The use of sonication treatment to completely decellularize blood arteries: a pilot study.

We have developed a novel sonication decellularization system to prepare completely decellularized bioscaffolds in a short treatment time. The aim of the study is to investigate the sonication decellularization efficiency and its relation with ultrasonic power output and dissolved oxygen (DO) concentration in different detergent solution. In the study, we used aorta samples to evaluate sonication decellularization efficiency, which assessed treatment duration, sonication power and SDS detergent with/without saline. The treated samples were evaluated histologically by Hematoxylin Eosin (HE) staining and scanning electron microscopic (SEM) photographs. The concentration of DO was monitored to identify the effect of sonication on cavitation-related DO concentration in the solution. From histological results, the sonication decellularization efficiency was better than the other preparation methods. Decellularization efficiency was tended to increase significantly when DO value decreasing after 6 hours of treatment. In conclusion, we conclude that sonication treatment can be used to prepare the complete decellularized scaffolds in short treatment time.

Coronary arterial calcification is associated with albuminuria in type 2 diabetic patient.

AIM: Although microalbuminuria has been suggested as an independent risk factor for ischemic heart disease, the relationship between diabetic nephropathy and macroangiopathy remains unclear. Previously, we reported that coronary artery calcification detected by electron beam computed tomography (EBCT) could indicate the degree of coronary atherosclerosis in type 2 diabetic patients. In this study, we examine the association between coronary arterial calcification and microalbuminuria and aortic calcification and microalbuminuria. METHODS: Two hundred and fifty-six patients, including 177 type 2 diabetic patients (106 patients with normoalbuminuria, 71 with microalbuminuria) and 79 non-diabetic patients were evaluated by assessing the urinary albumin excretion rate and using EBCT to determine a coronary calcification score (CCS) and an aortic calcification score (ACS). RESULTS: No differences were observed regarding age, smoking index or BMI. Diabetic patients exhibited a greater CCS than non-diabetic subjects (non-diabetes 33 +/- 75 vs. diabetes 203 +/- 467, p < 0.05). Diabetic patients with microalbuminuria exhibited the most advanced CCS (253 +/- 491, p < 0.05). In contrast, no difference was observed in ACS among three groups. Multiple regression analysis showed that CCS is significantly associated with urinary albumin excretion rate as well as age, duration of diabetes and serum creatinine (R(2) = 0.31), while ACS is strongly associated with age, smoking, serum creatinine, systolic blood pressure and low-density lipoprotein cholesterol level (R(2) = 0.29). CONCLUSION: Increased urinary albumin excretion is associated with coronary arterial calcification in diabetic patients.

Effect of exercise training on serum leptin levels in type 2 diabetic patients.

To evaluate the effect of exercise training on serum leptin levels 50 sedentary subjects with type 2 diabetes were enrolled in either 6 weeks of aerobic exercise training with diet therapy (n = 23) or diet therapy alone (n = 27). The training program consisted of walking and cycle ergometer exercise for 1 hour at least 5 times per week, with the intensity of exercise maintained at 50% of maximum oxygen uptake. Serum leptin levels decreased significantly in the exercise training (TR) group (7.2 +/- 3.6 to 4.6 +/- 2.5 ng/mL, P <.05), but not in the sedentary (SED) group (6.9 +/- 3.4 to 5.6 +/- 2.9 ng/mL). Leptin levels standardized for percentage body fat (dividing serum leptin level by percentage body fat) after treatment were lower in the TR subjects compared with the SED subjects. Body weight and percentage body fat decreased in all patients; however, no significant changes were observed in either group. Fasting concentrations of plasma insulin and cortisol and the urinary excretion of 17-hydroxycorticosteroid (17-OHCS) did not differ between the groups either before or after treatment. Fasting plasma glucose and hemoglobin A(1c) (HbA(1c)) improved significantly in both groups, although no significant differences were observed between the groups either before or after treatment. Ventilatory threshold increased significantly in the exercise training subjects. This study demonstrates that exercise training in type 2 diabetic subjects reduces serum leptin levels independent of changes in body fat mass, insulin, or glucocorticoids.

Functional expression of cholecystokinin-A receptor on ventromedial hypothalamic neurons in the immature rat brain.

Cholecystokinin-8 (CCK-8) dose-dependently increased the cytosolic Ca2+ concentration ([Ca]i) in ventromedial hypothalamic neurons acutely dissociated from the immature rat brain. The CCK-8 response was mimicked by caerulein, but not by CCK(B) agonists, and was often inhibited by CCK(A) receptor antagonists, but rarely by CCK(B) receptor antagonists. The response was dependent on external Ca2+ and Na+, and was inhibited by voltage-dependent Ca2+ channel blockers. The results suggest that CCK-8-induced depolarization via CCK(A) receptors increased Ca2+ influx through a voltage-dependent Ca2+ channel, which in turn increased [Ca]i.

The effect of the antipsychotic drug mosapramine on the expression of Fos protein in the rat brain: comparison with haloperidol, clozapine and risperidone.

In this study, we examined the effect of the acute p.o. administration of the antipsychotic drug mosapramine, as well as the antipsychotic drugs clozapine, haloperidol and risperidone, on the expression of Fos protein in the medial prefrontal cortex, nucleus accumbens and dorsolateral striatum of rat brain. The administration of mosapramine (1 or 3 mg/kg) significantly increased the number of Fos protein positive neurons in the medial prefrontal cortex, but not in the dorsolateral striatum. In addition, mosapramine (1, 3 or 10 mg/kg) produced a dose-dependent increase in the number of Fos protein positive neurons in the nucleus accumbens. The acute administration of 10 mg/kg of mosapramine significantly increased the number of Fos protein positive neurons in all brain regions. The acute administration of clozapine (30 mg/kg), similarly to mosapramine at lower doses (1 or 3 mg/kg), significantly increased the number of Fos protein positive neurons in the medial prefrontal cortex and nucleus accumbens, but not dorsolateral striatum. In contrast, haloperidol (0.3 mg/kg) significantly increased the number of Fos protein positive neurons in the nucleus accumbens and dorsolateral striatum, but not medial prefrontal cortex. The acute administration of risperidone (0.3 or 1 mg/kg) did not affect the number of Fos protein positive neurons in the medial prefrontal cortex, nucleus accumbens or dorsolateral striatum of rat brain, whereas a 3 mg/kg dose of risperidone significantly increased the number of Fos protein positive neurons in all brain regions. These results suggest that the ability of mosapramine to enhance expression of Fos protein in the medial prefrontal cortex may contribute to a clozapine-like profile with respect to actions on negative symptoms in schizophrenia. Furthermore, the lack of effect of low doses of mosapramine on Fos protein expression in the dorsolateral striatum, an area believed to play a role in movement, suggests that it may have a lower tendency to induce neurological side effects.

Activation of ATP receptor increases the cytosolic Ca(2+) concentration in nucleus accumbens neurons of rat brain.

ATP increased the cytosolic Ca(2+) concentration ([Ca](i)) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca(2+) and Na(+), and was blocked by voltage-dependent Ca(2+) channel blockers. The results suggest that the ATP-induced depolarization increases Ca(2+) influx resulting in the increase in [Ca](i).

Pharmacological hepatic preconditioning: involvement of 70-kDa heat shock proteins (HSP72 and HSP73) in ischaemic tolerance after intravenous administration of doxorubicin.

BACKGROUND: Pharmacological preconditioning may induce a stress response which protects liver against ischaemia-reperfusion injury (IRI). The aim of this study was to determine, in an animal model, whether intravenous administration of doxorubicin induces heat shock proteins (HSPs) in liver tissue and facilitates liver tolerance to subsequent warm IRI. METHODS: Male Wistar rats were used. Production of HSPs was determined in liver tissue sequentially after the injection of doxorubicin 1 mg/kg body-weight. Acquisition of tolerance for 30 min warm ischaemia and reperfusion of the liver was determined in animals pretreated (48 h beforehand) with doxorubicin, and in controls. Biochemical liver function and liver adenine nucleotide concentration 40 min after reperfusion and survival rate at 7 days after the ischaemic insult were recorded. RESULTS: Expression of HSP72 and HSP73 in the liver was confirmed 48 h after doxorubicin administration. Biochemical parameters and survival rates were significantly better in pretreated animals than in controls. CONCLUSION: These results indicate that doxorubicin has the potential to provide the liver with tolerance against IRI. A simultaneous increase of both HSP72 and HSP73 in liver tissue may explain the acquisition of tolerance following the administration of doxorubicin.

Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats.

Membrane lipids and cytosolic proteins are major targets of oxidative injury. This study examined the effect of heat-shock preconditioning associated with the induction of heat-shock protein 72 on liver injury, from the aspect of lipid peroxidation and protein denaturation after carbon tetrachloride (CCl4) administration in rats--one of the representative oxidative injuries. Male Wistar rats were divided into two groups, group HS (preconditioned by heat exposure) and group C (not preconditioned). Expression of HSP72 in the liver tissue was confirmed by Western blot analysis. After a 48-h recovery period, all rats were given CCl4 intragastrically. Liver damage was assessed by measuring serum liver-related enzyme levels and adenine nucleotide concentration in the liver tissue. Lipid peroxidation and protein denaturation were evaluated by measuring tiobarbituric acid reactive substances (TBARS) and by immunohistochemical staining of 4-hydroxy-2-nonenal(HNE)-modified proteins in the liver. Survival rates of the rats after CCl4 administration were also compared. Expression of HSP72 was clearly detected in group HS, but not in group C. Heat-shock preconditioning significantly improved the survival rate, suppressed the increase in liver-related enzyme levels and maintained adenosine triphosphate levels (P<0.01 each). HNE-modified proteins--denatured proteins by free radical attack--were significantly less stained in group HS than in group C (P<0.05). However, TBARS levels did not differ between groups. Because heat-shock preconditioning did not alter TBARS levels but reduced HNE-modified proteins in association with the expression of HSP72, it is suggested that HSP72 did not prevent lipid peroxidation but decreased the lipid peroxidation-induced denaturation of proteins. This seemed to be a mechanism of heat-shock preconditioning to ameliorate oxidative liver injury.

The augmentative effect of repeated heat shock preconditioning on the production of heat shock protein 72 and on ischemic tolerance in rat liver tissue.

OBJECTIVE: Heat shock pretreatment induces heat shock protein (HSP)72 strongly in rat livers and provides the tolerance against subsequent ischemia-reperfusion injury. In this study, the effects of repeated heat shock pretreatment on the production of HSP72 in rat livers and on subsequent ischemic tolerance were investigated. METHODS: Rats pretreated with repeated heat shock were compared with those that received a single heat shock pretreatment. The production of HSP72 was analysed using Western-blotting and densitometer. At 48 h after heat shock pretreatment, all rats were subjected to warm liver ischemia for 30 or 45 min and then reperfused. Survival rate of the animals and liver functions during reperfusion were analysed. RESULTS: The production of HSP72 increased in the repeated heat shock group more than in the single heat shock group. Although there were no significant differences in animal survival or in liver functions after a 30-min ischemia between the single heat shock group and the repeated heat shock group, animal survival and liver functions after a 45-min ischemia were significantly better in the repeated heat shock group. CONCLUSION: In rats, repetition of heat shock pretreatment augmented the production of HSP72 in liver tissue and protected the liver from ischemia-reperfusion injury.

Effects of geranyl-geranyl-acetone administration before heat shock preconditioning for conferring tolerance against ischemia-reperfusion injury in rat livers.

The effect of geranyl-geranyl-acetone (GGA) administration before heat shock preconditioning on heat shock protein (HSP) 72 induction and on the acquisition of tolerance against ischemia-reperfusion Injury was studied in rat livers. Male Wistar rats were divided into four groups: a control group (group C); a GGA group (group G); a simple heat shock group (group VH); and a heat shock with GGA premedication group (group GH). Five-, 10-, and 15-minute periods of heat shock preconditioning at 42 degrees C were performed in groups VH and GH. Subgroups were determined according to the period of heat shock exposure. After a 48-hour recovery, rats in groups C, VH5, VH15, and GH5 received a 30-minute period of hepatic ischemia. Induction of HSP72, survival rates, and changes in biochemical and histologic parameters were compared among the groups. Five-minute heat shock preconditioning was not enough to Induce HSP72. However, livers in group GH5 expressed approximately the same amount of HSP72 as those in group VH15. The expression of HSP72 in group GH15 was stronger than that found in group VH15. The degree and location of HSP72 expression were not different between groups GH5 and VH15. Seven-day survival was significantly better in groups GH5 (16/16) and VH15 (15/16) than in group C (8/16) or VH5 (9/16). The recovery of adenosine triphosphate in liver tissue was faster, and the release of liver-related enzymes during reperfusion was lower in groups GH5 and VH15 than in group C or VH5. Administration of GGA before heat shock preconditioning augmented the induction of HSP72 by decreasing the threshold for triggering the stress response.

Effects of antipsychotic drugs on neurotoxicity, expression of fos-like protein and c-fos mRNA in the retrosplenial cortex after administration of dizocilpine.

In this study, we examined the effect of clozapine, olanzapine, risperidone and haloperidol on the neuropathology (i.e. neuronal vacuolization) and the expression of Fos-like protein and c-fos mRNA in the retrosplenial cortex of female Sprague-Dawley rats induced by the NMDA receptor antagonist dizocilpine. Pretreatment (15 min) with clozapine or olanzapine, but not risperidone or haloperidol, blocked the neuronal vacuolization produced by dizocilpine (0.5 mg/kg, s.c.) in the rat retrosplenial cortex in a dose-dependent manner. Furthermore, pretreatment (15 min) with clozapine or olanzapine, but not risperidone or haloperidol, significantly attenuated the expression of Fos-like protein in the retrosplenial cortex induced by dizocilpine (0.5 mg/kg, s.c.) in a dose-dependent manner. The marked expression of c-fos mRNA in the rat retrosplenial cortex induced by the administration of dizocilpine (0.5 mg/kg, s.c.) was significantly attenuated by pretreatment (15 min) with clozapine (10 mg/kg) or olanzapine (10 mg/kg), but not risperidone (10 mg/kg) or haloperidol (10 mg/kg). The present results suggest that pharmacologically relevant doses of clozapine or olanzapine, but not risperidone or haloperidol, block the neuropathological changes and the expression of Fos-like protein and c-fos mRNA in the rat retrosplenial cortex elicited by the administration of dizocilpine. It is possible that the blockade of dizocilpine-induced neuropathological changes by clozapine and olanzapine may be related to the unique antipsychotic actions of these drugs in schizophrenic patients, although this remains to be verified.

Dizocilpine-induced neuropathological changes in rat retrosplenial cortex are reversed by subsequent clozapine treatment.

In this study, we examined the effect of post-treatment with clozapine on the neuropathological changes in the rat retrosplenial cortex induced by the administration of non-competitive NMDA receptor antagonist dizocilpine ((+)-MK-801). The maximal increase in vacuolized neurons, which are representative of neuropathology, was observed 4 hours after a single injection of dizocilpine (0.5 mg/kg s.c.), with a complete reversal of the neuropathology after 16-24 hours. The administration of clozapine (10 mg/kg, i.p.,) 4 hours after the administration of dizocilpine significantly decreased the number of vacuolized neurons in the retrosplenial cortex 6, 8 or 10 hours after administration of dizocilpine, compared to vehicle-treated animals. Furthermore, the administration of clozapine (5, 10 or 20 mg/kg i.p.) 4 hours after the administration of dizocilpine produced a significant decrease in the number of vacuolized neurons in the retrosplenial cortex in a dose-dependent manner when measure 6 hours post-dizocilpine. These results show that neuropathological changes in the rat retrosplenial cortex produced by dizocilpine can be attenuated by post-treatment with clozapine.

Formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins in rat liver after ischemia-reperfusion: distinct localization of the two oxidatively modified products.

Ischemia-reperfusion (IR) injury is an intractable process associated not only with therapeutic recanalization of vessels, but also with partial resection or transplantation of solid organs including liver. To develop methods for predicting the degree of hepatic IR injury and further to identify injured cells, we studied the formation of 8-hydroxy-2'-deoxy-guanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE)-modified proteins in the normothermic hepatic IR model of rats using immunohistochemistry, high-performance liquid chromatography (HPLC) determination and Western blot. The Pringle maneuver for either 15 or 30 min duration produced reversible or lethal damage, respectively. The levels of both products were significantly increased in proportion to ischemia duration 40 min after reperfusion, suggesting the involvement of hydroxyl radicals. Increased immunoreactivity of 8-OHdG was observed not only in the nuclei of hepatocytes but also in those of bile canalicular and endothelial cells. However, immunoreactivity of HNE-modified proteins was detected in the cytoplasm of hepatocytes, which was confirmed by Western blot, and in addition, in the nuclei of hepatocytes after severe injury. Thus, localization of the two oxidatively modified products was not identical. Our data suggest that these two products could be used for the assessment of hepatic IR injury in tissue, but that the biological significance of the two products might be different.

Heat shock preconditioning on mitochondria during warm ischemia in rat livers.

BACKGROUND: The aim of this study was to investigate the effects of stress tolerance from heat shock preconditioning on changes in mitochondrial functions during ischemia-reperfusion injury of the liver. MATERIALS AND METHODS: Rats were divided into a heat shock group (group HS) and a control group (group C). In group HS, rats received heat shock pretreatment 48 h prior to ischemia-reperfusion. Heat shock pretreatment was performed in a water bath at 42 degrees C for 15 min under general anesthesia. In group C, the same treatment was done with the water bath at 37 degrees C instead of at 42 degrees C. A 30-min warm ischemia by cramping the hepatoduodinal ligament (Pringle's maneuver) followed by a 60-min reperfusion was administered to all rats. Changes in membrane potential of hepatic mitochondria (MPM); mitochondrial respiratory function before ischemia (n = 5), after ischemia (n = 10), and after reperfusion (n = 10); and ATP recovery after reperfusion were compared between the groups. RESULTS: After a 30-min ischemia, MPM in group C decreased significantly and did not recover even after reperfusion. On the other hand, MPM in group HS was maintained even after a 30-min ischemia and 60 min into reperfusion as well. The respiratory control ratio (RCR) of the mitochondria in group C decreased to as low as 5.06 +/- 0.72 after a 30-min ischemia, but in group HS, RCR was maintained near a normal level. The ATP level recovered significantly earlier in group HS than in group C after reperfusion. CONCLUSIONS: Heat shock preconditioning of the liver protected mitochondria from loss of membrane integrity during ischemia and contributed to their ability to produce energy-rich phosphates during reperfusion.

Pharmacologic stimulation of adenosine A2 receptor supplants ischemic preconditioning in providing ischemic tolerance in rat livers.

BACKGROUND: Ischemic preconditioning (IPC) is a promising strategy for conferring ischemic tolerance. We confirmed the acquisition of ischemic tolerance in the liver immediately after IPC and the role of adenosine kinetics in this process. METHODS: Male Lewis rats were used. IPC was administered with a 10-minute ischemia followed by a 10-minute reperfusion. Ischemic tolerance was tested with a 45-minute ischemia. Changes in the adenosine concentrations in liver tissue were evaluated, and the effects of adenosine A1 or A2 receptor agonists or antagonists were examined either in place of or against IPC. RESULTS: The 7-day animal survival was significantly better in the IPC group than in the control group (87% vs 53%; n = 15, P < .05). The release of liver-related enzymes during reperfusion was suppressed better in the IPC group (P < .01). Recovery of adenosine triphosphate levels was faster in the IPC group (P < .01). After IPC, adenosine concentrations in liver tissue immediately increased to 1555 +/- 299 pmol/g wet tissue and were maintained at that level during a subsequent 45-minute ischemia. The ischemic tolerance generated by IPC was mimicked by the administration of adenosine A2 receptor agonist and opposed by adenosine A2 receptor antagonist. CONCLUSIONS: The ischemic tolerance of the liver immediately after IPC can be supplanted by selective pharmacologic stimulation of adenosine A2 receptors.

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes.

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.

Excitatory effect of Cd2+ on cat adrenal chromaffin cells.

To understand the mechanism(s) underlying the Cd2+- and Co2+-induced increases in the cytosolic free Ca2+ concentration ([Ca]i) in cat adrenal chromaffin cells, we used nystatin-perforated patch recording method and fura-2 microfluorometry. Under the current-clamp conditions, the external application of 5x10(-7) M Cd2+ slowly depolarized the cells resulting in the bursting of action potentials. Under the voltage-clamp conditions, Cd2+ evoked a slow inward current accompanied by a decrease of K+ conductance at a holding potential of -40 mV, and Co2+ mimicked Cd2+ action. In some cells (16%), Cd2+ evoked an additional rapid transient outward current associated with an increased K+ conductance and a successive slow inward current. The Cd2+-induced inward current was activated in a concentration-dependent manner with a half-maximum concentration of 9.3x10(-8) M. The Cd2+- and Co2+-induced [Ca]i increases measured with fura-2 microfluorometry were maximal at 10(-6) and 10(-5) M, respectively, and the higher concentrations of both cations caused the smaller responses. Additional transient increase in [Ca]i was often evoked upon the removal of relatively higher concentrations of these metals. It was concluded that the Cd2+-induced membrane depolarization due to the decrease in K+ conductances evoked the bursting firings resulting in the increase in [Ca]i, and consequently might stimulate the catecholamine secretion.