Citrin-deficient patient-derived induced pluripotent stem cells as a pathological liver model for congenital urea cycle disorders.

Citrin deficiency is a congenital secondary urea cycle disorder lacking useful disease models for effective treatment development. In this study, human induced pluripotent stem cells (iPSCs) were generated from two patients with citrin deficiency and differentiated into hepatocyte-like cells (HLCs). Citrin-deficient HLCs produced albumin and liver-specific markers but completely lacked citrin protein and expressed argininosuccinate synthase only weakly. In addition, ammonia concentrations in a medium cultured with citrin-deficient HLCs were higher than with control HLCs. Sodium pyruvate administration significantly reduced ammonia concentrations in the medium of citrin-deficient HLCs and slightly reduced ammonia in HLCs differentiated from control iPSCs, though this change was not significant. Our results suggest that sodium pyruvate may be an efficient treatment for patients with citrin deficiency. Citrin-deficient iPSCs are a pathological liver model for congenital urea cycle disorders to clarify pathogenesis and develop novel therapies.

Efficient protocol for the differentiation of kidney podocytes from induced pluripotent stem cells, involving the inhibition of mTOR.

The mechanistic/mammalian target of rapamycin (mTOR) is involved in a wide range of cellular processes. However, the role of mTOR in podocytes remains unclear. In this study, we aimed to clarify the role of mTOR in podocyte differentiation from human induced pluripotent stem cells (hiPSCs) and to establish an efficient differentiation protocol for human podocytes. We generated podocytes from hiPSCs by modifying protocol. The expression of the podocyte-specific slit membrane components nephrin and podocin was measured using PCR, western blotting, flow cytometry, and immunostaining; and the role of mTOR was evaluated using inhibitors of the mTOR pathway. Nephrin and podocin were found to be expressed in cells differentiated from hiPSCs, and their expression was increased by mTOR inhibitor treatment. S6, a downstream component of the mTOR pathway, was also found to be involved in podocyte differentiation. we evaluated its permeability to albumin, urea, and electrolytes. The induced podocytes were permeable to the small molecules, but only poorly permeable to albumin. We have shown that the mTOR pathway is involved in podocyte differentiation. Our monolayer podocyte differential protocol, using an mTOR inhibitor, provides a novel in vitro model for studies of kidney physiology and pathology.

Clinical Impact of Prehabilitation on Elective Laparoscopic Surgery in Frail Octogenarians With Colorectal Cancer.

BACKGROUND/AIM: The aim of the present study was to clarify the clinical impact of prehabilitation by the perioperative management center (PERIO) at our hospital in severely frail octogenarians with colorectal cancer. PATIENTS AND METHODS: We compared the clinicopathological characteristics of octogenarians who underwent surgery for colorectal cancer before the establishment of PERIO intervention (Control group) with those who received prehabilitation (PERIO group). All patients were classified as American Society of Anesthesiologists (ASA) class 3 or higher. The primary outcome was the incidence of postoperative complications. RESULTS: There were 21 patients in the Control group and 19 patients in the PERIO group. Operative time was significantly longer in the PERIO group (Control group, 200 min vs. PERIO group, 230 min; p=0.03) and blood loss was significantly higher in the PERIO group (Control group, 5 ml vs. PERIO group, 30 ml; p=0.02). Postoperative complications occurred in 10 patients (47.6%) in the Control group and 3 patients (15.8%) in the PERIO group and were significantly lower in the PERIO group (p=0.03). Postoperative hospital stay was 13 days (range=7-31 days) in the Control group and 11 days (range=8-70 days) in the PERIO group (p=0.39). The rate of discharge directly to home was 81% in the Control group and 93.3% in the PERIO group (p=0.29). CONCLUSION: In frail octogenarians with colorectal cancer of ASA class 3 or higher, the incidence of postoperative complications was significantly lower after PERIO intervention.

The Induction of Parathyroid Cell Differentiation from Human Induced Pluripotent Stem Cells Promoted Via TGF-α/EGFR Signaling.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

Decreased expression of hyaluronan synthase and loss of hyaluronan-rich cells in the anterior tibial fascia of the rat model of chemotherapy-induced peripheral neuropathy.

Introduction: Previous studies on chemotherapy-induced peripheral neuropathy (CIPN) have focused on neuronal damage. Although some studies have revealed that the fascia is an important sensory organ, currently, we do not know about chemotherapy drug-induced fascial dysfunction. Objectives: This study aimed to explore the fascia as a nonneural cause of mechanical hypersensitivity in CIPN by investigating the expression of hyaluronic acid synthase (HAS) and histology of the fascia in an animal model of CIPN. Methods: Rats were intraperitoneally administered with vincristine (VCR). Mechanical hypersensitivities of the hind paw and the anterior tibial muscle were assessed. The expression of HAS mRNA in the fascia of the anterior tibial muscles was quantitated using reverse transcription polymerase chain reaction. Immunohistochemistry was also performed for HAS2, hyaluronic acid-binding protein, and S100A4 in the fascia. Results: Vincristine administration significantly decreased mechanical withdrawal thresholds in the hind paw and the anterior tibial muscle after day 3. Quantitative polymerase chain reaction showed significant downregulation of HAS mRNAs in the fascia of VCR-treated rats. Immunohistochemical analysis showed that the number of cells with strong HAS2 immunoreactivity, classified as fasciacytes by morphology and colocalized marker S100A4, decreased significantly in the VCR group. Conclusion: Hyaluronic acid plays a critical role in somatic pain sensation. Damaged fascia could be a possible cause of musculoskeletal pain in patients with CIPN. This study suggests that fascia is a nonneural cause and novel therapeutic target for chemotherapy-induced "peripheral neuropathy."

The Japanese Epidemiologic Study for Perioperative Anaphylaxis, a prospective nationwide study: allergen exposure, epidemiology, and diagnosis of anaphylaxis during general anaesthesia.

BACKGROUND: Diagnosis of perioperative anaphylaxis is often challenging. This study describes the utility of a newly developed tool for identifying patients with a high possibility of anaphylaxis, and aimed to investigate the frequency of anaphylaxis with each drug during the perioperative period in Japan. METHODS: This study included patients with anaphylaxis of Grade 2 or higher severity during general anaesthesia at 42 facilities across Japan in 2019 and 2020. We developed and adopted a unique objective evaluation tool yielding a composite score for diagnosing anaphylaxis, which includes the results of skin tests and basophil activation tests, and clinical scores for perioperative anaphylaxis. The number of cases using each drug and the total number of anaphylaxis cases were investigated to calculate the frequency of anaphylaxis. RESULTS: General anaesthesia was performed in 218 936 cases, which included 55 patients with suspected perioperative anaphylaxis. The developed composite score diagnosed 43 of them with a high probability of anaphylaxis. The causative agent was identified in 32 cases. Plasma histamine levels showed high diagnostic accuracy for anaphylaxis. The top causative agents were rocuronium (10 cases in 210 852 patients, 0.005%), sugammadex (7 cases in 150 629 patients, 0.005%), and cefazolin (7 cases in 106 005 patients, 0.007%). CONCLUSIONS: We developed a composite tool to diagnose anaphylaxis, and found that the combination of tryptase levels, skin testing, and basophil activation testing results and clinical score improved the certainty of anaphylaxis diagnosis. The incidence of perioperative anaphylaxis in our study was 1 in about 5000 general anaesthesia cases. CLINICAL TRIAL REGISTRATION: UMIN000035350.

An Assessment of the Practice of Neuromuscular Blockade and the Association Between Its Prophylactic Use and Outcomes Among Postoperative Pediatric Cardiac Patients.

OBJECTIVES: The authors investigated the management of neuromuscular blocking agents (NMBAs) for pediatric patients after cardiac surgery, and compared the outcomes of patients who received prophylactic NMBA (pNMBA) infusions and patients without pNMBA infusions. DESIGN: A retrospective cohort study. SETTING: At a tertiary teaching hospital. PARTICIPANTS: Patients younger than 18, with congenital heart disease, who underwent cardiac surgery. INTERVENTIONS: Commencement of NMBA infusion in the first 2 hours after surgery MEASUREMENTS AND MAIN RESULTS: The primary endpoint was a composite of one or more of the following major adverse events (MAEs) that occurred within 7 days after surgery: death from any cause, a circulatory collapse that needed cardiopulmonary resuscitation, and requirement for extracorporeal membrane oxygenation. The secondary endpoints included the total duration of mechanical ventilation for the first 30 days after surgery. A total of 566 patients were included in this study. The MAEs occurred in 13 patients (2.3%). An NMBA was commenced within 2 hours after surgery in 207 patients (36.6%). There were significant differences in the incidence of postoperative MAEs between the pNMBA group and the non-pNMBA group (5.3% v 0.6%; p < 0.001). In multivariate regression models, pNMBA infusion was not significantly associated with the incidence of MAEs (odds ratio: 1.79, 95% CI: 0.23-13.93, p = 0.58), but was significantly associated with prolonged mechanical ventilation by 3.85 days (p < 0.001). CONCLUSIONS: Postoperative prophylactic neuromuscular blockade after cardiac surgery can be associated with prolonged mechanical ventilation, but has no association with MAEs among pediatric patients with congenital heart disease.

Intrathecal Administration of the α1 Adrenergic Antagonist Phentolamine Upregulates Spinal GLT-1 and Improves Mirror Image Pain in SNI Model Rats.

Mirror image pain (MIP) is a type of extraterritorial pain that results in contralateral pain or allodynia. Glutamate transporter-1 (GLT-1) is expressed in astrocytes and plays a role in maintaining low glutamate levels in the synaptic cleft. Previous studies have shown that GLT-1 dysfunction induces neuropathic pain. Our previous study revealed bilateral GLT-1 downregulation in the spinal cord of a spared nerve injury (SNI) rat. We hypothesized that spinal GLT-1 is involved in the mechanism of MIP. We also previously demonstrated noradrenergic GLT-1 regulation. Therefore, this study aimed to investigate the effect of an α1 adrenergic antagonist on the development of MIP. Rats were subjected to SNI. Changes in pain behavior and GLT-1 protein levels in the SNI rat spinal cords were then examined by intrathecal administration of the α1 adrenergic antagonist phentolamine, followed by von Frey test and western blotting. SNI resulted in the development of MIP and bilateral downregulation of GLT-1 protein in the rat spinal cord. Intrathecal phentolamine increased contralateral GLT-1 protein levels and partially ameliorated the 50% paw withdrawal threshold in the contralateral hind paw. Spinal GLT-1 upregulation by intrathecal phentolamine ameliorates MIP. GLT-1 plays a role in the development of MIPs.

Ketamine Improves Desensitization of µ-Opioid Receptors Induced by Repeated Treatment with Fentanyl but Not with Morphine.

The issue of tolerance to continuous or repeated administration of opioids should be addressed. The ability of ketamine to improve opioid tolerance has been reported in clinical studies, and its mechanism of tolerance may involve improved desensitization of µ-opioid receptors (MORs). We measured changes in MOR activity and intracellular signaling induced by repeated fentanyl and morphine administration and investigated the effects of ketamine on these changes with human embryonic kidney 293 cells expressing MOR using the CellKey™, cADDis cyclic adenosine monophosphate, and PathHunter® ß-arrestin recruitment assays. Repeated administration of fentanyl or morphine suppressed the second MOR responses. Administration of ketamine before a second application of opioids within clinical concentrations improved acute desensitization and enhanced ß-arrestin recruitment elicited by fentanyl but not by morphine. The effects of ketamine on fentanyl were suppressed by co-treatment with an inhibitor of G-protein-coupled receptor kinase (GRK). Ketamine may potentially reduce fentanyl tolerance but not that of morphine through modulation of GRK-mediated pathways, possibly changing the conformational changes of ß-arrestin to MOR.

Thermosensitive TRPV4 channels mediate temperature-dependent microglia movement.

Microglia maintain central nervous system homeostasis by monitoring changes in their environment (resting state) and by taking protective actions to equilibrate such changes (activated state). These surveillance and protective roles both require constant movement of microglia. Interestingly, induced hypothermia can reduce microglia migration caused by ischemia, suggesting that microglia movement can be modulated by temperature. Although several ion channels and transporters are known to support microglia movement, the precise molecular mechanism that regulates temperature-dependent movement of microglia remains unclear. Some members of the transient receptor potential (TRP) channel superfamily exhibit thermosensitivity and thus are strong candidates for mediation of this phenomenon. Here, we demonstrate that mouse microglia exhibit temperature-dependent movement in vitro and in vivo that is mediated by TRPV4 channels within the physiological range of body temperature. Our findings may provide a basis for future research into the potential clinical application of temperature regulation to preserve cell function via manipulation of ion channel activity.

Two cases of intraoperative hemodynamic instability during combined thoracoscopic-laparoscopic surgery for esophagogastric junction carcinoma.

BACKGROUND: Intraoperative complications during combined thoracoscopic-laparoscopic surgery for esophagogastric junction (EGJ) carcinoma have not been reported as compared to those during surgery for esophageal carcinoma. We present two cases which had surgery-related hemodynamic instability during laparoscopic proximal gastrectomy and intra-mediastinal valvuloplastic esophagogastrostomy (vEG) with thoracoscopic mediastinal lymphadenectomy for EGJ carcinoma. CASE PRESENTATION: In case 1, the patient fell into hypotension with hypoxemia during laparoscopic vEG due to pneumothorax caused by entry of intraabdominal carbon dioxide. In case 2, ventricular arrythmia and ST elevation occurred during laparoscopic vEG. Pericardium retraction to secure surgical field during reconstruction compressed the coronary artery, which caused coronary malperfusion. These two events were induced by the surgical procedure, characterized by the following: (1) connection of the thoracic and abdominal cavities and (2) cardiac displacement during vEG. CONCLUSION: These cases indicated tension pneumothorax and coronary ischemia are possible intraoperative complications specific to combined thoracoscopic-laparoscopic surgery for EGJ carcinoma.

Incorporation of one N-glycosylation-deficient subunit within a tetramer of HCN2 channel is tolerated.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are glycoproteins N-glycosylated at a specific asparagine residue in the S5-S6 linker region. Previous reports suggested that N-glycosylation-deficient HCN2 N380Q (NQ) channels fail to properly target to the plasma membrane and are unable to form functional ion channels. HCN channels are known to homo- and hetero-oligomerize and it is not known whether HCN2-NQ subunits can oligomerize with wild type (wt) N-glycosylated subunits to form a tetrameric assembly. In the present study, homomeric NQ-mutant resulted in no current, cRNA titration experiments controlling the amount of wt-to-NQ injected into Xenopus oocytes indicated that the observed currents were consistent with a model where presence of a single nonglycosylated subunit in a tetrameric oligomer is tolerated forming functional channels. The activation voltage-dependence described by half-activation voltage and slope factor, and the reversal potential of the wt-NQ oligomeric channels were identical to the wt only tetrameric channels. Further incorporation of the nonglycosylated subunit rendered the channels nonconductive or not incorporated into the plasma membrane.

Possible biased analgesic of hydromorphone through the G protein-over ß-arrestin-mediated pathway: cAMP, CellKey™, and receptor internalization analyses.

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for µ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the ß-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or ß-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone < morphine ≤ oxycodone, all also exhibiting full agonist responses. However, in the internalization assay, only fentanyl elicited a full agonist response. Hydromorphone had the strongest potency next to fentanyl; however, contribution of the ß-arrestin-mediated pathway was small, suggesting that its effect could be biased toward the G protein-mediated pathway. Based on these properties, hydromorphone could be chosen as an effective analgesic.

Anesthetic management of a patient with sodium-channel myotonia: a case report.

BACKGROUND: Sodium-channel myotonia (SCM) is a nondystrophic myotonia, characterized by pure myotonia without muscle weakness or paramyotonia. The prevalence of skeletal muscle channelopathies is approximately 1 in 100,000, and the prevalence of SCM is much lower. To our knowledge, this is the first report on anesthetic management of a patient with SCM. CASE PRESENTATION: A 23-year-old woman with congenital nasal dysplasia and SCM was scheduled to undergo rhinoplasty with autologous costal cartilage. Total intravenous anesthesia without muscle relaxants was administered followed by continuous intercostal nerve block. Although transient elevation of potassium level in the blood was observed during surgery, the patient did not show exacerbation of myotonic or paralytic symptoms in the postoperative period. CONCLUSION: Total intravenous anesthesia and peripheral nerve block can be administered safely to a patient with SCM. However, careful monitoring of the symptoms and electrolytes is recommended.

Incidence Rates of Postoperative Pulmonary Embolisms in Symptomatic and Asymptomatic Patients, Detected by Diagnostic Images　- A Single-Center Retrospective Study.

BACKGROUND: Pulmonary embolism (PE) is a serious complication during the perioperative period. However, because most previous studies on the incidence of postoperative PE are based on symptoms, asymptomatic occurrences of PE have been overlooked, and the absolute incidence of postoperative PE remains unknown. The aim of this study was to investigate the incidence of perioperative PE, regardless of its symptoms, by reviewing the clinical interpretations of the diagnostic images obtained during the postoperative period. Methods and Results: This study included all patients aged at least 18 years who underwent operations under general and/or neuraxial anesthesia in our institution from 2013 to 2016. We reviewed all interpretations of the diagnostic imaging performed in the postoperative period. We analyzed the 90-day cumulative incidence of postoperative PE and the characteristics, risk factors, and symptoms of patients with and without postoperative PE. Among 21,763 operations, postoperative diagnostic imaging was performed in 1,168 patients, which found PE in 217 patients. Symptoms appeared in 11.1% (24/217) of the PE patients, and 66.7% of these symptoms were decreased levels of SpO2alone. Mortality from PE was 0.5% (1/217). CONCLUSIONS: Diagnostic imaging found a number of postoperative PE cases, regardless of the presence of symptoms. Although symptomatic PE was not a frequent occurrence, these findings suggest that clinicians should be aware of postoperative PE even under current prophylaxis.

Prolonged-duration pulsed radiofrequency is associated with increased neuronal damage without further antiallodynic effects in neuropathic pain model rats.

AIM OF INVESTIGATION: Pulsed radiofrequency (PRF) is a safe and effective approach for treating neuropathic pain. However, the optimal treatment conditions and analgesic mechanisms of PRF remain unclear. The aim of our study was to assess the beneficial and adverse effects of prolonged-duration PRF and the analgesic mechanisms of PRF treatment with neuropathic pain rats. METHODS: Male Sprague Dawley rats received L5 spinal nerve ligation (SNL) for developing neuropathic pain. Fourteen days after L5 SNL surgery, they were divided into three groups according to duration of PRF current for 6 minutes, 12 minutes, and none. PRF current was delivered via direct visualization adjacent to the L5 dorsal root ganglion (DRG). Pain behavior was evaluated every week after L5 SNL surgery, until day 28. Seven days after PRF treatment, L5 DRG tissue was harvested to detect levels of activating translation factor 3 (ATF3; a marker of neuronal damage) and hyperpolarization-activated cyclic nucleotide (HCN)-gated cation channels (key factors in neuropathic pain) using quantitative PCR. RESULTS: Before PRF application, withdrawal thresholds were significantly lower than at baseline and did not differ significantly between the three groups. After PRF application, withdrawal thresholds in the PRF6 and PRF12 groups were significantly increased compared to those in the sham group. However, those in the PRF6 and PRF12 groups did not differ significantly. The expression level of ATF3 mRNA in the PRF12 group was significantly higher than that in the sham group (P<0.01), but the expression of HCN1 and HCN2 channels did not differ significantly between the three groups. CONCLUSION: Prolonged PRF exposure, from 6 to 12 minutes, was not only ineffective but also associated with increased neuronal damage. These findings do not support prolonged PRF exposure as a helpful treatment for neuropathic pain. In this study, the involvement of HCN channels in the antiallodynic effects of PRF was uncertain.

Quercetin Attenuates Neuropathic Pain in Rats with Spared Nerve Injury.

Quercetin is a flavonoid widely found in plants and marketed to the public as a supplement. Several studies have reported its effect on glial cells. This study aimed to examine the effect of quercetin on the development of neuropathic pain and the underlying mechanism in a spared nerve injury (SNI) rat model. Male Sprague-Dawley rats randomly assigned to the control or the quercetin group were subjected to SNI of the sciatic nerve. We measured pain behaviors on the hind paw and glial fibrillary acidic protein (GFAP) in the dorsal root ganglion (DRG) and spinal cord. Oral administration of 1% quercetin, begun before surgery, attenuated mechanical allodynia compared to the control group at days 7 and 10 after SNI. On the other hand, established pain was not attenuated in a post-dose group in which quercetin was begun 7 days after SNI. Quercetin inhibited GFAP in the satellite glial cells of the ipsilateral L5 DRG on day 7 compared to the control group. Quercetin suppressed the development of neuropathic pain through a mechanism partly involving the inhibition of satellite glial cells. As its safety is well established, quercetin has great potential for clinical use in pain treatment.