Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

LIN28 is essential for the maintenance of chicken primordial germ cells.

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

Differential modularity of the mammalian Engrailed 1 enhancer network directs sweat gland development.

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species' uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

Prime editing in chicken fibroblasts and primordial germ cells.

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

In ovo electroporation of chicken limb bud ectoderm: Electroporation to chick limb ectoderm: Electroporation to chick limb ectoderm.

BACKGROUND: Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. RESULTS: Here we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. CONCLUSION: Our method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.

Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution.

Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans' high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the Engrailed 1 (EN1) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification of more eccrine glands. Here, we show that regulation of EN1 has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, we identified a human EN1 skin enhancer, hECE18. We showed that multiple epistatically interacting derived substitutions in the human ECE18 enhancer increased its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 expression in the skin to induce the formation of more eccrine glands. Our study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change.

Evaluation of a new apparatus combining a mobile device and hand-held roller for the detection of spinal deformities: association with the sum of the right and left angles of trunk inclination.

PURPOSE: To investigate the intra- and inter-observer reliabilities of the newly developed i-Scolioroller for scoliosis screening, and to determine the optimal i-Scolioroller measurement cutoff values for identifying adolescent scoliosis with a Cobb angle ≥ 20°. METHODS: The i-Scolioroller displays the right- and left-side maximum inclination angle (Rmax, Lmax) during the forward bending test (FBT), as well as the angle of trunk inclination (ATI, i.e., whether the Rmax or Lmax is greater). Sum-ATI is defined as the sum of Rmax and Lmax. Intra-class correlation coefficients (ICC) of the ATI and sum-ATI measurements were calculated to analyze the intra- and inter-observer reliabilities for 10 plaster torsos in FBT positions obtained from patients with idiopathic scoliosis. The optimal cutoff values for scoliosis were determined using receiver operating characteristic (ROC) analysis of i-Scolioroller measurements versus Cobb angles obtained from the upright whole-spine radiographs of 112 adolescent outpatients. RESULTS: The intra-observer ICCs for the ATI/sum-ATI for 3 observers were 0.851/0.856, 0.786/0.900, and 0.772/0.796, respectively, while the corresponding inter-observer ICCs for all participants were 0.733/0.745. On ROC analysis, an ATI of 8° was the optimal cutoff value for scoliosis (sensitivity and specificity: 79.2% and 70.0%, respectively). The optimal cutoff value for sum-ATI was 11° (sensitivity and specificity: 86.1% and 82.5%, respectively). The areas under the ROC curves were 0.859 for ATI and 0.908 for sum-ATI. CONCLUSION: The optimal cutoff values for identifying scoliosis using the i-Scolioroller were a combination of 11° for the sum-ATI and 8° for the ATI.

L-type voltage-gated Ca2+ channel CaV1.2 regulates chondrogenesis during limb development.

All cells, including nonexcitable cells, maintain a discrete transmembrane potential (Vmem), and have the capacity to modulate Vmem and respond to their own and neighbors' changes in Vmem Spatiotemporal variations have been described in developing embryonic tissues and in some cases have been implicated in influencing developmental processes. Yet, how such changes in Vmem are converted into intracellular inputs that in turn regulate developmental gene expression and coordinate patterned tissue formation, has remained elusive. Here we document that the Vmem of limb mesenchyme switches from a hyperpolarized to depolarized state during early chondrocyte differentiation. This change in Vmem increases intracellular Ca2+ signaling through Ca2+ influx, via CaV1.2, 1 of L-type voltage-gated Ca2+ channels (VGCCs). We find that CaV1.2 activity is essential for chondrogenesis in the developing limbs. Pharmacological inhibition by an L-type VGCC specific blocker, or limb-specific deletion of CaV1.2, down-regulates expression of genes essential for chondrocyte differentiation, including Sox9, Col2a1, and Agc1, and thus disturbs proper cartilage formation. The Ca2+-dependent transcription factor NFATc1, which is a known major transducer of intracellular Ca2+ signaling, partly rescues Sox9 expression. These data reveal instructive roles of CaV1.2 in limb development, and more generally expand our understanding of how modulation of membrane potential is used as a mechanism of developmental regulation.

Coordination between body growth and tissue growth: Wolffian duct elongation and somitogenesis proceed in harmony with axial growth.

During embryogenesis, different tissues develop coordinately, and this coordination is often in harmony with body growth. Recent studies allow us to understand how this harmonious regulation is achieved at the levels of inter-cellular, inter-tissue, and tissue-body relationships. Here, we present an overview of recently revealed mechanisms by which axial growth (tail growth) drives a variety of morphogenetic events, with a focus on the coordinated progression between Wolffian (nephric) duct elongation and somitogenesis. We also discuss how we can relate this coordination to the events occurring during limb bud outgrowth, since the limb buds and tail bud are appendage anlagen acquired during vertebrate evolution, both of which undergo massive elongation/outgrowth.

Integration of Shh and Fgf signaling in controlling Hox gene expression in cultured limb cells.

During embryonic development, fields of progenitor cells form complex structures through dynamic interactions with external signaling molecules. How complex signaling inputs are integrated to yield appropriate gene expression responses is poorly understood. In the early limb bud, for instance, Sonic hedgehog (Shh) is expressed in the distal posterior mesenchyme, where it acts as a mediator of anterior to posterior (AP) patterning, whereas fibroblast growth factor 8 (Fgf8) is produced by the apical ectodermal ridge (AER) at the distal tip of the limb bud to direct outgrowth along the proximal to distal (PD) axis. Here we use cultured limb mesenchyme cells to assess the response of the target Hoxd genes to these two factors. We find that they act synergistically and that both factors are required to activate Hoxd13 in limb mesenchymal cells. However, the analysis of the enhancer landscapes flanking the HoxD cluster reveals that the bimodal regulatory switch observed in vivo is only partially achieved under these in vitro conditions, suggesting an additional requirement for other factors.

Early formation of the Müllerian duct is regulated by sequential actions of BMP/Pax2 and FGF/Lim1 signaling.

The Müllerian duct (MD) and Wolffian duct (WD) are embryonic tubular tissues giving rise to female and male reproductive tracts, respectively. In amniote embryos, both MD and WD emerge in both sexes, but subsequently degenerate in the males and females, respectively. Here, by using MD-specific gene manipulations in chicken embryos, we identify the molecular and cellular mechanisms that link early MD specification to tubular invagination. Early (pre-)specification of MD precursors in the coelomic epithelium requires BMP signaling and its downstream target Pax2 in a WD-independent process. Subsequently, the BMP/Pax2 axis induces Lim1 expression, a hallmark of MD specification, for which FGF/ERK and WD-derived signals are also required. Finally, the sequential actions of the BMP/Pax2 and FGF/Lim1 axes culminate in epithelial invagination to form a tubular structure driven by an apical constriction, where apical accumulation of phospho-myosin light chain is positively regulated by FGF/ERK signaling. Our study delineates mechanisms governing the early formation of the MD, and also serves as a model of how an epithelial cell sheet is transformed to a tubular structure, a process seen in a variety of developmental contexts.

Clinical, physical, and radiographic analyses of lumbar degenerative kyphosis and spondylolisthesis among community-based cohort.

PURPOSE: To investigate longitudinal radiographic changes, and physical characteristics of lumbar degenerative kyphosis (LDK) and spondylolisthesis (DS). METHODS: Two-hundred eighty nine community-based female subjects were recruited from population register and studied longitudinally for a mean 12.3 years. Upright entire spine radiographs were used to evaluate spinopelvic parameters, including lumbar lordosis (LL), pelvic incidence (PI), and vertebral slip (% slip). Physical measurements included lumbar range of motion (ROM), isometric trunk muscle strength, and photometric gait posture using change in trunk inclination angle (dTIA). RESULTS: Subjects' mean age (standard deviation: SD) was 56.9 (10.0) years at baseline and 68.5 (9.2) years at the final follow-up. Among 202 subjects who could perform instructed physical measurements, DS, defined as more than 5 % slip, was found in 50 subjects (24.8 %), and LDK, defined as LL of less than 1SD of mean value (<24.4°), was found in 24 subjects (11.9 %). DS subjects showed a significant weakness in trunk flexor strength (normal 282.5 ± 73.0 N vs. DS 245.5 ± 75.5 N, p = 0.0219), and LDK subjects showed significant differences in: trunk extensor strength (normal 493.4 ± 172.8 N vs. LDK 386.3 ± 167.6 N, p = 0.0066), ROM, and dTIA (normal 3.5° ± 2.7° vs. LDK 7.6° ± 4.8°, p < 0.0001). PI was significantly larger in DS and smaller in LDK than normal subjects (normal 53.8° ± 9.9° vs. DS 58.2° ± 10.6°, p = 0.0111; normal vs. LDK 48.4° ± 9.2°, p = 0.0191). CONCLUSIONS: Current study showed that DS was associated with reduced trunk flexor strength, which might increase pelvic anteversion, and LDK was associated with reduced extensor strength, ROM, and ambulatory kyphosis. Physical characteristics should be evaluated for the successful management of adult spinal deformity.

FGF8 coordinates tissue elongation and cell epithelialization during early kidney tubulogenesis.

When a tubular structure forms during early embryogenesis, tubular elongation and lumen formation (epithelialization) proceed simultaneously in a spatiotemporally coordinated manner. We here demonstrate, using the Wolffian duct (WD) of early chicken embryos, that this coordination is regulated by the expression of FGF8, which shifts posteriorly during body axis elongation. FGF8 acts as a chemoattractant on the leader cells of the elongating WD and prevents them from epithelialization, whereas static ('rear') cells that receive progressively less FGF8 undergo epithelialization to form a lumen. Thus, FGF8 acts as a binary switch that distinguishes tubular elongation from lumen formation. The posteriorly shifting FGF8 is also known to regulate somite segmentation, suggesting that multiple types of tissue morphogenesis are coordinately regulated by macroscopic changes in body growth.

Interepithelial signaling with nephric duct is required for the formation of overlying coelomic epithelial cell sheet.

In most organs of the body, epithelial tissues are supported by their own basement membrane and underlying stroma, the latter being regarded as a complex of amorphous cells, extracellular matrices, and soluble factors. We demonstrate here that an epithelial tube can serve as a component of stroma that supports the formation of epithelial cell sheet derived from a different origin. During development of the mesonephros in chicken embryos, the intermediate mesoderm (IMM), which contains the Wolffian duct (WD) and its associated tubules, is overlain by a sheet of epithelial cells derived from lateral plate (coelomic) mesoderm. We describe that in normal embryos, epitheliogenesis of IMM tubes and the adjacent coelomic cell sheet proceed in a coordinated manner. When the WD was surgically ablated, the overlying coelomic epithelium exhibited aberrant morphology accompanied by a punctated basement membrane. Furthermore, the WD-ablated coelomic epithelium became susceptible to latent external stress; electroporation of Rac1 resulted in epithelial-to-mesenchymal transitions (EMTs) within the coelomic epithelium. The distorted coelomic epithelium was rescued by implanting fibronectin-producing cells in place of the WD, suggesting that fibronectin provided by WD has an important role acting interepithelially. This notion was corroborated further by directly visualizing a translocation of EGFP-tagged fibronectin from fibronectin-producing to -receiving epithelia in vivo. Our findings provide a novel insight into interepithelial signaling that also might occur in adult tissues to protect against EMT and suggest a possible new target for anticancer therapeutic strategy.

Transgenesis of the Wolffian duct visualizes dynamic behavior of cells undergoing tubulogenesis in vivo.

Deciphering how the tubulogenesis is regulated is an essential but unsolved issue in developmental biology. Here, using Wolffian duct (WD) formation in chicken embryos, we have developed a novel method that enables gene manipulation during tubulogenesis in vivo. Exploiting that WD arises from a defined site located anteriorly in the embryo (pronephric region), we targeted this region with the enhanced green fluorescent protein (EGFP) gene by the in ovo electroporation technique. EGFP-positive signals were detected in a wide area of elongating WD, where transgenic cells formed an epithelial component in a mosaic manner. Time-lapse live imaging analyses further revealed dynamic behavior of cells during WD elongation: some cells possessed numerous filopodia, and others exhibited cellular tails that repeated elongation and retraction. The retraction of the tail was precisely regulated by Rho activity via actin dynamics. When electroporated with the C3 gene, encoding Rho inhibitor, WD cells failed to contract their tails, resulting in an aberrantly elongated process. We further combined with the Tol2 transposon-mediated gene transfer technique, and could trace EGFP-positive cells at later stages in the ureteric bud sprouting from WD. This is the first demonstration that exogenous gene(s) can directly be introduced into elongating tubular structures in living amniote embryos. This method has opened a way to investigate how a complex tubulogenesis proceeds in higher vertebrates.

Epidemiology of degenerative lumbar scoliosis: a community-based cohort study.

STUDY DESIGN: A 12-year prospective study of pre-existing and de novo degenerative lumbar scoliosis (DLS) among community-based female volunteers. OBJECTIVE: To investigate serial entire spine radiographs of healthy female volunteers and to clarify radiographical characteristics and predictors of pre-existing and de novo DLS. SUMMARY OF BACKGROUND DATA: DLS is among the most frequent spinal defomities in the aging spine; however, the onset or the natural course of this condition has not been elucidated. METHODS: A total of 144 female volunteers were recruited from population register. Radiographical measurements using entire spine radiographs included thoracic kyphosis (T4-12), lumbar lordosis (LL) (L1-5), sacral inclination angle, pelvic incidence, sagittal balance (C7 plumb), coronal L4 endplate angle (L4 tilt), and scoliotic angle by Cobb method. More than 10° of scoliosis was diagnosed as DLS. L4 vertebral size was measured (divided by body height) as well as lateral osteophyte formation and lateral disc wedge angle. RESULTS: Mean baseline age and follow-up period were 54.4 years and 12.1 years, respectively. Pre-existing DLS (pre-DLS) was found in 42 subjects (29.2%) at baseline. Among pre-DLS, 11 subjects (26%) showed more than 5° progression in scoliosis. De novo DLS has developed in 30 subjects (29.4%) among those without baseline scoliosis.Cox proportional hazards models revealed younger age, smaller L4 size, lower LL, greater DLS angle, and L4 tilt at baseline to be the risk factors of progression of pre-DLS and smaller L4 size, unilateral osteophyte formation, and lateral disc wedging to the risk of development of de novo DLS. CONCLUSION: This study indicated that younger age, smaller L4 size, lower LL, greater DLS angle, and L4 tilt at baseline should be evaluated as predictors of progression of pre-DLS. Early signs of asymmetric disc degeneration and smaller L4 size should also be evaluated as predictors of development of de novo DLS.

Radiographic analysis of newly developed degenerative spondylolisthesis in a mean twelve-year prospective study.

STUDY DESIGN: Prospective study of community-based female volunteers. OBJECTIVE: To investigate the incidence of newly developed degenerative spondylolisthesis (DS) among those without baseline deformity, and to clarify radiographic characteristics and predictors of DS. SUMMARY OF BACKGROUND DATA: There has been limited number of prospective studies of DS. Our on-going cohort study of healthy volunteers enabled long-term observation of highly susceptible perimenopause female subjects. METHODS: A final total of 142 female subjects without spondylolisthesis at baseline radiographs were included and followed up for more than 8 years. Standardized serial entire spine radiographs were used to measure spinopelvic alignment, including pelvic incidence (PI), vertebral inclination angle, disc height, vertebral size, and facet orientation. RESULTS: The incidence of newly developed DS was 12.7%. Comparison between DS and non-DS subjects demonstrated that DS subjects had significantly greater baseline lumbar lordosis, PI, vertebral inclination angle, and smaller vertebral size. Exaggerated lumbopelvic alignment was more prominent in L3-DS than in L4-DS, and L4-DS was associated with the decrease in L4/5 disc height. Multivariate analysis revealed that PI, L4 vertebral inclination, adjusted vertebral size, and facet sagittalization were independent predictors of the development of DS. CONCLUSION: This was the first study to confirm the relationship of PI and the development of DS in a long-term prospective observation. Proposed pathogenetic differences might explain the fact that L4-DS is far more prevalent than L3-DS. The development of DS could be predicted by baseline lumbopelvic morphology among the highly susceptible perimenopause women.

Cartilage degeneration is associated with augmented chemically-induced joint pain in rats: a pilot study.

BACKGROUND: Osteoarthritis arising from cartilage degeneration is the most common cause of joint pain. However, the relationship between joint pain and cartilage degeneration is not well understood. QUESTIONS/PURPOSES: We asked whether the inflammatory mediators participate in the joint pain in the presence of cartilage degeneration. METHODS: We observed electromyographic responses of hindlimb flexors to four inflammatory mediators (bradykinin, ATP, acetylcholine, and serotonin) injected in normal rat knees and in those with monosodium iodoacetate (MIA)-induced arthritis. RESULTS: Joint cartilage of all the rats with MIA-induced arthritis histologically showed severe degeneration. We observed greater magnitude and longer duration responses in the MIA-induced arthritis than normal joints with all four mediators. CONCLUSIONS: The data suggested nociceptors in osteoarthritic joints are more sensitive to inflammatory mediators than in normal joints. Such nociceptive sensitization to inflammatory mediators may participate in the joint pain in osteoarthritis.

Changes in the sagittal spinal alignment of the elderly without vertebral fractures: a minimum 10-year longitudinal study.

BACKGROUND: Thoracic hyperkyphosis, or loss of lumbar lordosis, is often equated with osteoporosis because vertebral fractures are assumed to be a major causative factor. However, recent evidence suggests that up to one-half of the patients with hyperkyphosis have no evidence of underlying vertebral fracture. The shape characteristics of the intervertebral discs and their role in determining kyphotic curvature have been investigated. The spinal sagittal parameters and segmental disc angles of elderly subjects were examined during a longitudinal follow-up. METHODS: A total of 53 subjects (20 men, 33 women) without vertebral fractures during a more than 10-year follow-up were included in this study, undergoing standing lateral radiographs of the spine using 36-inch film at baseline and final follow-up. The mean age of the subjects was 63 years (range 50-77 years) at baseline and 75 years (range 62-88 years) at follow-up; and the mean follow-up period was 11 years 11 months. RESULTS: The lumbar lordosis and the sacral inclination angle decreased and the C7-plumbline distance increased with age. Among a total of 664 discs, 70 discs (10.5%) showed anterior wedging over 5 degrees at follow-up. In contrast, 39 discs (5.9%) showed posterior wedging over 5 degrees . The subjects had only discs with anterior wedging, decreased total lumbar lordosis, and the C7 plumbline displaced anteriorly. However, when the subjects had discs with posterior wedging, the C7 plumbline and sagittal spinal balance tended to be maintained. This compensatory mechanism was seen in younger subjects. CONCLUSIONS: A decrease in the total lumbar lordosis and the sacral inclination angle occurred with age. Increasing age correlated with a more forward sagittal vertical axis, depending on a decrease in the total lumbar lordosis. The cause of loss of lumbar lordosis in the subjects without vertebral fracture was anterior wedging of the segmental discs. Posterior wedging of the thoracic and lumbar segmental discs then could occur, compensating for the loss of lumbar lordosis.

Effects of dry needling at tender points for neck pain (Japanese: katakori): near-infrared spectroscopy for monitoring muscular oxygenation of the trapezius.

BACKGROUND: Neck pain (katakori) is a common symptom in adult Japanese people. However, the pathophysiological aspect of this condition has not been well documented to date. The purpose of this study was to investigate the effects of tender point dry needling to the trapezius muscles and the resultant changes in muscular hemodynamics. METHODS: "Neck pain" patients were defined as those complaining of dull pain or discomfort mainly along the trapezius muscles without serious spinal or shoulder disorders. We used near-infrared spectroscopy to monitor the changes of oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb) of the trapezius muscles and a Visual Analogue Scale (VAS) to assess subjective neck pain intensity. Experiment I: Nine subjects with "neck pain" and four control subjects were recruited. Total hemoglobin (Hb) and SdO2 [= oxyHb/(oxyHb + deoxyHb)] were measured before and immediately after needling for 15 min. We compared these parameters and VAS before and immediately after needling. Experiment II: Thirteen subjects with "neck pain" were instructed to perform isometric contraction of their trapezius muscles for 1 min; the half-recovery time of SdO2 (defined as T(R)) was measured. After that, all subjects underwent needling. On the next day, we repeated the measurements of T(R) after the same voluntary contraction of the trapezius muscle in the same patients. We compared T(R) and VAS before and on the day after needling. RESULTS: Experiment I: All subjects with "neck pain" reported significant pain relief (P = 0.0147) measured by VAS immediately after needling, but total Hb and SdO2 exhibited no significant change after needling. Experiment II: T(R) was shortened on the day after needling in 10 of 13 patients (P = 0.0043), and neck pain was decreased in 12 patients (P = 0.0158). CONCLUSIONS: After dry needling, total Hb and SdO2 did not change in real time, but T(R) was shortened on the next day. These results showed that the shortening of T(R) would provide a measure by which to assess the effectiveness of treatment for neck pain.