Yoshiyasu Arai

A microRNA regulatory mechanism of osteoblast differentiation

Growing evidence shows that microRNAs (miRNAs) regulate various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; however, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast differentiation remains elusive. Here, through comprehensive analysis of miRNAs expression during osteoblast differentiation, we show that miR-206, previously viewed as a muscle-specific miRNA, is a key regulator of this process. miR-206 was expressed in osteoblasts, and its expression decreased over the course of osteoblast differentiation. Overexpression of miR-206 in osteoblasts inhibited their differentiation, and conversely, knockdown of miR-206 expression promoted osteoblast differentiation. In silico analysis and molecular experiments revealed connexin 43 (Cx43), a major gap junction protein in osteoblasts, as a target of miR-206, and restoration of Cx43 expression in miR-206-expressing osteoblasts rescued them from the inhibitory effect of miR-206 on osteoblast differentiation. Finally, transgenic mice expressing miR-206 in osteoblasts developed a low bone mass phenotype due to impaired osteoblast differentiation. Our data show that miRNA is a regulator of osteoblast differentiation.

Dynamic motion study of the whole lumbar spine by videofluoroscopy

Study Design. Dynamic lumbar flexion‐extension motion was assessed by videofluoroscopy. Objectives. To identify the motion patterns of the whole lumbar spine in normal subjects and in patients with low back pain or spondylolisthesis during actual movement. Summary of Background Data. Assessment of lumbar instability on terminal radiographs is controversial. Information regarding spinal kinematics during actual movementin vivo is scarce. Methods. Fluoroscopic lumbar sagittal motion videos were recorded in volunteers (n = 13; mean age, 22.3) and in patients with chronic low back pain (n = 8; mean age, 43.5) and degenerative spondylolisthesis(n = 8; mean age, 63.1) while the subjects bent forward from a standing neutral position(eccentric motion) and then returned to the original position (concentric motion). The videos recorded approximately 8 seconds of motion and were converted to still images at 5 frames per second. Disc angles from the horizontal line were measured to estimate sagittal rotation of each segment. Disc degeneration was evaluated on T2‐weighted midsagittal magnetic resonance image. Results. In the volunteer group, six exhibited sequentially spreading motion, four exhibited simultaneous motion, and three showed an altered motion‐spreading pattern in the eccentric phase. The first two patterns were considered normal. Six (67%) of the patients with chronic low back pain also showed normal patterns, but seven (88%) of the patients with degenerative spondylolisthesis showed disordered patterns. The order of motion in the concentric phase was also different among the three groups. Prolonged deflection of the slipped segment was observed more frequently in the patients with degenerative spondylolisthesis. Disc degeneration was not always associated with motion‐spreading order and the motion patterns. Conclusion. Segmental instability influences the whole lumbar motion in patients with degenerative spondylolisthesis. The patients with chronic low back pain did not show a significant difference when compared with the volunteers.

Evaluation of voltage-sensitive dyes for long-term recording of neural activity in the hippocampus.

Abstract. We searched for an optimal voltage-sensitive dye for optical measurements of neural activity in the hippocampal slice by evaluating several merocyanine-rhodanine and oxonol dyes. The wavelength dependence (action spectra), pharmacological effects of staining, signal size, signal-to-noise ratio, and the utility of the dyes for long-term continuous recording were examined for four merocyanine-rhodanine dyes (NK2761, NK2776, NK3224 and NK3225), which had been reported to be optimal in embryonic nervous systems, and for two oxonol dyes (NK3630 (RH482) and NK3041 (RH155)), which have been among the most popular potentiometric probes for the hippocampal slice preparation. NK2761, NK3224 and NK3225 provided large signal-to-noise ratios, and proved to be useful for optical recordings lasting several hours. NK3630 was most suitable for long-term recording, although the signal-to-noise ratio was slightly inferior to that of the merocyanine-rhodanines. Using NK3630 (RH482) on the hippocampal slice preparation, we demonstrate here that long-term potentiation can be monitored stably for more than 8 hr.

Middle-Term Results of a Prospective Comparative Study of Anterior Decompression With Fusion and Posterior Decompression With Laminoplasty for the Treatment of Cervical Spondylotic Myelopathy

Study Design. A clinical prospective study. Objective. To assess whether clinical and radiologic outcomes differ between anterior decompression and fusion (ADF) and laminoplasty (LAMP) in the treatment of cervical spondylotic myelopathy (CSM). Summary of Background Data. No reports to date have accurately and prospectively compared middle-term clinical outcomes after anterior and posterior decompression for CSM. Methods. We prospectively performed LAMP (n = 50) in 1996, 1998, 2000, and 2002, and ADF (n = 45) in 1997, 1999, 2001, and 2003. The Japanese Orthopedic Association (JOA) score, recovery rate, and each item of the JOA score were evaluated. For radiographic evaluation, the lordotic angle and range of motion (ROM) at C2–C7 and residual anterior compression to the spinal cord (ACS) after LAMP on magnetic resonance imaging were investigated. Results. Eighty-six patients (ADF n = 39; LAMP n = 47) could be followed for more than 5 years (follow-up rate 91.5%). Demographics were similar between the two groups. The mean JOA score and recovery rate in the ADF group were superior to those in the LAMP group from 2-year data collected after surgery. However, LAMP was safer and less invasive than ADF with respect to physical status and complications in the perioperative period. For individual items of the JOA score, the ADF group showed significantly more improvement of upper extremity motor function than the LAMP group (P < 0.05). There was a significant difference in maintenance of the lordotic angle in the ADF group compared with the LAMP group despite no difference in ROM. The LAMP group was divided into two subgroups: (1) LAMP(+) (n = 16) comprising patients who had ACS at 2 years after surgery, and (2) LAMP(–) (n = 31) comprising patients without ACS. Recovery rate differed significantly between the LAMP(+) and LAMP(−) groups despite there being no difference between the LAMP(−) and ADF groups. Conclusion. The recovery rate of the JOA score in the ADF group was better than that in the LAMP group. The clinical outcomes after LAMP could be influenced by ACS.

Optical illustration of glutamate-induced cell swelling coupled with membrane depolarization in embryonic brain stem slices

USING intrinsicand voltage-sensitive dye optical recordings, we have elucidated coupling of glutamateinduced depolarization and neuronal swelling in early embryonic chick brain stem slices. Twenty-four slices were prepared from 8-day old chick embryos, and stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). The pressure ejection of glutamate to one site within the preparation evoked changes in transmitted light intensity. With 700 nm incident light, three components were identified in glutamate-induced optical changes. The first component was wavelength dependent, while the second and third components were independent of the wavelength. With reference to the action spectrum of the merocyanine-rhodanine dye and osmotic changes in optical properties, we concluded that the first component reflects glutamate-induced depolarization of the membrane, and that the second component is an intrinsic light-scattering change resulting from neural cell swelling coupled with the membrane depolarization.

Optical mapping reveals the functional organization of the trigeminal nuclei in the chick embryo.

The functional organization of the trigeminal nuclei during embryogenesis was investigated using multiple-site optical recording with a fast voltage-sensitive dye. Brainstem preparations with three classified trigeminal nerve afferents, the ophthalmic, maxillary and mandibular nerves, together with motor nerve fibers, were dissected from five- to eight-day-old chick embryos. Electrical responses evoked by trigeminal nerve stimulations were optically recorded simultaneously from many loci of the stained preparations. We identified three response areas related to the trigeminal nerve: area I, located cephalic to the level of the trigeminal ganglion; area II, located caudal to the level of the trigeminal ganglion; and area III, located at the level of the trigeminal root. The neural responses in areas I and II were evoked by ophthalmic, maxillary or mandibular nerve stimulation, while the responses in area III were detected when the stimulation was applied to the trigeminal motor nerve. In comparison with the morphology indicated by DiI labeling, the results suggest that areas I, II and III correspond to the principal sensory nucleus of the trigeminal nerve, the spinal sensory nucleus of the trigeminal nerve and the trigeminal motor nucleus, respectively. We identified two components of the optical response: a fast and a slow signal. In five-day-old preparations, fast spike-like signals related to action potentials were recorded from the three response areas. In six-day-old preparations, slow optical signals which reflect glutamate-mediated excitatory postsynaptic potentials were detected from area II only when the ophthalmic nerve was stimulated: no slow signal was evoked by maxillary or mandibular nerve stimulation. In seven- and eight-day-old preparations, slow signals were detected from both areas I and II with every nerve stimulation. These results suggest that synaptic function is first generated in the spinal trigeminal nucleus by the six-day embryonic stage, and the developmental organization of synaptic function is not the same in the three trigeminal nerves or in the two sensory nuclei. Contour line maps of the signal amplitude revealed that the size and the area of the neural responses within the trigeminal nuclei changed dramatically with development. We compared the spatial distribution and temporal dynamics of the optical signals between the ophthalmic, maxillary and mandibular nerve stimulations, and we found that somatotopic organization is less clear in a rostrocaudal/mediolateral X-Y plane, although the areas of the maxillary and mandibular nerves appeared to separate in the lateral direction.

Hybrid grafting using bone marrow aspirate combined with porous β-tricalcium phosphate and trephine bone for lumbar posterolateral spinal fusion: a prospective, comparative study versus local bone grafting.

Study Design. A prospective, comparative study. Objective. We developed a hybrid graft (HBG) of porous &bgr;-tricalcium phosphate ceramics/percutaneously harvested bone sticks/autologous bone marrow aspirate for lumbar posterolateral fusion (PLF). The aim of this study was to investigate the efficacy of the HBG as a substitute for conventional corticocancellous iliac autografts. Summary of Background Data. Iliac crest bone graft (ICBG) has been traditionally used as the golden standard for lumbar spinal fusion. The significant complication rate associated with harvesting corticocancellous ICBG, however, has encouraged development of alternative graft substitutes. Methods. From September 2005, 61 consecutive patients underwent decompressive laminotomy and 1-level instrumented PLF. Each patient in this study had the constructs of the HBG placed on 1 side of the intertransverse process gutter. An autologous local bone graft (LBG) harvested during decompressive laminotomy was placed on the other side as a control. Radiographic evaluation was performed at 6 months, 1 year after surgery, and subsequently on an annual basis. The fusion statuses on either side of vertebra were compared. Results. The flexion-extension motion in the dynamic x-rays at the target level decreased over time. Only 1 case exhibited over 5° of angular motion 2 years after surgery. In the evaluation of fusion status, the fusion rate for the HBG side (68.9% at 6 months, 83.6% at 1 year, 93.5% at 2 years) was higher than that for the LBG side (49.2% at 6 months, 75.4% at 1 year, 89.1% at 2 years) with a significant difference at 6 months after surgery. No significant complications at the donor site were found postoperatively. Conclusion. The HBG promoted posterolateral spinal fusion without significant donor site morbidity. Because of its efficacy and safety, this hybrid construct seems promising as an alternative to conventional iliac bone grafts for lumbar spinal fusion.

Cervical Sagittal Imbalance is a Predictor of Kyphotic Deformity After Laminoplasty in Cervical Spondylotic Myelopathy Patients Without Preoperative Kyphotic Alignment.

Study Design. A retrospective cohort study. Objective. The aim of this study is to investigate the preoperative factors for postlaminoplasty kyphotic deformity in cervical spondylotic myelopathy (CSM) patients without preoperative kyphotic alignment focused on the cervical sagittal balance. Summary of Background Data. After laminoplasty (LAMP), appropriate decompression may be obtained when cervical lordosis is maintained to allow the posterior shift of the spinal cord. Therefore, LAMP is not suitable for patients with preoperative cervical kyphosis. However, we sometimes encounter patients who developed postoperative kyphosis despite normal preoperative alignment. The risk factors of postlaminoplasty kyphotic deformity for the patients without preoperative kyphotic alignment are not well known. Methods. A total of 174 consecutive patients who received a double-door LAMP for CSM without preoperative kyphotic alignment and completed a 1-year follow-up were enrolled. Cervical lateral X-ray images obtained in the standing position were measured at the preoperative stage and during a 1-year follow-up visit. The radiographic measurements included the following: (1) C2-7 lordotic angle (C2-7 angle), (2) C2-7 range of motion (C2-7 ROM), (3) CGH (center of gravity of the head)-C7 SVA, and (4) C7 slope. The clinical results were evaluated using the Japanese Orthopedic Association score system for cervical myelopathy (C-JOA score). Results. Postoperative kyphotic deformity was observed in 9 patients (5.2%). The recovery rates of the C-JOA scores at the 1-year follow-up period in the kyphotic deformity (+) group were inferior to those of the kyphotic deformity (−) group. The CGH-C7 SVA and advanced age were detected as preoperative risk factors using multivariate analysis. Cutoff values for predicting postlaminoplasty kyphotic deformity were a CGH-C7 SVA = 42 mm and an age of 75 years. Conclusion. Cervical sagittal imbalance and advanced age were the preoperative risk factors for kyphotic deformity after LAMP for CSM in patients without preoperative cervical kyphotic alignment. Level of Evidence: 4

Optical imaging of spreading depolarization waves triggered by spinal nerve stimulation in the chick embryo: possible mechanisms for large-scale coactivation of the central nervous system.

Using a multiple‐site optical recording technique with a voltage‐sensitive dye, we found that widely spreading depolarization waves were evoked by dorsal root stimulation in embryonic chick spinal cords. Spatiotemporal maps of the depolarization waves showed that the signals were mainly distributed in the ventral half of the slice, with the highest activity in the ventrolateral area. The propagation velocity of the waves was estimated to be in the order of mm/s. Depolarization waves were evoked in the ventral root‐cut preparation, but not in the dorsal root‐cut preparation, suggesting that the wave was triggered by synaptic inputs from the primary afferents, and that activation of the motoneurons was not essential for wave generation. In intact spinal cord–brain preparations, the depolarization wave propagated rostrally and caudally for a distance of several spinal segments in normal Ringers solution. In a Mg2+‐free solution, the amplitude and extent of the signals were markedly enhanced, and the depolarization wave triggered in the cervical spinal cord propagated to the brainstem and the cerebellum. The depolarization wave demonstrated here had many similarities with the vagus nerve‐evoked depolarization wave reported previously. The results suggest that functional cell‐to‐cell communication systems mediated by the depolarization wave are widely generated in the embryonic central nervous system, and could play a role in large‐scale coactivation of the neurons in the spinal cord and brain.

Multiple‐site optical recording reveals embryonic organization of synaptic networks in the chick spinal cord

We examined embryonic expression of postsynaptic potentials in stages 26–31 (E5 to E7) chick spinal cord slices. Slow optical signals related to the postsynaptic potentials which were evoked by electrical stimulation of afferent fibers were identified in the dorsal grey matter and the ventral motoneuronal area. In cervical spinal cord (C13) preparations, the dorsal slow signal appeared from stage 28 (E6), whilst the ventral slow signal was recognized from stage 29. At stages 26 and 27 (E5), no slow signal was observed in either the dorsal or ventral regions. On the other hand, in lumbosacral spinal cord (LS5) preparations, the dorsal, as well as ventral, slow signals appeared from stage 29; at stage 28 no slow signal was detected in the dorsal or ventral regions. These results suggest that there are differences in the ontogenetic expression of synaptic functions between the dorsal and ventral regions, and between the cervical and lumbosacral spinal cords. In embryos older than stage 29, removal of Mg2+ from the bathing solution markedly enhanced the amplitude and incidence of the ventral slow signal. In addition, in C13 preparations at stage 28, removal of Mg2+ elicited small slow signals in the ventral region in which no synaptic response was evoked in normal Ringers solution. The slow signals induced in the Mg2+‐free solution were blocked by 2‐amino‐5‐phosphonovaleric acid (APV), showing that they are attributable to N‐methyl‐d‐aspartate (NMDA) receptors. These results suggest that functional synaptic connections via polysynaptic pathways are already generated on motoneurons, but are suppressed by a Mg2+ block on the NMDA receptors at developmental stages when synaptic transmission from the primary afferents to the dorsal interneurons is initially expressed in the dorsal region.