Development of nephrotic ICGN mice--the origin, reproductive ability, and incidence of glomerulonephritis

ICGN is a strain of spontaneous nephrotic mice with nonproliferative glomerular lesions. It was derived from an outbred Yok: ICR colony in our laboratory. The renal disease constantly occurred in animals of the first to the tenth generations (greater than 13.0%; 70 days of age). When affected males were mated with unaffected females, the incidence of the disease in their offspring was 38.8% (n = 49) at 70 days after birth. When both parents were affected, their offspring were all affected (n = 12). The disease evenly progressed in both sexes. It usually began 40 to 150 days after birth and death occurred within two months after onset. The animals usually showed sufficient reproductive ability as long as unaffected females were used for mating.     

Chemopreventive effects of hydroxymatairesinol on uterine carcinogenesis in Donryu rats

Hydroxymatairesinol (HMR), obtained from the heartwood of spruce (Picea abies), has been demonstrated to exert chemo-preventive effects on the development of mammary tumors in rats. To examine the influence of HMR on uterine carcinogenesis, adult Donryu rats were initiated with a single intrauterine treatment of N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 11 weeks of age and fed thereafter 0, 200, or 600 ppm HMR mixed in the soy-containing diet until 15 months of age. Incidences of uterine adenocarcinoma in both 200 and 600 ppm HMR-dosed groups were significantly reduced to 11% and 15%, respectively, less than 50% of 0 ppm, at the end of the experiment (P < 0.05). A delay in the start of persistent estrus by HMR was observed at 8 months of age compared with controls given carcinogen alone. From urinalysis, HMR was metabolized mainly to enterolactone and hydroxyenterolactone. These findings suggest that HMR or its metabolites exert chemo-preventive effects in the rat ENNG-uterine carcinogenesis model.     

Reduced food consumption increases water intake and modulates renal aquaporin-1 and -2 expression in autoimmune prone mice

Aquaporin-1(AQP1) and AQP2 are members of the aquaporin family of cell membrane water channel transport proteins and have been implicated in the regulation of renal water excretion. We have previously shown that calorie restriction (CR) relative to ad libitum (AL) feeding extends lifespan and delays the onset of autoimmune kidney disease in lupus-prone (NZBxNZW)F1 (B/W) mice. To determine if AQP1 and/or AQP2 expression is influenced by CR, mice were fed an AL or CR (40% less food) diet until 4 (young) or 9 (old) months of age when mice were sacrificed. Kidneys were removed and the expression of AQP1 and AQP2 was determined at the protein and mRNA levels using western blotting and RT-PCR respectively. While age did not significantly increase AQP1 expression in the AL groups, CR did increase both the protein (1.4-fold) and mRNA (2.4-fold) levels. In old mice, AQP1 expression was higher (1.8-fold) in CR compared to the AL group while CR had no effect in young mice. In contrast, AQP2 showed an age related decrease (55%) in the AL groups and an increase in the protein (8.4-fold) and mRNA (1.7-fold) levels in the CR groups. Relative to AL, CR decreased AQP2 expression at the protein (90%) and mRNA (50%) levels in the young mice while an increase at the protein (2.9-fold) and mRNA (1.9-fold) levels was evident in the old mice. Interestingly, a significant increase in water intake per gram body weight was found in both young and old CR fed mice when compared to their AL counterparts which may contribute to the prevention of autoimmune disease with age and differences in longevity. These data show, for the first time, significant age and diet influences in renal AQP1 and AQP2 expression at both protein and mRNA levels in lupus-prone mice.     

Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis

Background

Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse).

Results

Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups.

Conclusion

Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA.     

Determination of muscimol and ibotenic acid in Amanita mushrooms by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry

A reliable analytical method was developed for the quantification and identification of muscimol (MUS) and ibotenic acid (IBO), the toxic constituents of Amanita muscaria and Amanita pantherina. MUS and IBO were extracted from mushrooms by aqueous methanol and derivatized with dansyl chloride (DNS-Cl). After extraction with ethyl acetate and evaporation of the solvent, the residue was ethylated with 1.25 M hydrogen chloride in ethanol. The resulting derivatives were quantified by high-performance liquid chromatography with UV detection and identified by liquid chromatography electrospray ionization tandem mass spectrometry. Calibration curves were linear in the range of 25-2500 ppm for MUS and 40-2500 ppm for IBO, respectively. This method was successfully applied to identify and quantify MUS and IBO in Amanita mushrooms naturally grown and circulated in the drug market.     

Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis)

In previous studies we observed that human umbilical cord blood (HUCB) could have a protective effect on the onset of disease and time of death in MRL Lpr/Lpr mice which have an autoimmune disease that may be considered similar to human lupus. We believed a temporary xenograph may have occurred in these animals with the disease process delayed and the life span markedly increased. When HUCB is stored at 4 degrees C in gas permeable bags, there is a decrease of the cell reaction in mixed lymphocyte cultures. The blood, however, maintains a significant number of cells capable of producing replatable colonies. This study attempted to determine the effect of HUCB on SOD1 mice (transgenic B6SJL-TgN(SOD1-G93A)1GUR), which have a mutation of the human transgene, (CuZn superoxide dismutase gene SOD1) that has been associated with amyotrophic lateral sclerosis. We previously developed evidence that the survival of lethally irradiated mice was related to the number of human mononuclear cells administered. In the present study, we decided to investigate the effect of a relatively large dose of human mononuclear cord blood cells on SOD1 mice subjected to a sublethal dose of irradiation preceded by antikiller sera (rabbit anti-asialo). The SOD1 mice show evidence of paralysis at 4 to 5 months. The average expected lifetime of these mice is reported to be 130 days (Jackson Laboratory). In this experiment, there were 23 mice. Two mice died before the onset of paralysis. The remainder were divided into three groups: group I: control group of 4 untreated mice; group II: an experimental group of 6 mice treated with antikiller sera, 800 cGy irradiation plus 5 x 10(6) congenic bone marrow mononuclear cells; group III: another experimental group of 11 mice treated with antikiller sera, 800 cGy irradiation plus 34.2-35.6 x 10(6) HUCB mononuclear cells, previously stored for 17-20 days at 4 degrees C in gas permeable bags. The results were as follows: the average age at death was: (I) 127 days for the untreated control group, (II) 138 days for the group that received 800 cGy of irradiation and congenic bone marrow (BM) and (III) 148 days for the group that received irradiation and HUCB. (P < 0.001 HUCB vs control, p < 0.01 HUCB vs BM). The longest surviving mouse in each group was 131, 153, and 182 days old respectively. In summary, large doses of HUCB mononuclear cells produced considerable delay in the onset of symptoms and death of SOD1 mice. These preliminary results may not only indicate that amyotrophic lateral sclerosis is an autoimmune disease, but may also indicate a possible treatment for a devastating disease and possibly others.     

Effect of feeding polychlorinated biphenyl (Aroclor 1260) on hepatic enzymes of rats

Significant increase in the activity of liver succinate dehydrogenase (SDH) was observed in male Wistar rats, fed Aroclor 1260 (PCB; polychlorinated biphenyl) at 50 and 100 ppm level for 120 days. Lactate dehydrogenase (LDH) activity increased in 50 ppm PCB fed animals and decreased in 100 ppm PCB fed rats. On the other hand, enzymes like alanine and aspartate aminotransferase, alkaline and acid phosphatase showed remarkable decrease in activity in PCB fed animals. Slab gel electrophoresis of LDH isozymes showed remarkable increase in LDH2 and LDH3 and to some extent increase in LDH1 isozymes of livers of 50 ppm PCB fed animals but not in 100 ppm PCB fed groups as compared to controls. In both the PCB fed groups, liver showed centrilobular hypertrophy, hepatocellular damage, hyperplasia, karyolysis and karyorrhexis.     

Effects of dietary iron overload on glutathione peroxidase knockout mice

Excess iron (Fe) intake has been associated with an increased risk of cardiovascular disease in humans, presumably the result of increased oxidative stress. Previous work by us has shown that feeding a high-Fe diet to selenium (Se)-deficient weanling mice for 4 wk resulted in elevated plasma cholesterol and triglycerides and increased hepatic thiobarbituric acid reactive substances (TBARS). Here, we report the effect of Fe overload in mice lacking cellular glutathione peroxidase (GPX1 knockout [KO] mice), the selenoenzyme thought to account for much of the antioxidant action of Se. Four groups of 9–13 weanling wild-type (WT) or GPX1 KO mice were randomly assigned, then fed either an Fe-adequate (35 ppm Fe) or high-Fe (1100 ppm Fe) casein-based diet for 4 wk. Iron was added as ferric citrate. Both diets also contained 0.2 ppm Se added as sodium selenite. As expected, liver GPX1 activity was essentially absent in the KO mice. Another Se parameter measured (hepatic thioredoxin reductase activity) did not vary across groups. Although liver Fe was elevated in mice fed the high-Fe diet, liver TBARS was largely unaffected either by mouse genotype or diet fed. Moreover, plasma lipids were not elevated in the Fe-overloaded GPX1 KO mice. Thus, decreased GPX1 activity cannot account for the pro-oxidant hyperlipidemic effects observed earlier in mice fed the high-Fe Se-deficient diet. This suggests that impairment of Se functions other than GPX1 activity may be responsible for the elevated plasma lipids and hepatic TBARS seen in the Fe-overloaded Se-deficient mice.     

Effects of feeding endophyte-infected tall fescue seed on the reproductive performance in male and female CD-1 mice by combination crosses

The present study was undertaken to assess the effects of endophyte-infected Acremonium coenophialum tall fescue (KY 31) seed (80% infected) on the reproductive performance of male and female CD-1 mice by combination crosses. Forty-eight male and 48 female 30-d-old mice were randomly allocated to two diet treatments. Twenty-four males and 24 females were fed Diet 1, consisting of 50% non-infected fescue and 50% mouse chow; the remaining animals were fed Diet 2, containing 50% infected fescue. At the end of 50 d, the animals were paired in groups (n = 12) as follows: 1) Diet 1 males and females; 2) Diet 2 males and Diet 1 females; 3) Diet 1 males and Diet 2 females; and 4) Diet 2 males and females. The pairs were maintained on the diets of their female partners and were allowed to cohabitate for 96 h. After this period the males were removed and the females were carried to term. Pregnancy rates among the four treatment groups were 100, 83.3, 75.0 and 54.5%, respectively. Similarly, the average number of pups born per litter among the four groups were 11.5, 8.0, 9.7, and 7.5, respectively. Also, the weights (grams) of pups born per litter were 17.5, 12.6, 12.4, and 9.8, respectively. The results point out that the reproductive capacity of both male and female mice are affected (P < 0.05) by the ingestion of endophyte-infected fescue seeds. Such negative effects were more severe in the female than the male (P < 0.05).     

The relationship between plasma fibronectin levels and autoimmune disease activity in MRL/l mice

Plasma fibronectin levels increased significantly over time in MRL/l mice with progressive autoimmune disease. At 100 and 120 days of age both male and female MRL/l mice exhibited significantly higher fibronectin (Fn) levels than the more resistant MRL/l controls. Male mice at early time points had Fn levels no greater than controls due perhaps to the later onset of disease in MRL/l males. In contrast, female MRL/l mice, when compared with MRL/n controls, had higher Fn levels from 40 days of age. The proteinuria in these animals was also above MRL/n controls from the first time point taken (Day 40). In a temporal study with female MRL/l mice, Fn levels peaked at age 120 days and reflected the pattern of the survival curve, indicating that plasma Fn levels have an association with disease activity.     

Seasonal changes in age class structure and reproductive status of house mice on Marion Island (sub-Antarctic)

Feral house mice on sub-Antarctic Marion Island become reproductively active (males scrotal, females with perforate vaginas or pregnant) at an age >60 days and breed until death, which may occur at more than 13 months. Breeding is strongly seasonal; pregnant or lactating females were found only from October to May. A substantial proportion of mice old enough to breed in one summer overwinters to form a significant component of the breeding population the following summer but it is unlikely that any survive a second winter. The onset of breeding is closely synchronized with increasing day length but occurs about 2 months before mean temperature at the ground surface starts to increase significantly. Cessation of breeding is more closely associated with declining temperatures in late summer. For both males and females, the best correlation between reproductive activity and any of the temperature parameters measured was with average maximum temperature 1 cm above the ground. Competition for macroinvertebrate prey increases sharply in early winter due to high mouse numbers. The breeding season in 1991/1992 and 1992/1993 was at least 2 months longer than in 1979/1980, because the mice started breeding earlier, and stopped breeding later, in 1991/1993. The later cessation of breeding in 1991/1993 was despite the fact that there was a greater competition for macroinvertebrate prey, and that mean air temperatures during the early winter months were lower, than in 1979/1980.     

Aggression toward groups of male and female OF1 mice by a male congenere and survival to acute hypoxia]

In OF1 mice, a 107 day-old mouse, isolated since 51 days, acts as an "aggressor" when introduced into a group of 10 males or into a group of 10 females of the age of 51 days. This results in fights and flights in the males' group and in copulations in the females' group. In 31 experiments, each performed on a total of 40 males and 40 females, it was observed that an acute DL50 hypoxia (inhalation of nitrogen) provokes a mortality significantly (p less than 0.001) higher in the male "aggressors" (95.16%) than in the groups of 10 aggressed mice (mortality: 64.26% for the males' groups and 52.65% for the females groups). Besides, compared to control groups without male "aggressor", the introduction of the male congener significantly (p less than 0.001) increases the acute hypoxic mortality in the females' but not in the males' groups.     

Age-dependent decrease in the lethal threshold of pentylenetetrazole in mice

We previously reported that the minimal effective concentrations in plasma and brain for inducing the maximal seizure after intraperitoneal injection of pentylenetetrazole (PTZ) significantly increased with age in mice. We also observed that some old mice died without the maximal seizure during the observation period of 15 min. To examine how the lethal dose changes with age in the mouse, in the present study we employed a continuous intravenous infusion of PTZ that enabled us to control the dose, either stopping the infusion after the maximal seizure or continuing it until the lethal dose was reached. This study has demonstrated that the lethal threshold of PTZ in the oldest mice (27 months for males and 30 months for females) was significantly lower than that for the respective younger groups. We conclude that our observation in old mice of death occurring before the appearance of maximal seizure is due to a decrease in the lethal threshold combined with an increase in the threshold for maximal seizure.     

Suppression of antibody response by excess dietary zinc exposure during certain stages of ontogeny

A study was made of the effects of excess dietary zinc on the antibody response to sheep red blood cells (SRBC) in mice. C57BL/6J mice were divided into 10 different dietary groups and exposed to diets containing zinc in normal (50 ppm) or excess (2000 ppm) concentrations during gestation/lactation/postweaning development in the sequences (1) 50/50/50; (2) 50/50/2000; (3) 2000/50/50; (4) 2000/2000/50; (5) 2000/50/2000; (6) 50/2000/50; (7) 50/2000/2000; (8) 2000/2000/2000; (9) 50/50/50 (pair-fed); and (10) chow/chow/chow. Mice in group 8 had severe signs of copper deficiency at 8 weeks of age, such as reduced plasma copper, lowered plasma hematocrit, and achromotrichia. Mice receiving 2000 ppm zinc during gestation had fewer offspring per litter (measured at 2 weeks of age) and more nonviable births than mice given 50 ppm zinc during gestation. The growth curve of mice exposed to excess zinc in the 50/50/2000 group was identical to that of the control (50/50/50) group. Growth curves for all other groups were reduced by varying amounts. The plaque-forming cell response to SRBC was reduced only in the groups receiving 50/2000/2000 and 2000/2000/2000 ppm zinc (P less than 0.05); this reduced response was not associated with atrophy of the lymphoid organs. Splenic cell surface markers and mitogenic responsiveness were similar in the 50/50/50 and 2000/2000/2000 groups. These results suggest that the immune response is more susceptible to dietary manipulation during development than after the immune response has been developed.     

Onset of puberty in CD-1 mouse pups exposed prenatally through weaning to endophyte-infected tall fescue seed

This study was undertaken to investigate the effects of feeding endophyte - infected (Acremonium coenophialum ) tall fescue seed to CD-1 mouse dams (P(1)) during gestation and lactation, and on the subsequent growth and sexual maturity (onset of puberty) of their male and female offspring (F(1)). Forty-eight 21 d old pups (24 male and 24 female F(1) mice) were weaned from dams fed one of two diets containing 50% rodent chow (w/w) and 50% KY-31 tall fescue (Festuca arundinacea ) seed. The seed in Diet 1 was noninfected, while the seed in Diet 2 was 80% endophyte-infected. At weaning (21 d), the F(1) pups were fed rodent chow, ad libitum throughout the remaining experimental period. At 24 d, they were paired with sexually mature non-treated virgin CD-1 mice (fed 100% rodent chow) for one parturition cycle. Male F(1) mice were sacrificed at 84 d to determine testicular development. The age at the birth of the first litter for Diet 2 F(1) male (76.8 +/- 2.2 d) and female (58.4 +/- 2.1 d) was significantly greater (P<0.05) than the age at parturition for Diet 1 male and female F(1) test mice (64.1 +/- 1.8 and 51.9 +/- 1.2 d, respectively). At parturition, the female F(1) mice showed no significant differences (P>0.05) in either mean parturition weight or number of F(2) pups born per litter. However, total F(2) litter wight (11.38 +/- 1.14 g) and mean weight per F(2) pup (1.40 +/- 0.04 g) for Diet 2 female F(1) mice litters were lower (P<0.05) when compared with Diet 1 females (14.53 +/- 0.57 g and 1.66 +/- 0.02 g, respectively). No significant differences were observed between the two male F(1) treatment groups, for total F(2) litter weight or the number of pups born per F(2) litter. Although Diet 2 F(1) males weighed significantly less (P<0.05) at weaning and at pairing, final body weights at sacrifice (84 d) were not different (P>0.05) from the Diet 1 males.     

Sex- and age-related differences in arterial pressure and albuminuria in mice

Background

Animal models have become valuable experimental tools for understanding the pathophysiology and therapeutic interventions in cardiovascular disease. Yet to date, few studies document the age- and sex-related differences in arterial pressure, circadian rhythm, and renal function in normotensive mice under basal conditions, across the life span. We hypothesized that mice display similar sex- and age-related differences in arterial pressure and renal function to humans.

Methods

Mean arterial pressure (MAP) and circadian rhythm of arterial pressure were measured over 3 days via radiotelemetry, in 3- and 5-month-old (adult) and 14- and 18-month-old (aged) FVB/N and in 5-month-old (adult) C57BL/6 male and female normotensive mice. In FVB/N mice, albuminuria from 24-h urine samples as well as body, heart, and kidney weights were measured at each age.

Results

Twenty-four-hour MAP was greater in males than females at 3, 5, and 14 months of age. A similar sex difference in arterial pressure was observed in C57BL/6 mice at 5 months of age. In FVB/N mice, 24-h MAP increased with age, with females displaying a greater increase between 3 and 18 months of age than males, such that MAP was no longer different between the sexes at 18 months of age. A circadian pattern was observed in arterial pressure, heart rate, and locomotor activity, with values for each greater during the active (night/dark) than the inactive (day/light) period. The night-day dip in MAP was greater in males and increased with age in both sexes. Albuminuria was greater in males than females, increased with age in both sexes, and rose to a greater level in males than females at 18 months of age.

Conclusions

Arterial pressure and albuminuria increase in an age- and sex-specific manner in mice, similar to patterns observed in humans. Thus, mice represent a useful model for studying age and sex differences in the regulation of arterial pressure and renal disease. Understanding the mechanisms that underlie the pathophysiology of cardiovascular disease may lead to new and better-tailored therapies for men and women.

     

A quantitative study of serum testosterone,sex accessory organ growth,and the development of intermale aggression in the mouse

Radioimmunoassay of serum testosterone (T) was used to characterize circulating T levels in mice from birth to sexual maturity. Until 25 days of age, serum T levels ranged from 1 to 4 ng/ml. A significant increase in T concentrations was observed in 30-day-old males, followed by a secondary rise in serum T between Days 45 and 50 of life. The latter increment was associated with the appearance of extreme individual variation in circulating T levels which was also observed in adult (120 days) males. The most rapid growth of accessory sex organs occurred between 30 and 50 days of age, the period preceding attainment of peak serum-T levels. The first incidence of intermale aggression coincided with a prepubertal rise in circulating T, but adult levels of fighting were present prior to the secondary increase in T observed between 45 and 50 days of age. Although animals involved in a fight did not differ with respect to weight of the accessory sex organs or serum T concentrations, the male that weighed more than his opponent usually won an encounter. Compared to males in encounters in which no fighting occurred, animals that won or lost an aggressive encounter showed significantly greater accessory sex organ development. While circulating T is required for the initiation and maintenance of intermale aggression, it is apparent that additional factors are related to the onset of fighting and the establishment of dominance/ subordinance relationships in mice.     

Effects of accelerating growth on the onset of puberty in male mice

Sexual maturation was evaluated in male mice subjected to prenatal and preweaning overnutrition induced by reduction of litter size in embryonic and post-natal life. From birth to adulthood body weight was higher in overfed males than in controls. Plasma and testicular testosterone levels followed a similar pattern in normally fed and overfed males. Adult gonadotrophin levels were attained at 30 (FSH) and 40 (LH) days in controls and as soon as 20 days in overfed males. First fertile matings occurred at the same age in both groups. Overnourished males attained puberty when weighing considerably more than controls (34.7 +/- 0.4 vs. 29.4 +/- 0.3 g). The 30-day-old overfed males, which have attained the critical pubertal body weight of controls, were sexually immature. The data showed that in male mice puberty does not seem to be triggered by the attainment of a critical body weight.