Mu opioid receptor antagonism in the nucleus accumbens shell blocks consumption of a preferred sucrose solution in an anticipatory contrast paradigm.

Binge eating, a central feature of multiple eating disorders, is characterized by excessive consumption occurring during discrete, often brief, intervals. Highly palatable foods play an important role in these binge episodes - foods chosen during bingeing are typically higher in fat or sugar than those normally consumed. Multiple lines of evidence suggest a central role for signaling by endogenous opioids in promoting palatability-driven eating. This role extends to binge-like feeding studied in animal models, which is reduced by administration of opioid antagonists. However, the neural circuits and specific opioid receptors mediating these effects are not fully understood. In the present experiments, we tested the hypothesis that endogenous opioid signaling in the nucleus accumbens promotes consumption in a model of binge eating. We used an anticipatory contrast paradigm in which separate groups of rats were presented sequentially with 4% sucrose and then either 20% or 0% sucrose solutions. In rats presented with 4% and then 20% sucrose, daily training in this paradigm produced robust intake of 20% sucrose, preceded by learned hypophagia during access to 4% sucrose. We tested the effects of site-specific infusions of naltrexone (a nonspecific opioid receptor antagonist: 0, 1, 10, and 50µg/side in the nucleus accumbens core and shell), naltrindole (a delta opioid receptor antagonist: 0, 0.5, 5, and 10µg/side in the nucleus accumbens shell) and beta-funaltrexamine (a mu opioid receptor antagonist: 0 and 2.5µg/side in the nucleus accumbens shell) on consumption in this contrast paradigm. Our results show that signaling through the mu opioid receptor in the nucleus accumbens shell is dynamically modulated during formation of learned food preferences, and promotes binge-like consumption of palatable foods based on these learned preferences.

Convergent, not serial, striatal and pallidal circuits regulate opioid-induced food intake.

Mu opioid receptor (MOR) signaling in the nucleus accumbens (NAcc) elicits marked increases in the consumption of palatable tastants. However, the mechanism and circuitry underlying this effect are not fully understood. Multiple downstream target regions have been implicated in mediating this effect but the role of the ventral pallidum (VP), a primary target of NAcc efferents, has not been well defined. To probe the mechanisms underlying increased consumption, we identified behavioral changes in rats' licking patterns following NAcc MOR stimulation. Because the temporal structure of licking reflects the physiological substrates modulating consumption, these measures provide a useful tool in dissecting the cause of increased consumption following NAcc MOR stimulation. Next, we used a combination of pharmacological inactivation and lesions to define the role of the VP in hyperphagia following infusion of the MOR-specific agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) into the NAcc. In agreement with previous studies, results from lick microstructure analysis suggest that NAcc MOR stimulation augments intake through a palatability-driven mechanism. Our results also demonstrate an important role for the VP in normal feeding behavior: pharmacological inactivation of the VP suppresses baseline and NAcc DAMGO-induced consumption. However, this interaction does not occur through a serial circuit requiring direct projections from the NAcc to the VP. Rather, our results indicate that NAcc and VP circuits converge on a common downstream target that regulates food intake.

Portal systemic shunt through the renal vein.

BACKGROUND: Despite the semi-routine use of color Doppler sonography for evaluating portal circulation abnormalities, there is a relative paucity of detailed color Doppler findings of portal systemic (P-S) shunt through the renal vein (P-SR shunt). METHODS: We reviewed the color Doppler findings of 18 patients with P-SR shunt to determine its clinical significance and appropriate scanning techniques for diagnosing accurately P-SR shunt. RESULTS: The splenorenal shunt was imaged as a highly tortuous vessel at the splenic hilum, which then coursed backward behind the spleen. Splenic vein flow was reversed or very slow. The gastrorenal shunt originated from the splenic vein, coursed backward, and joined the left renal vein. Flow direction in the splenic vein was always hepatopetal. The P-S shunt through the right renal vein originated from duodenal or jejunal varices, coursed posterolaterally, and joined the right renal vein at the renal hilum. CONCLUSION: Familiarity with these color Doppler findings will help increase the diagnostic confidence of P-SR shunt by color Doppler sonography.

Malignant fibrous histiocytoma of the spleen: report of a case and literature review.

There is a marked paucity of reports on malignant fibrous histiocytoma (MFH) of the spleen in the literature, and there are no previous reports of its color Doppler sonographic (US) and contrast-enhanced US findings. We report on an 82-year-old male with splenic MFH (inflammatory subtype), with an emphasis on color Doppler and contrast-enhanced US findings.

The role of mast cells in the development of 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in rats.

BACKGROUND: The role of mast cells in Crohn disease (CD) remains to be established. The aim of this study was to elucidate this in the development of CD-like colitis in rats by the use of mast-cell-deficient Ws/Ws and their control W+/W+ rats. METHODS: CD-like colitis was induced in both groups by an enema of 10 mg of 2,4, 6-trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Colonic damage, adhesion and colonic weight were measured at 7 and 14 days after the TNBS/ethanol enema. Rat mast cell protease-2 (RMCP-2) in the colonic tissue was also measured at 7 days after the enema. RESULTS: There was no significant difference between W+/W+ and Ws/Ws rats in terms of colonic damage, adhesion or colonic weight. The tissue content of RMCP-2 in Ws/Ws rats treated with either saline or TNBS/ethanol was only maintained at a much lower level than that in W+/W+ rats with the corresponding treatment. CONCLUSIONS: These results demonstrate that mast cells are not essential in the development of 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in rats.

Efficacy of postinfection treatment with anti-Shiga toxin (Stx) 2 humanized monoclonal antibody TMA-15 in mice lethally challenged with Stx-producing Escherichia coli.

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome. TMA-15 is a humanized monoclonal antibody against Stx2, a major pathogenic factor. In a mouse infection model that used B2F1, a virulent STEC strain, the efficacy of TMA-15 was assessed when it was administered after bacterial and toxin exposure. In this model, a time-course analysis of the serum Stx2 level showed that the toxin was detectable from 24 h after infection. In an evaluation of the time-dependent efficacy, treatment with TMA-15 up to 24 h after infection ameliorated the lethal challenge, although treatment at 48 h showed no efficacy. To determine the effective dose, escalating doses were administered at 24 h after infection. The number of mice that survived after doses of 0, 0.25, 0.5, 1.0, and 2.0 mg/kg were 0/20, 11/20, 17/20, 20/20, and 20/20, respectively. These findings suggest that TMA-15 shows potential for prevention of severe complications associated with STEC infection.

Activated protein C prevents multiple organ injury following extensive hepatectomy in cirrhotic rats.

BACKGROUND/AIMS: Extended hepatectomy for cirrhotic liver in patients with hepatocellular carcinoma often triggers posthepatectomy liver failure. It has been shown that the microcirculatory disturbance caused by microthrombus formation and sinusoidal endothelial cellular injury is one of the causes of post-hepatectomy liver dysfunction. We therefore investigated the effect of activated protein C (APC), a potent antithrombotic serine protease with anti-inflammatory effects, on posthepatectomy liver dysfunction and multiple organ injury in cirrhotic rats. METHODS/RESULTS: Dimethylnitrosamine-induced cirrhotic rats underwent 70% hepatectomy and received lipopolysaccharide (200 microg/kg) 48 h later to prepare a lethal posthepatectomy acute liver failure model. APC (1500 U/kg), given intravenously 15 min before and 1 h after endotoxin challenge, attenuated liver dysfunction and decreased serum tumor necrosis factor-alpha concentration. APC significantly improved the survival rate of rats at 12 h after endotoxin challenge. Histological examination revealed that APC treatment inhibited not only intrasinusoidal fibrin deposition and massive hepatocellular necrosis but also pulmonary injury and glomerular fibrin deposition. Immunohistochemically, expression of intercellular adhesion molecule-1 on sinusoidal cells and renal glomeruli was decreased in the APC-treated animals. CONCLUSIONS: APC administration prevented acute liver dysfunction and attenuated multiple organ injury following extended hepatectomy in cirrhotic rats, possibly via anticoagulant and anti-inflammatory effects.

Effect of activated human protein C on disseminated intravascular coagulation induced by lipopolysaccharide in rats.

Protein C is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating coagulation and fibrinolysis by inactivating not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). The aim of the present study was to examine the effect of a human APC product (designated as CTC-111), compared with that of heparin, on the disseminated intravascular coagulation (DIC) induced by lipopolysaccharide (LPS) in rats. LPS (1 mg/kg/h) infusion was performed through a femoral vein for 4 h. One-fifth amount of the total dosage of CTC-111 or heparin was injected into the other femoral vein, followed by a 4-h infusion of the remainder. Both CTC-111 (10,000-100,000 U/kg) and heparin (400-800 IU/kg) inhibited the decrease in platelet count and fibrinogen level equally. The prolonged activated partial thromboplastin time and prothrombin time observed in DIC rats were further elongated in both CTC-111- and heparin-treated rats. But, this prolongation was less in CTC-111-treated rats than in the heparin-treated ones. Heparin inhibited the increase in fibrin and fibrinogen degradation products more prominently than CTC-111. On the other hand, CTC-111 strongly inhibited the increase in PAI-1 activity but heparin did not. These results suggest that CTC-111 may enhance fibrinolysis through its direct inhibitory effect on PAI-1. The parameters for liver or renal damage, i.e., plasma glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine (Cre) and blood urea nitrogen (BUN), were significantly increased by LPS infusion. Both CTC-111 (100,000 U/kg) and heparin (800 IU/kg) decreased the increase in GOT and GPT levels significantly, whereas neither affected the increase in Cre or BUN. From these results, the activation of the blood coagulation system might partially contribute to the progression of liver damage caused by LPS, and might be less involved in the progression of renal damage in this model. In conclusion, CTC-111 showed both anticoagulant and profibrinolytic activity in the LPS-induced DIC model without excessive prolongation of coagulation time. From these results, CTC-111 is expected to be a useful remedy for DIC without the risk of bleeding.

Effect of activated human protein C on experimental venous thrombosis induced by stasis with operative invasion in mice.

Protein C (PC) is the zymogen of an anticoagulant serine protease and is converted to its active form (activated protein C: APC) by thrombin in the presence of thrombomodulin. APC plays an important role in regulating blood coagulation and fibrinolysis by inhibiting not only blood coagulation factors Va and VIIIa but also type-1 plasminogen activator inhibitor (PAI-1). In this study, it was reported that the antithrombotic effect of a human APC product (designated as CTC-111) compared with that of heparin and human PC on the deep venous thrombosis (DVT) model induced in mice by stasis caused by inferior vena cava ligation and operative invasion. Drugs were injected into a tail vein at -2, 30, 60, and 120 min after the inferior vena cava ligation. One-fifth amount of the total dosage of a given drug was injected at each time point. The wet weight of thrombus formed was reduced by APC or heparin administration, however, PC, which was equal to APC in protein amount, did not show any antithrombotic effect. To confirm whether human PC could be activated by mouse thrombin, PC was treated with mouse or human thrombin to measure the amount of APC formed. Mouse thrombin could activate human PC at a similar activation rate as human thrombin. These results suggest that externally administrated PC cannot exhibit antithrombotic effect in this DVT model due to slow activation rate to APC and that APC is a better antithrombic agent than PC for treating thrombotic diseases.

Elevation of tail skin temperature in ovariectomized rats in relation to menopausal hot flushes.

Menopausal hot flushes (HFs), which manifest as an increase in skin temperature, most frequently occur after menopause and cease with the passage of time. We designed this study to elucidate the characteristics of the elevation of tail skin temperature (TST) in ovariectomized (OVX) rats, which is relevant to human symptoms of HFs. First, we measured TST and rectal temperature (RT) and investigated the time course of their changes up to 20 wk after ovariectomy. The TST in OVX rats (28.4 +/- 0.3 degrees C) was significantly (P = 0.0035) elevated from 2 to 7 wk after the ovariectomy compared with that in sham-operated (Sham) rats (27.0 +/- 0.2 degrees C), whereas the RT in OVX rats was elevated from 8 to 20 wk. We next examined the therapeutic effects of estradiol (E(2)) on the elevation of the TST by continuous subcutaneous infusion. E(2) treatment (1.0 microg/day) completely (P = 0.0232) inhibited the elevation of the TST (28.4 +/- 0.3 degrees C for Sham rats, 29.3 +/- 0.3 degrees C for OVX rats, 28.2 +/- 0.4 degrees C for OVX + E(2) 1.0 microg/day rats). These results demonstrated that the elevation of TST in OVX rats was exhibited soon after the estrogen removal and diminished with time and that it was normalized with continuous E(2) replacement. These characteristics are similar to the symptoms of menopausal HFs in women.

Endotoxin and thrombin elevate rodent endothelial cell protein C receptor mRNA levels and increase receptor shedding in vivo.

The endothelial cell protein C receptor (EPCR) facilitates protein C activation by the thrombin-thrombomodulin complex. Protein C activation has been shown to be critical to the host defense against septic shock. In cell culture, tumor necrosis factor-alpha (TNF-alpha) down-regulates EPCR expression, raising the possibility that EPCR might be down-regulated in septic shock. We examined EPCR mRNA and soluble EPCR levels in mice and rats challenged with lethal dose 95 levels of endotoxin. Toxic doses of TNF-alpha failed to alter EPCR mRNA levels in mice. Rather than EPCR mRNA levels falling in response to endotoxin, as predicted from cell-culture experiments, they rose approximately 3-fold 6 hours after exposure to endotoxin before returning toward baseline levels at 24 hours after exposure. Soluble EPCR levels rose approximately 4-fold. Infusion of hirudin, a specific thrombin inhibitor, before endotoxin exposure almost completely blocked the increase in EPCR mRNA and soluble EPCR. Consistent with the idea that the responses were mediated by thrombin, thrombin infusion (5 U/kg of body weight for 3 hours) resulted in an approximately 2-fold increase in EPCR mRNA and soluble EPCR. Incubation of rat endothelial cells with thrombin or murine protease-activated receptor 1 agonist peptide resulted in a 2-fold increase in EPCR mRNA. These results indicate that thrombin plays a major role in up-regulating EPCR mRNA and shedding in vivo. (Blood. 2000;95:1687-1693)

Effects of progesterone on the elevation of tail skin temperature in ovariectomized rats.

We examined the effects of progesterone on the elevation of tail skin temperature (TST) in ovariectomized rats and compared them with those of estradiol. Progesterone showed only insignificant effects on the TST elevation, whereas estradiol showed complete inhibition. The TST elevation induced by ovariectomy is caused by estradiol deficiency, but progesterone plays little or no role.

[Anti-thrombotic effect of activated protein C].

Protein C (PC) is an important anticoagulant protein in blood and converted to its active form, activated protein C (APC), by thrombin bound with thrombomodulin. APC exhibits an anticoagulant effect by the inactivation of FV a and FVIII a. In addition, APC exerts a profibrinolytic effect by inactivation of PAI-1 and inhibition of TAFI activation. APC is strongly anti-thrombotic because of its anticoagulant and profibrinolytic effect. APC has gamma-carboxyglutamic acid residues that bind to acidic phospholipids expressed on activated platelet or injured endothelial cells. Thus APC works only at the site where clots are formed and has a weak effect in primary hemostasis; this means that the use of APC is expected not to have any hemorrhagic risk. In both DIC animal models and clinical studies, we confirmed safer amelioration by APC than heparin. Recently, a specific receptor for PC/APC was found on endothelial cell membrane and anti-inflammatory effects of APC were also reported. Thus APC is thought to play an important regulatory role in blood coagulation, fibrinolysis and inflammation, especially in thrombotic diseases.

Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain after transient focal ischemia in rats.

Chemokines have been shown to play an important role in leukocyte infiltration into ischemic lesions. Recently, the increased expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) was observed in experimental stroke models where infiltrated leukocytes were supposed to induce tissue injury, however, the protein level and time course of these chemokines have not been fully elucidated. Therefore, we analyzed the time-dependent production of MCP-1 and CINC in the rat brain after transient middle cerebral artery occlusion (MCAO) by means of specific enzyme-linked immunosorbent assay systems. The MCP-1 levels in the ipsilateral hemispheres increased from 6 h, peaked at 2 days, and thereafter gradually decreased. The peak MCP-1 concentration was 89.2+/-28.2 ng/g tissue wet weight (mean +/- SEM, n = 5, 49.3-fold greater than the contralateral value at the same time, P < 0.05), which is supposed to be high enough to exert its biological effects. In contrast, the maximum CINC concentration that corresponded to 2.9+/-0.7 ng/g tissue wet weight (mean +/- SEM, n = 5, 55.0-fold greater than the contralateral value at the same time, P < 0.05), was observed at 6 h. In addition, we confirmed the temporal profile of leukocyte subtypes that infiltrated into the ischemic brain, thus, neutrophil infiltration occurred at early stages (1-3 days), followed by massive infiltration of macrophages at later stages (2-7 days). These studies suggest that MCP-1 in cerebral ischemia actually plays a significant role in the migration of macrophages into the lesion and that the differential temporal production of these chemokines contributes to the regulation of infiltrated leukocyte subtypes.

Primary Sjögren's syndrome complicated by sarcoidosis.

Sjögren's syndrome and sarcoidosis share several common features, such as keratoconjunctivitis sicca, swelling of parotid glands, lung involvement, cutaneous anergy, T cell-mediated immunodeficiency, an increased CD4+/CD8+ lymphocyte ratio, and association with the human leucocyte antigen (HLA)-B 8 and DR 3 haplotypes. However, only five patients with primary Sjögren's syndrome and sarcoidosis have been previously reported in the English language literature. The rare case of a 49-year-old Japanese woman with primary Sjögren's syndrome complicated by sarcoidosis is described. The serum angiotensin-converting enzyme level was increased, and histological examination of lung and skin biopsies revealed noncaseating granulomas, indicating that her primary Sjögren's syndrome was complicated by sarcoidosis.

Comparison of hemorrhagic effect of heparin and human activated protein C with use of Thrombostat 4000.

The importance of bleeding as a complication of anticoagulant therapy is clearly recognized. We previously reported that amelioration of hemorrhage associated with disseminated intravascular coagulation by the human activated protein C (APC) was greater than that by heparin. In this study, we compared the bleeding complication of intravenously administered APC and heparin in rabbits, and also estimated primary hemostasis. When both anticoagulants were intravenously infused, the bleeding time from a punctured ear vein was prolonged dose-dependently. However, at doses which prolonged the activated partial thromboplastin time nearly equally, the prolongation of bleeding was greater in heparin-administered rabbits. Blood withdrawn from heparin-administered animals showed increases in in vitro bleeding parameters which correlated with the in vivo bleeding time. However, only small changes were observed in the blood withdrawn from APC-administered animals. Both drugs induced either no change or only a slight decrease in the platelet count, hematocrit and fibrinogen content. These observations suggest that APC may be a more useful anticoagulant than heparin since it causes less bleeding tendency.

Effects of a highly selective synthetic inhibitor of plasma kallikrein on disseminated intravascular coagulation in rats.

We found a new, highly selective plasma kallikrein inhibitor, trans-4-aminomethyl-cyclohexanecarbonylphenylalanine 4-carboxymethylanilide hydrochloride, called PKSI-527 in our laboratories. This study was conducted to evaluate PKSI-527, on thromboplastin (TP)- and endotoxin (LPS)-induced disseminated intravascular coagulation (DIC) in rats. PKSI-527 was infused intravenously at 0.1 mg/kg/min for 250 min. Three of the parameters of the coagulation and fibrinolysis system, fibrinogen level, platelet counts and fibrin(ogen) degradation products (FDP) level were assayed. PKSI-527 prevented the change in the coagulation and fibrinolysis system in LPS-induced DIC, however it was not clearly effective in TP-induced DIC. The parameters of organ failure, such as serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine phosphokinase (CPK), lactate, blood urea nitrogen and beta-glucuronidase, were assayed. Although the changes in the fibrinogen level, platelet counts and FDP level were almost the same in both models, the parameters of organ failure apparently increased in LPS-induced DIC more so than in TP-induced DIC. PKSI-527 significantly suppressed the increases in GOT and GPT in LPS-induced DIC. These results indicate that plasma kallikrein may play a significant role in LPS-induced DIC. Therefore, PKSI-527, as a synthetic plasma kallikrein inhibitor may be a valuable tool to explore the mechanism of DIC and the accompanying organ failure.

Species specificity of anticoagulant activity of activated human protein C: involvement of factor V as well as protein S.

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.