Nitric oxide modulates the firing rate of the rat supraoptic magnocellular neurons

In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect Of L-arginine, a precursor for NO synthesis, and N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 mu M) and DL-2-amino-5-phosphonovaleric acid (DL-AP5) (30 mu M) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 mu M) and saclofen (30 mu M) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 mu M L-arginine decreased the firing rate from 3.7 +/- 0.6 Hz to 1.3 +/- 0.3 Hz. Conversely, 100 mu M L-NAME increased the firing rate from 3.0 +/- 0.3 Hz to 5.8 +/- 0.4 Hz. All points histogram analysis showed changes in resting potential from -58.1 +/- 0.8 mV to -62.2 +/- 1.1 mV in the presence of L-arginine and from -59.8 +/- 0.7 mV to -56.9 +/- 0.8 mV by L-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4 +/- 1.2 mV to 16.8 +/- 0.7 mV by L-arginine, but without significant changes by L-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell ( due to the. ring of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K(ATP) channel conductance (G(K,ATP)) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC(50) of approximately 4 mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca(2+)](i) waves to spread with a speed of approximately 80 mu m s(-1), similar to that observed experimentally in confocal [Ca(2+)](i) imaging.

Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca(2+) Channels From Secretory Vesicles

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS-C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca(2+)](i)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca(2+)](i). The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca(2+)](i) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes. Diabetes 59:1192-1201, 2010

Potassium Current in Mature Bovine Spermatozoa

Ion channels have been assigned a pivotal importance in various sperm functions and are therefore promising targets for contraceptive development. The lack of data on channel functionality and pharmacology has hampered this goal. This is a consequence of technical problems of applying electrophysiological techniques to spermatozoa due to their small size and form. By using a laminin coating to increase adherence of spermatozoa and nystatin in the patch pipette for pore formation, we have adapted the whole-cell recording technique to study currents in mature uncapacitated bovine spermatozoa. Employing these conditions, in the head region, patched spermatozoa could be transferred into the whole-cell configuration. For the first time we document an outward rectifying current in mature bovine spermatozoa was blocked by tetraethyl ammonium (TEA) chloride. The observation of a shift in the reversal potential as a response to changes in the extracellular concentration of K+ ions allowed us to identify this current as K+ selective. This result shows that K+ channels in the head region of mature uncapacitated bovine spermatozoa can be suitably investigated using the whole-cell recording patch-clamp technique.

Rehabilitation of severely resorbed edentulous mandible using the modified visor osteotomy technique

The prosthetic rehabilitation of an atrophic mandible is usually unsatisfactory due to the lack of support tissues, mainly bone and keratinized mucosa for treatment with osseointegrated implants or even conventional prosthesis. The prosthetic instability leads to social and functional limitations and chronic physical trauma decreasing the patient's quality of life. A 53-year-old female patient sought care at our surgical service complaining of impairment of her masticatory function associated with the instability of the lower total prosthetic denture. The clinical and complementary exams revealed edentulism in both arches, while the mandibular arch presented severe reabsorption resulting in denture instability and chronic trauma to the oral mucosa. The proposed treatment plan consisted in the mandibular rehabilitation with osseointegrated implants and fixed Brånemark's protocol prosthesis after mandibular reconstruction applying the modified visor osteotomy technique. The proposed technique offered predictable results for reconstruction of the severely resorbed edentulous mandible and posterior rehabilitation with osseointegrated implants.

Medida da visão em candelas: description of a psychophysical technique to quantify luminous intensity

OBJETIVO: Desenvolver um método e um dispositivo para quantificar a visão em candela (cd). Os estudos de medida da visão são importantes para todas as ciências visuais. MÉTODOS: É um estudo teórico e experimental. Foram descritos os detalhes do método psicofísico e da calibração do dispositivo. Foram realizados testes preliminares em voluntários. RESULTADOS: É um teste psicofísico simples e com resultado expresso em unidades do sistema internacional de medidas. Com a descrição técnica será possível reproduzir o experimento em outros centros de pesquisa. CONCLUSÃO: Os resultados aferidos em intensidade luminosa (cd) são uma opção para estudo visual. Esses resultados possibilitarão extrapolar medidas para modelos matemáticos e para simular efeitos individuais com dados aberrométricos.

Biópsia hepática por laparotomia paracostal em bovinos e búfalos

A técnica de biópsia hepática em ruminantes tem importante valor no diagnóstico clínico de doenças tóxicas e metabólicas, em especial nos desequilíbrios minerais. As técnicas mais comumente utilizadas restringem análises devido ao limitado volume de tecido obtido. No presente trabalho, avaliou-se o uso de uma técnica de biópsia hepática por laparotomia paracostal em bovinos e búfalos. Foram utilizados 10 bovinos e 10 búfalos hígidos. Os animais foram mantidos em estação, sedados com xilazina e infiltrados localmente com lidocaína e epinefrina. O acesso à cavidade abdominal foi realizado por meio de uma incisão dorso-ventral de 15cm no flanco direito, iniciada ventralmente (cerca de 4-5cm) ao processo transverso da 2a ou 3a vértebra lombar e situada caudalmente (cerca de 4cm) e paralelamente à 13a costela, obtendo-se visualização do fígado. Foi então realizado pinçamento do bordo caudal do órgão com pinça Doyen para remoção de fragmento hepático (2 a 4g). Procedeu-se o fechamento da cavidade abdominal como de rotina. Foram analisados os parâmetros bioquímicos e hematológicos antes do procedimento (tempo zero) e após 24 horas, 48 horas, 5 dias e 10 dias após a biópsia. Todas as variáveis bioquímicas estudadas retornaram aos valores basais 5 e 10 dias após o procedimento nos bovinos e búfalos, respectivamente. O tempo médio de cirurgia por animal foi de 25 minutos. A biópsia hepática por laparotomia paracostal demonstrou ser uma técnica eficaz e de baixo risco à saúde dos animais, permitindo a coleta de suficiente quantidade de tecido hepática para realização de múltiplas análises.

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen

Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as "selective medium" and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10-1 to 10-4) between the selective and non-selective media at different dilution factors. At 1/10th dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100th dilution, (3%, 2/80) and (49%, 39/80) and at 1/1,000th dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

Damage assessment of the equine sperm membranes by fluorimetric technique

To validate a practical technique of simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in equine spermatozoa three fluorescent probes (PI, FITC-PSA and MITO) were associated. Four ejaculates from three stallions (n=12) were diluted in TALP medium and split into 2 aliquots, 1 aliquot was flash frozen in liquid nitrogen to induce damage in cellular membranes. Three treatments were prepared with the following fixed ratios of fresh semen: flash frozen semen: 100:0 (T100), 50:50 (T50), and 0:100 (T0). A 150-µL aliquot of diluted semen of each treatment was added of 2 µL of PI, 2 µL of MITO and 80 µL of FITC-PSA; incubated at 38.5ºC/8 min, and sperm cells were evaluated by epifluorescent microscopy. Based in regression analysis, this could be an efficient and practical technique to assess damage in equine spermatozoa, as it was able to determine the sperm percentage more representative of the potential to fertilize the oocyte.

Evaluation technique for determining wheel performance in the grinding of aerospace materials

Nickel-based super alloys are used in a variety of applications in which high-temperature strength and resistance to creep, corrosion, and oxidation are required, such as in aircraft gas turbines, combustion chambers, and automotive engine valves. The properties that make these materials suitable for these applications also make them difficult to grind. Grinding systems for such materials are often built around vitrified cBN (cubic boron nitride) wheels to realize maximum productivity and minimum cost per part. Conditions that yield the most economical combination of stock removal rate and wheel wear are key to the successful implementation of the grinding system. Identifying the transition point for excessive wheel wear is important. The aim of this study is to compare the performance of different cBN wheels when grinding difficult-to-grind (DTG) materials by determining the 'wheel wear characteristic curve', which correlates the G-ratio to the calculated tangential force per abrasive grain. With the proposed methodology, a threshold force per grit above which the wheel wear rate increases rapidly can be quickly identified. A comparison of performance for two abrasive product formulations in the grinding of three materials is presented. The obtained results can be applied for the development of grinding applications for DTG materials.

Computational adjustment technique for digital mammographic images based on the digitizer characteristic curve

We evaluated the performance of a novel procedure for segmenting mammograms and detecting clustered microcalcifications in two types of image sets obtained from digitization of mammograms using either a laser scanner, or a conventional ""optical"" scanner. Specific regions forming the digital mammograms were identified and selected, in which clustered microcalcifications appeared or not. A remarkable increase in image intensity was noticed in the images from the optical scanner compared with the original mammograms. A procedure based on a polynomial correction was developed to compensate the changes in the characteristic curves from the scanners, relative to the curves from the films. The processing scheme was applied to both sets, before and after the polynomial correction. The results indicated clearly the influence of the mammogram digitization on the performance of processing schemes intended to detect microcalcifications. The image processing techniques applied to mammograms digitized by both scanners, without the polynomial intensity correction, resulted in a better sensibility in detecting microcalcifications in the images from the laser scanner. However, when the polynomial correction was applied to the images from the optical scanner, no differences in performance were observed for both types of images. (C) 2008 SPIE and IS&T [DOI: 10.1117/1.3013544]

Pulse train fluorescence technique for measuring triplet state dynamics

We report on a method to study the dynamics of triplet formation based on the fluorescence signal produced by a pulse train. Basically, the pulse train acts as sequential pump-probe pulses that precisely map the excited-state dynamics in the long time scale. This allows characterizing those processes that affect the population evolution of the first excited singlet state, whose decay gives rise to the fluorescence. The technique was proven to be valuable to measure parameters of triplet formation in organic molecules. Additionally, this single beam technique has the advantages of simplicity, low noise and background-free signal detection. (C) 2011 Optical Society of America

Intrahepatic Glissonian Approach for Pure Laparoscopic Left Hemihepatectomy

Background: Recent advances in laparoscopic devices and experience with advanced techniques have increased the indications for laparoscopic liver. Aim: The aim of this work was to present a video with technical aspects of a pure laparoscopic left hemi-hepatectomy (segments 2, 3, and 4) by using the intrahepatic Glissonian approach and control of venous outflow without hilar dissection or the Pringle maneuver. Patient and Method: A 63-year-old woman with a 5-cm solitary liver metastasis was referred for treatment. Four trocars were used. The left lobe was pulled upward and the lesser omentum was divided, exposing Arantius' ligament. This ligament is a useful landmark for the identification of the main left Glissonian pedicle. A small anterior incision was made in front of the hilum, and a large clamp was introduced behind the Arantius' ligament toward the anterior incision, allowing control of the left main sheath. Ischemic discoloration of the left liver was achieved and marked with cautery. The vascular clamp was replaced by a stapler. If ischemic delineation was coincident with a previously marked area, the stapler was fired. The left hepatic vein was dissected and encircled. Parenchymal transection and vascular control of the hepatic veins were accomplished with a Harmonic scalpel and an endoscopic stapling device, as appropriate. All these steps were performed without the Pringle maneuver and without hand assistance. Results: Operative time was 220 minutes with minimum blood loss. Hospital stay was 4 days. Pathology showed free surgical margins. The patient is alive with no signs of recurrence 18 months after the operation. Conclusion: Totally laparoscopic left hemihepatectomy is safe and feasible in selected patients and should be considered for patients with benign or malignant liver neoplasms. The described technique, with the use of the intrahepatic Glissonian approach and control of venous outflow, may facilitate laparoscopic left hemihepatectomy by reducing the technical difficulties in pedicle control and may decrease bleeding during liver transection.

Thermodiffusion in a multicomponent lyotropic mixture in the vicinity of the critical micellar concentration by using the Z-scan technique

Thermodiffusion in a lyotropic mixture of water and potassium laurate is investigated by means of an optical technique (Z scan) distinguishing the index variations due to the temperature gradient and the mass gradients. A phenomenological framework allowing for coupled diffusion is developed in order to analyze thermodiffusion in multicomponent systems. An observable parameter relating to the mass gradients is found to exhibit a sharp change around the critical micellar concentration, and thus may be used to detect it. The change in the slope is due to the markedly different values of the Soret coefficients of the surfactant and the micelles. The difference in the Soret coefficients is due to the fact that the micellization process reduces the energy of interaction of the ball of amphiphilic molecules with the solvent.

Spectrally resolved femtosecond Maker fringes technique

We present a femtosecond third-harmonic generation Maker fringes technique capable of simultaneously providing the magnitude of the cubic nonlinearity and the refractive index dispersion of optical materials. This technique takes advantage of the high intensity and broad spectral band of femtosecond pulses, but requires the use of a spectrometer to deconvolute the information contained in Maker fringes produced by the broad band light. (C) 2008 American Institute of Physics.