Age-matched Animals Research Articles

Hepatic and renal cadmium accumulation is associated with mass-specific daily metabolic rate in the bank vole ( Clethrionomys glareolus)

Previous study has shown that photoperiod and age affect tissue accumulation of cadmium (Cd) in a small rodent, the bank vole. Since the body mass is also influenced by these factors, the present study was designed to determine whether mass-specific daily metabolic rate might be responsible for differential accumulation of Cd in the liver and kidneys of the short- and long-photoperiod bank voles as well as of the young and old animals. One- and five-month old male bank voles were held under short (8 h light / 16 h dark) or long (16 h light / 8 h dark) photoperiods and exposed to dietary Cd (100 μg/g) for 6 weeks. The bank voles raised under the short photoperiod and those injected subcutaneously with melatonin (7 μmol/kg/day) under the long photoperiod showed significantly higher concentrations of Cd in the liver (43–60%) and kidneys (40–47%) than the age-matched long-photoperiod animals. The old bank voles accumulated significantly less Cd in both organs than the young animals. These differences in Cd accumulation appeared not to be associated with the relative Cd intake. However, the hepatic and renal Cd levels followed a pattern similar to that of the mass-specific daily metabolic rate (or energy expenditure) and energy assimilation efficiency. These data indicate that mass-specific daily metabolic rate and energy assimilation efficiency (an indicative of digestive and absorptive processes) may be responsible for differential tissue Cd accumulation in the bank vole.

Genetic Deletion of the Repressor of Estrogen Receptor Activity (REA) Enhances the Response to Estrogen in Target Tissues In Vivo

We previously identified a coregulator, repressor of estrogen receptor activity (REA), that directly interacts with estrogen receptor (ER) and represses ER transcriptional activity. Decreasing the intracellular level of REA by using small interfering RNA knockdown or antisense RNA approaches in cells in culture resulted in a significant increase in the level of up-regulation of estrogen-stimulated genes. To elucidate the functional activities of REA in vivo, we have used targeted disruption to delete the REA gene in mice. The targeting vector eliminated, by homologous recombination, the REA exon sequences encoding amino acids 12 to 201, which are required for REA repressive activity and for interaction with ER. The viability of heterozygous animals was similar to that of the wild type, whereas homozygous animals did not develop, suggesting a crucial role for REA in early development. Female, but not male, heterozygous animals had an increased body weight relative to age-matched wild-type animals beginning after puberty. REA mRNA and protein levels in uteri of heterozygous animals were half that of the wild type, and studies with heterozygous animals revealed a greater uterine weight gain and epithelial hyperproliferation in response to estradiol (E2) and a substantially greater stimulation by E2 of a number of estrogen up-regulated genes in the uterus. Even more dramatic in REA heterozygous animals was the loss of down regulation by E2 of genes in the uterus that are normally repressed by estrogen in wild-type animals. Mouse embryo fibroblasts derived from heterozygous embryos also displayed a greater transcriptional response to E2. These studies demonstrate that REA is a significant modulator of estrogen responsiveness in vivo: it normally restrains estrogen actions, moderating ER stimulation and enhancing ER repression of E2-regulated genes.

Postischemic Reperfusion: Ultrastructural Blood-Brain Barrier and Hemodynamic Correlative Changes in an Awake Model of Transient Forebrain Ischemia

In nonrecovery models of cerebral ischemia, blood-brain barrier (BBB) and cerebral blood flow (CBF) changes are known to occur during reperfusion. It is unknown, however, whether those CBF and BBB alterations occur after brief, transient ischemia with neurological recovery. The purpose of this study was to characterize the time course of CBF and BBB ultrastructural changes during reperfusion in an awake, recovery model of transient global forebrain ischemia (GFI). Forty-five adult Sprague-Dawley rats were subjected, while awake, to 10 minutes of GFI by the nine-vessel occlusion method. Thirty-five age-matched animals composed a sham-operated group. Normal control (n = 5), sham-operated (n = 5), and nine-vessel occlusion/reperfusion (n = 15) rats were selected for ultrastructural analysis. Electroencephalography was performed, and CBF, mean arterial blood pressure, and intracranial pressure were measured during ischemia and reperfusion up to 24 hours. Quantitative morphological analysis of cortical BBB capillaries was performed by transmission electron microscopy at the same time points at which specific CBF changes occurred during reperfusion. CBF decreased to 6% of preocclusion values during GFI. This correlated with coma and decerebrate rigidity. During reperfusion, short-lived hyperemia (225 +/- 18%, P < 0.001) was characterized by increased intracranial pressure (28.3 +/- 2.6 mm Hg, P < 0.001) and isoelectric electroencephalogram. This was followed by hypoperfusion, which reached a nadir of 59.7% (59.7 +/- 8.8%, P < 0.01) from baseline by 90 minutes. At this time point, the electroencephalogram recovered, and intracranial pressure and mean arterial blood pressure showed no abnormalities. By 8.5 hours, CBF returned to normal, and this coincided with complete recovery of the animal. Ultrastructural BBB analysis revealed astrocyte end-foot process edema and patent capillaries during hyperemia. Severe interstitial BBB edema and capillary lumen collapse was observed during hypoperfusion. Detachment and migration of pericytes was observed during hypoperfusion and beyond. A biphasic CBF response is elicited during reperfusion after brief nonlethal GFI under awake conditions.

The effect of chronic physical exercise on succinic dehydrogenase activity in the heart muscle of old rats

Succinic dehydrogenase (SDH) activity, preferentially evidenced by cytochemical methods, has been measured by computer-assisted morphometry in the heart muscle of old sedentary and age-matched animals chronically undergone physical exercise (20 min, twice a day, 5 days a week). The area of the SDH-positive mitochondria (MA) and the overall area of the cytochemical precipitates due to SDH activity (PA) were semiautomatically measured and the ratios PA/MA as well as MA/overall myocardial tissue area analysed (MA/TA) were the parameters taken into account. No significant difference was found between the two groups investigated as regards PA/MA, whereas the MA/TA value is significantly increased in the animals undergone physical training. The present findings document that chronic physical exercise significantly increases the overall mitochondrial area involved in energy provision in the old myocardial tissue. Considering that myocardial function highly relies on mitochondrial metabolism, our results support a beneficial effect of chronic physical exercise on the old heart muscle.

Chronic inhibition of Na/H-exchanger attenuates cardiac hypertrophy and prevents cellular remodeling in heart failure

In patients with heart disease, the transition from compensatory hypertrophy to heart failure (HF) is associated with altered calcium handling. Up-regulated Na(+)/H(+)-exchanger (NHE-1) activity underlies increased [Na(+)](i) and disturbance of cellular calcium handling in HF. We hypothesize that chronic inhibition of NHE-1 activity prevents the hypertrophic response, cellular remodeling, and development of HF. Rabbits received a control or cariporide (inhibitor of NHE-1) diet for 3 months, starting after induction of combined volume and pressure overload. Age-matched animals served as control. Development of HF was examined echocardiographically and electrocardiographically after 3 months. [Na(+)](i), [Ca(2+)](i), pH(i), and action potentials were measured in left ventricular midmural myocytes with SBFI, indo-1, SNARF, and di-4-anepps. Sarcoplasmic reticulum calcium content was calculated from the response of [Ca(2+)](i) to rapid cooling. Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. Chronic treatment of rabbits with the specific Na(+)/H(+)-exchanger activity inhibitor cariporide greatly attenuated development of hypertrophy and entirely abolished development of HF; the heart/body weight ratio increased only little, no change in lung weight occurred, left ventricular dimensions and fractional shortening changed mildly, ascites was not present, QT duration did not increase, and sudden death did not occur. Chronic cariporide treatment also prevented cellular electrical and ionic remodeling. Myocyte dimensions were unaltered, action potentials were not prolonged, cytoplasmic sodium and NHE-1 activity did not increase, cytoplasmic and SR calcium handling remained undisturbed, and no increase of the incidence of calcium after-transient dependent delayed after depolarizations (DADs) occurred. We conclude that enhanced activity of NHE-1 underlies cardiac cellular electrical and ionic remodeling in experimental heart failure, and that chronic dietary treatment with cariporide attenuates hypertrophy, development of HF, and cellular remodeling.

Loss of IRF-4 Exacerbates the CML-Like Phenotype of IRF-8 Knockout Mice.

IRF-4 and IRF-8 are related interferon regulatory factors that are required for the development and function of myeloid and lymphoid cells. Mice deficient in IRF-8 develop a myeloproliferative disorder with many similarities to human chronic myeloid leukemia (CML). Our group has shown that in B-lymphocyte development, IRF-4 and 8 function redundantly to control the pre-B to B transition. Though expression of IRF-4 has been demonstrated in myeloid cells, its role in these lineages has not been defined. We hypothesized that, similar to their role in B-cells, IRF-4 and 8 may to some extent function redundantly in myeloid cells. To test this hypothesis, myelopoiesis was analyzed in IRF-4/8 double knockout (DKO) mice. We found that from seven weeks of age, the DKO animals have a more aggressive leukemic phenotype than the IRF-8 KO mice. The white blood cell (WBC) counts of DKO animals were typically in the range of 40,000 to 80,000 cells per microliter, campared to 15,000 to 20,000 for age-matched IRF-8 KO animals. FACS analysis showed that the increased WBCs in the DKO animals was due to a massive expansion of mature granulcytes. The absolute granulocyte counts of DKO animals were typically 7-fold to 10-fold higher than those of IRF-8 KO animals. In addition, histologic preparations showed that by 15 weeks of age the spleens, lymph nodes, and bone marrow of DKO animals were invaded by large numbers of mature granulocytes and pseudo-Gaucher cells, with complete effacement of the normal micro-architecture. Age-matched IRF-8 KO animals showed invasion to a lesser degree, with preservation of many of the normal architectural features. In conclusion, IRF-4 is an important myeloid tumor suppressor, whose loss augments the myeloproliferative disease manifested in IRF-8 deficient mice. This suggests the two transcription factors, similar to their role in B-cell development, function redundantly to control the normal maturation of myeloid-lineage cells.

Digenic inheritance of deafness caused by mutations in genes encoding cadherin 23 and protocadherin 15 in mice and humans

Mutations in genes coding for cadherin 23 and protocadherin 15 cause deafness in both mice and humans. Here, we provide evidence that mutations at these two cadherin loci can interact to cause hearing loss in digenic heterozygotes of both species. Using a classical genetic approach, we generated mice that were heterozygous for both Cdh23 and Pcdh15 mutations on a uniform C57BL/6J background. Significant levels of hearing loss were detected in these mice when compared to age-matched single heterozygous animals or normal controls. Cytoarchitectural defects in the cochlea of digenic heterozygotes, including degeneration of the stereocilia and a base-apex loss of hair cells and spiral ganglion cells, were consistent with the observed age-related hearing loss of these mice beginning with the high frequencies. In humans, we also have obtained evidence for a digenic inheritance of a USH1 phenotype in three unrelated families with mutations in CDH23 and PCDH15. Altogether, our data indicate that CDH23 and PCDH15 play an essential long-term role in maintaining the normal organization of the stereocilia bundle.

The anti‐obesity effect of rimonabant is associated with an improved serum lipid profile

We investigated the effects of chronic treatment with the CB1 receptor antagonist rimonabant (10 mg/kg/day p.o. for 10 weeks) in mice with established obesity (5-month high-fat diet). Untreated obese mice showed a weight gain of 46% (45.0 +/- 0.6 g vs. 30.8 +/- 0.5 g) compared with age-matched animals fed a standard diet. Rimonabant treatment, commencing after 5-month high-fat diet, produced a marked and sustained decrease in body weight (34.5 +/- 0.8 g vs. 47.2 +/- 0.5 g in the high-fat vehicle group, p < 0.001). The anti-obesity effect of rimonabant was similar to that obtained by switching obese mice from high-fat diet to standard laboratory diet during 10 weeks (final weight 33.7 +/- 0.6 g) and was associated with only transient (14 days) reduction in energy intake. Serum leptin, insulin and glucose levels were markedly elevated in obese animals. Rimonabant treatment significantly reduced these elevations (leptin -81%, insulin -78%, glucose -67%, p < 0.001 in all cases vs. high-fat vehicle group). In addition, rimonabant treatment modestly but significantly increased serum adiponectin levels (+18%, p < 0.05 vs. high-fat vehicle group). Obese mice demonstrated abnormal serum lipid profiles. Although rimonabant did not modify high-density lipoprotein cholesterol (HDLc) and had modest effects on total cholesterol, it significantly reduced triglycerides and low-density lipoprotein cholesterol (LDLc) and, notably, increased the HDLc/LDLc ratio (12.4 +/- 0.8 vs. 7.9 +/- 0.2 in high-fat vehicle group, p < 0.001). Therefore, in a model of established obesity, chronic rimonabant treatment produces a marked and sustained decrease in body weight (equivalent to that achieved by dietary change) which is associated with favourable modifications in serum biochemical and lipid profiles.

Distribution of viral antigen and tissue lesions in persistent and acute infection with the homologous strain of noncytopathic bovine viral diarrhea virus.

Viral distribution and lesions were compared between calves born with persistent infection (PI) and calves acutely infected with the same bovine viral diarrhea virus (BVDV) isolate. Two PI calves from 1 dairy herd were necropsied. The PI viruses from these calves were isolated, characterized by sequencing, and found to be identical. This virus strain, designated BVDV2-RS886, was characterized as a noncytopathic (ncp) type 2 BVDV. To establish acute infections, BVDV2-RS886 was used to inoculate clinically healthy, seronegative calves which were 3 weeks to 3 months old. Nine calves received 10(6)-10(7) tissue culture infective dose of BVDV2-RS886 intranasally. Four additional age-matched animals served as noninfected controls. Infected calves were necropsied at 3, 6, 9, or 13 days postinoculation (dpi). Viral antigen was detected by immunohistochemistry in frozen sections, and lesions were evaluated in hematoxylin eosin-stained paraplast sections. In the PI calves, a wide distribution of viral antigen was found in all tissues and was not associated with lesions. In the acutely infected calves, viral antigen was widespread in lymphoid tissues at 6 dpi but had been mostly eliminated at 9 and 13 dpi. Depletion of lymphoid tissues was seen at 6, 9, and 13 dpi and repopulation at 9 and 13 dpi. In 1 of the calves at 13 dpi, severe arteritis was present in lymph nodes and myocardium. This comparison shows that an ncp BVDV strain that causes no lesions in PI animals is able to induce marked depletion of lymphoid tissues in calves with acute infection. Therefore, the failure to eliminate PI cattle from a herd causes problems not only in pregnant cattle but may also affect other age groups.

Influence of corticosteroids on myonuclear domain size in the rat diaphragm muscle.

Skeletal muscle fibers are multinucleated. Each myonucleus regulates gene products and protein expression in only a restricted portion of the muscle fiber, the myonuclear domain (MND). In the rat diaphragm muscle (DIAm), corticosteroid (CoS) treatment causes atrophy of fibers containing myosin heavy chain (MHC): MHC2X and/or MHC2B. We hypothesized that DIAm fiber MND size is maintained during CoS-induced atrophy. Adult male rats received methylprednisolone for 11 days at 1 (CoS-Low, n = 8) or 8 mg x kg(-1) x day(-1) (CoS-High, n = 8). Age-matched (CTL-AgeM, n = 8), sham-operated (SHAM-AgeM, n = 8), and weight-matched (CTL-WtM, n = 8) animals served as controls. In single DIAm fibers, cross-sectional area (CSA), MND size, and MHC expression were determined. Fiber CSA and MND size were similar in CTL-AgeM and SHAM-AgeM groups. Only fibers containing MHCslow or MHC2A displayed smaller CSA in CTL-WtM than in CTL-AgeM and SHAM-AgeM groups, and MND size was reduced in all fibers. Thus fibers containing MHCslow and MHC2A maintain the number of myonuclei, whereas MHC2X or MHC2B fibers show loss of myonuclei during normal muscle growth. Both CoS groups displayed smaller CSA and MND size than CTL-AgeM and SHAM-AgeM groups. However, compared with CTL-WtM DIAm fibers, only fibers containing MHC2X or MHC2B displayed reduced CSA and MND size after CoS treatment. Thus little, if any, loss of myonuclei was associated with CoS-induced atrophy of MHC2X or MHC2B DIAm fibers. In summary, MND size does not appear to be regulated during CoS-induced DIAm atrophy.

Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-alpha activation via the mitogen-activated protein kinase pathway.

IntroductionPregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer.MethodsThree groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed.ResultsIGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D1 and transforming growth factor-β3 in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous rats.ConclusionWe argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade.

Mechanical ventilation effect on surfactant content, function, and lung compliance in the newborn rat.

Studies of ventilator-associated lung injury in adult experimental animal models have documented that high tidal volume (TV) results in lung injury characterized by impaired compliance and dysfunctional surfactant. Yet, there is evidence that, in neonates, ventilation with a higher than physiologic TV leads to improved lung compliance. The purpose of our study was to evaluate how lung compliance and surfactant was altered by high TV ventilation in the neonate. We utilized a new model (mechanically air-ventilated newborn rats, 4-8 d old), and used 40 or 10 mL/kg TV strategies. Age-matched nonventilated animals served as controls. In all animals, dynamic compliance progressively increased after initiation of mechanical ventilation and was significantly greater than basal values after 60 min (p < 0.01). Lung lavage total surfactant with both TV strategies (p < 0.05) and the large aggregate fraction (only in TV = 40 mL/kg; p < 0.01) were significantly increased by 60 min of mechanical ventilation, compared with control animals. Ventilation with 40 mL/kg TV for 60 min adversely affected the lung surfactant surface-tension lowering properties (p < 0.01). After 180 min of ventilation with 40 mL/kg TV, the lung total surfactant content and dynamic compliance values were no longer distinct from the nonventilated animals' values. We conclude that, in the newborn rat, mechanical ventilation with a higher than physiologic TV increases alveolar surfactant content and, over time, alters its biophysical properties, thus promoting an initial but transient improvement in lung compliance.

Gene therapy with human recombinant osteoprotegerin reverses established osteopenia in ovariectomized mice

Osteoporosis is a chronic condition that is typically treated by the long-term repeated administration of antiresorptive agents. Gene therapy has the potential to deliver protein-based antiresorptive agents without the need for repeated administration. Osteoprotegerin (OPG) is a naturally occuring protein that prevents bone resorption by inhibiting osteoclast formation, function and survival. We tested whether adeno-associated virus (AAV) could deliver OPG at levels that are sufficient to reverse established osteopenia in ovariectomized (OVX) mice without causing liver toxicity. Tibial bone mineral density (BMD) was measured by peripheral quantitative computed tomography (pQCT) in 12-week-old CDF1 mice prior to OVX or sham surgery. Six weeks later, BMD was significantly reduced in OVX mice compared to sham controls or pre-surgery values. Sham and OVX mice were then injected once IV with an AAV vector carrying cDNA for recombinant hOPG (AAV-OPG) or β-galactosidase (AAV-βGal). BMD and bone histomorphometry were assessed 10 weeks after treatment. A single injection of AAV-OPG led to the appearance of human OPG (hOPG) in the serum of mice within 7 days, and high serum levels of hOPG were maintained for the duration of the 10-week study. At the end of the study, OVX mice given AAV-OPG had significantly greater tibial BMD compared to age-matched OVX animals given AAV-βGal. In sham-operated mice, AAV-OPG also significantly increased tibial BMD compared to AAV-βGal. The increased BMD in AAV-OPG animals was accompanied by significantly increased bone volume and significantly reduced osteoclast surfaces in the proximal tibial metaphysis. Liver histology was normal, and circulating activities of hepatocyte cytosolic enzymes were unaffected by AAV exposure. In an accompanying experiment, young (3–4 weeks) C57BL/6 mice treated once IV with AAV-OPG maintained pharmacologically active levels of OPG in serum for at least 16 months. In summary, a single AAV-OPG treatment reversed established osteopenia in OVX mice without evidence of liver toxicity. AAV delivery appears to be a safe and effective method for producing sustained systemic exposure to OPG.

24ASSESSMENT OF TELOMERE LENGTH IN NUCLEAR TRANSFER DERIVED SHEEP CLONES, THEIR OFFSPRING, AND CONTROL ANIMALS

The technique of somatic cell nuclear transfer (SCNT) involves the transplantation of a cultured somatic cell into an enucleated oocyte. Following activation, the nucleus of the somatic cell should be reprogrammed to a state of totipotency, which involves the resetting of epigenetic modifications of the somatic genome and also rebuilding of the chromosomal ends (telomeres). This study was carried out to investigate whether the nuclear transfer technique could rebuild the telomeres in sheep clones and their offspring when compared to age-matched control animals. Skin and blood samples were collected from three SCNT-derived sheep clones, and three of their offspring generated by natural mating. Control samples were collected from age-matched animals (n=17), spanning an age range from 1 month to 36 months of age. Ovine genomic DNA was extracted from samples using a Qiagen DNA extraction kit according to the manufacturer’s instructions. TeloTAGGG telomere length chemiluminescent assay kit (Roche Diagnostics) was used to determine the telomere length of all samples. Samples of ovine genomic DNA (2mg) were digested at 37°C for 2h using Hinf 1 and Rsa 1 endonuclease master-mix at the rate of 40 IU per sample. The resulted terminal restriction fragments (TRF) were separated on 1.2% agarose gel by pulsed field electrophoresis at 6V with 0.1s switch time for 4h in 0.5% TBE buffer at 14°C. The DNA fragments were southern-transferred to a nylon membrane and hybridized with telomere-specific (TTAGGG)n, digoxigenin (DIG)-labeled hybridization probe at 42°C. Following post-hybridization washes the membrane was incubated with DIG-specific antibody covalently coupled to alkaline phosphatase. The immobilized telomere probe was visualized by adding a chemiluminecence substrate to the membrane and exposed to X-ray film. Mean TRF length was determined by comparing the telomere densitometry signals to a molecular weight standard. High resolution bands resulting from pulsed field gel electrophoresis revealed that ovine TRF size distribution was in the range of 10–19kb. Linear regression analysis resulted in significant (t=25.84, P&amp;lt;0.05) decrease of mean TRF size with increasing age, at a mean rate of 0.08kb per every 6 months. Mean TRF length of SCNT-derived sheep clones, Matilda (28 months old), Macather (14 months old) and Frida (13 months old) were 11.12kb, 11.40kb, and 13.41kb, respectively. Their mean TRF values were significantly (P&amp;lt;0.05) shorter than those of their age-matched control animals: 16.95kb, 14.76kb, and 14.76kb, respectively (n=3 per group). The mean TRF length of the 3 offspring (21 months old) derived from natural mating was not significantly (P&amp;lt;0.05) different from age-matched controls (17.75±0.69kb v. 16.28±0.72kb). These results demonstrate that sheep clones derived from cultured somatic cells have shorter telomere lengths compared to age-matched controls, but this telomere loss is reset in the subsequent generation through natural breeding of the clones. (Funded by NSERC, OMAFRA and International Council for Canadian Studies.)

Immunosuppression after injury in aged mice is associated with a TH1–TH2 shift, which can be restored by estrogen treatment

Aged mice are less likely to survive following traumatic injury and are more immunosuppressed than young mice who sustain comparable injuries. Immunosuppression in severely injured patients is associated with a TH1–TH2 shift. Young mice had robust delayed-type hypersensitivity (DTH) responses after receiving scald or sham injury, whereas the response was diminished in aged sham-injured mice ( P<0.05), and completely absent in aged burn-injured mice ( P<0.01). Production of interferon-γ (IFN-γ) did not differ between splenocytes from sham-injured young and aged mice. Splenocytes from burn-injured young and aged mice yielded similar (63–68%) decreases in IFN-γ, relative to sham-injured mice ( P<0.05). In the absence of injury, cells from aged mice produced 2-fold more interleukin-4 (IL-4) than cells from young ( P<0.01). Interestingly, after burn, less IL-4 was produced by cell from young and aged mice, when compared to age-matched sham-injured animals ( P<0.05). Further studies revealed that estrogen replacement in aged mice restored the post-injury DTH responses ( P<0.05). Interestingly, this restoration paralleled a recovery in IFN-γ production by splenocytes, but not IL-4 production. Additional studies will be required to determine if age-specific therapies are needed for the treatment of all trauma patients.

Impaired neuronal and vascular responses to angiotensin II in a rabbit congestive heart failure model.

Congestive heart failure (CHF) is characterised by activation of the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system (SNS). Both systems are known to interact and to potentiate each other s activities. We recently demonstrated that angiotensin II (Ang II) enhances sympathetic nerve traffic via prejunctionally-located AT1-receptors. At present, little is known about the effects of Ang II at the level of the sympathetic neurones in CHF. Accordingly, we investigated the effect of Ang II in the presence and absence of the AT1-receptor antagonist, eprosartan, on stimulation-induced nerve traffic in isolated thoracic aorta preparations obtained from rabbits suffering from experimentally-induced CHF. Control-preparations were obtained from age-matched animals. Sympathetic activity was assessed by a [3H]noradrenaline spill-over model. Additionally, Ang II constrictor responses were compared between CHF and control vessels in the presence and absence of eprosartan. Additionally, to study postjunctional facilitation, the effects of Ang II on postsynaptic a-adrenoceptor-mediated responses were studied using noradrenaline. Stimulation-evoked SNS-neurotransmission was similar in both groups (CHF versus control). Ang II (0.1 nM 0.1 M) caused a concentration-dependent increase of the stimulation-evoked sympathetic outflow in both groups, with a maximum at 10 nM (control [n=7], FR2/FR1 2.03+0.11 and CHF- preparations [n=7], FR2/FR1 1.71+0.07). The enhancement by Ang II was decreased in CHF- preparations compared with controls (p<0.05). Eprosartan concentration-dependently attenuated the Ang II-enhanced (10 nM) sympathetic outflow in both CHF- and control preparations. The sympatho-inhibitory potency of eprosartan was similar in both groups (control pIC50 8.81 0.31; CHF 8.65+0.42). Ang II (1 nM 0.3 M) concentration-dependently increased the contractile force in control preparations (Emax 21.64+3.86 mN, pD2 7.63+0.02, n=7). Eprosartan (1 nM 0.1 M) influenced the Ang II- contractions via a mixed form of antagonism. In CHF-preparations, Ang II caused impaired vascular contraction. The KCl-induced contraction was decreased in the CHF- compared with control preparations (13.02+0.64 mN versus 30.40+0.89 mN). The relative Ang II contraction (% of KCl) was also decreased (2.3% vs. 58.0%). Concentration-response curves to noradrenaline (%KCl) were similar (control pD2 6.93+0.05, Emax 131.0+2.7; CHF pD2 7.00+0.05, Emax 136.7+2.6) (p>0.05) and were not affected by Ang II. We conclude that Ang II-enhanced sympathetic neurotransmission is mediated by the prejunctional AT1-receptor in both control and CHF-preparations. The decreased facilitation of SNS effects by Ang II may be explained by down-regulation or desensitisation of the neuronal AT1-receptor. Additionally, the aortic contractile capacity in heart failure rabbits appears to be decreased, probably as a result of heart failure-associated neuroendocrine and functional changes.

Abnormal serotonergic development in a mouse model for the Smith–Lemli–Opitz syndrome: implications for autism

The Smith–Lemli–Opitz syndrome (SLOS) is a malformation/mental retardation syndrome resulting from an inborn error in 3β-hydroxysteroid Δ7-reductase (DHCR7), the terminal enzyme required for cholesterol biosynthesis. Using a targeting strategy designed to virtually eliminate Dhcr7 activity, we have created a SLOS mouse model that exhibits commissural deficiencies, hippocampal abnormalities, and hypermorphic development of serotonin (5-HT) neurons. The latter is of particular interest with respect to current evidence that serotonin plays a significant role in autism spectrum disorders and the recent clinical observation that 50% of SLOS patients present with autistic behavior. Immunohistochemical analyses have revealed a 306% increase in the area of 5-HT immunoreactivity (5-HT IR) in the hindbrains of mutant (Dhcr7−/−) mice as compared to age-matched wild type animals. Amount of 5-HT IR was measured as total area of IR per histological section. Additionally, a regional increase as high as 15-fold was observed for the most lateral sagittal hindbrain sections. In Dhcr7−/− mice, an expansion of 5-HT IR into the ventricular zone and floor plate region was observed. In addition, the rostral and caudal raphe groups exhibited a radial expansion in Dhcr7−/− mice, with 5-HT IR cells present in locations not seen in wild type mice. This increase in 5-HT IR appears to represent an increase in total number of 5-HT neurons and fibers. These observations may help explain the behavioral phenotype seen in SLOS, and provide clues for future therapeutic interventions that utilize pharmacological modulation of the serotonergic system.

Intravesicular glucose modulates magnesium2+ transport in liver plasma membrane from streptozotocin-treated rats

Plasma membrane vesicles purified from livers of 4-week-old streptozotocin-injected diabetic rats present an increased basal and cation-stimulated magnesium (Mg)2+ transport as compared with vesicles purified from age-matched nondiabetic animals. Furthermore, diabetic basolateral membranes are unable to accumulate extravesicular Mg2+ in exchange for intravesicular sodium (Na)+. Loading diabetic vesicles with varying concentrations of d-glucose, in addition to Mg2+, renormalizes basal and Na+- or calcium (Ca)2+-induced Mg2+ extrusion in a dose-dependent manner, but does not restore Na+/Mg2+ exchanger reversibility. A similar effect on Mg2+ extrusion is observed when d-glucose is replaced with 2-deoxy-glucose, amylopectin, or glycogen. The loading with 3-methyl-o-glucose or l-glucose, instead, affects basal and Na+-dependent Mg2+ extrusion, but not Ca2+-dependent Mg2+ fluxes. In contrast, loading the vesicles with hexoses other than glucose or varying extravesicular glucose concentration from 5 to 20 mmol/L does not modify basal or cation-stimulated Mg2+ fluxes. Taken together, these data indicate that basal and cation-stimulated Mg2+ transport across the hepatocyte plasma membrane is altered under diabetic conditions as a result of a decrease in intravesicular (intracellular) glucose.

Spontaneous ECL cell carcinomas in cotton rats: natural course and prevention by a gastrin receptor antagonist.

In our inbred strain of cotton rats (Sigmodon hispidus) 50% of the females develop spontaneous ECL cell-derived tumors in the acid-producing part of the stomach due to hypergastrinemia secondary to gastric hypoacidity. Although the mechanism behind the hypoacidity is unknown, the female cotton rat is an excellent model for studying ECL cell-related tumorigenesis. In this study we wanted to explore the malignancy potential of these tumors and the ability of a gastrin receptor antagonist (YF476) to prevent their development. First, nine hypergastrinemic female cotton rats (10 months of age) were diagnosed by laparotomy as having gastric tumors. They were killed 6 months later. Second, 18 female cotton rats (2 months of age) were dosed monthly for 6 months with YF476 (500 micro mol/kg body wt) by s.c. injection, while 21 age-matched animals received vehicle. Samples from each stomach were collected for histology, immunohistochemistry and northern blot analysis. The gastric tumors harbored cells with immunohistochemical features of ECL cells. The tumors were found at times to invade and penetrate the stomach wall and to metastasize to perigastric sites. ECL-derived tumor cells were discovered in peritoneal fluid. At death only 1 out of 18 animals given YF476 displayed carcinomas (invasive growth), compared with 7 out of 21 in the vehicle dosed control group (P = 0.048). The spontaneous gastric tumors in cotton rats derived from ECL cells. The tumors were able to penetrate the stomach wall and to metastasize by intracavital seeding. Gastrin receptor blockade lowered the incidence of such tumors. We propose that the tumors are ECL cell carcinomas and that gastrin is the driving force behind the transformation from normal to malignant ECL cells.

Response to re-infection with Brachylaima cribbi in immunocompetent and immunodeficient mice

The course of infection in C57BL/6J mice re-infected with Brachylaima cribbi was assessed by comparing faecal egg excretion of re-infected mice with age- and sex-matched mice receiving a primary infection only. For both male and female mice there was a significant reduction in the mean number of eggs per gram of faeces at the peak of infection 4 weeks after the challenge infection compared with mice receiving a primary infection only. There was no significant difference in the duration of the infection. This experiment was repeated using age-matched male mice but on this occasion all mice were killed and dissected 4 weeks after the challenge infection and mean eggs per gram of faeces, worm burden and fecundity determined. There was no significant difference in the worm burdens of the re-infected mice compared with age-matched animals receiving a primary infection only. However, there were significant differences in the mean faecal eggs per gram and worm fecundity with the challenge infection group having lower egg counts and reduced fecundity. An enzyme-linked immunosorbent assay using whole worm antigens was developed and used to determine mouse anti- B. cribbi serum antibody levels during the course of infection. Anti- B. cribbi serum antibody absorbance ratios increased six- to sevenfold by 4 weeks after a primary infection beyond which a constant level was maintained. The course of challenge infection in non-obese diabetic severe combined immunodeficient mice showed no significant differences in egg excretion, worm burden or fecundity when primary and challenge infections were compared. These results indicate that the immune response invoked by a previous B. cribbi infection in immunocompetent mice affects fecundity but does not affect the establishment or duration of infection.