Proprioceptors in extraocular muscles.

Proprioception is the sense that lets us perceive the location, movement and action of the body parts. The proprioceptive apparatus includes specialized sense organs (proprioceptors) which are embedded in the skeletal muscles. The eyeballs are moved by six pairs of eye muscles and binocular vision depends on fine-tuned coordination of the optical axes of both eyes. Although experimental studies indicate that the brain has access to eye position information, both classical proprioceptors (muscle spindles and Golgi tendon organ) are absent in the extraocular muscles of most mammalian species. This paradox of monitoring extraocular muscle activity in the absence of typical proprioceptors seemed to be resolved when a particular nerve specialization (the palisade ending) was detected in the extraocular muscles of mammals. In fact, for decades there was consensus that palisade endings were sensory structures that provide eye position information. The sensory function was called into question when recent studies revealed the molecular phenotype and the origin of palisade endings. Today we are faced with the fact that palisade endings exhibit sensory as well as motor features. This review aims to evaluate the literature on extraocular muscle proprioceptors and palisade endings and to reconsider current knowledge of their structure and function.

Ultrasound-guided thread versus ultrasound-guided needle release of the A1 pulley: a cadaveric study.

PURPOSE: To assess and compare two ultrasound-guided, minimally invasive procedures to release the A1-pulley (needle release and thread release) regarding efficacy and safety in an anatomical specimen model. MATERIALS AND METHODS: Twenty-one ultrasound-guided needle releases and 20 ultrasound-guided thread releases were performed on digits of Thiel-embalmed anatomical specimens. A scoring system was developed to assess ultrasound visibility, intervention outcome (incomplete, almost complete, or full transection of the A1 pulley), and injury to adjacent structures (neurovascular structures, tendons, A2 pulley). Statistical analysis was performed to compare the score of the two groups (group 1: needle release,group 2: thread release). A P-value of ≤ 0.05 was considered significant. RESULTS: Needle release was completely successful in 15 cases (71.5%), almost complete release was achieved in four cases (19%), and incomplete transection occurred in two cases (9.5%). Thread release was completely successful in 17 cases (85%), and almost complete transection was observed in the remaining three cases (15%). In both procedures no neurovascular structures were harmed. Slight injury of flexor tendons occurred in two cases (9.5%) in needle release and in five cases (25%) in thread release. There were no significant statistical differences between the groups regarding ultrasound visibility, intervention safety and outcome, (P > 0.05). CONCLUSION: Ultrasound-guided needle release and ultrasound-guided thread release have similar success of release, both being effective and safe techniques for the release of the A1 pulley.

Spinal cord from body donors is suitable for multicolor immunofluorescence.

Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level. Obtaining fresh neuronal tissue from human organ donors is difficult and sometimes impossible. In anatomical body donations, neuronal tissue is dedicated to research purposes and because of its easier availability, it may be an alternative source for research. In this study, we harvested spinal cord from a single organ donor 2 h (h) postmortem and spinal cord from body donors 24, 48, and 72 h postmortem and tested how long after death, valid multi-color immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry is possible. We used general and specific neuronal markers and glial markers for immunolabeling experiments. Here we showed that it is possible to visualize molecularly different neuronal elements with high precision in the body donor spinal cord 24 h postmortem and the quality of the image data was comparable to those from the fresh organ donor spinal cord. High-contrast multicolor images of the 24-h spinal cords allowed accurate automated quantification of different neuronal elements in the same sample. Although there was antibody-specific signal reduction over postmortem intervals, the signal quality for most antibodies was acceptable at 48 h but no longer at 72 h postmortem. In conclusion, our study has defined a postmortem time window of more than 24 h during which valid immunohistochemical information can be obtained from the body donor spinal cord. Due to the easier availability, neuronal tissue from body donors is an alternative source for basic and clinical research.

Structural and molecular characteristics of axons in the long head of the biceps tendon.

The innervation of the long head of the biceps tendon (LHBT) is not sufficiently documented. This is a drawback since pathologies of the LHBT are a major source of shoulder pain. Thus, the study aimed to characterize structurally and molecularly nervous elements of the LHBT. The proximal part of 11 LHBTs was harvested intraoperatively. There were 8 female and 3 male specimens. Age ranged from 66 to 86 years. For structural analyses, nervous elements were viewed in the transmission electron microscope. For molecular characterization, we used general neuronal markers including antibodies against neurofilament and protein gene product 9.5 (PGP9.5) as well as specific neuronal markers including antibodies against myelin basic protein (MBP), calcitonin gene-related product (CGRP), substance P (SP), tyrosine hydroxylase (TH), and growth-associated protein 43 (GAP43). Anti-neurofilament and anti-PGP9.5 visualized the overall innervation. Anti-MBP visualized myelination, anti-CGRP and anti-SP nociceptive fibers, anti-TH sympathetic nerve fibers, and anti-GAP43 nerve fibers during development and regeneration. Immunolabeled sections were analyzed in the confocal laser scanning microscope. We show that the LHBT contains unmyelinated as well as myelinated nerve fibers which group in nerve fascicles and follow blood vessels. Manny myelinated and unmyelinated axons exhibit molecular features of nociceptive nerve fibers. Another subpopulation of unmyelinated axons exhibits molecular characteristics of sympathetic nerve fibers. Unmyelinated sympathetic fibers and unmyelinated nociceptive fibers express proteins that are found during development and regeneration. Present findings support the hypothesis that ingrowth of nociceptive fibers are the source of chronic tendon pain.

Axons giving rise to the palisade endings of feline extraocular muscles display motor features.

Palisade endings are nerve specializations found in the extraocular muscles (EOMs) of mammals, including primates. They have long been postulated to be proprioceptors. It was recently demonstrated that palisade endings are cholinergic and that in monkeys they originate from the EOM motor nuclei. Nevertheless, there is considerable difference of opinion concerning the nature of palisade ending function. Palisade endings in EOMs were examined in cats to test whether they display motor or sensory characteristics. We injected an anterograde tracer into the oculomotor or abducens nuclei and combined tracer visualization with immunohistochemistry and α-bungarotoxin staining. Employing immunohistochemistry, we performed molecular analyses of palisade endings and trigeminal ganglia to determine whether cat palisade endings are a cholinergic trigeminal projection. We confirmed that palisade endings are cholinergic and showed, for the first time, that they, like extraocular motoneurons, are also immunoreactive for calcitonin gene-related peptide. Following tracer injection into the EOM nuclei, we observed tracer-positive palisade endings that exhibited choline acetyl transferase immunoreactivity. The tracer-positive nerve fibers supplying palisade endings also established motor terminals along the muscle fibers, as demonstrated by α-bungarotoxin. Neither the trigeminal ganglion nor the ophthalmic branch of the trigeminal nerve contained cholinergic elements. This study confirms that palisade endings originate in the EOM motor nuclei and further indicates that they are extensions of the axons supplying the muscle fiber related to the palisade. The present work excludes the possibility that they receive cholinergic trigeminal projections. These findings call into doubt the proposed proprioceptive function of palisade endings.

Biomechanical characteristics of suture anchor implants for flexor digitorum profundus repair.

PURPOSE: To determine strength and failure characteristics of 2 suture anchors used to repair simulated flexor digitorum profundus avulsions during passive mobilization protocol simulation. METHODS: We simulated avulsion of the flexor digitorum profundus tendon in 30 distal phalanges from fresh-frozen human cadavers. Repair was performed with a 1.3 × 3.7 mm Micro-Mitek suture anchor (3-0 Orthocord suture) and a 2.2 × 4.0-mm Corkscrew suture anchor (2-0 FiberWire suture). All specimens were loaded cyclically from 2 to 15 N at 5 N/s for a total of 500 cycles. Samples were tested to failure at the completion of 500 cycles. Load at failure, load at first noteworthy displacement (> 2 mm), elongation of the system, gap formation at the tendon-bone interface, and the mechanism of failure were assessed. RESULTS: Suture failure at maximum load was the prevalent failure mechanism in both groups. No statistically significant difference in elongation of the tendon-suture complex was observed. The Corkscrew suture anchor showed a significantly superior performance in load to failure, load at first significant displacement, and gap formation at the tendon-bone interface. CONCLUSIONS: The significantly higher load capacity at first displacement (> 2 mm) and the significance of a lower gap formation at the repair site seem to be the most relevant clinical parameters. Based on this concept, the Corkscrew anchor may be superior biomechanically to the Micro-Mitek when considering an early passive mobilization protocol. CLINICAL RELEVANCE: The choice of an appropriate implant may influence the postoperative mobilization protocol and thereby improve currently reported success rates. Defining a biomechanically superior implant will provide an essential basis for further studies in flexor tendon repair research.

Ultrasound-guided minimally invasive cubital tunnel thread release: An experimental study.

INTRODUCTION: Cubital Tunnel Syndrome is characterized by the compression of the ulnar nerve in the cubital tunnel and can cause restrictions to the activities of daily living. Traditional treatment includes conservative methods and, in more severe cases, different types of surgery. In these cases, common side effects of surgery may limit the clinical success. A new alternative could be a minimally invasive Ultrasound-guided (US-guided) thread release of cubital tunnel retinaculum also known as the Osborne's ligament. The aim of this study was to assess the efficacy and safety of ultrasound-guided thread cubital tunnel release (TCuTR) in an anatomical specimen model. METHODS: In this single-center prospective experimental study, US-guided thread cubital tunnel release (TCuTR) was performed on 11 softly embalmed anatomic specimens. Subsequently, the elbows were dissected to assess the transection of Osborne's ligament and potential damage to adjacent structures. RESULTS: Due to calcification, one specimen was ineligible for the intervention. In all remaining ten interventions, Osborne's ligament (OL) was completely transected, without any damage to the surrounding nerves, blood vessels, tendons, or muscles. CONCLUSION: US-guided decompression has demonstrated a high success rate in transecting Osborne's ligament while averting damage to neighboring structures. This emerging technique appears to present an efficient and secure alternative to existing procedures.

Ultrasound-guided minimally invasive thread release of Guyon's canal: initial experience in cadaveric specimens.

OBJECTIVE: Guyon's canal syndrome is caused by compression of the ulnar nerve at the wrist, occasionally requiring decompression surgery. In recent times, minimally invasive approaches have gained popularity. The aim of this study was to assess the efficacy and safety of ultrasound-guided thread release for transecting the palmar ligament in Guyon's canal without harming surrounding structures, in a cadaveric specimen model. METHODS: After ethical approval, thirteen ultrasound-guided thread releases of Guyon's canal were performed on the wrists of softly embalmed anatomic specimens. Cadavers showing injuries or prior operations at the hand were excluded. Subsequently, the specimens were dissected, and the outcome of the interventions and potential damage to adjacent anatomical structures as well as ultrasound visibility were evaluated with a score from one to three. RESULTS: Out of 13 interventions, a complete transection was achieved in ten cases (76.9%), and a partial transection was documented in three cases (23.1%). Irrelevant lesions on the flexor tendons were observed in two cases (15.4%), and an arterial branch was damaged in one (7.7%). Ultrasound visibility varied among specimens, but essential structures were delineated in all cases. CONCLUSION: Ultrasound-guided thread release of Guyon's canal has shown promising first results in anatomic specimens. However, further studies are required to ensure the safety of the procedure. RELEVANCE STATEMENT: Our study showed that minimally invasive ultrasound-guided thread release of Guyon's canal is a feasible approach in the anatomical model. The results may provide a basis for further research and refinement of this technique. KEY POINTS: • In Guyon's canal syndrome, the ulnar nerve is compressed at the wrist, often requiring surgical release. • We adapted and tested a minimally invasive ultrasound-guided thread release technique in anatomic specimens. • The technique was effective; however, in one specimen, a small anatomic branch was damaged.

The chemicals between us-First results of the cluster analyses on anatomy embalming procedures in the German-speaking countries.

Hands-on courses utilizing preserved human tissues for educational training offer an important pathway to acquire basic anatomical knowledge. Owing to the reevaluation of formaldehyde limits by the European Commission, a joint approach was chosen by the German-speaking anatomies in Europe (Germany, Austria, Switzerland) to find commonalities among embalming protocols and infrastructure. A survey comprising 537 items was circulated to all anatomies in German-speaking Europe. Clusters were established for "ethanol"-, formaldehyde-based ("FA"), and "other" embalming procedures, depending on the chemicals considered the most relevant for each protocol. The logistical framework, volumes of chemicals, and infrastructure were found to be highly diverse between the groups and protocols. Formaldehyde quantities deployed per annum were three-fold higher in the "FA" (223 L/a) compared to the "ethanol" (71.0 L/a) group, but not for "other" (97.8 L/a), though the volumes injected per body were similar. "FA" was strongly related to table-borne air ventilation and total fixative volumes ≤1000 L. "Ethanol" was strongly related to total fixative volumes >1000 L, ceiling- and floor-borne air ventilation, and explosion-proof facilities. Air ventilation was found to be installed symmetrically in the mortuary and dissection facilities. Certain predictors exist for the interplay between the embalming used in a given infrastructure and technical measures. The here-established cluster analysis may serve as decision supportive tool when considering altering embalming protocols or establishing joint protocols between institutions, following a best practice approach to cater toward best-suited tissue characteristics for educational purposes, while simultaneously addressing future demands on exposure limits.

Eye Movements But Not Vision Drive the Development of Palisade Endings.

Purpose: To test whether visual experience and/or eye movements drive the postnatal development of palisade endings in extraocular muscles. Methods: In three newborn cats, the right eye was covered until 30 days from postnatal (P) day 7 (before opening their eyes), and in three cats both eyes were covered until 45 days, also from P7. To block eye movements, another seven cats received a retrobulbar injection of botulinum neurotoxin A (BoNT-A) into the left orbit at birth and survived for 45 days (three cats) and 95 days (four cats). The distal third of the rectus muscles containing the palisade endings was used for whole-mount preparation and triple-fluorescence labeling with anti-neurofilament along with (1) anti-synaptophysin and phalloidin or (2) anti-growth associated protein 43 (GAP43) and phalloidin. Immunolabeled specimens were analyzed in the confocal laser scanning microscope. Results: After unilateral and bilateral dark rearing, palisade endings were qualitatively and quantitatively equal to those from age-matched controls. After BoNT-A induced eye immobilization for 45 or 95 days, palisade endings were absent in the superior rectus and lateral rectus muscles and only present in the inferior rectus and medial rectus muscle. These BoNT-A-treated palisade endings were rudimentary and reduced in number, and the expression of the neuronal developmental protein GAP43 was significantly reduced. Conclusions: This study demonstrates that eye immobilization, but not visual deprivation, affects palisade ending development. Palisade endings develop in the first month of life, and the present findings indicate that, during this time window, palisade endings are prone to oculomotor perturbations.

Comparison of Thiel preserved, fresh human, and animal liver tissue in terms of mechanical properties.

BACKGROUND: In medical training and research fresh human tissue is often replaced by preserved human or fresh animal tissue, due to availability and ethical reasons. Newer preservation approaches, such as the Thiel method, promise more realistic mechanical properties than conventional formaldehyde fixation. Concerning animal substitute material, porcine and bovine tissue is often chosen, as it is easily obtainable and certain similarity to human tissue is assumed. However, it has not been thoroughly investigated how Thiel preservation changes non-linear and viscoelastic behaviour of soft organ tissues. Furthermore, differences in these properties between animal tissue and human tissue have not been previously corroborated. METHODS: We conducted ramp and relaxation tensile tests on fresh human and Thiel preserved hepatic tissue, extracting strain-specific elastic moduli, and viscoelastic properties. The results for fresh human liver were then compared to corresponding results for Thiel preserved liver, as well as previously published results for porcine and bovine liver. RESULTS: Our results showed that Thiel preservation seems to be associated with increased stiffness as well as decreased viscoelastic damping behaviour. Porcine liver was stiffer than human liver with similar viscoelastic properties. Bovine liver exhibited similar stiffness as human liver, however lower viscoelastic damping. CONCLUSIONS: The differences between human and animal liver tissue, concerning their mechanical properties, can be explained by their characteristic histology. Changes in mechanical properties due to Thiel preservation might stem from altered protein cross-linking and dehydration. The results illustrate that appropriate materials for medical training systems must be selected based on which mechanical properties are relevant for the respective application.

Palisade Endings Have an Exocytotic Machinery But Lack Acetylcholine Receptors and Distinct Acetylcholinesterase Activity.

Purpose: The purpose of this work was to test whether palisade endings express structural and molecular features of exocytotic machinery, and are associated with acetylcholine receptors, and enzymes for neurotransmitter breakdown. Methods: Extraocular rectus muscles from six cats were studied. Whole-mount preparations of extraocular muscles (EOMs) were immunolabeled with markers for exocytotic proteins, including synaptosomal-associated protein of 25 kDa (SNAP25), syntaxin, synaptobrevin, synaptotagmin, and complexin. Acetylcholine receptors (AChRs) were visualized with α-bungarotoxin and with an antibody against AChRs, and acetylcholinesterase (AChE) was tagged with anti-AChE. Molecular features of multicolor labeled palisade endings were analyzed in the confocal scanning microscope, and their ultrastructural features were revealed in the transmission electron microscope. Results: All palisade endings expressed the exocytotic proteins SNAP25, syntaxin, synaptobrevin, synaptotagmin, and complexin. At the ultrastructural level, vesicles docked at the plasma membrane of terminal varicosities of palisade endings. No AChRs were associated with palisade endings as demonstrated by the absence of α-bungarotoxin and anti-AChR binding. AChE, the degradative enzyme for acetylcholine exhibited low, if any, activity in palisade endings. Axonal tracking showed that axons with multiple en grappe motor terminals were in continuity with palisade endings. Conclusions: This study demonstrates that palisade endings exhibit structural and molecular characteristics of exocytotic machinery, suggesting neurotransmitter release. However, AChRs were not associated with palisade endings, so there is no binding site for acetylcholine, and, due to low/absent AChE activity, insufficient neurotransmitter removal. Thus, the present findings indicate that palisade endings belong to an effector system that is very different from that found in other skeletal muscles.

How to visualize the innervation pattern in tendons: A methodical guide.

BACKGROUND: Tendon pathologies are common and several data suggests that the peripheral nervous system is involved in this disorder. Immunohistochemistry (IHC) is one of the pillars to characterize nervous structures and their implication in the pathogenesis of chronic tendon pain. Most commonly, formalin-fixed, paraffin-embedded (FFPE) tendons are used for immunohistochemical characterization of the innervation. However, FFPE specimens exhibit major disadvantages: First, antigens (proteins) are masked and antigen retrieval is necessary to restore antigenicity. Second, FFPE specimens involve immunolabeling with enzyme-conjugated antibodies but this approach has limitations when multiple antigens are of interest simultaneously. Consequently, there is a demand in the orthopedic community for an alternative immunohistochemical approach to visualize tendon innervations. RESULTS: Here, we present a guide how to visualize tendon innervation. This guide couples paraformaldehyde fixation, cryo-embedding, immunofluorescence, and confocal laser scanning microscopy. We demonstrate the utility of our approach in the long head of the biceps tendon. For nerve fiber characterization, we used different neuronal markers including antibodies against neurofilament, protein gene product 9.5, calcitonin gene related peptide, and substance P. We show that it is possible to collect high quality, multicolor images of the innervation pattern of tendons. To map immunolabeled structures and the anatomical structures of the tendon fluorescence images and bright field images were merged. CONCLUSION: For the orthopedic community our approach might be a convenient research tool to simultaneously utilize multiple neuronal markers on the same tissue section and to define with greater accuracy the heterogeneity of tendon innervation.

Introduction of a new repair technique in bony avulsion of the FDP tendon: A biomechanical study.

The purpose of this study was to determine the biomechanical characteristics of an innovative surgical technique based on a tension banding principle using a suture anchor in the repair of bony avulsions of the flexor digitorum profundus tendon. After injury simulation in 45 fresh frozen distal phalanges from human cadavers, repair was performed with minifragment screws, interosseous sutures and the innovative technique (15 per group). All repairs were loaded for a total of 500 cycles. Subsequently the specimens were loaded to failure. Load at failure, load at first noteworthy displacement (>2 mm), elongation of the system, gap formation at the avulsion site, and the mechanism of failure were assessed. The new techniques' superior performance in load at failure (mean: 100.5 N), load at first noteworthy displacement (mean 77.4 N), and gap formation (median 0 mm) was statistically significant, which implies a preferable rigidity of the repair. No implant extrusion or suture rupture during cyclic loading were recorded when the new technique was applied. This innovative repair technique is superior biomechanically to other commonly used surgical tendon reattachment methods, particularly with respect to an early passive mobilisation protocol. Further, due to its subcutaneous position, reduction of complications may be achieved.

Palisade Endings of Extraocular Muscles Develop Postnatally Following Different Time Courses.

Purpose: To analyze in a frontal-eyed mammal (cat) the postnatal development of palisade endings in extraocular muscles (EOMs) and to compare the spatiotemporal and quantitative patterns of palisade endings among individual rectus muscles. Methods: Cats of different ages ranging from birth to adult stage were studied. EOM whole-mount preparations were fluorescently labeled using six combinations of triple staining and analyzed in the confocal laser scanning microscope. Results: Palisade endings developed postnatally and passed in each rectus muscle through the same, three developmental steps but in a heterochronic sequence and to a different final density per muscle. Specifically, palisade ending development was first completed in the medial rectus and later in the inferior, lateral, and superior rectus. The highest density of palisade endings was observed in the medial rectus and the lowest in the lateral rectus whereas values for the inferior and superior rectus were in between. Palisade endings expressed high levels of growth associated protein 43 during development and were supplied by axons that established motor terminals. Conclusions: Cats open their eyes 7 to 10 days after birth and later develop a complex three-dimensional visuomotor climbing and jumping behavior depending on accurate binocular vision and fine tuning of the ocular movements. Our findings indicate that palisade ending development correlates with important landmarks in visuomotor behavior and provide support for our previous notion that palisade endings play an important role for convergence eye movements in frontal-eyed species.

Proprioception in the extraocular muscles of mammals and man.

This article summarizes the authors' previous studies on proprioceptors in extraocular muscles (EOMs) of mammals and man. They report on muscle spindles in the EOMs of man, Golgi tendon organs in the EOMs of even-toed ungulates, and palisade endings in the EOMs of the cat. Muscle spindles: Muscle spindles are present in the EOMs of some mammals and in the EOMs of man. Compared with muscle spindles in other skeletal muscles, those in human EOMs exhibit structural differences. These structural differences may indicate a special function. Golgi tendon organs: Golgi tendon organs are absent in human EOMs. Golgi tendon organs exhibiting a specific morphology are present in the EOMs of even-toed ungulates. Their high number and rich innervation indicate functional importance. Palisade endings: Palisade endings are nervous end organs confined to the EOMs of mammals and man. It is assumed that these organs have a proprioceptive function. The authors show that palisade endings are immunoreactive for antibodies against choline acetyltransferase. Neuromuscular contacts, if present in palisade endings, are alpha -bungarotoxin positive as well. Taken together, these results show that palisade endings exhibit molecular characteristics of effector organs.

Palisade Endings Are a Constant Feature in the Extraocular Muscles of Frontal-Eyed, But Not Lateral-Eyed, Animals.

PURPOSE: To test whether palisade endings are a general feature of mammalian extraocular muscles (EOMs). METHODS: Thirteen species, some frontal-eyed (human, monkey, cat, and ferret), and others lateral-eyed (pig, sheep, calf, horse, rabbit, rat, mouse, gerbil, and guinea pig) were analyzed. Palisade endings were labeled by using different combinations of immunofluorescence techniques. Three-dimensional reconstructions of immunolabeled palisade endings were done. RESULTS: In all frontal-eyed species, palisade endings were a consistent feature in the rectus EOMs. Their total number was high and they exhibited an EOM-specific distribution. In particular, the number of palisade endings in the medial recti was significantly higher than in the other rectus muscles. In the lateral-eyed animals, palisade endings were infrequent and, when present, their total number was rather low. They were only found in ungulates (sheep, calf, pig, and horse) and in rabbit. In rodents (rat, guinea pig, mouse, and gerbil) palisade endings were found infrequently (e.g., rat) or were completely absent. Palisade endings in frontal-eyed species and in some lateral-eyed species (pig, sheep, calf, and horse) had a uniform morphology. They generally lacked α-bungarotoxin staining, with a few exceptions in primates. Palisade endings in other lateral-eyed species (rabbit and rat) exhibited a simplified morphology and bound α-bungarotoxin. CONCLUSIONS: Palisade endings are not a universal feature of mammalian EOMs. So, if they are proprioceptors, not all species require them. Because in frontal-eyed species, the medial rectus muscle has the highest number of palisade endings, they likely play a special role in convergence.

Molecular characteristics suggest an effector function of palisade endings in extraocular muscles.

PURPOSE: To analyze palisade endings in cat extraocular muscles (EOMs) and to clarify whether these EOM-specific organs are sensory or motor. METHODS: Twelve cats aged between 1 and 16 years were analyzed. Whole EOM tendons were immunostained using four different combinations of triple fluorescence labeling. Triple labeling included antibodies against choline acetyltransferase (ChAT), neurofilament, synaptophysin, and alpha-bungarotoxin. Preparations were examined by confocal laser scanning microscopy. ChAT-labeled EOMs were also analyzed by immunoelectron microscopy. Three-dimensional reconstructions were made of palisade endings. RESULTS: Palisade endings were found in the distal and proximal myotendinous regions of cat EOMs. These endings arose from thin nerve fibers coming from the muscle and extending into the tendon. There, the nerve fibers turned back 180 degrees to divide into terminal branches around the muscle fiber tips. Terminal branches established numerous contacts with the tendon attached to the muscle fiber tip and only a few contacts with the muscle fiber. Often, nerve fibers forming palisade endings on muscle fiber tips were observed to establish multiple motor contacts on muscle fibers outside palisade endings. Three-dimensional reconstructions depicted the complex morphology of the palisade endings. All nerve fibers supplying palisade endings stained positively for ChAT and neurofilament. All nerve terminals in palisade endings were ChAT and synaptophysin positive. Only neuromuscular contacts in palisade endings were positive for alpha-bungarotoxin, as well. CONCLUSIONS: This study provides evidence that palisade endings in cat EOMs have effector function. The findings may be of significance for strabismus surgery because palisade endings are also found in human EOMs.

Palisade endings in extraocular muscles of the monkey are immunoreactive for choline acetyltransferase and vesicular acetylcholine transporter.

PURPOSE: To analyze palisade endings in extraocular muscles (EOMs) of a primate species and to examine our previous findings in cat that palisade endings are putative effector organs. METHODS: Eleven monkeys (Macaca fascicularis) of both sexes, between 4 and 6 years of age were analyzed. Whole EOM myotendons were immunostained with four combinations of triple-fluorescent labeling and examined by confocal laser scanning microscopy. Labeling included antibodies against choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neurofilament, and synaptophysin. Muscle fibers were counterstained with phalloidin. RESULTS: Palisade endings were observed in all monkey EOMs. Nerve fibers extended from the muscle into the tendon and looped back to divide into a terminal arborization (palisade ending) around a single muscle fiber tip. In approximately 30% of the cases, nerve fibers supplying palisade endings often established motor terminals outside the palisade complex. Nerve fibers forming palisade endings were ChAT-neurofilament positive. Axonal branches of palisade endings were ChAT-neurofilament positive as well. All palisade nerve terminals exhibited ChAT-synaptophysin immunoreactivity. Within the palisade complex, palisade nerve terminals exhibited VAChT immunoreactivity. All palisade nerve terminals were VAChT-synaptophysin immunoreactive. CONCLUSIONS: The results confirm that in the monkey, palisade endings contain acetylcholine and are therefore most likely effector organs. Palisade endings are also present in human EOMs and because of their location at the myotendinous junction, these organs are of crucial interest for strabismus surgery.

Four-dimensional analysis of early pelvic girdle development in Rana temporaria.

A key event in vertebrate evolution is the linkage of the appendicular to the axial skeleton. The present study investigates the developmental dynamics of pelvic girdle morphogenesis in Rana temporaria up until metamorphosis, with respect to its functional and spatio-temporal organization. The main questions to be addressed are: initial location and the number of elements contributing to pelvic girdle formation, mechanism of bridging between the pelvic anlage and the sacrum and arthrogenesis. Serial histological sections of specimens from Gosner Stages 30 to 41 were bright-field microscopically examined and 3D-reconstructed. 3D-models were merged to 4D-animations illustrating the complex developmental dynamics through time. The results reveal the initial formation of a single mesenchymal condensation located close to the appendicular skeleton, but far from the axial skeleton. In addition, our analysis detects a thin connective tissue strand in R. temporaria guiding the elongation of the ilium towards the sacrum. The 4D-visualization allows novel insight into the ilio-sacral bridging process and the reorientation of the pelvis.