The worldwide prevalence of neuromuscular disease is growing and now approximates that of other diseases such as Parkinson’s (1). Diagnosis, basic research, and therapeutic trials will benefit from ready access to human skeletal muscle samples from a wide range of subjects. Diagnosis of neuromuscular disorders often relies on clinical observations, but is accelerated with molecular studies of the muscle itself. Older adults may have muscle biopsies to investigate acute muscle weakness, but the unrelenting age-induced declines in muscle mass or function rarely prompt a muscle biopsy. This is despite our current lack of a clear etiology for muscle aging. The advent of therapeutic interventions for some neuromuscular diseases requires validated biomarkers of treatment efficacy or toxicity.
There is a growing interest in tissue and cellular heterogeneity, qualities that can only be assessed in primary tissue samples. Skeletal muscle is no exception as many pathological changes in muscle are characteristically focal or segmental e.g., fiber loss, fiber type switching, fibrosis, and group atrophy (2). Muscle stem cells are another focal phenomenon in skeletal muscle that rely on an understanding of the in vivo niche and, therefore, require primary tissue samples (3).
In the standard open muscle biopsy, the typical amount of muscle collected is approximately (5 to 550mg and sizes range from 0.5 x 1 cm to 5 × 2 cm)(4). This procedure is costly (>$3,000), requires an operating room, specially qualified personnel, and general or regional anesthesia, which may be of greater risk in younger and older individuals. The procedure and recovery times are lengthy and results in a considerable scar (e.g., ~30mm) (4). All of the above generally limit open biopsies to a single site in a single muscle and decrease the likelihood of later biopsies. A common alternative to open biopsy, particularly in the research setting, is needle biopsy using the Bergstrom or vacuum-enabled University College Hospital (UCH) needles (Figure 1). These needles allow for the use of local anesthetic in an office-based setting for the sampling of small amounts of human muscle. The sample size for the Bergstrom needle is typically ~100mg, but size can vary widely with operator skill (5-7). Recovery time from the needle biopsy is generally within a day with only a 1cm scar remaining. The challenges of Bergstrom needle biopsy include the technical difficulty, lack of sample consistency, and small sample size.
An additional challenge with human muscle biopsies, and muscle histological studies in general, is the effect of freeze/thaw damage on microscopic tissue morphology. It is thought that slow freezing of muscle tissue allows formation of ice crystals that disrupt the muscle fiber architecture in subsequent histological preparations. Prevention of freeze/thaw damage in muscle typically focuses on freezing muscle samples as quickly as possible. Liquid nitrogen (-170 Celsius) is the coldest available freezing substrate and is readily available in research facilities and clinical offices (where it is used by dermatologist for ablative procedures on the skin). The freezing rate of an object in liquid nitrogen is hampered by the formation of a gaseous layer of evaporated liquid nitrogen that surrounds the object. The gaseous layer effectively insulates the object from the liquid nitrogen and reduces heat transfer. To circumvent this, muscle samples are often frozen in a liquid nitrogen-chilled isopentane, an organic solvent with a freezing temperature of -160 Celsius. Because it does not form a gaseous layer around the sample, the isopentane transfers heat from the sample more quickly. Isopentane is not readily available, particularly in clinical settings, has a low flash point, and is difficult to maintain at the correct temperature when chilled in liquid nitrogen. In settings where large numbers of samples need to be processed quickly (e.g., mouse muscle harvest at a specific time point) or in clinical setting, the use of isopentane can be prohibitive. An ideal freezing protocol for muscle samples would: 1) optimize histological quality with the avoidance of freeze damage; 2) avoid the use of specialized reagents or equipment that are not readily available or have safety issues; 3) facilitate correct tissue orientation and sufficient embedding material to facilitate serial sections transverse to the long axis of the muscle fibers.
To simplify and improve the consistency of our human muscle biopsy protocol in older adults, we have deployed a self-contained, vacuum-assisted biopsy device (i.e., the Vacora Biopsy System) and developed a novel freezing protocol that uses a tissue cassette and freezing of samples directly in liquid nitrogen. The Vacora System greatly improves the amount, quality and consistency of the older human muscle biopsies while the new muscle freezing method is quicker and provides high quality histology. Combined, these approaches have broad potential utility in expanding skeletal muscle aging research by providing consistently high quality primary human tissues.