Microcalcifications in atherosclerotic lesion of apolipoprotein E‐deficient mouse

Evidence is accumulating that calcium-rich microdeposits in the vascular wall might play a crucial role in the onset and progression of atherosclerosis. Here we investigated an atherosclerotic lesion of the carotid artery in an established murine model, i.e. the apolipoprotein E-deficient (APOE^−/−) mouse to identify (i) the presence of microcalcifications, if any, (ii) the elemental composition of microcalcifications with special reference to calcium/phosphorus mass ratio and (iii) co-localization of increased concentrations of iron and zinc with microcalcifications. Atherosclerosis was induced by a flow-divider placed around the carotid artery resulting in low and high shear-stress regions. Element composition was assessed with a proton microprobe. Microcalcifications, predominantly present in the thickened intima of the low shear-stress region, were surrounded by areas with normal calcium levels, indicating that calcium-precipitation is a local event. The diameter of intimal microcalcifications varied from 6 to 70 μm. Calcium/phosphorus ratios of microcalcifications varied from 0.3 to 4.8, mainly corresponding to the ratio of amorphous calcium-phosphate. Increased iron and zinc concentrations commonly co-localized with microcalcifications. Our findings indicate that the atherosclerotic process in the murine carotid artery is associated with locally accumulated calcium, iron and zinc. The calcium-rich deposits resemble amorphous calcium phosphate rather than pure hydroxyapatite. We propose that the APOE^−/− mouse, in which atherosclerosis was evoked by a flow-divider, offers a useful model to investigate the pathophysiological significance of accumulation of elements such as calcium, iron and zinc.     

ApoE^-/-母体孕期高脂高胆固醇饮食对子代成年鼠动脉粥样硬化的影响

目的研究母体孕期高脂高胆固醇饮食对子代成年鼠动脉粥样硬化的发生影响。方法ApoE^-/-孕鼠分为高脂高胆固醇饮食组（G组）和普通饮食组（P组）,G组雄性子代成年鼠分为高脂高胆固醇饮食组（G—G）和普通饮食组（G—P）,P组子代雄性成年鼠继续喂养普通饲料（P—P）,60d后检测子代成年鼠总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDL）和低密度脂蛋白（LDL）浓度,HE染色观察血管形态学变化及动脉粥样硬化（atherosclerosis,AS）斑块形成情况。结果G—G组和G—P组的TC、TG、LDL和HDL浓度较P—P组明显升高,且与P—P组相比Tc浓度存在显著性差异（P〈0．05）,TG、LDL和HDL浓度存在极显著性差异（P〈0．01）;P—P组无AS斑块形成,G—G组与G．P组均有AS斑块形成,前者斑块形成率及斑块面积与血管管腔面积之比（PA／LA）都高于后者,两组PA／LA存在极显著性差异（P〈0．01）。结论母体孕期高脂高胆固醇饮食对子代成年鼠动脉粥样硬化的发生具有重要影响。     

In vivo imaging of atherosclerotic plaques in apolipoprotein E deficient mice using nonlinear microscopy

Structural proteins such as elastin and collagen can be readily imaged by using two-photon excitation and second-harmonic generation microscopic techniques, respectively, without physical or biochemical processing of the tissues. This time- and effort-saving advantage makes these imaging techniques convenient for determining the structural characteristics of blood vessels in vivo. Fibrillar collagen is a well-known element involved in the formation of atherosclerotic lesions. It is also an important component of the fibrous cap responsible for structural stability of atherosclerotic plaques. High resolution in vivo microscopic imaging and characterization of atherosclerotic lesions in animal models can be particularly useful for drug discovery. However, it is hindered by the limitations of regular microscope objectives to gain access of the tissues of interest and motional artifacts. We report a technique that facilitates in vivo microscopic imaging of carotid arteries of rodents using conventional microscope objectives, and at the same time avoids motional artifacts. As a result, collagen, elastin, leukocytes, cell nuclei, and neutral lipids can be visualized in three dimensions in live animals. We present and discuss in vivo imaging results using a flow cessation mouse model of accelerated atherosclerosis.     

Reduced brain edema after traumatic brain injury in mice deficient in P-selectin and intercellular adhesion molecule-1

Platelet (P-) selectin and intercellular adhesion molecule-1 (ICAM-1) mediate accumulation of neutrophils in brain. However, the mechanisms regulating neutrophil accumulation and damage after traumatic brain injury (TBI) are poorly defined. We hypothesized that mice deficient in both P-selectin and ICAM-1 (-/-) would have decreased brain neutrophil accumulation and edema, and improved functional and histopathological outcome after TBI compared with wild-type (+/+). In Protocol I, neutrophils and brain water content were quantified at 24 h after TBI. No difference in brain neutrophil accumulation was observed between groups; however, brain edema was decreased in dual P-selectin and ICAM-1 -/- (P < 0.05 vs. +/+ mice). In Protocol II, after TBI, tests of motor and memory function and histopathology were assessed over 21 days. No difference in motor or memory function or histopathological damage was observed between +/+ and -/- mice. A role for adhesion molecules in the pathogenesis of brain edema independent of leukocyte accumulation in brain is suggested.     

Ultrastructural identification of cells with dendritic cell appearance in atherosclerotic aorta of apolipoprotein E deficient mice

The present electron microscopic study was undertaken to see whether cells with a dendritic cell appearance accumulate in atherosclerotic lesions of apolipoprotein E (apoE) deficient mice. Atherosclerotic aortas from 7 eight-month old apoE deficient mice were examined. In atherosclerotic plaques as well as in the underlying media and adventitia, cells with a dendritic cell appearance including the presence of a unique tubulovesicular system were detected. The tubulovesicular system was most hypertrophied in their cellular processes where the continuous cisterns sometimes formed circular structures. The cells containing the tubulovesicular system lacked lysosomes and phagolysosomes and their cytoplasm was free of lipid inclusions. The present observations suggest that dendritic cells are involved in apoE deficient mouse atherosclerosis. ApoE deficient mice might be a useful model for investigating functions of dendritic cells in atherogenesis.     

Photoperiod, but not a high-fat diet, alters body fat in Shaw's jird

Many animal species living in temperate zones show annual body fat cycles. In an apparent regulation of total body fat, species showing naturally occurring decreases in adiposity in the fall (e.g., meadow voles and Siberian hamsters) are resistant to high-fat diet (HFD)-induced obesity in short, "winter-like" days (SDs), and in long, "summer-like" days (LDs) at their peak adiposity. SD-exposed Shaw's jirds (Meriones shawi) show SD-induced decreases in body fat; therefore, we predicted they also would be resistant to HFD-induced obesity. Male jirds were fed a standard chow diet or a HFD, and half of each group was exposed to LDs or SDs. SD-exposed jirds significantly decreased their carcass lipid content and testes mass compared with LD controls, but not body or WAT pad masses. HFD feeding in either photoperiod did not affect any of these measures, nor did it trigger overeating. Thus, it appears disadvantageous for this, and other species, that exhibit body fat peaks in the LDs of summer and nadirs in the SDs of winter, to fatten further or dampen their body fat losses, respectively, whereas species that exhibit body fat nadirs in the LDs of summer and peaks in the SDs of winter do become fat in LDs, and even fatter in SDs, when fed a HFD (e.g., Syrian hamsters). This dichotomous separation of HFD-induced body fat responses to HFD feeding among species showing opposite seasonal lipid mass peaks and nadirs may prove useful in understanding resistance or susceptibility to HFD-induced obesity, especially because they are naturally occurring.     

Modification of adriamycin toxicity in rats fed a high fat diet

Rats fed a diet containing a high percentage of butter, cholesterol, cholic acid and propyl thiouracil (HFD) showed weight loss and developed hyperlipidemia, marked fatty in filtration of the liver, moderate elevation of SGPT, degenerative changes of the heart muscle, bradycardia, alterations of the QRS complex in the electrocardiogram, and initial hemoconcentration followed by moderate anemia. Treatment with adriamycin (18×1 mg/kg i.p.) resulted in significant augmentation of the cardiotoxic effects of this drug demonstrated by electrocardiographic measurements and myocardial histopathology. Adriamycin-induced atrophy of the lymphatic tissue was seen only in rats fed HFD and not in animals receiving ground chow. Adriamycin levels in the heart after single i.p. injection were higher in rats receiving HFD. This effect was present already after 10 days on HFD. At this time histopathological liver changes were present and SGPT was elevated. It is concluded that the increase in adriamycin toxicity is, at least in part, due to diminished excretion by the liver. These experimental findings are in accordance with clinical observations which have identified liver disease as one of the important risk factors for the development of adriamycin cardiomyopathy.     

Pathology of lymphoid organs in chickens fed a diet deficient in zinc.

An experiment was conducted, including flow cytometry, to study the pathology of the lymphoid organs and peripheral blood T lymphocytes in zinc (Zn)-deficient chickens. One hundred 1-day-old broiler chickens were randomly divided into two groups and fed on diets with 100 mg/kg Zn (controls) or Zn-deficient diets (Zn, 23.63 mg/kg) for 7 weeks. The weight and growth index of the bursa of Fabricius, thymus and spleen were significantly reduced (P<0.05 or P<0.01) in Zn-deficient birds when compared with those of control broilers. The G0/G1 phase of the cell cycle of the bursa, thymus and spleen was much higher (P<0.01), and the S, G2+M phases and proliferating index lower (P<0.05 or P<0.01) in Zn-deficient broilers than in the controls. The acid alpha-naphthyl acetate esterase-positive ratio of the peripheral blood T lymphocytes and the CD4 and CD8 numbers were markedly reduced (P<0.05 or P<0.01), and the CD4/CD8 ratio increased. Histopathologically, lymphocytes of lymphoid organs were depleted and the reticular cells of the thymus were also degenerate or necrotic in the Zn-deficient birds. The results demonstrate that Zn deficiency seriously inhibited the development of lymphoid organs, impaired the progression of lymphocytes from the G0/G1 phase to the S phase, and caused pathological injury in the lymphoid organs. The results also showed that the effect of Zn deficiency on the primary lymphoid organs occurred earlier than on the secondary lymphoid organs. The effect of Zn deficiency was greatest on the bursa of Fabricius, followed by the thymus, and then the spleen. Potential mechanisms underlying the observations are discussed.     

Myocardial lesions in the offspring of female rats fed a copper deficient diet

Cardiac disease occurred in the offspring of dams fed a copper deficient diet from weaning. The usual age incidence at death for animals succumbing with heart failure was 5 to 7 weeks; the age of rapid growth when copper deficient offspring were slightly anemic. At necropsy the hearts were enlarged and usually pale. The most common histopathologic change was diffuse fatty change in the myocardium with small to large areas of necrosis in the ventricular and atrial musculature. Aneurysm of the apex developed in some severely involved hearts. Hemopericardium and hemothorax also occurred but specific rupture sites were not found. Myocardial cell hypertrophy with large vesicular nuclei was the only histopathologic change observed in some grossly enlarged hearts. Histochemical studies of the heart from copper deficient offspring demonstrated reductions in myocardial cytochrome oxidase activity especially in the myocardial sections from hearts with gross and histopathologic change. The activities of other mitochondrial oxidative enzymes, including NAD diaphorase, succinic, malic and glutamic dehydrogenase and glycolytic enzyme, lactic dehydrogenase, were increased. The myocardium from young control rats had little or no activity when stained for monoamine oxidase, however, the qualitative activity for this enzyme was consistently increased in the hearts from copper deficient offspring. Copper deficient offspring supplemented with copper had improved growth rate and there was evidence for return of myocardial enzyme activities to control levels.     

Reduced brain volumes in mice expressing APP-Austrian mutation but not in mice expressing APP-Swedish-Austrian mutations

We previously described two transgenic mouse lines expressing sub-endogenous levels of the 'Austrian' APP-T714I mutation (driven by the prenatally active PDGF-beta promoter; APP-Au mice) and showing intraneuronal Abeta pathology and reduced brain volumes on MRI at 12 and 20 months of age. To further investigate whether reduced brain sizes were caused by neurodegeneration or a neurodevelopmental defect, we now measured brain volumes as early as postnatal day 10. At this age, a distinguishable reduction in brain volumes was absent, indicating that brain volume deficits in APP-Au mice are not caused by a neurodevelopmental defect. To further study the association between intraneuronal Abeta and reduced brain volumes, we further generated and analyzed an APP transgenic mouse model expressing both Austrian and Swedish (K670N/M671L) mutations (APP-SwAu mice). APP-Swedish mutation is known to lead to altered APP processing in the secretory pathway, precluding its later processing in endosomal-lysosomal compartments, the site of intraneuronal Abeta accumulation. Also, to have higher levels of transgene expression only after birth, a murine Thy-1 promoter was utilized for APP-SwAu mouse lines. Despite having five times higher transgene APP levels compared to APP-Au mice, APP-SwAu mice showed significantly lower intraneuronal Abeta levels in the absence of reduced brain volumes, suggesting that intraneuronal Abeta accumulation is related to reduced brain volumes in APP-Au mice. These data also provide a first in vivo indication of altered processing of APP-Swedish at sub-endogenous levels, an effect not observed in mouse models expressing the APP-Swedish mutation in high amounts.     

Nucleolar-cytoplasmic transport of RNA in livers of rats fed a deficient diet.

Electron microscopic autoradiographic studies were conducted on livers of young female rats that were force-fed a complete or threonine-devoid diet for 3 days. Groups of control and experimental rats were killed on the fourth morning 15 or 60 min after intraperitoneally administering [³H]orotic acid. The electron microscopic autoradiographs of liver sections revealed grains attributed to RNA overlying or adjacent to several small nucleoli of hepatocytes of control rats. In contrast, the livers of the experimental rats revealed grains overlying single enlarged nucleoli and often they were present at the nucleolar-nuclear membrane junctions. Grains appearing in the cytoplasm of the livers of experimental rats were observed in the vicinity of the nucleolar base where it was attached to the nuclear membrane. Thus our results suggest that newly synthesized nucleolar RNA of the livers of rats force-fed a threonine-devoid diet migrates in the nucleolus toward its base at the nucleolar-nuclear membrane junction and then exits from the nucleus through the nuclear pores to enter the cytoplasm.     

Mallory body formation and amyloid deposition in the liver of aged mice fed a vitamin A deficient diet for a prolonged period

When twenty Swiss albino mice (male) were fed a vitamin A deficient diet for 18-24 months, Mallory bodies (MB(s] were observed. MBs were found by light microscopy and electron microscopy in the hepatocytes of six mice fed the vitamin A deficient diet and in one of the 15 control mice. Coincidentally, amyloid deposits were found in the space of Disse in the livers of 8 mice fed the deficient diet (three of the six mice with MBs). Amyloid was seen in two control mice suggesting that vitamin A enhanced amyloid deposition and MB formation in aging mice which spontaneously form amyloid and MBs. The possible role of vitamin A deficiency in the pathogenesis of MB formation is briefly discussed.     

Ultrastructure of gallstones produced in mice fed a high cholesterol diet

Mice fed a high cholesterol-cholic acid diet for two to six months develop gallstones; these were studied by transmission electron microscopy after glutaraldehyde-digitonin fixation. Examination of the contents of mouse gallbladders presents views of layered structures and surrounding amorphous material. We interpret these images of gallstones to suggest that they may arise by cohesion of material rich in cholesterol to form more ordered structures. Gallbladder contents of mice fed the diet for five to six months were found to contain occasional crystals and rectangular areas similar to those observed in thin sections of human gallstones (unpublished observations). Recent findings that human gallstones can be dissolved with chenodeoxycholic acid are discussed, with reference to their applicability to studies of gallstones in mice.     

CCK(1) receptor is essential for normal meal patterning in mice fed high fat diet

Cholecystokinin (CCK), released by lipid in the intestine, initiates satiety by acting at cholecystokinin type 1 receptors (CCK(1)Rs) located on vagal afferent nerve terminals located in the wall of the gastrointestinal tract. In the present study, we determined the role of the CCK(1)R in the short term effects of a high fat diet on daily food intake and meal patterns using mice in which the CCK(1)R gene is deleted. CCK(1)R(-/-) and CCK(1)R(+/+) mice were fed isocaloric high fat (HF) or low fat (LF) diets ad libitum for 18 h each day and meal size, meal frequency, intermeal interval, and meal duration were determined. Daily food intake was unaltered by diet in the CCK(1)R(-/-) compared to CCK(1)R(+/+) mice. However, meal size was larger in the CCK(1)R(-/-) mice compared to CCK(1)R(+/+) mice when fed a HF diet, with a concomitant decrease in meal frequency. Meal duration was increased in mice fed HF diet regardless of phenotype. In addition, CCK(1)R(-/-) mice fed a HF diet had a 75% decrease in the time to 1st meal compared to CCK(1)R(+/+) mice following a 6 h fast. These data suggest that lack of the CCK(1)R results in diminished satiation, causing altered meal patterns including larger, less frequent meals when fed a high fat diet. These results suggest that the CCK(1)R is involved in regulating caloric intake on a meal to meal basis, but that other factors are responsible for regulation of daily food intake.     

Neuron-specific transgene expression of Bcl-XL but not Bcl-2 genes reduced lesion size after permanent middle cerebral artery occlusion in mice.

Protective effects after focal cerebral ischemia were assessed in transgenic mice that overexpress in a neuron-specific fashion mouse Bcl-XL or human Bcl-2. Both Bcl genes were under the control of the same mouse Thy-1 regulatory sequences resulting in very similar expression patterns in cortical neurons. Furthermore, these sequences direct lateonset (i.e. around birth) expression in brain, thus minimizing effects of transgene expression during brain development. Effects on infarct volume were measured using MRI after permanent occlusion of the middle cerebral artery (MCA). When compared to their non-transgenic littermates, Thy1mbcl-XL mice showed a significant 21% reduction in infarct size whereas Thy1hbcl-2 mice did not reveal any reduction. These findings suggest a selective protective advantage of Bcl-XL as compared with Bcl-2 in this mouse model for human stroke.     

Effect of tea catechins on body fat accumulation in rats fed a normal diet

Although it is known that tea catechins exert potent effects in obese subjects, there is scant information concerning these effects on body weight gain and body fat accumulation in the non-obese. We studied normal rats fed a normal diet and water containing either 0.1% or 0.5% tea catechins to examine the effects on body fat content and serum cholesterol levels, as well as evaluating whether the effect is related to bile acids, which in recent years have emerged as an inducer of energy expenditure. The administration of 0.5% catechins decreased the accumulation of body fat and the serum levels of cholesterol and bile acids. These results indicate that tea catechins modulate lipid metabolism not only in obese subjects, but also in the non-obese.     

Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet.

Female, albino mice were fed a choline-deficient diet containing 0.5% DL-ethionine. All animals died within 5 days due to the development of an acute hemorrhagic pancreatis with fat necrosis throughout the peritoneal cavity. The apancreatitis was characterized by a massive necrosis of the exocrine parenchyma with intense hemorrhage and inflammatory reaction of the stroma. The sequence of histologic and ultrastructural alterations occurring in the acinar cells of the pancreas were studied in mice fed the diet for 1, 2, and 3 days. Major findings consited of accumulation of zymogen granules, vacuolation due to foci of cytoplasmic degradation, and alterations in the morphology of the zymogen granules. The pancreatitis appears to be due to intraparenchymal activation of zymogens, resulting from a synergistic action of choline deficiency with the basic toxicity of ethionine toward the acinar cells of the pancreas. The experimental model simulates closely the acute hemorrhagic pancreatitis with fat necrosis occurring in humans and may prove useful for exploring the pathogenesis of this condition.