Yuto Ogawa

Musashi1 : An evolutionally conserved marker for CNS progenitor cells including neural stem cells

In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form ‘neurospheres’ in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(–) cells, which included Tα1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(–). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.

Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats

Neural progenitor cells, including neural stem cells, are a potential expandable source of graft material for transplantation aimed at repairing the damaged CNS. Here we present the first evidence that in vitro‐expanded fetus‐derived neurosphere cells were able to generate neurons in vivo and improve motor function upon transplantation into an adult rat spinal‐cord‐contusion injury model. As the source of graft material, we used a neural stem cell‐enriched population that was derived from rat embryonic spinal cord (E14.5) and expanded in vitro by neurosphere formation. Nine days after contusion injury, these neurosphere cells were transplanted into adult rat spinal cord at the injury site. Histological analysis 5 weeks after the transplantation showed that mitotic neurogenesis occurred from the transplanted donor progenitor cells within the adult rat spinal cord, a nonneurogenic region; that these donor‐derived neurons extended their processes into the host tissues; and that the neurites formed synaptic structures. Furthermore, analysis of motor behavior using a skilled reaching task indicated that the treated rats showed functional recovery. These results indicate that in vitro‐expanded neurosphere cells derived from the fetal spinal cord are a potential source for transplantable material for treatment of spinal cord injury.

Long-term results of expansive open-door laminoplasty for cervical myelopathy--average 14-year follow-up study.

Study Design. Retrospective case series on long-term follow-up results of original expansive open-door laminoplasty for cervical myelopathy due to cervical spondylosis (CSM) and ossification of posterior longitudinal ligament (OPLL). Objectives. To elucidate efficacy and problems of original open-door laminoplasty to improve future surgical outcomes. Summary of Background Data. Little information is available on long-term outcomes of original open-door laminoplasty without grafts, implants, or instruments. Method. The study group included 80 patients who underwent original open-door laminoplasty and were followed for minimum 10 years. Clinical results, including Japanese Orthopedic Association scores, recovery rates, occurrences of complications, and long-term deterioration were investigated. Cervical alignments, type of OPLL, cervical range of motion, anteroposterior diameter of spinal canal, and progression of OPLL were assessed on plain radiographs. Spinal cord decompression was verified on magnetic resonance imaging. Results. Average Japanese Orthopedic Association score and recovery rate improved significantly until 3 years after surgery and remained at an acceptable level in both cervical spondylosis and OPLL patients with slight deterioration after 5 years. Segmental motor palsy developed in 8 patients. Late deterioration, mainly lower extremity motor score decline, developed in 8 CSM and 16 OPLL patients. Overall cervical range of motion decreased by 36%. Patients with cervical lordosis decreased gradually in both patient groups. Such changes in alignments did not affect surgical results in CSM patients, while OPLL patients with preoperative kyphosis had lower recovery rates than those with straight and lordotic alignments. OPLL progression that was detected in 66% of patients did not affect clinical results. Although infrequent, magnetic resonance imaging revealed atrophy of spinal cord, spinal cord compression at adjacent segments due to degenerative changes and OPLL progression. Conclusions. Long-term results of open-door laminoplasty without bone graft, graft substitutes, or instruments were satisfactory. However, segmental motor paralysis, kyphosis, established before and after surgery, OPLL progression, and late deterioration due to age-related degeneration remain challenging problems.

Transplantation of neural stem cells into the spinal cord after injury.

Recovery from central nervous system damage in adult mammals is hindered by their limited ability to replace lost cells and damaged myelin, and reestablish functional neural connections. However, recent progresses in stem cell biology are making it feasible to induce the regeneration of injured axons after spinal cord injury. Transplantation of in vitro expanded neural stem cells into rat spinal cord 9 days after contusion injury induced their differentiation into neurons and oligodendrocytes, and the functional recovery of skilled forelimb movement. It was partly because the microenvironment within the injured spinal cord at 9 days after injury was more favorable for grafted neural stem cells in terms of their survival and differentiation.

Reduced postoperative wound pain after lumbar spinous process–splitting laminectomy for lumbar canal stenosis: a randomized controlled study

OBJECT to reduce intraoperative damage to the posterior supporting structures of the lumbar spine during decompressive surgery for lumbar canal stenosis (LCS), lumbar spinous process-splitting laminectomy (LSPSL or split laminectomy) was developed. This prospective, randomized, controlled study was conducted to clarify whether the split laminectomy decreases acute postoperative wound pain compared with conventional laminectomy. METHODS forty-one patients with LCS were enrolled in this study. The patients were randomly assigned to either the LSPSL group (22 patients) or the conventional laminectomy group (19 patients). Questionnaires regarding wound pain (intensity, depth, and duration) and activities of daily living (ADL) were administered at postoperative days (PODs) 3 and 7. Additionally, the authors evaluated the pre- and postoperative serum levels of C-reactive protein and creatine phosphokinase, the amount of pain analgesics used during a 3-day postoperative period, and the muscle atrophy rate measured on 1-month postsurgical MR images. RESULTS data obtained in patients in the LSPSL group and in 16 patients in the conventional laminectomy group were analyzed. The mean visual analog scale for wound pain on POD 7 was significantly lower in the LSPSL group (16 ± 17 mm vs 34 ± 31 mm, respectively; p = 0.04). The mean depth-of-pain scores on POD 7 were significantly lower in the LSPSL group than in the conventional group (0.9 ± 0.6 vs 1.7 ± 0.8, respectively; p = 0.013). On POD 3, the mean serum creatine phosphokinase level was significantly lower in the LSPSL group (126 ± 93 U/L) than in the other group (207 ± 150 U/L) (p = 0.02); on POD 7, the mean serum C-reactive protein level was significantly lower in the LSPSL group (1.1 ± 0.6 mg/dl) than in the conventional laminectomy group (1.9 ± 1.5 mg/dl) (p = 0.04). The number of pain analgesics taken during the 3-day postoperative period was lower in the LSPSL group than in the conventional laminectomy group (1.7 ± 1.3 tablets vs 2.3 ± 2.4 tablets, respectively; p = 0.22). The mean muscle atrophy rate was also significantly lower in the LSPSL group (24% ± 15% vs 43% ± 22%; p = 0.004). CONCLUSIONS lumbar spinous process-splitting laminectomy for the treatment of LCS reduced acute postoperative wound pain and prevented postoperative muscle atrophy compared with conventional laminectomy, possibly because of minimized damage to the paraspinal muscles.

Targeted expression of anti-apoptotic protein p35 in oligodendrocytes reduces delayed demyelination and functional impairment after spinal cord injury

Functional impairment after spinal cord injury (SCI) is attributed to neuronal cell necrosis death and axonotmesis, with further worsening caused by the accompanying apoptosis of myelin‐forming oligodendrocytes (OLGs). However, it is unclear as to how much OLG apoptosis contributes to functional impairment. To address this issue, we used transgenic mice characterized by the targeted expression of p35, a broad‐spectrum caspase inhibitor, in OLGs using the cre/loxP system (referred to as cre/p35 transgenic mice). In this study, we examined the motor function and histopathologic changes after a contusive thoracic spinal cord injury in the cre/p35 transgenic mice. A larger number of OLGs and a lesser extent of demyelination were observed after SCI in the cre/p35 transgenic mice than in the control cre mice, which did not carry the p35 transgene. Furthermore, the motor function of the hindlimbs recovered to a significantly better degree in the cre/p35 transgenic mice than in the control cre mice. Thus, the inhibition of OLG apoptosis decreased the extent of functional impairment after SCI. These findings suggest that the inhibition of OLG apoptosis may be a potential treatment for SCI.

Expansive open-door laminoplasty for ossification of the posterior longitudinal ligament of the cervical spine: Surgical indications, technique, and outcomes

In 1968, Kirita devised a sophisticated extensive laminectomy technique in which the laminae were thinned and divided at the midline by a high-speed drill followed by en bloc resection of the laminae to achieve decompression of the spinal cord safely in patients with cervical spondylosis and ossifi cation of the posterior longitudinal ligament (OPLL) [1]. This technique added much safety to conventional laminectomy and signifi cantly reduced the rate of neurological complications. Hirabayashi et al. simplifi ed Kirita’s method, in which the bilateral bony gutters at the junction of the laminae and the facet joints were made using a high-speed drill followed by en bloc removal of the laminae [2]. The idea of open-door laminoplasty evolved when they noticed dural pulsation when one side of the laminae was lifted before their total removal. They performed the fi rst case using this technique in 1977, leaving the ventral cortex on one side as a hinge and lifting the lamina on the other side, just like opening a book cover; they named it “expansive open-door laminoplasty (ELAP)” [3,4]. The advent of ELAP has contributed signifi cantly to the establishment of the concept “laminoplasty,” which later became the treatment of choice for cervical myelopathy in Japan and has also led to the development of various technical modifi cations. Laminoplasty has become the treatment of choice for cervical myelopathy, and satisfactory results have been reported [2,5–7]. Although ELAP is not radical decompression surgery that directly removes the ossifi ed ligament, it has a total decompression effect induced by the dorsal shift of the spinal cord in addition to local decompression of the spinal cord by posterior displacement of the laminae (Fig. 1) [8]. Several studies have proven that the decompression effect of ELAP is virtually equivalent to that of laminectomy and anterior corpectomy and fusion [9]. The posterior structures, including the lamina and the supraspinous and interspinous ligaments, are preserved; and the cervical muscles are reattached to reconstruct the spinal canal, thereby maintaining the preoperative cervical alignments and at the same time restoring cervical stability. Herkowitz concluded from his biomechanical study that the stability of the cervical spine after ELAP was not signifi cantly different from that of the intact spine [10]. Indeed, kyphotic deformity or instability after ELAP that required salvage surgery has rarely been experienced in our institution [6,11]. Moreover, the incidence of adjacent-segment disease, which may also lead to a salvage operation, was much lower after ELAP than after anterior decompression and fusion [2].