Toshio Nakamae

MicroRNA-223 expression in neutrophils in the early phase of secondary damage after spinal cord injury

MicroRNA (miR)s are short non-coding RNAs that suppress the translation of target genes, and play an important role in gene regulation. Despite this prominence, there are few reports that refer to the expression of miRs after spinal cord injury (SCI). Previously, we reported on miR-223 expression after SCI in mice. The purpose of this study is to reveal the distribution of miR-223 and identify the cells that express miR-223 in the injured spinal cord. Quantitative polymerase chain reaction analysis revealed high expression of miR-223 at 12h after SCI. Double staining of in situ hybridization and immunohistochemistry showed that the signals of miR-223 merged with Gr-1 positive neutrophils. Our data indicate that miR-223 might regulate neutrophils in the early phase after SCI.

Expansive laminoplasty for cervical myelopathy with interconnected porous calcium hydroxyapatite ceramic spacers: comparison with autogenous bone spacers.

Study Design Expansive cervical laminoplasties with interconnected porous calcium hydroxyapatite ceramic (IP-CHA) spacers were performed in cervical myelopathy patients. Objectives To evaluate the usefulness and osteoconductive capability of IP-CHA spacers in expansive laminoplasty. Summary of Background Data Expansive laminoplasty for cervical myelopathy is designed to preserve the posterior structures, so as to prevent postoperative development of instability and cervical kyphosis. The technique requires successful reconstruction of the laminae of vertebral arches, as sinking or nonunion of the expanded laminae may induce neurologic regression, segmental motor paralysis, and postoperative axial pain. A novel IP-CHA with sufficient biocompatibility and mechanical strength was developed as an artificial bone substitute. Methods Expansive open-door laminoplasties were performed in 88 cervical myelopathy patients, and both autogenous bone spacers harvested from the spinous processes and IP-CHA spacers in combination with bone marrow were alternately grafted into the opened side of each lamina. All patients were followed up with computerized tomography scans, and bonding rates for both the IP-CHA and autogenous spacers, bone fusion rates of the hinges of the laminae, and complications associated with the implants were examined. Results Clinical symptoms significantly improved in all patients without major complications related to the procedure. The IP-CHA spacers demonstrated comparable bone bonding to the autogenous spacers on postoperative computerized tomography scans. The expanded laminae withstood expanded positions without sinking or floating throughout the followups, and the hinges completely fused in more than 95% of patients in both groups within 1 year. Conclusions The IP-CHA spacer contributed to high bone fusion rates of the spacers and hinges of the laminae, and there were no complications associated with their use. Cervical laminoplasty with the IP-CHA spacers is a safe and simple method that yields sufficient fixation strength and provides sufficient bone bonding within a short period of time after operation.

Chondroitinase ABC promotes corticospinal axon growth in organotypic cocultures

Study design:Organotypic coculture model using brain cortex and spinal cord of neonatal rats was used to test the effect of chondroitinase ABC (ChABC) on corticospinal axon growth.Objective:Chondroitin sulfate proteoglycan (CSPG) is neurite outgrowth inhibitory factor that combines with reactive astrocyte at the lesion site to form a dense scar that acts as a barrier to regenerating axons. ChABC is a bacteria enzyme that digests the glycosaminoglycan side chain of CSPG. We investigated the effect of ChABC on corticospinal axon growth quantitatively using the organotypic cocultures of brain cortex and spinal cord.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University.Method:We used organotypic cocultures with neonatal brain cortex and spinal cord as an in vitro assay system for assessing axon growth. After administering ChABC, we counted the number of axons passing through a reference line running parallel to the junction between the brain cortex and spinal cord 500 and 1000 μm from the junction. The immunoreactivity of CSPG was assessed.Result:The average number of axons after ChABC administration was significantly greater than in the control group. Administration of ChABC decreased CSPG expression in this coculture system.Conclusion:ChABC induces axonal regeneration by degrading CSPG after central nerve system injury. ChABC has great potential for future therapeutic use in spinal cord-injured patients.

Pharmacological inhibition of tumor necrosis factor may reduce pain behavior changes induced by experimental disc puncture in the rat: an experimental study in rats.

Study Design. Pain behavior assessment in rats following disc puncture (DP) and simultaneous tumor necrosis factor (TNF) inhibition. Objective. To assess if treatment with TNF inhibition could reduce the pain behavior changes induced by DP in the rat. Summary of Background Data. Anular tears with leakage of nucleus pulposus have been suggested to be one possible cause of low back pain (LBP). In an experimental model, it was recently shown that DP might induce specific pain behavior changes. The aim of the present study was to a study if inhibition of TNF might reduce such pain behavior changes. Methods. Sixty rats underwent facetectomy and puncture of the fourth lumbar disc. The rats were simultaneously treated with doxycycline locally at 0.3 and 3.0 mg/kg and systemically at 3.0 mg/kg, or infliximab locally at 0.5 and 5.0 mg/kg, and systemically at 5.0 mg/kg, (n ∇ 10 for each subseries). The rats were videotaped at 1, 3, 7, 14, and 21 days after surgery. The videos were analyzed regarding presence of wet-dog shakes (WDS). Data from a previous study with sham surgery and DP without treatment were included for comparison. Results. All groups treated with doxycycline resulted in a statistically significant reduction of WDS compared to the group without treatment (DP). In infliximab treated animals, WDS decreased with statistically significance compared to the nontreated DP group at all analyzed days except for the group with high dose local treatment where a statistically significant reduction was obtained only at days 14 and 21. Conclusion. The present study showed that TNF inhibition induced a marked reduction of wet dog shakes. It is not fully understood if wet-dog shakes may relate to LBP, but in view of recent clinical findings one may consider clinical studies of TNF inhibition for the treatment of LBP.

Medium-term clinical results of microsurgical lumbar flavectomy that preserves facet joints in cases of lumbar degenerative spondylolisthesis: comparison of bilateral laminotomy with bilateral decompression by a unilateral approach.

Study Design: A retrospective study of medium-term results. Objective: To describe a technique for posterior decompression using microsurgical lumbar flavectomy (MLF) without facetectomy, which is based on the anatomic features of the ligamentum flavum, and to examine the clinical outcomes of patients with lumbar spinal spondylolisthesis with lower extremity symptoms rather than low back pain, who underwent this procedure by 2 different approaches. Summary of Background Data: Posterior decompression with fusion has been the optimal and standard operative treatment for lumbar degenerative spondylolisthesis. Alternatively, minimally invasive procedures have been used for the treatment of lumbar degenerative spondylolisthesis with favorable outcomes. Methods: A bilateral laminotomy (BL group) was performed on 44 consecutive patients, and bilateral decompression by a unilateral approach (BDU group) was performed on 23 consecutive patients. The mean follow-up period was 7.0 years. The Japanese Orthopaedic Association score and recovery rate were obtained, and radiographic assessment was performed using plain radiograms on the lateral view while standing in flexion, neutral, and extension postures before surgery and at the final follow-up. Results: The Japanese Orthopaedic Association score at the final follow-up was improved in the BL and BDU groups, compared with that before MLF. The mean recovery rate was 72.4% and 68.4%, respectively. The mean % slip increased at the final follow-up, compared with that before surgery in both groups, except for the % slip in the extension posture in the BDU group. However, there was no significant difference in the dynamic % slip in the flexion-extension posture between before surgery and at the final follow-up. Conclusions: Clinical and radiologic parameters were not significantly different between the 2 groups. This technique of MLF using either approach did not increase the dynamic % slip and showed favorable medium-term clinical results in cases of lumbar degenerative spondylolisthesis.

The effect of an external magnetic force on cell adhesion and proliferation of magnetically labeled mesenchymal stem cells

BackgroundAs the strategy for tissue regeneration using mesenchymal stem cells (MSCs) for transplantation, it is necessary that MSCs be accumulated and kept in the target area. To accumulate MSCs effectively, we developed a novel technique for a magnetic targeting system with magnetically labeled MSCs and an external magnetic force. In this study, we examined the effect of an external magnetic force on magnetically labeled MSCs in terms of cell adhesion and proliferation.MethodsMagnetically labeled MSCs were plated at the bottom of an insert under the influence of an external magnetic force for 1 hour. Then the inserts were turned upside down for between 1 and 24 hours, and the number of MSCs which had fallen from the membrane was counted. The gene expression of MSCs affected magnetic force was analyzed with microarray. In the control group, the same procedure was done without the external magnetic force.ResultsAt 1 hour after the inserts were turned upside down, the average number of fallen MSCs in the magnetic group was significantly smaller than that in the control group, indicating enhanced cell adhesion. At 24 hours, the average number of fallen MSCs in the magnetic group was also significantly smaller than that in control group. In the magnetic group, integrin alpha2, alpha6, beta3 BP, intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1) were upregulated. At 1, 2 and 3 weeks after incubation, there was no statistical significant difference in the numbers of MSCs in the magnetic group and control group.ConclusionsThe results indicate that an external magnetic force for 1 hour enhances cell adhesion of MSCs. Moreover, there is no difference in cell proliferation after using an external magnetic force on magnetically labeled MSCs.

Investigation of segmental motor paralysis after cervical laminoplasty using intraoperative spinal cord monitoring with transcranial electric motor-evoked potentials.

Study Design A retrospective study of segmental motor paralysis after cervical laminoplasty for cervical myelopathy. Objective The objective of this study was to use transcranial electric motor-evoked potentials during cervical laminoplasty, to monitor and investigate the intraoperative electrophysiologic change in patients with cervical myelopathy, who subsequently develop postoperative segmental motor paralysis potentials. Summary of Background Data There have been several reports about segmental motor paralysis after cervical laminoplasty for patients with cervical myelopathy. However, the pathogenesis of segmental motor paralysis of C5 myotome, so-called C5 palsy, remains unclear. Methods The cases of 184 consecutive patients who underwent cervical laminoplasty were analyzed to clarify the incidence of postoperative segmental motor paralysis and to monitor intraoperative change. Postoperative C5 palsy was defined as development of postoperative motor palsy of the deltoid and biceps muscles in the upper extremity by at least 1 grade in the manual muscle test without sensory disturbance or impairment of the lower extremities. All patients underwent cervical laminoplasty under intraoperative spinal cord monitoring with transcranial electric motor-evoked potentials. The evoked potentials were recorded over the deltoid, biceps, and triceps muscles in the bilateral upper extremities. Results Postoperative C5 palsy developed in 6 patients (3.3%, 5 males and 1 female) but there were no abnormal changes monitored. The incidence of C5 palsy involved 4 of 124 (3.2%) cervical spondylotic myelopathy patients, 2 of 31 (6.5%) patients with cervical ossification of the posterior longitudinal ligament. No patients with cervical disc herniation or cervical spondylotic amyotrophy developed C5 palsy. Conclusions There were no abnormal findings on transcranial electric motor-evoked potential monitoring even in those patients who developed postoperative C5 palsy. These results indicate that the development of postoperative segmental motor paralysis after cervical laminoplasty occurs even if there are no abnormal findings during intraoperative monitoring.

Quantitative assessment of myelopathy patients using motor evoked potentials produced by transcranial magnetic stimulation

Motor evoked potentials (MEPs) study using transcranial magnetic stimulation (TMS) may give a functional assessment of corticospinal conduction. But there are no large studies on MEPs using TMS in myelopathy patients. The purpose of this study is to confirm the usefulness of MEPs for the assessment of the myelopathy and to investigate the use of MEPs using TMS as a screening tool for myelopathy. We measured the MEPs of 831 patients with symptoms and signs suggestive of myelopathy using TMS. The MEPs from the abductor digiti minimi (ADM) and abductor hallucis (AH) muscles were evoked by transcranial magnetic brain stimulation. Central motor conduction time (CMCT) is calculated by subtracting the peripheral conduction time from the MEP latency. Later, 349 patients had surgery for myelopathy (operative group) and 482 patients were treated conservatively (nonoperative group). CMCTs in the operative group and nonoperative group were assessed. MEPs were prolonged in 711 patients (86%) and CMCTs were prolonged in 493 patients (59%) compared with the control patients. CMCTs from the ADM and AH in the operative group were significantly more prolonged than that in the nonoperative group. All patients in the operative group showed prolongation of MEPs or CMCTs or multiphase of the MEP wave. MEP abnormalities are useful for an electrophysiological evaluation of myelopathy patients. Moreover, MEPs may be effective parameters in spinal pathology for deciding the operative treatment.

CD133(+) cells from human umbilical cord blood reduce cortical damage and promote axonal growth in neonatal rat organ co-cultures exposed to hypoxia

To evaluate the effect of CD133+ cells (endothelial progenitor cells) on the hypoxia‐induced suppression of axonal growth of cortical neurons and the destruction of blood vessels (endothelial cells), we used anterograde axonal tracing and immunofluorescence in organ co‐cultures of the cortex and the spinal cord from 3‐day‐old neonatal rats. CD133+ cells prepared from human umbilical cord blood were added to the organ co‐cultures after hypoxic insult, and axonal growth, vascular damage and apoptosis were evaluated. Anterograde axonal tracing with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate was used to analyze axonal projections from the cortex to the spinal cord. Immunolabeling co‐cultured tissues of the cortex and the spinal cord were used to investigate the effect of CD133+ cells on the survival of blood vessels and apoptosis in the brain cortex. Hypoxia remarkably suppressed axonal growth in organ co‐cultures of the cortex and the spinal cord, and this suppression was significantly restored by the addition of CD133+ cells. CD133+ cells also reduced the hypoxia‐induced destruction of the cortical blood vessels and apoptosis. CD133+ cells had protective effects on hypoxia‐induced injury of neurons and blood vessels of the brain cortex in vitro. These results suggest that CD133+ cell transplantation may be a possible therapeutic intervention for perinatal hypoxia‐induced brain injury.

CD133+ cells from human peripheral blood promote corticospinal axon regeneration.

To assess the effect of endothelial progenitor cells on the axon growth of cortex neurons, we transplanted CD133+ cells derived from human peripheral blood to an organotypic coculture system consisting of spinal cord and cortex from neonatal rats. The axon growth from cortex to spinal cord was significantly promoted in cultures after CD133+ cells transplantation compared with that of the control cultures. In addition, real-time reverse transcription-PCR showed a significant upregulation of vascular endothelial growth factor mRNA in the spinal cord of the cultures containing CD133+ cells. In contrast, the transplanted cells did not differentiate into endothelial cells. These data suggest that CD133+ cells may promote axonal regeneration by upregulating vascular endothelial growth factor mRNA in spinal cord tissues.