Masashi Yamazaki

Mechanisms for the Enhancement of Fracture Healing in Rats Treated With Intermittent Low-Dose Human Parathyroid Hormone (1–34)†

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1–34) [PTH(1–34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low‐dose treatment with human PTH(1–34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 μg/kg of PTH(1–34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH‐treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)‐positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH‐treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COL1A1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH‐treated group and accompanied by enhanced expression of insulin‐like growth factor (IGF)‐I mRNA during the early stages of healing (days 4–7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14–21) despite a lack of up‐regulation of IGF‐I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1–34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low‐dose PTH(1–34) to enhance fracture healing are indicated.

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.

The Extent of Ossification of Posterior Longitudinal Ligament of the Spine Associated with Nucleotide Pyrophosphatase Gene and Leptin Receptor Gene Polymorphisms

Study Design. A case-control study using radiograph findings and the PCR assay with regard to the susceptibility and the severity of ossification of posterior longitudinal ligament of the spine (OPLL). Objective. To analyze whether polymorphisms of the nucleotide pyrophosphatase (NPPS) gene and the leptin receptor gene predispose to an increased frequency and severity of OPLL. Summary of Background Data. The NPPS gene is responsible for ectopic ossification in the ttw mouse, an animal model for OPLL. The Zucker fatty rat, another animal model for OPLL, has a missense mutation in the leptin receptor gene. Methods. Analysis of 172 OPLL patients and 93 non-OPLL controls was performed. Radiographs of the cervical, thoracic and lumber spine were analyzed to determine whether OPLL was present and to what degree. Genomic DNA was extracted from all participants. Polymorphisms of the NPPS gene and the leptin receptor gene were analyzed using the PCR assay. The association of the polymorphisms with the development and extent of OPLL were statistically evaluated. Results. No significant association was found between the polymorphisms and the existence of OPLL in both the NPPS and the leptin receptor genes. However, the IVS20–11delT variant in the NPPS gene and the A861G variant in the leptin receptor gene were more frequent in patients with OPLL in the thoracic spine compared with patients whose OPLL was restricted to the cervical spine. Conclusion. The present results suggest that the IVS20–11delT variant of the NPPS gene and the A861G variant of the leptin receptor gene are associated with more extensive OPLL, but not with the frequency with which it occurs.

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 18 h, 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.

Spinal root avulsion-induced upregulation of osteopontin expression in the adult rat spinal cord

Osteopontin (OPN) is a secretory adhesive glycoprotein that is expressed in various tissues and plays a role in inflammation and tissue repair. It has been suggested that OPN plays a role in inflammation and wound healing after spinal cord injury; however, the expression of OPN and its function in the spinal cord under normal conditions and following spinal motoneuron injury have not been well characterized. Here we examined the expression of OPN mRNA before and after spinal root avulsion. OPN mRNA was detected at a low level in the normal spinal cord in a Northern blot analysis, but dramatically increased following avulsion. In situ hybridization and immunohistochemical studies demonstrated that OPN was present only in a subset of spinal motoneurons before avulsion. After avulsion, the number of OPN-expressing motoneurons increased, although the total number of motoneurons was reduced. OPN expression also became apparent in activated microglia/macrophages and astrocytes. These data suggest that the upregulation of OPN after spinal root avulsion is involved in two events, the protection of neurons and the post-traumatic inflammatory response in microglia/macrophages and astrocytes.