Number, distribution, and morphologic particularities of encapsulated proprioceptors in pig extraocular muscles.

PURPOSE: To analyze qualitatively and quantitatively the total complement of encapsulated proprioceptors (Golgi tendon organs [GTOs] and neuromuscular spindles) in pig extraocular muscles (EOMs). METHODS: EOMs of four pigs of different ages were prepared for light microscopic histochemical and immunohistochemical analysis and for transmission electron microscopy. RESULTS: GTOs and muscle spindles were numerous in pig EOMs. GTOs were found to be distributed in aponeurotic expansions of the distal and proximal EOM tendons, being more numerous in the distal aponeurosis than in the proximal aponeurosis. The total number of GTOs was higher in the recti EOMs (100-128) than in the oblique EOMs (45-61). Spindles were distributed over the entire muscle length. In each EOM the number of muscle spindles (142-333) exceeded those of GTOs. The morphology of the GTOs was variable. In addition to collagen bundles, approximately one third of the GTOs contained intracapsular muscle fibers that resembled the multiply innervated fiber type. Intracapsular muscle fibers entered the poles of the GTOs and either terminated inside the receptors in collagen bundles or exited the GTOs at the opposite poles. Nerve terminals were numerous in each GTO and established intimate contacts with collagen fibrils. CONCLUSIONS: Most structural particularities formerly observed in GTOs of rhesus monkey and sheep EOMs are also present in GTOs of pig EOMs. The high number of GTOs with their typical nerve terminals indicates functional importance. During muscle activity, afferent signals from GTOs and muscle spindles may provide sufficient information about eye position.

Presence and structure of innervated myotendinous cylinders in rabbit extraocular muscle.

Innervated myotendinous cylinders (IMCs) in rabbit extraocular muscles (EOMs) were identified for the first time. The nature of IMC nerve terminals was demonstrated by means of electron microscopy and double fluorescent staining. The distal EOM portions of four rabbits of different age and sex were prepared for transmission electron microscopy and for double-fluorescent labelling. Antibody against neurofilament H and alpha-bungarotoxin were applied on longitudinal cryostat sections of distal myotendinous junction. IMCs were consistently and frequently observed at the distal myotendons of each EOM. More than 30 IMCs were counted in two medial recti of a 6 month and 3 year old rabbit. IMCs were enveloped by two to three layered capsules of fibrocytes. Each IMC contained the terminal portion of one multiply-innervated muscle fibre and its corresponding tendon. The tendon compartment of an IMC was entered by a single myelinated nerve fibre (2-3 microm in diameter). Inside the IMC, this nerve fibre ramified into up to four preterminal branches. Nerve terminals exclusively established contacts with the muscle fibre at its junction with the tendon fibrils. Nerve terminals contained mitochondria and a multitude of clear vesicles. Within the synaptic cleft a basal lamina was always present. alpha-Bungarotoxin labelled the muscle side of these myoneural contacts. Nerve terminals exhibited neither age nor sex differences. Among all species so far investigated, rabbit IMCs are unique by exhibiting exclusively myoneural terminal contacts. Based on fine structure and alpha-bungarotoxin binding, myoneural contacts in rabbit IMCs are almost certainly motor, as previously observed only in human IMCs. Thus, rabbit IMCs are supposed to have a predominant effector function.

Innervated myotendinous cylinders in human extraocular muscles.

PURPOSE: To analyze palisade endings and their end organs, the so-called innervated myotendinous cylinders (IMCs), of human extraocular muscle (EOM) in more detail and to clarify with the help of double-fluorescent labeling and electron microscopy whether terminals in IMCs are sensory, serving proprioception. METHODS: EOMs obtained from a donated cadaver (66 years) and distal parts of EOMs from multiorgan donors (35, 53 years) were processed for double-fluorescent labeling. Antibodies against the protein gene product 9.5 and alpha-bungarotoxin labeling were used on cryostat sections of distal myotendons. EOMs from multiorgan donors (2, 17 years) were prepared for electron microscopy. RESULTS: Palisade endings investing muscle fiber tips established contacts with tendon fibrils and the muscle fiber attached. Alpha-bungarotoxin bound to myoneural contacts but not to axonal varicosities in the tendon compartment. Ultrastructural analysis revealed that palisade endings form IMCs, which were associated exclusively with multiply innervated global layer muscle fibers. IMCs consisted of a muscle fiber tendon junction, tightly enclosed by fibrocytes, and a supplying axon with preterminals and terminals. Terminals contained mitochondria, few neurotubuli, few neurofilaments, and accumulations of clear vesicles of uniform size. A basal lamina always intervened between axolemma and tendon fibrils as well as between axolemma and muscle fiber cell membrane. CONCLUSIONS: Palisade endings of human EOM form IMCs as in cat, monkey, and sheep. In contrast to animals, myoneural contacts in human IMCs are almost certainly motor, whereas terminals contacting tendon fibrils are arguably sensory. Thus, IMCs might be best described as "propriocept-effectors."

Presence and morphological variability of Golgi tendon organs in the distal portion of sheep extraocular muscle.

This study was undertaken to demonstrate the presence of Golgi tendon organs (GTOs) in the distal portion of sheep extraocular muscle (EOM) and to describe the morphological variability of these receptors. Extraocular muscles of a young and an old sheep were perfusion fixed and/or immersion fixed. Tissue was prepared for light microscopy and transmission electron microscopy. Immunohistochemistry was done to demonstrate the myosin pattern of the intracapsular muscle fibers of the GTOs. All GTOs in the distal portions of the sheep EOMs were located in a distinct muscle layer which was designated in a former investigation as the so-called peripheral patch layer. Each EOM of the young sheep contained GTOs; between four and 15 GTOs were counted in the rectus EOMs. Eight GTOs were found in the superior rectus of the old sheep. Golgi tendon organs in EOMs of the young and the old sheep did not differ in their morphology. In the young sheep the mean length of the GTOs was 447 +/- 132 microm (n = 60) and their mean width 101 +/- 26 microm (n = 60). In the old sheep values were 576 +/- 188 microm (mean length, n = 8) and 103 +/- 18 microm (mean width, n = 8). The GTOs were encapsulated by perineurial cells. In 12 GTOs, only collagen bundles were inside. In the remaining GTOs (56), intracapsular muscle fibers were present. Muscle fibers entered the proximal poles of the GTOs and either terminated inside the receptors or muscle fibers left the GTOs at their distal poles. These intracapsular muscle fibers were of the multiply-innervated type. In the GTOs variably shaped nerve terminals were found which contained a high number of mitochondria. In two GTOs, additionally, nerve terminals with aggregates of densely packed vesicles were present.

Muscle fiber types of human extraocular muscles: a histochemical and immunohistochemical study.

PURPOSE: To classify muscle fibers of human extraocular muscle (hEOM) and to compare them to previous studies on hEOM, as well as to nonhuman EOM classification schemes and skeletal muscle fiber types. METHODS: Muscle fibers cut in different muscle planes were followed on consecutive cross sections and typed with regard to their oxidative profile in combination with their myosin-immunohistochemical characteristics. RESULTS: Three zones were observed. In the global layer three muscle fiber types were observed: global layer singly innervated granular fibers, 79.4 +/- 8.1 microm (perimeter [values at midmuscle region] +/- SD); 59%; global layer singly innervated coarse fibers (80.3 +/- 10.8 microm; 21%); and global layer multiply innervated muscle fibers (4.1 +/- 9.7 microm; 21%). Two muscle fiber types were detected in the orbital layer: orbital layer singly innervated muscle fibers (54.1 +/- 8.5 microm; 83%) and orbital layer multiply innervated muscle fibers (53.5 +/- 7.6 microm; 17%). Three muscle fiber types were differed in the marginal zone: marginal zone singly innervated muscle fibers (83.1 +/- 15.8 microm; 56%), marginal zone multiply innervated low oxidative muscle fibers (84.4 +/- 23.3 microm; 7%), and marginal zone multiply innervated high oxidative muscle fibers (88.4 +/- 14.5 microm; 37%). Coexpressions of developmental myosin heavy chain isoforms and fast myosin heavy chain isoform were detected mainly in the marginal zone. CONCLUSIONS: hEOMs resemble mammalian EOM with regard to their organization. However, in addition to an inner global layer and an orbital layer an external marginal zone was described for the first time in hEOM in the present study.

Observations on the number, distribution and morphological peculiarities of muscle spindles in the tensor tympani and stapedius muscle of man.

Although the middle ear muscles have been described for the first time more than four hundred years ago their role in modulation and transmission of sound is not yet fully understood. Surprisingly very little is known about proprioceptors in these muscles, especially in man, although this seems to be the key to the understanding of their various functions. Therefore, the question for proprioceptive sensory organs in these muscles is still relevant. The tensor tympani and stapedius muscles of four women who had donated their bodies to our institute were taken. Complete serial sections of these muscles were made which were either impregnated with silver, stained with ferric oxide for acidic polysaccharides or incubated with antibodies against S-100 protein. Thereby four to eight (mean five) muscle spindles distributed along the whole muscle could be detected in the tensor tympani muscles. These spindles contain one to three intrafusal muscle fibres and their length ranges from 140 to 4270 microm (mean 1492.8 microm). Furthermore, in three stapedius muscles one to two (mean 1.7) muscle spindles were found. They were from 350 to 500 microm (mean 482 microm) long and contained only one intrafusal muscle fiber. Regarding the diameter of intrafusal muscle fibers in both, the tensor tympani as well as the stapedius muscle, no difference to extrafusal muscle fibers of these muscles could be detected. The structure of these spindles differs considerably from those found in skeletal muscles. The morphological findings presented strongly suggest that muscle spindles occur regularly in both middle ear muscles. The results presented herein are consistent with clinical findings obtained from electromyographic studies and may help to elucidate all functions the middle ear muscles might serve in man.

Fine structural analysis of extraocular muscle spindles of a two-year-old human infant.

PURPOSE: To clarify whether structural peculiarities formerly described in extraocular muscle (EOM) spindles of aged persons are already present in EOM spindles of a 2-year-old infant. METHODS: Distal halves of two EOMs obtained from a 2-year-old multiorgan donor were immersion-fixed and prepared for electron microscopy. The fine structure of 10 muscle spindles and of 1 "false spindle" was investigated. RESULTS: Extraocular muscle spindles of an infant 2 years of age had 2- to 4-layered outer capsules, 376 microm (range, 217-606 microm) long and 97 microm (range, 55-140 microm) wide. In 10 EOM spindles, 4 to 16 intrafusal muscle fibers (mean, 7.9) were present. From a total of 79 intrafusal fibers, 43 (54%) were nuclear chain fibers, and 8 of the 43 exhibited posttraumatic degenerative changes. Thirty-six (46%) intrafusal fibers indistinguishable from extrafusal fibers were called anomalous fibers. No nuclear bag fibers were found. Each muscle spindle contained a variable number of chain fibers and at least one anomalous fiber. Sensory nerve terminals were restricted to the 35 normal chain fibers but were absent from damaged chain fibers and from anomalous fibers. One "false spindle" without a periaxial space was composed of three anomalous fibers and one chain fiber, all of them devoid of sensory terminals. CONCLUSIONS: Most structural particularities of human EOM spindles described in aged persons are already found in the infant. They cannot be interpreted as age-related changes, but rather they represent specific features of human EOM spindles.

Carbocyanine postmortem neuronal tracing. Influence of different parameters on tracing distance and combination with immunocytochemistry.

Carbocyanines (DiI, DiA, DiO) are able to travel along membranes by diffusion and therefore have been used as postmortem neuronal tracers in aldehyde-fixed tissues. Surprisingly, detailed data on the influence of different parameters on tracing distances are still missing. This study was carried out to optimize tracing procedures and to reveal the validity of the combination of postmortem tracing with immunocytochemistry. Carbocyanine crystals were applied to the cervical spinal cord, sciatic nerves, and brachial plexuses of humans and guinea pigs. Incubation in the dark at 37C for 12-15 weeks proved optimal to achieve longest tracing distances (28.9 +/- 2.2 mm) in human and animal tissues. Longer incubation times and incubation temperatures higher than 37C did not result in longer tracing distances. No differences were evident between adult and newborn animals and between central and peripheral nervous system. The diffusion coefficient for DiI was calculated to be 2.5 x 10(-7) cm2 sec-1. After application of DiI to nerves of guinea pig extraocular muscles, DiI-positive afferent perikarya were observed in the anteromedial part of the trigeminal ganglion. These perikarya were identified by calcitonin gene-related peptide immunoreactivity (CGRP-IR). The percentage of CGRP-IR neurons after tracing was concordant with the percentage of CGRP-IR in trigeminal ganglia exclusively processed for CGRP-IR without previous postmortem tracing. These results demonstrate carbocyanines to be specific tracers for exact neuronal mapping studies. (J Histochem Cytochem 46:901-910, 1998)

Presence and structure of innervated myotendinous cylinders in sheep extraocular muscle.

Innervated myotendinous cylinders (IMCs) were for the first time described in a sheep extraocular muscle (EOMs). They were found at the distal myotendinous junction of a medial rectus and were investigated by light and transmission electron microscopy. The IMCs are enveloped by a multi-layered capsule of fibrocytes and each contains the terminal portion of one multiply-innervated muscle fibre and its corresponding tendon. The tendinous compartment of the IMC is entered by a single nerve fibre which, inside, spreads into several terminal branches. Numerous terminal branches were found among the collagen fibrils but few on the muscle fibre tips. Nerve terminals contain mitochondria and are full of clear vesicles. Within the nerve terminals, vesicles are often concentrated in an area where the axolemma exhibits dense patches. Innervated myotendinous cylinders of sheep EOMs exhibit the same ultrastructural features as those earlier described as palisade endings or myotendinous cylinders in cat, monkey and man.

Sensory innervation of the guinea pig extraocular muscles: a 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate tracing and calcitonin gene-related peptide immunohistochemical study.

The sensory apparatus of the extraocular muscles attains special interest because of the great variation among different species with respect to the proprioceptors. The sensory innervation of the guinea pig extraocular muscles, lacking both muscle spindles and tendon organs, was investigated with a fluorescence double-labelling method. Primary sensory perikarya were assessed by postmortem application of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (Di-I) to the extraocular muscle nerves. Traced neurons were found in the ipsilateral ophthalmic part of the trigeminal ganglion. This is in line with findings in other species. Calcitonin gene-related peptide (CGRP) was detected immunohistochemically within the trigeminal ganglion. No somatotopic organization was observed for CGRP-like immunoreactive perikarya. Small (maximal diameter below 30 microm), medium (maximal diameter between 30 and 50 microm), and large (maximal diameter larger than 50 microm) trigeminal ganglion cells were found among the primary afferent perikarya from extraocular muscles. Among CGRP-like immunoreactive cells, only small and medium cells were observed. Double-labelling experiments indicated the CGRP content of primary afferents of the guinea pig extraocular muscles. The relationship to former morphological categories of ganglion cells is discussed. Primary afferent neurons with CGRP-like immunoreactivity might have efferent functions and might also be involved in inflammatory processes of extraocular muscles.

[Proprioception of extra-ocular muscles in the human: on the morphology of muscle spindles]. / Propriozeption aus äusseren Augenmuskeln des Menschen: Zur Morphologie ihrer Muskelspindeln.

BACKGROUND: Increasing importance is attributed to the proprioceptive innervation of extraocular muscles for the development of binocular vision. In the literature, data on proprioceptors in human extraocular muscles are rare and inconsistent. Therefore a detailed morphological analysis of spindles in human extraocular muscles appeared indicated. MATERIAL AND METHODS: Complete serial sections of all extraocular muscles of 3 human individuals, 67, 72, and 83 years of age, were alternately impregnated with silver, stained following Mowry and immunohistochemically stained for S100 or PGP 9.5. Distal parts of extraocular muscles from multiorgan donors, 2, 17, and 34 years of age, were prepared for electron microscopy. RESULTS: Spindles contained 1 to 18 intrafusal muscle fibers, 55% of which were nuclear chain fibers, 2% nuclear bag fibers, and 43% fibers exhibiting the same morphological features as extrafusal fibers. The latter had been previously described as "anomalous fibers" (Ruskell). This intrafusal type exclusively occurs in human extraocular muscle spindles. Ultrastructural analysis revealed morphologically normal sensory terminals on all 3 types of intrafusal fibers. Findings in a two-year-old individual were similar to those in aged individuals. CONCLUSIONS: Spindles in human extraocular muscles are specifically structured. Their special morphological features are also present during the development of binocular vision. Spindles are supposed to play a role in the (fine) control of eye movements.

Two fibromuscular transverse ligaments related to the levator palpebrae superioris: Whitnall's ligament and an intermuscular transverse ligament.

BACKGROUND: This study was performed to reinvestigate the detailed anatomy of the connective tissues related to the levator palpebrae superioris (LPS). MATERIALS AND METHODS: One hundred and twenty human orbits were prepared macroscopically or with the help of an operational microscope. Connective tissues related to the LPS were measured, removed, and prepared for histology. Different staining procedures including immunohistochemistry for alpha-smooth muscle actin were applied to analyze the quality of these tissues. RESULTS: Preparation consistently revealed two definite fibromuscular transverse ligaments related to the LPS, i.e., Whitnall's ligament (WL) and a definite intermuscular transverse ligament (ITL) underlying the LPS. The ITL exhibited a greater anterio-posterior extension than WL. In sagittal section, the ITL was observed slightly more posterior in the orbit than WL. Histologically, both ligaments consisted of collagenous fibers, elastic fibers, and smooth muscle fibers. WL was connected with the LPS by sagittal fibroelastic connecting strands. CONCLUSIONS: Two transverse ligaments of fibromuscular tissue, WL and the ITL, form a sleeve superior and inferior to the LPS. This sling is supposed to assist the LPS in its action and should be preserved at ptosis surgery.

Number and distribution of neuromuscular spindles in human extraocular muscles.

PURPOSE: To examine the number and distribution of muscle spindles in all extraocular muscles (EOMs) in humans. METHODS: Thirty-six EOMs were obtained after death from three persons 67, 72, and 83 years of age. Serial sections were made throughout the length of these muscles. Consecutive sections were stained with different methods. To discriminate true spindles from false spindles, light microscopic criteria were defined and were subject to ultrastructural investigation. A distal portion of a single EOM was gained from a multiorgan donor 17 years of age, processed for electron microscopy, and analyzed. RESULTS: Spindles were observed in all muscles studied, with the medial rectus exhibiting a mean of 18.8 spindles +/- 3.0 (+/- standard deviation), the lateral rectus 19.3 +/- 1.9, the superior rectus 15.8 +/- 2.5, the inferior rectus 34.0 +/- 4.4, , the superior oblique 27.3 +/- 8.2, and the inferior oblique 4.3 +/- 1.8 per muscle [corrected]. For each different human EOM, a typical distribution of spindles was observed in the persons examined. The ultrastructural investigation revealed sensory endings in structures primarily identified as spindles. CONCLUSIONS: By comparing 1 g of tissue, spindles are found to be at least as frequent in human EOM as in skeletal muscles known to have a high density of spindles. This fact and the peculiar distribution of spindles in human EOMs suggest that spindles are functionally important proprioceptors in EOM.