Ca2+-imaging and photo-manipulation of the simple gut of zebrafish larvae in vivo.

Zebrafish larval gut could be considered as an excellent model to study functions of vertebrate digestive organs, by virtue of its simplicity and transparency as well as the availability of mutants. However, there has been scant investigation of the detailed behavior of muscular and enteric nervous systems to convey bolus, an aggregate of digested food. Here we visualized peristalsis using transgenic lines expressing a genetically encoded Ca2+ sensor in the circular smooth muscles. An intermittent Ca2+ signal cycle was observed at the oral side of the bolus, with Ca2+ waves descending and ascending from there. We also identified a regular cycle of weaker movement that occurs regardless of the presence or absence of bolus, corresponding likely to slow waves. Direct photo-stimulation of circular smooth muscles expressing ChR2 could cause local constriction of the gut, while the stimulation of a single or a few neurons could cause the local induction or arrest of gut movements. These results indicate that the larval gut of zebrafish has basic features found in adult mammals despite the small number of enteric neurons, providing a foundation for the study, at the single-cell level in vivo, in controlling the gut behaviors in vertebrates.

Iron-Dependent Mitochondrial Dysfunction Contributes to the Pathogenesis of Pulmonary Fibrosis.

Although the pathogenesis of pulmonary fibrosis remains unclear, it is known to involve epithelial injury and epithelial-mesenchymal transformation (EMT) as a consequence of cigarette smoke (CS) exposure. Moreover, smoking deposits iron in the mitochondria of alveolar epithelial cells. Iron overload in mitochondria causes the Fenton reaction, leading to reactive oxygen species (ROS) production, and ROS leakage from the mitochondria induces cell injury and inflammation in the lungs. Nevertheless, the mechanisms underlying iron metabolism and pulmonary fibrosis are yet to be elucidated. In this study, we aimed to determine whether iron metabolism and mitochondrial dysfunction are involved in the pathogenesis of pulmonary fibrosis. We demonstrated that administration of the iron chelator deferoxamine (DFO) reduced CS-induced pulmonary epithelial cell death, mitochondrial ROS production, and mitochondrial DNA release. Notably, CS-induced cell death was reduced by the administration of an inhibitor targeting ferroptosis, a unique iron-dependent form of non-apoptotic cell death. Transforming growth factor-ß-induced EMT of pulmonary epithelial cells was also reduced by DFO. The preservation of mitochondrial function reduced Transforming growth factor-ß-induced EMT. Furthermore, transbronchial iron chelation ameliorated bleomycin-induced pulmonary fibrosis and leukocyte migration in a murine model. Our findings indicate that iron metabolism and mitochondrial dysfunction are involved in the pathogenesis of pulmonary fibrosis. Thus, they may be leveraged as new therapeutic targets for pulmonary fibrosis.

Effective ferroptotic small-cell lung cancer cell death from SLC7A11 inhibition by sulforaphane.

Small-cell lung cancer (SCLC) is a highly aggressive cancer with poor prognosis, due to a lack of therapeutic targets. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables and has shown anticancer effects against numerous types of cancer. However, its anticancer effect against SCLC remains unclear. The present study aimed to demonstrate the anticancer effects of SFN in SCLC cells by investigating cell death (ferroptosis, necroptosis and caspase inhibition). The human SCLC cell lines NCI-H69, NCI-H69AR (H69AR) and NCI-H82 and the normal bronchial epithelial cell line, 16HBE14o- were used to determine cell growth and cytotoxicity, evaluate the levels of iron and glutathione, and quantify lipid peroxidation following treatment with SFN. mRNA expression levels of cystine/glutamate antiporter xCT (SLC7A11), a key component of the cysteine/glutamate antiporter, were measured using reverse transcription-quantitative PCR, while the levels of SLC7A11 protein were measured using western blot analysis. Following the addition of SFN to the cell culture, cell growth was significantly inhibited, and cell death was shown in SCLC and multidrug-resistant H69AR cells. The ferroptotic effects of SFN were confirmed following culture with the ferroptosis inhibitor, ferrostatin-1, and deferoxamine; iron levels were elevated, which resulted in the accumulation of lipid reactive oxygen species. The mRNA and protein expression levels of SLC7A11 were significantly lower in SFN-treated cells compared with that in the control cells (P<0.0001 and P=0.0006, respectively). These results indicated that the anticancer effects of SFN may be caused by ferroptosis in the SCLC cells, which was hypothesized to be triggered from the inhibition of mRNA and protein expression levels of SLC7A11. In conclusion, the present study demonstrated that SFN-induced cell death was mediated via ferroptosis and inhibition of the mRNA and protein expression levels of SLC7A11 in SCLC cells. The anticancer effects of SFN may provide novel options for SCLC treatment.

Increased extracellular vesicle miRNA-466 family in the bronchoalveolar lavage fluid as a precipitating factor of ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening disease; however, its treatment has not yet been fully established. The progression of ARDS is considered to be mediated by altered intercellular communication between immune and structural cells in the lung. One of several factors involved in intercellular communication is the extracellular vesicle (EV). They act as carriers of functional content such as RNA molecules, proteins, and lipids and deliver cargo from donor to recipient cells. EVs have been reported to regulate the nucleotide-binding oligomerization like receptor 3 (NLRP3) inflammasome. This has been identified as the cellular machinery responsible for activating inflammatory processes, a key component responsible for the pathogenesis of ARDS. METHODS: Here, we provide comprehensive genetic analysis of microRNAs (miRNAs) in EVs, demonstrating increased expression of the miRNA-466 family in the bronchoalveolar lavage fluid of a mouse ARDS model. RESULTS: Transfection of bone marrow-derived macrophages (BMDMs) with miRNA-466 g and 466 m-5p resulted in increased interleukin-1 beta (IL-1ß) release after LPS and ATP treatment, which is an established in vitro model of NLRP3 inflammasome activation. Moreover, LPS-induced pro-IL-1ß expression was accelerated by miRNA-466 g and 466 m-5p in BMDMs. CONCLUSIONS: These findings imply that miRNA-466 family molecules are secreted via EVs into the airways in an ARDS model, and this exacerbates inflammation through the NLRP3 inflammasome. Our results suggest that the NLRP3 inflammasome pathway, regulated by extracellular vesicle miRNA, could act as a therapeutic target for ARDS.

Distinguishing analgesic drugs from non-analgesic drugs based on brain activation in macaques with oxaliplatin-induced neuropathic pain.

The antineoplastic agent oxaliplatin is a first-line treatment for colorectal cancer. However, neuropathic pain, characterized by hypersensitivity to cold, emerges soon after treatment. In severe instances, dose reduction or curtailing treatment may be necessary. While a number of potential treatments for oxaliplatin-induced neuropathic pain have been proposed based on preclinical findings, few have demonstrated efficacy in randomized, placebo-controlled clinical studies. This failure could be related, in part, to the use of rodents as the primary preclinical species, as there are a number of distinctions in pain-related mechanisms between rodents and humans. Also, an indicator of preclinical pharmacological efficacy less subjective than behavioral endpoints that is translatable to clinical usage is lacking. Three days after oxaliplatin treatment in Macaca fascicularis, a significantly reduced response latency to cold (10 °C) water was observed, indicating cold hypersensitivity. Cold-evoked bilateral activation of the secondary somatosensory (SII) and insular (Ins) cortex was observed with functional magnetic resonance imaging. Duloxetine alleviated cold hypersensitivity and significantly attenuated activation in both SII and Ins. By contrast, neither clinically used analgesics pregabalin nor tramadol affected cold hypersensitivity and cold-evoked activation of SII and Ins. The current findings suggest that suppressing SII and Ins activation leads to antinociception, and, therefore, could be used as a non-behavioral indicator of analgesic efficacy in patients with oxaliplatin-induced neuropathic pain.

Stochastic Ca(2+) waves that propagate through the neuroepithelium in limited distances of the brain and retina imaged with GCaMP3 in zebrafish embryos.

Ca(2+) plays important roles in animal development and behavior. Various Ca(2+) transients during development have been reported in non-neuronal tissues, mainly by using synthesized calcium indicators. Here we used GCaMP3, a genetically encoded calcium indicator, to monitor stochastic Ca(2+) waves, in zebrafish embryos. To express GCaMP3 systemically throughout the body, its mRNA was injected into fertilized eggs. In the neuroepithelium of developing anterior brain and retina at 12-20 hours post-fertilization, we found spontaneously occurring stochastic Ca(2+) waves. Each Ca(2+) wave typically appeared in a randomly distributed spot, spread for 5-60 sec to form an area whose position and size varied each time with a diameter ranging from 10 to 160 µm, and then shrank and decreased to 50% brightness in 4-67 sec. A precise examination of the cellular distribution using Nipkow disk multibeam confocal laser scanning indicated that the Ca(2+) waves spread cell by cell. 2-APB, IP3-receptor inhibitor, but not carbenoxolone, a gap junction blocker, inhibit these Ca(2+) waves. Stronger fluorescence was found in the cytoplasm compared to the nuclei in the resting cells, and localized fluorescence was observed at the spindle poles in dividing cells. Ca(2+) waves also spread through the dividing cells. Our results reveal a novel type of cell-to-cell communication through the neuroepithelium in the developing zebrafish brain and retina, distinct from communication through neuron-neuron circuits. Our findings also indicated that GCaMP3 was useful for monitoring both stochastic and behavior-related Ca(2+) waves in the nervous system and skeletal muscles in zebrafish embryos.

Positive effect on bone fusion by the combination of platelet-rich plasma and a gelatin ß-tricalcium phosphate sponge: a study using a posterolateral fusion model of lumbar vertebrae in rats.

We developed a novel method for bone fusion by combining platelet-rich plasma (PRP) and a gelatin ß-tricalcium phosphate (ß-TCP) sponge. The PRP is an autologous concentration of platelets that includes several growth factors. The gelatin ß-TCP sponge comprises gelatin and ß-TCP, thus enabling the sustained release of growth factors and osteoconduction. To evaluate this method, we generated a posterolateral fusion model of lumbar vertebrae in rats and divided it into five groups by implanting the following materials between transverse processes of vertebrae, (1) the gelatin ß-TCP sponge with PRP (PRP sponge), (2) the gelatin ß-TCP sponge with platelet-poor plasma, (3) gelatin hydrogel with PRP, (4) autologous iliac bone (autograft), and (5) no material was implanted as a control. The assessment of bone fusion by a radiographic assessment, a biomechanical test, microcomputed tomography, and histological evaluations demonstrated that there were no significant differences between the PRP sponge and the autograft groups regarding the osteogenic effect. Subsequent examinations revealed that no significant differences existed between the PRP sponge and the autograft groups in either biomechanical stiffness or the bone volume over time; whereas the radiographic and histological composition underwent similar changes in the fusion process. These results indicate that the PRP sponge could, therefore, be potentially useful as an attractive and less invasive method for bone fusion.

A displaced stress fracture of the femoral neck in an adolescent female distance runner with female athlete triad: A case report.

This report presents a case of a displaced stress fracture of the femoral neck in an adolescent female distance runner with amenorrhea. Both reduction and internal fixation were performed early after the injury. At 24 months postoperatively, magnetic resonance imaging and bone scintigraphy showed no positive signs of femoral head necrosis and bone union was confirmed on plain X-ray. A medical examination for the presence of the signs of the female athlete triad by checking weight, calorie intake and menstrual cycles is most important to prevent such stress fractures. Athletes as well as their coaches or parents therefore need to understand female athlete triad.

Characterization of in vivo effects of platelet-rich plasma and biodegradable gelatin hydrogel microspheres on degenerated intervertebral discs.

We have previously shown that administration of platelet-rich plasma-impregnated gelatin hydrogel microspheres (PRP-GHMs) into a degenerated intervertebral disc (IVD) markedly suppresses progression of IVD degeneration. In the current study, we characterized the in vivo effects of PRP-GHM treatment in a degenerated IVD model in rabbit. On magnetic resonance images, the IVD height was significantly greater after treatment with PRP-GHMs compared with phosphate-buffered saline-impregnated GHMs, PRP without GHMs, and needle puncture only. Water content was also preserved in PRP-GHM-treated IVDs. Consistent with this observation, the mRNA expression of proteoglycan core protein and type II collagen was significantly higher after PRP-GHM treatment compared with other treatment groups. No proliferating cells were found in the nucleus pulposus and inner annulus fibrosus in any groups, but the number of apoptotic cells in the nucleus pulposus after PRP-GHM treatment was significantly lower than that after other treatments. These results provide an improved understanding of the therapeutic effects of PRP-GHM treatment of degenerated IVDs.