Muscles from euthanized mice (under IACUC protocol approved by the University of Pennsylvania) were used in two different sets of experiments. One was in determining the effects of two different high potassium solutions on the ultrastructure of intact muscle fibers, and the second in devising the best approach for the preservation of needle biopsy material. First, the extensor digitorum longus (EDL) muscles were carefully dissected, pinned to a Sylgard dish, exposed to a solution that replaced extracellular sodium with potassium and chelated extracellular calcium. Two different solutions were used: either a ‘KCl’ solution [150 mM KCl, 5 mM MgCl2, 3 mM ethylene glycol tetraacetic acid (EGTA), 10 mM phosphate buffer, pH 7.2] or a ‘K acetate’ solution (150 mM K acetate, 5 mM MgSO4, 10 mM EGTA, 10 mM phosphate buffer, pH 7.2). Muscles were kept for ~3 min at room temperature in one of these two solutions and then fixed in freshly prepared 3.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for ~1 h. Secondly, using a small size needle (semiautomatic 14-gauge needle biopsy device: Vantage GS, Zamar S.r.l., Suzzara, Italy; Precisa 1310, HS, Precisa Medico Corp., Latina, Italy) () biopsies were taken from the gastrocnemius and the masseter. The latter muscle was more suitable as the needle was relatively large for the leg muscle of the mouse. The samples that were expelled directly from the needle into the ‘K acetate’ solution at room temperature contained several small coherent bundles of fibers with different orientations and the fibers appeared mostly wavy. Individual bundles of parallel fibers were separated by gentle teasing, straightened out by ‘combing’ and/or stretching out with tweezers or syringe needles while viewing under a dissecting microscope and then immersed in fixative solution as described above. The fixed bundles were stored at 4°C, for different periods of time.
Figure 1 Semiautomatic needle biopsy device for TPNB. Shown is the semiautomatic Tru-cut type system for performing multiple biopsies on skeletal muscle. The external cutting cannula has been removed in order to take the muscle specimen. The insert (upper left (more ...)
We further obtained three small biopsy samples from the vastus lateralis (VL) muscle from 32–66-year-old male healthy volunteers (indicated as M32, 32 years old; M33, 41 years old; M34, 66 years old, respectively) using the TPNB procedure as described in our previous study [Pietrangelo et al
)]. The study was approved by the Ethics Committee of the University of Chieti-Pescara (approval protocol no. 1233/06 COET). Each subject provided written informed consent. Biopsy was performed under local anesthesia using 2 ml carbocaine (20 mg/ml; AstraZeneca S.P.A., Basiglio, Italy) following skin sterilization with betadine. We used semiautomatic needle biopsy devices as for the mouse, but 13-gauge in size. The cylinder of the muscle thus obtained had a cross-sectional area of ~3 mm2
and a length of ~4 mm. Despite the small needle diameter, the TPNB VL muscle specimens were of adequate size and good quality. The specimens were immediately immersed in ‘K acetate’ solution and then treated as for the mouse.
All fixed muscles were further rinsed in buffer, post-fixed in buffered 2% osmium tetroxide (OsO4), and the block was then stained in saturated uranyl acetate and embedded in Epon 812. Sections (~40 nm thick) were cut using a Leica Ultracut R microtome (Leica Microsystems, Vienna, Austria) using a Diatome diamond knife (Diatome Ltd., Biel, Switzerland) and stained with lead citrate solution. The sections were imaged in using a Phillips 410 electron microscope (Philips Electron Optics, Mahwak, NJ, USA) with a Hamamatsu C4742-95 digital camera (Advanced Microscopy Techniques, Chazy, NY, USA).
Some fibers were separated from the mouse K-acetate treated biopsy bundles following osmium fixation. After gentle teasing to separate them, the fibers were whole-mounted in 100% glycerol under a coverslip and imaged using a Nikon microscope equipped with phase contrast optics and a Nikon Digital Sight DS-Fi1 camera.
The canonical number of three individuals for the human samples is sufficient to assess the quality of structural preservation based on the criteria shown.