Mice Of Various Ages Research Articles

Immunohistochemical Demonstration of Leptin in Pancreatic Islets of Non-Obese Diabetic and CD-1 mice: Co-localization in Glucagon Cells and its Attenuation at the Onset of Diabetes

Leptin is a 16 kD polypeptide hormone produced predominantly by white adipose tissue and exerts profound effects on food intake and energy balance. More recent studies have shown extra sites of leptin production in human and rodent tissues and have ascribed additional roles for the hormone, e.g., in immune and reproductive functions. A role for the hormone has also been implicated in insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse. However, whether leptin originates from islet cells of the mouse is not known. Here dual-label immunohistochemistry was employed to examine leptin expression in islet cells, and its distribution and cellular sources in pancreatic sections of female NOD/Ak and CD-1 mice of various ages. For comparison, leptin immunolabelling was examined in adult pancreatic sections from male NOD/Ak CD-1, Balb/c and FVB/N mice and female severe combined immunodeficient CB. 17 mice. Pancreatic tissues from adult female guinea pig, sheep and cattle and neonatal pigs were also studied. Our results show that in the day 1 NOD and CD-1 mice, leptin immunolabelling was observed in selective glucagon cells within the developing islets while at days 15 and 22, it became more intense and co-incident. This pattern of staining was maintained at days 40, 90, 150 and 250. In the female NOD mouse, leptin was absent in intra-islet immune cells. Its expression was variable in islets from male NOD and CD-1 mice. In spontaneously diabetic female NOD mice and following acceleration of diabetes with cyclophosphamide, despite the persistence of strong immunolabelling for glucagon in the re-distributed alpha cells, leptin expression was either absent, diminished or present in only a proportion of alpha cells. The reduction in leptin labelling was often associated with diabetic islets which had insulitis in association with only a small number of residual beta cells. Leptin expression was absent in guinea pig, ovine, bovine and neonatal porcine islet cells, despite the expression of intensely labelled glucagon cells. The present results demonstrate leptin co-localization in glucagon cells of the mouse islet. Its expression diminishes in the presence of inadequate insulin. Leptin produced within the mouse islet may have bi-directional influences on leptin and insulin regulation and may play local functions in islet development and metabolism.

Mating attenuates aging-associated increase of lipid peroxidation activity in C57BL/6J mice.

In the frame of the free-radical hypothesis of aging and literature data on increased life span of mated animals, we evaluated brain, kidney, and liver lipid peroxidation in C57Bl/6J mice of various ages and compared lipid peroxidation activity in mated and non-mated mice of both genders. An aging-associated increase (from 3 to 12 months of age) of lipid peroxidation, as measured by malonaldehyde and 4-hydroxyalkenals (MDA + HAE) levels, was observed in the liver and kidney, but not in the brain. Tissue MDA + HAE levels were lower in 12-month old mated mice (housed with animals of opposite gender from three to five months of age) than in 12-month old non-mated animals. There were no gender differences in the observed effect. It is suggested that mating might attenuate the increase of lipid peroxidation associated with aging.

Age-related base excision repair activity in mouse brain and liver nuclear extracts.

To assess DNA repair activity relative to age, in vitro base excision repair assays were performed using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old mice showed a decreased abundance of DNA polymerase-beta, but the addition of purified protein did not restore base excision repair activity. Abundances of other tested base excision repair proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA repair could contribute to increased levels of DNA damage and mutagenesis.

The Role of Aging, High Fat Diet and Blue Light Exposure in an Experimental Mouse Model for Basal Laminar Deposit Formation

We sought to investigate the role of aging as a susceptibility factor for the capacity of dietary fat intake to increase the development of subretinal deposits. Mice of various ages (2, 9 and 16 months) were fed a normal diet or a diet high in saturated and unsaturated fats for a total four and a half months. Some eyes were also exposed to non-phototoxic levels of blue-green light. The outer retina and choroid were examined by light and transmission electron microscopy, and the characteristics, frequency and severity of subRPE deposits was determined. Aged mice fed normal diets developed only very mild subretinal deposits. However, many eyes of mice aged 9 months or older at the time of initiation of diet developed frequent basal laminar deposits of moderate severity, and only 16 month old mice developed more severe deposits after exposure to blue-green light. Some eyes in this older group also developed endothelial invasion into Bruch's membrane. None of the eyes developed classic drusen or linear deposits. These observations demonstrate that age increases the capacity of dietary fat, especially in the presence of environmental light, to induce subRPE deposits.

Apparent mesonephric duct (rete anlage) origin for cysts and proliferative epithelial lesions in the mouse ovary.

Ovaries and adjacent parovarian (mesovarial) tissues of CD-1 mice of various ages were examined to characterize cystic and proliferative epithelial lesions of the ovary and parovarian tissues. Ovaries and adjacent tissues from 6 mice each at approximately 3 and 8 months of age were processed for light microscopy and step-sections (50-micron intervals) of the entire tissue were examined. Tissues were collected from 40 mice each at 16 through 24 months of age and 3 step sections per mouse were examined. Mesonephric duct remnants were found in all mice at 3 and 8 months. Ducts were usually in the mesovarial adipose tissue and near or within the ovarian hilus and were often loosely associated with smooth muscle of the mesovarial ligament. The epithelium of the ducts varied from low cuboidal to columnar, occasional individual cells were ciliated, and small papillary configurations of epithelium were occasionally present. Ducts were dilated (> 1 mm) more often in mice at 8 months compared to 3 months, and some were continuous with cystic spaces within the ovaries. As mice aged (16-24 months), dilation and cystic change in ducts and associated compression of ovarian tissue away from the ovarian hilus became common. The epithelium of dilated ducts was generally flattened, but foci of cuboidal to columnar epithelium and/or occasional ciliated cells were present. Many ducts contained foci of hyperplastic and/or hypertrophic epithelium, and papillary projections of epithelium were occasionally found. Some of the latter lesions were consistent with a diagnosis of papillary cystadenoma. Hyperplasia of associated fibromuscular stroma was limited to a few apparently extraovarian ducts. The results of this study indicate that remnant mesonephric ductular structures are common in and adjacent to the ovaries of CD-1 mice. As mice age, these ducts become dilated and, in some, the epithelium becomes hyperplastic and/or hyperptrophic. These mesonephric duct remnants appear to be a common source of ovarian and parovarian cyts cysts and epithelial neoplasms of the ovary in mice.

Postnatal development of blood pressure and baroreflex in mice

Postnatal development of blood pressure, heart rate and their regulation by arterial baroreceptor reflex in mice was examined. We first confirmed that simultaneous recordings of pulsatile blood pressure by the “servo null” method and the conventional catheter method gave almost identical tracings in halothane-anesthetized adult mice. We then measured blood pressure by servo null method together with electrocardiograph in mice of various ages from newborn to adult. Mean blood pressure increased progressively with age from 19±2 mm Hg in P0 newborn to 74±1 in adult mice, while heart rate initially increased from 365±12 bpm in newborn to 441±15 in infant (7 days old), and then decreased to 337±15 in adult mice. Between 1 and 2 weeks of age, gain of arterial baroreceptor reflex abruptly increased from a newborn value of 0.3 to a near adult value of 1.1 ms/mm Hg. On the other hand, sensitivity to anesthesia did not differ except for P1 and P2 newborns. We conclude that pulsatile blood pressure can be accurately measured by the servo null method even in the newborn mice and that baroreflex heart rate control mature at around 2 weeks after birth in the mice.

Dietary modulation of age-related changes in cerebral pro-oxidant status

It has been proposed that senescence may be associated with changes associated with oxidative damage to macromolecules. Levels of cerebellar nitric oxide synthase (NOS) and rates of generation of cortical reactive oxygen species (ROS), have been determined in mice of various ages. Both of these parameters were significantly reduced in mice aged 9 months relative to 3-month-old mice. In order to determine whether dietary manipulation can modulate these changes, the effect of exposure of mice to differing diets incorporating various antioxidants, was examined. These diets were given to 3-month-old mice for a total period of 6 further months. The presence of melatonin (40 ppm) in the basal diet restored both NOS and ROS levels to the corresponding values found in the younger (3-month-old) group of mice while lipoic acid (1650 ppm) also restored levels of NOS to those found in 3-month-old animals. Addition of coenzyme Q (ubiquinone), 200 ppm or α-tocopherol (1000 ppm) to the basal diet had no effect on either NOS levels or ROS generation. These data suggest that dietary supplementation may aid in delaying onset of metabolic changes characteristic of the older brain. In behavioral testing, older (9-month-old) animals exhibited reduced motor activity and diminished recall ability on the second day of exposure to the test paradigm. While no diet altered motor activity or improved recall of older animals, lipoic acid or tocopherol treatment adversely affected place recall familiarity.

Reduced incidence of thrombosis in mice with hereditary spherocytosis following neonatal treatment with normal hematopoietic cells

Thrombosis is a life-threatening complication of hemolytic anemia in humans. Cardiac thrombi are present in all adult alpha-spectrin-deficient (sph/sph) mice with severe hereditary spherocytosis, providing a model for events preceding thrombosis. The current study evaluated (1) the timing of thrombosis initiation and (2) the effect of postnatal transplantation of normal cells on life span and thrombotic incidence in adult mice. Thrombi are detected histologically following necropsy in untreated sph/sph mice of various ages and are not observed until 6 weeks of age. Thrombotic incidence increases from 50% at 6 to 7 weeks of age to 100% at 9 weeks of age. As a potential therapy, nonablated sph/sph neonates were transfused with either genetically marked normal peripheral blood (PB), bone marrow (BM), or both and assessed for donor cells and thrombosis. A single transfusion of PB, with or without BM, significantly increases the percentage of sph/sph mice that survive to weaning (4 weeks of age). Replacement in all sph/sph recipients is limited to red blood cells (RBCs). RBCs derived from donor PB are lost within 5 weeks. PB plus BM prolongs high-level donor PB cell production better than BM alone. Thrombotic incidence is significantly reduced in all sph/sph mice treated with PB, BM, or both. Hence, the presence of normal blood cells in the peripheral circulation of neonatal and adult sph/sph mice rescues the former and abrogates the development of thrombosis in the latter. (Blood. 2001;97:3972-3975)

Organ-specific distribution of AP-1 in AP-1 luciferase transgenic mice during the maturation process.

Activator protein-1 (AP-1), a dimeric complex consisting of proteins encoded by the jun and fos gene families, is a transcription factor induced by a variety of signals including those eliciting proliferation, differentiation, and neoplastic transformation. Although AP-1 has been widely studied in the last decade, physiological levels of AP-1 in different tissues are unclear. In the present study, we analyzed AP-1 activity in several organs (liver, kidney, brain, lung, spleen, heart, skin) of AP-1-luciferase transgenic mice of various ages. Results of these studies indicate that the level of AP-1 in young mice is much higher than that in older mice, and, second, that the skin contains considerably higher levels of AP-1 than other organs. The level of phosphorylated extracellular signal-regulated protein kinase (ERK) in skin was higher in 1- and 2-day-old mice than in mice of other ages. In addition, phosphorylated p38 kinase was high in 2-day-old and 1-wk-old mice, but phosphorylated c-Jun NH(2)-terminal kinase was not detected at any age. AP-1 activity and level of phosphorylated ERKs declined with maturation. These results imply that AP-1 activity mediated through an ERKs-dependent pathway may be involved in skin development.

Naturally acquired anti-αGal antibodies in a murine allograft model similar to delayed xenograft rejection

Antibodies directed against galactose-alpha1,3-galactose (alphaGal) are believed to play an important role in the pathogenesis of delayed xenograft rejection (DXR). This study was designed to determine whether alpha1,3-galactosyltransferase-deficient (Gal KO) mice can naturally acquire a sufficient anti-alphaGal titre to cause the delayed type rejection of alphaGal-expressing hearts. Gal KO mice of various ages were assessed for anti-alphaGal antibody levels. alphaGal-expressing hearts were transplanted heterotopically into these mice and monitored daily. Rejecting and surviving hearts were evaluated histologically. In Gal KO mice greater than 6-month-old, 64% had an anti-alphaGal antibody titre above the background level. When wild-type alphaGal-expressing hearts were transplanted into this group, 45% of grafts rejected within 5 to 13 days. Histological examination of the rejected hearts displayed marked tissue damage and an inflammatory infiltrate of predominantly macrophage/monocytes. Surviving grafts showed preserved morphology. Like humans, Gal KO mice naturally develop anti-alphaGal antibodies with age. The titre in these mice was sufficient to cause a "delayed-type" rejection of a significant proportion of alphaGal-expressing cardiac grafts. This model thus provides an opportunity to investigate the role of naturally acquired anti-alphaGal antibodies in the pathogenesis of DXR.

Influence of ageing on blastogenesis of mouse thymocytes in response to concanavalin A.

The influence of ageing on the blastogenic response of mouse thymocytes to a mitogen was investigated. Thymocytes from mice of various ages (10-70 days) were incubated with the mitogen, concanavalin A, to induce blastogenesis. The thymocytes were then incubated with [3H]thymidine, and the radioactivity of [3H]thymidine taken up by the cells was measured. The stimulation index (ratio of radioactivity uptake by cells incubated with and without mitogen) was used as a measure of blastogenesis of thymocytes. The stimulation index of thymocytes from 10-day-old mice was 2.9; it increased with age, reaching a peak of 33.7 in 40-day-old mice and then gradually decreasing to 8.2 in 70-day-old mice. The blastogenesis of thymocytes, an indicator of thymus function, was thus found to change considerably with age.

Mitochondrial DNA deletions are associated with ischemia and aging in Balb/c mouse brain

Deletions in the mitochondrial DNA (mtDNA) of Balb/c mouse cerebrums, resembling deletions found in elderly humans or in patients with certain disorders, were detected by PCR. Analysis was carried out on mice of various ages and on mice in which the bilateral common carotid arteries had been incompletely ligated to reconstruct cerebral ischemia. A 3,867 bp mtDNA deletion was present only in old or ischemic mouse groups. Among the non-ischemic groups, it was found in 0 of 12 weaning, 0 of 12 young, and four of eight old mice. Among the ischemic groups, it was found in 12 of 17 young and 11 of 11 old mice. Moreover, the percentage of total mtDNA containing deletions was 22% for the old non-ischemic group, 37% for the young ischemic group, and 69% for the old ischemic group. In addition, PCR analysis detected two other deletions of 3,726 bp and 4,236 bp in 4 of the 11 old ischemic cerebrums. The results indicate that mtDNA deletions are associated with aging, that ischemia increases the incidence of mtDNA deletions, and that mtDNA deletions resulting from ischemia are more likely to occur in old mice than in young mice.

Involvement of Renal γ-Glutamyltranspeptidase in Differences in the Renal Uptake of Mercuric Mercury by Male and Female Mice of Various Strains and Ages

Involvement of renal γ-glutamyltranspeptidase (γ-GTP) in differences in the renal uptake of Hg2+ by male and female mice of various ages was examined using five strains of mice, namely, BALB/cA, C57BL/6N, CBA/JN, C3H/HeN and ICR. We observed strain-related and gender-related differences in the renal accumulation of Hg<2+> 30min after the administration of mercuric chloride (1μmol/kg, s.c.). Renal γ-GTP activity also varied among the tested strains, and the activity in males was about twice that in females. A significant correlation was recognized between renal γ-GTP activity and the renal accumulation of Hg<2+>. Both renal uptake of Hg<2+> and renal γ-GTP activity increased gradually with age in male ICR mice from 2 to 8 weeks after birth but remained relatively constant in ICR females. Significant gender-related differences in both renal accumulation of Hg<2+> and γ-GTP activity were observed 4 weeks after birth and thereafter. Castration of male ICR mice decreased both renal accumulation of Hg<2+> and γ-GTP activity to the levels in females. Injection of testosterone increased both renal accumulation of Hg<2+> and γ-GTP activity in castrated male mice and in normal female mice to the levels in control male mice. These results suggest that strain-related, gender-related and age-related differences in the renal accumulation of Hg<2+> in mice might be due to differences in renal γ-GTP activity and, furthermore, that renal γ-GTP activity might be controlled, at least to some extent, by testosterone.

Modulation of stress-induced murine lymphoid tissue involution by age, sex and strain: role of bone marrow

C57BL/6 and Balb C male and female mice of various ages were stressed by immobilization for 1 h/day (1, 2, 3, 5, 8, 11 or 14 consecutive days). The animals were then killed for determination of total body weight and the weights of the thymus, spleen and axillary lymph nodes. In addition, the total number of cells in the thymus and the proportion of lymphoid cells in the bone marrow cell population was defined. The effects of stress were modulated by age, sex and strain. Stress-induced involution of the thymus was generally more pronounced in older animals, while for the spleen was the opposite. Involution of the thymus was higher in males than in females, but there were no marked differences between the sexes in the response of the spleen. In general C57BL/6 mice were more sensitive to stress than Balb C mice. However, for the involution induced by stress on lymph nodes there were not a clear trend with age, sex or strain. In male and female mice of all ages and both strains, stress led to statistically significant reductions in the absolute number of cells inside the thymus and spleen and in the proportion of lymphoid cells in the bone marrow.

Crown morphology, enamel distribution, and enamel structure in mouse molars.

Biomolecular research and genetic manipulations have stressed the importance of thorough knowledge of normal organ morphology. Mouse molar teeth are convenient models for studying basic interactions in organ development and morphogenesis. The aim of the present study was to provide basic information on their morphology. Intact and sectioned/ground molars of mice of various ages were observed with SEM. Enamel-free areas (EFA) were present on cusp tips at time of eruption. The dominating structural configuration in enamel was prism decussation in inner enamel and parallel prisms in outer enamel. Prism decussation tended to be absent at cusp ridges and in the bottom of grooves. In the former location, the distinction between prisms and interprism was often obscured in the middle enamel zone due to decreased difference in orientation of their crystals. A thin layer of enamel, often aprismatic, covered the distal aspect of cusps in maxillary molars and the mesial aspect of cusps in mandibular molars. The enamel abutting on EFA was often aprismatic. Aprismatic enamel exhibited incremental lines with a periodicity of about 1 microm and was often traversed by cracks. The enamel surface was porous in the bottom of grooves. Parts of mouse molar enamel were incompletely mineralized at the time of eruption. SEM is a convenient method for combined studies of crown morphology and enamel structure. Based on morphological criteria, a modification of the cusp nomenclature is proposed. Enamel thickness and structure in mouse molars show regional variations. Fundamental similarities exist between mouse molar cusps and mouse incisors. Mouse molar enamel undergoes posteruptive maturation.

Thymic stromal-cell abnormalities and dysregulated T-cell development in IL-2-deficient mice.

The role that interleukin-2 (IL-2) plays in T-cell development is not known. To address this issue, we have investigated the nature of the abnormal thymic development and autoimmune disorders that occurs in IL-2-deficient (IL-2–/–) mice. After 4 to 5 weeks of birth, IL-2–/– mice progressively develop a thymic disorder resulting in the disruption of thymocyte maturation. This disorder is characterized by a dramatic reduction in cellularity, the selective loss of immature CD4-8- (double negative; DN) and CD4+8+ (double positive; DP) thymocytes and defects in the thymic stromal-cell compartment. Immunohistochemical staining of sections of thymuses from specific pathogen-free and germ-free IL-2–/– mice of various ages showed a progressive ,loss of cortical epithelial cells, MHC class II-expressing cells, monocytes, and macrophages. Reduced numbers of macrophages were apparent as early as week after birth. Since IL-2–/– thymocyte progenitor populations could mature normally on transfer into a normal thymus, the thymic defect in IL-2–/– mice appears to be due to abnormalities among thymic stromal cells. These results underscore the role of IL-2 in maintaining functional microenvironments that are necessary to support thymocyte growth, development, and selection.

Degenerative Hairlets on the Vestibular Sensory Cells in Mutant Bustling (BUS/Idr) Mice

The bustling mouse (BUS/Idr: bus) is a mutant mouse strain which exhibits deafness, bustling/hyperkinetic behaviour and functional disorders seemingly related to the vestibular system. This phenotype develops in homozygous (bus/bus) mice and has been shown from cross experiments to be genetically induced by a single autosomal recessive gene. We previously detected, with light and electron microscopy, post-natal degeneration of the inner ear sensory cells in homozygotes. In the present study, we examined, by electron microscopy, the development of pathological changes in the sensory epithelia of the macula acustica and crista ampullaris of homozygous mice of various ages, paying special attention to the detailed morphology of the sensory hairlets. The homozygous mice exhibited specific pathological changes: a decrease in the number of hairs; disarrangement of the kinocilium-stereocilia pattern; and, fused and/or very large stereocilia. Homozygotes also frequently exhibited apical cytoplasmic herniation, or bleb of hair cells, as well as a degenerated kinocilium in the sensory epithelium. Heterozygotes showed similar changes, but to a lesser degree and frequency. As for the vestibular organs, similar pathological changes had developed at day 17 of gestation. These pathological findings and onset suggest that the BUS mouse may be a mutant mouse strain distinct from other reported strains which display similar behaviour, and may be a useful animal model for the study of human degenerative vestibular disorders.

Polysaccharide of Astragali radix enhances IgM antibody production in aged mice.

The effect of Astragali Radix (AR) on IgM antibody production in mice of various ages (10 weeks, 36 weeks and 60 weeks) was examined. The antibody production levels in the 36- and 60-week-old mice were significantly decreased to about 70 and 60% of that in the 10-week-old mice. The enhancement effect of a crude polysaccharide AR fraction on the antibody production was nil in the 10-week-old mice, but significant enhancement effects were observed in the 36- and 60-week-old mice, compared to the age-matched control. Two polysaccharides active in the enhancement of the IgM antibody production in the aged mice were isolated from the high molecular weight fraction of AR by cetavlon precipitation, ion-exchange and gel permeation chromatography. The molecular masses of these polysaccharides were calculated by HPLC in salt solution. Only one major peak was observed for each, and their molecular masses were estimated to be 1.2 x 10(4) and 2.2 x 10(4). The major components of these polysaccharides were neutral carbohydrates (89.3 and 95.5%), followed by uronic acid and protein; glucose was the predominant sugar component.

The relationship of aging to endotoxin shock and to production of TNF-alpha.

Aged people are considered prone to gram-negative bacteremia and septic shock. This relationship was tested in murine endotoxin shock. Balb/c mice of various ages (1.4-13.4 months) were intraperitoneally injected with lipopolysaccharide (LPS), and rates of survival were observed. The production of TNF-alpha in vivo induced by LPS was measured. The survival rates were the smallest in the oldest and youngest groups. Production of TNF-alpha attained a maximum at 2 h after LPS injection and was smaller in the oldest group; it had a reciprocal relationship to survival rates in each group except the youngest group. Old and young mice had smaller rates of survival and greater production of TNF-alpha following endotoxin shock induced by LPS.

β-Cell Destruction May Be a Late Consequence of the Autoimmune Process in Nonobese Diabetic Mice

The NOD mouse is an animal model of IDDM that shows many of the characteristics of human IDDM. It has been proposed that beta-cell destruction in IDDM progresses over time in a linear manner. Recently, we and others have demonstrated that T helper type 1 (Th1) cells have pathogenic roles in the NOD model and proposed that cytokine balances change as the disease progresses. However, it has not been demonstrated how or when the cytokine balances change or how the beta-cell destruction progresses. We have recently demonstrated that the cytokine profiles of CD45RB(low) CD4+ cells correlate either with their pathogenic or with their protective roles in the NOD mouse. To further analyze this apparent correlation between the shift in cytokine level and IDDM, we examined the anti-CD3-induced cytokine profiles of this subset from NOD mice of various ages compared with that from age-matched I-Ak transgenic NOD and BALB/c mice as controls. A significantly higher ratio of anti-CD3-induced interferon-gamma/interleukin-4 was found in diabetic NOD mice (P < 0.0001) but not in age-matched nondiabetic NOD mice. This cytokine ratio did not change significantly until the onset of diabetes in NOD mice. Based upon these results, we propose that IDDM in the NOD mouse progresses as a predominant inflammatory beta-cell dysfunction without actual beta-cell destruction until late in the disease process. This supports the possibility that late-stage immunotherapy may preserve islet beta-cell mass.