Isamu Murata

Characterization of systemic and histologic injury after crush syndrome and intervals of reperfusion in a small animal model.

BACKGROUND Prolonged compression of limb muscles and subsequent decompression are important in the development of crush syndrome (CS). We applied a simple rubber tourniquet to rat hind limbs to create a CS model. METHODS Anesthetized rats were subjected to bilateral hind limb compression for 5 hours followed by decompression and reperfusion for 0 hour, 1 hour, 3 hours, and 24 hours under monitoring of arterial blood pressure and electrocardiography. Blood and tissue samples were collected for histology, biochemical analysis, and tissue myeloperoxidase activity assessment. RESULTS The survival rates of the CS-model groups remained at 100% until 3 hours, however, dropped to 25% at 24 hours after reperfusion mainly because of hyperkalemia and consequent hypotension observed at 1 hour and deteriorated at 3 hours after reperfusion. Rhabdomyolysis evaluated by circulating and histologic markers of injury was found as early as 1 hour and more marked at 3 hours, resulting in impaired renal function 24 hours after reperfusion. Myeloperoxidase activities increased with incremental periods after reperfusion not only in injured limb muscles but also in kidney and lung, suggesting an abnormal interaction between the vascular endothelium and circulating leukocytes after rhabdomyolysis, possibly causing subsequent multiple organ dysfunction frequently encountered in CS. CONCLUSION The findings from this study demonstrate the feasibility of a novel small animal model of extremity crush injury. By using this model, the impact of incremental periods of reperfusion on mortality and remote organ dysfunctions can be characterized. Future studies are necessary to better define a threshold for this injury pattern and the impact of other factors underlying this syndrome.

Nitrite reduces ischemia/reperfusion-induced muscle damage and improves survival rates in rat crush injury model.

BACKGROUND Nitrite is an intrinsic signaling molecule with potential therapeutic implications in mammalian ischemia/reperfusion (I/R) injury of the heart, liver, and kidney. Although limb muscle compression and subsequent reperfusion are the causative factors in developing crush syndrome (CS), there has been no report evaluating the therapeutic effects of nitrite on CS. We therefore tested whether nitrite could be a therapeutic agent for the treatment of CS. METHODS To create a CS model, anesthetized rats were subjected to bilateral hind limb compression with rubber tourniquets for 5 hours, followed by reperfusion for 0 hour to 6 hours while monitoring blood pressure. Saline for the CS group or sodium nitrite (NaNO2-100, 200, and 500 &mgr;mol/kg) for the nitrite-treated CS groups was intravenously administered immediately before reperfusion. Blood and tissue samples were collected for biochemical analysis. RESULTS Tissue nitrite levels in injured muscles were significantly reduced in the CS group compared with the sham group during I/R injury. Nitrite administration to CS rats restored nitric oxide bioavailability by enhancing nitrite levels of the muscle, resulting in a reduction of rhabdomyolysis markers such as potassium, lactate dehydrogenase, and creatine phosphokinase. Nitrite treatment also reduced plasma levels of interleukin-6 and myeloperoxidase activities in muscle and lung tissues, finally resulting in a dose-dependent improvement of survival rate from 24% (CS group) to 36% (NaNO2-100 group) and 64% (NaNO2-200 and 500 groups). CONCLUSION These results indicate that nitrite reduces I/R-induced muscle damage through its cytoprotective action and contributes to improved survival rate in a rat CS model.

A comparison of the physicochemical properties and a sensory test of Acyclovir creams

In external medicine, types and ratios of additives are not necessarily the same for well-known brand-name drugs and generic drugs. This study sought to compare the physicochemical properties and sensory test results of a brand-name Acyclovir (ACV) cream and two generic ACV creams. Near-infrared (NIR) spectroscopy revealed changes in absorption spectra attributed to differences in the oil and water content of the 3 creams. In addition, ACV-B and ACV-C had similar NIR absorption spectra. Microscopic examination revealed crystallization in each of the creams and droplets in ACV-C. Powder X-ray diffraction measurement revealed diffraction peaks due to ACV for ACV-A and ACV-B. Assessment of viscoelasticity indicated that stress of subjection to 35 °C caused no changes in the viscoelasticity of ACV-B and ACV-C in comparison to stress of subjection to 25 °C but it did cause the viscoelasticity of ACV-A to decrease. ACV-A had a greater tolerance to stress and a higher viscosity, tan δ, and yield value than the other 2 creams. Results of a sensory test revealed significant differences in adhesiveness, spreadability, and feel for ACV-A in comparison to ACV-B and ACV-C. Thus, differences in the viscosity and elasticity of the creams due to differences in types and ratios of additives were noted. These differences are surmised to be differences in physical properties. In addition, results suggested that viscoelasticity and spreadability in the sensory test reflected differences in physical properties.

Acute lethal crush-injured rats can be successfully rescued by a single injection of high-dose dexamethasone through a pathway involving PI3K-Akt-eNOS signaling.

BACKGROUND Crush syndrome (CS) is characterized by ischemia/reperfusion–induced rhabdomyolysis and the subsequent onset of systemic inflammation. CS is associated with a high mortality, even when patients are treated with conventional therapy. We hypothesized that treatment of lethal CS rat model with dexamethasone (DEX) have therapeutic effects on the laboratory findings and clinical course and outcome. METHODS To create a CS model, anesthetized rats were subjected to bilateral hind limb compression with rubber tourniquets for 5 hours and randomly divided into three groups as follows: saline-treated CS group, CS groups treated with low (0.1 mg/kg) and high doses (5.0 mg/kg) of DEX. Saline for the CS group or DEX for the DEX-treated CS groups was intravenously administered immediately before reperfusion. Under continuous monitoring and recording of arterial blood pressures, blood and tissue samples were collected for histologic and biochemical analysis at designated period before and after reperfusion. RESULTS Ischemic compression of rat hind limbs reduced the nitrite content in the crushed muscle, and the subsequent reperfusion induced reactive oxygen species–mediated circulatory collapse and systemic inflammation, finally resulting in a mortality rate of 76% by 48 hours after reperfusion. A single injection of high-dose DEX immediately before reperfusion activated endothelial nitric oxide synthase (eNOS) by sequential phosphorylation through the nongenomic phosphoinositide 3-kinase (PI3K)–Akt–eNOS signaling pathway. DEX also exhibited anti-inflammatory effects by modulating proinflammatory and anti-inflammatory mediators, consequently suppressing myeloperoxidase activities and subsequent systemic inflammation, showing a complete recovery of the rats from lethal CS. CONCLUSION These results indicate that high-dose DEX reduces systemic inflammation and contributes to the improved survival rate in a rat CS model.

Low-Dose Sodium Nitrite Fluid Resuscitation Prevents Lethality From Crush Syndrome by Improving Nitric Oxide Consumption and Preventing Myoglobin Cytotoxicity in Kidney in A Rat Model

Objective: Crush syndrome (CS) is a serious medical condition characterized by muscle cell damage resulting from pressure. CS has a high mortality, even when patients receive fluid therapy. We examined whether administration of NaNO2-containing fluid can improve survival in a rat model of CS. Design: The CS model was generated by subjecting anesthetized rats to bilateral hind limb compression with a rubber tourniquet for 5 h. Rats were then randomly divided into six groups: sham; CS with no treatment; CS with normal saline treatment; CS with normal saline + 25 mEq/L bicarbonate treatment; and CS with normal saline + 200 or 500 &mgr;mol/kg NaNO2. Measurements and Main Results: Blood and tissue samples were collected for histological and biochemical analyses at predetermined time points before and after reperfusion. Ischemic compression of rat hind limbs reduced nitrite content in the crushed muscle, and subsequent reperfusion resulted in reactive oxygen species-induced circulatory dysfunction and systemic inflammation. Rats treated with 200 &mgr;mol/kg NaNO2 showed increased nitric oxide (NO) levels, blood circulation, and neoangiogenesis, decreased generation of reactive oxygen species, and suppression of the inflammatory response, leading to complete recovery. Conclusions: Treatment with 200 &mgr;mol/kg NaNO2 prevents muscle damage induced by ischemia reperfusion via the protective effects of NO and suppression of systemic inflammation, thereby increasing survival rates in CS.

Pharmacokinetics characteristics of dexamethasone in Crush syndrome model rats

Crush syndrome (CS) is characterized by ischemia/reperfusion-induced rhabdomyolysis and subsequent systemic inflammation and has a high mortality rate, even when treated with conventional therapy. In previous studies, we demonstrated that treatment of rats with acute lethal CS using dexamethasone (DEX) had therapeutic effects in laboratory findings and improved the clinical course of CS. However, because the application of DEX in CS therapy is unknown, evaluation of the pharmacokinetic parameters of DEX was considered essential to support its clinical use. Here, we investigated the pharmacokinetic characteristics of DEX in a rat model of CS. Anesthetized rats were subjected to bilateral hind limb compression using rubber tourniquets for 5 h, followed by reperfusion for 0 to 24 h. Rats were divided randomly into 4 groups: saline-treated sham (S) and CS groups and 5.0 mg/kg DEX-treated S (S-DEX) and CS (CS-DEX) groups. Blood and tissue samples were collected for HPLC analysis. In the CS-DEX group, the pharmacokinetic parameters of the area under the concentration-time curve, mean residence time, and distribution volume levels increased significantly compared to the S-DEX group, whereas total body clearance, elimination rate constant, and renal clearance levels decreased significantly. Moreover, decrease of muscle tissue DEX concentration and of CYP3A activity were observed in the CS-DEX group. These results show the pharmacokinetic characteristics of DEX in the rat CS model and support the potential use of DEX in disaster medical care.

Effect of low glycemic index food and postprandial exercise on blood glucose level, oxidative stress and antioxidant capacity

Low glycemic index (GI) food and postprandial exercise are non-drug therapies for improving postprandial hyperglycemia. The present randomized, crossover study investigated the effect of low GI food combined with postprandial exercise on postprandial blood glucose level, oxidative stress and antioxidant capacity. A total of 13 healthy subjects were each used in four experiments: i) rice only (control), ii) salad prior to rice (LGI), iii) exercise following rice (EX) and iv) salad prior to rice and exercise following rice (MIX). The blood glucose level, oxidative stress and antioxidant capacity were then measured. At 60 min after the meal, the blood glucose level was observed to be increased in the MIX group compared with that in the LGI group. Furthermore, at 180 min, the antioxidant capacity was found to be reduced in the MIX group compared with those of the LGI and EX groups. These findings suggest that low GI food combined with postprandial exercise does not improve postprandial hyperglycemia. It may be necessary to establish optimal timing and intensity when combining low GI food with postprandial exercise to improve postprandial hyperglycemia.

Evaluation of formulation properties and skin penetration in the same additive-containing formulation

The aim of this study is to examine the physicochemical properties of the external preparation, the effect on the skin permeability and the human senses. Miconazole nitrate cream formulation (MCZ-A: bland name and MCZ-B, −C, −D: generics) to measure the physicochemical properties, was performed by the skin permeation test and human sensory test. The flattening, viscoelasticity, and water content of each cream were measured and each cream was subjected to near-infrared (NIR) absorption spectroscopy and human sensory testing. The yield value was calculated based on measured flattening and was 734.8 dynes/cm2 for MCZ-A, 1198.9 dynes/cm2 for MCZ-B, 461.3 dynes/cm2 for MCZ-C and 3112.3 dynes/cm2 for MCZ-D. Measurement of viscoelasticity and viscosity revealed that MCZ-C had a smaller tanδ than the other 3 creams at 25 °C. NIR absorption spectroscopy revealed that MCZ-A had the highest absorption peak due to hydroxyl groups, followed by MCZ-C, −B, and then −D. Measurement of water content revealed that MCZ-A had a water content of 65.9%, MCZ-B, −C, and −D had a water content of around 56.3%. Human sensory testing revealed differences between MCZ-A and MCZ-C and between MCZ-B and MCZ-D in terms of spreadability and feel. These findings indicate that differences in water and oil content and emulsification resulted in the creams having different physical properties, such as flattening, internal structure, and dynamic viscoelasticity. NIR absorption spectroscopy, which allows non-destructive measurement of a sample’s physicochemical properties, and measurement of viscoelasticity and viscosity, which allows measurement of a sample’s dynamic viscoelasticity, revealed differences in the physical properties of creams. The skin permeation test, skin MCZ amount was 7.48 µg/cm2 for MCZ-A, 5.11 µg/cm2 for MCZ-B, 12.08 µg/cm2 for MCZ-C and 3.75 µg/cm2 for MCZ-D. In addition, since the drug spread is good about the skin migration, spreadability is affecting the potential dermal transfer.

Effect of antioxidant activity of caffeic acid with cyclodextrins using ground mixture method

Graphical Abstract Caffeic acid/α-cyclodextrin (molar ratio = 1/1), the vinylene group of the caffeic acid molecule appears to be included from the wider to the narrower rim of the ring of α-cyclodextrin. Caffeic acid /β-cyclodextrin (molar ratio = 1/1), the aromatic ring of the caffeic acid molecule appears to be included from the wider to the narrower rim of the ring of βCD. This suggests that forms of inclusion differed in the caffeic acid /α-cyclodextrin (molar ratio = 1/1) and the caffeic acid /β-cyclodextrin (molar ratio = 1/1).Unlabelled image

Early Therapeutic Intervention for Crush Syndrome: Characterization of Intramuscular Administration of Dexamethasone by Pharmacokinetic and Biochemical Parameters in Rats

Crush syndrome (CS) is the systemic manifestation of muscle cell damage resulting from pressure and crushing. It is associated with a high mortality rate, even when patients are treated with conventional therapy. We demonstrated the utility of intramuscular administration of dexamethasone (DEX) in disaster medical care by using a model of CS to characterize the pharmacokinetics and biochemical parameters. We compared intravenous (IV) and intramuscular (IM) injection. The IM sites were the right anterior limb (AL), bilateral hind limbs (bHL), and unilateral hind limb (uHL). DEX (5.0 mg/kg) was administered in sham-operated (sham, S-IV, S-AL, S-bHL, S-uHL groups) and CS rats (control, C-IV, C-AL, C-bHL, C-uHL groups). The survival rate in the IM groups was lower than that in the C-IV group. Survival was highest in the C-AL group, followed by the C-uHL and C-bHL groups. The blood DEX concentration of the C-AL group was similar to that in the C-IV group. The C-bHL and C-uHL groups had decreased blood DEX concentrations. Moreover, inhibition of inflammation was related to these changes. Administration of DEX to non-injured muscle, as well as IV administration, increased the survival rate by modulating shock and inflammatory mediators, consequently suppressing myeloperoxidase activity and subsequent systemic inflammation, resulting in a complete recovery of rats from lethal CS. These results demonstrate that injection DEX into the non-injured muscle is a potentially effective early therapeutic intervention for CS that could easily be used in transport to the hospital.