Masao Koda

Brain-Derived Neurotrophic Factor Suppresses Delayed Apoptosis of Oligodendrocytes after Spinal Cord Injury in Rats

We evaluated the effect of brain-derived neurotrophic factor (BDNF) on cell death after spinal cord injury. A rat spinal cord injury model was produced by static load, and continuous intrathecal BDNF or vehicle infusion was carried out either immediately or 3 days after the injury. Cell death was examined by nuclear staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After injury, typical apoptotic cells were observed. Double staining with TUNEL and specific cell markers revealed that, soon after the injury, the apoptotic or necrotic cells at the injury site were neurons and microglia. One week after the injury, apoptotic oligodendrocytes, but not apoptotic astrocytes, were observed in the white matter rostral and caudal to the injury site, whereas few apoptotic cells were found in the gray matter. The immediate BDNF treatment significantly reduced the number of TUNEL-positive cells in the adjacent rostral site 1 and 2 weeks after the injury, and in the adjacent caudal site 3 days and 1 week after the injury, even though there was no significant difference between BDNF-treated and control rats at the injury site itself. In addition, similar antiapoptotic effects were observed in these regions 1 week after injury in rats that received BDNF treatment from the third day after injury. These findings suggest that BDNF suppresses delayed apoptosis of oligodendrocytes after spinal cord injury, for which even delayed injections are effective. BDNF administration may therefore be useful for the clinical treatment of spinal cord injury through the suppression of secondary events.

Anomalous vertebral artery at the extraosseous and intraosseous regions of the craniovertebral junction: analysis by three-dimensional computed tomography angiography.

Study Design. This study examined the extraosseous and intraosseous anomalies of vertebral arteries in patients who underwent surgery of the craniovertebral junction. Objectives. To describe the usefulness of three-dimensional computed tomography angiography for evaluating vertebral artery anomalies before surgery. Summary of Background Data. Previous studies using catheter angiograms have identified anomalous courses of the vertebral artery at the craniovertebral junction. Studies using computed tomography reconstruction also showed deviation of the vertebral artery groove at the C2 isthmus, demonstrating a risk of vertebral artery injury for C1–C2 transarticular screw placement. These analyses provided us with useful information for identifying anomalies of the vertebral artery, but they could not visualize the artery and its circumferential osseous tissue simultaneously, nor could they analyze the reciprocal anatomy of both tissues. Methods. Thirty-one consecutive patients who submitted to surgery at the craniovertebral junction were evaluated before surgery by three-dimensional computed tomography angiography. Eleven of the patients had congenital osseous anomalies at the craniovertebral junction including os odontoideum and ossiculum terminale. Anomalous vertebral arteries at the extraosseous region were visualized by three-dimensional reconstruction images, and the intraosseous deviation of the vertebral artery at the C2 isthmus was evaluated by multiplanar reconstruction images. Results. Extraosseous and/or intraosseous vertebral artery anomalies were detected in 9 cases. Eight of the 9 cases had osseous anomalies at the craniovertebral junction. Abnormal courses of the vertebral artery at the extraosseous region were detected in 4 cases: 2 had fenestration and 2 had persistent first intersegmental artery. Asymmetry of bilateral vertebral arteries was found in 5 cases: the right was dominant in 3 cases and the left in 2 cases. A high-riding vertebral artery at the C2 isthmus was detected in 5 cases. Based on these findings, we modified our surgical approach and the screw placement; consequently, no vertebral artery injury occurred. Conclusions. In patients having osseous anomalies at the craniovertebral junction, the frequency of vertebral artery anomalies at the extraosseous and intraosseous regions is increased. With preoperative three-dimensional computed tomography angiography, we can precisely identify the anomalous vertebral artery and reduce the risk of intraoperative injury to the vertebral artery, in advance.

Adenovirus Vector-Mediated In Vivo Gene Transfer of Brain-Derived Neurotrophic Factor (BDNF) Promotes Rubrospinal Axonal Regeneration and Functional Recovery after Complete Transection of the Adult Rat Spinal Cord

Neurotrophins have been shown to promote axonal regeneration, but the techniques available for delivering neurotrophins have limited effectiveness. The aim of this study was to evaluate the effect of adenovirus vector mediated gene transfer of brain-derived neurotrophic factor (BDNF) on axonal regeneration after spinal cord injury. We prepared adenovirus vectors encoding either beta-galactosidase (AxCALacZ) or BDNF (AxCABDNF). AxCALacZ was used to assess infection levels of the adenovirus BDNF produced by AxCABDNF was detected by Western blotting and its bioactivity was confirmed by bioassay. As a model of spinal cord injury, the rat spinal cord was completely transected at the T8 level. Immediately after transection, the vectors were injected into both stumps of the spinal cord. Axonal regeneration after transection was assessed by retrograde and anterograde tracing. In AxCALacZ-injected rats, adenovirus-infected cells were observed not only at the injected site but also in brainstem nuclei, as shown by LacZ expression. After the injection of the retrograde tracer fluorogold (FG) distal portion to the transection, AxCABDNF-injected rats showed FG-labeled neurons in the red nucleus. The anterograde tracer biotinylated dextran amine (BDA) injected into the red nucleus was also found in regenerating rubrospinal fibers distal to the transection. These tracing experiments demonstrated the regeneration of descending axons. In addition, rats of the AxCABDNF group showed significant locomotor recovery of hindlimb function, which was completely abolished by re-transection. These results indicate that the recovery was caused by regeneration of rubrospinal axons, not by simple enhancement of the central pattern generator.

Up-regulation of macrophage migration-inhibitory factor expression after compression-induced spinal cord injury in rats

Macrophage migration inhibitory factor (MIF) is a multipotential protein that acts as a pro-inflammatory cytokine, pituitary hormone, immunoregulator, and mitogen. To elucidate function of MIF in spinal cord injury, we examined expression of MIF after compression-induced spinal cord injury using Northern blot analysis, in situ hybridization and immunohistochemistry. The MIF mRNA was up-regulated in injured spinal cord, peaking 3xa0days after injury shown by Northern blot analysis. In situ hybridization revealed up-regulation of MIF in microglia accumulating in the lesion epicenter 3xa0days after injury and astrocytes around the cystic cavity 1xa0week after injury. Double staining showed co-localization of MIF and tomato lectin in the lesioned site, indicating that microglia accumulating to the lesion epicenter express MIF. The time course of MIF expression is different from that of previous reports about cytokine expression peaking at earlier time points; thus, it is unlikely that MIF acts as a pro-inflammatory factor in the present study. The MIF may contribute to proliferation of astrocytes around the lesioned site in spinal cord injury because of its cell proliferation-promoting property.

Upregulation of Osteopontin Expression in Rat Spinal Cord Microglia after Traumatic Injury

Osteopontin is a noncollagenous extracellular matrix protein that is expressed in various tissues. Recent studies have shown the upregulation of osteopontin expression in the ischemic cortex after cerebral infarction. We demonstrate here the upregulation of osteopontin expression in the spinal cord after compression injury. Laboratory rats were used in a compression model of spinal cord injury (30-g load for 5 min). Northern blot analysis showed that osteopontin mRNA expression levels reached a peak 3 days after injury (sevenfold; p < 0.05). In situ hybridization demonstrated osteopontin mRNA expression in necrotic areas from 24 h, peaking 3 days after injury. Immunohistochemistry detected osteopontin protein immunoreactivity from 12 h, peaking at 3 days. The peak time and distribution of osteopontin protein expression were coincident with those of osteopontin mRNA expression. Osteopontin expression in our model preceded that shown in the previously reported cerebral infarction models. Osteopontin protein was found in the cytoplasm at 3 days and secreted into the extracellular matrix at 7 days. Triple immunolabeling showed that osteopontin was localized in activated microglia surrounded by astrocytes.

Transient paraparesis after laminectomy for thoracic myelopathy due to ossification of the posterior longitudinal ligament: a case report.

Study Design. Case report. Objectives. We report a case with thoracic myelopathy due to ossification of the posterior longitudinal ligament (OPLL) of the spine, in which neurologic deterioration progressed after laminectomy and was markedly reversed after additional posterior instrumented fusion. Summary of Background Data. Many different surgical procedures may be used in the treatment of thoracic OPLL. However, the possibility of postoperative paraplegia remains a major risk, and consistent protocols and procedures for surgical correction of thoracic OPLL have not been established. Methods. The patient was a 53-year-old man with continuous OPLL at T3–T8 that compressed the spinal cord anteriorly. Anterior decompression surgery employing a posterior approach was initiated, but during OPLL extirpation electrophysiologic monitoring of spinal cord activity showed abnormalities. As a result, the procedure was converted to a wide laminectomy. Over the next 4 weeks, kyphosis of the thoracic spine increased and myelopathy worsened, producing severe paraparesis. Results. Four weeks after surgery, posterior instrumented fusion (T1–L1) was performed without correction of the kyphosis. After the fusion, neurologic deficits gradually recovered and the patient was fully recovered after 10 months. At follow-up 15 years after the fusion, no neurologic deterioration was seen despite the presence of residual anterior impingement of spinal cord by OPLL. Conclusions. The present case suggests that kyphosis and instability are major factors that affect the severity of thoracic myelopathy due to OPLL, and posterior fusion with spinal instrumentation is a safe and effective adjunct procedure for surgical treatment of thoracic OPLL.

Transient paraparesis after laminectomy for thoracic ossification of the posterior longitudinal ligament and ossification of the ligamentum flavum

Study design:Case report.Objectives:To report a case with thoracic myelopathy caused by ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), in which postoperative paralysis occurred after laminectomy and was reversed after an additional posterior instrumented fusion.Setting:A University Hospital in Japan.Case report:A 71-year-old woman, with a spastic palsy of both lower extremities, had OPLL and OLF at T10–T11, which pinched the spinal cord anteriorly and posteriorly. She underwent a laminectomy at T10–T11, and no further neurological deterioration was seen immediately after surgery. Over the next 18u2009h, however, myelopathy worsened, showing severe paraparesis. An additional posterior instrumented fusion at T7–L1 was performed without correction of the kyphosis. After fusion, neurological deficits gradually recovered, despite the presence of residual anterior impingement of spinal cord by the OPLL.Conclusions:The present case provides evidence for the possibility that laminectomy alone produces postoperative paralysis for combined thoracic OPLL and OLF, and we recommend that a posterior instrumented fusion should be added when posterior decompression is performed for this disorder.

Gene expression profiling of cathepsin D, metallothioneins-1 and -2, osteopontin, and tenascin-C in a mouse spinal cord injury model by cDNA microarray analysis

The purpose of this study was to use a cDNA microarray to identify new genes involved in healing of spinal cord injury. C57BL/6 mice (7–8xa0weeks, male) were subjected to spinal cord compression injury (SCI) at the T7/8 level (20xa0g, 5xa0min; SCI group). For the control group, mice underwent only laminectomy. Mice were killed at 1, 3 and 7xa0days. cDNA transcribed from mRNA was hybridized to NIA mice 15K microarrays at each time point. We found 84 genes showing significant expressional changes, including higher and lower expression levels in the SCI groups than in the control [more than 1.0 or less than −1.0 using log ratio (base 2)]. Five genes were selected for further quantitative gene expression analysis by real-time reverse transcription (RT)-PCR. For histological examination, we applied in situ hybridization and fluorescence immunohistochemistry. Cathepsin D, metallothionein-1 (MT-1), metallothionein-2 (MT-2), osteopontin (OPN), and tenascin-C were selected for quantitative and histological analysis. Microarray analysis revealed that SCI led to the up-regulation of OPN and cathepsin D expression at 7xa0days and also of MT-1, MT-2, and tenascin-C expression at 1xa0day. Tenascin-C was re-up-regulated at 7xa0days. These values agreed with those of real-time RT-PCR analysis. By double labeling with in situ hybridization and fluorescence immunohistochemistry, MT-1, MT-2 and tenascin-C expression was observed in neurons and glial cells at 1xa0day, whereas at 7xa0days the main MT-2 and tenascin-C expression was found in fibronectin-positive fibroblasts. The main cathepsin D and OPN expression was observed in activated macrophages/microglia at 3 and 7xa0days. The five genes picked up by microarray gene expression profiling were shown to exhibit temporal and spatial changes of expression after SCI. This system is potentially useful for identifying genes that are involved in the response to SCI.

Regulation of semaphorin 3A expression in neurons of the rat spinal cord and cerebral cortex after transection injury

Semaphorin 3A (Sema3A) is a secreted repulsive axon guidance protein. It appears to play important roles in axon fasciculation, branching, neuronal migration, and tissue differentiation during embryonic development. In adults, Sema3A is expressed in spinal motoneurons and in some neurons in the brain. Here, we demonstrate changes in Sema3A expression in the spinal cord after complete transection and in the brain after spinal cord hemisection at the Th8 level in laboratory rats. Semi-quantitative reverse transcriptase-PCR analysis showed that the expression of Sema3A mRNA, which was present in the normal spinal cord, rapidly decreased after transection, reaching its lowest level 1xa0day after injury. Thereafter, Sema3A expression levels recovered and reached four-fifths of the normal level at 28xa0days. Double staining by in situ hybridization and fluorescence immunohistochemistry showed that Sema3A was expressed in NeuN-positive neurons, but not in glia in the spinal cord. Sema3A expression was up-regulated in the contralateral cerebral cortex and in the ipsilateral spinal trigeminal nucleus 1–3xa0days after spinal cord hemisection. It is likely that the up-regulation occurred in neurons whose descending fibers were transected. These results suggest that Sema3A is regulated differently in spinal motoneurons and brain neurons following axonal injury.