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PubMed1986

Histamine-induced coronary spasm in regions of intimal thickening in miniature pigs: roles of serum cholesterol and spontaneous or induced intimal thickening.

The pathogenesis of histamine-induced coronary spasm was examined angiographically and morphometrically in Göttingen miniature pigs. In five of 36 consecutive pigs that were 4 to 5 months of age, coronary spasm was provoked by the intracoronary administration of histamine, and the left coronary arteries were examined histologically without endothelial denudation (group 1). Endothelial balloon denudation of the major branch of the left coronary artery was performed in 31 of 36 pigs and five died during the procedure. The remaining 26 pigs were randomly allotted to one of two groups, one fed a cholesterol-supplemented (group 2, n = 13) and one fed a regular low-cholesterol diet (group 3, n = 13). After 3 months, serum cholesterol increased significantly from 57 +/- 6 to 222 +/- 27 mg/dl (p less than .01) in group 2, but remained unchanged (48 +/- 5 to 55 +/- 6 mg/dl) in group 3. Percent narrowing of the coronary diameter induced by 10 micrograms/kg ic histamine after administration of the H2 blocker cimetidine (60 mg/kg iv) was 39 +/- 3% and 24 +/- 2% (p less than .05 between groups 2 and 3) at the nondenuded site and 78 +/- 3% and 74 +/- 4% at the denuded site in groups 2 and 3, respectively (p less than .01 between nondenuded and denuded sites). Histamine-induced percent narrowing of the coronary diameter after cimetidine in group 1, 2, and 3 pigs correlated well with the degree of intimal thickness on an exponential curve (r = .92, p less than .001). Since percent narrowing at the intact site was 27% (n = 19) in all three groups, predicted histamine-induced percent narrowing at the spastic site, applying the geometric theory, was 33 +/- 3%. Accordingly, enhanced constriction of the coronary artery with intimal thickening in response to histamine can largely be explained by the acquired hyperresponsiveness of the vascular wall to autacoids. This phenomena, not related to the level of serum cholesterol, may be uniquely linked to the basic pathology of evolution of atherosclerosis.

Animals↗
PubMed1991

Transvascular intracardiac applications of a miniaturized phased-array ultrasonic endoscope. Initial experience with intracardiac imaging in piglets.

BACKGROUND: Recent advances in miniaturization of phased-array and mechanical ultrasound devices have resulted in exploration of alternative approaches to cardiac and vascular imaging in the form of transesophageal or intravascular imaging. Preliminary efforts in adapting phased-array endoscopes designed for transesophageal use to a transvascular approach have used full-sized phased-array devices introduced directly into the right atrium in open-chested animals. The purpose of this study was to assess the feasibility of using a custom-made, very small phased-array endoscope for intracardiac imaging introduced intravascularly through a jugular venous approach in young piglets. METHODS AND RESULTS: Experimental atrial septal defects created in four piglets (3-4 weeks old) had been closed with a buttoned atrial septal defect closure device consisting of an occluder in the left atrium and a counteroccluder in the right atrium. Five to 15 days after atrial septal defect closure, the piglets were returned to the experimental laboratory, where a 6.3-mm, 17-element, 5-MHz phased-array probe mounted on a 4-mm endoscope was introduced through a cutdown incision of the external jugular vein and advanced to the right atrium. From the right atrium all four cardiac chambers, their inflows and outflows, and all four valves were well imaged with minimal superior and inferior rotation. High-resolution imaging of the atrial septum defined with anatomical accuracy, later verified by autopsy, the exact placement of both the occluder and counteroccluder in the left and right sides of the atrial septal defects and the absence of any shunting across the atrial septum in any of the four animals. CONCLUSIONS: Our efforts indicate that transvascular passage of small phased-array probes can be easily accomplished and is a promising technique for detailed visualization of cardiac structures. This approach may provide an alternative to transesophageal echocardiography, particularly for guiding interventional procedures such as placement of transcatheter closure devices in pediatric patients.

Animals↗
PubMed1987

Attenuation of endothelium-related relaxation and enhanced responsiveness of vascular smooth muscle to histamine in spastic coronary arterial segments from miniature pigs.

Mechanism of coronary spasm was examined regarding endothelium-related relaxation and contraction produced by smooth muscle cells of spastic vessels isolated from Göttingen miniature pigs. In these pigs, coronary artery spasm was documented angiographically in vivo three months after endothelial denudation, and spastic and control segments of the coronary artery were suspended in organ chambers at their optimal length for generating tension. Applications of KCl (118 mM), acetylcholine(10(-9) to 10(-4) M), and PGF2 alpha (10(-8) to 3 X 10(-5) M) produced similar tension, at the respective doses, in both the spastic and control coronary arteries. During increasing concentrations of histamine (10(-8) to 3 X 10(-4) M; n = 14) and serotonin (10(-9) to 10(-5) M; n = 13), the maximum tension of the spastic vessel was 136 +/- 6 and 97 +/- 4%, respectively, of the tension produced by 118 mM KCl. That is significantly larger than seen in the control vessels: 98 +/- 4 and 74 +/- 4%, respectively. The ED50 to histamine and serotonin was also significantly less in the spastic vessels. After mechanical removal of the endothelium, the tension generated during the cumulative administration of histamine (n = 8) but not serotonin (n = 8) was larger in the spastic than the control vessels, thereby suggesting the presence of augmented responses of the smooth muscle to histamine in the spastic vessels. The increase in maximum tension after mechanical denudation was greater in the control than the spastic vessels in cases of histamine and serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Cocaine-induced microvascular spasm in Yucatan miniature swine. In vivo and in vitro evidence of spasm.

The purpose of the present study was to determine the maximal coronary flow reserve (CFR) before and after the administration of successive cocaine doses (0.1, 0.5, 3, and 7 mg/kg IV) for 2 minutes at 10-minute intervals in eight miniature swine. CFR was assessed by the administration of adenosine (0.03, 0.3, and 3 mg IC). Hemodynamic and flow measurements were performed 3 minutes after each dose. Coronary flow (CF) was measured with a Doppler-tipped wire in the proximal left anterior descending coronary artery (LAD). Also, microvessels were dissected, and vessel diameters were measured by a videoelectronic dimension analyzer. In vivo, LAD CF increased fourfold, CFR increased twofold, and coronary vascular resistance (CVR) decreased fourfold after the administration of adenosine. In contrast, LAD CF decreased threefold, CFR decreased onefold, and CVR increased sixfold 3 minutes after the administration of cocaine. Adenosine (3 mg) was repeated 4 minutes after the administration of cocaine, and LAD CF increased 1.4-fold, CVR increased 2.5-fold, and CFR decreased onefold. Thus, adenosine partially reversed the potent cocaine constrictor effect. In vitro, 10(-9) mol/L cocaine decreased the diameter of the coronary microvessels from 129 +/- 12 to 127 +/- 12 microns, and 10(-4) mol/L cocaine decreased coronary microvessel diameter to 114 +/- 15 microns (P < .05). In conclusion, cocaine in vivo decreases CFR, and consistent with the in vivo effect, cocaine in vitro produced constriction of vessels < 200 microns. These results indicate that cocaine can produce profound microvascular spasm. This may contribute to the ischemia/infarction reported in patients who abuse cocaine and who are subsequently found to have normal epicardial coronary arteries.

Adenosine↗
PubMed1991

Effects of cocaine on epicardial coronary artery reactivity in miniature swine after endothelial injury and high cholesterol feeding. In vivo and in vitro analysis.

The purpose of this study was to determine the effects of cocaine on vasoreactivity in the swine model. Eight miniature pigs underwent regional endothelial denudation of the left anterior descending coronary artery and were then fed a high cholesterol diet. Cross-sectional area (CSA) of coronary arteries was measured by quantitative angiography. Before denudation, intravenous cocaine (1, 3, and 10 mg/kg) decreased CSA of epicardial vessels by 19-44%. At 3 mo after the denudation, the percent reduction in CSA of the denuded vessels induced by the 10 mg/kg dose was significantly augmented compared to nondenuded vessels (59 +/- 5% vs. 48 +/- 4%, P less than 0.05). Under in vitro conditions where isometric force of isolated ring segments was measured, methoxamine (an alpha 1 agonist) or BHT-920 (an alpha 2 agonist) produced similar degrees of contraction of denuded and control vessels; however, cocaine in concentrations up to 3 x 10(-3) M did not produce contraction. These responses were unaffected by removal of the endothelium. Histologically, myointimal thickening was noted at the denuded site. The present study demonstrates an enhanced vasoreactivity of atherosclerotic coronary arteries to cocaine in vivo, the mechanism of which appears to be mediated by endogenous vasoactive substances rather than by a direct action of cocaine on vascular smooth muscle.

Animals↗
PubMed1993

Pulp capping with Bioglass and autologous demineralized dentin in miniature swine.

The purpose of this study was to compare the responses of mechanically exposed dental pulps which had been capped with three dissimilar materials: a bioactive ceramic (Bioglass), autologous demineralized dentin matrix (DDM), and a calcium hydroxide product (Life), with Teflon discs as controls. Mechanical dental pulp exposures were made after preparation of deep buccal Class V cavities in 48 teeth in four miniature swine. The exposures were capped and the cavity preparations restored with zinc oxide-eugenol (IRM) cement. The animals were killed after 90 days, the coronal 2/3 of the teeth removed, and sections prepared for either histological or microradiographic examination. The pulpal inflammatory reactions and the degree of reparative dentin formation were assessed from demineralized serial sections. A qualitative assessment of the degree of mineralization of the reparative dentin was made from microradiographs of undecalcified sections. The observations suggest that reparative dentin formation occurs under a variety of pulp-capping materials, but the structure of the reparative dentin varies with the material and the condition of the underlying pulp.

Analysis of Variance↗
PubMed1993

Effects of oral sensory afferents on mastication in the miniature pig.

The performance of mastication is presumed to be influenced by afferent stimulation, but little information is available from awake, intact animals. Thirteen experiments were carried out with four miniature pigs. Electromyographic signals from the jaw muscles were recorded simultaneously with jaw movements during natural chewing of foods of differing hardnesses. Harder foods were found to be associated with higher activity levels of jaw-closing muscles and greater lateral deviation, but reduced jaw opening. The sensory supply from one side of the oral cavity was then removed by injected local anesthetic, and the recordings repeated. In addition to decreased activity levels, the jaw-closing muscles showed reduced ability to adjust to different foods. The frequency of mastication fell slightly, and the animals preferred to chew on the uninjected side. Lateral deviation was reduced, but at the same time the jaw-opening muscles usually became more active and accordingly the jaw opened more widely. These findings provide new information on the role of oral sensory afferents in regulating natural chewing patterns. The decreased activities of the jaw-closing muscles are consistent with, although they do not demonstrate, the existence of a positive feedback from periodontal afferents.

Afferent Pathways↗
PubMed1989

A comparative study of minoxidil-induced myocardial lesions in beagle dogs and miniature swine.

Minoxidil, a long-acting vasodilating antihypertensive agent, reduces arterial blood pressure by a direct action on arteriolar smooth muscle. The present studies examined the gross anatomic, histologic and ultrastructural myocardial alterations produced by 2 daily doses of minoxidil in beagle dogs (0.5, 1, or 3 mg/kg) and miniature swine (1, 3, or 10 mg/kg). Both myocardial necroses and hemorrhages were observed in dogs and pigs 24 hr after the last dose of minoxidil. In both species, the necroses were most frequent in the left ventricular papillary muscles, particularly the posterior one, but were less severe in swine. An ischemic origin of the necroses was suggested by the location of the lesions and by the pharmacologic effects of minoxidil. Gross epicardial or endocardial hemorrhages involving the atria and, to a lesser extent, the ventricles were observed in both species but were more severe in dogs. The atrial lesions were manifested grossly by diffuse redness and microscopically by interstitial edema, extravasated erythrocytes and infiltration of mononuclear cells. Hemorrhages, occurring on the epicardial surfaces, were often associated with lesions that involved small arteries having 3-6 layers of medial smooth muscle cells and were characterized by endothelial injury, intramural accumulations of erythrocytes and platelets, periadventitial hemorrhage, fibrin deposits and an inflammatory cell reaction. These lesions preferentially involved the right atrium in dogs and the left atrium in pigs. Pretreatment of beagle dogs with pyribenzamine (3 doses of 6 mg/kg every 8 hr), cimetidine (3 doses of 15 mg/kg every 8 hr), or both, had no effect on the incidence or severity of minoxidil-induced hemorrhagic lesions. The hemorrhagic lesions may be caused by overstretching of the vascular walls due to excessive, prolonged vasodilatation.

Animals↗
PubMed2001

Single-molecule detection technologies in miniaturized high throughput screening: binding assays for g protein-coupled receptors using fluorescence intensity distribution analysis and fluorescence anisotropy.

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

Drug Evaluation, Preclinical↗
PubMed2004

Development and implementation of a highly miniaturized confocal 2D-FIDA-based high-throughput screening assay to search for active site modulators of the human heat shock protein 90beta.

The beta isoform of the heat shock protein 90 (Hsp90beta) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90beta are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90beta. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90beta inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90beta. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

Biological Assay↗
PubMed2005

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes.

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

Cytochrome P-450 Enzyme Inhibitors↗
PubMed2004

Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression.

BACKGROUND: RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array) procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. RESULTS: RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. CONCLUSION: RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

Animals↗
PubMed2006

Genomic presence of recombinant porcine endogenous retrovirus in transmitting miniature swine.

The replication of porcine endogenous retrovirus (PERV) in human cell lines suggests a potential infectious risk in xenotransplantation. PERV isolated from human cells following cocultivation with porcine peripheral blood mononuclear cells is a recombinant of PERV-A and PERV-C. We describe two different recombinant PERV-AC sequences in the cellular DNA of some transmitting miniature swine. This is the first evidence of PERV-AC recombinant virus in porcine genomic DNA that may have resulted from autoinfection following exogenous viral recombination. Infectious risk in xenotransplantation will be defined by the activity of PERV loci in vivo.

Amino Acid Sequence↗
PubMed2003

Miniaturization in cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) remains the key technology for more complex cardiac operations. The perfusion equipment used nowadays has seen tremendous progress since its introduction into clinical practice 50 years ago. However, overall, CPB is still far from perfect. Major haemodilution is not only a problem for red cell-dependent gas transport, but also for the platelet and humeral factor-dependent coagulation, the protein-dependent intravascular oncotic pressure and so forth. Reduction of the priming volume through further miniaturization of CPB equipment is, therefore, the most obvious next step. A systematic approach needs to optimize all CPB components, including pumps, oxygenator/heat exchanger structures, filters, reservoirs, cardiotomy suction, tubings and cannulas. This report provides an update of already commercially available low prime perfusion devices (e.g., the CORx integrated pump-oxygenator) as well as promising prototypes like the smart suction system and the smartcanula.

Cardiopulmonary Bypass↗
PubMed1984

Effect of different total parenteral nutrition fuel mixes on the body composition of infant miniature pigs.

At 10 d of age miniature pigs were randomized to receive either of two total parenteral nutrition fuel mixes; oral feedings were discontinued. Both groups received 170 kcal X kg-1 X d-1 and 11 g X kg-1 X d-1 of synthetic amino acids. Nonprotein energy was supplied as glucose in group A, whereas in group B, it was divided equally between glucose and fat. Blood samples were drawn on the second and eighth postoperative days for hematologic, biochemical, and hormonal measurements. On the ninth postoperative day, total body water was determined and the animals were killed for carcass analysis. The animals tolerated the intravenous nutrition without ill effects as indicated by both clinical and biochemical parameters. Group A had significantly elevated levels of insulin and a higher insulin/glucagon ratio than group B. Cortisol levels did not differ significantly between groups. Total body fat, nitrogen, ash, K, Na, Cl, Ca, and P were similar between groups. TBW was significantly greater in group A compared with group B. Extracellular space calculated from body Cl and plasma Cl was similar between groups.

Adipose Tissue↗
PubMed1988

The miniature pig as an animal model for the study of intestinal enzyme development.

Studies of intestinal enzyme development and regulation relevant to the human infant require an animal model with a rate of maturation similar to that of the human infant. Hanford miniature pigs were weaned at 3 days of age to a standard swine weaning formula. At 1, 2, 3, 4, 5, and 6 wk of age, duodenal jejunal, and ileal segments were analyzed for protein content and lactase, sucrase, maltase, glucoamylase, and acid beta-galactosidase activities. Protein content of the small intestine changed significantly with age only in the ileum (p less than 0.05). Lactase activity fell significantly with age in all segments of the small intestine (p less than 0.001); activity was highest in the jejunum. Sucrase and maltase activities were present in all segments of the small intestine at 1 wk of age. Sucrase increased significantly (2-fold, p less than 0.02) with age only in the ileum and maltase increased significantly with age in the jejunum (by 50%, p less than 0.05) and the ileum (3-fold, p less than 0.001). Activities were highest in the jejunum. Glucoamylase activity was present at 1 wk of age and showed a small but significant increase with age only in the duodenum (p less than 0.005). Acid beta-galactosidase activity demonstrated small but significant decreases with age in all small intestinal segments. Glucoamylase and acid beta-galactosidase activities were similar in all segments. In the 6-wk-old pigs, activities of all the enzymes tested were similar to those found in young human infants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Bone mineral content reflects total body calcium in neonatal miniature piglets.

We measured bone mineral content (BMC) in 18 neonatal miniature piglets by single photon absorptiometry, total body calcium (TBC) by total body neutron activation analysis, growth, and serum indices of mineral status (calcium, phosphorus, alkaline phosphatase activity). Measurements were begun on day 6, when the piglets were weaned, and were continued to day 19. After weaning, the piglets were assigned randomly to receive one of three diets which differed only in their concentrations of calcium and phosphorus: 100% of the recommended level (diet A), 60% (diet B), and 20% (diet C). No differences were observed among groups during the 19-day study, either in weight gain (48 +/- 2 g/day) or increment in crown-rump length (2.4 +/- 0.2 cm/wk). BMC correlated significantly (p less than 0.001) with TBC at 6 (r = 0.83), 13 (r = 0.77), and 19 (r = 0.93) days. BMC correlated significantly (p less than 0.001) with the ash weight (r = 0.87) and calcium content (r = 0.90) of the corresponding tibial bone segment. Anthropometric parameters and serum indices of mineral status did not predict TBC as accurately as did BMC measurements. We observed a range in BMC measurements in this study that was similar to the range reported for infants in the 1st yr of life. The high correlation between BMC and TBC suggested that BMC is useful in the assessment of mineral status in infants.

Animals↗
PubMed1983

Thyroid hormone regulation of glucose homeostasis in the miniature pig.

The effect of different thyroid states on glucose homeostasis was investigated during metabolic adaptation to starvation in the conscious unrestrained miniature pig. Moderate hyperthyroidism increased the rate of glucose turnover, whereas hypothyroidism was without effect. Glucose recycling was elevated in hyperthyroid pigs, and reduced after thyroidectomy. Supplementary doses of T4 normalized total glucose recycling. Glucose metabolic clearance rate and pool size were unaffected by thyroid hormones. During starvation serum insulin showed a similar decrease in all thyroid states; glucagon increased in euthyroid and hypothyroid pigs, although it was already elevated in the hyperthyroid fed state. Serum cortisol levels although varying were enhanced in hyperthyroid and hypothyroid-T4-treated pigs. Glucogenic precursor concentration and cumulative urinary N-excretion were increased in hyperthyroid pigs. It is concluded that 1) even a moderate hyperthyroidism produces an increase in glucose turnover and a concomitant acceleration in protein breakdown, and 2) thyroid hormone is essential for the starvation-induced total glucose recycling.

Animals↗