Longitudinal micro-ultrasound assessment of the ob/ob mouse model: evaluation of cardiovascular, renal and hepatic parameters.

BACKGROUND/OBJECTIVES: Obesity is reaching epidemic proportions and is associated with increased risk factors for cardiovascular disease. Leptin-deficient mice (ob/ob) are widely employed to investigate obesity. Aim of this study was to provide a micro-ultrasound (µUS) longitudinal evaluation of the ob/ob mouse model in terms of cardiovascular effects, consequences for renal microcirculation and liver fat accumulation. SUBJECTS/METHODS: Sixteen wild-type (wt) and eleven ob/ob male mice were studied at 8 (T0) and 25 (T1) weeks of age with a µUS system (Vevo2100) and B-mode and Doppler images were acquired. Cardiac output (CO), ejection fraction (EF), stroke volume (SV), fractional shortening (FS) and E/A ratio were measured from cardiac images. Mean diameter (Dmabd, Dmcar), relative distension (relDabd and relDcar) and pulse wave velocity (PWVabd and PWVcar) were obtained for both abdominal aorta and common carotid. As regards renal microcirculation, renal resistivity and pulsatility index (RI and PI) were assessed. The ratio between grey levels related to liver and kidney (Steato-Score) was used as index of hepatic steatosis. RESULTS: At T0, ob/ob mice showed reduced SV, EF, CO and relDabd values and increased LVmass, PWVabd, RI, PI and Steato-score measurements. The same comparison repeated at T1 highlighted similar results for SV, EF, CO, RI, PI and Steato-Score; furthermore, obese mice showed reduced Dmabd and Dmcar measurements in comparison with lean controls. The longitudinal analysis showed an increase in LVmass and Dmabd and a reduction of FS, EF, CO, relDabd and relDcar for wt animals, while no differences were found for the ob/ob group. CONCLUSIONS: ob/ob mice presented a premature cardiac dysfunction without a further age-related deterioration and a reduction in the abdominal aorta and carotid artery mean diameter in adult age. The proposed analysis can represent a valid approach for longitudinal studies aimed at testing new therapeutic strategies or for characterizing other mouse models.

Quantification of thyroxine and 3,5,3'-triiodo-thyronine in human and animal hearts by a novel liquid chromatography-tandem mass spectrometry method.

Assaying tissue T3 and T4 would provide important information in experimental and clinical investigations. A novel method to determine tissue T3 and T4 by HPLC coupled to mass spectrometry is described. The major difference vs. previously described methods lies in the addition of a derivatization step, that is, to convert T3 and T4 into the corresponding butyl esters. The yield of esterification was Ì´ 100% for T3 and 80% for T4. The assay was linear (r>0.99) in the range of 0.2-50 ng/ml, accuracy was in the order of 70-75%, and the minimum tissue amount needed was in the order of 50 mg, that is, about one order of magnitude lower than observed with the same equipment (AB Sciex API 4000 triple quadrupole mass spectrometer) if derivatization was omitted. The method allowed detection of T3 and T4 in human left ventricle biopsies yielding concentrations of 1.51±0.16 and 5.94±0.63 pmol/g, respectively. In rats treated with different dosages of exogenous T3 or T4, good correlations (r>0.90) between plasma and myocardial T3 and T4 concentrations were observed, although in specific subsets different plasma T4 concentrations were not associated with different tissue content in T4. We conclude that this method could provide a novel insight into the relationship between plasma and tissue thyroid hormone levels.

New insights into mechanisms of cardioprotection mediated by thyroid hormones.

Heart failure represents the final common outcome in cardiovascular diseases. Despite significant therapeutic advances, morbidity and mortality of heart failure remain unacceptably high. Heart failure is preceded and sustained by a process of structural remodeling of the entire cardiac tissue architecture. Prevention or limitation of cardiac remodeling in the early stages of the process is a crucial step in order to ameliorate patient prognosis. Acquisition of novel pathophysiological mechanisms of cardiac remodeling is therefore required to develop more efficacious therapeutic strategies. Among all neuroendocrine systems, thyroid hormone seems to play a major homeostatic role in cardiovascular system. In these years, accumulating evidence shows that the "low triiodothyronine" syndrome is a strong prognostic, independent predictor of death in patients affected by both acute and chronic heart disease. In experimental models of cardiac hypertrophy or myocardial infarction, alterations in the thyroid hormone signaling, concerning cardiac mitochondrion, cardiac interstitium, and vasculature, have been suggested to be related to heart dysfunction. The aim of this brief paper is to highlight new developments in understanding the cardioprotective role of thyroid hormone in reverting regulatory networks involved in adverse cardiac remodeling. Furthermore, new recent advances on the role of specific miRNAs in thyroid hormone regulation at mitochondrion and interstitial level are also discussed.

Oxidative and genotoxic damage after radio-iodine therapy of Graves' hyperthyroidism.

PURPOSE: To evaluate genetic damage and oxidative stress following a single therapeutic dose of 131I in Graves' disease patients monitored up to 180 days after treatment. MATERIALS AND METHODS: Genetic damage induction was estimated as the increase in micronuclei in peripheral lymphocytes of patients. As indicators of radiogenic oxidative stress, vitamin E and lipoperoxide levels were assessed in the plasma of patients, as well as the release of plasmic clastogenic factors measured by the induction of micronuclei in vitro in peripheral lymphocytes of a healthy donor. RESULTS: Vitamin E depletion lasted at least 3 days and the basal level was restored within 7 days. No statistically significant variations were observed in lipoperoxide plasma levels. A sharp increase of micronuclei in the peripheral lymphocytes of patients was correlated (p < 0.001) with the release of clastogenic factor in the plasma. The highest micronucleus value was negatively correlated (p < 0.03) with the lowest vitamin E level observed in each patient. CONCLUSIONS: Micronuclei induction was the direct consequence not only of the energy deposition of 131I on the genetic material, but also of oxidative stress, likely via the release of clastogenic factor.

Oxidative stress in fulminant hepatic failure: comparison of two pig models with and without liver necrosis.

BACKGROUND/AIMS: No experimental study has clearly demonstrated how liver necrosis worsens the evolution of fulminant hepatic failure. Considering that several types of liver injury are associated with oxidative stress, we decided to measure plasma oxidative markers in two pig models of fulminant hepatic failure without and with liver necrosis. METHODOLOGY: Fulminant hepatic failure was produced in two groups of six pigs each by either total hepatectomy or complete hepatic devascularization. The following parameters were recorded before and during the course of hepatic failure: electrocerebral activity, plasma vitamin E, malondialdehyde and fluorescent protein-aldehyde adducts, total cholesterol, lactate-dehydrogenase, creatine phosphokinase, and ammonium. RESULTS: Despite comparable survival periods, hepatic necrosis was associated with earlier electrocerebral deterioration. Plasma concentration of malondialdehyde and fluorescent protein-aldehyde adducts rose and vitamin E content decreased in both groups. However, while in the group without liver necrosis the rates of cholesterol and vitamin E decay were identical, in the group with liver necrosis cholesterol concentration decreased less than vitamin E concentration, strongly indicating a true intravascular oxidation of vitamin E. Interestingly, in both models the rise of oxidative parameters preceded the development of cell injury. CONCLUSIONS: Oxidative stress, although present in both models, was significantly higher in the group with liver necrosis.

The antioxidant drug dipyridamole spares the vitamin E and thiols in red blood cells after oxidative stress.

OBJECTIVE: To test the antioxidant effect of therapeutic doses of dipyridamole on cellular membranes, human erythrocytes were chosen as an appropriate model to study oxidative stress induced by cumene hydroperoxide because of their high content in heme-Fe(2+). METHODS: The oxidative stress was induced by incubation with 160 micromoll(-1) cumene hydroperoxide and expressed by three main factors: lipid peroxidation by means of kinetics of decrease in fluorescence emission of the probe incorporated in the cell membranes, vitamin E oxidation and intracellular thiol content. The concentrations of dipyridamole tested (2-20 micromoll(-1)) did not exceed pharmacological doses. RESULTS: After 7 min of incubation at 25 degrees C with the oxidant and 20 micromoll(-1) dipyridamole thiol content was 50.1%+/-2.6 compared with 31.5%+/-2.4 in the absence of the drug. After 12 min vitamin E content was 88.3%+/-2.3 compared with 64.7%+/-3.4 of untreated cells in the absence of dipyridamole. Dipyridamole added 5 min after the oxidation reaction suppressed the fluorescence decrease for a time proportional to the drug concentration. CONCLUSIONS: Thus, at clinically realistic doses dipyridamole shows a concentration-dependent antioxidant effect. It protects membranes from oxidation and spares the antioxidant power of erythrocytes.

Antioxidant effect of oral dipyridamole during cerebral hypoperfusion with human carotid endarterectomy.

We sought to test the hypothesis of an antioxidant effect of dipyridamole in vivo in a model of cerebral hypoperfusion. Twenty-one patients (65+/-10 years, 11 men) undergoing carotid endarterectomy were allocated in two groups (group 1, 10 with dipyridamole, 200 mg p.o., 3-4 h before surgery; group II, 11 with placebo) in a double-blind placebo-controlled randomized design. Blood was sampled from ipsilateral jugular bulb, and plasma vitamin E content was assayed before, after 15 and 30 min of clamp, and 2 and 10 min after declamping. In 12 of them, lipoperoxides were assayed. Vitamin E plasma content decreased significantly in group II (rest, 3.71+/-0.22 mmol/mol of cholesterol, 100%) after clamp (91.5% of rest, p < 0.01) and remained unchanged during declamping (90.9% of rest, p < 0.01), but did not change in group I (rest, 3.5+/-0.44 mmol/mol of cholesterol, 100%) during clamping (99.9% of rest; p = NS) and after declamping (97.6% of rest; p = NS). Lipoperoxide concentration did not change in group I (rest, 302+/-8 a.u.; clamp, 296+/-13 a.u.; p = NS vs. rest; declamp, 304+/-8 a.u.; p = NS vs. rest), and increased significantly in group II (rest, 313+/-5 a.u.; clamp, 352+/-9 a.u.; p < 0.01 vs. rest; declamp, 343+/-6 a.u.; p < 0.05 vs. rest). Cerebral oxidative stress associated with human carotid endarterectomy can be attenuated by pretreatment with oral dipyridamole.

Morphology and spectral sensitivities of retinal and extraretinal photoreceptors in freshwater teleosts.

Fish eyes possess a complicated morphological and neural organisation of retinal and extra-retinal receptors. Features such as photoreceptor mosaic array and photoreceptor grouping are unique among vertebrates. Spectral sensitivities of these photoreceptors range from UV to the red portion of the visible spectrum. Moreover, these sensitivities can change with the age of the animals. In this review we will examine thoroughly the morphology, and the spectral sensitivities of retinal and extra-retinal receptors and the influence upon them of factors such as hormones, ageing, season, habitat light conditions, and migration.

A new technique for total hepatectomy in the pig for testing liver support devices.

BACKGROUND: A large animal model of total hepatectomy is suitable to test the efficacy of any system designed to support patients in hepatic coma. The models previously described in the pig entail a significant degree of surgical trauma, which might alter the evolution of the ensuring hepatic failure and compromise the reproducibility of the model. METHODS: Twenty-eight pigs underwent a total hepatectomy according to a new technique. A model was considered satisfactory when it required no blood transfusions and when hematologic and hemodynamic parameters determined before, during, and until 4 hours after hepatectomy showed no significant variations. Moreover, to revive the pattern of hepatic coma produced in the anhepatic model, 7 pigs were monitored until brain death occurred. RESULTS: Twenty-five pigs (89%) underwent a smooth total hepatectomy with minimal variations of the selected parameters. They constituted a highly homogeneous group. Survival of the 7 pigs, followed up until brain death occurred, ranged from 625 to 1595 minutes (mean 1013.57 minutes). The animals remained stable until a few hours before brain death, an event heralded by a final sharp increase of the serum ammonia level and by a well-evident decline of both arterial pressure and liver-dependent clotting factors. CONCLUSIONS: This technique of total hepatectomy allows the construction of a reproducible model of anhepaty suitable to test the efficacy of any system conceived to temporarily replace hepatic functions.

Vitamin E consumption induced by oxidative stress in red blood cells is enhanced by melatonin and reduced by N-acetylserotonin.

The effect of melatonin and its precursor N-acetylserotonin was studied in a model of lipid peroxidation induced in human red blood cells by incubation with cumene hydroperoxide (CHP) and H2O2. The oxidative stress was expressed as vitamin E consumption in the presence of melatonin or N-acetylserotonin (concentration ranging from 0.3 to 400 microM): incubation with melatonin not only lacked any protective effect but it induced a dose-dependent extra vitamin E consumption with both CHP and H2O2. On the contrary, N-acetylserotonin showed a strong antioxidant effect at concentrations between 100 and 400 microM. The hydrogen-donating capacity of melatonin and N-acetylserotonin was also evaluated from the decay of the ESR signal of galvinoxyl radical used as hydrogen abstractor. Lack of hydrogen-donating capacity was observed with melatonin, whereas N-acetylserotonin showed a significant hydrogendonating capacity although inferior to vitamin E, thus suggesting that N-acetylserotonin acts by the classical antioxidant mechanism of hydrogen donation. The measurement of the oxidation potential and the specific molecular structure suggest that the vitamin E consumption effect observed with melatonin could be due to the interactions of its radical cation or derivatives on vitamin E.

Binocular field in pigeons: behavioral measures of stimulus detection and coding.

The extension of the binocular visual field of pigeons is measured using a pecking behavioral method. Our estimate of the binocular field derived at the animal near point of accommodation indicates a 50 degrees width on the horizontal axis and more than 50 degrees on the vertical axis. Within the binocular field there is a core of about 24 degrees, named coding area, in which the stimulus is detected and coded by only one head fixation before accomplishing the pecking response, in both binocular and monocular viewing conditions. Outside the coding area, lies the detection area, in which the stimulus can be detected, but it needs a second head fixation to be coded in all viewing conditions. Our results are in agreement with the hypothesis that the coding area corresponds to the red field projection of each retina. In binocular vision an overlap of the two areas occurs, thus promoting the image fusion useful for a stereoscopic vision during the pecking to the seed.

Cell physiology of the pineal body.

The results from recent experiments on the cellular physiology of the trout pineal photoreceptors are briefly reviewed. The arguments are mainly concerned with pineal phototransduction. These studies have stimulated further research on melatonin, a molecule produced in pineal as well as in retinal photoreceptors. A discussion follows on our actual research object, that is a study of the influences of endogenous melatonin upon retinal receptor cells activities.

The electrical responses of the trout pineal photoreceptors to brief and prolonged illumination.

Intracellular recordings from 103 photoreceptors in the excised pineal body of adult trouts were obtained by using single electrode current- and voltage-clamp techniques. The photoresponses to brief flashes showed the same polarity but a slower time course than those previously recorded from retinal photoreceptors of lower vertebrates. Pineal photoreceptors showed spectral sensitivity peaks at about 495 and 521 nm and absolute sensitivity comparable to retinal cone cells of the same species. The photoreceptor membrane conductance, measured under voltage clamp during moderate illumination was about 10% lower than in the dark, and the extrapolated reversal potential of the response was at 60 mV above the dark membrane potential. The addition of 3-isobutyl-1-methylxanthine (IBMX) to the perfusate was followed by a receptor depolarization in the dark and by a slow-down of the response kinetic. Pineal receptor cells produce constant amplitude responses during steady illumination, without displaying the delayed slow depolarization typically associated with light adaptation of retinal photoreceptors. Photoresponses to brief flashes superimposed on a steady illumination are decreased in amplitude by an amount directly related to the background intensity. Increase of the background intensity leads to threshold increments without significant changes of the saturation intensity, resulting in a gradual compression of the cell dynamic range. These results were discussed relative to light adaptation in retinal photoreceptors. The conclusion can be drawn that the response properties of pineal photoreceptors during steady illumination are part of an unknown, self-regulating mechanism to lock the rate of metabolism and secretion of indolamines to the absolute level of diurnal light.

Binocular probability summation in a choice reaction-time task in pigeons.

We present evidence for binocular summation in pigeons in a choice reaction time paradigm. Considering binocular vision as the result of a race between the equivalent eyes, pigeons can adopt both a 'subject by subject' and a 'trial by trial' race model, and binocular advantage can be attributed merely to a probability summation. Pigeons were tested on a visual discrimination task in which the time for detection was gradually reduced and a quick response was required. The time presentation of a small black spot could vary from 2000 ms to the shortest value (157-226 ms) allowing maintenance of a satisfactory performance criterion.

An algorithm comparing the two mononuclear curves of choice reaction times in pigeons.

A simple and fast algorithm allowing the matching between frequency distributions is developed. The procedure consists in a class by class mapping of two distributional curves in order to derive a class transformation function that specifies how a class from one distribution is modified to produce a class in another distribution. Being interested in investigating the equivalence between the eyes, we have compared left and right monocular curves of choice reaction times to three different visual discrimination tasks in intact and surgically manipulated pigeons by means of the present technique of analysis. The results indicate that RTs distributions of the eyes belong to the same family of functions. It is possible, however, to demonstrate a difference in the range of time responses between the two monocular curves during one of the three visual discriminations used, particularly in the lesioned group.

Membrane resistance properties of pineal photoreceptors in the trout.

Intracellular recordings were obtained from pineal photoreceptors of the trout Salmo irideus. Illumination produced graded hyperpolarizing responses whose time course resembled that of retinal cones in lower vertebrates. Current-voltage relations were derived by using a voltage-clamp technique in the dark and during illumination. Membrane resistance values obtained in the dark were about 550 M omega, and they increased during illumination. The extrapolated reversal potential of the photocurrent is about 55 mV above the dark membrane potential, indicating that an ionic mechanism similar to that in retinal photoreceptors may be involved.

Binocular interactions measured by choice reaction times in pigeons.

In 7 pigeons binocular interactions were studied by recording monocular and binocular choice reaction times (RTs) to a pattern discrimination task. In all the animals binocular responses were found to be significantly faster than monocular RTs. In order to assess whether binocular advantage might be ascribed to a neural interaction between the two eyes, monocular and binocular performances were evaluated according to a probability summation model. The results indicate that in the great majority of animals, probability summation alone can account for binocular superiority.