Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy

We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 µm²) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 µg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion.

Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages

Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 µg/ml and 50 µg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 µg/ml and 1250 µg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

Food restriction induces in vivo ventricular dysfunction in spontaneously hypertensive rats without impairment of in vitro myocardial contractility

Cardiac structures, function, and myocardial contractility are affected by food restriction (FR). There are few experiments associating undernutrition with hypertension. The aim of the present study was to analyze the effects of FR on the cardiac response to hypertension in a genetic model of hypertension, the spontaneously hypertensive rat (SHR). Five-month-old SHR were fed a control or a calorie-restricted diet for 90 days. Global left ventricle (LV) systolic function was evaluated in vivo by transthoracic echocardiogram and myocardial contractility and diastolic function were assessed in vitro in an isovolumetrically beating isolated heart (Langendorff preparation). FR reduced LV systolic function (control (mean ± SD): 58.9 ± 8.2; FR: 50.8 ± 4.8%, N = 14, P < 0.05). Myocardial contractility was preserved when assessed by the +dP/dt (control: 3493 ± 379; FR: 3555 ± 211 mmHg/s, P > 0.05), and developed pressure (in vitro) at diastolic pressure of zero (control: 152 ± 16; FR: 149 ± 15 mmHg, N = 9, P > 0.05) and 25 mmHg (control: 155 ± 9; FR: 150 ± 10 mmHg, N = 9, P > 0.05). FR also induced eccentric ventricular remodeling, and reduced myocardial elasticity (control: 10.9 ± 1.6; FR: 9.2 ± 0.9%, N = 9, P < 0.05) and LV compliance (control: 82.6 ± 16.5; FR: 68.2 ± 9.1%, N = 9, P < 0.05). We conclude that FR causes systolic ventricular dysfunction without in vitro change in myocardial contractility and diastolic dysfunction probably due to a reduction in myocardial elasticity.

Comparison of the homeostasis model assessment and quantitative insulin sensitivity check index with data from forearm metabolic studies for the in vivo assessment of insulin sensitivity

The present study was designed to compare the homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) with data from forearm metabolic studies of healthy individuals and of subjects in various pathological states. Fifty-five healthy individuals and 112 patients in various pathological states, including type 2 diabetes mellitus, essential hypertension and others, were studied after an overnight fast and for 3 h after ingestion of 75 g of glucose, by HOMA, QUICKI and the forearm technique to estimate muscle uptake of glucose combined with indirect calorimetry (oxidative and non-oxidative glucose metabolism). The patients showed increased HOMA (1.88 ± 0.14 vs 1.13 ± 0.10 pmol/l x mmol/l) and insulin/glucose (I/G) index (1.058.9 ± 340.9 vs 518.6 ± 70.7 pmol/l x (mg/100 ml forearm)-1), and decreased QUICKI (0.36 ± 0.004 vs 0.39 ± 0.006 (µU/ml + mg/dl)-1) compared with the healthy individuals. Analysis of the data for the group as a whole (patients and healthy individuals) showed that the estimate of insulin resistance by HOMA was correlated with data obtained in the forearm metabolic studies (glucose uptake: r = -0.16, P = 0.04; non-oxidative glucose metabolism: r = -0.20. P = 0.01, and I/G index: r = 0.17, P = 0.03). The comparison of QUICKI with data of the forearm metabolic studies showed significant correlation between QUICKI and non-oxidative glucose metabolism (r = 0.17, P = 0.03) or I/G index (r = -0.37, P < 0.0001). The HOMA and QUICKI are good estimates of insulin sensitivity as data derived from forearm metabolic studies involving direct measurements of insulin action on muscle glucose metabolism.

Effect of Sapindus trifoliatus on hyperalgesic in vivo migraine models

Phytotherapies have offered alternative sources of therapy for migraine and gained much importance in prophylactic treatment. Sapindus trifoliatus is a medium-sized deciduous tree growing wild in south India that belongs to the family Sapindaceae. The pericarp is reported for various medicinal properties. A thick aqueous solution of the pericarp is used for the treatment of hemicrania, hysteria or epilepsy in folklore medicine. We have investigated the antihyperalgesic effects of the lyophilized aqueous extract of S. trifoliatus in animal models predictive of experimental migraine models using morphine withdrawal-induced hyperalgesia on the hot-plate test and on 0.3% acetic acid-induced abdominal constrictions in adult male Swiss albino mice. The extract significantly (N = 10, P < 0.05) increased the licking latency in the hot-plate test when administered ip at 10 mg/kg (6.70 ± 0.39 s in saline control vs 18.76 ± 0.96 s in S. trifoliatus-treated animals) and significantly (N = 10, P < 0.001) reduced the abdominal constrictions when administered ip at 2 and 10 mg/kg (40.20 ± 1.36 in saline control vs 30.20 ± 1.33 and 23.00 ± 0.98 for 2 and 10 mg/kg, ip, respectively, in S. trifoliatus-treated animals). Furthermore, when administered ip at 20 and 100 mg/kg, the extract significantly (N = 10, P < 0.05) inhibited the apomorphine-induced climbing behavior in mice (climbing duration 15.75 ± 5.0 min for saline control vs 11.4 ± 1.28 and 3.9 ± 1.71 min for 20 and 100 mg/kg, respectively, in S. trifoliatus-treated animals). In receptor radioligand-binding studies, the extract exhibited affinity towards D2 receptors. The findings suggest that dopamine D2 antagonism could be the mechanism involved in the antihyperalgesic activity of the aqueous extract of S. trifoliatus.

Lycopene and ß-carotene protect in vivo iron-induced oxidative stress damage in rat prostate

It has been suggested that iron overload may be carcinogenic. In the present study, we evaluated the effect of plasma and prostate carotenoid concentration on oxidative DNA damage in 12-week-old Wistar rats treated with intraperitoneal (ip) ferric nitrilotriacetate (Fe-NTA) (10 mg Fe/kg). Plasma ß-carotene and lycopene concentrations were measured as a function of time after ip injection of carotenoids (10 mg kg-1 day-1 ß-carotene or lycopene) in rats. The highest total plasma concentration was reached 3 and 6 h after ip injection of lycopene or ß-carotene, respectively. After 5 days of carotenoid treatment, lycopene and ß-carotene were present in the 0.10-0.51 nmol/g wet tissue range in the prostate. Using a sensitive method to detected 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by HPLC/EC, the level of 8-oxodGuo in rat prostate DNA was significantly higher (6.3 ± 0.6 residues/10(6) dGuo) 3 h after Fe-NTA injection compared with control rats (1.7 ± 0.3 residues/10(6) dGuo). Rats supplemented with lycopene or ß-carotene for 5 days prior to Fe-NTA treatment showed a reduction of about 70% in 8-oxodGuo levels to almost control levels. Compared with control rats, the prostate of Fe-NTA-treated animals showed a 78% increase in malondialdehyde accumulation. Lycopene or ß-carotene pre-treatment almost completely prevented lipid damage. Epidemiological studies have suggested a lower risk of prostate cancer in men reporting a higher consumption of tomato products. However, before associating this effect with tomato sauce constituents, more information is required. The results described here may contribute to the understanding of the protective effects of carotenoids against iron-induced oxidative stress.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg-1 day-1 intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3% for piplartine and 55.1 and 56.8% for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

Translational models of coronary artery disease, myocardial infarction and heart failure. Development, validation and in vivo imaging studies using positron emission tomography

Coronary artery disease is an atherosclerotic disease, which leads to narrowing of coronary arteries, deteriorated myocardial blood flow and myocardial ischaemia. In acute myocardial infarction, a prolonged period of myocardial ischaemia leads to myocardial necrosis. Necrotic myocardium is replaced with scar tissue. Myocardial infarction results in various changes in cardiac structure and function over time that results in “adverse remodelling”. This remodelling may result in a progressive worsening of cardiac function and development of chronic heart failure. In this thesis, we developed and validated three different large animal models of coronary artery disease, myocardial ischaemia and infarction for translational studies. In the first study the coronary artery disease model had both induced diabetes and hypercholesterolemia. In the second study myocardial ischaemia and infarction were caused by a surgical method and in the third study by catheterisation. For model characterisation, we used non-invasive positron emission tomography (PET) methods for measurement of myocardial perfusion, oxidative metabolism and glucose utilisation. Additionally, cardiac function was measured by echocardiography and computed tomography. To study the metabolic changes that occur during atherosclerosis, a hypercholesterolemic and diabetic model was used with [18F] fluorodeoxyglucose ([18F]FDG) PET-imaging technology. Coronary occlusion models were used to evaluate metabolic and structural changes in the heart and the cardioprotective effects of levosimendan during post-infarction cardiac remodelling. Large animal models were used in testing of novel radiopharmaceuticals for myocardial perfusion imaging. In the coronary artery disease model, we observed atherosclerotic lesions that were associated with focally increased [18F]FDG uptake. In heart failure models, chronic myocardial infarction led to the worsening of systolic function, cardiac remodelling and decreased efficiency of cardiac pumping function. Levosimendan therapy reduced post-infarction myocardial infarct size and improved cardiac function. The novel 68Ga-labeled radiopharmaceuticals tested in this study were not successful for the determination of myocardial blood flow. In conclusion, diabetes and hypercholesterolemia lead to the development of early phase atherosclerotic lesions. Coronary artery occlusion produced considerable myocardial ischaemia and later infarction following myocardial remodelling. The experimental models evaluated in these studies will enable further studies concerning disease mechanisms, new radiopharmaceuticals and interventions in coronary artery disease and heart failure.

Nucleotide-sugar transporters: structure, function and roles in vivo

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5% (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats

The present study was carried out in order to compare the effects of administration of organic (methylmercury, MeHg) and inorganic (mercury chloride, HgCl 2 ) forms of mercury on in vivo dopamine (DA) release from rat striatum. Experiments were performed in conscious and freely moving female adult Sprague-Dawley (230-280 g) rats using brain microdialysis coupled to HPLC with electrochemical detection. Perfusion of different concentrations of MeHg or HgCl 2 (2 µL/min for 1 h, N = 5-7/group) into the striatum produced significant increases in the levels of DA. Infusion of 40 µM, 400 µM, or 4 mM MeHg increased DA levels to 907 ± 31, 2324 ± 156, and 9032 ± 70% of basal levels, respectively. The same concentrations of HgCl 2 increased DA levels to 1240 ± 66, 2500 ± 424, and 2658 ± 337% of basal levels, respectively. These increases were associated with significant decreases in levels of dihydroxyphenylacetic acid and homovallinic acid. Intrastriatal administration of MeHg induced a sharp concentration-dependent increase in DA levels with a peak 30 min after injection, whereas HgCl 2 induced a gradual, lower (for 4 mM) and delayed increase in DA levels (75 min after the beginning of perfusion). Comparing the neurochemical profile of the two mercury derivatives to induce increases in DA levels, we observed that the time-course of these increases induced by both mercurials was different and the effect produced by HgCl 2 was not concentration-dependent (the effect was the same for the concentrations of 400 µM and 4 mM HgCl 2 ). These results indicate that HgCl 2 produces increases in extracellular DA levels by a mechanism differing from that of MeHg.

Tympanometry and Spectral Gradient Acoustic Reflectometry in the Diagnosis of Otitis Media in Young Children

Acute otitis media (AOM) is the most prevalent bacterial infection among children. Tympanometry and spectral gradient acoustic reflectometry (SG-AR) are adjunctive diagnostic tools to pneumatic otoscopy. The aim was to investigate the diagnostic accuracy and success rates of tympanometry and SG-AR performed by physicians and nurses. The study populations comprised 515 (I-II), 281 (III), and 156 (IV) outpatients (6-35 months). Physicians performed 4246 tympanometric (I) and SG-AR (II) examinations. Nurses performed 1782 (III) and 753 (IV) examinations at symptomatic and asymptomatic visits, respectively. Pneumatic otoscopy by the physician was the diagnostic standard. The accuracy of test results by physicians or nurses (I-IV) and the proportion of visits with accurate exclusive test results from both ears (III-IV) were analyzed. Type B tympanogram and SG-AR level 5 (<49˚) predicted middle ear effusion (MEE). At asymptomatic visits, type A and C1 tympanograms (peak pressure > -200 daPa) and SG-AR level 1 (>95˚) indicated healthy middle ear. Negative predictive values of type A and C1 tympanograms by nurses in excluding AOM at symptomatic and MEE at asymptomatic visits were 94% and 95%, respectively. Nurses obtained type A or C1 tympanogram from both ears at 94/459 (20%) and 81/196 (41%) of symptomatic and asymptomatic visits, respectively. SG-AR level 1 was rarely obtained from both ears. Type A and C1 tympanograms were accurate in excluding AOM at symptomatic and MEE at asymptomatic visits. However, nurses obtained these tympanograms from both ears only at one fifth of symptomatic visits and less than half of asymptomatic visits.

Clinical utility of standard base excess in the diagnosis and interpretation of metabolic acidosis in critically ill patients

The aims of this study were to determine whether standard base excess (SBE) is a useful diagnostic tool for metabolic acidosis, whether metabolic acidosis is clinically relevant in daily evaluation of critically ill patients, and to identify the most robust acid-base determinants of SBE. Thirty-one critically ill patients were enrolled. Arterial blood samples were drawn at admission and 24 h later. SBE, as calculated by Van Slyke's (SBE VS) or Wooten's (SBE W) equations, accurately diagnosed metabolic acidosis (AUC = 0.867, 95%CI = 0.690-1.043 and AUC = 0.817, 95%CI = 0.634-0.999, respectively). SBE VS was weakly correlated with total SOFA (r = -0.454, P < 0.001) and was similar to SBE W (r = -0.482, P < 0.001). All acid-base variables were categorized as SBE VS <-2 mEq/L or SBE VS <-5 mEq/L. SBE VS <-2 mEq/L was better able to identify strong ion gap acidosis than SBE VS <-5 mEq/L; there were no significant differences regarding other variables. To demonstrate unmeasured anions, anion gap (AG) corrected for albumin (AG A) was superior to AG corrected for albumin and phosphate (AG A+P) when strong ion gap was used as the standard method. Mathematical modeling showed that albumin level, apparent strong ion difference, AG A, and lactate concentration explained SBE VS variations with an R² = 0.954. SBE VS with a cut-off value of <-2 mEq/L was the best tool to diagnose clinically relevant metabolic acidosis. To analyze the components of SBE VS shifts at the bedside, AG A, apparent strong ion difference, albumin level, and lactate concentration are easily measurable variables that best represent the partitioning of acid-base derangements.

Evaluation of a 1-h 75-g oral glucose tolerance test in the diagnosis of gestational diabetes

In order to evaluate the performance of a 1-h 75-g oral glucose tolerance test (OGTT) for the diagnosis of gestational diabetes mellitus (GDM), a cohort of 4998 women, 20 years or older, without previous diabetes being treated in prenatal care clinics in Brazil answered a questionnaire and performed a 75-g OGTT including fasting, 1-h and 2-h glucose measurements between their 24th and 28th gestational weeks. Pregnancy outcomes were transcribed from medical registries. GDM was defined according to WHO criteria (fasting: ≥126 mg/dL; 2-h value: ≥140 mg/dL) and macrosomia as a birth weight equal to or higher than 4000 g. Areas under the receiver operator characteristic curve (AUC) were compared and diagnostic properties of various cut-off points were evaluated. The AUCs for the prediction of macrosomia were 0.606 (0.572-0.637) for the 1-h and 0.589 (0.557-0.622) for the 2-h plasma glucose test. Similar predictability was demonstrable regarding combined adverse outcomes: 0.582 (0.559-0.604) for the 1-h test and 0.572 (0.549-0.595) for the 2-h test. When the 1-h glucose test was evaluated against a diagnosis of GDM defined by the 2-h glucose test, the AUC was 0.903 (0.886-0.919). The cut-off point that maximized sensitivity (83%) and specificity (83%) was 141 mg/dL, identifying 21% of the women as positive. A cut-off point of 160 mg/dL, with lower sensitivity (62%), had higher specificity (94%), labeling 8.6% as positive. Detection of GDM can be done with a 1-h 75-g OGTT: the value of 160 mg/dL has the same diagnostic performance as the conventional 2-h value (140 mg/dL). The simplification of the test may improve coverage and timing of the diagnosis of GDM.