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Abstract Background Fentanyl is widely used as an analgesic and sedative for patients with severe burn injuries in intensive care units. However, pharmacokinetic (PK) data for fentanyl, particularly for continuous intravenous infusion during the acute phase of burn injuries, are limited. Here, we report the clinical course and changes in blood fentanyl concentrations during the acute phase in a patient with severe burns treated with continuous intravenous infusion of fentanyl. Case presentation A woman in her 40s, with burns caused by a gas cylinder explosion, was transported to our hospital. The patient had burn wounds on face, neck, shoulders, and all four extremities, with a total burn area of 39.0%. For pain relief, the patient received a continuous infusion of 0.01 mg/mL fentanyl (20–30 µg/h) with a target blood concentration of 1.0–1.5 ng/mL, but continued to suffer from pain due to burning during the acute phase. We measured the blood fentanyl concentrations and found that all concentrations obtained during the acute phase were subtherapeutic. Notably, during the burn shock stage, blood concentrations of fentanyl were 0.50 ng/mL on day 1 and 0.66 ng/mL on day 2, indicating that the blood concentration did not rise sufficiently for the dosage. From days 0 to 2, the patient was administered a massive fluid load for burn shock. After the burn shock stage resolved, fentanyl concentrations gradually approached the target range, and the pain rating scale improved, even though the fentanyl administration rate remained unchanged (30 µg/h). Conclusions Major changes in the fluid volumes of body compartments that occur with large burns might increase the volume of fentanyl distribution, thereby lowering its concentration when a standard dose is administered. Our findings indicate that the PK of fentanyl in patients with severe burns can be substantially affected, especially during the shock phase, implying the importance of titrating analgesics for clinical efficacy in the acute phase.

Abstract Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative pluripotent stem cells present in the bone marrow, peripheral blood, and organ connective tissues. We assessed the homing and therapeutic effects of systemically administered nafimestrocel, a clinical-grade human Muse cell-based product, without immunosuppressants in a neonatal hypoxic–ischemic (HI) rat model. HI injury was induced on postnatal day 7 (P7) and was confirmed by T2-weighted magnetic resonance imaging on P10. HI rats received a single dose nafimestrocel (1 × 106 cells/body) or Hank’s balanced salt solution (vehicle group) intravenously at either three days (on P10; M3 group) or seven days (on P14; M7 group) after HI insult. Radioisotope experiment demonstrated the homing of chromium-51-labeled nafimestrocel to the both cerebral hemispheres. The cylinder test (M3 and M7 groups) and open-field test (M7 group) showed significant amelioration of paralysis and hyperactivity at five weeks of age compared with those in the vehicle group. Nafimestrocel did not cause adverse events such as death or pathological changes in the lung at ten weeks in the both groups. Nafimestrocel attenuated the production of tumor necrosis factor-α and inducible nitric oxide synthase from activated cultured microglia in vitro. These results demonstrate the potential therapeutic benefits and safety of nafimestrocel.

Abstract Background In Japan, the use of risperidone in combination with adrenaline is contraindicated, except in cases of anaphylaxis. Therefore, there is limited clinical evidence regarding the interaction of these two drugs. Here, we report the clinical course of a case of adrenaline-resistant anaphylactic shock induced by a contrast medium injection after a risperidone overdose. Case presentation A man in his 30s was transported to our hospital after attempting suicide by taking 10 mg of risperidone and jumping from a height of 10 m. To determine the location and severity of his injuries, he was injected with an iodinated contrast medium, after which he developed generalized erythema and hypotension and was diagnosed with anaphylactic shock. A 0.5 mg dose of adrenaline was administered with no improvement, followed by another 0.5 mg dose that did not change his blood pressure. After infusion of a sodium bicarbonate solution (8.4%), administration of fresh frozen plasma, and additional administration of adrenaline (0.6–1.2 µg/min), his blood pressure improved, and he recovered from the anaphylactic shock. Conclusions This was a rare case of a risperidone overdose followed by adrenaline-resistant anaphylactic shock. The resistance is likely associated with the high blood concentration of risperidone. Our findings indicate that the potential for decreased adrenergic responsiveness should be considered in patients undergoing risperidone treatment in the event of anaphylactic shock.

Abstract Background Postmenopausal osteoporosis is a widespread health concern due to its prevalence among older adults and an associated high risk of fracture. The downregulation of bone regeneration delays fracture healing. Activated fibroblast growth factor receptor 3 (FGFR3) accelerates bone regeneration at juvenile age and downregulates bone mineralization at all ages. However, the impact of FGFR3 signaling on bone regeneration and bone mineralization post-menopause is still unknown. This study aimed to evaluate the impact of FGFR3 signaling on bone regeneration and bone mineralization during menopause by developing a distraction osteogenesis (DO) mouse model after ovariectomy (OVX) using transgenic mice with activated FGFR3 driven by Col2a1 promoter (Fgfr3 mice). Methods The OVX or sham operations were performed in 8-week-old female Fgfr3 and wild-type mice. After 8 weeks of OVX surgery, DO surgery in the lower limb was performed. The 5-day-latency period followed by performing distraction for 9 days. Bone mineral density (BMD) and bone regeneration was assessed by micro-computed tomography (micro-CT) scan and soft X-ray. Bone volume in the distraction area was also evaluated by histological analysis after 7 days at the end of distraction. Osteogenic differentiation and mineralization of bone marrow-derived mesenchymal stem cells (BMSCs) derived from each mouse after 8 weeks of the OVX or sham operations were also evaluated with and without an inhibitor for FGFR3 signaling (meclozine). Results BMD decreased after OVX in both groups, and it further deteriorated in Fgfr3 mice. Poor callus formation after DO was also observed in both groups with OVX, and the amount of regenerated bone was further decreased in Fgfr3 mice. Similarly, histological analysis revealed that Fgfr3 OVX mice showed lower bone volume. Osteogenic differentiation and mineralization of BMSCs were also deteriorated in Fgfr3 OVX mice. An inhibitor for FGFR3 signaling dramatically reversed the inhibitory effect of OVX and FGFR3 signaling on BMSC mineralization. Conclusion Upregulated FGFR3 decreased newly regenerated bone after DO and BMD in OVX mice. FGFR3 signaling can be a potential therapeutic target in patients with postmenopausal osteoporosis.

Abstract The proton pump inhibitor lansoprazole has been previously identified to upregulate the expression and transcriptional activity of runt-related transcription factor 2 (Runx2) that promotes lineage commitment and differentiation of osteoprogenitor cells. We could not elicit the expected efficacy of insoluble lansoprazole in enhancing osteogenesis when combined with beta-tricalcium phosphate (β-TCP) bone substitutes. This study aimed to evaluate the effects of soluble lansoprazole on in vitro osteoblastogenesis and new bone formation in vivo. Commercially available human mesenchymal stem cells or patient-derived bone marrow-derived stromal cells were treated with 20 µM of soluble lansoprazole at the beginning of osteogenic induction. Soluble lansoprazole-impregnated β-TCP materials were embedded in the cortical bone defect model of rabbits. Rabbits were sacrificed four weeks postoperatively and undecalcified bone specimens were prepared for evaluation of intra-material new bone formation. Only a 1-day treatment with soluble lansoprazole facilitated osteoblastic differentiation and matrix calcium deposition when added to undifferentiated human mesenchymal stromal cells at the beginning of the osteogenic differentiation. Soluble lansoprazole dose-dependently accelerated intra-material new bone formation when being impregnated with porous β-TCP artificial bones. Local use of soluble lansoprazole can be applicable for fracture and bone defect repair when combined with porous β-TCP scaffolds.

Abstract Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.

Study Design Single-center retrospective study in pediatric patients. Purpose The purpose of the study was to investigate the effects of the presence and severity of leg-length discrepancy (LLD) on scoliosis in children. Overview of Literature LLD is a common orthopedic problem that can result in standing imbalance, low back pain, and stress fractures. It may cause structural changes in the spine over time, but the exact effect of LLD on scoliosis is unknown. Methods The subjects were 23 children with LLD ≥20 mm (range, 27–65 mm) treated at Nagoya University Hospital between 2007 and 2017. Whole spine posteroanterior standing radiographs and whole lower limb radiographs in a supine position were recorded. Data were collected for demographics, LLD, Cobb angle, pelvic obliquity, and Nash/Moe index. Scoliosis was defined as a Cobb angle ≥10°. Leg length was measured from the top of the femoral head to the middle of the tibial plafond, and LLD was defined as the difference between the left and right leg lengths. Results The patients (nine males and 14 females) had a mean age of 14.0 years (range, 5–18 years). The average LLD was 44.3±17.2 mm, with LLDs of 20 to 39 mm, 40 to 59 mm, and ≥60 mm in 13, five, and five subjects, respectively. The average Cobb angle was 13.0°±7.0°, and 15 subjects (65%) had scoliosis. Convexity of the scoliosis was to the short leg side in all cases. The Cobb angle was significantly related to the severity of the LLD (R=0.736, p

Abstract This article reports a girl with Bardet‐Biedl syndrome (BBS) having a novel causative mutation who developed Legg‐Calvé‐Perthes disease (LCPD). There exists a possibility that the prognosis of LCPD had been adversely affected by the concomitant BBS.

Background HMGB1 (high‐mobility group box 1) is known to worsen the functional prognosis after cerebral ischemia. Hp (haptoglobin) binds and sequesters HMGB1. Furthermore, Hp‐HMGB1 complexes are rapidly cleared by scavenger receptors on macrophages/microglia and modulate polarization of macrophages/microglia toward the M2 phenotype. Therefore, Hp may prevent aggravation by HMGB1 after cerebral ischemia and promote tissue repair by M2 macrophages/microglia. The aim of this study was to investigate the effects of Hp on ischemic brain damage induced by a high systemic HMGB1 level in mice subjected to 4 hours of middle cerebral artery occlusion (MCAO). Methods and Results One day after MCAO, Hp was administered intraperitoneally at a dose of 20 or 200 U/kg once daily for 7 days. Neurological scores, motor coordination, and plasma HMGB1 levels were measured 1, 3, and 7 days after MCAO. Expression of M1 and M2 macrophage/microglia markers, such as CD16/32 and CD206, were evaluated by immunostaining 7 days after MCAO. Treatment with Hp for 7 days improved the neurological score, motor coordination, and survival and prevented brain damage after MCAO. The systemic HMGB1 level increased 1 to 7 days after MCAO and was higher at 7 days than at day 1. Hp significantly decreased the systemic HMGB1 level and increased the M2 phenotype when compared with the M1 phenotype after MCAO. Conclusions Hp improved functional outcomes, including survival, motor function, and brain damage by binding to HMGB1 and modulating the polarization of macrophages/microglia. Hp may be an effective option in the treatment of cerebral ischemia.

Achondroplasia (ACH) is the most common form of short-limbed skeletal dysplasia. Patients with ACH sometimes undergo lower limb lengthening to get functional and psychological achievements. The periosteal resection (PR) is a known mechanism to increase longitudinal bone growth without osteotomy, although the results are not predictable. It could be alternative for limb lengthening in a minimally invasive technique. The purpose of this study is to evaluate the effect of PR on acceleration of bone growth in a mouse model of ACH (Fgfr3ach). We performed a circumferential resection of periosteum on the proximal tibia to both wild-type and Fgfr3ach mice at the age of four weeks. The second PR was done one week later in each mouse, which was subsequently sacrificed at the age of six weeks for micro-computed tomography (micro-CT) scan and histological examinations. We measured tibial bone length, bone volume, and metaphyseal trabecular bone parameters, including bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) by reconstructed micro-CT images. We also quantified the entire width of the growth plate of the proximal tibial from the sections stained with hematoxylin and eosin. Tibial bone length and bone volume of the PR side were significantly larger than the sham side in wild-type mice, while they were not statistically significant in Fgfr3ach mice. The BV/TV and Tb.N in the metaphysis were significantly decreased in the PR side of both mice. The histological analysis revealed that the growth plate of the proximal tibia was significantly wider in the PR side of wild-type mice while it showed no difference in width between the PR side and the sham side in Fgfr3ach mice. PR promoted longitudinal bone growth in wild-type mice, but it exhibited only a marginal effect on bone growth in Fgfr3ach mice.

Abstract Background Achondroplasia (ACH), the most common form of short‐limbed skeletal dysplasia, is caused by gain‐of‐function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. More than 97% of patients result from a heterozygous p.G380R mutation in the FGFR3 gene. We present here a child who had two de novo variants in the FGFR3 on the same allele, a common p.G380R mutation and a novel p.S378N variant. Methods A 3‐year‐old Japanese girl born from non‐consanguineous healthy parents showed more severe clinical and radiological phenotypes than classic ACH, including severe short‐limbed short stature with marked ossification defects in the metaphysis and epiphysis, hydrocephalus and cervicomedullary compression due to foramen magnum stenosis, prolonged pulmonary hypoplasia, and significant delay in the gross motor development. Genomic DNA was extracted from the proband and whole‐exome sequencing was performed. The variants were subsequently confirmed by Sanger sequencing. Results Mutation analysis demonstrated that the proband had p.S378N (c.1133G>A) and p.G380R (c.1138G>A) variants in the FGFR3 gene. Both variants were not detected in her parents and therefore considered de novo. An allele‐specific PCR was developed in order to determine whether these mutations were on the same allele (cis) or on different alleles (trans). The c.1138G>A mutation was found in the PCR product generated with the primer for the mutant 1133A, but it was not detected in the product with the wild‐type 1133G, confirming that p.S378N and p.G380R variants were located on the same allele (cis). Conclusion This is the second case who had two FGFR3 variants in the transmembrane domain on the same allele. The p.S378N variant may provide an additive effect on the activating receptor with the p.G380R mutation and alter the protein function, which could be responsible for the severe phenotype of the present case.

Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. We identified that meclizine hydrochloride inhibited FGFR3 signaling in various chondrocytic cells and promoted longitudinal bone growth in mouse model of ACH. Meclizine has safely been used for more than 50 years, but it lacks the safety data for repeated administration and pharmacokinetics (PK) when administered to children. We performed a phase Ia study to evaluate the PK and safety of meclizine administered orally to ACH children. Twelve ACH children aged from 5 to younger than 11 years were recruited, and the first 6 subjects received once a day of meclizine in the fasted condition, subsequent 6 subjects received twice a day of meclizine in the fed condition. Meclizine was well tolerated in ACH children with no serious adverse events. The mean Cmax, Tmax, AUC0-24h, t1/2 during 24 hours in the fasted condition were 130 ng/mL, 1.7 hours, 761 ng·h/mL, and 8.5 hours respectively. The simulation of repeated administration of meclizine for 14 days demonstrated that plasma concentration apparently reached steady state around 10 days after the first dose both at once a day and twice a day administration. The AUC0-10h of the fasting and fed condition were 504 ng·h/mL and 813 ng·h/mL, respectively, indicating exposure of meclizine increased with the diet. Although higher drug exposure was confirmed in ACH children compared to adults, a single administration of meclizine seemed to be well tolerated.

The role of the GABAB receptor in the anterior cingulate cortex (ACC) of neuropathic pain is unclear. Injection of a GABAB receptor antagonist CGP35348 into the ACC induced mechanical hypersensitivity in normal rats. Activation of the GABAB receptor injected by a GABAB receptor agonist baclofen into the ACC attenuated mechanical hypersensitivity in partial sciatic nerve ligation (PSNL) rats. Co-microinjection of CGP35348 with a muscarinic M1 receptor agonist McN-A-343 into the ACC significantly inhibited McN-A-343-induced antihypersensitivity in PSNL rats. These results suggest that the GABAB receptor in the ACC contributes to mechanical hypersensitivity and is involved in muscarinic M1 receptor-mediated antihypersensitivity. Keywords: GABAB receptor, Anterior cingulate cortex, Partial sciatic nerve ligation model

Previously, we reported that ovariectomy (OVX) combined with β-amyloid peptide (Aβ) impaired spatial memory by decreasing extracellular acetylcholine (ACh) levels in the dorsal hippocampus. Here, we investigated the effect of tokishakuyakusan (TSS), a Kampo medicine, on the impairment of spatial memory induced by OVX combined with Aβ in rats. Repeated administration of TSS (300 mg/kg, p.o.) significantly decreased the number of errors in the eight-arm radial maze test. Though TSS had no effect on extracellular ACh levels at baseline, TSS significantly increased extracellular ACh levels in the dorsal hippocampus. These results suggest that TSS improves the impairment of spatial memory induced by OVX combined with Aβ by (at least in part) increasing extracellular ACh levels in the dorsal hippocampus. Keywords: Tokishakuyakusan, Ovariectomy, β-Amyloid, Memory, Acetylcholine

Abstract Achondroplasia (ACH) is the most common short-limbed skeletal dysplasia caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3). No effective FGFR3-targeted therapies for ACH are currently available. By drug repositioning strategies, we identified that meclozine, which has been used as an anti-motion-sickness, suppressed FGFR3 signaling in chondrocytes and rescued short-limbed phenotype in ACH mouse model. Here, we conducted various pharmacological tests for future clinical application in ACH. Pharmacokinetic analyses demonstrated that peak drug concentration (Cmax) and area under the concentration-time curve (AUC) of 2 mg/kg of meclozine to mice was lower than that of 25 mg/body to human, which is a clinical usage for anti-motion-sickness. Pharmacokinetic simulation studies showed that repeated dose of 2 mg/kg of meclozine showed no accumulation effects. Short stature phenotype in the transgenic mice was significantly rescued by twice-daily oral administration of 2 mg/kg/day of meclozine. In addition to stimulation of longitudinal bone growth, bone volume and metaphyseal trabecular bone quality were improved by meclozine treatment. We confirmed a preclinical proof of concept for applying meclozine for the treatment of short stature in ACH, although toxicity and adverse events associated with long-term administration of this drug should be examined.

The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)–p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

Localized scleroderma, also known as morphea, is a rare condition characterized by progressive sclerosis of the skin and associated atrophy of the underlying tissues. The linear type of localized scleroderma is the most frequent form in childhood, usually affecting unilateral extremities. Fibrosclerosis of the fasciae and muscles can spread across joints and impair the range of motion of the joint. Dysplastic and/or atrophic bones of the affected lower extremity can lead to clinically significant leg length discrepancy (LLD). Limb reconstruction surgery has rarely been indicated for LLD in patients with linear morphea. We report on a case of extensive bone lengthening for appreciable LLD in a pediatric patient with linear morphea. A Japanese girl with linear morphea underwent staged simultaneous lengthening of the femur and tibia twice at seven and eleven years of age using a unilateral external fixator. A healing index exceeded 100 days/cm except for the first femoral lengthening that was complicated by regenerate fracture. At the final follow-up, LLD of 38 mm remained, but she could walk independently without a brace or a crutch. Due to soft tissue tightness and poor regenerative ability in the affected limb, cautions should be taken to prevent regenerate fracture and/or malalignment of the limb.

In the present study, we examined the effect of N-acetyl-l-cysteine (NAC), a glutamate-modulating agent, on marble-burying behavior in mice. Fluvoxamine (30 mg/kg, p.o.) and mirtazapine (3 mg/kg, i.p.) significantly inhibited marble-burying behavior without affecting locomotor activity. Similarity, NAC (150 mg/kg, i.p.) significantly inhibited marble-burying behavior without affecting locomotor activity. On the other hand, the antioxidant α-tocopherol (10, 30, and 100 mg/kg, p.o.) had no effect on the marble-burying behavior. These results suggest that the glutamatergic system is involved in the marble-burying behavior, and NAC may be useful for treatment of OCD. Keywords:: marble-burying behavior, N-acetyl-l-cysteine, obsessive-compulsive disorder

Abstract.: In the present study, we investigated the effect of the Kampo medicine Yokukansan (YKS) on pentobarbital-induced sleep in group-housed and socially isolated mice. Socially isolated mice showed shorter sleeping time than the group-housed mice. YKS (300 mg/kg, p.o.) prolonged the pentobarbital-induced sleeping time in socially isolated mice without affecting pentobarbital sleep in group-housed mice. The prolongation of sleeping time by YKS was reversed by bicuculline (3 mg/kg, i.p.) and flumazenil (3 mg/kg, i.p.), but not WAY100635. These findings suggest that the GABAA – benzodiazepine receptor complex, but not 5-HT1A receptors, is involved in the reversal effect of YKS on the decrease of pentobarbital sleep by social isolation. Keywords:: sleep, social isolation, Yokukansan

Abstract.: Citidine-5-diphosphocholine or citicoline (CDP-choline) is used as a neuroprotective and memory-enhancing drug in cerebral stroke, Alzheimer’s disease, and other neurovascular diseases. Non-clinical studies have demonstrated the neuroprotective effects of CDP-choline in ischemic animal models. However, the relationship between the neuroprotective effect and the memory enhancing effect of CDP-choline is still unknown. No studies have demonstrated the ameliorative effect on impaired spatial memory and the suppressive effect on neuronal cell death of CDP-choline in the same model. In this study, we examined the effect of CDP-choline on impaired spatial memory and hippocampal CA1 neuronal death in rats subjected to repeated cerebral ischemia, and we compared the mechanism of CDP-choline to that of donepezil. Seven days post administration of CDP-choline (100, 300, 1000 mg/kg per day, p.o.) or donepezil increased correct choices and reduced error choices in an eight-arm radial maze task in a dose-dependent manner. Neuronal cell death of caspase-3 protein–positive neurons in the hippocampus were reduced by repeated administration of CDP-choline at the highest dose. These results suggest that CDP-choline has ameliorative effects on the impairment of spatial memory via hippocampal neuronal cell death in a rat model of cerebral ischemia. Keywords:: citidine-5-diphosphocholine (CDP-choline), spatial memory, repeated ischemia, apoptosis

Cannabis contains the psychoactive component delta9-tetrahydrocannabinol (delta9-THC), and the non-psychoactive components cannabidiol (CBD), cannabinol, and cannabigerol. It is well-known that delta9-THC and other cannabinoid CB1 receptor agonists are neuroprotective during global and focal ischemic injury. Additionally, delta9-THC also mediates psychological effects through the activation of the CB1 receptor in the central nervous system. In addition to the CB1 receptor agonists, cannabis also contains therapeutically active components which are CB1 receptor independent. Of the CB1 receptor-independent cannabis, the most important is CBD. In the past five years, an increasing number of publications have focused on the discovery of the anti-inflammatory, anti-oxidant, and neuroprotective effects of CBD. In particular, CBD exerts positive pharmacological effects in ischemic stroke and other chronic diseases, including Parkinson’s disease, Alzheimer’s disease, and rheumatoid arthritis. The cerebroprotective action of CBD is CB1 receptor-independent, long-lasting, and has potent anti-oxidant activity. Importantly, CBD use does not lead to tolerance. In this review, we will discuss the therapeutic possibility of CBD as a cerebroprotective agent, highlighting recent pharmacological advances, novel mechanisms, and therapeutic time window of CBD in ischemic stroke.

Endocannabinoids have been shown to activate reward-related feeding and to promote astrocytic differentiation. We investigated whether high-fat diet (HFD) intake produced a preference for HFD via an endocannabinoid-dependent mechanism. In the conditioned place preference test, the 2-week HFD–intake group showed preference for HFD and had increased expression of a marker for reactive astrocytes, glial fibrillary acid protein (GFAP), in the hypothalamus. The cannabinoid CB1–receptor antagonist O-2050 reduced the preference for HFD and expression of GFAP in the hypothalamus. These results suggested that HFD intake led to the development of a preference for HFD via astrocytic CB1 receptors in the hypothalamus. Keywords:: preference for high-fat diet, endocannabinoid, astrocyte

In the present study, we investigated the neuroprotective effects of Kangen-karyu (KGK) in a repeated cerebral ischemia model (2 × 10 min, 1-h interval). A 21-day pre- and postischemic treatment with KGK (10 – 300 mg/kg) and aspirin (5 mg/kg) improved the spatial memory impairment and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia. However, a 7-day post-ischemic treatment with KGK did not attenuate the spatial memory impairment and neuronal death in this model. To determine the mechanism of action of KGK, we investigated the effects of a 14-day pre-ischemic treatment with KGK on cerebral blood flow in the hippocampal area of the repeated cerebral ischemia model using laser Doppler flowmetry. The 14-day pre-ischemic treatment with KGK increased the cerebral blood flow during reperfusion. These results suggest that a 21-day pre- and post-ischemic treatment with KGK can protect against brain damage caused by cerebral ischemia by increasing the cerebral blood flow in the hippocampal area. Keywords:: Kangen-karyu, repeated cerebral ischemia, cerebral blood flow, neuronal death

Arginine vasopressin (AVP) is a neurohypophyseal peptide best known as an antidiuretic hormone. AVP receptors have been classified into three subtypes: V1a, V1b, and V2 receptors. V1a receptor (V1aR) and V1b receptor (V1bR) are widely distributed in the central nervous system, including the septum, cortex, hippocampus, and hypothalamus. Clinical studies have demonstrated an involvement of AVP in psychiatric disorders. In the present study, we examined the performance of V1aR or V1bR knockout (KO) mice compared to wild-type (WT) mice in behavioral tests. V1aR and V1bR KO mice exhibited deficits of social behavior and prepulse inhibition in comparison to WT mice. Moreover, V1aR KO mice exhibited reduced anxiety-like behavior and impairment of spatial learning. These results suggest that V1aR and V1bR play an important role in psychological and cognitive functions. Keywords:: arginine vasopressin, V1a receptor, V1b receptor, behavior, psychiatric disorder, knockout mice

In the present study, we examined the effects of calcium-channel antagonists on marble-burying behavior, which is an animal model of obsessive-compulsive disorder. Amlodipine (10 mg/kg, i.p.), cilnidipine (10 mg/kg, i.p.), nilvadipine (3 and10 mg/kg, i.p.), and flunarizine (30 mg/kg, i.p.) significantly inhibited marble-burying behavior in mice. These results suggest that calcium channels may be involved in the marble-burying behavior. Keywords:: marble-burying behavior, calcium-channel antagonist, obsessive-compulsive disorder

The purpose of the present study was to examine the effect of beta-amyloid (Aβ ) oligomers, not the fibrils that make up Aβ plaques, on spatial memory and the cholinergic system in rats. Recently, several researchers have suggested that small assemblies of Aβ, Aβ oligomers, caused memory loss during the early stages of Alzheimer’s disease without showing cell death. In the present study, the combination of Aβ oligomers and cerebral ischemia, but not cerebral ischemia alone, significantly impaired spatial memory without apoptosis in the CA1 region of the hippocampus. Donepezil, an acetylcholinesterase inhibitor, ameliorated this memory impairment. Therefore we examined acetylcholine (ACh) release from the dorsal hippocampus. A microdialysis study showed that spontaneous release of ACh was not significantly decreased by the combination of Aβ oligomers and cerebral ischemia; however, high K+-evoked ACh release was decreased. These results suggest that a combination of Aβ oligomers and cerebral ischemia induces memory impairment by cholinergic synapse dysfunction without apoptosis. This model may be useful for developing new drugs for the treatment of early-phase Alzheimer’s disease. Keywords:: Alzheimer’s disease, oligomer, acetylcholine, memory impairment, behavior

We investigated the involvement of γ-aminobutyric acidA (GABAA) receptors in the neuroprotective effect of γ-glutamylethylamide (theanine), a component of Japanese green tea, following a 4-h middle cerebral artery (MCA) occlusion in mice. Theanine (1 mg/kg) reduced the size of the cerebral infarct and alterations of NeuN, GFAP, and Iba 1 expression levels at 24 h after MCA occlusion. This neuroprotective effect of theanine was prevented by bicuculline (GABAA-receptor antagonist, 10 mg/kg) but not 3-mercaptopropionic acid (glutamate decarboxylase inhibitor). These results suggest that the neuroprotective effect of theanine is mediated, at least in part, by GABAA receptors. Keywords:: theanine, focal cerebral ischemia, γ-aminobutyric acidA (GABAA) receptor

The effect of the serotonin 5-HT2A/2C-receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on ∆9-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization was studied in mice. DOI (0.3 and 1 mg/kg, i.p.) significantly inhibited the catalepsy-like immobilization induced by THC (10 mg/kg, i.p.). In contrast, the selective 5-HT2C-receptor agonist 8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one (WAY 161503) had no effect on this catalepsy-like immobilization. Moreover, the 5-HT2A-receptor antagonist ketanserin (0.3 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization caused by DOI (1 mg/kg), whereas the selective 5-HT2C-receptor antagonist 6-chloro-2,3-dihydro-5-methyl-N-[6-(2-methyl-3-pyridinyl)oxyl]-3-pyridinyl]-1H-indole-1-carboxyamide (SB 242084) did not affect this inhibitory effect of DOI. On the other hand, ketanserin (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). Thus, on the basis of these results, it appears that 5-HT2A-receptor mechanisms might be responsible for the inhibitory effect of DOI on THC-induced catalepsy-like immobilization. Keywords:: ∆9-tetrahydrocannabinol, cannabinoid, catalepsy, 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-HT2A receptor

In order to determine the differential effects of flavonoids on cerebral ischemia, we investigated the effects of (−)-epigallocatechin gallate (EGCG), catechin, rutin, and quercetin on spatial memory impairment and neuronal death induced by repeated cerebral ischemia in rats. Both rutin and quercetin (50 mg/kg × 2) improved spatial memory impairment in the 8-arm radial maze task and neuronal death in the hippocampal CA1 area; however, catechin (200 mg/kg × 2) and EGCG (50 mg/kg × 1) did not. Administration of EGCG (50 mg/kg × 2) resulted in a high mortality rate. These results suggest that in this repeated cerebral ischemia model, the 4-oxo group and the 2,3-double bond in the C ring of rutin and quercetin are related to their neuroprotective action. Keywords:: (−)-epigallocatechin gallate (EGCG), catechin, rutin, quercetin, repeated cerebral ischemia model

In addition to their role in physiological activities, ionotropic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) play an important role in neuronal death, especially that following ischemic insults. In this study, we examined the effect of single (SI) and twice repeated (RI)-4-vessel occlusion-ischemia on rat performance in the 8-armed radial maze test. Moreover, the effects of SI and RI on the AMPARs subunits glutamate receptor (GluR) 1 and GluR2 flip and flop variants composition in the CA1 subregion of the hippocampus were investigated using RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed as their ratios to the latter. The results showed that SI and RI impaired the maze performance by decreasing correct choices and increasing the error choices, but RI increased error choices to a greater extent than the SI. The SI reduced only GluR1 flip/GAPDH on day 1. The SI did not alter ratios of GluR2 variants to those of GluR1. On the other hand, the RI decreased GluR2 flip and flop variants after 1 and 3 days, respectively, whereas after 7 days, it increased the flip variant of both GluR1 and GluR2. Moreover, the RI reduced ratios of GluR2 variants to those of GluR1. These results reveal the differential effects of the SI and RI on memory and expression of the AMPARs subunits GluR1 and GluR2 and their flip and flop variants in the CA1. Keywords:: AMPA GluR1 GluR2 flip and flop, hippocampus CA1, single- and repeated-ischemia, memory, Alzheimer’s disease

Perospirone is a novel atypical antipsychotic drug with dopamine (DA) D2- and serotonin (5-hydroxytryptamine, 5-HT) 5-HT2A-receptor antagonist, and 5-HT1A-receptor agonist properties. In the present study, we examined the effect of perospirone on marble-burying behavior, which has been considered an animal model of obsessive-compulsive disorder (OCD), compared with the effects of other antipsychotics such as haloperidol and risperidone. Perospirone at a dose of 10 mg/kg (p.o.) inhibited marble-burying behavior without affecting the locomotor activity in mice. On the other hand, haloperidol (0.1 mg/kg, i.p.) and risperidone (1 mg/kg, p.o.) showed significant suppression of locomotor activity at the dose that inhibited marble-burying behavior. Furthermore, the inhibition of marble-burying behavior by perospirone was antagonized by WAY100135 (10 mg/kg, i.p.), a selective 5-HT1A-receptor antagonist. WAY100135 at the same dose also antagonized the inhibition of marble-burying behavior by 8-OH-DPAT (3 mg/kg, i.p.), a selective 5-HT1A-receptor agonist. These findings suggest that perospirone may exhibit anti-OCD activity in clinical use and that 5-HT1A-receptor agonistic activity may be involved in the inhibition of marble-burying behavior by perospirone. Keywords:: marble-burying behavior, perospirone, antipsychotic drug, 5-HT1A receptor, obsessive-compulsive disorder

In the present experiment, we studied the action of buckwheat polyphenol (BWP, from Fagopyrum esculentum MOENCH) in a repeated cerebral ischemia model, which induced a strong and long-lasting impairment of spatial memory in 8-arm radial maze with hippocampal CA1 cell death in rats. BWP (600 mg/kg, continuous 21-day p.o.) significantly ameliorated not only the impairment of spatial memory in the 8-arm radial maze, but also necrosis and TUNEL-positive cells in the hippocampal CA1 area subjected to repeated cerebral ischemia (10 min × 2 times occlusion, 1-h interval) in rats. In order to investigate the mechanism of BWP protective action, we measured the release of glutamate and NOx- (NO2- +NO3-) production induced by repeated cerebral ischemia in the rat dorsal hippocampus using microdialysis. A 14-day BWP treatment significantly inhibited the excess release of glutamate after the second occlusion. In addition, the BWP remarkably suppressed a delayed increase in NOx- (NO2- +NO3-) induced by repeated cerebral ischemia in the dorsal hippocampus as determined in vivo by microdialysis. However, the 14-day treatment did not affect hippocampal blood flow in either intact rats or rats subjected to repeated ischemia measured by lasser Doppler flowmeter. These results suggested that BWP might ameliorate spatial memory impairment by inhibiting glutamate release and the delayed generation of NOx- in rats subjected to repeated cerebral ischemia. Keywords:: buckwheat polyphenol, repeated cerebral ischemia, spatial memory, glutamate, nitric oxide

The improving effects of various components of Toki-Shakuyaku-San (TSS) and fractions isolated from Angelica acutiloba Radix (Toki) on scopolamine-induced spatial memory impairment were investigated in eight-armed radial maze. The scopolamine-induced memory impairment was characterized by prominent increase of error choices in addition to decreased correct choices. Toki, Cnidium officinale Rhizoma (Senkyu), Poria cocos Hoelen (Bukuryo), Alisma orientale Rhizoma (Takusha), and Atractylodes lancea Rhizoma (Sojutsu) increased the correct choices, while only the Toki, Sojutsu, and Takusha decreased the error choices. No effect was produced by Paeonia lactiflora Radix (Shakuyaku). Investigation of effects of fractions isolated from Toki revealed that its activity mainly resided in the butanol layer and its contents of N-methyl-β-carboline-3-carboxamide and amines. Moreover, the alkaloid, internal and external solutions (containing poly-, di-, and monosaccharides) obtained by dialysis with Visking cellophane tubing also improved the memory. However, no improving properties were detected for methanol and hexanol layers, L-(-)-tryptophan, L-arginine, L-(-)-lysine, and choline chloride. The results showed that the TSS components could improve the reference and working memory impaired by scopolamine. The improving effect of TSS is produced greatly by the Toki component, the activity of which was greatly produced by the fraction extracted by butanol. Keywords:: Toki-Shakuyaku-San, Angelica acutiloba, N-methyl-β-carboline-3-carboxamide, scopolamine, spatial memory

ABSTRACT: We investigated the effects of nilvadipine and amlodipine on the cerebral ischemia-induced impairment of spatial memory in 8-arm radial maze performance and hippocampal CA1 apoptosis in rats. Single cerebral ischemia impaired memory without inducing apoptosis. In these rats, neither nilvadipine nor amlodipine at 3.2 mg/kg, i.p. improved the impaired memory. On the other hand, repeated cerebral ischemia (10 min ischemia × 2, 1 h interval) impaired spatial memory and induced hippocampal apoptosis 7 days after the final occlusion/reperfusion. Moreover, repeated ischemia increased the apoptotic cell number, an effect observed after 3 days and peaked after 7 days. However, mRNA expression of the apoptosis-related early oncogene bax and CPP 32 (caspase-3) was observed after 24 h. In these rats, nilvadipine, but not amlodipine, significantly improved memory, concomitantly decreased hippocampal apoptosis, and suppressed both bax and CPP 32 expression. These results suggest that nilvadipine improved the memory impairment in repeated ischemia by reducing bax and CPP 32 expression and suppressing the induction of apoptosis in the hippocampus. Nilvadipine may have a neuroprotective effect and could be a useful pharmacotherapeutic agent for cerebrovascular dementia.

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

The astrocyte is a major glial cell type of the brain, and plays key roles in the formation, maturation, stabilization and elimination of synapses. Thus, changes in astrocyte condition and age can influence information processing at synapses. However, whether and how aging astrocytes affect synaptic function and maturation have not yet been thoroughly investigated. Here, we show the effects of prolonged culture on the ability of astrocytes to induce synapse formation and to modify synaptic transmission, using cultured autaptic neurons. By 9 weeks in culture, astrocytes derived from the mouse cerebral cortex demonstrated increases in β-galactosidase activity and glial fibrillary acidic protein (GFAP) expression, both of which are characteristic of aging and glial activation in vitro. Autaptic hippocampal neurons plated on these aging astrocytes showed a smaller amount of evoked release of the excitatory neurotransmitter glutamate, and a lower frequency of miniature release of glutamate, both of which were attributable to a reduction in the pool of readily releasable synaptic vesicles. Other features of synaptogenesis and synaptic transmission were retained, for example the ability to induce structural synapses, the presynaptic release probability, the fraction of functional presynaptic nerve terminals, and the ability to recruit functional AMPA and NMDA glutamate receptors to synapses. Thus the presence of aging astrocytes affects the efficiency of synaptic transmission. Given that the pool of readily releasable vesicles is also small at immature synapses, our results are consistent with astrocytic aging leading to retarded synapse maturation.

In this study, we examined alterations in the hypothalamic reward system related to high-fat diet (HFD) preferences. We previously reported that hypothalamic 2-arachidonoylglycerol (2-AG) and glial fibrillary acid protein (GFAP) were increased after conditioning to the rewarding properties of a HFD. Here, we hypothesized that increased 2-AG influences the hypothalamic reward system.The conditioned place preference test (CPP test) was used to evaluate HFD preferences. Hypothalamic 2-AG was quantified by gas chromatography-mass spectrometry. The expression of GFAP was examined by immunostaining and western blotting.Consumption of a HFD over either 3 or 7 days increased HFD preferences and transiently increased hypothalamic 2-AG levels. HFD consumption over 14 days similarly increased HFD preferences but elicited a long-lasting increase in hypothalamic 2-AG and GFAP levels. The cannabinoid 1 receptor antagonist O-2050 reduced preferences for HFDs after 3, 7, or 14 days of HFD consumption and reduced expression of GFAP after 14 days of HFD consumption. The astrocyte metabolic inhibitor Fluorocitrate blocked HFD preferences after 14 days of HFD consumption.High levels of 2-AG appear to induce HFD preferences, and activate hypothalamic astrocytes via the cannabinoid system. We propose that there may be two distinct stages in the development of HFD preferences. The induction stage involves a transient increase in 2-AG, whereas the maintenance stage involves a long lasting increase in 2-AG levels and activation of astrocytes. Accordingly, hypothalamic 2-AG may influence the development of HFD preferences.