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Significance: Image reconstruction of fNIRS data is a useful technique for transforming channel-based fNIRS into a volumetric representation and managing spatial variance based on optode location. We present an innovative integrated pipeline for image reconstruction of fNIRS data using either MRI templates or individual anatomy. Aim: We demonstrate a pipeline with accompanying code to allow users to clean and prepare optode location information, prepare and standardize individual anatomical images, create the light model, run the 3D image reconstruction, and analyze data in group space. Approach: We synthesize a combination of new and existing software packages to create a complete pipeline, from raw data to analysis. Results: This pipeline has been tested using both templates and individual anatomy, and on data from different fNIRS data collection systems. We show high temporal correlations between channel-based and image-based fNIRS data. In addition, we demonstrate the reliability of this pipeline with a sample dataset that included 74 children as part of a longitudinal study taking place in Scotland. We demonstrate good correspondence between data in channel space and image reconstructed data. Conclusions: The pipeline presented here makes a unique contribution by integrating multiple tools to assemble a complete pipeline for image reconstruction in fNIRS. We highlight further issues that may be of interest to future software developers in the field.

Purpose: Reconstruction of patient anatomy is critical to patient-specific instrument (PSI) design in total joint replacement (TJR). Conventionally, computed tomography (CT) and magnetic resonance imaging (MRI) are used to obtain the patient anatomy as they are accurate imaging modalities. However, computing anatomical landmarks from the patient anatomy for PSIs requires either high-resolution CT, increasing time of scan and radiation exposure to the patient, or longer and more expensive MRI scans. As an alternative, reconstruction from single-plane fluoroscopic x-ray provides a cost-efficient tool to obtain patient anatomical structures while allowing capture of the patient’s joint dynamics, important clinical information for TJR. Approach: We present a three-dimensional (3D) reconstruction scheme that automatically and accurately reconstructs the 3D knee anatomy from single-plane fluoroscopic x-ray based on a nonlinear statistical shape model called kernel principal component analysis. To increase robustness, we designed a hybrid energy function that integrated feature and intensity information as a similarity measure for the 3D reconstruction. Results: We evaluated the proposed method on five subjects during deep knee bending: the root-mean-square accuracy is 1.19±0.36 mm for reconstructed femur and 1.15±0.17 mm for reconstructed tibia. Conclusions: The proposed method demonstrates reliable 3D bone model reconstruction accuracy with successful elimination of prior 3D imaging and reduction of manual labor and radiation dose on patient as well as characterizing joints in motion. This method is promising for applications in medical interventions such as patient-specific arthroplasty design, surgical planning, surgical navigation, and understanding anatomical and dynamic characteristics of joints.

Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.

Background Reduced susceptibility of mosquito vectors to currently used insecticides hampers control interventions. Recently pyriproxyfen, an insect growth regulator has been demonstrated to effectively reduce the reproductive potential in vector mosquitoes. Methods Pyriproxyfen (PPF), in different concentrations (0.75%, 0.075%, 0.0075%, and 0.00075%) was applied on papers and Indian wild type Aedes aegypti female mosquitoes (N ≥ 20 for each treatment) were exposed onto it as per WHO guidelines, to study the reproductive disruption. PPF concentration on treated papers was quantitatively cross-determined using HPLC method. Reduction in fecundity, fertility and adult emergence in exposed female Ae. aegypti was determined. Abnormal development in ovary and eggs of exposed females was studied microscopically after different time intervals. Results Eggs laid, eggs hatched, pupae formed and adults emerged per female exposed in both before blood meal and after blood meal groups declined significantly from lowest to highest concentration of PPF (F ≥ 5.2; p < 0.02). Adult emergence inhibition in females exposed to PPF before and after blood meal groups ranged from 58.8% [OR = 0.18 (95% CI = 0.09–0.36)] to 79.2% [OR = 0.04 (95% CI = 0.02–0.10)] and 64.4% [OR = 0.12 (95% CI = 0.05–0.28)] to 77.1% [OR = 0.05 (95% CI = 0.02–0.14)] respectively in different concentrations. The probit model used suggested that FI50 (50% fertility inhibition) and EI50 (50% emergence inhibition) were 0.002% (p = 0.82) and 0.0001% (p = 0.99) for females exposed before blood meal, while 0.01% (p = 0.63) and, Author summary Development of resistance against insecticides has challenged mosquito control programmes globally and prompted the research of alternative options that can complement insecticides. An insect growth regulator, pyriproxyfen (PPF) usage against mosquitoes can effectively reduce the vector population. PPF mainly inhibits the metamorphosis of mosquito larvae into pupae and prevent the adult emergence, therefore it is generally applied in mosquito breeding habitats. PPF has been shown to exhibit delayed residual effect that may impair the reproductive capacity by affecting the survival, fecundity and fertility of adult mosquito exposed through tarsal contact. Presently, the effectiveness of different concentrations of PPF intended to be delivered through contact have been evaluated against dengue vector Ae. aegypti. Results suggested that very low PPF concentration treated surfaces drastically reduce the fecundity, fertility and adult emergence in mosquitoes. Study suggests that control interventions based on PPF-treated surfaces could provide an additional route to target mosquito vector control by overall population density reduction.

Significance: Diffuse optical spectroscopic imaging (DOSI) measures quantitative functional and molecular information in thick tissue in a noninvasive manner using near-infrared light. DOSI may be useful for diagnosis and prognosis of bone pathologies including osteosarcoma and Ewing’s sarcoma, but little is currently known about DOSI-derived parameters in bony anatomic locations where this disease occurs. Aim: Our goal is to quantify the optical characteristics and chromophore content of bony anatomic locations of healthy volunteers and assess differences due to anatomic region, age, sex, ethnicity, race, and body fat. Approach: Fifty-five healthy volunteers aged 4 to 72 were enrolled in the study. The optical properties and quantitative tissue concentrations of oxyhemoglobin, deoxyhemoglobin, water, and lipids were assessed at the distal humerus, distal femur, and proximal tibia. Body fat was assessed using skinfold calipers. One volunteer underwent a more comprehensive body scan from neck to foot to explore chromophore distributions within an individual. Regression analysis was used to identify the most important sources of variation in the measured data set. Results: Statistical differences between bony locations were found for scattering, water, and lipids, but not for hemoglobin. All chromophores had statistical differences with sex, but there were no significant age-related correlations. Regression analysis revealed that body fat measured with skinfold calipers was the most important predictor of oxy-, deoxy-, total hemoglobin, and lipids. Hemoglobin and lipid levels were highly correlated (ρ≥0.7) over the subject population and within the single-subject body scan. Conclusions: DOSI can successfully measure bony anatomic sites where osteosarcomas and Ewing’s sarcomas commonly occur. Future studies of bone pathology using DOSI should account for the variation caused by anatomic region, sex, race and ethnicity, and body fat as these cause substantial variations in DOSI-derived metrics.

Modern interpretation of head conformation in the Cavalier King Charles spaniel (CKCS) has favoured a smaller, more exaggerated, brachycephalic type than originally described in the 1929 breed standard. Recent research studies identified brachycephaly and reduced hind cranium as two conformational (dysmorphic) features that increase risk for symptomatic Chiari-like malformation and secondary syringomyelia (SM). A prospective pilot study investigated the hypothesis that dysmorphic head features could be assessed visually and correlated with risk of SM. Thirteen CKCS, selected from anonymised photographic evidence, were physically appraised by authorised Kennel Club judges using a head shape checklist. These subjective evaluations were then matched with objective measurements of the cranium (cephalic index and rostrocaudal doming) and their subsequent MRI. A positive correlation (P=0.039) between the judges’ checklist score and rostrocaudal doming (hindskull ratio) and a positive correlation between the cephalic index and hindskull ratio (P=0.042) were identified. Five CKCS had no SM and their status tallied with 62 per cent of the judges’ evaluation. Although the ability of adjudicators to identify differences in head conformation varied, there was sufficient association between the dysmorphic parameters and the risk of SM to cause concern and propose a larger study in CKCS breed.

Objective (1) To compare temporal bone anatomy comprehension taught to speech therapy students with or without a papercraft model. (2) To explore the effect of papercraft simulation on the understanding of surgical approaches in first-year residents. Methods (1) One-hundred and ten speech therapy students were divided into three classes. The first class was taught with a lecture only. The students in the second class were given a lecture and a papercraft modeling task without instruction. The third class modeled a papercraft with instruction after the lecture. The students were tested on their understanding of temporal bone anatomy. (2) A questionnaire on the understanding of surgical approaches was completed by 10 residents before and after the papercraft modeling. The papercraft models were cut with scissors to simulate surgical approaches. Results (1) The average scores were 4.4/8 for the first class, 4.3/8 for the second class, and 6.3/8 for the third class. The third class had significantly better results than the other classes (p

Poor cell retention and limited cell survival after grafting are major limitations of cell therapy. Recent studies showed that the use of matrices as vehicles at the time of cell injection can significantly improve cell engraftment by providing an appropriate structure and physical support for the injected cells. Properly designed matrices can also promote the organization of the cells into a functioning cardiac-like tissue and enhance integration between the host and the engrafted tissue. Furthermore, the use of an injectable biomaterial provides an opportunity to release in situ bioactive molecules that can further enhance the beneficial effects of cell transplantation. In this article we review a large variety of biologically derived synthetic and hybrid materials that have been tested as matrices for cardiac repair. We summarize the optimal parameters required for an ideal matrix including biocompatibility, injectability, degradation rate, and mechanical properties. Using an in vivo subcutaneous grafting model, we also provide novel data involving a side-by-side comparison of six synthetic matrices derived from maltodextrin. By systematically varying polymer molecular weight, cross-link density, and availability of cell adhesion motifs, a synthetic matrix was identified that supported skeletal myotube formation similar to Matrigel™. Our results emphasize not only the need to have a range of tunable matrices for cardiac cell therapy but also the importance of further characterizing the physical properties required for an ideal injectable matrix.

The heterogeneity of vascular smooth muscle cells (SMCs) is related to their different developmental origins such as the neural crest and mesoderm. Derivation of SMCs from different origins will provide valuable in vitro models for the investigation of vascular development and diseases. From the perspective of regenerative medicine and tissue engineering, an expandable cell source of SMCs is required for the construction of tissue-engineered blood vessels. In this study, we developed a robust protocol to derive neural crest stem cells (NCSCs) from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). NCSCs derived from ESCs and iPSCs were expandable with similar cell doubling times. NCSCs were capable of differentiating into neural and mesenchymal lineages. TGF-β1 induced the expression of SMC markers calponin-1, SM22α, and smooth muscle myosin heavy chain and resulted in the assembly of smooth muscle α-actin, calponin-1, and SM22α into stress fibers. This work provides a basis for using iPSCs to study SMC biology and deriving vascular cells for tissue engineering.

The major objective of successful development of tissue-engineered vascular grafts is long-term in vivo patency. Optimization of matrix, cell source, surface modifications, and physical preconditioning are all elements of attaining a compatible, durable, and functional vascular construct. In vitro model systems are inadequate to test elements of thrombogenicity and vascular dynamic functional properties while in vivo implantation is complicated, labor-intensive, and cost-ineffective. We proposed an ex vivo ovine arteriovenous shunt model in which we can test the patency and physical properties of vascular grafts under physiologic conditions. The pressure, flow rate, and vascular diameter were monitored in real-time in order to evaluate the pulse wave velocity, augmentation index, and dynamic elastic modulus, all indicators of graft stiffness. Carotid arteries, jugular veins, and small intestinal submucosa-based grafts were tested. SIS grafts demonstrated physical properties between those of carotid arteries and jugular veins. Anticoagulation properties of grafts were assessed via scanning electron microscopy imaging, en face immunostaining, and histology. Luminal seeding with endothelial cells greatly decreased the attachment of thrombotic components. This model is also suture free, allowing for multiple samples to be stably processed within one animal. This tunable (pressure, flow, shear) ex vivo shunt model can be used to optimize the implantability and long-term patency of tissue-engineered vascular constructs.

Considerable progress has been made toward a molecular understanding of how cells form lumen and tube structures in three-dimensional (3D) extracellular matrices (ECM). This progress has occurred through work performed with endothelial and epithelial cell models using both in vitro and in vivo approaches. Despite the apparent similarities between endothelial and epithelial cell lumen and tube formation mechanisms, there are clear distinctions that directly relate to their functional differences. This review will focus on endothelial cell (EC) lumen formation mechanisms which control blood vessel formation during development and postnatal life. Of great interest is that an EC lumen signaling complex has been identified which controls human EC lumen and tube formation in 3D matrices and which coordinates integrin-ECM contacts, cell surface proteolysis, cytoskeletal rearrangements, and cell polarity. This complex consists of the collagen-binding integrin α2β1, the collagen-degrading membrane-type 1 matrix metalloproteinase (MT1-MMP), junction adhesion molecule (Jam)C, JamB, polarity proteins Par3 and Par6b, and the Rho GTPase Cdc42-GTP. These interacting proteins are necessary to stimulate 3D matrix-specific signaling events (including activation of protein kinase cascades that regulate the actin and microtubule cytoskeletons) to control the formation of EC lumens and tube networks. Also, EC lumen formation is directly coupled to the generation of vascular guidance tunnels, enzymatically generated ECM conduits that facilitate EC tube remodeling and maturation. Mural cells such as pericytes are recruited along EC tubes within these tunnel spaces to control ECM remodeling events resulting in vascular basement membrane matrix assembly, a key step in tube maturation and stabilization.

Dacron® (polyethylene terephthalate) and Goretex® (expanded polytetrafluoroethylene) vascular grafts have been very successful in replacing obstructed blood vessels of large and medium diameters. However, as diameters decrease below 6 mm, these grafts are clearly outperformed by transposed autologous veins and, particularly, arteries. With approximately 8 million individuals with peripheral arterial disease, over 500,000 patients diagnosed with end-stage renal disease, and over 250,000 patients per year undergoing coronary bypass in the USA alone, there is a critical clinical need for a functional small-diameter conduit [Lloyd-Jones et al., Circulation 2010;121:e46–e215]. Over the last decade, we have witnessed a dramatic paradigm shift in cardiovascular tissue engineering that has driven the field away from biomaterial-focused approaches and towards more biology-driven strategies. In this article, we review the preclinical and clinical efforts in the quest for a tissue-engineered blood vessel that is free of permanent synthetic scaffolds but has the mechanical strength to become a successful arterial graft. Special emphasis is given to the tissue engineering by self-assembly (TESA) approach, which has been the only one to reach clinical trials for applications under arterial pressure.

Damaged heart muscle has only a minimal ability for regeneration following myocardial infarction in which cardiomyocytes are lost to ischemia. The most clinically promising approach to regeneration of cardiac muscle currently under investigation is that of injecting cardiogenic repair cells or implanting a preformed tissue-engineered patch. While major advances are being made in the derivation of functional human cardiomyocytes and the development of tissue-engineering modalities for cardiac repair, the host environment into which the repair cells are placed is largely overlooked. Within seconds of myocardial ischemia, hypoxia sets in in the myocardium and the inflammatory response starts, characterized by rapid deployment of circulating cells and the release of paracrine and autocrine signals. Therefore, the inflammatory conditions under which these interactions take place, the design of the scaffold material used, and the maturity of the implanted cells will determine the outcomes of any stem cell-based therapy. We discuss here the interactions between implanted and inflammatory cells of the host, which are critical for the design of effective heart repair therapies.

DMP1 has been shown to play many roles in osteogenesis. We recently demonstrated that calcium-mediated stress kinase activation by DMP1 leads to osteoblast differentiation. In this study we demonstrate that DMP1 can also activate the extracellular signal-regulated kinase (ERK)-MAPK pathway. This activation was mediated through the RGD integrin-binding domain in DMP1. Further, we demonstrate that Runx2, an essential transcription factor, is stimulated by the ERK-MAPK pathway.

Since first being proposed as a tandem gene family in 2001, the relatedness of the 5 SIBLING proteins (BSP, DMP1, DSPP, MEPE, and SPP1/OPN) has predominantly depended on arguments involving shared intron/exon properties as well as conserved protein biochemical properties (e.g. unstructured and acidic) and specific peptide motifs (e.g. phosphorylation and integrin-binding RGD). This report discusses the evidence that an ancient DMP1 gene underwent a simple duplication in the common ancestor of mammals and reptiles and then separately evolved into DSPP-like paralogs in the 2 classes. Genomic sequence analyses show that different copies of the original DMP1 duplication process were selected by mammalian and reptilian (anole lizard) classes to acquire genetically different but biochemically similar phosphoserine-rich repeat domains by convergent evolution. Mammals, for example, expanded phosphoserine motifs encoded exclusively using motifs containing AGC/T serine codons while the reptile line’s repeats also used TCN-encoding serine codons. A similar analysis of the origins of the other 4 SIBLINGs will require even more detailed analysis as genome sequences of various fish and amphibia become available.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2flox/flox mice. Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric and open forked incisors. Microradiographs revealed broken incisor tips and dental pulp chamber exposure. The enamel layer of incisors and molars was thin with hypomineralization. Scanning electron microscopy analysis showed that the enamel surface was rough with chipping and the enamel lacked a typical prismatic architecture. These results demonstrate that Bmp2 is essential for enamel formation.

The motor system is capable of preserving the trajectories during locomotion of task level variables such as limb length and limb orientation in the face of paralysis of major muscle groups. This compensation is accomplished by the adjustment of the kinematics of joints other than the one most affected by the paralysis. The conservation of these task level variables could be accomplished quickly by feedback regulation or intrinsic mechanics, or by a longer-term adaptive process. We investigated the immediate effects of denervation of the triceps surae muscles in one limb of stepping, decerebrate cats to determine whether task level variables were preserved by short-term regulatory or intrinsic mechanisms. We further investigated the effects of disruption of the crural fascia in conjunction with denervation of the triceps surae muscles to determine whether the system consisting of multi-articular muscles of the thigh and crural fascia provided some contribution toward the preservation of limb length and orientation. Denervation led to substantial increases in ankle yield during stance, as previously observed, but also to significant decreases in limb length during early stance. Disruption of the crural fascia did not lead to increased ankle yield but, instead, to evidence for decreased propulsion. The results suggest that the preservation of task level variables observed in other studies does not result from online error correction or intrinsic properties of the musculoskeletal system but, by inference, from longer-term neural adaptation.

Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

The collagen 2.3 and 3.6 promoters have been used to drive Cre expression for generation of conditional transgenic mutant mice. Within the bone, Col3.6 is expressed by mesenchymal precursor cells and their downstream progeny, while Col2.3 is more osteoblast specific. Our generation of transgenic mice with Col2.3-Cre- and Col3.6-Cre-driven deletion of the long-form leptin receptor (ObRb) necessitated a thorough analysis of the nonspecific expression of these promoters in the central nervous system. Both Col2.3 and Col3.6 were capable of forcing loxP recombination in the brain as demonstrated by EGFP expression in ROSA reporter mice. Expression of Col2.3 was limited to the central base of the brain near the third ventricle. In contrast, robust expression of Col3.6 was noted throughout the brain, centering near the distal third ventricle, third ventricle, and aqueduct. We subsequently analyzed the colocalization of leptin-responsive P-Stat3 neurons with Col3.6-expressing neurons. Approximately 5–10% colocalization was noted in leptin-responsive brain areas such as the arcuate nucleus, dorsal medial hypothalamus, ventral premammillary nucleus, and lateral hypothalamus. Injection of 3.6Cre+F/F ObRb knockout mice with leptin confirmed the presence of an intact P-Stat3 response that was dampened in the lateral hypothalamus (p < 0.050). This test was done to explore the contribution of neural leptin signaling to the bone phenotype of the 3.6Cre+F/F mice. Our analysis indicates that neural ObRb deletion, while present, is likely not the sole driver of femoral changes through traditional sympathetic circuits.

Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoADN) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoADN fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoADN-8), intermediate (TgEGFP-RhoADN-2), or high (TgEGFP-RhoADN-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoADN expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride.

The goal of this investigation was to ascertain whether bone cells undergo autophagy and to determine if this process is regulated by environmental factors. We showed that osteocytes in both murine and human cortical bone display a punctuate distribution of microtubule-associated protein light chain 3, indicative of autophagy. In addition, we noted a basal level of autophagy in preosteocyte-like murine long bone-derived osteocytic (MLO)-A5 cells. Autophagy was upregulated following nutrient deprivation and hypoxic culture, stress conditions that osteocytes encounter in vivo. Furthermore, in response to calcium stress, the transcription factor hypoxia inducible factor 1 regulated MLO-A5 autophagy. Finally, we showed that the more differentiated MLO-Y4 osteocyte-like cells exhibited a significant basal autophagic flux. Based on these findings, we suggest that raising the level of autophagic flux is a mechanism by which differentiated bone cells survive in a stressful environment.

Purpose: To ensure the efficiency and safety of transplanted human embryonic stem cell (hESC)-derived osteoblast-like cells (hESC-OS) for bone regeneration, this study was designed to determine the effects of continuous cell expansion on the osteoblastic differentiation stability, pluripotency, and tumorigenic potential of long-term expanded hESC-OS. Methods: hESCs manually harvested as cell aggregates or enzymatically dissociated as single cells were directly incubated in osteogenic medium and serially passaged to passage 25. Expression of osteoblast-related genes, pluripotent regulator genes, and genes related to tumorigenesis were examined at the primary passage and every 5 passages thereafter. hESC-OS were subcutaneously transplanted into nude mice for 4–24 weeks to test for teratoma formation. hESC-OS were recultivated in hESC culture conditions to evaluate the extent to which reverse differentiation back to the undifferentiated stage may occur. Results: hESC-OS derived from hESC aggregates and dissociated cells exhibited comparable osteoblast differentiation patterns. Expression levels of osteoblast-related genes reached plateau levels at passages 5–10 before declining in higher passages. Expression of tumor-associated genes was not significantly increased. Only hESC-OS at primary and first passages formed teratomas after 4 weeks in vivo. The hESC-OS were not able to revert to hESCs. Conclusions: Expanded hESC-OS demonstrated lineage-specific differentiation stability, did not maintain the pluripotency of hES cells, and were genetically stable. Thus, hESC-OS may be considered for large animal preclinical studies.

This study was undertaken to determine whether periosteum from different bone sources in a donor results in the same formation of bone and cartilage. In this case, periosteum obtained from the cranium and mandible (examples of tissue supporting intramembranous ossification) and the radius and ilium (examples of tissues supporting endochondral ossification) of individual calves was used to produce tissue-engineered constructs that were implanted in nude mice and then retrieved after 10 and 20 weeks. Specimens were compared in terms of their osteogenic and chondrogenic potential by radiography, histology, and gene expression levels. By 10 weeks of implantation and more so by 20 weeks, constructs with cranial periosteum had developed to the greatest extent, followed in order by ilium, radius, and mandible periosteum. All constructs, particularly with cranial tissue although minimally with mandibular periosteum, had mineralized by 10 weeks on radiography and stained for proteoglycans with safranin-O red (cranial tissue most intensely and mandibular tissue least intensely). Gene expression of type I collagen, type II collagen, runx2, and bone sialoprotein (BSP) was detectable on QRT-PCR for all specimens at 10 and 20 weeks. By 20 weeks, the relative gene levels were: type I collagen, ilium >> radial ≧ cranial ≧ mandibular; type II collagen, radial > ilium > cranial ≧ mandibular; runx2, cranial >>> radial > mandibular ≧ ilium; and BSP, ilium ≧ radial > cranial > mandibular. These data demonstrate that the osteogenic and chondrogenic capacity of the various constructs is not identical and depends on the periosteal source regardless of intramembranous or endochondral ossification. Based on these results, cranial and mandibular periosteal tissues appear to enhance bone formation most and least prominently, respectively. The appropriate periosteal choice for bone and cartilage tissue engineering and regeneration should be a function of its immediate application as well as other factors besides growth rate.

Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5–15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30–40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function.

Fourier transform infrared imaging spectroscopy (FTIRI)-assessed bone composition parameters (mineral content, collagen maturity, crystal size and perfection, and carbonate content) describe bone quality and correlate to bone fracture risk. The challenge with studying bone quality in patients treated with antiresorptive drugs such as bisphosphonates (e.g., alendronate) and selective estrogen receptor modulators (SERMs) (e.g. raloxifene) is being able to test bone mechanical performance and material properties pre- and posttreatment. The purpose of this study was to evaluate the FTIRI changes in a large animal model of osteoporosis (female sheep with dietary induced metabolic acidosis; MA). Previous studies have investigated the relationship between bone material properties and bone strength in humans and smaller animals and have shown that changes in compositional properties influence fracture risk. Here we characterize the MA model at 6 and 12 months, demonstrate the loss of bone and changes in compositional properties, and show that 6 months of treatment with both antiresorptives ameliorate the bone loss as assessed by bone mineral density and FTIRI. This preliminary data suggest that the MA sheep model allows investigation of whether drug treatments preserve bone properties that exist at the time of treatment or if they induce further beneficial changes.

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

Nerve regeneration was examined in 108 mouse sciatic nerves in a prospective morphometric study in which a new anastomotic technique using nitrocellulose paper was compared with conventional suturing and simple sectioning. Mice were randomly allocated to one of the three arms of the trial and sciatic nerves were examined following surgery at time intervals up to 8 weeks. Serial sections were taken for qualitative and quantitative morphometric analyses. Counts demonstrated that the rate of regeneration was faster in the nitrocellulose group than following nerve section alone, but that the suture group was even better. However, 8 weeks after surgery, there were no significant differences in myelinated axon counts 4mm distal to the site of section between any of the experimental groups. The superlative regenerative capacity of rodent axons may limit the applicability of this model to human nerve tissue.

We have examined and compared the effects of various differentiation-inducing media on mineralization, cell morphology and expression of pOBCol3.6GFP (3.6-GFP) in primary dental pulp cultures derived from 3.6-GFP transgenic mice. Our results show that media containing ascorbic acid only could not induce mineralization in primary dental pulp cultures. On the other hand, media containing ascorbic acid and β-glycerophosphate induced formation of mineralized matrix-containing dentin. The amount of mineralized matrix was increased by addition of dexamethasone. Cells treated with ascorbic acid and β-glycerophosphate were fibroblast like and cells treated with dexamethasone were cuboidal. In all culture conditions, high levels of 3.6-GFP were expressed in areas of mineralization

The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.

The enamel matrix proteins (amelogenin, enamelin and ameloblastin) are degraded by matrix metalloproteinase-20 and kallikrein-4 during enamel development and mature enamel is virtually protein free. The precise mechanism of removal and degradation of the enamel protein cleavage products from the matrix, however, remains poorly understood. It has been proposed that receptor-mediated endocytosis allows for the cleaved proteins to be removed from the matrix during enamel formation and then transported to the lysosome for further degradation. This study aims to identify lysosomal proteases that are present in maturation-stage enamel organ. RNA from first molars of 11-day-old mice was collected and expression was initially assessed by RT-PCR and then quantified by qPCR. The pattern of expression of selected proteases was assessed by immunohistochemical staining of demineralized mouse incisors. With the exception of cathepsin G, all lysosomal proteases assessed were expressed in maturation-stage enamel organ. Identified proteases included cathepsins B, D, F, H, K, L, O, S and Z. Tripeptidyl peptidases I and II as well as dipeptidyl peptidases I, II, III and IV were also found to be expressed. Immunohistochemical staining confirmed that the maturation-stage ameloblasts express cathepsins L and S and tripeptidyl peptidase II. Our results suggest that the ameloblasts are enriched by a large number of lysosomal proteases at maturation that are likely involved in the degradation of the organic matrix.

Biglycan (BGN) and WISP-1 are 2 extracellular matrix proteins that bind to each other and colocalize in mineralizing tissue. Here we show that WISP-1 abrogates the repression of proliferation in bone marrow stromal cells induced by BGN. We also demonstrate that WISP-1 and its variant WISP-1va can alleviate the repressed osteogenic differentiation caused by the absence of BGN. These preliminary data suggest that WISP-1 and BGN may functionally interact and control each other’s activity, thus regulating the differentiation and proliferation of osteogenic cells.

The challenges in engineering bone scaffolds reflect the complexity of bone as an organ. The organic-inorganic hybrid system design aims to provide signals within a conductive apatite layer to promote cell adhesion, proliferation and ultimately differentiation into bone tissue. Dual functioning peptides designed to specifically adhere to the apatite layer, while promoting cell adhesion via cell recognition sequences, may increase cell adhesion, leading to increased osteogenesis. The aim of this study is to identify peptide sequences with preferential adsorption towards apatite-based materials. Bone-like mineral films and hydroxyapatite disks were panned with a phage library to elucidate peptide sequences with favorable adsorption. Peptide sequences were analyzed using the web-based biotechnology tool RELIC and validated with a modified ELISA, in addition to being investigated using a newly developed method of high-throughput computational modeling. Peptides having the highest affinity and greatest potential to be incorporated into a dual functioning peptide design are APWHLSSQYSRT, VTKHLNQISQSY and STLPIPHEFSRE. These experiments provide a method of rationally designing peptides that adhere to apatite and that may improve bone tissue regeneration. This work also provides structure for investigating peptide/protein adsorption on apatite substrates with varied carbonate, or other impurity, content.

Fibroblast growth factor (FGF) signaling plays a critical role in skeletal development, yet the mechanism by which FGFs affect bone mineralization is not well understood. Review of the literature investigating effects of FGF signaling on bone mineralization indicates that FGFs may stimulate expression of factors that prevent mineralization in the short term and enhance mineralization in the long term. Pyrophosphate is an ideal example of a factor that, dependent upon environment, has the capacity to inhibit or enhance mineralization. PC-1 is the primary generator of pyrophosphate in osteoblastic cells; therefore, regulated expression of PC-1 by FGFs may be a principal mechanism by which FGF signaling affects bone mineralization. We previously showed that FGF2 induces PC-1 expression in preosteoblastic cells and that this induction is differentiation stage dependent. In order to more directly investigate the mechanism by which PC-1 expression is regulated, we have cloned a 2.8-kb region of the PC-1 gene promoter and constructed a PC-1 gene promoter/firefly luciferase reporter construct. Results indicate that this construct is specifically responsive to FGF2 or ascorbate (an inducer of osteoblast differentiation). Promoter responsiveness to FGF2 is significantly diminished upon osteoblast differentiation, and increases in promoter activity that occur with osteoblast differentiation are inhibited by FGF2 treatment. These results indicate that the mechanism of PC-1 induction by FGF2 in preosteoblastic cells is distinct from the mechanism of induction that occurs with osteoblast differentiation. These results also indicate that PC-1 may play multiple and distinct roles in the development of mineralized tissues.

Dentin matrix protein 1 (DMP1) has been shown to be critical for the formation of dentin and bone. However, the precise pathway by which DMP1 participates in dentinogenesis and osteogenesis remains to be clarified. DMP1 is present in the extracellular matrix of dentin and bone as processed NH2- and COOH-terminal fragments. The NH2-terminal fragment occurs as a proteoglycan, whereas the COOH-terminal fragment is highly phosphorylated. The differences in biochemical properties suggest that these fragments may have different tissue and cell distribution in association with distinct functions. In this study, we analyzed the distribution of the NH2- and COOH-terminal fragments of DMP1 in tooth, bone, osteocytes as well as MC3T3-E1 and HEK-293 cells. Immunohistochemical analyses were performed using antibodies specific to the NH2- or COOH-terminal region of DMP1. Clear differences in the distribution of these fragments were observed. In the teeth and bone, the NH2-terminal fragment was primarily located in the nonmineralized predentin and cartilage of the growth plate, while the COOH-terminal fragment accumulated in the mineralized zones. In osteocytes, the NH2-terminal fragment appeared more abundant along cell membrane and processes of osteocytes, while the COOH-terminal fragment was often found in the nuclei. This pattern of distribution in cellular compartments was further confirmed by analyses on MC3T3-E1 and HEK-293 cells transfected with a construct containing DMP1 cDNA. In these cell lines, the COOH-terminal fragment accumulated in cell nuclei, while the NH2-terminal fragment was in the cytosol. The different distribution of DMP1 fragments indicates that these DMP1 variants must perform distinct functions.

Objective: X linked spinobulbar muscular atrophy (Kennedy disease (KD)), which is clinically characterised mainly by neuromuscular and endocrine symptoms, has to be considered as a multisystem disorder. Based on clinical evidence of central nervous system involvement, potential KD associated cerebral volume alterations were analysed in vivo. Methods: Whole brain based analysis of optimised voxel based morphometry (VBM) was applied to three dimensional MRI data from 18 genetically confirmed KD patients and compared with age matched controls. Results: Subtle decreases in grey matter volume, mainly localised in frontal areas, were found, but extensive white matter atrophy was observed, particularly in frontal areas, but also involving multiple additional subcortical areas, the cerebellar white matter and the dorsal brainstem from the midbrain to the medulla oblongata. Conclusion: The VBM results demonstrated a morphological correlate of central nervous system involvement in KD, in agreement with aspects of the clinical phenotype (behavioural abnormalities, central–peripheral axonopathy) and with pathohistological findings.

While some regard the flail arm syndrome as a variant of amyotrophic lateral sclerosis (ALS), others have argued that it is a distinct clinical entity. Consequently, the present study applied novel central and peripheral nerve excitability techniques to further explore disease pathophysiology in flail arm syndrome.Cortical and peripheral nerve excitability studies were undertaken in 11 flail arm patients, defined by muscle weakness limited to the proximal aspects of the upper limbs for at least 24 months.Mean age at disease onset (60.3 years) was similar to other ALS phenotypes (58.3 years), with strong male predominance (male:female distribution: flail arm 10:1; ALS 1.5:1; p0.05) and prolonged disease duration (flail arm 62.5 months; ALS 15.8 months; p0.05). There was evidence of cortical hyperexcitability in flail arm patients, similar to findings in ALS, with reduction in short interval intracortical inhibition (flail arm 0.8 (0.6)%; ALS 4.1 (1.1)%; controls 8.5 (1.0)%; p0.0001) and resting motor threshold (flail arm 53.4 (2.8)%; ALS 56.6 (1.8)%; controls 60.7 (1.5)%; p0.05), along with an increase in motor evoked potential amplitude (flail arm 49.5 (9.0)%; ALS 44.4 (4.9)%; controls 25.8 (2.8)%; p0.05). Peripheral nerve excitability studies demonstrated changes consistent with upregulation in persistent Na+ currents and reduction of slow K+ conductances, similar to findings in ALS.This study has demonstrated the presence of cortical hyperexcitability in flail arm syndrome, along with abnormalities in peripheral nerve excitability, findings consistent with previous studies in other ALS phenotypes. By demonstrating the presence of upper motor neuron dysfunction, the present study suggests that the flail arm syndrome is an unusual variant of ALS.

Background: Laing early onset distal myopathy (MPD1) is an autosomal dominant myopathy caused by mutations within the slow skeletal muscle fibre myosin heavy chain gene, MYH7. It is allelic with myosin storage myopathy, with the commonest form of familial hypertrophic cardiomyopathy, and with one form of dilated cardiomyopathy. However, the clinical picture of MPD1 is distinct from these three conditions. Objective: To collate and discuss the histological features reported in the muscle biopsies of MPD1 patients and to outline the clinical features. Results: The phenotype of MPD1 was consistent, with initial weakness of great toe/ankle dorsiflexion, and later development of weakness of finger extension and neck flexion. Age of onset was the only variable, being from birth up to the 20s, but progression was always very slow. The pathological features were variable. In this retrospective series, there were no pathognomonic diagnostic features, although atrophic type I fibres were found in half the families. Rimmed vacuoles are consistently seen in all other distal myopathies with the exception of Myoshi distal myopathy. However, they were found in a minority of patients with MPD1, and were not prominent when present. Immunohistochemical staining for slow and fast myosin showed co-expression of slow and fast myosin in some type I fibres, possibly indicating a switch to type II status. This may be a useful aid to diagnosis. Conclusions: The pathological findings in MPD1 are variable and appear to be affected by factors such as the specific muscle biopsied, the age of the patient at biopsy, and the duration of disease manifestations.

Objective: To correlate morphological findings of idiopathic carpal tunnel syndrome (CTS) with the function of the median nerve. Methods: In this study, 105 wrists of 105 women patients with idiopathic CTS, and 36 wrists of 36 female volunteers were subjected to nerve conduction studies and MRI. Cross sectional area, signal intensity ratio, and the flattening ratio of the median nerve, carpal tunnel area, flexor tendon area, synovial area, and intersynovial space, and the palmar bowing of the transverse carpal ligament (TCL) were quantified by MRI and correlated with the severity of the disease determined by nerve conduction studies. Results: Cross sectional areas of the median nerve, flexor tendons, and carpal tunnel, and the palmar bowing of the TCL of the CTS groups were greater than in the control group, but differences were not detected among the CTS groups for the area of the flexor tendons and the carpal tunnel. Enlargement, flattening, and high signal intensity of the median nerve at the distal radioulnar joint level were more significant in the advanced than in the earlier stages of the disease. Increase in palmar bowing of the TCL was less prominent in the most advanced group. Linear correlation between the area of the carpal tunnel and palmar bowing of the TCL was noted. Conclusion: Severity of the disease could be judged by evaluating not only longitudinal changes of signal intensity and configuration of the median nerve, but also palmar bowing of the TCL. Increased palmar bowing of the TCL was found to be associated with an increase in the area of the carpal tunnel.

Objective: To clarify age related changes in the clinicopathological features of hereditary neuropathy with liability to pressure palsy (HNPP) in Japanese patients with deletion of 17p11.2, particularly concerning axonal abnormalities. Methods: Forty eight proband patients from 48 HNPP families were assessed as to clinical, electrophysiological, and histopathological features, including age associated changes beyond those in controls. Results: Motor conduction studies showed age associated deterioration of compound muscle action potentials in nerves vulnerable to repetitive compression (median, ulnar, and peroneal nerves), but not in others such as the tibial nerve. Sensory conduction studies revealed more profound reduction of action potentials than motor studies with little age related change. Large myelinated fibre loss was seen in the sural nerve irrespective of age at examination. Conclusions: Irreversible axonal damage may occur at entrapment sites in motor nerves in HNPP patients, progressing with aging. Sensory nerves may show more profound axonal abnormality, but without age association. The electrophysiological features of HNPP are presumed to be a mixture of abnormalities occurring from early in life and acquired features caused by repetitive insults at entrapment sites. Unlike Charcot-Marie-Tooth disease type 1A, age associated axonal damage may not occur unless the nerves are subjected to compression.

Objective: To assess whether different types of malformation of cortical development (MCD) are associated with specific patterns of hippocampal abnormalities. Methods: A total of 122 consecutive patients with MRI diagnosis of MCD (53 males, age range 1–58 years) were included in the study. Hippocampal measurements were made on 1–3 mm coronal T1-weighted MRIs and compared with MRIs of normal controls. Results: A total of 39 patients had focal cortical dysplasia, 5 had hemimegalencephaly, 5 had lissencephaly-agyria-pachygyria, 11 had SLH, 11 had PNH, 12 had bilateral contiguous PNH, 5 had schizencephaly, and 34 had polymicrogyria. The frequency of hippocampal abnormalities in these patients with MCD was 29.5%. A small hippocampus was present in all types of MCD. Only patients with lissencephaly and SLH had an enlarged hippocampus. Abnormalities in hippocampal rotation and shape were present in all types of MCD; however, these predominated in PNH. None of the patients with lissencephaly-agyria-pachygyria or SLH had hyperintense signal on T2 or FLAIR images or abnormal hippocampal internal architecture. Conclusion: A small hippocampus was present in all types of MCD; however, the classic MRI characteristics of hippocampal sclerosis were often lacking. Abnormal enlargement of the hippocampus was associated with only diffuse MCD due to abnormal neuronal migration (lissencephaly-agyria-pachygyria and SLH).

Objectives: To examine vestibular control of balance in those who recovered the ability to stand after middle cerebral artery (MCA) stroke. Methods: Sixteen patients with MCA stroke were compared with 10 age matched controls. Two additional patients were studied with isolated corticospinal tract lesions, one each at the level of the pons and medulla. Vestibular evoked postural responses were obtained using galvanic vestibular stimulation (GVS) while patients stood with their eyes closed and head facing forwards, equally loading both legs. The GVS response was characterised by measuring the amplitude of the stimulus evoked lateral forces acting through each leg and the lateral displacement of the axial skeleton. Results: Lateral displacement and net lateral force following GVS were significantly larger after stroke. Unlike controls, the lateral forces in the stroke group were asymmetrical, being enhanced on the side of the non-paretic limb and small on the side of the paretic limb. The degree of GVS evoked asymmetry correlated with corticospinal damage assessed using transcranial magnetic stimulation. A similar asymmetrical response was seen in the patient with the pontine lesion but not the patient with the medullary lesion. Conclusions: MCA stroke may disrupt corticobulbar projections to brainstem output pathways involved in vestibular control of balance. These projections are either collaterals of the corticospinal tract or lie close to that tract and terminate in the pons/upper medulla. This hypothesis accounts for the association between corticospinal tract damage and GVS response asymmetry, and the lack of GVS evoked asymmetry with corticospinal lesions below the rostral medulla.

Aims: The dural and arachnoid hole caused by lumbar puncture needles is a determining factor in triggering headaches. The aim of this study is to assess the dimensions and morphological features of the dura mater and arachnoids when they are punctured by a 22 gauge Quincke needle having its bevel either in the parallel or in the transverse position. Methods: Fifty punctures were made with 22 gauge Quincke needles in the dural sac of four fresh cadavers using an “in vitro” model especially designed for this purpose. The punctures were performed by needles with bevels parallel or perpendicular to the spinal axis and studied under scanning electron microscopy. Results: Thirty five of the 50 punctures done by Quincke needles (19 in the external surface and 16 in the internal) were used for evaluation. When the needle was inserted with its bevel parallel to the axis of the dural sac (17 of 35), the size of the dura-arachnoid lesion was 0.032 mm 2 in the epidural surface and 0.037 mm 2 in the subarachnoid surface of the dural sac. When the needle’s bevel was perpendicular to the axis (18 of 35) the measurement of the lesion size was 0.042 mm 2 for the external surface and 0.033 mm 2 for the internal. There were no statistical significant differences between these results. Conclusions: It is believed that the reported lower frequency of postdural puncture headache when the needle is inserted parallel to the cord axis should be explained by some other factors besides the size of the dura-arachnoid injury.

Background: Congenital bilateral perisylvian syndrome (CBPS) is characterised by bilateral perisylvian polymicrogyria and suprabulbar paresis. Mild tetraparesis, cognitive impairment, and epilepsy are frequently associated. Sensory deficits are surprisingly rare, even though polymicrogyria often extends to auditory and sensorimotor cortex. Objectives: To study the sensorimotor and auditory cortex function and location in CBPS patients. Methods: We mapped the sensory and motor cortex function onto brain magnetic resonance images in six CBPS patients and seven control subjects using sources of somatosensory and auditory evoked magnetic fields, and of rhythmic magnetoencephalographic (MEG) activity phase-locked to surface electromyogram (EMG) during voluntary hand muscle contraction. Results: MEG-EMG coherence in CBPS patients varied from normal (if normal central sulcus anatomy) to absent, and could occur at abnormally low frequency. Coherent MEG activity was generated at the central sulcus or in the polymicrogyric frontoparietal cortex. Somatosensory and auditory evoked responses were preserved and also originated within the polymicrogyric cortex, but the locations of some source components could be grossly shifted. Conclusion: Plastic changes of sensory and motor cortex location suggest disturbed cortex organisation in CBPS patients. Because the polymicrogyric cortex of CBPS patients may embed normal functions in unexpected locations, functional mapping should be considered before brain surgery.

Most afferent signals from the viscera do not give rise to conscious experience and yet they participate in the complex neural control of visceral functions. Surprisingly little information is available on the origin, morphology, and receptor functional characteristics of the nerve endings of most primary afferent neurones to the digestive tract. This review deals with the morphological nature of the afferent neurones that supply the gastrointestinal tract specifically.

Objective: The objective of this study was to assess the volumes of medial temporal lobe structures using high resolution magnetic resonance images from patients with chronic refractory medial temporal lobe epilepsy (MTLE). Methods: We studied 30 healthy subjects, and 25 patients with drug refractory MTLE and unilateral hippocampal atrophy (HA). We used T1 magnetic resonance images with 1 mm isotropic voxels, and applied a field non-homogeneity correction and a linear stereotaxic transformation into a standard space. The structures of interest are the entorhinal cortex, perirhinal cortex, parahippocampal cortex, temporopolar cortex, hippocampus, and amygdala. Structures were identified by visual examination of the coronal, sagittal, and axial planes. The threshold of statistical significance was set to p

Objectives: To investigate if intraoperative focused high frequency repetitive transcranial magnetic stimulation (rTMS) can localise the primary motor cortex without exposure of the cortical surface. Methods: A high frequency train (357 Hz) of four suprathreshold magnetic stimuli was delivered transcranially to the region of the rolandic area during brain tumour operations in 12 patients. To induce a focal magnetoelectric field, the flat figure of eight coil (outer diameter of each loop 7 cm) was used. Motor evoked potentials (MEP) were recorded in eight muscles of the upper and lower contralateral extremities. The first stimulation site was 2.5 cm behind the bregma, the second site 2 cm, and the third site 4 cm dorsal to the first stimulation site. If no MEP were obtainable, stimulation was repeated in anteroposterior direction at more laterally located sites. Using neuronavigation, each positive stimulation site was correlated with the underlying cortical anatomy. Results: Stimulation was performed at a total of 42 sites (in two patients, maximum stimulation at the three initial sites failed to evoke a motor response). In four patients, MEP were obtained only from one stimulation site. This site exactly overlayed the primary motor cortex. In eight patients, MEP could be elicited from more than one stimulation site. In seven of the eight patients, the site from which MEP with peak amplitudes were elicited, corresponded to the primary motor cortex. In total, the primary motor cortex was correctly identified on the basis of electrophysiological findings in 11 of 12 patients (92 %). In two patients, only the more lateral stimulation sites permitted MEP recording. Conclusion: Intraoperative focused rTMS is highly sensitive for localisation of the primary motor cortex. Focused rTMS as a localising instrument alleviates the need of motor cortex exposure and, thereby, can contribute to minimise the surgical approach to brain tumours in the rolandic area.

Background: The subthalamic nucleus is the preferred target for deep brain stimulation in patients with advanced Parkinson’s disease. The site of permanent stimulation is the subject of ongoing debate, as stimulation both within and adjacent to the subthalamic nucleus may be effective. Objective: To assess the position of active electrode contacts in relation to the dorsal margin of the subthalamic nucleus as determined by intraoperative microrecordings and magnetic resonance imaging (MRI). Methods: In 25 patients suffering from severe levodopa sensitive parkinsonism, deep brain stimulating electrodes (n = 49) were implanted following mapping of the subthalamic nucleus by microrecording and microstimulation along five parallel tracks. Postoperative stereotactic radiography and fusion of pre- and postoperative MRI studies were used to determine the stereotactic position relative to the midcommissural point of the most effective electrode contacts selected for permanent stimulation (n = 49). Intraoperative microrecordings were analysed retrospectively to define the dorsal margin of the subthalamic nucleus. In cases where the dorsal margin could be defined in at least three microrecording tracks (n = 37) it was correlated with the position of the active contact using an algorithm developed for direct three dimensional comparisons. Results: Stimulation of the subthalamic nucleus resulted in marked improvement in levodopa sensitive parkinsonian symptoms and levodopa induced dyskinesias, with significant improvement in UPDRS III scores. In several instances, projection of the electrode artefacts onto the T2 weighted MRI visualised subthalamic nucleus of individual patients suggested that the electrodes had passed through the subthalamic nucleus. When the actual position of active electrode contacts (n = 35) was correlated with the dorsal margin of the subthalamic nucleus as defined neurophysiologically, most contacts were located either in proximity (± 1.0 mm) to the dorsal border of the subthalamic nucleus (32.4%) or further dorsal within the subthalamic region (37.8%). The other active contacts (29.7%) were detected within the dorsal (sensorimotor) subthalamic nucleus. The average position of all active contacts (n = 49) was 12.8 mm (± 1.0) lateral, 1.9 mm (± 1.4) posterior, and 1.6 mm (± 2.1) ventral to the midcommissural point. Conclusions: Subthalamic nucleus stimulation appears to be most effective in the border area between the upper subthalamic nucleus (sensorimotor part) and the subthalamic area containing the zona incerta, fields of Forel, and subthalamic nucleus projections.

Objectives: Despite many hypotheses, the pathophysiology of syringomyelia is still not well understood. In this report, the authors propose a hypothesis based on analysis of cerebrospinal fluid dynamics in the spine. Methods: An electric circuit model of the CSF dynamics of the spine was constructed based on a technique of computational fluid mechanics. With this model, the authors calculated how a pulsatile CSF wave coming from the cranial side is propagated along the spinal cord. Results: Reducing the temporary fluid storage capacity of the cisterna magna dramatically increased the pressure wave propagated along the central canal. The peak of this pressure wave resided in the mid-portion of the spinal cord. Conclusions: The following hypotheses are proposed. The cisterna magna functions as a shock absorber against the pulsatile CSF waves coming from the cranial side. The loss of shock absorbing capacity of the cisterna magna and subsequent increase of central canal wall pressure leads to syrinx formation in patients with Chiari I malformation.

Background: Structural alterations in the association cortices as well as in the corpus callosum (CC) have been described in schizophrenia, and have been considered to reflect developmental abnormalities. Areas of primary and association cortices have been topographically mapped in the CC. Objective: To investigate whether, in schizophrenia, there are alterations in CC subdivisions that connect association, but not primary, cortices, and also to see if the normative, developmentally mediated increase in CC size with age is absent in this disorder. Methods: The midsagittal magnetic resonance imaging scans of 31 first episode, neuroleptic naive, schizophrenic patients, 12 non-schizophrenic, psychotic patients, and 31 healthy controls were compared. The total area of CC as well as that of anterior, middle and posterior genu, body, isthmus, and anterior, middle, and posterior splenii were measured. Results: Patients with schizophrenia as a group had a smaller CC, anterior genu, anterior body, isthmus, and anterior splenium than normal controls. Furthermore, the age related increase in CC size seen in normal subjects was absent in the patients. Conclusions: The observed reductions in size in selected regions of CC suggest a reduction in axonal connections between the heteromodal association cortices, which typically involve small diameter fibres. Furthermore, the absence of an age related increase in CC size in patients with schizophrenia suggests a neurodevelopmental abnormality that may extend into adolescence and early adulthood.

Objectives: To clarify the clinical significance of positional down beat nystagmus (pDBN). Methods: A discussion of the neuro-otological findings in 50 consecutive patients with pDBN. Results: In 38 patients there was evidence of CNS disease (central group) but in 12 there was not (idiopathic group). In the CNS group, presenting symptoms were gait, speech, and autonomic dysfunction whereas in the idiopathic group patients mostly reported positional vertigo. The main neurological and oculomotor signs in the CNS group were explained by cerebellar dysfunction, including 13 patients with multiple system atrophy. In patients with multiple system atrophy with a prominent extrapyramidal component, the presence of pDBN was helpful in the differential diagnosis of atypical parkinsonism. No patient with pDBN had the Arnold-Chiari malformation, a common cause of constant down beat nystagmus (DBN). In the idiopathic group, the pDBN had characteristics which suggested a peripheral labyrinthine disorder: vertigo, adaptation, and habituation. In six patients an additional torsional component was found (concurrently with the pDBN in three). Features unusual for peripheral disorder were: bilateral positive Dix-Hallpike manoeuvre in nine of 12 patients and selective provocation by the straight head-hanging manoeuvre in two Conclusion: It is argued that some patients with idiopathic pDBN have benign paroxysmal positional vertigo (BPPV) with lithiasis of the anterior canal. The torsional component may be weak, because of the predominantly sagittal orientation of the anterior canal, and may not be readily seen clinically. Nystagmus provocation by bilateral Dix-Hallpike and straight head-hanging may be explained by the vertical upwards orientation of the ampullary segment of the anterior canal in the normal upright head position. Such orientation makes right-left specificity with the Dix-Hallpike manoeuvre less important than for posterior canal BPPV. This orientation requires a further downwards movement of the head, often achieved with the straight head-hanging position, to provoke migration of the canaliths. The straight head-hanging manoeuvre should be carried out in all patients with a history of positional vertigo and a negative Dix-Hallpike manoeuvre.