Interest of submucosal dissection knife for endoscopic treatment of Zenker's diverticulum.

BACKGROUND: Dual-Knife(®) (Olympus) and Hydride-Knife(®) are new needle knives frequently used for submucosal dissection because of their safety and precision. In this study we aimed to evaluate the efficacy and safety of such devices in the diverticulopexy by flexible endoscopy. METHODS: From February 2009 to March 2013, 42 patients (25 men), mean age 74.5, with symptomatic Zenker's diverticulum, were included in a non-randomized prospective multicenter study. The symptoms described by all patients include dysphagia, regurgitation and/or swallowing disorders. The diverticulopexy was performed with the Dual-Knife(®) or Hydrid-Knife(®), after septum exposure with the diverticuloscope, and terminated with distal tip clips positioning. All complications were noted. Patients' symptoms were regularly assessed during follow-up visits or telephone interviews. RESULTS: The first endoscopy treatment was successful for all patients. Thirty-seven patients (88%) had symptoms improvement after the first treatment. The recurrence rate was 14% (6 patients); a second endoscopic treatment was required 12 months on average after the first treatment, with 100% efficiency. Mid-term (16 months) efficiency was 91.67% after 1 to 3 endoscopic treatments. A total of 55 procedures were performed without perforation or significant bleeding and 3 patients underwent surgery. In multivariate analysis, the diverticulum size and the type of dissection knife were not risks factors for recurrence. CONCLUSIONS: Endoscopic diverticuloscope-assisted diverticulotomy with submucosal dissection knives is a safe and effective alternative treatment for patients with a symptomatic Zenker's diverticulum measuring between 2 and 10 cm.

Enzyme-linked immuno-filtration assay applied to accelerated immunodetection of gel transferred proteins on immobilizing matrices.

The enzyme-linked immunofiltration assay (ELIFA) has been used for the rapid detection of electrophoresed proteins transferred from gels to immobilizing matrices. By controlled filtration, ELIFA permits the saturation of nylon or nitrocellulose membranes and the immunodetection of blotted antigens to be carried out in 15 min. The method is simple, can be automated and requires no handling of membranes. It complements the well standardized steps of gel electrophoresis and semi-dry horizontal electroblotting which can themselves be carried out in less than an hour. The sensitivity is at least 1-5 ng. The same process can be extended to the accelerated characterization of glycoproteins using appropriate ligands, or to the identification of antigens in a variety of biological fluids.

Hypothalamic neurons. Mechanisms of sensitivity to temperature.

Rostral hypothalamic neurons are influenced by endogenous factors that affect thermoregulation and fever. Intracellular recordings reveal the synaptic and intrinsic mechanisms responsible for neuronal thermosensitivity. Many temperature-sensitive and temperature-insensitive neurons display a depolarizing prepotential that precedes action potentials. Temperature has little effect on the prepotential of insensitive neurons; however, in warm-sensitive neurons, the prepotential's depolarization is elevated by warming, and this increases the firing rate. Intracellular cAMP can increase neuronal thermosensitivity by enhancing the thermal response of the prepotential, most likely by thermosensitive ionic conductances. Warm-sensitive neurons also receive inhibitory synaptic input (IPSPs) from temperature-insensitive neurons, enhancing the thermosensitivity of some neurons, because cooling increases IPSP amplitude and duration. Therefore, even though IPSP frequencies do not change, cooling can decrease firing rates by increasing IPSP amplitudes. Because endogenous factors change neuronal firing rate and thermosensitivity, these changes likely occur both post- and presynaptically as well as by ionic conductances that determine the time interval between action potentials.

A diencephalic slice preparation and chamber for studying neuronal thermosensitivity.

An in vitro slice preparation and electrophysiological recording chamber for studying neuronal thermosensitivity throughout the hypothalamus are described. A series of eight, 300 microns thick, horizontal tissue slices encompassing most of the hypothalamus are prepared from male Sprague-Dawley rats. Horizontal slice maps showing the major nuclei and fiber tracts are provided. Horizontal tissue slices contain many hypothalamic nuclei as well as the medial forebrain bundle, a large fiber tract interconnecting these nuclei. Three water-perfused thermodes directly beneath the tissue slices are used to produce discrete thermal stimulations of rostral, middle, and caudal nuclear regions. Fine thermocouples monitor slice temperature over each thermode. Limiting microelectrode explorations to regions directly over a thermode eliminates the problems of temperature gradients and permits more accurate manipulation of temperature at the recording site. While this preparation is ideal for characterizing hypothalamic neuronal thermosensitivity, it is also appropriate for electrophysiological studies of other hypothalamic functional systems.

Altered brainstem structure of spontaneously hypertensive (SHR) rats.

Computerized morphometric analysis of soma cross-sectional areas of single neurons in selected brainstem nuclei revealed that significant structural differences exist between spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Neuronal sizes were significantly reduced in 5 of 9 brainstem regions of SHR's compared to WKY rats. Differences in cell densities were also found.

Dopamine effects on thermosensitive neurons in hypothalamic tissue slices.

To understand the role of hypothalamic dopamine in thermoregulation, single unit activity was recorded in vitro, from constantly perfused tissue slices of rat preoptic area and anterior hypothalamus, PO/AH. The firing rate and thermosensitivity of individual PO/AH neurons were determined before, during and after tissue perfusion with media containing dopamine. Dopamine excited 41% of the warm-sensitive neurons, inhibited 100% of the cold-sensitive neurons, and had no effect on 83% of the temperature-insensitive neurons. In addition, dopamine decreased the local thermosensitivity of most cold-sensitive neurons. There were no major differences between these neuronal types in terms of the time course or latency of dopamine's effect. These results are consistent with the hypothesis that dopamine is involved in hypothalamic synapses controlling thermoregulatory responses which oppose increases in body temperature.

Altered CNS neuroanatomical organization of spontaneously hypertensive (SHR) rats.

Compared to Wistar-Kyoto (WKY) normotensive control rats, spontaneously hypertensive (SHR) rats have significantly reduced brain weights (-10.6%) and brain volumes (-11.8%). Computerized morphometric analysis of soma cross-sectional areas of single neurons in 12 selected hypothalamic regions revealed significant differences between SHR and WKY animals. Neurons from the periventricular, medial and lateral preoptic nuclei and ventromedial hypothalamus show significantly increased soma cross-sectional areas in SHR animals when compared to normotensive controls. Cells located in the two circumventricular organs, organ vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), also showed significantly greater cross-sectional areas in the SHR. In contrast, neurons in the paraventricular and arcuate nuclei and dorsomedial hypothalamus were significantly smaller in spontaneously hypertensive rats when compared to normotensive controls. Only neurons in supraoptic nucleus, lateral and anterior hypothalamus have equivalent cross-sectional areas in WKY and SHR animals. Differences also exist in the number of cells in certain nuclei in SHR animals. Cell densities in periventricular preoptic nucleus, paraventricular nucleus, arcuate nucleus, ventromedial and anterior hypothalamus, organ vasculosum lamina terminalis and subfornical organ were reduced in SHR animals compared to WKY controls. Because of decreased brain weight and volume along with observed morphometric differences in individual neuronal soma size and cell densities, it is suggested that the SHR brain differs significantly from normotensive control rats. The differences may underlie some of the abnormalities in cardiovascular and endocrine regulation associated with neurogenic hypertension.

Neuronal thermosensitivity and survival of rat hypothalamic slices in recording chambers.

Several studies have examined the activity of neurons in hypothalamic tissue slices. The present experiments studied relationships between neuronal activity (firing rate and thermosensitivity) and tissue survival as a function of time and slice thickness. Rat hypothalamic tissue slices were sectioned at different thicknesses (350, 450, and 600 microm) and maintained in an oxygenated interface chamber which was perfused with artificial cerebrospinal fluid (ACSF). Electron and light microscopy were used to examine tissue morphology at different depths from the slice surfaces, and extracellular recordings were used to measure each cell's spontaneous activity and response to changes in temperature. Tissue damage was most evident at tissue layers nearest the gas-exposed surface. At 9 h in the chamber, 350 microm thick slices showed subtle changes in morphology with little difference between the gas-exposed and ACSF-exposed surfaces. In the 450 and 600 microm thick slices, tissue degeneration became more evident with increased damage at the gas-exposed surface. This damage extended fully into the tissue of the 600 microm section. There were no differences in firing rate or thermosensitivity between 350 and 450 microm slices; but in 600 microm slices, there were fewer spontaneously active neurons, although these neurons had a higher mean thermosensitivity. Based on the incidence of spontaneous activity and morphological integrity, the results suggest that electrophysiological experiments using 350 microm slices are preferable to experiments using thicker slices.

Neuronal sensitivities in preoptic tissue slices: interactions among homeostatic systems.

The preoptic area participates in many homeostatic systems, which include the regulation of body temperature, fluid and metabolite balance, and reproduction. Some preoptic neurons have been shown to be sensitive to either temperature, osmotic pressure, glucose, testosterone or estradiol. While previous studies have treated these as separate and distinct neuronal populations, this paper reviews recent experiments which show that many neurons have multiple sensitivities to these endogenous factors. Neurons in preoptic tissue slices were tested for their responses to changes in temperature, as well as various perfusion media containing 30 pg/ml testosterone or estradiol, low glucose (1.0 mM) or increased osmotic pressure (309 mosmol/kg). The steroid-sensitive, osmosensitive and glucosensitive neurons were not confined to the temperature insensitive neurons; but instead nearly half of the thermosensitive neurons responded to these nonthermal stimuli. In addition, many osmosensitive neurons showed glucosensitivity and steroid-sensitivity. This suggests that, even at the neuronal level, there is a basis for interactions between homeostatic systems.

Oxygen consumption of hypothalamic tissue slices after varying incubation periods.

In order to quantitate possible time-related changes in the viability of rat hypothalamic tissue slices, tissue oxygen consumption was measured after incubation periods ranging from 0-4 hours. There were no significant differences in mean tissue oxygen consumption between the various incubation periods; nor was there any trend indicating that oxygen consumption gradually decreases over time. Moreover, no regional differences were observed among the various rostral hypothalamic slices. One obvious trend, however, was that during the first two hours of each experiment, tissue oxygen consumption decreased briefly and then returned to normal higher levels. The exact occurrence of this transitory decrease varied from experiment to experiment; but the subsequent recovery in oxygen consumption was always complete by two hours of incubation. This initial transient decrease in tissue oxygen consumption may reflect the initial period of electrophysiological inactivity reported in several in vitro studies.

A slice chamber for intracellular and extracellular recording during continuous perfusion.

The design of a tissue slice perfusion system is described, and examples are given showing the stability of this system for intracellular and extracellular recordings during changes in perfusion media. The stability of this system is attributed to several features. Mini-drips serve to cushion transient changes in flow rate when switching from one medium to another. Solenoid valves are used to quickly switch perfusion media with minimal mechanical movement. A finely-controlled adjustable flow valve provides a uniform flow rate for all media. Constant tissue temperature is maintained by media perfusion through a thermoelectric Peltier assembly. In addition, a filter paper wick insures that the perfusate is constantly removed without movement in the tissue slices. With this design, the slices are supported on a net at the interface between the perfusion medium and a humidified, oxygenated atmosphere. This arrangement appears to be conducive to tissue viability and facilitates the placement of microelectrodes in the slices.

Properties of hypothalamic temperature sensitive and insensitive neurones.

Intracellular recordings show that some hypothalamic neurones are inherently warm sensitive and have branching dendrites that allow synaptic integration of different afferent pathways.

Laparoscopic myotomy for primary esophageal achalasia: prospective evaluation.

BACKGROUND/AIM: The feasibility and safety of the laparoscopic myotomy having been previously demonstrated, the purpose of this prospective study was to evaluate its effectiveness. MATERIALS AND METHODS: Eight patients with primary esophageal achalasia underwent a laparoscopic modified Heller's myotomy with a posterior fundoplication. Early post-operative course has been uneventful in all cases. Clinical, endoscopic, and manometric prospective evaluations were performed with a median follow-up of 21 months (range 4-40). RESULTS: Excellent or good clinical results were present in all cases. Endoscopic studies were normal in all cases and the post-operative esophageal manometry (n = 7) showed that the median pressure of the lower esophageal sphincter decreased to 8.5 mmHg (range 3-9) which was significant compared to the median pre-operative value of 35 mmHg (p < 0.01). CONCLUSION: Though this experience is limited, these mean-term results suggest that the laparoscopic myotomy is effective to treat achalasia. It combines the efficacy of surgery and the minimally invasive aspect of dilatations. Thus, a prospective controlled trial comparing laparoscopic myotomy and dilatations is needed.

Intrinsic membrane properties of pre-oromotor neurons in the intermediate zone of the medullary reticular formation.

Neurons in the lower brainstem that control consummatory behavior are widely distributed in the reticular formation (RF) of the pons and medulla. The intrinsic membrane properties of neurons within this distributed system shape complex excitatory and inhibitory inputs from both orosensory and central structures implicated in homeostatic control to produce coordinated oromotor patterns. The current study explored the intrinsic membrane properties of neurons in the intermediate subdivision of the medullary reticular formation (IRt). Neurons in the IRt receive input from the overlying (gustatory) nucleus of the solitary tract and project to the oromotor nuclei. Recent behavioral pharmacology studies as well as computational modeling suggest that inhibition in the IRt plays an important role in the transition from a taste-initiated oromotor pattern of ingestion to one of rejection. The present study explored the impact of hyperpolarization on membrane properties. In response to depolarization, neurons responded with either a tonic discharge, an irregular/burst pattern or were spike-adaptive. A hyperpolarizing pre-pulse modulated the excitability of most (82%) IRt neurons to subsequent depolarization. Instances of both increased (30%) and decreased (52%) excitability were observed. Currents induced by the hyperpolarization included an outward 4-aminopyridine (4-AP) sensitive K+ current that suppressed excitability and an inward cation current that increased excitability. These currents are also present in other subpopulations of RF neurons that influence the oromotor nuclei and we discuss how these currents could alter firing characteristics to impact pattern generation.

Role of the preoptic-anterior hypothalamus in thermoregulation and fever.

Lesion and thermal stimulation studies suggest that temperature regulation is controlled by a hierarchy of neural structures. Effector areas for specific thermoregulatory responses are located throughout the brain stem and spinal cord. The preoptic region, in and near the rostral hypothalamus, acts as a coordinating center and strongly influences each of the lower effector areas. The preoptic area contains neurons that are sensitive to subtle changes in hypothalamic or core temperature. Preoptic thermosensitive neurons also receive a wealth of somatosensory input from skin and spinal thermoreceptors. In this way, preoptic neurons compare and integrate central and peripheral thermal information. As a result of this sensory integration and its control over lower effector areas, the preoptic region elicits the thermoregulatory responses that are the most appropriate for both internal and environmental thermal conditions. Thermosensitive preoptic neurons are also affected by endogenous substances, such as pyrogens. By reducing the activity of warm-sensitive neurons and increasing the activity of cold-sensitive neurons, pyrogens cause fever, a state in which all thermoregulatory responses have elevated set-point temperatures.