An Integrated Platform for in vivo Electrophysiology in Spatial Cognition Experiments.

Spatial cognition research requires behavioral paradigms that can distinguish between different navigational elements, such as allocentric (map-like) navigation and egocentric (e.g., body centered) navigation. To fill this need, we developed a flexible experimental platform that can be quickly modified without the need for significant changes to software and hardware. In this paper, we present this inexpensive and flexible behavioral platform paired with software which we are making freely available. Our behavioral platform serves as the foundation for a range of experiments, and though developed for assessing spatial cognition, it also has applications in the non-spatial domain of behavioral testing. There are two components of the software platform, 'Maze' and 'Stim Trigger'. Both programs can work in conjunction with electrophysiology acquisition systems, allowing for precise time stamping of neural events with behavior. The Maze program includes functionality for automatic reward delivery based on user defined zones. 'Stim Trigger' permits control of brain stimulation via any equipment that can be paired with an Arduino board. We seek to share our software and leverage the potential by expanding functionality in the future to meet the needs of a larger community of researchers.

Cue polarization and representation in mouse home base behaviors.

The nature of the representation guiding spatial navigation has been investigated extensively; however, most of this work has used behavioral tasks that involved learning the location of food reward or an escape platform. In contrast, relatively few studies have focused on the spatial representation of a home base, a ubiquitous feature of open-field behavior, and its ability to be encoded relative to environmental cues. The current set of experiments investigated acquisition and retention of the location of home base establishment. In general, proximal cues anchored the position of the home base during acquisition sessions across all four experiments. Although mice established a home base during retention sessions, previous experience did not influence its position during retention sessions. These observations demonstrate that stimulus control of home base position depends on access to proximal cues. Further work is needed to determine the extent that home base establishment may provide a framework to encode goal-directed spatial behaviors.

Leydig cell insufficiency in hypospermatogenesis: a paracrine effect of activin-inhibin signaling?

Clinical findings and a variety of experimental models indicate that Leydig cell dysfunction accompanies damage to the seminiferous tubules with increasing severity. Most studies support the idea that intratesticular signaling from the seminiferous tubules to Leydig cells regulates steroidogenesis, which is disrupted when hypospermatogenesis occurs. Sertoli cells seem to play a pivotal role in this process. In this review, we summarize relevant clinical and experimental observations and present evidence to support the hypothesis that testicular activin signaling and its regulation by testicular inhibin may link seminiferous tubular dysfunction to reduced testosterone biosynthesis.

In vitro aerodynamic characterization of the dose emitted during nebulization of tobramycin high strength solution by novel and jet nebulizer delivery systems.

BACKGROUND: Chronic infections with Pseudomonas aeruginosa are a leading cause of morbidity in patients with cystic fibrosis (CF). The aim of tobramycin inhalation therapy in CF patients with chronic pulmonary infection is to deliver high amounts of drug directly to the site of infection. TOBI(®) is a tobramycin nebulizer solution (300 mg/5 ml) approved by FDA for maintenance therapy for patient with CF. The 20% tobramycin sulfate solution was reported as the optimal and maximal concentration. METHODS: Nebulization of high strength tobramycin solution (20% tobramycin sulfate) (HSTS) has been assessed in this study by using different selected high performance nebulizer delivery systems: two different designs of jet nebulizers, and three new nebulizers based on vibrating mesh technology. The aerosol particle size distribution and output characteristics were measured for in vitro performance assessment of the nebulizer systems. The methodology was adapted from the current European standard, EN 13544-1:2001E. RESULTS: The particle size distribution characteristic measurements showed that all tested nebulizers may be suitable for inhalation of HSTS. The mean (SD) of highest percentage of fine particles (<5 µm) was 77.64 (2.3) % for Sidestream(®), at flow rate 16 L/min. The highest respirable inhaled mass was for Pari LC Plus(®) combined with PariBoyN(®) compressor, with mean (SD) 90.85 (8.6) mg. The mean (SD) of highest drug wastage percentage was 63.9 (3.9) % for Sidestream(®) jet nebulizer combined with compressed air cylinder at flow rate 16 L/min, while the lowest was 2.3 (0.26) % for NE-U22 Omron(®) (high frequency). CONCLUSIONS: The HSTS can be nebulized by all tested nebulisers but the high frequency NE-U22 Omron(®) and Aeroneb Go(®) are more efficient. When the HSTS compared to TOBI(®), the respirable inhaled dose was increased to more than 73%.

Microemulsion high performance liquid chromatography (MELC) method for the determination of terbutaline in pharmaceutical preparation.

A robust and sensitive microemulsion HPLC (MELC) method using oil-in-water microemulsion mobile phase was developed and used for the determination of terbutaline in Bricanyl(®) Turbuhaler. The applicability of microemulsion as an eluent for reversed phase HPLC was examined. In addition, the effect of operating parameters on the separation behaviour was studied. The samples were injected into C18 Spherisorb (250mm×4.6mm×5µm) columns at 25°C using a flow rate of 1ml/min. The mobile phase was 95.5% aqueous orthophosphate buffer (adjusted to pH 3 with orthophosphoric acid), 0.5% ethyl acetate, 1.5% Brij35, and 2.5% 1-butanol, all w/w. The terbutaline peak was detected by fluorescence, using excitation and emission wavelengths of 267 and 313nm, respectively. The accuracy of method was >99% and the calibration curve was linear (r(2)=0.99). The limit of detection (LOD) and limit of quantitation (LOQ) were 8µg/L and 26µg/L, respectively. The intra-day and inter-day precisions (in term of % coefficient of variation) were<1.46% and <0.97%, respectively. The influence of the composition of the microemulsion system was also studied and the method was found to be robust with respect to some changes of the microemulsion components. The microemulsion HPLC method has been applied to determine the content of the emitted dose and the fine particle dose of terbutaline in a Bricanyl(®) Turbuhaler.

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate.

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.

Validation of high-performance liquid chromatography assay for quantification of formoterol in urine samples after inhalation using UV detection technique.

A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb (250 mm x 4.6 mm x 5 microm) analytical column maintained at 30 degrees C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r2=0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n=6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n=10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.

Optimising reversed-phase liquid chromatographic separation of an acidic mixture on a monolithic stationary phase with the aid of response surface methodology and experimental design.

An optimization strategy for the separation of an acidic mixture by employing a monolithic stationary phase is presented, with the aid of experimental design and response surface methodology (RSM). An orthogonal array design (OAD) OA(16) (2(15)) was used to choose the significant parameters for the optimization. The significant factors were optimized by using a central composite design (CCD) and the quadratic models between the dependent and the independent parameters were built. The mathematical models were tested on a number of simulated data set and had a coefficient of R(2) > 0.97 (n = 16). On applying the optimization strategy, the factor effects were visualized as three-dimensional (3D) response surfaces and contour plots. The optimal condition was achieved in less than 40 min by using the monolithic packing with the mobile phase of methanol/20 mM phosphate buffer pH 2.7 (25.5/74.5, v/v). The method showed good agreement between the experimental data and predictive value throughout the studied parameter space and were suitable for optimization studies on the monolithic stationary phase for acidic compounds.

Experimental design for a basic mixture on a fluorinated packing. The effect of composition of the mobile phase.

An optimization methodology is introduced for investigating the separation and the retention behavior of analytes on a new fluorinated reversed-phase packing. Ten basic compounds were selected as test probes to study the predictive models developed by using SPSS and MATLAB software. A two-level orthogonal array design (OAD) was used to extract significant parameters. The significant factors were optimised using a central composite design to obtain the quadratic relationship between the dependent and the independent variables. Using this strategy, response surfaces were derived as the 3D and contour plots, and mathematical models were defined for the separation. The models had a satisfactory coefficient (R(2) > 0.97, n = 16). For the test compounds, the best separation condition was: MeCN/30 mM phosphate buffer pH 7.1(55.5:44.5, v/v) and 10 basic solutes were resolved in 22 min. The significant influence of the concentration of buffer shows that different mechanisms of separation for basic compounds on the fluorinated packing exist compared with a common ODS stationary phase.

Modeling the electrophoretic mobility of analytes in binary solvent electrolyte systems in capillary electrophoresis using an artificial neural network.

An artificial neural network (ANN) methodology was used to model the electrophoretic mobility of basic analytes in binary solvent electrolyte systems. The electrophoretic mobilities in pure solvent electrolytes, and the volume fractions of the solvents in mixtures were used as input. The electrophoretic mobilities in mixed solvent buffers were employed as the output of the network. The optimized topology of the network was 3-3-1. 32 experimental mobility data sets collected from the literature were employed to test the correlation ability and prediction capability of the proposed method. The mean percentage deviation (MPD) between the experimental and calculated values was used as an accuracy criterion. The MPDs obtained for different numerical analyses varied between 0.21% and 13.74%. The results were also compared with similar calculated mobilities which were derived from the best multiple linear model from the literature. From these results it was found that the ANN methodology is superior to the multiple linear model.

Determination of the bioavailability of gentamicin to the lungs following inhalation from two jet nebulizers.

AIMS: To determine the bioavailability of gentamicin to the lung following inhalation from two jet nebulizers. METHODS: Serial urine samples were obtained from 10 volunteers after a 80 mg dose given orally, nebulized from a Pari LC + (PARI) and MicroNeb III (MN) devices, or after a 40 mg intravenous dose. In vitro aerodynamic characteristics of the nebulized doses were also determined. RESULTS: The mean (SD) absolute gentamicin lung bioavailalibility following delivery by PARI and MN devices was 1.4 (0.4) and 1.7 (0.5) %. The mass median aerodynamic diameter (MMAD) of the drug particles from the PARI and MN systems was 8.6 (0.6) and 6.7 (0.5) microm and the corresponding fine particle doses (FPD) were 10.2 (2.8) and 11.7 (1.5) mg. CONCLUSIONS: The MMAD and FPD data reflect the poor lung deposition of gentamicin identified by urinary excretion.

Electrophoretic behavior of alprenolol in mixed solvent electrolyte systems.

The electrophoretic mobilities of alprenolol have been determined in a mixed solvent background electrolyte system containing sodium acetate (40 mM)+acetic acid (40 mM) as buffering agent and different volume fractions of water, methanol and ethanol using capillary electrophoresis. The mobility of alprenolol has been used to test the prediction capability of a model trained by previously reported mobility data of five beta-blocker drugs at the same electrophoretic conditions. The average percentage mean deviations (APMD) between experimental and predicted values were used as an accuracy criterion. The APMD (+/-SD) obtained for alprenolol data in binary/ternary solvent electrolyte system employing the mobility values in mono-solvent buffers was 4.37 (+/-3.50)% and the corresponding value for an ab initio prediction method was 7.65 (+/-4.30)%.

Mathematical representation of electrophoretic mobility of basic drugs in ternary solvent buffers in capillary zone electrophoresis.

The electrophoretic mobilities of two beta-blocker drugs, i.e., labetalol and atenolol, have been determined in a mixed solvent background electrolyte system containing sodium acetate+acetic acid as buffering agent and different volume fractions of water, methanol and ethanol using capillary electrophoresis. The produced data and three other sets collected from a recent work are employed to study the accuracy and prediction capability of a mathematical model to calculate the electrophoretic mobility with respect to the volume fractions of the solvents in the mixture. The results show that the proposed model is able to correlate/predict the mobility within an acceptable error range and it is possible to use the model in industry to achieve the optimum solvent composition for the buffer where using a ternary solvent system is required. The average percentage deviations (APDs) obtained for correlated and predicted data points are 0.71-2.48 and 1.72-4.39%, respectively. The accuracy of the proposed model is compared with that of a mixture response surface method and the results show that the proposed model is superior from both correlation and prediction points of view. The possibility of calculation of the mobility of chemically related drugs in water-methanol-ethanol mixtures using the proposed model is also shown and the produced prediction APD is approximately 8%.

Calculation of electrophoretic mobility in ternary solvent electrolyte systems.

Electrophoretic mobility of salmeterol and phenylpropanolamine in capillary electrophoresis has been determined using acetate buffer containing different concentrations of water, methanol and acetonitrile. Maximum electrophoretic mobilities for salmeterol and phenylpropanolamine have been observed at water-methanol-acetonitrile (5:50:45, v/v) and (3:60:37, v/v), respectively, while minimum mobilities of both compounds occurred at methanol-acetonitrile (30:70, v/v). The generated experimental data have been used to evaluate a mathematical model to compute the electrophoretic mobility of the analytes. The proposed model reproduced the mobility data with mean percentage deviations within 1-4%.

Calculation of electrophoretic mobility in mixed solvent buffers in capillary zone electrophoresis using a mixture response surface method.

The electrophoretic mobilities of three beta-blocker drugs, practolol, timolol and propranolol, have been measured in electrolyte systems with mixed binary and ternary water-methanol-ethanol solvents with acetic acid/sodium acetate as buffer using capillary electrophoresis. The highest mobilities for the analytes studied have been observed in pure aqueous, the lowest values in ethanolic buffers. The measured electrophoretic mobilities have been used to evaluate the accuracy of a mathematical model based on a mixture response surface method that expresses the mobility as a function of the solvent composition. Mean percentage error (MPE) has been computed considering experimental and calculated mobilities as an accuracy criterion. The obtained MPE for practolol, timolol and propranolol in the binary mixtures are between 0.9 and 2.6%, in the ternary water-methanol-ethanol solvent system the MPE was about 2.7%. The MPE values resulting from the proposed equation lie within the experimental relative standard deviation values and can be considered as an acceptable error.

Nonaqueous capillary electrophoretic analysis of hexetidine in a commercial liquid formulation.

A non-aqueous capillary electrophoresis method has been proposed to analyse hexetidine and its degradation products in a commercial liquid pharmaceutical formulation. The separation was achieved using a 37 cm fused silica capillary with a 75 microns internal diameter, an electrolyte of 120 mM sodium acetate buffer in methanol, at 16 kV and 25 degrees C. The results show that the proposed method is able to resolve hexetidine from its degradation products and/or impurities.

Sarcoma of the mitral valve causing coronary arterial occlusion in children.

Primary tumors of the cardiac valves are rare. One of the most common reasons that left-sided cardiac tumors come to clinical attention is embolization to the systemic circulation. We present two children who suffered left coronary arterial occlusion due to embolization of a sarcoma of the mitral valve. A 6-year-old female who had been admitted to the hospital after cerebrovascular embolization of a fragment of sarcoma of the mitral valve experienced sudden cardiovascular collapse due to occlusion of the left coronary artery. She was placed on extracorporeal membrane oxygenation, and underwent coronary embolectomy and resection of the tumor from the mitral valve and its tendinous cords. Left ventricular function did not improve, and she underwent orthotopic heart transplantation. On follow-up 32 months after transplant, the patient is well, with no evidence of recurrence of or metastasis from the tumor. The tumor arose from the leaflets and tendinous cords of the mitral valve, and was composed grossly of multiple white nodules. Histopathologic evaluation disclosed fragments composed predominantly of peripheral spindle cells in an extensive fibromyxoid stroma. The mildly pleomorphic cells of the tumor gradually blended with adjacent pieces of the mitral valvar leaflet and tendinous cords. Immunohistochemical studies revealed strong staining for vimentin, smooth muscle actin, muscle specific actin, and myoglobin, suggesting myogenic differentiation. The other patient was a 2 1/2-year-old female who died suddenly at home. Grossly and histologically, the tumor was essentially identical to the first case, and there was a 3 cm string-like extension passing into the orifice of the left coronary artery. To put the cases in context, we compare them with other descriptions of this rare type of tumor.