Kazuyoshi Nakanishi

BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures.

Study Design. Experimental study of spinal cord injury using an organotypic slice culture. Objective. To clarify the mechanism of corticospinal axon regeneration following transplantation of neural progenitor cells (NPCs) in the injured spinal cord. Summary of Background Data. Several mechanisms underlying central nervous system regeneration after transplantation of NPCs have been proposed; however, the precise mechanism has not been clarified. Previously, we demonstrated that transplanted NPCs secreted humoral factors that in turn promoted corticospinal axon growth using the unique organotypic coculture system involving brain cortex and spinal cord from neonatal rats. Methods. Cultured NPCs were immunostained with antibodies against neurotrophic factors including brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, nerve growth factor (NGF), and ciliary neurotrophic factor (CNTF) both before and after differentiation. To evaluate corticospinal axon growth quantitatively, we used the organotypic coculture system. The dissected brain cortex and spinal cord obtained from neonatal rats were aligned next to each other and cultured on a membrane. NPCs were transplanted onto the cocultures. Furthermore, neutralizing antibodies against BDNF, NT-3, NGF, or CNTF were added to the cocultures. Axon growth from the brain cortex into the spinal cord was assessed quantitatively using anterograde axon tracing with DiI. Results. The cultured NPCs were positively immunostained by antibodies against BDNF, NT3, NGF, and CTNF both before and after differentiation. Transplantation of NPCs promoted axon growth from the brain cortex into the spinal cord. The axon growth promoted by NPCs was significantly suppressed by the addition of neutralizing antibodies against BDNF, NT-3, and NGF but not CNTF. Conclusion. The neurotrophic factors, BDNF, NT-3, and NGF, secreted by transplanted NPCs, were involved in the promotion of corticospinal axon growth after transplantation of NPCs.

Responses of microRNAs 124a and 223 following spinal cord injury in mice.

Study design:We investigated microRNA (miRNA) expression after spinal cord injury (SCI) in mice.Objectives:The recent discovery of miRNAs suggests a novel regulatory control over gene expression during plant and animal development. MiRNAs are short noncoding RNAs that suppress the translation of target genes by binding to their mRNAs, and play a central role in gene regulation in health and disease. The purpose of this study was to examine miRNA expression after SCI.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University.Methods:We examined the expression of miRNA (miR)-223 and miR-124a in a mouse model at 6 h, 12 h, 1 day, 3 days and 7 days after SCI using quantitative PCR. The miRNA expression was confirmed by in situ hybridization.Results:Quantitative PCR revealed two peaks of miR-223 expression at 6 and 12 h and 3 days after SCI. MiR-124a expression decreased significantly from 1 day to 7 days after SCI. In situ hybridization demonstrated the presence of miR-223 around the injured site. However, miR-124a, which was present in the normal spinal cord, was not observed at the injured site.Conclusion:Our results indicate a time-dependent expression pattern of miR-223 and miR-124a in a mouse model of SCI. In this study, the time course of miRNA-223 expression may be related to inflammatory responses after SCI, and the time course of decreased miR-124a expression may reflect cell death.

Postoperative segmental C5 palsy after cervical laminoplasty may occur without intraoperative nerve injury: a prospective study with transcranial electric motor-evoked potentials.

Study Design. Intraoperative neurophysiologic monitoring with transcranial electric motor-evoked potentials was performed on patients who underwent cervical laminoplasty at a university hospital in a prospective study. Objective. To evaluate the usefulness of intraoperative spinal cord monitoring with transcranial electric motor-evoked potentials for prediction of the occurrence of segmental motor paralysis after cervical laminoplasty. Summary of Background Data. Segmental motor paralysis occasionally occurs among patients who undergo expansive laminoplasty for cervical myelopathy, and it has been attributed to nerve root lesions caused by either a traumatic surgical technique or a tethering effect after decompression. Methods. Sixty-two consecutive patients (47 men and 15 women; mean age 64 years [range 32–89]) who were scheduled to undergo cervical laminoplasty under intraoperative spinal cord monitoring with transcranial electric motor-evoked potentials were included in this study. Transcranial electrical stimulations were delivered through pin-type electrodes, and the evoked potentials were recorded over the deltoid, biceps, and triceps muscles in the bilateral upper extremities and thoracic spinal cord. Results. Intraoperative evoked potentials were successfully recorded in all muscles in 57 patients (92%), and incomplete evoked potentials were recorded in the remaining 5 patients. No critical decrease in the amplitude of the evoked potentials was observed in any of the 62 patients. All patients showed sufficient postoperative recovery from their clinical symptoms; however, postoperative transient C5 palsy occurred in 3 patients. Conclusions. No abnormalities were observed on transcranial electric motor-evoked potential monitoring, even in those patients who developed postoperative transient C5 palsy. These results suggest that the development of postoperative C5 palsy after cervical laminoplasty is not associated with intraoperative injury of the nerve root or the spinal cord, although the precise mechanism of its development is still unclear. Surgeons should be aware that C5 palsy is a possible complication of cervical laminoplasty, even in the absence of intraoperative nerve injury.

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.

Lnk Deletion Reinforces the Function of Bone Marrow Progenitors in Promoting Neovascularization and Astrogliosis Following Spinal Cord Injury

Lnk is an intracellular adaptor protein reported as a negative regulator of proliferation in c‐Kit positive, Sca‐1 positive, lineage marker‐negative (KSL) bone marrow cells. The KSL fraction in mouse bone marrow is believed to represent a population of hematopoietic and endothelial progenitor cells (EPCs). We report here that, in vitro, Lnk−/− KSL cells form more EPC colonies than Lnk+/+ KSL cells and show higher expression levels of endothelial marker genes, including CD105, CD144, Tie‐1, and Tie2, than their wild‐type counterparts. In vivo, the administration of Lnk+/+ KSL cells to a mouse spinal cord injury model promoted angiogenesis, astrogliosis, axon growth, and functional recovery following injury, with Lnk−/− KSL being significantly more effective in inducing and promoting these regenerative events. At day 3 following injury, large vessels could be observed in spinal cords treated with KSL cells, and reactive astrocytes were found to have migrated along these large vessels. We could further show that the enhancement of astrogliosis appears to be caused in conjunction with the acceleration of angiogenesis. These findings suggest that Lnk deletion reinforces the commitment of KSL cells to EPCs, promoting subsequent repair of injured spinal cord through the acceleration of angiogenesis and astrogliosis. STEM CELLS 2010;28:365–375

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.

Administration of human peripheral blood-derived CD133+ cells accelerates functional recovery in a rat spinal cord injury model

Study Design. Magnetically isolated, peripheral blood-derived CD133+ cells were used as the therapeutic agent of spinal cord injury (SCI). A rat model was used to investigate the hypothesis that the cell therapy using this clinically accessible cell fraction could be an attractive option for injured spinal cord. Objective. Given the capacity for the peripheral blood-derived CD133+ cells in vivo to produce neurogenesis via vasculogenesis as the feasible candidate for SCI in the clinical setting, the focus of the experiment was to investigate whether the cells could contribute to histologic and functional recovery of SCI after transplantation. Summary of Background Data. No evidence for peripheral blood-derived CD133+ cells application to SCI and no experimental studies showed functional recovery from SCI using this cell fraction have been published. Methods. Contusion SCI was induced by placing a 25-g rod onto the spinal cord for 90 seconds in athymic nude rats. CD133+ cells or phosphate-buffered saline was administered intravenously immediately after SCI. The animals were analyzed at specific times after transplantation by several methods to examine histologic vasculogenesis and neurogenesis and to confirm functional recovery from SCI. Results. After cell transplantation, intrinsic angiogenesis and axonal regeneration were enhanced, and cavity formation was reduced in injured spinal cord, histologically, with significant functional recovery. Gene expression of vascular endothelial growth factor increased in the cell-administrated group. Conclusion. The administration of CD133+ cells has a therapeutic potential to a rat spinal cord injury model and could be an optional treatment for spinal cord injury in the clinical settings.

Surgical outcome of posterior decompression for cervical spondylosis with unilateral upper extremity amyotrophy.

Study Design. Case studies of patients with cervical spondylosis with unilateral upper extremity amyotrophy. Objective. To clarify the surgical outcome of posterior decompression for this amyotrophy. Summary of Background Data. Cervical spondylosis sometimes causes a characteristic severe muscular atrophy without sensory disturbance or lower-extremity dysfunction, which is the so-called “cervical spondylotic amyotrophy.” However, response to treatment, especially to posterior decompression, has not been well understood. Method. This study included 32 patients. All underwent posterior cervical laminoplasty, and 22 patients had an additional foraminotomy. Preoperative and postoperative muscle power and results of imaging and electrophysiologic studies were evaluated. The follow-up period averaged 78 months. Whether impingement was against the ventral nerve root (VNR) or anterior horn (AH) in the spinal cord was assessed according to these findings. These cases were divided into proximal type and distal type according to the most severely atrophic muscle and compared statistically. Results. Severe preoperative muscle atrophy was observed in the deltoid and biceps muscles of 24 patients (proximal type) and in the forearm and hand muscles of 8 patients (distal type). Impingements against the VNR and AH were observed in 21 and 28 cases, respectively, and 17 cases had impingement of both the VNR and AH. Improvements in muscle atrophy after surgery were observed in 25 cases. In proximal-type patients, muscle power improved in 92% of cases but was improved in only 38% of the distal-type cases. Conclusions. Laminoplasty and foraminotomy were effective in the treatment of most patients with this syndrome, although the outcome in the distal type was inferior to that in the proximal type.

Therapeutic effects with magnetic targeting of bone marrow stromal cells in a rat spinal cord injury model.

Study Design. Experimental rat animal study using a new cell delivery system. Objective. To investigate the therapeutic effects with magnetic targeting of bone marrow stromal cells (BMSCs) in a rat spinal cord injury (SCI) model. Summary of Background Data. Several methods to deliver therapeutic agents have been used for the treatment of SCI in animal studies. However, the most appropriate administration method for clinical application has not been established. Previously, we reported the development of a new cell delivery system using magnetic targeting. This system has potential as a clinical application for a minimally invasive and efficient transplant method in SCI. Methods. Contusion SCI was induced by placing a 25 g rod onto the spinal cord for 90 seconds in adult SD rats. A neodymium magnet was placed in the paravertebral muscles at the T7 level in the magnet group, whereas a nonmagnet metal was placed at the same spinal cord level in the nonmagnet group. Magnetically labeled BMSCs were injected into the subarachnoid space in both the magnet and nonmagnet group. Results. Aggregations of the BMSCs were detected on the surface of the injured spinal cord in the magnet group, whereas few BMSCs were observed in the nonmagnet group. Hindlimb motor function of the magnet group demonstrated significant improvement compared with that of the nonmagnet group. Conclusion. This cell delivery system may be a useful method for future clinical application in the treatment of SCI.

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.