Kayo Nemoto

The novel selective toll-like receptor 4 signal transduction inhibitor tak-242 prevents endotoxaemia in conscious Guinea-pigs.

1 TAK‐242 is a novel compound that suppresses nitric oxide and cytokine production by selectively inhibiting intracellular signals from toll‐like receptor (TLR)‐4. In the present study, we investigated the effectiveness of TAK‐242 against sepsis using an endotoxaemia model in conscious and unrestricted guinea‐pigs. Measures examined included muscle tension paralysis of the intestine, blood pressure, high morbidity group box (HMGB)‐1 levels and survival rate. 2 Tension of the longitudinal muscle of the colon was monitored continuously by telemetry. Arterial blood pressure was monitored via a carotid artery catheter. TAK‐242 was administered intravenously through a jugular vein catheter. Guinea‐pigs were divided into a control group, given vehicle (placebo emulsion), and the experimental group, administered 3 or 10 mg/kg TAK‐242, 1 h before administration of 10 mg/kg lipopolysaccharide (LPS). 3 In the control group, the tension of the longitudinal muscle of the colon decreased in a time‐dependent manner and blood pressure was reduced, with maximal effects observed 1–3 h after administration of LPS. In the TAK‐242‐treated group, LPS‐induced relaxation of the intestine and hypotension were significantly inhibited. In the control group, HMGB‐1 levels were increased after LPS administration and this reaction was significantly blocked in the TAK‐242‐treated group. Importantly, survival rate was increased after TAK‐242 treatment. 4 In conlusion, the results of the present study show that TAK‐242 inhibited the symptoms associated with endotoxaemia in a guinea‐pig model of sepsis and that it may, therefore, be an effective treatment for sepsis.

Fasudil, a Rho-kinase inhibitor, attenuates lipopolysaccharide-induced vascular hyperpermeability and colonic muscle relaxation in guinea pigs

BACKGROUND Rho-associated coiled coil-forming protein kinase (Rho-kinase), a downstream target effector of the small GTP-binding protein Rho, plays a key role in cell adhesion, motility, and contraction. The goal of the present study was to determine the role of the Rho/Rho-kinase signal pathway in the pathogenesis of lipopolysaccharide (LPS)-induced vascular hyperpermeability using the Rho-kinase inhibitor fasudil. METHODS To evaluate plasma leakage, fasudil (3 or 10 mg/kg) or saline was intravenously administered 30 min before LPS injection. LPS (100, 300, and 1,000 μg/0.1 mL/site) and saline (0.1 mL/site) were administered intracutaneously in the dorsum of guinea pigs. Vascular permeability was measured on the dorsal skin by the local accumulation of Evans Blue dye after intracutaneous injection of LPS (100-1000 μg/site) from Escherichia coli. For the measurement of colonic muscle tension, fasudil (3 mg/kg) or saline was intravenously administered 30 min before LPS injection. LPS (1 mg/kg) was administered intravenously. RESULTS Dye leakage in the skin increased significantly 2 h after the injection of LPS. This LPS-induced dye leakage was significantly suppressed by fasudil (3 and 10 mg/kg). LPS caused a transient decrease in colonic muscle tension, which peaked 2.5 h after the injection. This decrease in muscle tension was significantly suppressed by pretreatment with fasudil (3 mg/kg). CONCLUSIONS The Rho/Rho-kinase pathway might play an important role in the pathogenesis of LPS-induced endotoxemia, and fasudil could attenuate LPS-induced microvascular permeability, leading to inhibition of endotoxemia.

Mesenteric Lymph Flow in Endotoxemic Guinea Pigs

BACKGROUND Lipopolysaccharide (LPS) is a structural component of the outer membrane of gram-negative bacteria. LPS activates the host cells, leading to the production and release of proinflammatory cytokines. Given the induction duration for the release of cytokines, the initial mechanisms that produce LPS action on a timescale of minutes are not fully understood. We studied the effect of initial LPS-induced action on the lymphatic system by measuring the time-dependent changes in mesenteric lymph flow in guinea pigs in vivo. In addition, we determined the leakage of plasma protein into the lymphatic system using Evans blue dye. METHODS AND RESULTS The mesenteric lymphatic vessel was cannulated with a polyethylene catheter. We administered drugs via a catheter in jugular vein. The control animals received vehicle intravenously (i.v.). The experimental group received 1 mg/kg or 10 mg/kg LPS i.v. Twenty minutes before injection of the vehicle or LPS, Evans blue dye (5 mg/kg i.v.) was administered. Lymph output was measured every 20 min. The amount of Evans blue in the lymph was determined by spectrophotometry. The mesenteric lymph showed a steady flow rate of approximately 290 μL/kg/20 min. The lymph flow immediately increased after the administration of LPS and reached 3.4-fold and 7.4-fold after 1 h of 1 mg/kg and 10 mg/kg LPS injection, respectively. The albumin content in lymph significantly increased in proportion to the increased lymph volume. CONCLUSIONS These results suggest that the early increase in mesenteric lymph flow rate in guinea pigs produced by LPS is mediated by vascular hyperpermeability and plasma albumin leakage.

Elimination of 2-Arachidonoylglycerol Action by Direct Hemoperfusion Through Immobilized Polymyxin B Fibers: An Experimental Study in Conscious Guinea Pigs

Abstract:  Direct hemoperfusion through a network of immobilized polymyxin B fibers is used for the treatment of septic shock, but the mechanism underlying the clinical benefits remains unclear. The aims of the present study were to assess the actions of direct hemoperfusion through immobilized polymyxin B fibers on effects of exogenous endotoxin or 2‐arachidonoylglycerol in conscious guinea pigs; and to examine the underlying mechanisms. In the present study, colonic motion was monitored continuously by telemetry using a force transducer attached to the taenia caecum, while blood pressure was monitored with a carotid artery catheter. To establish a hemoperfusion circuit in a freely moving, conscious guinea pig, catheters were implanted in the carotid artery and the jugular vein, tunneled subcutaneously, exteriorized at the back of the neck in contact with a lightweight tethering spring, and attached to a swivel device at the top of the cage. On the day after the operation, lipopolysaccharide (Escherichia coli, O111:B4; 1 mg/kg) or 2‐arachidonoylglycerol (1 mg/kg) was given intraperitoneally (i.p.) and direct hemoperfusion through immobilized polymyxin B fibers was carried out for 2 h. The results showed that in guinea pigs treated with direct hemoperfusion through immobilized polymyxin B fibers, relaxation of colonic longitudinal muscle caused by lipopolysaccharide or 2‐arachidonoylglycerol was significantly suppressed, as were decreases in blood pressure. We conclude that the actions following exogenous administration of 2‐arachidonoylglycerol were eliminated by direct hemoperfusion through immobilized polymyxin B fibers. These results suggest that effectiveness of direct hemoperfusion through immobilized polymyxin B fibers in endotoxemia involves elimination of 2‐arachidonoylglycerol action. 

CONTINUOUS INFUSION OF SIVELESTAT SODIUM HYDRATE PREVENTS LIPOPOLYSACCHARIDE-INDUCED INTESTINAL PARALYSIS AND HYPOTENSION IN CONSCIOUS GUINEA-PIGS

1 Sivelestat sodium hydrate (sivelestat), a neutrophil elastase inhibitor, is used to treat acute lung injury associated with systemic inflammatory response syndrome, but its effects have not been described for endotoxaemia. In the present study, we examined the effects of a continuous infusion of sivelestat on intestinal mechanical activity and blood pressure using an endotoxaemic model in conscious, unrestrained guinea‐pigs. 2 Guinea‐pigs underwent laparotomy while anaesthetized and were implanted with a force transducer sutured onto the taenia caecum. With this transducer, changes in tension in the intestinal longitudinal muscle were measured continuously via telemetry. Catheters were inserted into the carotid artery and jugular vein, were tunnelled subcutaneously and were accessed from the back of the neck. These catheters were connected to a cannula swivel and were used to monitor arterial pressure as well as to administer drugs i.v. in conscious, unrestrained guinea‐pigs. Twenty hours after surgery, guinea‐pigs received a single dose of lipopolysaccharide (LPS; 0.3 mg/kg, i.p.) 10 min after the start of a continuous 2 h i.v. infusion of sivelestat (30 mg/kg per h) or vehicle (saline). Elastase activity before and after sivelestat or vehicle administration was measured spectrometrically using a specific synthetic substrate. 3 We confirmed that intestinal longitudinal muscle tension decreased 2–3 h after LPS administration in the control group, with a concurrent decline in blood pressure. In guinea‐pigs treated with sivelestat, the LPS‐induced decreases in muscle tension and blood pressure were significantly reduced. In LPS‐treated control guinea‐pigs, serum elastase activity was elevated and this increase was significantly attenuated by administration of sivelestat. 4 The findings from the present study suggest that sivelstat can effectively reduce intestinal dysfunction and attenuate LPS‐induced decreases in blood pressure in endotoxaemia.

EFFECTS OF DIRECT HAEMOPERFUSION THROUGH FIBRES IMMOBILIZING POLYMYXIN B AND NAFAMOSTAT MESILATE ON ENDOTOXAEMIA IN CONSCIOUS GUINEA-PIGS

1 Direct haemoperfusion through a network of fibres immobilizing polymyxin B (PMX‐B) is used for the treatment of septic shock, but the mechanism underlying its clinical benefits remains unclear. The aims of the present study were to assess the actions of direct haemoperfusion through fibres immobilizing PMX‐B (PMX‐DHP) on the effects of exogenously administered endotoxin in conscious guinea‐pigs and to examine the difference in the effects of heparin compared with nafamostat mesilate (NM) used as an anticoagulant. Although nafamostat is widely used in Japan, the agent cannot necessarily be used elsewhere in the world. Therefore, the study aimed to investigate and elucidate the effectiveness of NM compared with heparin. 2 Colonic motion was monitored continuously via telemetry using a force transducer attached to the taenia caecum, whereas blood pressure was monitored using a carotid artery catheter. To establish a haemoperfusion circuit in each freely moving and conscious guinea‐pig, catheters were implanted in the carotid artery and the jugular vein, tunnelled subcutaneously, exteriorized at the back of the neck in contact with a lightweight tethering spring and attached to a swivel device at the top of the cage. On the day after the operation, lipopolysaccharide (LPS; Escherichia coli, O111:B4; 1 mg/kg) was administered i.v. and PMX‐DHP was conducted for 2 h. Heparin (50 IU/h) or NM (0.4 mg/h) was used as the anticoagulant. Furthermore, guinea‐pigs were administered a lethal dose of LPS (10 mg/kg) and the survival rate was examined for animals undergoing PMX‐DHP compared with control animals. 3 In guinea‐pigs treated with PMX‐DHP, relaxation of colonic longitudinal muscle caused by LPS was significantly suppressed, as were decreases in blood pressure. Of the two anticoagulants used, NM was more effective than heparin. In addition, PMX‐DHP significantly increased the survival rate of guinea‐pigs that received potentially lethal doses of LPS. 4 In ca onscious and unrestrained guinea‐pig endotoxaemia model, PMX‐DHP significantly improved intestinal paralysis and decreases in blood pressure. These effects were augmented more by NM than by heparin when an anticoagulant was used in the perfusion process. These findings suggest that haemoperfusion using PMX and NM performed in the early stage of endotoxaemia is an effective treatment.