Role of KATP channels in reduced antinociceptive effect of morphine in streptozotocin-induced diabetic mice

The nociceptive effect was measured using withdrawal latency in tail flick test in mice rendered diabetic by administering streptozotocin (200 mg/kg, i.p.). The antinociceptive effect of morphine (4 and 8 mg/kg, s.c.) and cromakalim, a KATP channel opener, (0.3, 1 and 2 micrograms, i.c.v.) was significantly reduced in diabetic mice. Moreover, co-administration of cromakalim(0.3 microgram) did not alter the reduced antinociceptive effect of morphine(4 mg/kg) in diabetic mice. Spleenectomy in diabetic mice restored the decrease in antinociceptive effect of morphine and cromakalim. Multiple dose treatment with insulin to maintain euglycaemia for 3 days in diabetic mice prevented the decrease in antinociceptive effect of morphine and cromakalim. However, hyperglycaemic tyrode's buffer did not alter the pD2 value of morphine in isolated guinea pig ileum suggesting that hyperglycaemia does not interfere with mu receptor mediated responses in vitro. The results suggest that hyperglycaemia induced decrease in antinociceptive effect of morphine and cromakalim may be due to alteration in KATP channels. Some unknown factor from spleen in diabetic mice may be responsible for this alteration in KATP channels in diabetic mice.     

Low-dose aspirin prevents age-related endothelial dysfunction in a mouse model of physiological aging

The age-related impairment of endothelium-dependent vasodilatation contributes to increased cardiovascular risk in the elderly. For primary and secondary prevention, aspirin can reduce the incidence of cardiovascular events in this patient population. The present work evaluated the effect of low-dose aspirin on age-related endothelial dysfunction in C57B/J6 aging mice and investigated its protective antioxidative effect. Age-related endothelial dysfunction was assessed by the response to acetylcholine of phenylephrine-induced precontracted aortic segments isolated from 12-, 36-, 60-, and 84-wk-old mice. The effect of low-dose aspirin was examined in mice presenting a decrease in endothelial-dependent relaxation (EDR). The effects of age and aspirin treatment on structural changes were determined in mouse aortic sections. The effect of aspirin on the oxidative stress markers malondialdehyde and 8-hydroxy-2'-deoxyguanosine (8-OhdG) was also quantified. Compared with that of 12-wk-old mice, the EDR was significantly reduced in 60- and 84-wk-old mice (P < 0.05); 68-wk-old mice treated with aspirin displayed a higher EDR compared with control mice of the same age (83.9 +/- 4 vs. 66.3 +/- 5%; P < 0.05). Aspirin treatment decreased 8-OHdG levels (P < 0.05), but no significant effect on intima/media thickness ratio was observed. The protective effect of aspirin was not observed when treatment was initiated in older mice (96 wk of age). It was found that low-dose aspirin is able to prevent age-related endothelial dysfunction in aging mice. However, the absence of this effect in the older age groups demonstrates that treatment should be initiated early on. The underlying mechanism may involve the protective effect of aspirin against oxidative stress.     

Selenium modifies glutathione peroxidase activity and glutathione concentration in mice exposed to ozone-provoked oxidative stress

The aim of this study was to show the direct effect of selenium on glutathione peroxidase (GSH-Px) activity and GSH/GSSG concentrations in 3- and 6-month-old mice. An ozone-oxygen mixture was used to provoke an oxygen stress. To measure the Se-effect mice were gavaged with sodium selenite. GSH-Px activity and total glutathione concentrations were determined in serum and in the postnuclear fraction of liver and lungs. Additionally glutathione concentrations were determined in whole blood. Both ozone and selenium, administered separately, reduced GSH-Px activity in lungs of 6-month-old animals, while in young mice an opposite effect of Se was observed. Ozone administered jointly with Se did not influence GSH-Px activity in 6-month-old mice, while in young, 3-month-old mice, a stimulatory effect in lungs was observed. There were no significant changes in GSH-Px activity in the liver of 6-month-old mice, but the stimulatory effect occurred in young mice treated with Se and Se & ozone jointly. In young mice, ozone (also ozone with Se) augmented glutathione concentrations. The response to ozone and selenium strictly depended on age and the antagonism between selenium and ozone was observed only in a few cases.     

Link between intestinal CD36 ligand binding and satiety induced by a high protein diet in mice

CD36 is a ubiquitous membrane glycoprotein that binds long-chain fatty acids. The presence of a functional CD36 is required for the induction of satiety by a lipid load and its role as a lipid receptor driving cellular signal has recently been demonstrated. Our project aimed to further explore the role of intestinal CD36 in the regulation of food intake. Duodenal infusions of vehicle or sulfo-N-succinimidyl-oleate (SSO) was performed prior to acute infusions of saline or Intralipid (IL) in mice. Infusion of minute quantities of IL induced a decrease in food intake (FI) compared to saline. Infusion of SSO had the same effect but no additive inhibitory effect was observed in presence of IL. No IL- or SSO-mediated satiety occurred in CD36-null mice. To determine whether the CD36-mediated hypophagic effect of lipids was maintained in animals fed a satietogen diet, mice were subjected to a High-Protein diet (HPD). Concomitantly with the satiety effect, a rise in intestinal CD36 gene expression was observed. No satiety effect occurred in CD36-null mice. HPD-fed WT mice showed a diminished FI compared to control mice, after saline duodenal infusion. But there was no further decrease after lipid infusion. The lipid-induced decrease in FI observed on control mice was accompanied by a rise in jejunal oleylethanolamide (OEA). Its level was higher in HPD-fed mice than in controls after saline infusion and was not changed by lipids. Overall, we demonstrate that lipid binding to intestinal CD36 is sufficient to produce a satiety effect. Moreover, it could participate in the satiety effect induced by HPD. Intestine can modulate FI by several mechanisms including an increase in OEA production and CD36 gene expression. Furthermore, intestine of mice adapted to HPD have a diminished capacity to modulate their food intake in response to dietary lipids.     

The effect of Bordetella pertussis lymphocytosis-promoting factor (LPF) on antibody response in mice: its enhancing and suppressive effects

The effect of lymphocytosis-promoting factor (LPF) on antibody response in mice was estimated under different sets of experimental conditions. Four- and 6-week-old mice were intravenously inoculated with LPF. Three days later these mice were inoculated either intraperitoneally or intravenously with sheep red blood cell (SRBC) or human serum albumin (HSA) as an antigen. The adjuvant effect of LPF was demonstrated on antibody response in 6-week old mice to intraperitoneally inoculated SRBC but not to intravenously-inoculated one. When 4-week-old mice were immunized, hemagglutinin production in response to intraperitoneally inoculated SRBC was not enhanced by LPF. In addition, a rather suppressive effect of LPF at a comparatively high dose was demonstrated on hemagglutinin production in response to intravenously inoculated SRBC. Anti-HSA production was enhanced by inoculation of LPF in any combination of the mouse age and the route of antigen administration. These findings indicate that the adjuvant effect of LPF on antibody response in mice depends upon experimental conditions: the age of mice, the quality of antigen and the route of antigen administration used for immunization.     

Protection by dexamethasone from a lethal infection with Listeria monocytogenes in mice

Abstract The effects of dexamethasone (DEX) on a lethal infection with Listeria monocytogenes were studied in mice. Mice were completely protected against the lethal infection when treated with 3.3 mg per kg of DEX. The effect was observed only when DEX was injected before infection. The control mice died from day 3 to day 5 of infection, whereas DEX-treated mice could eliminate L. monocytogenes cells from the organs by day 11 of infection. High titres of endogenous tumour necrosis factor (TNF), interleukin-6 (IL-6) and gamma interferon (IFN-γ) were induced in the bloodstreams and organs of the drug-free mice. DEX suppressed IL-6 production, but augmented TNF and IFN-γ production within 24 h of infection, whereas production of all three endogenous cytokines was suppressed in the DEX-treated mice on day 3 of infection when the control mice began to die. These results suggest that DEX shows a protective effect on a lethal infection with L. monocytogenes in mice and that regulation of production of endogenous cytokines might be involved in the effect of DEX.     

Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10-12 weeks. Leptin acutely decreased food intake by approximately 30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased alpha2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited alpha2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters alpha2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.     

Body and organ weight, sperm acrosomal status and reproduction after genistein and diethylstilbestrol treatment of CD1 mice in a multigenerational study

The effect of genistein (GEN) and diethylstilbestrol (DES) on body weight, weight of different organs, sperm acrosomal status and in vivo fertility of CD1 mice was tested in a multigenerational study. The adult parental generation of mice and F1 and F2 generations were exposed to selected drugs for all their life. GEN had effect on different body parameters of 30-day-old mice, but not of adult mice in the first generation. Contrary to that, treatment by DES had a strong effect on body weight, other body parameters and on the levels of serum hormones. In the first generation only sterile pairs of mice were observed. Monoclonal antibody against mouse intra-acrosomal sperm protein was used for analysis of the acrosome state and as biomarkers of sperm damage. In the control groups, about 93% of acrosome-reacted sperm was found, acrosome staining decreased to 78-84% (P<0.01). However, the GEN had no effect on fertility of CD1 mice. On the other hand, the fertility of mice exposed to DES was disrupted, especially in the first generation.     

Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice that were also deficient in iNOS. ApoE-/- and iNOS-/- mice were cross-bred to produce apoE-/-/iNOS-/- mice and apoE-/-/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30-50% reduction in apoE-/-/iNOS-/- mice compared with apoE-/-/iNOS+/+ mice. Although the size of the lesions in apoE-/-/iNOS-/- mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45-70% reductions in both male and female apoE-/-/iNOS-/- mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.     

Effect of immune heterozygous spleen cell transfer on resistance to mouse hepatitis virus infection in nude mice

The role of cell mediated immune response to mouse hepatitis virus (MHV) infection in mice was studied by transferring spleen cells from immune heterozygous littermates (nu/+). A suppressive effect on viral growth was seen in infected nude (nu/nu) mice, whereas immune nu/+ serum transfer had no effect. The protective effect of immune nu/+ spleen cells was significantly reduced by treatment with anti-theta serum plus complement but not with anti-Ig serum. In infected nu/nu mice which received transfers of immune nu/+ cells, neutralizing antibody appeared although the titer was not high enough to protect nu/nu mice from fatal infection. Histopathologically, lymphocyte infiltration in hepatic lesions was evident in infected nu/nu mice with nu/+ cell transfer, while it was slight without nu/+ cell transfer.     

Prevention of type I diabetes by low-dose gamma irradiation in NOD mice

Pretreatment with nonlethal, low-dose irradiation has been shown to have a protective effect against oxidative injury in animal tissues. Since oxidative injury of tissues is known to be a major cause of many human diseases, we examined the effect of low-dose irradiation on the progression of type I diabetes in mice. Nonobese diabetic (NOD) mice were treated with gamma irradiation and the progression of the disease was monitored. An elevated level of glucose in urine was first detected at 15 weeks of age in the control NOD mice, whereas the detection was delayed as long as 7 weeks when the mice received a single dose of 0.5 Gy total-body irradiation between 12 and 14 weeks of age. The greatest effect was observed in the mice irradiated at 13 weeks of age. The increase in blood glucose and decrease in blood insulin were effectively suppressed by irradiation at 13 weeks of age. Both suppression of cell death by apoptosis and an increase in superoxide dismutase (SOD) activity were observed in the pancreas 1 week after irradiation. The results indicate that treatment with 0.5 Gy gamma rays suppresses progression of type I diabetes in NOD mice. This is the first report on the preventive effect of low-dose irradiation on disease progression.     

Reduced stress- and cold-induced increase in energy expenditure in interleukin-6-deficient mice

Interleukin-6 (IL-6) deficient (-/-) mice develop mature onset obesity. Pharmacological studies have shown that IL-6 has direct lipolytic effects and when administered centrally increases sympathetic outflow. However, the metabolic functions of endogenous IL-6 are not fully elucidated. We aimed to investigate the effect of IL-6 deficiency with respect to cold exposure and cage-switch stress, that is, situations that normally increase sympathetic outflow. Energy metabolism, core temperature, heart rate, and activity were investigated in young preobese IL-6-/- mice by indirect calorimetry together with telemetry. Baseline measurements and the effect of cage-switch stress were investigated at thermoneutrality (30 degrees C) and at room temperature (20 degrees C). The effect of cold exposure was investigated at 4 degrees C. At 30 degrees C, the basal core temperature was 0.6 +/- 0.24 degrees C lower in IL-6-/- compared with wild-type mice, whereas the oxygen consumption did not differ significantly. The respiratory exchange ratio at 20 degrees C was significantly higher and the calculated fat utilization rate was lower in IL-6-/- mice. In response to cage-switch stress, the increase in oxygen consumption at both 30 and 20 degrees C was lower in IL-6-/- than in wild-type mice. The increase in heart rate was lower in IL-6-/- mice at 30 degrees C. At 4 degrees C, both the oxygen consumption and core temperature were lower in IL-6-/- compared with wild-type mice, suggesting a lower cold-induced thermogenesis in IL-6-/- mice. The present results indicate that endogenous IL-6 is of importance for stress- and cold-induced energy expenditure in mice.     

Population density and growth rate in laboratory mice

Home Office guidelines recommend an area of 60 cm2 per mouse for growing mice up to 30 g. However, the overall growth rate, and final adrenal weight of weanling BALB/c and MF1 strain mice was not affected by being housed at a density of down to 27 cm2 per mouse, though there was some evidence of strain differences in ability to tolerate such dense housing. The presence of cage accessories had no effect on growth rate of BALB/c and female mice, but reduced growth of MF1 and male mice, though the effect was small. It is concluded that present Home Office guidelines make a generous provision for the space requirements of growing laboratory mice, and that the use of cage accessories of varying design may be worth exploring in more detail.     

Host-mediated antiviral activity of Lactobacillus casei against cytomegalovirus infection in mice

The protective effect of heat-killed Lactobacillus casei (LC) against murine cytomegalovirus (MCMV) infection was examined. ICR mice treated once with LC 1 day or 2 days before challenge survived lethal infection, but untreated or Lactobacillus fermentum (LF)-treated mice did not. The protective effect was evidenced by an increase in plaque-forming units (PFU) per 50% lethal dose (LD50) and a decrease in titers of infectious viruses replicated in the target organs. This was further confirmed by severity of histopathological damage to the target organs, especially the liver. LC neither inactivated MCMV nor inhibited its replication in mouse embryonic fibroblasts (MEF). The spleen cells from LC-treated mice inhibited its replication in MEF on co-cultivation. Augmentation by LC of splenic natural killer (NK) cell activity correlated with survival of mice from otherwise lethal MCMV infection. Cytotoxic activity of peritoneal cells and level of serum interferon (IFN) were elevated after MCMV infection, but they were not associated with survival of mice nor with treatment of LC. The protective effect of LC was not clear in NK-deficient beige mutant (bgJ/bgJ) mice, when compared with that in their littermate (bgJ/+) mice. Poor protection of bgJ/bgJ mice by LC treatment correlated with failure to induce NK cell activity by LC treatment in the mutant mice. Thus, it is likely that LC protects mice from MCMV infection by augmentation of NK cell activity.     

The effect of irradiation in air or in hyperbaric oxygen on the Fib/T tumor in WHT mice pretreated with a hypoxic gas mixture

The effect of exposing WHT mice bearing the Fib/T tumor to a low-oxygen environment (8, 10, and 15% oxygen) for 48 h or 72 h before irradiation was compared, using an in vitro colony-forming excision assay, to the effect obtained when mice were pretreated with air. The response of the Fib/T tumor to radiation delivered in air was improved both by a 48-h and by a 72-h exposure of the animals to 8, 10, and 15% oxygen. However, the greatest tumor sensitization was achieved when mice were kept in 8% oxygen for 48 h before irradiation. These results are interpreted and discussed in relation to increases in the 2,3-DPG concentration, which were shown to occur when mice were exposed to a reduced oxygen environment. The relative importance of two models proposed to explain these findings is assessed. If mice pretreated with air were irradiated in hyperbaric oxygen, a similar tumor response was observed compared to that when mice were exposed to 8% oxygen for 48 h and then irradiated in air.     

Effect of metformin on nitric oxide synthase in genetically obese (ob/ob) mice.

Genetically obese (ob/ob) mice were employed for the study of the effect of metformin on activity and expression of nitric oxide synthase (NOS ) in vitro and in vivo. For in vitro analysis, mouse liver extracts were used. For the in vivo study, (ob/ob) and their control litter mates (ob/c) mice were injected with specified amounts of metformin and the expression of NOS in the adipose tissue and hypothalamus was measured by Western blotting. Results show that metformin exhibited a biphasic effect on NOS activity in vitro. Expression of metformin was differentially altered in the hypothalamus and adipose tissues of the normal and ob/ob animals that were treated with metformin. Further, a significant decrease in food intake occurred in the (ob/ob) mice that received metformin. This decrease in food intake was not accompanied by changes in serum glucose. At inhibitory concentrations, hypothalamic NOS expression changes differentially in normal and ob/ob mice. In normal mice, metformin stimulated NOS expression, while in ob/ob mice there was an inhibition. NOS expression increased in brown adipose tissue of metformin treated control mice, while no such increase was observed in ob/ob mice. No effect of metformin was observed in white adipose tissue of control or obese mice. Thus, metformin may produce anorectic effects through modulation of NOS.     

Effects of deoxycoformycin in mice. I. Suppression and enhancement of in vivo antibody responses to thymus-dependent and -independent antigens

The effect of 2'-deoxycoformycin (DCF) on the PFC responses of AKR mice to SE, TNP-Ficoll, and TNP-B. abortus was examined. Subcutaneous injection of DCF 4 days before antigen caused suppression of all three responses by 70 to 78%. In contrast, injection of DCF 1 day after antigen caused enhancement of both the anti-SE and the anti-TNP-Ficoll responses. Although a single high dose of cortisone acetate injected 4 days before antigen caused a similar suppression, the effect of DCF was not mediated via a steroid release, inasmuch as DCF also suppressed the immune response in adrenalectomized mice. The response of BALB/c mice to TNP-Ficoll was also inhibited by DCF pretreatment and enhanced by injection of DCF after antigen. In contrast, in athymic mice DCF caused suppression of the anti-TNP-Ficoll PFC response, whether injected before or after antigen. These results are interpreted as suggesting that DCF causes suppression primarily via an effect on B cells. The enhancement seen in normal but not in athymic mice may possibly be ascribed to an effect on suppressor T cells. Apparently the enhancement of both TD and TI responses caused by DCF injected 1 day after antigen in normal mice is the net result of these two opposing effects. The results imply that helper T cells are resistant to DCF.     

Gender differences in myogenic tone in superoxide dismutase knockout mouse: animal model of oxidative stress

Oxidative stress mediated by prooxidants has been implicated in the pathogenesis of vascular disorders. However, the effect of prooxidants on myogenic regulation of vascular function and the differential influence of gender is not known. SOD, an intracellular enzyme, restricts excess prooxidant levels and may limit vascular dysfunction. We therefore tested the effects of Cu,Zn SOD deficiency on vascular tone in both male and female SOD knockout (SOD-/-) mice. We hypothesized that myogenic tone would be enhanced in SOD-/- mice by excess prooxidants compared with wild-type control mice. Indeed, resistance-sized mesenteric arteries from SOD-/- mice exhibited enhanced myogenic tone compared with control mice. Myogenic tone was lower in female than male control mice. Interestingly, this gender effect was absent in SOD-/- mice, such that myogenic tone of mesenteric arteries from females was equated to that of arteries from males. Furthermore, the pathways that modulate myogenic tone were diverse. In both male and female control mice, inhibition of prostaglandin H synthase (PGHS) and nitric oxide synthase (NOS) pathways enhanced myogenic tone. In female SOD-/- mice, inhibition of PGHS and NOS pathways enhanced myogenic tone to a greater extent compared with control mice. Conversely, in male SOD-/- mice, NOS and PGHS inhibition did not alter tone and only inhibition of gap junctions enhanced myogenic tone. In conclusion, this study revealed enhanced myogenic tone in SOD-/- mice compared with control mice. Furthermore, Cu,Zn SOD deficiency particularly enhanced myogenic tone in female mice such that their vascular tone attained the level of male SOD-/- mice, possibly mediated by prooxidants.     

Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice

A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract. This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile. The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine. When C. difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C. perenne became established even in the presence of high concentrations of copper.