Pharmacological evaluation of E2730, a novel selective uncompetitive GAT1 inhibitor, on epileptiform activities in resected brain tissues from human focal cortical dysplasia ex vivo.

Focal cortical dysplasia (FCD) is an important etiology of focal epilepsy in children and adults. However, only a few preclinical models sufficiently reproduce the characteristic histopathologic features of FCD. To improve the success rate of clinical trials for antiseizure medications (ASMs) in patients with FCD, more human-relevant preclinical models are needed, and epileptic foci resected from patients are a powerful tool for this purpose. Here, we conducted ex vivo studies using epileptic foci resected from patients with FCD type II to evaluate the pharmacologic effects of the ASM candidate E2730, a selective uncompetitive inhibitor of γ-aminobutyric acid transporter 1. We used the same ex vivo assay system to assess carbamazepine (CBZ), an ASM often prescribed for focal epilepsy, as a reference. At the higher dose tested (200 µM), both E2730 and CBZ suppressed spontaneous epileptiform activities almost completely. At the lower dose (100 µM), CBZ reduced the area of brain tissue showing epileptiform activity, whereas E2730 significantly decreased the number of epileptiforms. These findings suggest that E2730-both as a single agent and in combination with CBZ-merits evaluation in clinical trials involving patients with FCD.

Frequency and pathogenesis of periprosthetic atypical femoral fractures associated with total knee arthroplasty: A multicenter prospective study with complementary histopathological and biomechanical analysis.

INTRODUCTION: Although the diagnostic criteria for atypical femoral fracture (AFF) exclude periprosthetic fractures, reports of periprosthetic femoral fractures with characteristics of AFF are rapidly increasing. In this study, we investigated the frequency and pathogenesis of periprosthetic AFF associated with total knee arthroplasty (TKA) based on a theory of AFF subtypes that divides AFFs into two main types: fragility stress fractures of the bowed femoral shaft in the mid-shaft and "typical" subtrochanteric AFFs due to suppression of bone turnover (e.g., by bisphosphonates). PATIENTS AND METHODS: This multicenter prospective study of AFFs was conducted from 2015 through 2022. Clinical, pathological, and morphological characteristics were investigated in patients with periprosthetic AFFs associated only with non-stem TKA. Then, biomechanical investigation of the periprosthetic AFF was performed by computer tomography-based finite element analysis (CT/FEA) using two models with different load axes to examine how the correction of lower limb alignment by TKA influences the tensile stress distribution of the femur and the location of the AFF. RESULTS: Four of 61 AFFs (6.6%) were identified to be periprosthetic AFF (1 mid-shaft; 3 subtrochanteric). Periprosthetic AFFs had characteristics including mechanical stress due to bowing deformity and potentially suppressed bone turnover due to long-term exposure to specific drugs (e.g., bisphosphonates and glucocorticoids). Although 2 periprosthetic AFFs appeared to involve a bowed femur, one with both of the aforementioned characteristics occurred in the subtrochanteric region, which would be an unusual site for a bowed AFF, and it was demonstrated histologically to have biological activity at the fracture site, suggesting a stress fracture. Furthermore, CT/FEA revealed that tensile stress distribution changed proximally as load axis was shifted laterally according to correction of lower limb alignment by TKA. CONCLUSION: Orthopedic surgeons should recognize the presence of TKA-associated periprosthetic AFF caused by various factors including specific drugs, bowing deformity, and lower limb alignment. X-rays of the full-length femurs should be checked regularly after TKA, especially in patients with bowed femurs or long-term exposure to specific drugs.

Identification of a Biallelic Missense Variant in Gasdermin D (c.823G > C, p.Asp275His) in a Patient of Atypical Gorham-Stout Disease in a Consanguineous Family.

Gorham-Stout disease (GSD), also called vanishing bone disease, is a rare osteolytic disease, frequently associated with lymphangiomatous tissue proliferation. The causative genetic background has not been noted except for a case with a somatic mutation in KRAS. However, in the present study, we encountered a case of GSD from a consanguineous family member. Whole-exome sequencing (WES) analysis focusing on rare recessive variants with zero homozygotes in population databases identified a homozygous missense variant (c.823G > C, p.Asp275His) in gasdermin D (GSDMD) in the patient and heterozygous in his unaffected brother. Because this variant affects the Asp275 residue that is involved in proteolytic cleavage by caspase-11 (as well as -4 and -5) to generate an activating p30 fragment required for pyroptotic cell death and proinflammation, we confirmed the absence of this cleavage product in peripheral monocytic fractions from the patient. A recent study indicated that a shorter p20 fragment, generated by further cleavage at Asp88, has a cell-autonomous function to suppress the maturation of osteoclasts to resorb bone matrix. Thus, the present study suggests for the first time the existence of hereditary GSD cases or novel GSD-like diseases caused by GSDMD deficiency. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

E2730, an uncompetitive γ-aminobutyric acid transporter-1 inhibitor, suppresses epileptic seizures in a rat model of chronic mesial temporal lobe epilepsy.

OBJECTIVE: More than one third of mesial temporal lobe epilepsy (MTLE) patients are resistant to current antiseizure medications (ASMs), and half experience mild-to-moderate adverse effects of ASMs. There is therefore a strong need to develop and test novel ASMs. The objective of this work is to evaluate the pharmacokinetics and neurological toxicity of E2730, a novel uncompetitive inhibitor of γ-aminobutyric acid transporter-1, and to test its seizure suppression effects in a rat model of chronic MTLE. METHODS: We first examined plasma levels and adverse neurological effects of E2730 in healthy Wistar rats. Adult male rats were implanted with osmotic pumps delivering either 10, 20, or 100 mg/kg/day of E2730 subcutaneously for 1 week. Blood sampling and behavioral assessments were performed at several timepoints. We next examined whether E2730 suppressed seizures in rats with chronic MTLE. These rats were exposed to kainic acid-induced status epilepticus, and 9 weeks later, when chronic epilepsy was established, were assigned to receive one of the three doses of E2730 or vehicle for 1 week in a randomized crossover design. Continuous video-electroencephalographic monitoring was acquired during the treatment period to evaluate epileptic seizures. RESULTS: Plasma levels following continuous infusion of E2730 showed a clear dose-related increase in concentration. The drug was well tolerated at all doses, and any sedation or neuromotor impairment was mild and transient, resolving within 48 h of treatment initiation. Remarkably, E2730 treatment in chronically epileptic rats led to seizure suppression in a dose-dependent manner, with 65% of rats becoming seizure-free at the highest dose tested. Mean seizure class did not differ between the treatment groups. SIGNIFICANCE: This study shows that continuous subcutaneous infusion of E2730 over 7 days results in a marked, dose-dependent suppression of spontaneous recurrent seizures, with minimal adverse neurological effects, in a rat model of chronic MTLE. E2730 shows strong promise as an effective new ASM to be translated into clinical trials.

Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

OBJECTIVE: As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. METHODS: Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. RESULTS: E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-|fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. SIGNIFICANCE: E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

A Comparison of Spinopelvic Alignment and Quality of Life between Farmers and Non-Farmers: A Cross-Sectional Population-Based Study in a Japanese Rural Area.

BACKGROUND: It may be difficult to define what would constitute an abnormal spinal sagittal alignment. The same degree of malalignment may be found both in patients with pain and disability and in asymptomatic individuals. This study focuses on elderly farmers who characteristically have a kyphotic spine, in addition to local residents. It questions whether these patients experience cervical and lower back symptoms, respectively, more often than elderly people who never worked on a farm and do not have a kyphotic deformity. Previous research could have been biased by sampling patients who came to a spine clinic for treatment, whereas this study sampled asymptomatic elderly who may or may not have had kyphosis. METHODS: We studied 100 local residents at their annual health checkup (22 farmers and 78 non-farmers) with a median age of 71 years (range 65-84 years). Spinal radiographs were used to measure sagittal vertical axis, lumbar lordosis, thoracic kyphosis and other measurements of sagittal malalignment. Back symptoms were measured using Oswestry Disability Index (ODI) and Neck Disability Index (NDI). The association between alignment measures and back symptoms were calculated by bivariate comparison between patient groups and by Pearson's correlation. RESULTS: About 55% of farmers and 35% of non-farmers had abnormal radiographs (i.e., vertebral fracture). Farmers had higher measurements of sagittal vertical axis (SVA), compared to non-farmers, when measured from C7 (median 24.4 mm vs. 9.15 mm, p = 0.04) and from C2 (47.65 vs. 25.3, p = 0.03). Lumbar lordosis (LL) and thoracic kyphosis (TK) were significantly decreased in farmers vs. non-farmers (37.5 vs. 43.5, p = 0.04 and 32.5 vs. 39, p = 0.02, respectively). The ODI was likely to be higher among farmers compared to non-farmers while NDI scores showed no significant difference between farmers and non-farmers (median 11.7 vs. 6.0, p = 0.06 and median 13 vs. 12, p = 0.82, respectively). In terms of correlation among spinal parameters, LL had a higher correlation with SVA, but TK had less correlation with SVA among farmers compared to non-farmers. There was no significant correlation between disability scores and measurements of sagittal alignment. CONCLUSIONS: Farmers had higher measurements of sagittal malalignment, characterized by loss of LL, decreased TK and an increased forward translation of cervical vertebrae relative to sacrum. ODI was likely to higher in farmers compared to non-farmers although the association did not reach a significant level. These results probably indicate that the gradual development of spinal malalignment in agricultural workers does not result in excess morbidity compared to controls.

Evaluation of Ulnar Setting of Volar Locking Plates for Distal Radius Fractures Using Modified Skyline View.

Purpose: Volar lunate facet fragments in distal radius fractures are located at the center of the load in the wrist joint and, therefore, should be properly supported by the distal ulnar setting of volar locking plates to obtain better postoperative outcomes. This study evaluated the usefulness of the modified skyline view (MSV) in intraoperative fluoroscopy for the ulnar setting of volar locking plates by comparing it with that of the anteroposterior view (APV). Methods: Sixty-five patients with distal radius fractures who underwent open reduction and plate fixation as well as follow-up intraoperative fluoroscopy and postoperative computed tomography (CT) at our institution between April 2019 and March 2022 were included. The distance between the ulnar edge of the plate and the distal radius (d-value) was measured retrospectively using intraoperative fluoroscopy (distance measured from MSV and distance measured from APV) or postoperative CT (distance measured using postoperative CT). The distance measured from MSV and that measured from APV were compared with those measured using CT as the true values. Each measurement was performed twice by 2 examiners at an interval of 1 month. The comparison scores were evaluated using the intraclass correlation coefficient. Results: The distance measured from MSV showed a difference of 0.6 ± 0.5 mm from that measured using CT, which was significantly smaller than that measured from APV (1.2 ± 0.9 mm; P < .001). Neither postoperative volar subluxation nor dislocation of bone fragments was found during the study period. Both intraclass correlation coefficient (1,1) and intraclass correlation coefficient (2,1) reliabilities were substantial to almost perfect. Conclusions: Modified skyline view is an effective and versatile imaging method for estimating the ulnar setting of volar locking plates; it provides measurements more similar to those provided by postoperative CT compared with APV. The use of MSV may reduce postoperative complications, such as volar subluxation and dislocation of bone fragments, especially in cases with volar lunate facet fragments in which the ulnar setting of the plate is significant. Type of study/level of evidence: Diagnostic III.

Impact of romosozumab on serum calcium concentration and factors predicting the fluctuations in calcium concentration upon romosozumab administration: A multicenter retrospective study.

Objectives: As romosozumab has both bone anabolic and antiresorptive effects, it is not clear which patient groups are more likely to have decreased calcium concentrations when treated with romosozumab. The aim of this study was to investigate the impact of romosozumab treatment on serum calcium concentration in patients with osteoporosis with a high risk of fractures and identify factors that might be associated with, or even predict, a fluctuation in calcium concentration upon romosozumab administration. Materials and methods: In total, 47 patients were included in this retrospective study. We performed a Wilcoxon signed-rank test to identify differences in the calcium concentration before and 1 month after romosozumab initiation. Associations between baseline variables and changes in serum calcium concentration were investigated with a multiple-linear regression model using a forward-backward stepwise procedure. Results: Romosozumab administration reduced the serum calcium concentration by an average of 3.1 % after 1 month. No patient complained of symptoms of hypocalcemia during the first month after treatment. Univariate regression analysis showed that age and calcium concentration were significantly associated with the decrease in serum calcium concentrations by romosozumab administration. In addition, stepwise regression analysis identified age and calcium concentrations as independent factors associated with the decrease in calcium concentration by romosozumab. Conclusion: Romosozumab administration caused a modest but significant decrease in serum calcium concentration. Older age and higher baseline calcium concentrations were associated with a greater decrease in calcium concentrations by romosozumab administration. Although the likelihood of severe hypocalcemia from romosozumab administration may be low, physicians prescribing romosozumab to patients with osteoporosis should be aware of the symptoms of hypocalcemia and promptly evaluate calcium levels if patients complain of these symptoms.

The Real-World Effect of 12 Months of Romosozumab Treatment on Patients With Osteoporosis With a High Risk of Fracture and Factors Predicting the Rate of Bone Mass Increase: A Multicenter Retrospective Study.

Excluding clinical trials, there is limited evidence on the effect of 12 months of romosozumab treatment on bone mineral density (BMD) increase in real-world clinical practice because its use has only been approved recently. Thus, this study aimed to investigate the real-world effect of 12 months of romosozumab treatment on BMD increase and identify factors that predict the rate of BMD increase after 12 months of romosozumab treatment. We retrospectively investigated 106 patients who completed a 12-month romosozumab treatment for osteoporosis with a high risk of fractures at four hospitals from March 2020 to March 2022. The univariate and multiple regression analyses were performed to analyze the concurrent effects of various factors on the BMD increase after the 12-month romosozumab treatment. After 1 year of treatment, the lumbar spine BMD increased by 14.6%, and femoral neck BMD increased by 5.1%. Univariate regression analysis found that male sex, high tartrate-resistant acid phosphatase 5b (TRACP-5b) value before romosozumab administration, absence of osteoporosis medications before romosozumab administration, and low baseline lumbar spine BMD were associated with the extent of lumbar spine BMD increase. Moreover, stepwise multiple regression analysis found that the TRACP-5b value before romosozumab administration was a significant predictor of the rate of lumbar spine BMD increase after 1 year of romosozumab administration. In conclusion, our results demonstrated the effectiveness of the 12-month romosozumab treatment for osteoporosis with a high risk of fractures and the TRACP-5b value before romosozumab administration was a significant predictor of the rate of lumbar spine BMD increase after 1 year of romosozumab administration. Our findings could help establish more efficient treatment strategies for patients with osteoporosis at a high risk of fracture. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The necessity of routine screening for deep vein thrombosis before surgery.

Background: Pulmonary embolism (PE) from deep venous thrombosis (DVT) can be a fatal postoperative complication. Preventive measures for venous thromboembolism (VTE) was evaluated in this hospital. Materials and methods: Preoperative DVT screening following surgery under general anesthesia in 2009-2016 was examined, and then, 217 patients diagnosed with DVT by preoperative leg-ultrasound (US) between 2014 and 2016 were retrospectively analyzed. Results: There were 24,826 operations under general anesthesia in the study period. Preoperative leg-US was performed in 5345 (21.5%) patients, and 648 (12.1% of patients, 2.6% of total operations) were diagnosed with DVT. In 2014-2016, 217 patients, which is 11.7% of patients undergoing leg-US, were diagnosed with DVT. DVT was found in the proximal veins (upper popliteal vein) in 86 (39.6%) patients. A total of 143 (62%) patients were considered to have organized thrombi, no patient developed pulmonary embolism, and 133 (58%) patients were discharged without follow-up examination for DVT. Ninety-six patients were evaluated for changes on leg-US, with no difference in the results with and without anticoagulant use. On multivariate logistic regression analysis, anticoagulants appeared effective for non-organized thrombi, higher D-dimer levels (≥10 µg/mL), or orthopedic surgery. Conclusion: Preoperative screening for DVT did not appear useful, and treatment of asymptomatic DVT was not always necessary.

Impact of body mass index on surgical outcomes and complications in adult spinal deformity.

BACKGROUND: Several studies have reported that overweightness and obesity are associated with higher complication rates in lumbar spine surgery. However, little is known about the effect of obesity on postoperative complications in adult spinal deformity (ASD) surgery, especially in the elderly. This study aimed to examine the effect of body mass index (BMI) on surgical outcomes and postoperative complications in elderly ASD patients undergoing surgical correction in Japan. METHODS: We conducted a retrospective, multicenter, observational study of 234 consecutive patients diagnosed with ASD who underwent corrective surgery. Patients were divided into two groups according to BMI, BMI <25 (153 patients, mean age 71.9 years) and BMI ≥ 25 (overweight/obese, 81 patients, mean age 73.3 years). Radiographic results and perioperative complications were compared between the two groups. RESULTS: Surgical complications occurred in approximately 20% of patients in each group; complications did not significantly differ between the two groups. A greater proportion of patients in the BMI ≥ 25 group experienced mechanical failure and DJK, although the difference was not significant. Preoperative mean lumbar lordosis (LL), pelvic incidence (PI) minus LL, sacral slope (SS) and sagittal vertical axis (SVA) were similar in the BMI < 25 and BMI ≥ 25 groups. However, the BMI ≥25 group had lower mean LL (p = 0.015) and higher PI minus LL (p = 0.09) postoperatively. The BMI ≥25 groups also had significantly smaller LL (p = 0.026), smaller SS (p = 0.049) and higher SVA (p = 0.041) at the final follow-up, compared to the BMI < 25 group. CONCLUSIONS: In the present study, no difference in medical or surgical complications after ASD surgery was found between overweight/obese patients (BMI ≥ 25) and those with BMI < 25. However, correction of LL and SVA was smaller in patients with overweight/obese patients.

Comparison of Lateral Lumbar Interbody Fusion and Posterior Lumbar Interbody Fusion as Corrective Surgery for Patients with Adult Spinal Deformity-A Propensity Score Matching Analysis.

Lateral lumbar interbody fusion (LLIF) is increasingly performed as corrective surgery for patients with adult spinal deformity (ASD). This paper compares the surgical results of LLIF and conventional posterior lumbar interbody fusion (PLIF)/transforaminal lumbar interbody fusion (TLIF) in ASD using a propensity score matching analysis. We retrospectively reviewed patients with ASD who received LLIF and PLIF/TLIF, and investigated patients' backgrounds, radiographic parameters, and complications. The propensity scores were calculated from patients' characteristics, including radiographic parameters and preoperative comorbidities, and one-to-one matching was performed. Propensity score matching produced 21 matched pairs of patients who underwent LLIF and PLIF/TLIF. All radiographic parameters significantly improved in both groups at the final follow-up compared with those of the preoperative period. The comparison between both groups demonstrated no significant difference in terms of postoperative pelvic tilt, lumbar lordosis (LL), or pelvic incidence-LL at the final follow-up. However, the sagittal vertical axis tended to be smaller in the LLIF at the final follow-up. Overall, perioperative and late complications were comparable in both procedures. However, LLIF procedures demonstrated significantly less intraoperative blood loss and a smaller incidence of postoperative epidural hematoma compared with PLIF/TLIF procedures in patients with ASD.

Comparative analysis of clinical factors associated with pedicle screw pull-out during or immediately after surgery between intraoperative cone-beam computed tomography and postoperative computed tomography.

BACKGROUND: No studies to date have elucidated the clinical factors associated with pedicle screw pull-out during or immediately after surgery. The aim of this study was to assess the frequency of pedicle screw pull-out by comparing intraoperative scans obtained using cone-beam computed tomography (CBCT) with postoperative scans obtained using computed tomography (CT). We also sought to determine the incidence of pedicle screw pull-out and identify relevant risk factors. METHODS: This was a retrospective analysis of prospectively collected data for 742 pedicle screws placed in 76 consecutive patients who underwent at least triple-level posterior fixation for thoracic or lumbar spinal injury, spinal metastasis, or pyogenic spondylitis between April 2014 and July 2020. Pedicle screw pull-out distance in the axial and sagittal planes was compared between CT scans obtained 2 days postoperatively and CBCT images acquired intraoperatively. Risk factors associated with pedicle screw pull-out were investigated by multivariate logistic regression analysis. RESULTS: Pedicle screw pull-out was seen with 58 pedicle screws (7.8%) in 26 patients (34.2%). There were significant differences in age, number of fused segments, frequency of diffuse idiopathic skeletal hyperostosis (DISH), and medical history of osteoporosis for pedicle screw pull-out. Risk factors for pedicle screw pull-out were older age (odds ratio 1.07, 95% confidence interval 1.02-1.130) and a diagnosis of DISH (odds ratio 3.35, 95% confidence interval 1.12-10.00). Several cases suggest that use of connecting rods was an important factor in intraoperative pedicle screw pull-out. CONCLUSIONS: Our findings suggest that age, number of fused segments, presence of DISH, and medical history of osteoporosis are risk factors for pedicle screw pull-out, with the greatest being older age and DISH.

Inhibitory effect of anti-seizure medications on ionotropic glutamate receptors: special focus on AMPA receptor subunits.

OBJECTIVE: The purpose of the current analysis was to investigate the direct inhibitory effects of perampanel and other anti-seizure medications (ASMs) on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartic acid (NMDA), and kainate glutamate receptor subtypes using electrophysiological assessments. METHODS: AMPA receptor subunit-expressing cell lines (hGluA1-4, including two kinds of Q/R RNA-editing variants of hGluA2), NMDA receptor-expressing cells (hNR1/hNR2B), and kainate receptor-expressing cells (hGluK2) were developed in house. The effects of perampanel, and other ASMs including topiramate, phenobarbital, lamotrigine, gabapentin, carbamazepine, valproate, levetiracetam, and lacosamide, on AMPA, NMDA, and kainate receptors were evaluated by automated patch-clamp technique. In the same way, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) and GYKI 52466 were evaluated as reference compounds of AMPA receptor antagonists. For the AMPA receptor functional assay, AMPA currents were elicited by AMPA in the presence of cyclothiazide. NMDA with glycine was used as a stimulant for the NMDA receptor assays, while glutamate was used for the kainate receptor assays. The mean 50 % inhibitory concentration (IC50) values were determined based on sigmoidal-curve fitting using GraphPad Prism software. RESULTS: Perampanel inhibited functions of hGluA1-4, but did not inhibit hNR1/hNR2B and hGluK2 up to 25 µM, the maximum soluble concentration. The IC50 values were 660 nM for hGluA1, 780 nM for hGluA2(R), 1200 nM for hGluA2(Q), 1200 nM for hGluA3, and 1800 nM for hGluA4. NBQX and GYKI 52466 also inhibited the function of all AMPA receptor subunits, but did not inhibit hNR1/hNR2B and hGluK2. The IC50 values for NBQX were 880 nM for hGluA1, 290 nM for hGluA2(R), 310 nM for hGluA2(Q), 330 nM for hGluA3, and 630 nM for hGluA4. For GYKI 52466, IC50 values were 25,000 nM for hGluA1, 30,000 nM for hGluA2(R), 42,000 nM for hGluA2(Q), 28,000 nM for hGluA3, and 53,000 nM for hGluA4. Phenobarbital inhibited hGluA2(R) at an IC50 value of 730,000 nM. The majority of other ASMs evaluated in this study did not show a direct inhibitory effect on almost any of the glutamate receptor functions examined up to 1 M. However, lamotrigine and carbamazepine inhibited hNR1/hNR2B function at IC50 values of 930,000 and 1,000,000 nM, respectively. SIGNIFICANCE: Only a few ASMs evaluated in this study showed direct interaction with ionotropic glutamate receptors. Perampanel is the only ASM that had a potent inhibitory effect on all AMPA receptor subtypes, but did not inhibit NMDA or kainate receptor subunits; while phenobarbital inhibited GluA2(R), and carbamazepine and lamotrigine inhibited the NMDA receptor at high concentration ranges.

Biological activity is not suppressed in mid-shaft stress fracture of the bowed femoral shaft unlike in "typical" atypical subtrochanteric femoral fracture: A proposed theory of atypical femoral fracture subtypes.

BACKGROUND: We have investigated mid-shaft stress fractures of the bowed femoral shaft (SBFs), well before the first report of an association between suppression of bone turnover and atypical femoral fractures (AFFs). Although all cases of SBF meet the criteria for AFF, SBFs can also occur in patients with no exposure to bone turnover suppression-related drugs (e.g., bisphosphonates). Using bone morphometry and biomechanical analyses, we devised a theory of AFF subtypes, dividing AFFs into fragility SBFs in the mid-shaft and "typical" subtrochanteric AFFs caused by suppressed bone turnover. The aim of this multicenter prospective study was to provide evidence for this novel concept in terms of biological activity. METHODS: The study was conducted at 12 hospitals in Japan from 2015 through 2019. Thirty-seven elderly women with AFF were included and classified according to location of the fracture into a mid-shaft AFF group (n = 18) and a subtrochanteric AFF group (n = 19). Patient demographics and clinical characteristics were investigated to compare the two groups. The main focus was on histological analysis of the fracture site, and bone metabolism markers were evaluated to specifically estimate biological activity. RESULTS: All patients in the subtrochanteric AFF group had a history of long-term (>3 years) exposure to specific drugs that have been reported to cause AFF, but 5 of the 18 patients in the mid-shaft AFF group had no history of exposure to such drugs. Femoral bowing was significantly greater in the mid-shaft AFF group (p < 0.001). In the histological analysis, active bone remodeling or endochondral ossification was observed in the mid-shaft AFF group, whereas no fracture repair-related biological activity was observed in the majority of patients in the subtrochanteric AFF group. Levels of tartrate-resistant acid phosphatase-5b and undercaroxylated osteocalcin were significantly lower in the subtrochanteric AFF group (p < 0.05). CONCLUSION: The possibility of our devised AFF subtype theory was demonstrated. Biological activity tends not to be suppressed in mid-shaft SBFs unlike in "typical" subtrochanteric AFFs involving bone turnover suppression. Although validation of the proposed theory in other populations is needed, we suggest that the pathology and treatment of AFFs be reconsidered based on its subtype.

Potential bone fragility of mid-shaft atypical femoral fracture: Biomechanical analysis by a CT-based nonlinear finite element method.

INTRODUCTION: The authors previously reported a CT-based nonlinear finite element analysis (nonlinear CT/FEA) model to investigate loading stress distribution in the femoral shaft of patients with atypical femoral fractures (AFFs). This showed that stress distribution, influenced primarily by femoral bowing, may determine the location of AFF. Here, we demonstrate the locational characteristics associated with AFFs in an Asian, specifically Japanese, population regarding bone strength. This is the second report from our multicentre research project suggesting a possible new concept of diagnostic criteria or treatment according to AFF subtype. PATIENTS AND METHODS: A multicentre prospective study was conducted at 12 hospitals in Japan from August 2015 through June 2018. We recruited three study groups composed of elderly females over the age of 60 years-the mid-shaft AFF group (n = 14; 80.0 ±â€¯6.5 years), the subtrochanteric AFF group (n = 15; 73.9 ±â€¯6.8 years), and the control group who had sustained unilateral hip fracture (n = 21; 82.1 ±â€¯7.1 years)-and analysed femoral neck bone density and strength. Bone strength of the femoral neck was predicted with an evaluation method using nonlinear CT/FEA in both standing and falling configuration. RESULTS: Femoral neck bone density and strength were significantly higher in the subtrochanteric AFF group compared with the mid-shaft AFF and control groups (p <  0.0001). No significant difference was seen in bone strength between the mid-shaft AFF and control groups (standing, p =  0.7616; falling, p =  0.3803). CONCLUSIONS: AFF has different features, in terms of bone strength, depending on fracture location. At the very least, Japanese patients with mid-shaft AFF could be at high risk of hip fracture because of bone fragility, in contrast to the firm bone of subtrochanteric AFF. For internal fixation of mid-shaft AFF using an IM nail, cervical screw insertion toward the femoral head might be recommended to prevent possible hip fracture.

Airway and circulatory collapse due to retropharyngeal hematoma after blunt vertebral artery injury.

Retropharyngeal hematoma following blunt cervical spine injury is a known cause of airway obstruction, but it is not known to cause hemorrhagic shock. We report the case of a massive retropharyngeal hematoma caused by a blunt vertebral artery transection leading simultaneously to airway obstruction and hemorrhagic shock. An 83-year-old woman was injured in a motorcycle accident. In the field, the patient exhibited paradoxical breathing with no breath sounds, and her blood pressure could not be measured. Therefore, emergency intubation and fluid resuscitation were initiated and the patient was transferred to the emergency department. Computed tomography angiography revealed a massive retropharyngeal hematoma with contrast extravasation from the right vertebral artery, which caused airway obstruction and hemorrhagic shock. The right vertebral artery was transected at the C5 level, which was associated with C4/C5 dislocation. Vertebral artery transection was successfully treated by endovascular embolization, which was followed by complication of asymptomatic posterior circulation stroke. Blunt vertebral artery transection can cause massive retropharyngeal hematoma, which can rapidly expand and lead to hemorrhagic shock in addition to airway obstruction. In cases of massive retropharyngeal hematoma with hemorrhagic shock following blunt cervical spine injury, blunt vertebral artery transection should be suspected. If blunt vertebral artery transection is detected and hemorrhagic shock is persistent, endovascular embolization should be performed immediately in addition to emergency intubation.

Functional Characterization of Acetylcholine Receptors Expressed in Human Neurons Differentiated from Hippocampal Neural Stem/Progenitor Cells.

Neurotransmission mediated by acetylcholine receptors (AChRs) plays an important role in learning and memory functions in the hippocampus. Impairment of the cholinergic system contributes to Alzheimer's disease (AD), indicating the importance of AChRs as drug targets for AD. To improve the success rates for AD drug development, human cell models that mimic the target brain region are important. Therefore, we characterized the functional expression of nicotinic and muscarinic AChRs (nAChRs and mAChRs, respectively) in human hippocampal neurons differentiated from hippocampal neural stem/progenitor cells (HIP-009 cells). Intracellular calcium flux in 4-week differentiated HIP-009 cells demonstrated that the cells responded to acetylcholine, nicotine, and muscarine in a concentration-dependent manner (EC50 = 13.4 ± 0.5, 6.0 ± 0.4, and 35.0 ± 2.5 µM, respectively). In addition, assays using subtype-selective compounds revealed that major AD therapeutic target AChR subtypes-α7 and α4ß2 nAChRs, as well as M1 and M3 mAChRs-were expressed in the cells. Furthermore, neuronal network analysis demonstrated that potentiation of M3 mAChRs inhibits the spontaneous firing of HIP-009 neurons. These results indicate that HIP-009 cells are physiologically relevant for AD drug screening and hence are loadstars for the establishment of in vitro AD models.

Functional Comparison of Neuronal Cells Differentiated from Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Different Oxygen and Medium Conditions.

Because neurons are difficult to obtain from humans, generating functional neurons from human induced pluripotent stem cells (hiPSCs) is important for establishing physiological or disease-relevant screening systems for drug discovery. To examine the culture conditions leading to efficient differentiation of functional neural cells, we investigated the effects of oxygen stress (2% or 20% O2) and differentiation medium (DMEM/F12:Neurobasal-based [DN] or commercial [PhoenixSongs Biologicals; PS]) on the expression of genes related to neural differentiation, glutamate receptor function, and the formation of networks of neurons differentiated from hiPSCs (201B7) via long-term self-renewing neuroepithelial-like stem (lt-NES) cells. Expression of genes related to neural differentiation occurred more quickly in PS and/or 2% O2 than in DN and/or 20% O2, resulting in high responsiveness of neural cells to glutamate, N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ( S)-3,5-dihydroxyphenylglycine (an agonist for mGluR1/5), as revealed by calcium imaging assays. NMDA receptors, AMPA receptors, mGluR1, and mGluR5 were functionally validated by using the specific antagonists MK-801, NBQX, JNJ16259685, and 2-methyl-6-(phenylethynyl)-pyridine, respectively. Multielectrode array analysis showed that spontaneous firing occurred earlier in cells cultured in 2% O2 than in 20% O2. Optimization of O2 tension and culture medium for neural differentiation of hiPSCs can efficiently generate physiologically relevant cells for screening systems.

Establishment of a Human Neuronal Network Assessment System by Using a Human Neuron/Astrocyte Co-Culture Derived from Fetal Neural Stem/Progenitor Cells.

Using human cell models mimicking the central nervous system (CNS) provides a better understanding of the human CNS, and it is a key strategy to improve success rates in CNS drug development. In the CNS, neurons function as networks in which astrocytes play important roles. Thus, an assessment system of neuronal network functions in a co-culture of human neurons and astrocytes has potential to accelerate CNS drug development. We previously demonstrated that human hippocampus-derived neural stem/progenitor cells (HIP-009 cells) were a novel tool to obtain human neurons and astrocytes in the same culture. In this study, we applied HIP-009 cells to a multielectrode array (MEA) system to detect neuronal signals as neuronal network functions. We observed spontaneous firings of HIP-009 neurons, and validated functional formation of neuronal networks pharmacologically. By using this assay system, we investigated effects of several reference compounds, including agonists and antagonists of glutamate and γ-aminobutyric acid receptors, and sodium, potassium, and calcium channels, on neuronal network functions using firing and burst numbers, and synchrony as readouts. These results indicate that the HIP-009/MEA assay system is applicable to the pharmacological assessment of drug candidates affecting synaptic functions for CNS drug development.