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.

Fig. 1

Posterior view of a harvested adult human hemipharynx processed with Sihler's stain showing entire pharyngeal plexus. Note that the nerves supplying the pharyngeal muscles were divided at the level of the nodose ganglion (N) of the vagus (X) nerve. Individual nerve branches can be traced from their origins to their terminals. CP, cricopharyngeus muscle; ESLN, external superior laryngeal nerve; IPC, inferior pharyngeal constrictor muscle; ISLN, internal superior laryngeal nerve; L-IX, lingual branch of the glossopharyngeal (IX) nerve; MPC, middle pharyngeal constrictor muscle; Ph-IX, pharyngeal branches of the IX nerve; Ph-X, pharyngeal branches of the X nerve; SPC, superior pharyngeal constrictor muscle. 3 ×.

Fig. 2

Posterior view of a Sihler's stained incompletely harvested adult human pharynx. Note that nerves forming the pharyngeal plexus were not transected at the level of the nodose ganglion of the cranial nerve X. Therefore, it is impossible to know which nerve branches come from the cranial nerve X or from other nerves. The abbreviations used in this figure are the same as those in Fig. 1. 3 ×.

Fig. 3

Ventral view of the anterior adult human tongue processed for Sihler's stain. Note that the human tongue is supplied by the hypoglossal (XII; motor), lingual (LN; sensory), and glossopharyngeal (IX; sensory) nerves, which are organized in a complex manner. 3 ×.

Fig. 4

A pair of human geniohyoid (GH) muscles processed with Sihler's stain. Note that the human geniohyoid on each side is composed of two neuromuscular compartments, medial (M) and lateral (L), that are arranged in parallel. Note that each of the geniohyoid compartments is supplied by a distinct nerve branch derived from the hypoglossal (XII) nerve. C, caudal; R, rostral. 3 ×.

Fig. 5

A pair of canine geniohyoid (GH) muscles processed with Sihler's stain. Note that the canine geniohyoid is composed of two neuromuscular compartments, rostral (R) and caudal (C) that are separated by a fibrous septum (S) and arranged in series. Note that each of the geniohyoid compartments is supplied by a distinct primary nerve branch derived from the hypoglossal (XII) nerve. 3 ×.

Fig. 6

Sihler's stained adult human hemipharynx showing that the pharyngeal muscles are composed of neuromuscular compartments arranged in layers, inner and outer. Note that both muscle layers are supplied by different nerve branches from the pharyngeal plexus. 6 ×.

Fig. 7

Dorsal mucosa of human posterior tongue processed with Sihler's stain showing the neural connection (arrow) between two sensory nerves, the lingual (LN) and glossopharyngeal (IX) nerves. FP, foliate papillae. 12 ×.

Fig. 8

Sihler's stained adult human interarytenoid (IA) muscle. Note that the interarytenoid muscle is innervated bilaterally by recurrent laryngeal nerve (RLN). 9 ×.

Fig. 9

Magnification of the rostral compartment within the canine geniohyoid muscle as shown in Fig. 5. Note that this compartment is innervated bilaterally. The nerve branches on both sides cross the midline and connect with each other to form a network. 9 ×.

Fig. 10

Sihler's stained adult human epiglottis. Note that the mucosa covering the laryngeal surface is supplied bilaterally by the internal superior laryngeal nerve. The terminal branches connect with each other to form a dense network. 9 ×.

Fig. 11

Microdissected dorsal mucosa of the anterior canine tongue processed with Sihler's stain showing that the lingual nerve terminals innervate taste buds (arrows). 9 ×.

Fig. 12

Sihler's stained posterior adult human tongue. Note that the glossopharyngeal (IX) nerve terminals innervate the circumvallate papillae (arrows). 9 ×.

Fig. 13

An adult human biceps brachii muscle processed with Sihler's stain. Note that Sihler's stain shows the intramuscular nerve branching and distribution in this large human limb muscle.

Fig. 14

Sihler's stained adult human cricothyroid muscle showing the neural connections (arrows) between two primary motor branches derived from a single nerve, the external superior laryngeal nerve (ESLN). Note that both primary nerve branches supply the rectus (R) and oblique (O) bellies of the cricothyroid muscle, respectively. 6 ×.

Fig. 15

Sihler's stained adult human posterior cricoarytenoid (PCA) muscle showing a neural connection (arrow) between two motor nerve branches, posterior cricoarytenoid branch and interarytenoid (IA) branch that are derived from a single nerve, the recurrent laryngeal nerve (RLN). 6 ×.

Fig. 16

Adult human cricopharyngeus (CP) muscle processed with Sihler's stain showing the neural communications (arrows) between two different motor nerves, recurrent laryngeal nerve (RLN) and pharyngeal plexus (PP). 9 ×.

Fig. 17

Sihler's stained adult human laryngeal muscles showing the neural connection (arrow) between two different motor nerves, external superior laryngeal nerve (ESLN) and recurrent laryngeal nerve (RLN). Note that one of the external superior laryngeal nerve branches passed through the cricothyroid (CT) muscle and gave off a branch to connect with the recurrent laryngeal nerve. TA, thyroarytenoid muscle. 6 ×.

Fig. 18

Sihler's stained adult human interarytenoid (IA) muscle showing the neural communications (arrows) between a motor nerve, recurrent laryngeal nerve (RLN) and the traditionally described sensory nerve, internal superior laryngeal nerve (ISLN). 6 ×.

Fig. 19

Sihler's stained canine posterior tongue illustrating the neural communications (arrows) between a sensory nerve (lingual nerve; LN) and a motor nerve (hypoglossal nerve; XII). IX, glossopharyngeal nerve; VP, circumvallate papillae. 6 ×.

Fig. 20

Sihler's stained adult human posterior tongue showing the neural connection (arrow) between two major nerve branches derived from a single sensory nerve, glossopharyngeal nerve (IX). VP, vallate papillae. 9 ×.

Fig. 21

Sihler's stained canine posterior tongue showing the neural connection (arrow) between two branches derived from different sensory nerves, lingual (LN) and glossopharyngeal (IX) nerves. 6 ×.

Fig. 22

Sihler's stained adult human pharynx showing the neural communications (arrows) surrounding the epiglottis (E) between two different sensory nerves, glossopharyngeal (IX) and internal superior laryngeal nerves (ISLN). 9 ×.