Dexmedetomidine improves acute lung injury by activating autophagy in a rat hemorrhagic shock and resuscitation model.

Dexmedetomidine (DEX) can reduce lung injury in a hemorrhagic shock (HS) resuscitation (HSR) model in rats by inhibiting inflammation. Here, we aimed to investigate if these effects of DEX are due to autophagy activation. Therefore, we established HSR rat models and divided them into four groups. HS was induced using a blood draw. The rats were then resuscitated by reinjecting the drawn blood and saline. The rats were sacrificed 24 h after resuscitation. Lung tissues were harvested for histopathological examination, determination of wet/dry lung weight ratio, and detection of the levels of autophagy-related marker proteins LC3, P62, Beclin-1, and the ATG12-ATG5 conjugate. The morphological findings of hematoxylin and eosin staining in lung tissues and the pulmonary wet/dry weight ratio showed that lung injury improved in HSR + DEX rats. However, chloroquine (CQ), an autophagy inhibitor, abolished this effect. Detecting the concentration of autophagy-related proteins showed that DEX administration increased LC3, ATG12-ATG5, and Beclin-1 expression and decreased P62 expression. The expression levels of these proteins were similar to those in the HSR group after CQ + DEX administration. In summary, DEX induced autophagic activation in an HSR model. These findings suggest that DEX administration partially ameliorates HSR-induced lung injury via autophagic activation.

Therapeutic effect of carbon monoxide-releasing molecule-3 on acute lung injury after hemorrhagic shock and resuscitation.

Hemorrhagic shock and resuscitation (HSR) induces a pulmonary inflammatory response and frequently causes acute lung injury. Carbon monoxide-releasing molecule-3 (CORM-3) has been reported to liberate and deliver CO under physiological conditions, which exerts organ-protective effects during systemic insults. The present study aimed to determine whether the administration of CORM-3 following HSR exerts a therapeutic effect against HSR-induced lung injury without any detrimental effects on oxygenation and hemodynamics. To induce hemorrhagic shock, rats were bled to a mean arterial blood pressure of 30 mmHg for 45 min and then resuscitated with the shed blood. CORM-3 or a vehicle was intravenously administered immediately following the completion of resuscitation. The rats were divided into four groups, including sham, HSR, HSR/CORM-3 and HSR/inactive CORM-3 groups. Arterial blood gas parameters and vital signs were recorded during HSR. The histopathological changes to the lungs were evaluated using a lung injury score, while pulmonary edema was evaluated on the basis of the protein concentration in bronchoalveolar lavage fluid and the lung wet/dry ratio. We also investigated the pulmonary expression levels of inflammatory mediators and apoptotic markers such as cleaved caspase-3 and transferase-mediated dUTP-fluorescein isothiocyanate nick-end labeling (TUNEL) staining. Although HSR caused significant lung histopathological damage and pulmonary edema, CORM-3 significantly ameliorated this damage. CORM-3 also attenuated the HSR-induced upregulation of tumor necrosis factor-α, inducible nitric oxide synthase and interleukin-1ß genes, and the expression of interleukin-1ß and macrophage inflammatory protein-2. In addition, the expression of interleukin-10, an anti-inflammatory cytokine, was inversely enhanced by CORM-3, which also reduced the number of TUNEL-positive cells and the expression of cleaved caspase-3 following HSR. Although CORM-3 was administered during the acute phase of HSR, it did not exert any influence on arterial blood gas analysis data and vital signs during HSR. Therefore, treatment with CORM-3 ameliorated HSR-induced lung injury, at least partially, through anti-inflammatory and anti-apoptotic effects, without any detrimental effects on oxygenation and hemodynamics.

A case of perioperative glucose control by using an artificial pancreas in a patient with glycogen storage disease.

A 57-year-old woman was diagnosed with type I glycogen storage disease in her twenties. She had undergone hepatectomy under general anesthesia with epidural anesthesia. Fifty minutes after the induction of anesthesia, a 20-gauge venous catheter was inserted in the patient's right hand, and an artificial pancreas (STG-55, Nikkiso Co., Tokyo, Japan) was connected for continuous glucose monitoring and automatic glucose control. Insulin was infused when the blood glucose level reached 120 mg/dL or higher, and glucose was infused when the level fell to 100 mg/dL or lower. After the Pringle maneuver, the blood glucose level increased, and insulin was administered automatically via an artificial pancreas. Hypoglycemia did not occur during the operation. After total parenteral nutrition was started in the intensive care unit (ICU), the blood glucose level increased, and the artificial pancreas controlled the blood glucose level through automatic insulin administration. Thirty-four hours after admission to the ICU, the artificial pancreas was removed because the blood sampling failed. After the removal of the artificial pancreas, blood glucose level was measured every 2 h until extubation. During the ICU stay, hypoglycemia never occurred, with the average blood glucose level being 144 mg/dL. In conclusion, the use of an artificial pancreas for perioperative blood glucose management in a patient with glycogen storage disease had the beneficial effect of enabling the management of blood glucose levels without hypoglycemia.

Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.

Involvement of intracellular Ca(2+) in the regulatory volume decrease after hyposmotic swelling in MDCK cells.

We examined the source of Ca(2+) involved in the volume regulation of Madin-Darby canine kidney (MDCK) cells with confocal microscopy and fluoroprobes. Hyposmosis induced a transient increase in cell volume, as well as cytoplasmic Ca(2+), which peaked at 3 to 5 min and gradually decreased to reach the initial value within about 30 min. This late decrease in cell volume, as well as the transient rise in cytoplasmic Ca(2+), was reduced in Ca(2+)-free solution and was abolished by pretreatment with thapsigargin. In conclusion, Ca(2+) released from the intracellular store contributes to the regulatory volume decrease following hyposmotic swelling in MDCK cells.

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion.

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen-negative (Lin-) cells resulted in the generation of the cells that displayed cardiomyocyte-specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)-labeled BM Lin-ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host-derived CFP. We further reconstituted human hemopoietic- and immune systems in mice by injecting human cord blood (CB)-derived Lin-CD34+CD38- hematopoietic stem cells (HSCs) into neonatal T cell(-)B cell(-)NK cell- immune-deficient NOD/SCID/IL2rgamma(null) mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic- and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.

Simultaneous real-time detection of initiator- and effector-caspase activation by double fluorescence resonance energy transfer analysis.

Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-alpha-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-alpha-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.