Sihler's Staining Research Articles

Phrenic Nerve Distribution in the Rabbit Diaphragm and Morphometric Analysis of Nerve Branches

The best method to evaluate the pathogenesis of diaphragmatic disorders is to demonstrate the distribution pattern of the phrenic nerve in the diaphragm. For this purpose the branching pattern and the microanatomic features of the phrenic nerve were observed in six rabbits. All diaphragms were stained by using Sihler's stain method. The phrenic nerve divided into three to four branches when entering the diaphragm. These branches were classified as sternal, anterolateral, posterolateral and crural. The crural branches were the thickest whereas the anterolateral branches were the thinnest. Knowledge about the distribution pattern of the phrenic nerve may be important in surgical approach to the diaphragm.

Observation of the Relationship between the Shape of Skeletal Muscles and Their Nerve Distribution Patterns: A Transparent and Microanatomic Study

There are many gaps in the understanding of the neuroanatomy of skeletal muscles with regards to the nerve distribution pattern and shape of the muscles. This study was designed to examine the entire intramuscular nerve-distribution patterns of various human skeletal muscles. The relationships among nine skeletal muscles with various architecture (rhomboid major, biceps brachii, flexor pollicis longus, rectus femoris, sternohyoid, trapezius, masseter, digastric muscles) and their nerve-distribution patterns were investigated in four fetal cadavers using the Sihler staining method. The diameter and number of extramuscular (main) and major nerve branches, the number of minor nerve branches, and anastomoses were examined and evaluated statistically. With regards to the number of extramuscular (main) nerve branches, the rhomboid major muscle resembled the flexor pollicis longus, trapezius, masseter, and sternohyoid muscles, and the anterior belly of the digastricus muscle (p > 0.05), whereas it was significantly different from the rectus femoris, the posterior belly of digastricus, and the long and short heads of the biceps brachii (p < 0.05). Trapezius and masseter muscles were different from all of the skeletal muscles that were studied with regards to the diameter of main branches (p < 0.05). The masseter muscle had the largest diameter (p < 0.05). With regards to the number of minor nerve branches, the sternohyoid muscle was significantly different from all the skeletal muscles that were studied (p < 0.05) except the short head of the biceps brachii, rectus femoris, and the posterior belly of digastricus (p > 0.05). As for the number of neural anastomoses, the sternohyoid muscle was statistically different from all skeletal muscles that were studied (p < 0.05) except the masseter and trapezius muscles (p > 0.005). A surgeon's thorough knowledge of the relationship between the shape and nerve distribution pattern of skeletal muscles is important in successful reinnervation and regeneration of these muscles. It might also be useful in the field of muscle transplantation.

Intrinsic Properties of the Adult Human Mylohyoid Muscle: Neural Organization, Fiber-Type Distribution, and Myosin Heavy Chain Expression

The mylohyoid (MH) muscle plays a critical role in chewing, swallowing, respiration, and phonation. The present study was designed to test the hypothesis that the functional properties of the MH are reflected by its intrinsic specializations, including the neural organization, fiber-type distribution, and myosin heavy chain (MHC) expression. Adult human MH muscles were investigated to determine the nerve supply pattern using Sihler's stain, banding pattern and types of motor endplates using acetylcholinesterase (AChE) staining and silver impregnation, and muscle fiber type and MHC composition using immunocytochemical and immunoblotting techniques. The adult human MH was found to have the following neuromuscular specializations. First, the muscle was innervated by several branches of the MH nerve derived from the mandibular division of the trigeminal nerve. Each of the nerve branches supplied a distinct region of the muscle, forming a segmental innervation pattern. Second, the MH had a single motor endplate band which was located in the middle of the muscle length. Both en plaque and en grappe types of motor endplates were identified on the MH muscle fibers. Finally, the adult human MH fibers expressed unusual MHC isoforms (i.e., slow-tonic, alpha-cardiac, embryonic, and neonatal) which coexisted with the major MHC isoforms (i.e., slow type I, fast type IIa, and fast type IIx), thus forming various major/unusual (or m/u) MHC hybrid fiber types. The m/u hybrid fibers (84% of the total fiber population) were the predominant fiber types in the adult MH muscle. Determination of the neuromuscular specializations of the MH is helpful for better understanding of the muscle functions and for development of strategies to treat MH-related upper airway disorders.

Intrinsic muscles and distribution of the recurrent laryngeal nerve in the pig larynx.

To use the pig larynx in studies of laryngeal reinnervation, it is essential to have a clear understanding of its anatomy. We aimed to define the macroscopic anatomy of the intrinsic muscles and the course of the recurrent laryngeal nerve (RLN) in the pig larynx. Twelve large white pig larynges were used. Five larynges were preserved in formalin, then dissected to study the anatomy of the intrinsic muscles. Seven larynges were stained using the modified Sihler's staining technique, which results in nerves being stained dark purple while the remainder of the larynx is rendered translucent. The intrinsic muscles of the pig larynx were similar to those in the human. The RLN gives off a branch that enters the posterior cricoarytenoid muscle (PCA) on its deep surface and supplies the entire muscle, although the branching pattern of the nerve within the muscle varies considerably. These results facilitate detailed reinnervation studies in the pig laryngeal transplant model.

Intramuscular innervation of upper-limb skeletal muscles.

We studied 150 skeletal muscles from 8 upper limbs using the modified Sihler's staining technique. Based on the pattern of the intramuscular innervation and shape, the muscles were grouped into trapezoidal-shaped (Class I), spindle-shaped (Class II), and muscles that were combinations of these two classes (Class III). Such distinctions are clinically important for limb reconstruction procedures. Bipennate, spindle-shaped muscles with the aponeurosis of the tendons of insertion extending proximally into the muscle belly and Class III muscles with multiple tendons of origin may be split for separate independent functional transfers.

Distribution pattern of the human lingual nerve.

The tongue is an intricate organ with many functions. Despite the knowledge of the presence of muscular and neural connections in the tongue, a detailed neuroanatomical depiction of the nerves' topography in the tongue has not been demonstrated. The topography, branching patterns and neuronal interconnections of the lingual nerve were studied in five postmortem human tongues. They were stained with Sihler's stain, a technique that renders most of the tongue tissue translucent while counterstaining nerves. The lingual nerve reaches the tongue posterolaterally. There are two main branches off of the main trunk: the medial branch sends 2-4 small branches to the medial part of the ventrolateral tongue and the lateral branch runs along the lateral tongue border and sends 3-4 large branches to the anterior tip of tongue. Each subdivision gives off 2-5 distal branches. Both medial and lateral branches have interconnections with the proximal part of the hypoglossal nerve. One of the unexpected discoveries in this study was the high density of nervous fibers in the lateral aspect of the tongue as compared to the midline region. The average diameter of the main trunk of the lingual nerve is 3.5 mm. The medial and lateral branches average 1 mm in diameter, the more distal subdivisions measure 0.5-0.75 mm, and the lingual-hypoglossal interconnections measure 0.125-0.250 mm. In summary, this study provides the first detailed depiction of the topography of the human lingual nerve and its branches in situ, confirmation of lingual-hypoglossal nerve connection, and the first depiction of the high density of lingual nerve innervation in the lateral tongue.

Demonstration of the nerve distribution of the extraocularmuscles in rabbits (Oryctolagus cuniculus)

Purpose. The intramuscular nerve distribution of the extraocular muscles may be of utmost importance for better understanding of their physiologic and pathologic reactions. The aim of this study was to determine the entire intramuscular nerve distribution pattern of rabbit extraocular muscles by utilizing Sihler's staining technique. Methods. Six New Zealand rabbits were used in order to demonstrate the intramuscular nerve distribution of the extraocular muscles by using Sihler's staining method. Results. The number of extramuscular and intramuscular major nerve branches were higher in the inferior oblique muscle while the number of intramuscular minor nerve branches were higher in the superior oblique muscle when compared with the other extraocular muscles. The smallest number of extramuscular branch and intramuscular both major and minor branches were found in the medial rectus muscle. More complex anastomoses and a branching pattern were observed in the superior oblique and superior rectus muscle. The anastomosing nerve branches were observed to run in a "Y", "I" or "U"-shaped pattern in all of the extra-ocular muscles. Of all the extraocular muscles, the longest major nerve branches were observed in the retractor bulbi muscles. However, these branches had the smallest diameter. No morphological difference was observed between the two sides with regard to all the characteristics of the extraocular muscles. Conclusion. Sihler's neural staining technique could be quite useful in the demonstration of the intramuscular nerve distribution of extraocular muscles.

Branching patterns of rabbit oculomotor and trochlear nerves demonstrated by Sihler's stain technique

A modified Sihler's stain technique was used to visualize the branching patterns of oculomotor and trochlear nerves. The levator palpebrae, superior rectus, inferior rectus, medial rectus, inferior oblique, superior oblique and tensor trochlea muscles were isolated from the eyes of normal rabbits and processed using modified Sihler's technique. The distributions and terminal ramifications of the oculomotor and trochlear nerves were observed. Two distinct divisions and terminal branches of the oculomotor nerve were shown in detail together with the trochlear nerve distribution. The application of Sihler's technique enables researchers to trace nerve branching within relatively transparent muscles, whereas the nerve fibers are counterstained and clearly visible. This technique could be useful for detailed studies of the motor control of extraocular muscles.

Branching patterns of rabbit oculomotor and trochlear nerves demonstrated by Sihler's stain technique

Branching patterns of rabbit oculomotor and trochlear nerves demonstrated by Sihler's stain technique

Neuromuscular compartments and fiber-type regionalization in the human inferior pharyngeal constrictor muscle.

The inferior pharyngeal constrictor (IPC) muscle functions during swallowing, respiration, and vocalization. The most-caudal portion of the IPC is believed to be part of the functional upper esophageal sphincter (UES). We hypothesized that the caudal fibers of the human IPC may have enzyme-histochemical characteristics similar to those of the cricopharyngeus muscle, a major component of the UES. In this study, human IPC muscles obtained from autopsy were studied using Sihler's stain to examine innervation patterns, and using myofibrillar ATPase, NADH tetrazolium reductase (NADH-TR), and succinic dehydrogenase (SDH) techniques to investigate the distribution and oxidative capacity of the slow- (type I) and fast- (type II) twitch fibers in the muscle. The results showed that the human IPC consists of at least two neuromuscular compartments (NMCs): rostral and caudal. Each of the NMCs was innervated by a separate nerve branch derived from the pharyngeal branch of the vagus nerve. The rostral NMC is faster (39% type I, 61% type II) than the caudal NMC (70% type I, 30% type II). In addition, two histochemically-delineated fiber layers were identified in the human IPC: a slow inner layer (SIL) with predominantly type I fibers (66%), and a fast outer layer (FOL) with predominantly type II fibers (62%) (P < 0.01). However, the dimensions of both fiber layers and proportions of the muscle fiber types varied with the NMCs. Specifically, the ratio of the thickness of the SIL to FOL was approximately 2:1 for the caudal NMC and approximately 1:2 for the rostral NMC, respectively. In the SIL the type I fibers accounted for 84% for the caudal NMC and 69% and 44% for the lower and upper portions of the rostral NMC. In contrast, the type II fibers in the FOL accounted for 46% for the caudal NMC and 67% and 74% for the lower and upper portions of the rostral NMC, respectively (P < 0.01). The caudal NMC of the IPC shared histochemical characteristics with the cricopharyngeus muscle, in that it contained predominantly slow oxidative fibers. Overall, the caudal NMC and the SIL in the IPC had high NADH-TR and SDH activities. However, different patterns of oxidative enzyme activity were identified in both type I and type II fibers. This study provided histochemical evidence for the concept that the caudal NMC within the IPC contributes to the functional UES. In addition, the two histochemically-defined fiber layers in the IPC may be a specialized adaptation in humans to enable different upper-airway functions during respiration, swallowing, and speech.

Sensory nerve supply of the human oro- and laryngopharynx: a preliminary study.

To date, the details of human sensory innervation to the pharynx and upper airway have not been demonstrated. In this study, a single human oro- and laryngopharynx obtained from autopsy was processed with a whole-mount nerve staining technique, Sihler's stain, to determine its entire sensory nerve supply. The Sihler's stain rendered all mucosa and soft tissue translucent while counterstaining nerves. The stained specimen was then dissected and the nerves were traced from their origins to the terminal branches. It was found that the sensory innervation of the human pharynx is organized into discrete primary branches that innervate specific areas, although these areas are often connected by small neural anastomoses. The density of innervation varied, with some areas receiving almost no identifiable nerve supply (e.g., posterior wall of the hypopharynx) and certain areas contained much higher density of sensory nerves: the posterior tonsillar pillars; the laryngeal surface of the epiglottis; and the postcricoid and arytenoid regions. The posterior tonsillar pillar was innervated by a dense plexus formed by the pharyngeal branches of the IX and X nerves. The epiglottis was densely innervated by the internal superior laryngeal nerve (ISLN) and IX nerve. Finally, the arytenoid and postcricoid regions were innervated by the ISLN. The postcricoid region had higher density of innervation than the arytenoid area. The use of the Sihler's stain allowed the entire sensory nerve supply of the pharyngeal areas in a human to be demonstrated for the first time. The areas of dense sensory innervation are the same areas that are known to be the most sensitive for triggering reflex swallowing or glottic protection. The data would be useful for further understanding swallowing reflex and guiding sensory reinnervation of the pharynx to treat neurogenic dysphagia and aspiration disorders.

Neuromuscular specializations of the pharyngeal dilator muscles: II. Compartmentalization of the canine genioglossus muscle.

The genioglossus (GG) muscle is divided into horizontal and oblique compartments that are the main protrusor and depressor muscles of the tongue, respectively. In humans the GG plays an important role in speech articulation, swallowing, and inspiratory dilation of the pharynx. At present, little is known about the neuromuscular specializations of the GG in any mammal. This study examined the specializations of these compartments in the canine tongue using a variety of anatomical and histochemical techniques. Six canine GG muscles were sectioned and stained for myofibrillar ATPase to study muscle fiber types; five whole-mount GG muscles were stained for acetylcholinesterase (AChE) to study the distribution of motor endplates; and eight whole mount GG muscles were processed with Sihler's stain to study the entire nerve supply pattern. In addition, the arrangement of muscle fibers of the GG within the tongue was also determined (N = 3). The most notable difference between the compartments of the GG was their proportions of fast and slow twitch muscle fibers: the horizontal compartment contained 64% slow twitch muscle fibers compared to 41% in the oblique compartment. In addition, although the oblique compartment appeared to be grossly homogeneous, it could be divided into thirds by significant differences in the percentages of slow twitch fibers: posterior (23%), middle (15%), and anterior (56%; P < 0.05). The muscle fibers of the oblique GG within the tongue were found to be divided into medial and lateral layers that run vertically and transversely, respectively. The nerve supply to each third of the oblique GG formed a plexus with the anterior third being the densest. The innervation pattern of the oblique GG was also notable as terminal nerve branches coursing parallel to the muscle fascicles gave off perpendicular secondary branches along each motor endplate band. These secondary nerve branches connected the primary nerves and formed a regularly spaced grid throughout the compartment. Evidently, the two compartments of the GG exhibited different anatomical specializations. The horizontal had a slow muscle fiber profile and simple innervation pattern; these qualities are possibly related to its single force vector and respiratory related activity. The oblique compartment had a relatively fast muscle fiber profile with evidence for three separate functional subdivisions. The most anterior part was noticeably different, and was presumably specialized for fine motor control of the tip of the tongue. The vertically oriented fibers of the oblique GG within the tongue body may function as a midline depressor of the tongue, whereas its transversely oriented fibers could play a role in narrowing the tongue during other motor tasks.

Functional anatomy of the hypoglossal innervated muscles of the rat tongue: A model for elongation and protrusion of the mammalian tongue

This anatomical investigation in the rat was designed to illustrate the detailed organization of the tongue's muscles and their innervation in order to elucidate the actions of the muscles of the higher mammalian tongue and thereby clarify the protrusor subdivision of the hypoglossal-tongue complex. The hypoglossal innervated, extrinsic styloglossus, hyoglossus, and genioglossus and the intrinsic transversus, verticalis and longitudinalis linguae muscles were observed by microdissection and analysis of serial transverse-sections of the tongue. Sihler's staining technique was applied to whole rat tongues to demonstrate the hypoglossal nerve branching patterns. Dissections of the tongue demonstrate the angles at which the extrinsic muscles act on the base of the tongue. The Sihler stained hypoglossal nerves demonstrate branches to the styloglossus and hyoglossus emanating from its lateral division while branches to the genioglossus muscle exit from its medial division. The largest portions of both XIIth nerve divisions can be seen to enter the body of the tongue to innervate the intrinsic muscles. Transverse sections of the tongue demonstrate the organization of the intrinsic muscle fibers of the tongue. Longitudinal muscle fibers run along the entire circumference of the tongue. Alternating sheets of transverse lingual and vertical lingual muscles can be observed to insert into the circumference of the tongue. Most importantly in clarifying tongue protrusion, we demonstrate the transversus muscle fibers enveloping the most superior and inferior portions of the longitudinalis muscles. Longitudinal muscle fascicles are completely encircled and thus are likely to be compressed by transverse muscle fascicles resulting in elongation of the tongue. We discuss our findings in relation to biomechanical studies, that describe the tongue as a muscular hydrostat and thereby define the "elongation-protrusion apparatus" of the mammalian tongue. In so doing, we clarify the functional organization of the hypoglossal-tongue complex.

Neuromuscular organization of the canine tongue.

The tongue manipulates food while chewing and swallowing, dilates the airway during inspiration, and shapes the sounds of speech in humans. While performing these functions the tongue morphs through many complex shapes. At present it is not known how the muscles of the tongue perform these complex shape changes. The difficulty in understanding tongue biomechanics is partly due to gaps in our knowledge regarding the complex neuromuscular anatomy of the tongue. In this study the motor and sensory nerve anatomy of four canine tongues was studied with Sihler's stain, a technique that renders most of the tongue tissue translucent while counterstaining nerves. An additional tongue specimen was serially sectioned to provide a reference for the muscle structure of the tongue. The hypoglossal nerve (XII) has approximately 50 primary nerve branches that innervate all intrinsic and extrinsic tongue muscles. Two extrinsic muscles, the styloglossus and hyoglossus, are innervated by about three to four branches from the lateral division of the XII. The third extrinsic muscle, the genioglossus, is composed of oblique and horizontal compartments, which receive about ten nerve branches from the medial division of the XII. The intrinsic muscles are composed of many neuromuscular compartments. On each side, the superior longitudinal muscle had an average of 40 distinct muscle fascicles that spanned the length of the tongue. Each of the fascicles is supplied by a nerve branch. The inferior longitudinal muscle had a similar organization. Each of the transverse and vertical muscles is composed of over 140 separate muscle sheets, and every sheet is innervated by a separate terminal nerve. The muscle sheets from the vertical and transverse alternate their orientation 90 degrees throughout the length of the tongue. It is concluded that the intrinsic canine tongue muscles are actually composed of groups of neuromuscular compartments that are arranged in parallel (longitudinal muscles) or in a precise alternating sequence (transverse and vertical muscles). This arrangement suggests that the compartments from the different tongue muscles could cooperate to control the three-dimensional contractile state of their local area. This hypothesis could explain how many different tongue shapes are formed, and is supported by physiologic evidence.

Anatomy of the human internal superior laryngeal nerve.

The mucosa of the larynx contains one of the most dense concentrations of sensory receptors in the human body. This sensitivity is used for reflexes that protect the lungs, and even momentary loss of this function is followed rapidly by life-threatening pneumonia. The internal superior laryngeal nerve (ISLN) supplies the innervation to this area, and, to date, the distribution and branching pattern of this nerve is unknown. Five adult human larynges were processed by using Sihler's stain, a technique that clears soft tissue while counterstaining nerves. The whole-mount specimens were then dissected to demonstrate the branching of the ISLN from its main trunk down to the level of terminal axons. The human ISLN is divided into three divisions: The superior division supplies mainly the mucosa of the laryngeal surface of the epiglottis; the middle division supplies the mucosa of the true and false vocal folds and the aryepiglottic fold; and the inferior division supplies the mucosa of the arytenoid region, subglottis, anterior wall of the hypopharynx, and upper esophageal sphincter. Several dense sensory plexi that cross the midline were seen on the laryngeal surface of the epiglottis and arytenoid region. The human ISLN also appears to supply motor innervation to the interarytenoid (IA) muscle. A detailed map is presented of the distribution of the ISLN within the human larynx. The areas seen to receive the greatest innervation are the same areas that have been shown by physiological experiments to be the most sensate: the laryngeal surface of the epiglottis, the false and true vocal folds, and the arytenoid region. The observation that the human ISLN appears to supply motor innervation to the IA muscle is contrary to current concepts of the ISLN as a purely sensory nerve. These findings are relevant to understanding how the laryngeal protective reflexes work during activities like swallowing. The nerve maps can be used to guide surgical attempts to reinnervate the laryngeal mucosa when sensation is lost due to neurological disease.

Neuromuscular organization of the human upper esophageal sphincter.

The upper esophageal sphincter (UES) is a key component of swallowing, and yet, its anatomy and function are still incompletely understood. The UES is a functional entity that is composed of three muscles: the cricopharyngeal (CP) muscle, the inferior pharyngeal constrictor (IPC) muscle, and the upper esophageal (UE) muscle. This study compared the anatomy of the three muscles of the UES in nine human autopsy specimens. The variables examined included the pattern of motor end plates (acetylcholinesterase stain), the proportion of fast- and slow-twitch muscle fibers (myofibrillar adenosinetriphosphatase), and the details of their nerve supply (Sihler's stain). The results demonstrated that each variable is different in the three muscles. For example, the IPC muscle is innervated by the pharyngeal plexus, the CP muscle by both the pharyngeal plexus and the recurrent laryngeal nerve (RLN), and the UE muscle by the RLN. The IPC and CP muscles showed distinct motor end plate bands, while the horizontal part of the CP muscle also contained small and randomly scattered end plates. This latter pattern was present throughout the UE muscle. Analysis of the muscle fiber types of the UES revealed a type I (slow) predominance (89%) in the CP and UE muscles and a type II (fast) predominance (62%) in the IPC muscle. However, the IPC muscle is composed of two layers: a fast, thick, outer layer (90% type II) and a slow, thin, inner layer (85% type I). The implications of these findings for the diagnosis and treatment of UES dysfunction will be discussed.

Innervation pattern of the temporalis muscle.

The purpose of this article is to describe the neural anatomy of the temporalis muscle as dissected along the intramuscular temporal fascial plane. This sagittal plane is a natural cleavage plane of the muscle, which is explored along with its relationship to the deep temporal nerve. Eight temporalis muscle specimens were removed in their entirety from 8 preserved cadavers. The muscles were selected based on whether they were grossly intact prior to procurement for processing. The muscle specimens were then processed over a 3-month period using Sihler's staining technique. Muscle dissection was performed along the intramuscular fascial plane under an operating microscope, taking care to preserve the underlying nerve and arterial anatomy. Dissections demonstrated an anterior and posterior division of the deep temporal nerve running within the deep portion of the muscle below the intramuscular fascial plane. This fascial layer provided a natural dissection plane to expose and evaluate the underlying nerve and arterial anatomy. In all specimens the deep temporal artery originated with the anterior temporal nerve and then branched into an anterior and posterior division. The innervation density and nerve caliber of the anterior portion of the muscle was much greater than that of the posterior, correlating with a greater anterior muscle bulk. This may have implications in differences in fiber type and functional regionalization of the muscle. The results of this anatomic study support the finding of an anterior and posterior division of the deep temporal nerve within the deep portion of the temporalis muscle. In addition, differences in the innervation density and muscle bulk lend credence to the possibility of regional muscle specialization. The natural cleavage plane of the intramuscular temporal fascia may have clinical ramifications for temporalis myofascial flaps while preserving the underlying neural anatomy to allow for normal residual temporalis muscle function.

Neuromuscular specializations of the pharyngeal dilator muscles: I. Compartments of the canine geniohyoid muscle.

Little is known about the structure and innervation of the geniohyoid muscle (GH), which is an important pharyngeal dilator muscle activated in swallowing and respiration. The neuromuscular specializations of the canine GH were studied in detail by using a combination of histological, histochemical, and anatomical techniques. First, hematoxylin and eosin staining, Gomori's rapid one-step trichrome stain, and silver impregnation were used to determine the terminations of muscle fibers and existence of fibrous septa within the muscle (n = 8). Second, myofibrillar ATPase staining was employed to document the muscle fiber type distribution (n = 8). Finally, Sihler's stain (n = 10) and wholemount acetylcholinesterase staining (n = 8) were used to examine the distribution of the nerve supply within the muscle. The canine GH is divided into rostral and caudal compartments, which are arranged in series and separated by a transverse fibrous septum. Each compartment receives its own primary nerve branch, which supplies a separate motor endplate zone. The rostral compartment is innervated bilaterally, whereas the caudal compartment is innervated ipsilaterally. The rostral compartment was composed of significantly more type I (slow twitch) muscle fibers (56%) than the caudal compartment (25%). The canine GH is composed of two in-series neuromuscular compartments rather than a single muscle as traditionally believed. This anatomical finding suggests that these two compartments may function independently under different physiological conditions.

Respiratory activity of the rat posterior cricoarytenoid muscle.

An anatomic and electrophysiological study of the rat posterior cricoarytenoid (PCA) muscle is described. The intramuscular nerve distribution of the PCA branch of the recurrent laryngeal nerve was demonstrated by a modified Sihler's stain. The nerve to the PCA was found to terminate in superior and inferior branches with a distribution that appeared to be confined to the PCA muscle. Electromyography (EMG) recordings of PCA muscle activity in anesthetized rats were obtained under stereotaxic control together with measurement of phrenic nerve discharge. A total of 151 recordings were made in 7 PCA muscles from 4 rats. Phasic inspiratory activity with a waveform similar to that of phrenic nerve discharge was found in 134 recordings, while a biphasic pattern with both inspiratory and post-inspiratory peaks was recorded from random sites within the PCA muscle on 17 occasions. The PCA EMG activity commenced 24.6 +/- 2.2 milliseconds (p < .0001) before phrenic nerve discharge. The results are in accord with findings of earlier studies that show that PCA muscle activity commences prior to inspiratory airflow and diaphragmatic muscle activity. The data suggest that PCA and diaphragm motoneurons share common or similar medullary pre-motoneurons. The earlier onset of PCA muscle activity may indicate a role for medullary pre-inspiratory neurons in initiating PCA activity.

Modified Sihler's technique for studying the distribution of intramuscular nerve branches in mammalian skeletal muscle.

A largely forgotten technique initially designed by Sihler for staining nerve tissue has not been fully explored for staining intramuscular nerve branches in skeletal muscles. Fresh, long heads of triceps from locally bred New Zealand white rabbits were used for this study. Immediately after their removal, the muscles with their motor nerve branches from the radial nerve were fixed in 10% unneutralized formalin, followed by maceration and depigmentation in 3% aqueous potassium hydroxide, decalcification in Sihler's solution I, micro-dissection, staining in Sihler's solution II, destaining in Sihler's solution I, neutralization in 0.05% lithium carbonate, and clearance in increasing concentrations of glycerin. A clear three-dimensional orientation of the distribution of the intramuscular nerve branches within the muscle belly was visualized. It was found in all specimens that the long head of triceps in the rabbit was constantly innervated by three main intramuscular nerve branches and each of them supplied different amounts of muscle fibers with some variation. The Sihler's neural staining technique can be applied to the study of the distribution of intramuscular nerve branches in limb skeletal muscles. Extension of the technique may be utilised in the identification of neuromuscular compartments in skeletal muscles. Such information may be usefully applied in free muscle transfer of segments of skeletal muscle.