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Despite extensive research on primate cognitive function, understanding how anatomical connectivity at a neural circuit level relates to information transformation across different cortical areas remains primitive. New technology is needed to visualize inter-areal anatomical connectivity in living monkeys and to tie this directly to neurophysiological function. Here, we developed a novel method to investigate this structure-function relationship, by combining optical intrinsic signal imaging (OISI) with optogenetic stimulation in living monkeys (opto-OISI). The method involves expressing channelrhodophsin-2 in one area (source) followed by optical imaging of optogenetic activations in the other area (target). We successfully demonstrated the potential of the method with interhemispheric columnar projection patterns between V1/V2 border regions. Unlike the combination of optogenetics and functional magnetic resonance imaging (opto-fMRI), opto-OISI has the advantage of enabling us to detect responses of small clusters of neurons, even if the clusters are sparsely distributed. We suggest that opto-OISI can be a powerful approach to understanding cognitive function at the neural circuit level, directly linking inter-areal circuitry to fine-scale structure and function.

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., Koda et al. present the results of a randomized phase 3 trial comparing granulocyte colony-stimulating factor versus placebo in patients with acute spinal cord injury. While the primary endpoint was not met, a sub-analysis revealed a trend towards superior efficacy of G-CSF versus placebo in an elderly population.

Purpose To elucidate the independent preoperative factors that have a significant impact on poor surgical outcome after laminoplasty for K-line (+) ossification of the posterior longitudinal ligament (OPLL). Analyses in K-line (+) patient population can exclude the influence by mal-alignment and thick OPLL, both of which are well known two major factors that have significant impact on clinical outcome. Methods The present study included 72 patients (50 male and 22 female) who underwent laminoplasty for K-line (+) cervical OPLL and were followed-up for at least 1 year. Recovery of Japanese Orthopedic Association score (JOA score) for cervical myelopathy was used as the measure of clinical outcome. For radiographic assessment, the type of OPLL, the maximum OPLL occupation ratio, the C2-C7 angle, and the segmental range of motion at the peak of OPLL (segmental ROM) were assessed. To elucidate the factors that are significantly associated with a poor clinical outcome after laminoplasty for K-line (+) OPLL, statistical analyses were conducted. Results The mean preoperative JOA score was 8.9 points and improved to 12.8 points after surgery. The recovery of JOA score was 47 ± 35%. Stepwise logistic regression following univariate analyses revealed that preoperative segmental ROM at the peak of OPLL is an independent factor associated with a poor outcome (p = 0.04, odds ratio = 1.15). Conclusions Large preoperative segmental ROM at the peak of the OPLL is an independent factor that has significant impact on poor surgical outcome after laminoplasty for K-line (+) OPLL.

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.

Study design A retrospective multicenter survey. Objective To investigate the perioperative complications of oblique lateral interbody fusion (OLIF) surgery. Summary of background data OLIF has been widely performed to achieve minimally invasive, rigid lumbar lateral interbody fusion. The associated perioperative complications are not yet well described. Methods The participants were patients who underwent OLIF surgery under the diagnosis of degenerative lumbar diseases between April 2013 and May 2015 at 11 affiliated medical institutions. The collected data were classified into intraoperative and early-stage postoperative (≤1 mo) complications. The intraoperative complications were then subcategorized into organ damage (neural, vertebral, vascular, and others) and other complications, mainly related to instrumental failure. The collected data were also divided and analyzed based on whether the surgeon was certified to perform the surgery and the incidence of complications in the early (April 2013-March 2014) and late stages (April 2014-May 2015) of OLIF introduction. Results In the 155 included patients, 75 complications were reported (incidence rate, 48.3%). The most common complication was endplate fracture/subsidence (18.7%), followed by transient psoas weakness and thigh numbness (13.5%) and segmental artery injury (2.6%). Almost all these complications were transient, except for three patients who had permanent damage: one had ureteral injury and two had neurological injury. Postoperative complications included surgical site infection (1.9%) and reoperation (1.9%). Whether the primary operator was experienced did not affect the incidence of complications. Regarding the introductory stage, the incidence of complications was 50% in the early stage and 38% in the late stage. Conclusion The overall incidence of perioperative complications of OLIF surgery reached 48.3%, of which only 1.9% resulted in permanent damage. Our analysis based on surgeon experience indicated that the OLIF procedure could be performed without increasing incidence of complications, under the guidance of experienced supervisors. Level of evidence 3.

The guidance of axonal projections to ipsilateral and contralateral regions is essential for integration of bilateral sensory information and coordination of movement. In the development of olivocerebellar projections, newborn neurons of inferior olivary (IO) nuclei ventrally migrate from the hindbrain rhombic lip to the floor plate (FP). The cell bodies of IO neurons cannot cross the FP but their axons can, and thus IO neurons project their axons only to the contralateral cerebellar cortex. The molecular mechanisms determining the contralateral axonal projections of IO neurons, however, are obscure. The IO neurons and their axons express EphA4, whereas the FP expresses an EphA4 ligand, EphrinB3, from embryonic day 12.5. Therefore, we tested whether EphA4-deficient mice (EphA4−/−) would show impairment in the development of olivocerebellar projections. We found that, in EphA4−/− embryos, some of the IO neurons projected their axons to the ipsilateral cerebellar cortex because the cell bodies of the IO neurons abnormally crossed the FP. Furthermore, even in adults, EphA4−/− cerebella were bilaterally innervated by unilateral IO subnuclei. These observations indicate that EphA4 is involved in the contralateral axonal projections of IO neurons by preventing their cell bodies from crossing the midline FP. J. Comp. Neurol. 520:1702–1720, 2012. © 2011 Wiley Periodicals, Inc.

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.

The adult cerebellum is organized into longitudinal compartments that are revealed by specific axonal projections (olivocerebellar and corticonuclear projections). These compartments in the adult cerebellum are closely correlated with the striped expression of zebrin II (aldolase C), a late-onset marker of Purkinje cells. Similarly, the embryonic cerebellum is organized into longitudinal compartments that are revealed by striped expression of other genes (early-onset markers). The cerebellar compartments are thought to be the basic and functional subdivisions of the cerebellum. However, the relationship between the embryonic (early-onset) and the adult (late-onset) compartments has remained unknown, because the pattern of the embryonic compartments is distinct from that of the adult compartments. To examine this issue, we labeled Purkinje cells (PCs) born at embryonic day (E) 10.5, E11.5, and E12.5 by using an adenoviral vector and traced their fated positions in the adult cerebellum. By comparing the striped distribution of each cohort of birth date-related PCs with the striped pattern of zebrin II immunoreactivity (zebrin II bands) in the entire adult cerebellum, we found that the striped distribution of PCs correlated strikingly with zebrin II bands. Generally, a single early-onset compartment was transformed directly into a single late-onset compartment. Therefore, our observation also indicated the close correlation between the compartments formed by birth date-related PCs and olivocerebellar projections. Furthermore, we found that the cerebellum was composed of three units showing lateral-to-medial developmental gradients, as revealed by the birth dates of PCs. The results suggest that PC birth dates play an important role in organizing cerebellar compartmentalization.

We describe a low-cost, small, remotely triggerable LED device for wireless control of transcranial optical stimulation of cortical neurons, for use in freely moving mice. The device is easily mountable on the head of a mouse with a high-polymer block. Using the Thy1–ChR2–YFP transgenic mice, we demonstrate that the device is capable of remotely triggering muscle twitches upon activation of the primary motor cortex in freely moving conditions.

Sonic hedgehog (Shh) signaling regulates cell differentiation and proliferation during brain development. However, the role of Shh in neurogenesis during late gestation (embryonic day 13.5–18.5) remains unclear. Herein, we used a genetic approach and in utero electroporation to investigate the role of mouse Shh and patched homolog 1 (Ptch1), the putative receptor for Shh. Proliferating cortical intermediate (basal) progenitor cells (IPCs) were severely reduced in Shh mutant mice, suggesting that endogenous Shh signaling could play an essential role in cortical IPC development. During cortical neurogenesis, strong upregulation of Shh signaling enhanced the transition from ventricular zone (VZ) progenitors to ventralized IPCs, while low levels of signaling enhanced the generation and proliferation of cortical IPCs in the subventricular zone. The effects of Shh upregulation in this study were consistent with a phenotype of conditional loss of function of Ptch1, and the phenotype of a hypomorphic allele of Ptch1, respectively. These data indicated that endogenous Ptch1 mediates the broad effects of Shh on the transition from VZ progenitors to IPCs and activation of proliferation of the IPCs in the cortex during late gestational stages.

We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

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/m 2 /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.

Postoperative C5 palsy is a common complication after cervical spine decompression surgery. However, the incidence, prognosis, and etiology of C5 palsy after anterior decompression with spinal fusion (ASF) have not yet been fully established. In the present study, we analyzed the clinical and radiological characteristics of patients who developed C5 palsy after ASF for cervical degenerative diseases. The cases of 199 consecutive patients who underwent ASF were analyzed to clarify the incidence of postoperative C5 palsy. We also evaluated the onset and prognosis of C5 palsy. The presence of high signal changes (HSCs) in the spinal cord was analyzed using T2-weighted magnetic resonance images. C5 palsy occurred in 17 patients (8.5%), and in 15 of them, the palsy developed after ASF of 3 or more levels. Among ten patients who had a manual muscle test (MMT) grade ≤2 at the onset, five patients showed incomplete or no recovery. Sixteen of the 17 C5 palsy patients presented neck and shoulder pain prior to the onset of muscle weakness. In the ten patients with a MMT grade ≤2 at the onset, nine patients showed HSCs at the C3-C4 and C4-C5 levels. The present findings demonstrate that, in most patients with severe C5 palsy after ASF, pre-existing asymptomatic damage of the anterior horn cells at C3-C4 and C4-C5 levels may participate in the development of motor weakness in combination with the nerve root lesions that occur subsequent to ASF. Thus, when patients with spinal cord lesions at C3-C4 and C4-C5 levels undergo multilevel ASF, we should be alert to the possible occurrence of postoperative C5 palsy.

Object The authors assessed the clinical course in patients with a narrowed cervical spinal canal caused by ossification of the posterior longitudinal ligament (OPLL), but who have no or only mild myelopathy. Additionally, the authors analyzed the factors contributing to the development and aggravation of myelopathy in patients with OPLLinduced spinal canal stenosis. Methods Between 1997 and 2004, the authors selected treatments for patients with cervical OPLL in whom the residual space available for the spinal cord was ≤ 12 mm. Treatment decisions were based on the severity of myelopathy at presentation. Twenty-one patients with no or mild myelopathy (defined as a Japanese Orthopaedic Association [JOA] scale score ≥ 14 points) received conservative treatment, with a mean follow-up period of 4.5 years. In 20 patients with moderate or severe myelopathy (JOA scale score < 14 points), the authors performed surgery via an anterior approach. The clinical course in these patients was assessed with the JOA scale and the OPLL types were classified. The authors evaluated the range of motion between C-1 and C-7, the developmental segmental sagittal diameter, the percentage of spinal canal diameter occupied by the OPLL (% ratio), and the residual space available for the spinal cord on cervical radiographs; T2-weighted MR images were examined for high signal changes (HSCs). Results In the conservative treatment group, 8 patients showed improvement, 12 remained unchanged, and 1 patient's condition became slightly worse during the observation period. Fifteen patients in this group had mixedtype, 3 had continuous-type, 2 had localized-type, and 1 had a segmental-type OPLL. In the surgically treated group, there were 12 patients with segmental-type, 10 patients with mixed-type, and 1 with localized-type OPLL. The mean range of motion at C1–7 was 36.4° in the conservatively treated group and 46.5° in the surgical group (p < 0.05). No significant difference was seen between the groups in terms of developmental segmental sagittal diameter, % ratio, or residual space available for the cord. No HSCs were noted in the conservative group, while 17 patients in the surgical group had HSCs (p < 0.05). Conclusions In the present study, the authors demonstrate that the mobility of the cervical spine and the type of OPLL are important factors contributing to the development and aggravation of myelopathy in patients with OPLLinduced spinal canal stenosis. The authors advocate conservative treatment in most patients with OPLLs who have no or only mild myelopathy, even in the presence of spinal canal narrowing.

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.

Vomeronasal receptors from the V1R and V2R gene families mediate the detection of chemical stimuli such as pheromones via the vomeronasal organ (VNO). The differential expression of vomeronasal receptors might contribute in part to a variety of pheromonal effects, which are different sexually, developmentally and even individually. However, little is known about the mechanisms controlling vomeronasal receptor expression. Cultured vomeronasal sensory neurons (VSNs) bear phenotypic resemblance to the intact VNO but they remain immature. Because indices of VSN maturation are increased by coculture with the target cells for VSNs, accessory olfactory bulb (AOB) neurons, AOB neurons may regulate vomeronasal receptor expression and functional maturation in VSNs. To test this hypothesis, we examined the expression of V2R-type vomeronasal receptors (VR1 and VR4) and chemosensory responsiveness in VNOs cocultured with AOB neurons. Immunoblot and immunocytochemical analysis revealed that the coculture of VNOs with AOB neurons resulted in a greater expression of VR1 and VR4 after 10 days than VNOs cultured alone. Moreover, calcium imaging analysis showed that cocultured VNOs responded to urine components applied iontophoretically into their cavities with a time course similar to the V2R expression, in contrast to singly cultured VNOs that displayed no response. These results demonstrate that AOB neurons induce the expression of vomeronasal receptors in VSNs, allowing them to function.

The serine/threonine kinase p21-acti- vated kinase 1 (Pak1) modulates actin and microtubule dynamics. The neuronal functions of Pak1, despite its abundant expression in the brain, have not yet been fully delineated. Previously, we reported that Pak1 mediates initiation of dendrite formation. In the present study, the role of Pak1 in dendritogenesis, spine forma- tion and maintenance was examined in detail. Overex- pression of constitutively active-Pak1 in immature corti- cal neurons increased not only the number of the pri- mary branching on apical dendrites but also the number of basal dendrites. In contrast, introduction of dominant negative-Pak caused a reduction in both of these mor- phological features. The length and the number of sec- ondary apical branch points of dendrites were not sig- nificantly different in cultured neurons expressing these mutant forms, suggesting that Pak1 plays a role in den- dritogenesis. Pak1 also plays a role in the formation and maintenance of spines, as evidenced by the altered spine morphology, resulting from overexpression of mutant forms of Pak1 in immature and mature hippocampal neurons. Thus, our results provide further evidence of the key role of Pak1 in the regulation of dendritogenesis, dendritic arborization, the spine formation, and main- tenance. ' 2007 Wiley Periodicals, Inc. Develop Neurobiol 67: 655- 669, 2007

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.

The bit error rate (BER) and signal-to-noise ratio (SNR) of perpendicular recording media were investigated with a software channel and magnetic clustering based microstructure model of irregular magnetization transitions. The calculations show that the BER is not only determined by the mean cluster size, but also by the fractional standard deviation, as is the SNR. However, the BER performance does not correlate well with the SNR. Reducing the cluster size distribution is more effective at improving the BER than the SNR. An explanation for this discrepancy can be found by considering the distribution of transition position fluctuations and transition percolation.

Study design Case report of a Down syndrome patient with right vertebral artery fenestration and abnormalities of the craniovertebral junction. Objectives Describe the utility of 3-dimensional computed tomography angiography for evaluating vertebral artery anomalies before surgery. Summary of background data Previous reviews evaluating catheter angiograms identified various anomalies of vertebral artery at the craniovertebral junction. The frequency of vertebral artery anomalies is increased in patients having osseous anomalies at the craniovertebral junction. Down syndrome is associated with a high incidence of bone abnormalities at the craniovertebral junction, but there have been no published reports of vertebral artery anomalies per se at the craniovertebral junction. Methods A 16-year-old woman with trisomy 21 presented with gait abnormalities and myelopathy in association with bone abnormalities at the craniovertebral junction, including hypoplastic odontoid and ossiculum terminale. Computed tomography angiography showed that right vertebral artery bifurcated after exiting the C2 transverse foramen with one branch passing through the C1 transverse foramen, whereas the other turned posteromedially and entered the spinal canal between C1 and C2. Results Occipito-C2 posterior fusion was performed with a rod and screw system. Intraoperatively, the course of the anomalous right vertebral artery was identified by Doppler angiography, and the surgical approach was modified to allow safe pedicle screw insertion while avoiding vertebral artery injury. After surgery, myelopathy resolved within 3 months. Conclusions Before corrective surgery of craniovertebral junction anomalies in patients with Down syndrome, the possibility of vertebral artery anomalies associated with abnormal craniovertebral junction anatomy should be considered. With preoperative 3-dimensional computed tomography angiography, we can precisely identify the anomalous vertebral artery and modify the surgical approach to reduce the possible risk of intraoperative vertebral artery injury in advance.

The multilayered structure of the cerebral cortex has been studied in detail. Early-born neurons migrate into the inner layer and late-born neurons migrate into more superficial layers, thus establishing an inside-out gradient. The progenitor cells appear to acquire layer-specific properties at the time of neuronal birth; however, the molecular mechanisms of cell-fate acquisition are still unclear, because it has been difficult to identify a cohort of birthdate-related progenitor cells. Using replication-defective adenoviral vectors, we successfully performed “pulse gene transfer” into progenitor cells in a neuronal birthdate-specific manner. When adenoviral vectors were injected into the midbrain ventricle of mouse embryos between embryonic day 10.5 (E10.5) and E14.5, the adenoviral vectors introduced a foreign gene into a specific cohort of birthdate-related progenitor cells. The virally infected cohorts developed normally into cortical neurons and formed the canonical cortical layers in an inside-out manner. This technique allows us to distinguish a cohort of birthdate-related progenitor cells from other progenitor cells with different birthdates and to introduce a foreign gene into specific subsets of cortical layers by performing adenoviral injection at specific times. This adenovirus-meditated gene transfer technique will enable us to examine the properties of each subset of progenitor cells that share the same neuronal birthdate.

The adult cerebellum is functionally compartmentalized into clusters along the mediolateral axis (M-L clusters), and a variety of molecular makers are expressed in specific subsets of M-L clusters. These M-L clusters appear to be the basic structure in which cerebellar functions are performed, but the mechanisms by which cerebellar mediolateral compartmentalization is established are still unclear. To address these questions, we examined the development of M-L clusters using replication-defective adenoviral vectors. The adenoviral vectors effectively introduced foreign genes into the neuronal progenitor cells of the cerebellum in a birth date-specific manner, allowing us to observe the native behavior of each cohort of birth date-related progenitor cells. When the adenoviral vectors were injected into the midbrain ventricle of mouse embryos on embryonic days 10.5 (E10.5), E11.5, and E12.5, the virally infected cerebellar progenitor cells developed into Purkinje cells. Notably, the Purkinje cells that shared the same birth date formed specific subsets of M-L clusters in the cerebellum. Each subset of M-L clusters displayed nested and, in part, mutually complementary patterns, and these patterns were unchanged from the late embryonic stage to adulthood, suggesting that Purkinje cell progenitors are fated to form specific subsets of M-L clusters after their birth between E10.5 and E12.5. This study represents the first such direct observation of Purkinje cell development. Moreover, we also show that there is a correlation between the M-L clusters established by the birth date-related Purkinje cells and the domains ofengrailed-2, Wnt-7B, L7/pcp2, and EphA4 receptor tyrosine kinase expression.

To clarify the mechanism of cerebellar development, we have cloned a gene, named zic, encoding a zinc finger protein that is expressed abundantly in granule cells throughout development of the cerebellum. zic has a significant homology to the zinc finger domain of the Caenorhabditis elegans tra1 gene, the Drosophila cubitus interruptus Dominant gene, and the human GLI oncogene. An in situ hybridization study revealed that zic showed a restricted expression pattern in the granule cells and their putative precursor cells. It is also expressed at an early embryonic stage in the dorsal half of the neural tube. The expression pattern and nuclear localization were confirmed by immunohistochemical study. Furthermore, the bacterially expressed zic protein containing the zinc finger domains bound to the GLI-binding sequence. These findings suggest that zic is one of a number of nuclear factors involved in both differentiation in early development and maintenance of properties of the cerebellar granule cells.

Cell-mediated cytotoxicity may be involved in delayed and/or chronic xenograft rejection in which apoptosis is induced in the grafted cells via the Fas/Fas-ligand (FasL) and perforin/granzyme pathways. One barrier to the potential use of xeonogenic grafts for humans may be Fas/FasL-mediated apoptosis, which would be blocked by the gene expression of cytokine response modifier A (CrmA), a cowpox virus gene product. The purpose of this study is to explore whether crmA is an effective candidate gene for inhibiting apoptosis in an in vitro model of xenograft rejection, using Fas-expressing non-primate cells cultured with a soluble recombinant human FasL (sFasL). A recombinant adenovirus vector expressing CrmA (AxCALNLCrmA) was successfully generated with a Cre-mediated switching system. PK15 cells, derived from a porcine kidney and infected with AxCALNLCrmA and/or AxCANCre at a multiplicity of infection (MOI) ranging from 0.1 to 100, were cultured with human sFasL derived from KFL74.18, a human FasL-overexpressed cell line. The gene-expression level of the PK15 cells was confirmed by CrmA-immune staining. Approximately 70% of the control PK15 cells showed induced apoptosis when cultured with sFasL. In contrast, the apoptosis was dramatically reduced in crmA-gene-transduced PK15 cells. The inhibitory effect of apoptosis increased with an increase in the infection dose of AxCANCre. In addition, the activity of caspases 3 and 8 was significantly inhibited in the crmA-transduced cells. These results indicate that CrmA is an effective gene product for inhibiting Fas/FasL-mediated apoptosis, which suggests the potential therapeutic use of its gene transduction to protect against graft damage due to delayed and/or chronic xenograft rejection.

Solute carrier (SLC) transporters play important roles in absorption and disposition of drugs in cells; however, the expression pattern of human SLC transporters in the skin has not been determined. In the present study, the expression patterns of 28 human SLC transporters were determined in the human skin. Most of the SLC transporter family members were either highly or moderately expressed in the liver, while their expression was limited in the skin and small intestine. Treatment of human keratinocytes with a reactive metabolite of ibuprofen significantly reduced cell viability. Expression array analysis revealed that S100 calcium binding protein A7A (S100A7A) was induced nearly 50-fold in dermal cells treated with ibuprofen acyl-glucuronide. Determination of the expression of drug-metabolizing enzymes as well as drug transporters prior to the administration of drugs would make it possible to avoid the development of idiosyncratic skin diseases in individuals.

Posttranscriptional regulation plays essential roles in the wide variety of events that occur during animal development, including the localization of maternal-effect gene products within oocytes, cell fate determination, and cell polarity formation by controlling alternative splicing, mRNA stability, RNA transport, and/or the translation of existing mRNAs (11, 57, 66). Among developing tissues, the nervous system in particular uses a variety of posttranscriptional means to regulate the vast cellular diversity and synaptic plasticity that is generated. Neural RNA-binding proteins are likely to play essential roles in mediating these processes (44). Two classes of neural RNA-binding proteins with ribonucleoprotein-type RNA recognition motifs (RRMs), the Elav and Musashi subfamilies, have been proposed (44). The Elav subfamily includes the mammalian Elav homologue, Hu proteins, whose members are expressed in postmitotic neurons and have been proposed to be required for the survival or terminal differentiation of these cells (1, 2, 34, 45, 64, 69). Intensive study in this area revealed that Hu proteins regulate the gene expression involved in neuronal differentiation by controlling RNA stabilization or translation (2, 13, 35, 48, 64). On the other hand, the Musashi subfamily proteins are expressed in neural precursor cells rather than postmitotic neurons (15, 28, 44, 49, 52). Musashi1 (Msi1) was isolated as a mammalian homologue of Drosophila Musashi (d-Msi), which is required for the asymmetric cell division of sensory neural precursor cells (38). In Drosophila, genes that are responsible for the proper asymmetric cell division of sensory organ precursor cells or central nervous system neuroblasts have been identified and extensively investigated. Interestingly, many of these genes have been shown to be involved in the regulation of Notch signaling, including numb, tramtrack, Notch, and Delta (reviewed by Jan and Jan [23]). Recently, our laboratory showed that d-Msi represses the expression of tramtrack, which encodes a transcriptional repressor, at the level of translation (21, 43). We also identified the mammalian homologue of Drosophila Musashi, Musashi1, which is highly enriched in neural progenitor cells in the developing mouse embryonic central nervous system (28, 52, 53). Msi1 expression is gradually down-regulated during the course of neural differentiation. The Msi1 protein consists of 362 amino acid (aa) residues, and it contains two conserved RRMs in its N-terminal half and a putative nuclear export signal in its C-terminal half, which is consistent with its observed localization of the cytoplasm in embryonic neural progenitor cells (28, 52). To help elucidate the role of Msi1 protein in neural progenitor cells, we sought to determine (i) the RNA sequences that bind to Msi1, (ii) an in vivo target RNA of Msi1, and (iii) the mechanism of action of Msi1 on the expression of its downstream target genes. To this end, in the present study we identified the RNA sequence for Msi1 and demonstrated putative translational repression of a likely in vivo target gene mammalian numb (m-numb).

手術を行った頸髄症56例に対し簡易上肢機能検査(STEF)を行い,術前後の上肢運動機能を評価した.STEFは術前後で有意に改善し,手術およびリハビリテーションの効果を評価するのに有用であると思われた.10項目の検査のうち特に検査8～10において,術前は低値を示したが,術後の改善が著しかった.これらの検査は母指と示指による細かいもののピンチ動作を要求され,手指の巧緻運動性を定量的に評価するのに有用であると思われた.STEFとJOA scoreの上肢運動機能項目は術前はSpearmanの相関係数0.70(p

Hyperimmune response via Fas/Fas-ligand and perforin/granzyme pathways may be essential in pathogenesis of virus-induced fulminant hepatitis. CrmA inhibits activation of caspases and granzyme B, suggesting it may block these pathways. We investigated whether CrmA expression would inhibit Fas-associated lethal hepatitis in mice. We successfully generated AxCALNLCrmA, a recombinant adenovirus expressing CrmA gene with a Cre-mediated switching cassette. We increased CrmA expression level in the liver transfected with AxCALNLCrmA (10(9) pfu) by increasing administration dose (10(7)-10(9) pfu) of AxCANCre, a recombinant, adenovirus-expressing Cre gene. Injection of anti-Fas antibody into the control mice rapidly led to animal death due to massive liver apoptosis, while the apoptosis was dramatically reduced in the CrmA-expressed mice. The animal survival increased with an increase of CrmA expression. The formation of active caspase-3 was markedly inhibited in the crmA-transfected hepatocytes in vitro. These results suggest that crmA is an effective gene that can inhibit immune-related liver apoptosis.

Caspase-8 is a member of the family of caspases, which are involved in the execution of apoptosis. To investigate whether caspase-8 can be used for gene therapy of gliomas, we transduced A-172 and U251 glioma cells with the caspase-8 gene via an adenoviral vector (Adv) controlled by the chicken beta-actin (CA) promoter (Advcaspase-8), and found that a similar level of caspase-8 protein induced A-172 cells to undergo necrotic cell death and U251 cells to undergo apoptotic cell death. Neither Bcl-XL nor Bcl-2, which play important roles in antiapoptotic mechanisms in gliomas, protected glioma cells from apoptosis induced by overexpression of caspase-8. Injection of Adv-caspase-8 suppressed the in vivo growth of U251 xenografts, in which apoptotic cell death remarkably increased as revealed by TUNEL analysis. Finally, we assessed whether gene therapy with a tissue-specific promoter, the myelin basic protein (MBP) promoter, is applicable to gliomas. Adv for caspase-8 controlled by the MBP promoter induced drastic apoptosis in U251 and U-373MG glioma cells, whereas it did not induce apoptosis in human endothelial cells, fibroblasts, and nerve growth factor-treated PC12 cells. These results indicate that Adv for caspase-8 effectively induced cell death in gliomas, and that this approach may be a useful modality for gene therapy of gliomas.

Gliomas express a higher amount of Fas than normal brain tissue. It is of interest to know whether expression of the Fas receptor is unfavorable to the antiapoptotic pathways in gliomas. In this study, we introduced the Fas gene via an adenovirus vector (Adeno-Fas) into the A-172, U251, and U-373 MG glioma cell lines, each of which expresses Fas on the cell surface. Infection of Adeno-Fas induced apoptosis in each glioma cell line. In U251 cells and A-172 cells that express the same level of Fas as a result of infection with Adeno-Fas, a much higher percentage of U251 cells underwent apoptosis than did A-172 cells. This suggests that each glioma cell line has its own threshold of Fas expression, above which apoptosis is induced, and that the constitutive expression of Fas is below the level of this threshold. It was found that the constitutive expression of the anti-apoptotic gene Bcl-X(L) is higher in A-172 cells than in U251 cells. Adenovirus-mediated transduction of the Bcl-X(L) gene into U251 cells effectively suppressed Adeno-Fas-mediated apoptosis. These data indicate that the Bcl-X(L) gene is one of the important determinants of the threshold for Fas-mediated apoptosis. When U251 and U-373 MG cells were transduced with the Fas gene controlled by the myelin basic protein promoter, which had been shown to be active in gliomas but not in neural tissues, the cells underwent markedly enhanced apoptosis. Taken together, these results indicate that the overexpression of Fas alone induced apoptosis in each glioma cell line. The degree of Fas-mediated apoptosis was attenuated by the expression of an anti-apoptotic gene, Bcl-X(L). The adenovirus-mediated induction of Fas gene controlled by a tissue-specific promoter (e.g., myelin basic protein promoter) would be a promising therapeutic approach for malignant glioma.

Colloidal crystals are formed in dispersions at very low ionic strengths. Various techniques have been used to study the mechanism of colloidal crystal formation, such as the ultra-small-angle technique, whose usefulness has been demonstrated previously. Recently, the use of ultra-small-angle X-ray scattering (USAXS) showed that the nearest interparticle distance in colloidal crystals has a maximum as a function of added salt concentration. This finding could not be explained by classical colloidal theories and was not consistent with the observations based on the microscopic technique. In the present study, we applied the USAXS technique using synchrotron radiation, which provides monochromatic X-rays with a high flux density, to investigate the effect of gravity on colloidal crystal formation. The high flux density of the synchrotron radiation enables one to investigate the structure of colloidal crystals in polystyrene (PS) latex dispersions with a mixture of H 2 O and D 2 O as a medium designed to match the density of the PS latex particle. The synchrotron USAXS study also revealed that the effect of gravity on the colloidal crystal formation is negligible for poly(methylmethacrylate) (PMMA) latex dispersions, in which we observed the novel behavior of the interparticle distance as a function of added salt concentration. We also found the destruction of colloidal crystals due to the continuous irradiation of synchrotron radiation. In addition, we applied ultra-small-angle neutron scattering (USANS) to study colloidal alloy structures by incorporating the contrast matching method. The USANS study confirmed that the structure of colloidal crystals in a mixture of two latex dispersions changed drastically with the physicochemical condition of the mixture.

An instrument for ultra small angle neutron scattering has been constructed at the beam line C1-3 of JRR-3M. Performance tests show that the instrument fulfills its function within the q range of 10 −5 –10 −2 A −1 at temperatures ranging at least from −20 to 100°C.

We have previously reported that human amniotic epithelial (HAE) cells expressed neuronal and glial cell markers using immunostaining and western blotting. To study the expression system of these cell markers in HAE cells, we investigated the expression of mRNA for oligodendrocyte markers in HAE cells by reverse-transcriptase-polymerase chain reaction (RT-PCR) and northern blotting. Neural cell-type-specific expression system was used to examine the transcriptional activity of myelin basic protein (MBP). Oligodendrocyte markers were expressed such as CNPase, MBP and proteolipid protein (PLP and DM-20). PLP gene transcripts in the cells were in a lower level than DM-20, compared with those of human brain. Neural cell-type-specific expression system disclosed HAE cells were about 20% positive for β-Gal using AdexMBP-NL-LacZ. This strongly indicates that HAE cells have MBP-specific gene expressing cells.

Ultra-small-angle neutron scattering (USANS) experiments on one- and two-component aqueous colloidal dispersions were carried out at the Bonse-Hart double crystal diffractometers installed at the High Flux Isotope Reactor (Oak Ridge, TN) and at the JRR-3M reactor (Tokai, Japan). The USANS curves from low-concentration suspensions of hydrogenated poly(methyl methacrylate) (h-PMMA) latices in D 2 O show scattering patterns typical for isolated solid spheres, which change dramatically after adding the ion-exchange resin to remove ionic impurities. This additive significantly enhances electrostatic interaction between particles, and thus the diffraction pattern from 2.5% h-PMMA latices in D 2 O + ion-exchange resin corresponds to a colloidal crystal with the fcc structure. The USANS from hydrogenated and deuterated component of the colloidal alloy ofh- and d-polystyrene (h-PS and d-PS) latices was measured separately by matching the scattering length density of h-PS and d-PS particles in H 2 O/D 2 O mixture. It was found that the minority component of large diameter latices were randomly distributed in the alloy dispersion, whereas the majority component of small diameter particles formed colloidal crystals.

Candidate mammalian odorant receptors were first cloned some 6 years ago. The physiological function of these receptors in initiating transduction in olfactory receptor neurons remains to be established. Here, a recombinant adenovirus was used to drive expression of a particular receptor gene in an increased number of sensory neurons in the rat olfactory epithelium. Electrophysiological recording showed that increased expression of a single gene led to greater sensitivity to a small subset of odorants.

We demonstrated neural cell-type-specific gene expression using an adenovirus vector, which is useful for delivering foreign genes into quiescent neural cells. We produced eight recombinant replication-defective adenoviruses carrying the lacZ reporter gene driven by various promoters, including those of the L7/PCP2 gene (highly restricted expression in cerebellar Purkinje cells), and the myelin basic protein (in oligodendrocytes) gene. We demonstrated in vitro and in vivo promoter-driven, neural cell-type-specific gene expression by recombinant adenoviruses. The genes were transferred into these recombinant adenoviruses and expressed with high levels of efficiency in vitro and in vivo. In primary culture, the recombinant adenoviruses AdexL7-NL-LacZ and AdexMBP-NL-LacZ appeared to be expressed in a Purkinje cell and oligodendrocyte-specific manner. Introduction of 10(8) pfu of these viruses into the adult rat cerebellum by stereotactic injection yielded neural cell-type-specific expression without apparent toxicity to the animals. Thus, adenovirus vectors are useful for cell-type-specific therapeutic uses and in studies requiring neural cell-type-specific gene expression.

The pressure equilibrium and steady state of z-pinches trapping a small amount of axial magnetic flux are studied. The Bennett relation and the Pease–Braginskii-current are modified, taking into account the superposed axial field. The line energy density decreases in the modified Bennett relation, but the decrease is only of the order e2, where e = (the axial field strength at the axis)/(the azimuthal field strength at the plasma periphery) ≪ 1. On the other hand, the current in the steady state can increase without being limited by the Pease–Braginskii-current. Hence, the radiation collapse is prevented. The decrease of line energy density in the modified Bennett relation is almost canceled in the steady state.

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.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is clinically used to treat neutropenia. G-CSF also has non-hematopoietic functions and could potentially be used to treat neuronal injury. To confirm the safety and feasibility of G-CSF administration for acute spinal cord injury (SCI), we have initiated a phase I/IIa clinical trial of neuroprotective therapy using G-CSF.The trial included a total of 16 SCI patients within 48 h of onset. In the first step, G-CSF (5 μg/kg/day) was intravenously administered for 5 consecutive days to 5 patients. In the second step, G-CSF (10 μg/kg/day) was similarly administered to 11 patients. We evaluated motor and sensory functions of patients using the American Spinal Cord Injury Association (ASIA) score and ASIA impairment scale (AIS) grade.In all 16 patients, neurological improvement was obtained after G-CSF administration. AIS grade increased by one step in 9 of 16 patients. A significant increase in ASIA motor scores was detected 1 day after injection (P0.01), and both light touch and pin prick scores improved 2 days after injection (P0.05) in the 10 μg group. No severe adverse effects were observed after G-CSF injection.These results indicate that intravenous administration of G-CSF (10 μg/kg/day) for 5 days is essentially safe, and suggest that some neurological recovery may occur in most patients. We suggest that G-CSF administration could be therapeutic for patients with acute SCI.

Based on the neuroprotective effects of granulocyte colony-stimulating factor (G-CSF) on experimental spinal cord injury, we initiated a clinical trial that evaluated the safety and efficacy of neuroprotective therapy using G-CSF for patients with worsening symptoms of compression myelopathy.We obtained informed consent from 15 patients, in whom the Japanese Orthopaedic Association (JOA) score for cervical myelopathy decreased two points or more during a recent 1-month period. G-CSF (5 or 10 μg/kg/day) was intravenously administered for five consecutive days. We evaluated motor and sensory functions of the patients and the presence of adverse events related to G-CSF therapy.G-CSF administration suppressed the progression of myelopathy in all 15 patients. Neurological improvements in motor and sensory functions were obtained in all patients after the administration, although the degree of improvement differed among the patients. Nine patients in the 10-μg group (n=10) underwent surgical treatment at 1 month or later after G-CSF administration. In the 10-μg group, the mean JOA recovery rates 1 and 6 months after administration were 49.9±15.1 and 59.1±16.3%, respectively. On the day following the start of G-CSF therapy, the white blood cell count increased to more than 22,700 cells/mm3. It varied from 12,000 to 50,000 and returned to preadministration levels 3 days after completing G-CSF treatment. No serious adverse events occurred during or after treatment.The results indicate that G-CSF administration at 10 μg/kg/day is safe for patients with worsening symptoms of compression myelopathy and may be effective for their neurological improvement.

We thank Dr. Yoshihara for his comments on our manuscript and below provide our responses to his remarks. It appears that cervical myelopathy is more prevalent in Japan than in the US [1]. This is partly because the Japanese people have narrower spinal canal diameter [2] and a higher incidence of patients have ossification of the posterior longitudinal ligament [3]; both of which are among the major risk factors for cervical myelopathy. In fact, in our 199 cases of anterior cervical surgery series, patients with cervical radiculopathy comprised only 6 cases, while there were as many as 193 patients with cervical myelopathy. In our series there were no cases of postoperative C5 palsy amongst patients with cervical radiculopathy. All cases of the postoperative C5 palsy were in patients who had cervical myelopathy. What we would like to emphasize here, is that pre-existing asymptomatic damage of anterior horn cells in the grey mater at C3–C4 and C4–C5 disc levels may contribute to the development of postoperative C5 palsy. There were no cases of postoperative C5 palsy in patients with single-level anterior cervical discectomy and fusion. Fifteen of the 17 C5 palsy cases occurred after two or more anterior corpectomy and spinal fusion procedures. We reported that the greater the number of corpectomy levels involved, the more likely the occurrence of postoperative C5 palsy. Ikenaga et al. [4] reviewed postoperative C5 palsy cases after anterior corpectomy and fusion and postulated that the extent of anterior dural expansion might have enhanced the incidence of postoperative C5 palsy. That mechanism may be somewhat different from the tethering effect of the nerve rootlet after cervical laminoplasty described by Tsuzuki et al. [5], but also has much in common in that postoperative C5 palsy occurs after longitudinal continuous and extended decompression of the cervical spinal cord. As Dr. Yoshihara mentions in his letter, some postoperative C5 palsy cases may resolve spontaneously. Postoperative C5 palsy cases with MMT grades of 3 or more have a tendency to fully recover without any additional treatment, as reported in our manuscript. It is probable that even patients with postoperative C5 palsy are themselves less aware of the onset and recovery of the muscle weakness. In our series radiating neck and shoulder pain was recognized 1–7 days (average 3.6) after surgery and muscle weakness developed 2–23 days (average 7.2) after surgery. So we are convinced that in order to detect the onset of postoperative C5 palsy accurately, it is essential to see the patients every day after surgery for at least 1 week. The etiology of postoperative C5 palsy remains controversial. We hope that our recently published paper that proposed a “double-lesion” hypothesis may contribute to elucidating the etiology of postoperative C5 palsy. We would like to conduct further studies of postoperative C5 palsy and shed light on this clinically important and unsolved issue.

Adenoviral (Ad) vectors are useful for in vivo gene transfer into the brain. If Ad vectors are injected into the ventricle of mouse embryonic brain, Ad vectors introduce a foreign gene into neural progenitor cells on the surface of ventricle. However, Ad vectors were unable to deliver a foreign gene to a targeted region of the embryonic brain because Ad vectors evenly infected the neural progenitor cells on the surface of ventricle. Therefore, the Ad infection to the neural progenitor cells was uncontrollable. To develop a directional gene-transfer with Ad vector, we generated Ad vector tagged with magnetic nanoparticles (Ad-mag) by linking a biotinylated adenovirus vector with a streptavidin-conjugated magnetic nanoparticle. Ad-mags were attracted by magnetic force in vitro and in vivo. When Ad-mags were injected into the ventricle of mouse embryo and a strong magnet was attached to the head of the embryo, Ad-mags were attracted to the restricted direction or region where the magnet was placed. As a result, Ad-mags efficiently introduced a foreign gene into the restricted region of the brain.

Leiomyomas or benign tumors of smooth muscle origin are extremely rare in the larynx. A review of the literature revealed only 15 confirmed cases of leiomyoma, eight leiomyosarcomas, and no cases of leiomyoblastoma. An unusual case of "bizarre" laryngeal leiomyoma arising from the vocal cord, which is believed to be the first reported case in the literature is presented herein.