Randomized trial of granulocyte colony-stimulating factor for spinal cord injury.

Attenuation of the secondary injury of spinal cord injury (SCI) can suppress the spread of spinal cord tissue damage, possibly resulting in spinal cord sparing that can improve functional prognoses. Granulocyte colony-stimulating factor (G-CSF) is a haematological cytokine commonly used to treat neutropenia. Previous reports have shown that G-CSF promotes functional recovery in rodent models of SCI. Based on preclinical results, we conducted early phase clinical trials, showing safety/feasibility and suggestive efficacy. These lines of evidence demonstrate that G-CSF might have therapeutic benefits for acute SCI in humans. To confirm this efficacy and to obtain strong evidence for pharmaceutical approval of G-CSF therapy for SCI, we conducted a phase 3 clinical trial designed as a prospective, randomized, double-blinded and placebo-controlled comparative trial. The current trial included cervical SCI [severity of American Spinal Injury Association (ASIA) Impairment Scale (AIS) B or C] within 48 h after injury. Patients are randomly assigned to G-CSF and placebo groups. The G-CSF group was administered 400 µg/m2/day × 5 days of G-CSF in normal saline via intravenous infusion for five consecutive days. The placebo group was similarly administered a placebo. Allocation was concealed between blinded evaluators of efficacy/safety and those for laboratory data, as G-CSF markedly increases white blood cell counts that can reveal patient treatment. Efficacy and safety were evaluated by blinded observer. Our primary end point was changes in ASIA motor scores from baseline to 3 months after drug administration. Each group includes 44 patients (88 total patients). Our protocol was approved by the Pharmaceuticals and Medical Device Agency in Japan and this trial is funded by the Center for Clinical Trials, Japan Medical Association. There was no significant difference in the primary end point between the G-CSF and the placebo control groups. In contrast, one of the secondary end points showed that the ASIA motor score 6 months (P = 0.062) and 1 year (P = 0.073) after drug administration tend to be higher in the G-CSF group compared with the placebo control group. Moreover, in patients aged over 65 years old, motor recovery 6 months after drug administration showed a strong trend towards a better recovery in the G-CSF treated group (P = 0.056) compared with the control group. The present trial failed to show a significant effect of G-CSF in primary end point although the subanalyses of the present trial suggested potential G-CSF benefits for specific population.

A refined method of en bloc open-door laminoplasty for resection of intradural spinal tumors in the thoracic and lumbar spine.

BACKGROUND: The open-door laminoplasty has been used to treat cervical spondylotic myelopathy. This technique has been applied to the surgical treatment of thoracic and lumbar spinal canal tumors instead of simple laminectomy or hemilaminectomy. However, previously reported laminoplasty methods did not keep posterior supporting elements intact such as the laminae and the spinous processes with supraspinous and interspinous ligaments, and almost all of them needed instruments for the fixation of reconstructed laminae. The purpose of this paper is to introduce our open-door laminoplasty method, which keep all posterior supporting elements intact and reconstruct the laminae without instrument. METHODS: Eight patients (mean age 61 years) underwent en bloc open-door laminoplasty in the thoracic and lumbar spine for resection of intradural spinal tumors. Two grooves are made bilaterally on the laminae just medial side of the facet joints. One-half of each spinous process of the adjacent vertebrae above and below the laminoplasty is cracked diagonally to create a green stick fracture and bent to the hinged side for sufficient elevation of the laminar flap. After tumor resection, the laminar flap is restored to its original site, resulting in the complete preservation of the posterior supporting elements. RESULTS: Operative exposure was good and permitted complete resection. No complications such as postoperative spinal canal stenosis or kyphosis were observed. Computed tomography(CT) indicated that bony fusion occurred in all cases. CONCLUSION: The supraspinous and interspinous ligaments above and below laminoplasty were kept intact during surgery in our method. Therefore, the continuity of posterior supporting elements (laminae and spinous processes connected by supraspinous and interspinous ligaments) were completely preserved.

Additional decompression at adjacent segments leads to adjacent segment degeneration after PLIF.

PURPOSE: Adjacent segment degeneration (ASD) is one of the major complications of lumbar fusion. Several previous retrospective studies reported ASD after PLIF. However, few reports evaluated whether decompression surgery combined with fusion surgery increases the rate of complications in adjacent segments. The purpose of the current study was to investigate the degeneration in decompressed adjacent segments after PLIF. METHODS: A total of 23 patients (12 men, 11 women; average age, 58.6) who underwent PLIF surgery [1 level (n = 9), 2 levels (n = 8), 3 levels (n = 4), 4 levels (n = 2)] were included. Additional adjacent decompression above or below the level of interbody fusion was performed at 25 levels and no adjacent decompression was performed at 15 levels. We retrospectively investigated ASD by X-ray films of all 40 adjacent segments (above and below fusion level) and clinical outcomes of all 23 cases. RESULTS: Of the 40 adjacent segments, 19 (47.5%) showed ASD and 9 (22.5%) showed symptomatic ASD. In the 19 segments with ASD, ASD occurred in 16 of 25 (64.0%) segments at decompressed sites compared with 3 of 15 (20.0%) non-decompressed sites. The ratio of ASD in adjacent segments was significantly higher at decompressed sites than at non-decompressed sites (p < 0.01). CONCLUSION: ASD occurs frequently in association with additional decompression above or below the level of PLIF. In cases in which the adjacent segments require decompression, a surgical strategy that preserves as much of the posterior complex as possible should be selected.

Postmortem findings in a woman with history of laminoplasty for severe cervical spondylotic myelopathy.

CONTEXT: We report the autopsy of a 65-year-old woman who underwent a C3-C7 laminoplasty 4 years after the diagnosis of cervical spondylotic myelopathy (CSM). Her sensory disturbance, spasticity, and vesicorectal disturbance, which corresponded to long tract sign, had improved after surgery. FINDINGS: Cross sections at the C4-C5 level showed a triangular shape because of atrophied ventral gray matter. Moreover, despite the scarce glial scar formation around the cystic cavity, regeneration of gray matter had not occurred. In the white matter, the posterior and lateral funiculi were shrunken including three to four segments. CONCLUSION: Pathological change of white matter did not coincide with relief of clinical symptoms in this case. These findings indicate that it may be better to operate earlier in cases of CSM, because delay may lead to irreversible histological change.

Brain-derived neurotrophic factor suppresses anoikis-induced death of Schwann cells.

Anoikis is a type of apoptosis due to the detachment from the extracellular matrix and neighboring cells. In case of cell transplantation therapy for spinal cord injury, preparation of graft cells includes dissociation of cultured cells, which may cause anoikis-induced cell death. Thus suppression of anoikis may increase survival of grafted cells. Here we tested the effect of brain-derived neurotrophic factor (BDNF) on anoikis-induced cell death of cultured Schwann cells. Schwann cells were collected and cultured from sciatic nerves of neonatal Wistar rats. Schwann cells were plated upon a non-adherent polyhydroxyethyl methacrylate substrate to induce anoikis. BDNF was added into the culture medium at various concentrations. Twenty-four hours after non-adherent culture, approximately 40% of Schwann cells died and BDNF significantly decreased the number of dead cells in that culture condition. Next, Schwann cells were transplanted with or without BDNF treatment into contused rat spinal cord 1 week after injury. Five weeks after transplantation, immunohistochemistry revealed that the number of transplanted cells was significantly larger in the BDNF-treated group than that of the non-treated group. Suppression of anoikis may increase survival of grafted cells in case of cell therapy for spinal cord injury.

Reduction of cystic cavity, promotion of axonal regeneration and sparing, and functional recovery with transplanted bone marrow stromal cell-derived Schwann cells after contusion injury to the adult rat spinal cord.

OBJECT: The authors previously reported that Schwann cells (SCs) could be derived from bone marrow stromal cells (BMSCs) in vitro and that they promoted axonal regeneration of completely transected rat spinal cords in vivo. The aim of the present study is to evaluate the efficacy of transplanted BMSC-derived SCs (BMSC-SCs) in a rat model of spinal cord contusion, which is relevant to clinical spinal cord injury. METHODS: Bone marrow stromal cells were cultured as plastic-adherent cells from the bone marrow of GFPtransgenic rats. The BMSC-SCs were derived from BMSCs in vitro with sequential treatment using beta-mercaptoethanol, all-trans-retinoic acid, forskolin, basic fibroblast growth factor, platelet derived-growth factor, and heregulin. Schwann cells were cultured from the sciatic nerve of neonatal, GFP-transgenic rats. Immunocytochemical analysis and the reverse transcriptase-polymerase chain reaction were performed to characterize the BMSC-SCs. For transplantation, contusions with the New York University impactor were delivered at T-9 in 10- to 11-week-old male Wistar rats. Four groups of rats received injections at the injury site 7 days postinjury: the first received BMSCSCs and matrigel, a second received peripheral SCs and matrigel, a third group received BMSCs and matrigel, and a fourth group received matrigel alone. Histological and immunohistochemical studies, electron microscopy, and functional assessments were performed to evaluate the therapeutic effects of BMSC-SC transplantation. RESULTS: Immunohistochemical analysis and reverse transcriptase-polymerase chain reaction revealed that BMSC-SCs have characteristics similar to SCs not only in their morphological characteristics but also in their immunocytochemical phenotype and genotype. Histological examination revealed that the area of the cystic cavity was significantly reduced in the BMSC-SC and SC groups compared with the control rats. Immunohistochemical analysis showed that transplanted BMSCs, BMSC-SCs, and SCs all maintained their original phenotypes. The BMSC-SC and SC groups had a larger number of tyrosine hydroxilase-positive fibers than the control group, and the BMSC-SC group had more serotonin-positive fibers than the BMSC or control group. The BMSC-SC group showed significantly better hindlimb functional recovery than in the BMSC and control group. Electron microscopy revealed that transplanted BMSC-SCs existed in association with the host axons. CONCLUSIONS: Based on their findings, the authors concluded that BMSC-SC transplantation reduces the size of the cystic cavity, promotes axonal regeneration and sparing, results in hindlimb functional recovery, and can be a useful tool for spinal cord injury as a substitute for SCs.

Study protocol for the G-SPIRIT trial: a randomised, placebo-controlled, double-blinded phase III trial of granulocyte colony-stimulating factor-mediated neuroprotection for acute spinal cord injury.

INTRODUCTION: Granulocyte colony-stimulating factor (G-CSF) is generally used for neutropaenia. Previous experimental studies revealed that G-CSF promoted neurological recovery after spinal cord injury (SCI). Next, we moved to early phase of clinical trials. In a phase I/IIa trial, no adverse events were observed. Next, we conducted a non-randomised, non-blinded, comparative trial, which suggested the efficacy of G-CSF for promoting neurological recovery. Based on those results, we are now performing a phase III trial. METHODS AND ANALYSIS: The objective of this study is to evaluate the efficacy of G-CSF for acute SCI. The study design is a prospective, multicentre, randomised, double-blinded, placebo-controlled comparative study. The current trial includes cervical SCI (severity of American Spinal Injury Association (ASIA) Impairment Scale B/C) within 48 hours after injury. Patients are randomly assigned to G-CSF and placebo groups. The G-CSF group is administered 400 µg/m2/day×5 days of G-CSF in normal saline via intravenous infusion for 5 consecutive days. The placebo group is similarly administered a placebo. Our primary endpoint is changes in ASIA motor scores from baseline to 3 months. Each group includes 44 patients (88 total patients). ETHICS AND DISSEMINATION: The study will be conducted according to the principles of the World Medical Association Declaration of Helsinki and in accordance with the Japanese Medical Research Involving Human Subjects Act and other guidelines, regulations and Acts. Results of the clinical study will be submitted to the head of the respective clinical study site as a report after conclusion of the clinical study by the sponsor-investigator. Even if the results are not favourable despite conducting the clinical study properly, the data will be published as a paper. TRIAL REGISTRATION NUMBER: UMIN000018752.

Granulocyte colony-stimulating factor attenuates neuronal death and promotes functional recovery after spinal cord injury in mice.

Granulocyte colony-stimulating factor (G-CSF) is a protein that stimulates differentiation, proliferation, and survival of granulocytic lineage cells. Recently, a neuroprotective effect of G-CSF was reported in a model of cerebral infarction. The aim of the present study was to elucidate the potential therapeutic effect of G-CSF for spinal cord injury (SCI) in mice. We found that G-CSF is neuroprotective against glutamate-induced cell death of cerebellar granule neurons in vitro. Moreover, we used a mouse model of compressive SCI to examine the neuroprotective potential of G-CSF in vivo. Histologic assessment with cresyl violet staining revealed that the number of surviving neurons in the injured spinal cord was significantly increased in G-CSF-treated mice. Immunohistochemistry for neuronal apoptosis revealed that G-CSF suppressed neuronal apoptosis after SCI. Moreover, administration of G-CSF promoted hindlimb functional recovery. Examination of signaling pathways downstream of the G-CSF receptor suggests that G-CSF might promote functional recovery by inhibiting neuronal apoptosis after SCI. G-CSF is currently used in the clinic for hematopoietic stimulation, and its ongoing clinical trial for brain infarction makes it an appealing molecule that could be rapidly placed into trials for patients with acute SCI.

Granulocyte colony-stimulating factor (G-CSF) mobilizes bone marrow-derived cells into injured spinal cord and promotes functional recovery after compression-induced spinal cord injury in mice.

The aim of the present study was to elucidate the effects of granulocyte colony-stimulating factor (G-CSF)-mediated mobilization of bone marrow-derived stem cells on the injured spinal cord. Bone marrow cells of green fluorescent protein (GFP) transgenic mice were transplanted into lethally irradiated C57BL/6 mice. Four weeks after bone marrow transplantation, spinal cord injury was produced by a static load (20 g, 5 min) at T8 level. G-CSF (200 microg/kg/day) was injected subcutaneously for 5 days. Immunohistochemistry for GFP and cell lineage markers was performed to evaluate G-CSF-mediated mobilization of bone marrow-derived cells into injured spinal cord. Hind limb locomotor recovery was assessed for 6 weeks. Immunohistochemistry revealed that G-CSF increased the number of GFP-positive cells in injured spinal cord, indicating that bone marrow-derived cells were mobilized and migrated into injured spinal cord. The numbers of double positive cells for GFP and glial markers were larger in the G-CSF treated mice than in the control mice. Luxol Fast Blue staining revealed that G-CSF promoted white matter sparing. G-CSF treated mice showed significant recovery of hind limb function compared to that of the control mice. In conclusion, G-CSF showed efficacy for spinal cord injury treatment through mobilization of bone marrow-derived cells.

The use of hemopoietic stem cells derived from human umbilical cord blood to promote restoration of spinal cord tissue and recovery of hindlimb function in adult rats.

OBJECT: The use of human umbilical cord blood (HUCB) cells has been reported to improve functional recovery in cases of central nervous system injuries such as stroke, traumatic brain injury, and spinal cord injury (SCI). The authors investigated the effects of hemopoietic stem cells that were derived from HUCB and transplanted into the injured spinal cords of rats. METHODS: One week after injury, an HUCB fraction enriched in CD34-positive cells was transplanted into the experimental group. In control animals, vehicle (Matrigel) was transplanted. Recovery of motor functions was assessed using the Basso-Beattie-Bresnahan Locomotor Scale, and immunohistochemical examinations were performed. Cells from HUCB that were CD34 positive improved functional recovery, reduced the area of the cystic cavity at the site of injury, increased the volume of residual white matter, and promoted the regeneration or sparing of axons in the injured spinal cord. Immunohistochemical examination revealed that transplanted CD34-positive cells survived in the host spinal cord for at least 3 weeks after transplantation but had disappeared by 5 weeks. The transplanted cells were not positive for neural markers, but they were positive for hemopoietic markers. There was no evidence of an immune reaction at the site of injury in either group. CONCLUSIONS: These results suggest that transplantation of a CD34-positive fraction from HUCB may have therapeutic effects for SCI. The results of this study provide important preclinical data regarding HUCB stem cell-based therapy for SCI.

Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice.

We compared the effects of hematopoietic stem cell and marrow stromal cell transplantation for spinal cord injury in mice. From green fluorescent protein transgenic mouse bone marrow, lineage-negative, c-kit- and Sca-1-positive cells were sorted as hematopoietic stem cells and plastic-adherent cells were cultured as marrow stromal cells. One week after injury, hematopoietic stem cells or marrow stromal cells were injected into the lesioned site. Functional recovery was assessed and immunohistochemistry was performed. In the hematopoietic stem cell group, a portion of green fluorescent protein-positive cells expressed glial marker. In the marrow stem cell group, a number of green fluorescent protein and fibronectin-double positive cells were observed. No significant difference was observed in the recovery between both groups. Both hematopoietic stem cells and marrow stromal cells have the potential to restore the injured spinal cord and to promote functional recovery.

Multicenter prospective nonrandomized controlled clinical trial to prove neurotherapeutic effects of granulocyte colony-stimulating factor for acute spinal cord injury: analyses of follow-up cases after at least 1 year.

STUDY DESIGN: An open-labeled multicenter prospective nonrandomized controlled clinical trial. OBJECTIVE: To confirm the feasibility of using granulocyte colony-stimulating factor (G-CSF) for treatment of acute spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: We previously reported that G-CSF promotes functional recovery after compression-induced SCI in mice. On the basis of these findings, we conducted a multicenter prospective controlled clinical trial to assess the feasibility of G-CSF therapy for patients with acute SCI. METHODS: The trial ran from August 2009 to March 2011, and included 41 patients with SCI treated within 48 hours of onset. Informed consent was obtained from all patients. After providing consent, patients were divided into 2 groups. In the G-CSF group (17 patients), G-CSF (10 µg/kg/d) was intravenously administered for 5 consecutive days, and in the control group (24 patients), patients were similarly treated except for the G-CSF administration. We evaluated motor and sensory functions using the American Spinal Cord Injury Association score and American Spinal Cord Injury Association impairment scale at 1 week, 3 months, 6 months, and 1 year after onset. RESULTS: Only 2 patients did not experience American Spinal Cord Injury Association impairment scale improvement in the G-CSF group. In contrast, 15 patients in the control group did not experience American Spinal Cord Injury Association impairment scale improvement. In the analysis of increased American Spinal Cord Injury Association motor score, a significant increase in G-CSF group was detected from 1 week after the administration compared with the control group. After that, some spontaneous increase of motor score was detected in control group, but the significant increase in G-CSF group was maintained until 1 year of follow-up. CONCLUSION: Despite the limitation that patient selection was not randomized, the present results suggest the possibility that G-CSF administration has beneficial effects on neurological recovery in patients with acute SCI. LEVEL OF EVIDENCE: 3.

Neuroprotective therapy using granulocyte colony-stimulating factor for patients with worsening symptoms of thoracic myelopathy: a multicenter prospective controlled trial.

STUDY DESIGN: An open-labeled multicenter prospective controlled clinical trial. OBJECTIVE: To confirm the feasibility of granulocyte colony-stimulating factor (G-CSF) administration for patients with thoracic myelopathy. SUMMARY OF BACKGROUND DATA: Although G-CSF is best known as an important cytokine commonly used to treat neutropenia, it also has nonhematopoietic functions. Previous experimental studies have shown that G-CSF can enhance tissue regeneration of several organs, such as the heart and the brain. We previously reported that G-CSF promotes functional recovery after spinal cord injury in rodents. On the basis of those findings, we started a clinical trial of neuroprotective therapy, using G-CSF for patients with worsening symptoms of thoracic myelopathy. METHODS: Patients whose Japanese Orthopaedic Association (JOA) score for thoracic myelopathy had decreased 2 points or more during a recent 1-month period were eligible for entry. After giving informed consent, patients were assigned to G-CSF and control groups. The G-CSF group (n = 10) received G-CSF 10 µg/kg per day intravenously for 5 consecutive days. The control group (n = 14) received similar treatments as the G-CSF group except for G-CSF administration. The primary outcome was JOA recovery rate at 1 month after G-CSF administration or initial treatment. RESULTS: There was greater improvement in neurological functioning between baseline and 1-month follow-up in the G-CSF group (JOA recovery rate: 29.1 ± 20.5%) than in the control group (JOA recovery rate: 1.1 ± 4.2%) (P < 0.01). No serious adverse events occurred during or after the G-CSF administration. CONCLUSION: The results provide evidence that G-CSF administration caused neurological recovery in patients with worsening symptoms of thoracic compression myelopathy.

Infection-related atlantoaxial subluxation (Grisel syndrome) in an adult with Down syndrome.

STUDY DESIGN: Case report. OBJECTIVE: To report an adult case with Down syndrome, in whom infection-related atlantoaxial subluxation (Grisel syndrome) developed. SUMMARY OF BACKGROUND DATA: Atlantoaxial instability is a common complication in Down syndrome patients; however, there have been limited reports of adult-onset atlantoaxial subluxation with myelopathy. Grisel syndrome has been characterized as a nontraumatic atlantoaxial subluxation associated with pharyngeal infection. It usually affects children, and the subluxation can be successfully reduced by conservative treatments in most cases. METHODS: A 26-year-old man with Down syndrome suffered from retropharyngeal infection, after which his atlantoaxial subluxation was aggravated and myelopathy developed. He was treated with administration of antibiotics and application of a halo-vest. RESULTS: The conservative treatments failed to reduce the atlantoaxial subluxation. We performed a C1 laminectomy and posterior occipitocervical fusion, which successfully relieved his symptoms. CONCLUSION.: This experience suggests that when Down syndrome patients have retropharyngeal infection, we should consider the possible aggravation of atlantoaxial instability and development of myelopathy, even if the patient is an adult.

Abnormal course of the vertebral artery at the craniovertebral junction in patients with Down syndrome visualized by three-dimensional CT angiography.

INTRODUCTION: We determined the incidence of vertebral artery (VA) anomalies at the craniovertebral junction (CVJ) in patients with Down syndrome, and characterized the VA anomalies. METHODS: The course of the VA in 46 consecutive patients who were due to undergo posterior arthrodesis surgery at the CVJ were evaluated by three-dimensional CT angiography (3DCTA). Included were five patients with Down syndrome who suffered from myelopathy due to atlantoaxial subluxation. All five patients with Down syndrome also had a simultaneous congenital skeletal anomaly, either os odontoideum or ossiculum terminale. RESULTS: Of the five patients with Down syndrome, three had VA anomalies at the CVJ, two had fenestration and one had a persistent first intersegmental artery. Of the other 41 patients without Down syndrome, five had VA anomalies at the CVJ. The incidence of VA anomalies at the CVJ was much higher in patients with Down syndrome than in those without Down syndrome. CONCLUSION: In planning surgery in patients with Down syndrome with symptomatic atlantoaxial subluxation and a congenital skeletal anomaly at the CVJ, we should consider the possible presence of VA anomalies. Preoperative 3DCTA allows us to precisely identify an anomalous VA and evaluate the possible risk of intraoperative VA injury in advance.