Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

Background Many neuronal and glial diseases have been associated with changes in metabolism. Therefore, metabolic reprogramming has become an important area of research to better understand disease at the cellular level, as well as to identify targets for treatment. Model systems are ideal for interrogating metabolic questions in a tissue dependent context. However, while new tools have been developed to study metabolism in cultured cells there has been less progress towards studies in vivo and ex vivo. New Method We have developed a method using newly designed tissue restraints to adapt the Agilent XFe96 metabolic analyzer for whole brain analysis. These restraints create a chamber for Drosophila brains and other small model system tissues to reside undisrupted, while still remaining in the zone for measurements by sensor probes. Results This method generates reproducible oxygen consumption and extracellular acidification rate data for Drosophila larval and adult brains. Single brains are effectively treated with inhibitors and expected metabolic readings are observed. Measuring metabolic changes, such as glycolytic rate, in transgenic larval brains demonstrates the potential for studying how genotype affects metabolism. Comparison with Existing Methods and Conclusions Current methodology either utilizes whole animal chambers to measure respiration, not allowing for targeted tissue analysis, or uses technically challenging MRI technology for in vivo analysis that is not suitable for smaller model systems. This new method allows for novel metabolic investigation of intact brains and other tissues ex vivo in a quick, and simplistic way with the potential for large-scale studies.

SUMMARY1. A study was made ofthe effectiveness ofsynapses formed by foreign and original nerves during reinnerv -,tion of skeletal muscle of the newt Triturus cri8tatu-8. The extensor cranialis nerve (e.c.n.) of the forelimb was implanted into the humeroantebrachialis muscle (biceps) which was denervated by cutting or crushing the forelimb flexor nerve (f.f.n.).2. Although biceps became innervated by the implanted nerve, neuromuscular transmission was abnormal. The ratio ofthe tensions developed by biceps during single and repetitive (50 Hz) stimulation of e.c.n. was lower than either that obtained in normal biceps or during stimulation of f.f.n. after it had regenerated. Similarly, the mean quantal content of e.p.p.s evoked in biceps during stimulation of e.c.n. were lower (m = 17 1) than those evoked in normal muscles (m = 74 6) or during stimulation of the regenerated f.f.n. (m = 40 4).3. Although the implanted e.c.n. had innervated biceps, after 2-3 months a sprout had grown out of the side of the nerve to reinnervate the extensor digitorum communis muscle (e.d.c.) of the forearm. The mean quantal content of e.p.p.s evoked in this muscle by stimulation of e.c.n. (m = 32 2) was higher than that of those e.p.p.s evoked by stimulation of the same nerve in biceps (m = 17'1). 4. The results suggest that the synapses formed when a muscle is innervated by an inappropriate nerve are less effective than those formed when reinnervation by the correct nerve occurs. This may account for the tendency of the inappropriate synapses to regress following reinnervation by the correct nerve. In addition however, in the newt there seem to exist mechanisms which ensure that regenerating nerves reinnervate their correct muscles.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community's investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer's two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.