Sihler's whole mount nerve staining technique: a review

Abstract

Sihler's stain is a whole mount nerve staining technique that renders other soft tissue translucent or transparent while staining the nerves. It permits mapping of entire nerve supply patterns of organs, skeletal muscles, mucosa, skin, and other structures after the specimens are fixed in neutralized formalin, macerated in potassium hydroxide, decalcified in acetic acid, stained in Ehrlich's hematoxylin, destained in acetic acid, and cleared in glycerin. The unique advantage of Sihler's stain over other anatomical methods is that all the nerves within the stained specimen can be visualized in their three-dimensional positions. To date, Sihler's stain is the best tool for demonstrating the precise intramuscular branching and distribution patterns of skeletal muscles, which are important not only for anatomists, but also for physiologists and clinicians. Advanced knowledge of the neural structures within mammalian skeletal muscles is critical for understanding muscle functions, performing electrophysiological experiments and developing novel neurosurgical techniques. In this review, Sihler's stain is described in detail and its use in nerve mapping is surveyed. Special emphasis is placed on staining procedures and troubleshooting, strengths and limitations, applications, major contributions to neuroscience, physiological and clinical significance, and areas for further technical improvement that deserve future research.

Activation of peripheral nerve fibers by electrical stimulation in the sole of the foot

“…Sihler’s technique [17] was used to stain the myelin of the nerves in the sole of the foot. Innervation density at the heel, arch, and forefoot (under the distal part of the second metatarsal) was assessed (Figure 1).…”

“…The maceration (immersion in KOH) took 5–6 weeks. Following the maceration the specimens were rinsed and decalcified in Sihler’s solution 1 (acetic acid solution [17]). The decalcification took 2–3 weeks.…”

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures

“…However, one major disadvantage is that supra-vital MB staining reveals different neural structures non-specifically, bleaches rapidly, is highly temperature sensitive and is unstable following chemical fixation (Müller, 1989). On the other hand, Sihler's whole mount staining technique is considered a prominent tool for demonstrating intramuscular nerve branching and distribution patterns in skeletal muscles, skin, and mucosa (Mu and Sanders, 2010). Sihler's method stains axons in dark blue and the rest non-nervous tissues remain translucent, thus making the nerve branches visible in whole mount preparations.…”

The clinical anatomy of the musculotendinous part of the diaphragm

“…An et al used Sihler's stain to more accurately map the phrenic nerve distribution of the diaphragm. Sihler's stain highlights nerves in dark blue while making the surrounding muscle tissue translucent, therefore, allowing easy visualization of the full network of nerves [13]. Knowing the metromorphic estimations of a patient's muscular and tendinous regions can be beneficial to a surgeon following Merendino's guidelines in determining where and how much to incise in order to prevent any catastrophic neuronal injury.…”

“…The formation of a phrenic nerve distribution map allows for maximum prevention of diaphragmatic function impairment [10]. Traditionally, gross dissection has been used to describe the innervation of skeletal muscles [13]; however, the diameter of the nerve decreases quickly as it branches distally, and it becomes difficult to distinguish nerve from blood vessels [1]. An et al used Sihler's stain to more accurately map the phrenic nerve distribution of the diaphragm.…”