significant risk predictor of poor functional outcome. Moreover, a multiple linear regression analysis showed hyperglycemia at admission to be a powerful independent risk factor for a poor motor outcome, even after excluding patients with diabetes mellitus with chronic hyperglycemia (regression coefficient, -1.37; 95% confidence interval, -2.65 to -0.10; P < 0.05). Manipulating blood glucose during acute SCI in hyperglycemic mice rescued the exacerbation of pathophysiology and improved motor functional outcomes. Our findings suggest that hyperglycemia during acute SCI may be a useful prognostic factor with a negative impact on motor function, highlighting the importance of achieving tight glycemic control after central nervous system injury., (Copyright © 2014, American Association for the Advancement of Science.)

The transplantation of neural stem/precursor cells (NSPCs) is a promising therapeutic strategy for many neurodegenerative disorders including spinal cord injury (SCI) because it provides for neural replacement or trophic support. This strategy is now being extended to the treatment of chronic SCI patients. However, understanding of biological properties of chronically transplanted NSPCs and their surrounding environments is limited. Here, we performed temporal analysis of injured spinal cords and demonstrated their multiphasic cellular and molecular responses. In particular, chronically injured spinal cords were growth factor-enriched environments, whereas acutely injured spinal cords were enriched by neurotrophic and inflammatory factors. To determine how these environmental differences affect engrafted cells, NSPCs transplanted into acutely, subacutely, and chronically injured spinal cords were selectively isolated by flow cytometry, and their whole transcriptomes were compared by RNA sequencing. This analysis revealed that NSPCs produced many regenerative/neurotrophic molecules irrespective of transplantation timing, and these activities were prominent in chronically transplanted NSPCs. Furthermore, chronically injured spinal cords permitted engrafted NSPCs to differentiate into neurons/oligodendrocytes and provided more neurogenic environment for NSPCs than other environments. Despite these results demonstrate that transplanted NSPCs have adequate capacity in generating neurons/oligodendrocytes and producing therapeutic molecules in chronic SCI microenvironments, they did not improve locomotor function. Our results indicate that failure in chronic transplantation is not due to the lack of therapeutic activities of engrafted NSPCs but the refractory state of chronically injured spinal cords. Environmental modulation, rather modification of transplanting cells, will be significant for successful translation of stem cell-based therapies into chronic SCI patients., (Copyright © 2013 AlphaMed Press.)

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C(+) Ly6G(-) immature monocyte fraction exhibited the same characteristics as myeloid-derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C(+) Ly6G(-) fraction prior to injury by anti-Gr-1 antibody (clone: RB6-8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo-generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C(+) Ly6G(-) fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC-based strategy that can be applied to acute inflammatory diseases., (© 2012 International Society for Neurochemistry.)

Study Design: An in vivo animal study to examine the influence of pre-existing or concurrent spinal canal stenosis (SCS) on the functional recovery after spinal cord injury (SCI)., Objectives: To clarify whether spinal cord compression before or after SCI results in less favorable neurological recovery., Summary of Background Data: The influence of spinal cord compression on the neurological recovery after SCI remains unclear., Methods: We created mice with SCS using an extradural spacer before or after producing SCI and statistically analyzed the correlation between the extent of SCS and neurological outcomes. The extent of SCS was calculated by micro-computed tomography, and the spinal cord blood flow (SCBF) was measured serially with laser Doppler flowmetry. Molecular and immunohistochemical examinations were performed to evaluate the neovascularization at the site of cord compression., Results: Spacer placement (<300 μm) alone in the control mouse resulted in no neurological deficits. Even with spacer placement that caused asymptomatic SCS, the functional recovery after SCI was progressively impaired as spacer sizes increased in the mice with SCS co-occurring with SCI, whereas no significant impact was observed in the mice with pre-existing SCS, irrespective of the spacer sizes. The SCBF progressively decreased immediately after SCS was produced, but it fully recovered at the later time points. Angiogenesis-related genes were upregulated, and neovascular vessels were observed after producing the SCS. We found that concurrent SCS resulted in a significant reduction and impaired the subsequent recovery of the SCBF, whereas pre-existing SCS did not affect the hemodynamics of the spinal cord after SCI., Conclusion: The dynamic reduction of the SCBF occurring immediately after spinal cord compression is a significant factor that impairs the neurological recovery after SCI, whereas pre-existing SCS is not always an impediment due to the potentially restructured SCBF.

Study Design: An animal study using myeloperoxidase-knockout (MPO-KO) mice to examine the in vivo role of myeloperoxidase (MPO) in spinal cord injury (SCI)., Objective: To clarify the influence of MPO on inflammatory cell infiltration, tissue damage, and functional recovery after SCI., Summary of Background Data: MPO is considered to be important in spreading tissue damage after SCI because it generates strong neurotoxic oxidant hypochlorous acid (HOCl). However, the direct involvement of MPO in the pathophysiology of SCI remains to be elucidated., Methods: To compare the inflammatory reaction, tissue damage, and neurological recovery after SCI, a moderate contusion injury was created at the ninth thoracic level in MPO-KO mice and wild-type mice. A HOCl-specific probe solution was injected into the lesion epicenter to assess the spatiotemporal production of MPO-derived HOCl. Inflammatory reactions were quantified by flow cytometry and quantitative real-time polymerase chain reaction, and tissue damage was evaluated by an immunohistochemical analysis. The motor function recovery was assessed by the open-field locomotor score., Results: Prominent production of HOCl was observed during the hyperacute phase of SCI at the lesion site in the wild-type mice; however, little expression was observed in the MPO-KO mice. In this phase, the number of infiltrated neutrophils was significantly reduced in the MPO-KO mice compared with the wild-type mice. In addition, significant differences were observed in the expression levels of proinflammatory cytokines and apoptosis-related genes between 2 groups. In the histological sections, fewer terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells and more spared myelin were observed at the lesion site in MPO-KO mice. Consistent with these results, better functional recovery was observed in the MPO-KO mice than in the wild-type mice after SCI., Conclusion: These results clearly indicated that MPO exacerbated secondary injury and impaired the functional recovery not only by generating strong oxidant HOCl, but also by enhancing neutrophil infiltration after SCI.

Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation.

Previous experimental and clinical studies have suggested that the behavioral and pathological outcomes of spinal cord injury (SCI) are affected by the individual's age at the time of injury. However, the underlying mechanism responsible for these differences remains elusive because it is difficult to match injuries of similar severities between young and adult animals due to differences in the sizes of their respective spinal cords. In this study, the spinal cord size-matched young (4-week-old) and adult (10-week-old) mice were compared to evaluate their locomotor functions and inflammatory cellular/molecular responses after standardized contusion SCI. During the acute phase of SCI, young mice showed better functional recovery and lower pro-inflammatory cytokines/chemokines compared to adult mice. Flow-cytometric analysis revealed that the time courses of leukocyte infiltration were comparable between both groups, while the number of infiltrating neutrophils significantly decreased from 6 h after SCI in young mice. By combining flow-cytometric isolation and gene expression analysis of each inflammatory cell fraction, we found that microglial cells immediately initiate the production of several cytokines in response to SCI, which serve as major sources of IL-6, TNFa, and CXCL1 in injured spinal cord. Interestingly, the secretion of pro-inflammatory cytokines/chemokines but not anti-inflammatory cytokines by microglia was significantly lower in young mice compared to that in adult mice at 3 h after SCI, which will be attributed to the attenuation of the subsequent neutrophil infiltration. These results highlight age-related differences in pro-inflammatory properties of microglial cells that contribute to the amplification of detrimental inflammatory responses after SCI., (Copyright © 2011 Wiley Periodicals, Inc.)

Neural stem/progenitor cell (NSPC) transplantation is a promising treatment for various neurodegenerative disorders including spinal cord injury, however, no direct analysis has ever been performed on their in vivo profile after transplantation. Here we combined bioimaging, flow-cytometric isolation and ultra-high-throughput RNA sequencing to evaluate the cellular properties of engrafted NSPCs. The acutely transplanted NSPCs had beneficial effects on spinal cord injury, particularly neuroprotection and neurohumoral secretion, whereas their in situ secretory activity differed significantly from that predicted in vitro. The RNA-sequencing of engrafted NSPCs revealed dynamic expression/splicing changes in various genes involved in cellular functions and tumour development depending on graft environments. Notably, in the pathological environment, overall transcriptional activity, external signal transduction and neural differentiation of engrafted NSPCs were significantly suppressed. These results highlight the vulnerability of engrafted NSPCs to environmental force, while emphasizing the importance of in situ analysis in advancing the efficacy and safety of stem cell-based therapies.

Due to the complex cellular heterogeneity of the central nervous system (CNS), it is relatively difficult to reliably obtain molecular descriptions with cell-type specificity. In particular, comparative analysis of epigenetic regulation or molecular profiles is hampered by the lack of adequate methodology for selective purification of defined cell populations from CNS tissue. Here, we developed a direct purification strategy of neural nuclei from CNS tissue based on fluorescence-activated cell sorting (FACS). We successfully fractionated nuclei from complex tissues such as brain, spinal cord, liver, kidney, and skeletal muscle extruded mechanically or chemically, and fractionated nuclei were structurally maintained and contained nucleoproteins and nuclear DNA/RNA. We collected sufficient numbers of nuclei from neurons and oligodendrocytes using FACS with immunolabeling for nucleoproteins or from genetically labeled transgenic mice. In addition, the use of Fab fragments isolated from papain antibody digests, which effectively enriched the specialized cell populations, significantly enhanced the immunolabeling efficacy. This methodology can be applied to a wide variety of heterogeneous tissues and is crucial for understanding the cell-specific information about chromatin dynamics, nucleoproteins, protein-DNA/RNA interactions, and transcriptomes retained in the nucleus, such as non-coding RNAs.

Background: Reports on ossification of the posterior longitudinal ligament (OPLL) of the lumbar spine have so far been limited., Objective: To evaluate surgically documented cases of lumbar OPLL at our facility to clarify its characteristics and analyze clinical outcomes., Methods: During the past 27 years, 6192 patients underwent operations for degenerative lumbar spine diseases. Of these, we selected surgically treated lumbar OPLL patients from our database to analyze the clinical and radiological disease characteristics. Surgical results were classified according to the Japanese Orthopaedic Association scale., Results: Only 10 patients underwent surgery for lumbar OPLL: 6 men and 4 women (mean age 44.1 years). Among these, OPLL developed in 4 patients at multiple vertebral body levels and in 6 at a single level. Coexisting lumbar disc herniation was observed in 4 patients. Although the rate of maximum canal stenosis brought about by OPLL was relatively high (mean 45.1%), unilateral radicular symptoms were the most frequently observed, and only 2 patients exhibited typical lumbar claudication caused by the canal stenosis. Two patients underwent surgery via an anterior approach and 8 via a posterior approach. The mean preoperative Japanese Orthopaedic Association scale score was 7.9, which improved to 17.8 postoperatively. No neurological deterioration caused by surgery was observed in any case., Conclusion: Although the frequency of lumbar OPLL requiring surgical treatment was remarkably low, its clinical condition varies greatly among patients depending on the localization and degree of ossification. To achieve a better surgical outcome, precise diagnosis with computed tomography and an appropriate surgical approach are important.

Human heat shock cognate protein 70 (Hsc70), also known as Hsp73 and Hsp70-8, is a molecular chaperone. The human Hsp70 family comprises at least eight different molecular groups with strong homology. Among them, Hsc70 and Hsp72 share 86% homology. Both Hsp72 and Hsc70 localize in the cell cytoplasm and the nucleus. While Hsp72 expression is enhanced by stress, Hsc70 is constitutively expressed, suggesting that Hsc70 is critically involved in cell functions other than the stress response. Hsc70 has cell-specific and tissue-specific functions, such as cellular signaling, but its functions are not well understood. To further study the functions of Hsc70, we established a monoclonal antibody specific for Hsc70 using a rat medial iliac lymph node method. Immunoblot analysis with this antibody revealed that it specifically recognizes Hsc70. Immunocytochemical staining using this newly established antibody revealed that Hsc70 localizes predominantly in the cytoplasm in unstressed cells, whereas oxidative stress produced by H2O2 induces Hsc70 to translocate into the nucleus. This monoclonal antibody will be useful for further studies of Hsc70, including changes in its intracellular location, binding molecules, and functions.

Traumatic injury in the central nervous system induces inflammation; however, the role of this inflammation is controversial. Precise analysis of the inflammatory cells is important to gain a better understanding of the inflammatory machinery in response to neural injury. Here, we demonstrated that leukotriene B4 plays a significant role in mediating leukocyte infiltration after spinal cord injury. Using flow cytometry, we revealed that neutrophil and monocyte/macrophage infiltration peaked 12 hours after injury and was significantly suppressed in leukotriene B4 receptor 1 knockout mice. Similar findings were observed in mice treated with a leukotriene B4 receptor antagonist. Further, by isolating each inflammatory cell subset with a cell sorter, and performing quantitative reverse transcription-PCR, we demonstrated the individual contributions of more highly expressed subsets, ie, interleukins 6 and 1beta, tumor necrosis factor-alpha, and FasL, to the inflammatory reaction and neural apoptosis. Inhibition of leukotriene B4 suppressed leukocyte infiltration after injury, thereby attenuating the inflammatory reaction, sparing the white matter, and reducing neural apoptosis, as well as inducing better functional recovery. These findings are the first to demonstrate that leukotriene B4 is involved in the pathogenesis of spinal cord injury through the amplification of leukocyte infiltration, and provide a potential therapeutic strategy for traumatic spinal cord injury.

Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function.

Brm is a subunit of the SWI/SNF complex that has a ATPase activity. It is well known that the complex plays a major role in cell processes, such as proliferation, differentiation, and DNA repair of cells. Here we report the production of monoclonal antibody (1H7A10) against Brm by rat medial iliac lymph node method. Immunoblot analysis with the antibody revealed the specific recognition of Brm and increase of Brm protein level in skeletal muscle differentiation. Immunocytochemistry analysis shows nuclear localization in myoblast C2C12 and involvement of transcription in the late stages of differentiation.

Tyrosine kinase 2 (Tyk2), a member of the JAK family, is involved in IL-12- and IL-23-mediated signaling. In the present study, we examined the roles of Tyk2 in the development of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by using Tyk2 knockout (KO) mice. In vitro differentiation of Th1 but not Th17 cells was severely impaired in Tyk2 KO CD4 T cells, although Tyk2 KO Th17 cells did not respond to IL-23. Tyk2 KO mice showed complete resistance against EAE with no infiltration of CD4 T cells in the spinal cord. Surprisingly, the number of MOG-specific Th17 cells in the periphery was comparable between KO and wild-type (WT) mice, whereas Th1 cells were greatly reduced in Tyk2 KO mice. Adoptive transfer of MOG-primed WT T cells induced EAE in Tyk2 KO recipients, indicating that Tyk2 in the environment was dispensable for the infiltration of effector T cells into the spinal cord. A reduced but significant number of Tyk2 KO T cells were detected in the spinal cord of mice with EAE, which had been reconstituted with bone marrow cells of WT and KO mice. Furthermore, MOG-immunized Tyk2 KO mice developed EAE after adoptive transfer of MOG-primed WT Th1 cells, which might trigger local inflammation that recruits Th17 cells. Taken together, these results indicate that Tyk2 is critically involved in the pathogenic CD4 T cell responses and thus could be a target molecule for the treatment of autoimmune diseases.

Pax7 is a nuclear localization protein, well known as a member of the paired box family. It is expressed at a very early stage of muscle differentiation and is also found in muscle satellite cells that are recognized as muscle stem cells. Pax7 is also recognized as a tumor cell marker since it is greatly expressed in various types of tumor cells. Pax7 has homology among other paired family members and is not easy to distinguish one from the others. In this study, we report on the establishment of monoclonal antibodies (MAb) against Pax7 using a rat medial iliac lymph node method. The quality of the antibody was examined by immunoblotting analysis. It was confirmed that the antibody can specifically recognize the Pax7 protein. It was also revealed that the MAb antibody successfully recognizes the nuclear localized Pax7 protein in Ewing's sarcoma cells by immunocytochemistry. The antibody can clearly show the regions of euchromatin and heterochromatin where hoechst is positive.

Study Design: Retrospective outcome measurement study., Objectives: The purpose of this study is to assess whether ossification of the posterior longitudinal ligament (OPLL) affects neurologic outcomes in patients with acute cervical spinal cord injury (SCI)., Summary of Background Data: There have so far been few reports examining the relationship between OPLL and SCI and there is controversy regarding the deteriorating effects of OPLL-induced canal stenosis on neurologic outcomes., Methods: To obtain a relatively uniform background, patients nonsurgically treated for an acute C3-C4 level SCI without any fractures or dislocations of the spinal column were selected, resulting in 129 patients. There were 110 men and 19 women (mean age was 61.1 years), having various neurologic conditions on admission (American Spinal Injury Association [ASIA] impairment scale A, 43; B, 16; C, 58; D, 12). The follow-up period was the duration of their hospital stay and ranged from 50 to 603 days (mean, 233 days). The presence of OPLL, the cause of injury, the degree of canal stenosis (both static and dynamic), and the neurologic outcomes in motor function, including improvement rate, were assessed., Results: Of the 129 patients investigated in this study, OPLL was identified at the site of the injury in 13 patients (10.1%). In this OPLL+ group, the static and dynamic canal diameters at C3 and C4 were significantly smaller than those of the remaining 116 patients (OPLL- group). However, no significant difference was observed between the 2 groups in terms of ASIA motor score both at the time of administration and discharge, and the mean improvement rate in ASIA motor score was 55.5 +/- 9.0% in OPLL+ group, while it was 43.1 +/- 2.8% in the OPLL-group. Furthermore, no significant correlation was observed between the static/dynamic canal diameters and neurologic outcome in all 129 patients., Conclusion: No evidence was found for OPLL to have any effect on the initial neurologic status or recovery in motor function after traumatic cervical cord injury, suggesting that the neurologic outcome is not significantly dependent on canal space.