Non-invasive muscle contraction assay to study rodent models of sarcopenia.

BACKGROUND: Age-related sarcopenia is a disease state of loss of muscle mass and strength that affects physical function and mobility leading to falls, fractures, and disability. The need for therapies to treat age-related sarcopenia has attracted intensive preclinical research. To facilitate the discovery of these therapies, we have developed a non-invasive rat muscle functional assay system to efficiently measure muscle force and evaluate the efficacy of drug candidates. METHODS: The lower leg muscles of anesthetized rats are artificially stimulated with surface electrodes on the knee holders and the heel support, causing the lower leg muscles to push isometric pedals that are attached to force transducers. We developed a stimulation protocol to perform a fatigability test that reveals functional muscle parameters like maximal force, the rate of fatigue, fatigue-resistant force, as well as a fatigable muscle force index. The system is evaluated in a rat aging model and a rat glucocorticoid-induced muscle loss model RESULTS: The aged rats were generally weaker than adult rats and showed a greater reduction in their fatigable force when compared to their fatigue-resistant force. Glucocorticoid treated rats mostly lost fatigable force and fatigued at a higher rate, indicating reduced force from glycolytic fibers with reduced energy reserves. CONCLUSIONS: The involuntary contraction assay is a reliable system to assess muscle function in rodents and can be applied in preclinical research, including age-related sarcopenia and other myopathy.

Development of a Platform to Enable Fully Automated Cross-Titration Experiments.

In the triage of hits from a high-throughput screening campaign or during the optimization of a lead compound, it is relatively routine to test compounds at multiple concentrations to determine potency and maximal effect. Additional follow-up experiments, such as agonist shift, can be quite valuable in ascertaining compound mechanism of action (MOA). However, these experiments require cross-titration of a test compound with the activating ligand of the receptor requiring 100-200 data points, severely limiting the number tested in MOA assays in a screening triage. We describe a process to enhance the throughput of such cross-titration experiments through the integration of Hewlett Packard's D300 digital dispenser onto one of our robotics platforms to enable on-the-fly cross-titration of compounds in a 1536-well plate format. The process handles all the compound management and data tracking, as well as the biological assay. The process relies heavily on in-house-built software and hardware, and uses our proprietary control software for the platform. Using this system, we were able to automate the cross-titration of compounds for both positive and negative allosteric modulators of two different G protein-coupled receptors (GPCRs) using two distinct assay detection formats, IP1 and Ca2+ detection, on nearly 100 compounds for each target.

A synchronized emissive probe for time-resolved plasma potential measurements of pulsed discharges.

A pulsed emissive probe technique is presented for measuring the plasma potential of pulsed plasma discharges. The technique provides time-resolved data and features minimal disturbance of the plasma achieved by alternating probe heating with the generation of plasma. Time resolution of about 20 ns is demonstrated for high power impulse magnetron sputtering (HIPIMS) plasma of niobium in argon. Spatial resolution of about 1 mm is achieved by using a miniature tungsten filament mounted on a precision translational stage. Repeated measurements for the same discharge conditions show that the standard deviation of the measurements is about 1-2 V, corresponding to 4%-8% of the maximum plasma potential relative to ground. The principle is demonstrated for measurements at a distance of 30 mm from the target, for different radial positions, at an argon pressure of 0.3 Pa, a cathode voltage of -420 V, and a discharge current of about 60 A in the steady-state phase of the HIPIMS pulse.