The role of adrenergic receptors in the motility of duodenum and choledochoduodenal junction in the pig

The role of adenergic receptors in the motility of duodenum and choledochoduodenal junction in the pig. Acta Physiol. Pol., 1977, 28 (6): 521-528. The choldeochoduodenal junction in the Vietnamese pig is functionally and anatomically a part of duodenal wall. In view of this, investigations were carried out for establishing the role of adrenergic receptors in the development of motor function of this part of the intestinal tract. The experiments were performed on domestic Vietnamese pigs (Sus scrofa domestica) and they showed that after stimulation of alpha and beta adrenergic receptors the motor activity of the duodenal muscular coat and the choledochoduodenal junction is inhibited. The obtained results suggest similar reactions of the adrenergic receptors in both examined parts of the intestinal tract in the pig.     

Three-dimensional traction force microscopy: a new tool for quantifying cell-matrix interactions

The interactions between biochemical processes and mechanical signaling play important roles during various cellular processes such as wound healing, embryogenesis, metastasis, and cell migration. While traditional traction force measurements have provided quantitative information about cell matrix interactions in two dimensions, recent studies have shown significant differences in the behavior and morphology of cells when placed in three-dimensional environments. Hence new quantitative experimental techniques are needed to accurately determine cell traction forces in three dimensions. Recently, two approaches both based on laser scanning confocal microscopy have emerged to address this need. This study highlights the details, implementation and advantages of such a three-dimensional imaging methodology with the capability to compute cellular traction forces dynamically during cell migration and locomotion. An application of this newly developed three-dimensional traction force microscopy (3D TFM) technique to single cell migration studies of 3T3 fibroblasts is presented to show that this methodology offers a new quantitative vantage point to investigate the three-dimensional nature of cell-ECM interactions.     

Modulated polarization microscopy: a promising new approach to visualizing cytoskeletal dynamics in living cells

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.     

A technique for quantifying the bending moment acting on the lumbar spine in vivo

The bending properties of cadaveric lumbar spines were measured and used to convert in vivo measurements of lumbar flexion into bending moments ('stresses'). Forty-two lumbar motion segments were subjected to complex physiological loading and graphs were obtained of bending moment vs flexion angle. Variability was reduced by expressing both variables as a percentage of their values at the elastic limit. Data were averaged for each lumbar level, and a composite bending curve was compiled for the lumbar spine, L1-S1. A linear relationship was established between lumbar flexion measured in vitro and in vivo. This enabled values of 'per cent lumbar flexion' measured in vivo to be converted into 'per cent maximum bending moment' with a maximum likely error of about +/- 8%, which is equivalent to about +/- 5 Nm at L5-S1 for an average person. The technique was applied to 28 subjects, using dynamic measurements of lumbar flexion obtained with the '3-Space Isotrack' system. The bending moment at L5-S1 was 12 Nm on average when picking a pen up off the floor. Highly significant increases in bending moment were observed when heavier and bulkier objects were lifted.     

Fasciola hepatica: a technique for monitoring in vitro motility

An apparatus utilizing a force and displacement transducer is described for the direct and long-term recording of the motility in vitro of Fasciola hepatica. Normal movement is typically rhythmical, with bursts of more powerful contractions alternating with periods of lesser activity. Such rhythms and the overall level of activity are maintained for more than 30 hr. The fluke remains active for much longer periods of time: recordings of fluke movements have been made for up to 4 days. Potential damage to the fluke caused by the attachment system within the recording apparatus has been determined by the Evans' Blue Technique and scanning electron microscopy. It is restricted to the attachment sites, and does not spread to other parts of the body over the 30-hr normal activity period. Transmission electron microscope studies have shown that the tegument retains its structural and functional integrity over this period of time. There are advantages of the recording apparatus over previous kymographic methods for studying fluke motility.     

Music notation: a new method for visualizing social interaction in animals and humans

Background

Researchers have developed a variety of techniques for the visual presentation of quantitative data. These techniques can help to reveal trends and regularities that would be difficult to see if the data were left in raw form. Such techniques can be of great help in exploratory data analysis, making apparent the organization of data sets, developing new hypotheses, and in selecting effects to be tested by statistical analysis. Researchers studying social interaction in groups of animals and humans, however, have few tools to present their raw data visually, and it can be especially difficult to perceive patterns in these data. In this paper I introduce a new graphical method for the visual display of interaction records in human and animal groups, and I illustrate this method using data taken on chickens forming dominance hierarchies.

Results

This new method presents data in a way that can help researchers immediately to see patterns and connections in long, detailed records of interaction. I show a variety of ways in which this new technique can be used: (1) to explore trends in the formation of both group social structures and individual relationships; (2) to compare interaction records across groups of real animals and between real animals and computer-simulated animal interactions; (3) to search for and discover new types of small-scale interaction sequences; and (4) to examine how interaction patterns in larger groups might emerge from those in component subgroups. In addition, I discuss how this method can be modified and extended for visualizing a variety of different kinds of social interaction in both humans and animals.

Conclusion

This method can help researchers develop new insights into the structure and organization of social interaction. Such insights can make it easier for researchers to explain behavioural processes, to select aspects of data for statistical analysis, to design further studies, and to formulate appropriate mathematical models and computer simulations.     

Confocal laser scanning microscopy, a new in vivo diagnostic tool for schistosomiasis

Background

The gold standard for the diagnosis of schistosomiasis is the detection of the parasite''s characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM) permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates.

Methodology/Principal Findings

The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality). Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine.

Conclusion/Significance

We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable.     

Visualizing and quantifying the suppressive effects of glucocorticoids on the tadpole immune system in vivo

A challenging topic in undergraduate physiology courses is the complex interaction between the vertebrate endocrine system and the immune system. There are relatively few established and accessible laboratory exercises available to instructors to help their students gain a working understanding of these interactions. The present laboratory module was developed to show students how glucocorticoid receptor activity can be pharmacologically modulated in Xenopus laevis tadpoles and the resulting effects on thymus gland size visualized and quantified in vivo. After treating young tadpoles with a cortisol receptor agonist (dexamethasone) for 1 wk, students can easily visualize the suppressive effects of glucocorticoids on the intact thymus gland, which shrinks dramatically in size in response to this steroid hormone analog. However, the suppressive effect of dexamethasone is nullified in the presence of the glucocorticoid receptor antagonist RU-486, which powerfully illustrates the specific effects of glucocorticoid receptor inhibition on the immune system. Image analysis and statistics software are used to quantify the effects of glucocorticoid modulation on thymus size.     

A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver.

A method is described for both visualization and quantification of the total complement of rat liver free and membrane-bound ribosomes, undegraded by nucleolysis and unaggregated by pelleting. The method involves: (a) differential centrifugation of liver homogenate which separates free and membrane-bound ribosomes; (b) treatment of the fractions with detergents to solubilize membranes and remove nuclei; (c) centrifugation of a portion of each fraction to remove all the ribosomes; (d) sedimentation of the samples and blanks on sucrose gradients; and (e) difference photometric scanning of the gradients, sample minus ribosome-free blank, to detect the ribosomes free of interference from nonribosomal materials. The use of the SW 56 rotor in the initial centrifugation and of a high Mg²⁺ concentration (20 mm) in the medium used to suspend the bound fraction prior to detergent treatment were found to be essential in obtaining bound polysomes of large size (～19-somes). The difference scanning technique is shown to be a sensitive, accurate, and reproducible means of eliminating interference from nonribosomal materials, principally detergents and protein, and of quantifying ribosomes in both fractions. The method is rapid (3.5 h), simple to perform, and well suited for the analysis of multiple liver samples. It can be used to assess the concentration, distribution, organization, and average size of the total complement of rat liver free and membrane-bound ribosomes in a single experiment.     

Discovery and in vivo evaluation of new melanocortin-4 receptor-selective peptides

The melanocortin-4 receptor (MC4R) is involved in several physiological processes, including body weight regulation and grooming behaviour in rats. It has also been suggested that the MC4R mediates the effects of melanocortin ligands on neuropathic pain. Selective compounds are needed to study the exact role of the MC4R in these different processes. We describe here the development and evaluation of new melanocortin compounds that are selective for the MC4R as compared with the other centrally expressed receptors, MC3R and MC5R. First, a library of 18 peptides, in which a melanocortin-based sequence was systematically point-mutated, was screened for binding to and activity on the MC3R, MC4R and MC5R. Compound Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH(2) (JK1) appeared to be the most selective MC4R compound, based on affinity. This compound is 90- and 110-fold selective for the MC4R as compared to the MC3R and MC5R, respectively. Subsequent modification of JK1 yielded compound Ac-Nle-Gly-Lys-D-Nal(2)-Arg-Trp-Gly-NH(2) (JK7)(,) a selective MC4R antagonist with 34-fold MC4R/MC3R and 109-fold MC4R/MC5R selectivity. The compounds were active in vivo as determined in a grooming assay and a model for neuropathic pain in rats. Intravenous (i.v.) injections suggested that they were able to pass the blood-brain barrier.The compounds identified here will be useful in further research on the physiological roles of the MC4R.     

Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents

In this study, we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs because they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and BSA, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands, thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context.     

Predicting to new environments: tools for visualizing model behaviour and impacts on mapped distributions

Data limitations can lead to unrealistic fits of predictive species distribution models (SDMs) and spurious extrapolation to novel environments. Here, we want to draw attention to novel combinations of environmental predictors that are within the sampled range of individual predictors but are nevertheless outside the sample space. These tend to be overlooked when visualizing model behaviour. They may be a cause of differing model transferability and environmental change predictions between methods, a problem described in some studies but generally not well understood. We here use a simple simulated data example to illustrate the problem and provide new and complementary visualization techniques to explore model behaviour and predictions to novel environments. We then apply these in a more complex real‐world example. Our results underscore the necessity of scrutinizing model fits, ecological theory and environmental novelty.     

Algorithm and validation of a computer method for quantifying attachment locus of glenohumeral ligament in vivo

The aim of this work is to validate an algorithm that quantifies the locus of glenohumeral ligament (GHL) attachments on glenohumeral joint (GHJ) bones. A computed tomography scan of a GHJ was segmented to reconstruct the humerus, scapula, anatomical neck (AN) and glenoid rim (GR) into 3D meshes of interconnecting nodal vectors. These were applied to construct a 'clock face' coordinate system in which 3 o'clock points anteriorly. Based on the assigned clock face coordinate frame and the fitted plane, the error between the fitted plane and the actual bony node was quantified through manual data extraction. This was tested on 50 specimens. Mean algorithm quantification errors for GHL attachments were 4.8 (SD 2.2?mm) and 4.5?mm (1.7?mm) for the humerus and glenoid, respectively. Further studies would apply this to investigate GHL length changes during function and may suggest how these structures should be handled during surgical repairs.     

Algorithm and validation of a computer method for quantifying attachment locus of glenohumeral ligament in vivo

The aim of this work is to validate an algorithm that quantifies the locus of glenohumeral ligament (GHL) attachments on glenohumeral joint (GHJ) bones.

A computed tomography scan of a GHJ was segmented to reconstruct the humerus, scapula, anatomical neck (AN) and glenoid rim (GR) into 3D meshes of interconnecting nodal vectors. These were applied to construct a ‘clock face’ coordinate system in which 3 o'clock points anteriorly.

Based on the assigned clock face coordinate frame and the fitted plane, the error between the fitted plane and the actual bony node was quantified through manual data extraction. This was tested on 50 specimens.

Mean algorithm quantification errors for GHL attachments were 4.8 (SD 2.2 mm) and 4.5 mm (1.7 mm) for the humerus and glenoid, respectively. Further studies would apply this to investigate GHL length changes during function and may suggest how these structures should be handled during surgical repairs.     

In vivo modulation of intestinal motility and sites of opioid effects in the rat

The effects of subcutaneous (s.c.), intrathecal (i.t.) and intracerebroventricular (i.c.v.) injection of fentanyl and D-Ala2,D-Leu5-enkephalin (DADLE) on intestinal myoelectrical activity were examined in fed rats. In rats with chronically implanted electrodes on the small and large bowel, i.c.v. fentanyl and DADLE restored the 'fasted' pattern of duodenal activity, i.e. the migrating myoelectric complex (MMC) for 8-12 h at a dose as small as 1 nM/kg. In addition, the colonic pattern of activity evaluated as the number of migrating spike bursts (MSB) per min was nearly halved for 1 h following i.c.v. fentanyl (10 nM/kg). Pretreatment with naloxone, but not methylnaloxone prevented these effects on the small and large bowel. Fentanyl (100 nM/kg s.c.) significantly reduced small and large bowel motility, but DADLE (100 nM/kg s.c.) which induced a transient 'fasted pattern' on the duodenum strongly stimulated colonic motor activity. Pretreatment with methylnaloxone prevented the inhibitory effects of s.c. fentanyl but not the colonic excitatory effects of DADLE. The i.t. administration of fentanyl and DADLE did not modify the activity pattern of the bowel. Again, i.t. DADLE stimulated the colon, even after methylnaloxone treatment and at doses 100 times less than the smallest active s.c. dose. The long-lasting changes in small bowel motility and the important delay following DADLE and fentanyl i.c.v., reinforces the hypothesis of a central opioid control of the gastrointestinal motor pattern with possible involvement of released substances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feeding regulatory peptides: hopes and limits for the treatment of obesities

Excessive weight gain is directly related to a positive energy balance which is due to both a decreased physical activity and overeating. Obesity prevalence is increasing since thirty years and the treatment of obesities is particularly necessary to solve this public health and economical problem. The receptors of numerous hypothalamic neuropeptides are potential targets for such drug treatments. Hormones of the gastro-intestinal tract or produced by the adipose tissue directly interact with these central pathways to regulate feeding behavior. The use of leptin, an adipose tissue hormone that inhibits food intake, has not been conclusive because of the development of leptin resistance in obese subjects. Similar disappointing results have been obtained with antagonists of neuropeptide Y (NPY), one of the most potent orexigenic peptide. This was linked to the complexity and redundancy of the circuits involved in feeding regulation. Consequently, a multitherapy targeting several pathways simultaneously is probably the best option to cure obesity. Among these pathways, PYY 3-36 has been tested in man and some encouraging data have been obtained in animals with antagonists of some other orexigenic peptides such as orexins and melanin-concentrating hormone. A few gene therapy trials in the rat brain have restored interest for the leptin and NPY pathways. Their general use is however not planed in a next future. According to the type of obesity, these new treatments might be associated with either current (or almost current) drugs acting either on serotoninergic/catecholaminergic or cannabinoid pathways, or with surgery. Behavioral changes (food intake, exercise) and preventive actions during early life (in utero, young children) will remain for a while the best solutions to limit overweight development. The new treatments will help obese people to adhere to these behavioral changes by improving their efficiency to induce weight loss.