Publications by authors named "Roland Blumer"

25 Publications

  • Page 1 of 1

Selective Denervation of the Facial Dermato-Muscular Complex in the Rat: Experimental Model and Anatomical Basis.

Front Neuroanat 2021 22;15:650761. Epub 2021 Mar 22.

Clinical Laboratory for Bionic Extremity Reconstruction, Department of Plastic, Reconstructive and Aesthetic Surgery, Medical University of Vienna, Vienna, Austria.

The facial dermato-muscular system consists of highly specialized muscles tightly adhering to the overlaying skin and thus form a complex morphological conglomerate. This is the anatomical and functional basis for versatile facial expressions, which are essential for human social interaction. The neural innervation of the facial skin and muscles occurs via branches of the trigeminal and facial nerves. These are also the most commonly pathologically affected cranial nerves, often requiring surgical treatment. Hence, experimental models for researching these nerves and their pathologies are highly relevant to study pathophysiology and nerve regeneration. Experimental models for the distinctive investigation of the complex afferent and efferent interplay within facial structures are scarce. In this study, we established a robust surgical model for distinctive exploration of facial structures after complete elimination of afferent or efferent innervation in the rat. Animals were allocated into two groups according to the surgical procedure. In the first group, the facial nerve and in the second all distal cutaneous branches of the trigeminal nerve were transected unilaterally. All animals survived and no higher burden was caused by the procedures. Whisker pad movements were documented with video recordings 4 weeks after surgery and showed successful denervation. Whole-mount immunofluorescent staining of facial muscles was performed to visualize the innervation pattern of the neuromuscular junctions. Comprehensive quantitative analysis revealed large differences in afferent axon counts in the cutaneous branches of the trigeminal nerve. Axon number was the highest in the infraorbital nerve (28,625 ± 2,519), followed by the supraorbital nerve (2,131 ± 413), the mental nerve (3,062 ± 341), and the cutaneous branch of the mylohyoid nerve (343 ± 78). Overall, this surgical model is robust and reliable for distinctive surgical deafferentation or deefferentation of the face. It may be used for investigating cortical plasticity, the neurobiological mechanisms behind various clinically relevant conditions like facial paralysis or trigeminal neuralgia as well as local anesthesia in the face and oral cavity.
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http://dx.doi.org/10.3389/fnana.2021.650761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019738PMC
March 2021

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

Invest Ophthalmol Vis Sci 2020 12;61(14):31

Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain.

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.
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http://dx.doi.org/10.1167/iovs.61.14.31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774060PMC
December 2020

Molecular Pattern and Density of Axons in the Long Head of the Biceps Tendon and the Superior Labrum.

J Clin Med 2019 Dec 3;8(12). Epub 2019 Dec 3.

AUVA Trauma Center Vienna Meidling, Kundratstraße 37, 1120 Vienna, Austria.

The type II superior labrum anterior to posterior (SLAP) repair is a viable option in young and demanding patients, although a prolonged period of pain after surgery is described in the literature. The reason for this fact remains unknown. Thus, the purpose of this study was to investigate the molecular pattern of the biceps tendon anchor, where the sutures for repair are placed. The long head of the biceps tendon (LHBT), including the superior labrum, was dissected in the setting of reverse total shoulder arthroplasty. Immunohistochemical staining was performed using neurofilament (NF) and protein gene product (PGP) 9.5 as general markers for axons and calcitonin gene-related peptide (CGRP) and substance P for nociceptive transmission. A quantitative assessment was performed according to the two regions of interest (ROIs), i.e., the anterosuperior (ROI I) and the posterosuperior labrum (ROI II). Eleven LHBTs with a mean age of 73 years (range: 66-87 years) were harvested intraoperatively. Six LHBTs were gained in osteoarthrosis and five in fractures. We found an inhomogeneous distribution of axons in the anterosuperior and posterosuperior parts of the labrum in all the specimens irrespective of the age, gender, and baseline situation. There was a significantly higher number ( < 0.01) as well as density ( < 0.001) of NF-positive axons in ROI I compared to ROI II. Nociceptive fibers were always found along the NF-positive axons. Thus, our results indicate that the biceps tendon anchor itself is a highly innervated region comprising different nerve qualities. The anterosuperior labrum contains a higher absolute number and density of axons compared to the posterosuperior parts. Furthermore, we were able to prove the presence of nociceptive fibers in the superior labrum. The results obtained in this study could contribute to the variability of pain after SLAP repair.
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http://dx.doi.org/10.3390/jcm8122129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947398PMC
December 2019

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

Cell Tissue Res 2020 Apr 7;380(1):43-57. Epub 2019 Dec 7.

AUVA Trauma Center Vienna Meidling, A-1120, Vienna, Austria.

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.
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http://dx.doi.org/10.1007/s00441-019-03141-4DOI Listing
April 2020

Histologic Evaluation of Nonvisual Afferent Sensory Upper Eyelid Proprioception.

Ophthalmic Plast Reconstr Surg 2020 Jan/Feb;36(1):7-12

TOC Eye and Face.

Purpose: Recent research has suggested a possible role for proprioception in ipsilateral frontalis activation in the setting of ptosis; however, there has not been any robust histologic or anatomic evidence to support this theory. To further elucidate proprioceptive structures in the eyelid, this investigation uses validated histologic techniques to explore the presence of proprioceptive structures or afferent neural networks in the Levator Palpebrae Superioris (LPS) and Müller muscle.

Methods: Müller muscle and LPS samples were evaluated by a laboratory with extensive experience with the histology of extraocular muscle proprioception. Immunofluorescence and confocal laser scanning microscopy were used to analyze the tissue samples.

Results: Thirty-four Müller muscle samples and 10 LPS samples were analyzed. Golgi tendon bodies and muscle spindles were not identified in the Müller muscle and LPS samples. This result is expected in the Müller muscle given that these structures are not typically present in smooth muscle, but noteworthy in the skeletal muscle of the LPS. Previously undescribed synaptophysin-positive free nerve terminals within the intermuscular connective tissue of the Müller muscle were identified.

Conclusions: The nerve terminals identified are anatomically consistent with free nerve endings present in the extraocular muscles that have been implicated in proprioception. These findings advance our current knowledge of the ultrastructure of Müller muscle and the LPS and suggest a possible mechanism for proprioception in the upper eyelid that may have a role in ipsilateral brow elevation in the setting of ptosis.The authors describe proprioception in the upper eyelid: A histologic analysis.
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http://dx.doi.org/10.1097/IOP.0000000000001424DOI Listing
March 2021

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

Ann Anat 2019 Sep 11;225:21-27. Epub 2019 Jun 11.

AUVA Trauma Center Vienna Meidling, A-1120 Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.aanat.2019.05.009DOI Listing
September 2019

Functional diversity of motoneurons in the oculomotor system.

Proc Natl Acad Sci U S A 2019 02 13;116(9):3837-3846. Epub 2019 Feb 13.

Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, 41012 Sevilla, Spain;

Extraocular muscles contain two types of muscle fibers according to their innervation pattern: singly innervated muscle fibers (SIFs), similar to most skeletal muscle fibers, and multiply innervated muscle fibers (MIFs). Morphological studies have revealed that SIF and MIF motoneurons are segregated anatomically and receive different proportions of certain afferents, suggesting that while SIF motoneurons would participate in the whole repertoire of eye movements, MIF motoneurons would contribute only to slow eye movements and fixations. We have tested that proposal by performing single-unit recordings, in alert behaving cats, of electrophysiologically identified MIF and SIF motoneurons in the abducens nucleus. Our results show that both types of motoneuron discharge in relation to eye position and velocity, displaying a tonic-phasic firing pattern for different types of eye movement (saccades, vestibulo-ocular reflex, vergence) and gaze-holding. However, MIF motoneurons presented an overall reduced firing rate compared with SIF motoneurons, and had significantly lower recruitment threshold and also lower eye position and velocity sensitivities. Accordingly, MIF motoneurons could control mainly gaze in the off-direction, when less force is needed, whereas SIF motoneurons would contribute to increase muscle tension progressively toward the on-direction as more force is required. Anatomically, MIF and SIF motoneurons distributed intermingled within the abducens nucleus, with MIF motoneurons being smaller and having a lesser somatic synaptic coverage. Our data demonstrate the functional participation of both MIF and SIF motoneurons in fixations and slow and phasic eye movements, although their discharge properties indicate a functional segregation.
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http://dx.doi.org/10.1073/pnas.1818524116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397535PMC
February 2019

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

Invest Ophthalmol Vis Sci 2017 10;58(12):5105-5121

Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain.

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.
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http://dx.doi.org/10.1167/iovs.17-22643DOI Listing
October 2017

Axonal components of nerves innervating the human arm.

Ann Neurol 2017 Sep 8;82(3):396-408. Epub 2017 Sep 8.

Christian Doppler Laboratory for Restoration of Extremity Function, Medical University of Vienna, Vienna, Austria.

Objective: Axons traveling within the brachial plexus are responsible for the dexterous control of human arm and hand movements. Despite comprehensive knowledge on the topographical anatomy of nerves innervating the human upper limbs, the definite quantity of sensory and motor axons within this neural network remains elusive. Our aim was to perform a quantitative analysis of the axonal components of human upper limb nerves based on highly specific molecular features from spinal cord level to the terminal nerves at wrist level.

Methods: Nerve specimen harvest at predefined harvesting sites (plexus roots and cords as well as major nerves originating from the brachial plexus innervating the arm and hand) was performed in 9 human heart-beating organ donors. Double immunofluorescence staining using antibodies against choline-acetyltransferase and neurofilament was performed to differentiate motor and sensory axons on nerve cross sections.

Results: Three hundred fifty thousand axons emerge from the spinal cord to innervate the human upper limb, of which 10% are motor neurons. In all nerves studied, sensory axons outnumber motor axons by a ratio of at least 9:1. The sensory axon contribution increases when moving distally, whereas only 1,700 motor axons reach the hand to innervate the intrinsic musculature.

Interpretation: Our results suggest that upper limb motor execution, and particularly dexterous coordination of hand movement, require an unexpectedly low number of motor neurons, with a large convergence of afferent input for feedback control. Ann Neurol 2017;82:396-408.
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http://dx.doi.org/10.1002/ana.25018DOI Listing
September 2017

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

Invest Ophthalmol Vis Sci 2016 Feb;57(2):320-31

Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain.

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.
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http://dx.doi.org/10.1167/iovs.15-18716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826744PMC
February 2016

The secretome of apoptotic human peripheral blood mononuclear cells attenuates secondary damage following spinal cord injury in rats.

Exp Neurol 2015 May 19;267:230-42. Epub 2015 Mar 19.

Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria; Department of Thoracic Surgery, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. Electronic address:

After spinal cord injury (SCI), secondary damage caused by oxidative stress, inflammation, and ischemia leads to neurological deterioration. In recent years, therapeutic approaches to trauma have focused on modulating this secondary cascade. There is increasing evidence that the success of cell-based SCI therapy is due mainly to secreted factors rather than to cell implantation per se. This study investigated peripheral blood mononuclear cells as a source of factors for secretome- (MNC-secretome-) based therapy. Specifically, we investigated whether MNC-secretome had therapeutic effects in a rat SCI contusion model and its possible underlying mechanisms. Rats treated with MNC-secretome showed substantially improved functional recovery, attenuated cavity formation, and reduced acute axonal injury compared to control animals. Histological evaluation revealed higher vascular density in the spinal cords of treated animals. Immunohistochemistry showed that MNC-secretome treatment increased the recruitment of CD68(+) cells with concomitant reduction of oxidative stress as reflected by lower expression of inducible nitric oxide synthase. Notably, MNC-secretome showed angiogenic properties ex vivo in aortic rings and spinal cord tissue, and experiments showed that the angiogenic potential of MNC-secretome may be regulated by CXCL-1 upregulation in vivo. Moreover, systemic application of MNC-secretome activated the ERK1/2 pathway in the spinal cord. Taken together, these results indicate that factors in MNC-secretome can mitigate the pathophysiological processes of secondary damage after SCI and improve functional outcomes in rats.
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http://dx.doi.org/10.1016/j.expneurol.2015.03.013DOI Listing
May 2015

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

J Neurosci 2013 Feb;33(7):2784-93

Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, A-1090 Vienna, Austria.

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.
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http://dx.doi.org/10.1523/JNEUROSCI.4116-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811327PMC
February 2013

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

J Morphol 2011 Mar 15;272(3):287-301. Epub 2010 Dec 15.

Center for Anatomy and Cell Biology, Integrative Morphology Group, Medical University of Vienna, Vienna 1090, Austria.

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.
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http://dx.doi.org/10.1002/jmor.10913DOI Listing
March 2011

Evidence that the extraocular motor nuclei innervate monkey palisade endings.

Neurosci Lett 2011 Feb 7;489(2):89-93. Epub 2010 Dec 7.

Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, Waehringer Strasse 13, A-1090 Vienna, Austria.

Palisade endings are found in the extraocular muscles (EOMs) of almost every mammalian species, including primates. These nerve specializations surrounding the muscle fiber insertion have been postulated to be the proprioceptors of the EOMs. However, it was recently demonstrated that palisade endings have a cholinergic nature, which reopened the question of whether palisade endings are motor or sensory structures. In this work, we examined whether the cell bodies of palisade endings lie in EOM motor nuclei by injecting an anterograde tracer, biotinylated dextran amine, into the abducens nucleus of a macaque monkey. Tracer visualization in the lateral rectus muscle was combined with choline acetyltransferase (ChAT) and α-bungarotoxin staining. Analysis of the samples was performed by conventional light microscopy and confocal laser scanning microscopy. About 30% of the nerve fibers innervating the muscle were tracer positive. These were ChAT positive as well. Tracer positive nerve fibers established motor contacts on singly and multiply innervated muscle fibers, which were confirmed by α-bungarotoxin staining. At the transition between muscle and distal tendon, we found palisade endings that contained tracer. Palisade endings exhibited the classic morphology: axons arising from the muscle extend onto the tendon, then turn back 180° and terminate in a cuff of terminals around an individual muscle fiber tip. This finding suggests that the cell bodies of palisade endings lie in the EOM motor nuclei, which complements prior studies demonstrating a cholinergic, and possibly motor, phenotype for palisade endings.
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http://dx.doi.org/10.1016/j.neulet.2010.11.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3028275PMC
February 2011

Palisade endings are present in canine extraocular muscles and have a cholinergic phenotype.

Neurosci Lett 2009 Nov 17;465(3):199-203. Epub 2009 Sep 17.

Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, Waehringer Strasse 13, A-1090 Vienna, Austria.

Classical proprioceptors, like Golgi tendon organs and muscle spindles are absent in the extraocular muscles (EOMs) of most mammals. Instead, a nerve end organ was detected in the EOMs of each species including sheep, cat, rabbit, rat, monkey, and human examined so far: the palisade ending. Until now no clear evidence appeared that palisade endings are also present in canine EOMs. Here, we analyzed dog EOMs by confocal laser scanning microscopy, 3D reconstruction, and transmission electron microscopy. In EOM wholemount preparations stained with antibodies against neurofilament and synaptophysin we could demonstrate typical palisade endings. Nerve fibers coming from the muscle extend into the tendon. There, the nerve fibers turn 180 degrees and return to branch into preterminal axons which establish nerve terminals around a single muscle fiber tip. Fine structural analysis revealed that each palisade ending in dog EOMs establish nerve terminals on the tendon. In some palisade endings we found nerve terminals contacting the muscle fiber as well. Such neuromuscular contacts have a basal lamina in the synaptic cleft. By using an antibody against choline acetyltransferase (ChAT) we proved that canine palisade endings are ChAT-immunoreactive. This study shows that palisade endings are present in canine EOMs. In line with prior findings in cat and monkey, palisade endings in dog have a cholinergic phenotype.
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http://dx.doi.org/10.1016/j.neulet.2009.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811328PMC
November 2009

Ultrastructural and molecular biologic comparison of classic proprioceptors and palisade endings in sheep extraocular muscles.

Invest Ophthalmol Vis Sci 2009 Dec 24;50(12):5697-706. Epub 2009 Jun 24.

Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, Vienna, Austria.

Purpose: To analyze and compare the structural and molecular features of classic proprioceptors like muscle spindles and Golgi tendon organs (GTOs) and putative proprioceptors (palisade endings) in sheep extraocular muscle (EOMs).

Methods: The EOMs of four sheep were analyzed. Frozen sections or wholemount preparations of the samples were immunohistochemically labeled and analyzed by confocal laser scanning microscopy. Triple labeling with different combinations of antibodies against neurofilament, synaptophysin, and choline acetyltransferase (ChAT), as well as alpha-bungarotoxin and phalloidin, was performed. Microscopic anatomy of the nerve end organs was analyzed by transmission electron microscopy.

Results: The microscopic anatomy demonstrated that muscle spindles and GTOs had a perineural capsule and palisade endings a connective tissue capsule. Sensory nerve terminals in muscle spindles and GTOs contained only a few vesicles, whereas palisade nerve terminals were full of clear vesicles. Likewise, motor terminals in the muscle spindles' polar regions were full of clear vesicles. Immunohistochemistry showed that sensory nerve fibers as well as their sensory nerve terminals in muscle spindles and GTOs were ChAT-negative. Palisade endings were supplied by ChAT-positive nerve fibers, and the palisade complexes including palisade nerve terminals were also ChAT-immunoreactive. Motor terminals in muscle spindles were ChAT and alpha-bungarotoxin positive.

Conclusions: The present study demonstrated in sheep EOMs that palisade endings are innervated by cholinergic axons exhibiting characteristics typical of motoneurons, whereas muscle spindles (except the polar regions) and GTOs are supplied by noncholinergic axons. These results raise the question of whether palisade endings are candidates for proprioceptors in EOMs.
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http://dx.doi.org/10.1167/iovs.09-3902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811329PMC
December 2009

Palisade endings: cholinergic sensory organs or effector organs?

Invest Ophthalmol Vis Sci 2009 Mar 20;50(3):1176-86. Epub 2008 Oct 20.

Center of Anatomy and Cell Biology, Medical University Vienna, Vienna, Austria.

Purpose: This study aims to complement the authors' prior findings on palisade endings in extraocular muscles (EOMs) of monkeys, and to clarify whether palisade endings are cholinergic motor or cholinergic sensory.

Methods: Macaque monkeys (Macaca fascicularis, n = 10) of both sexes were analyzed using three-dimensional (3D) reconstructions, confocal laser scanning microscopy (CLSM), and conventional/immuno transmission electron microscopy (TEM). For CLSM, we used three combinations of triple fluorescent labeling. EOM wholemounts were labeled with cholinergic markers, including choline acetyltransferase (ChAT), choline transporter (ChT), vesicular acetylcholine transporter (VAChT), and a classical postsynaptic marker for motor terminals, namely alpha-bungarotoxin. Muscle fibers were counterstained with phalloidin. 3D reconstructions were done of triple-labeled palisade endings. For immuno TEM, tissue was labeled with antibody against ChAT.

Results: Concordant with prior findings, the authors demonstrated that palisade endings at the muscle fiber tips arose from nerve fibers that are ChAT-positive. In 25% of the cases, axons forming palisade endings established multiple neuromuscular contacts outside the palisade complex. Such additional neuromuscular contacts were motor terminals, as demonstrated by alpha-bungarotoxin binding. All palisade endings established nerve terminals on the tendon. In 40% of the palisade endings, nerve terminals were observed on the muscle fiber as well. Neurotendinous contacts and neuromuscular contacts in palisade endings were ChT/ChAT/VAChT-immunoreactive. Neuromuscular contacts exhibited structural features of motor terminals and were also alpha-bungarotoxin positive.

Conclusions: The present study ascertained that palisade endings are cholinergic motor organs. Therefore, it was concluded that palisade endings are not candidates to provide eye-position signals.
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http://dx.doi.org/10.1167/iovs.08-2748DOI Listing
March 2009

Proprioception in the extraocular muscles of mammals and man.

Strabismus 2006 Jun;14(2):101-6

Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University of Vienna, Austria.

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.
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http://dx.doi.org/10.1080/09273970600701192DOI Listing
June 2006

Combined enzymatic and antioxidative treatment reduces ischemia-reperfusion injury in rabbit skeletal muscle.

J Surg Res 2006 Jun 3;133(2):150-8. Epub 2006 Feb 3.

Department of Vascular Surgery, Medical University of Vienna, Vienna, Austria.

Background: Ischemia/reperfusion (I/R) injury is characterized by the production of oxygen-free radicals leading to disturbances in vasomotility (microvascular constriction) and microvascular permeability (interstitial edema formation). The objective was to evaluate the effect of the combined antioxidative and enzymatic preparation Phlogenzym on I/R injury of skeletal muscle.

Materials And Methods: A rabbit hindlimb model of I/R (2.5/2 h) was used (IR group). Phlogenzym, containing rutin, trypsin, and bromelain, was applied enterally (60 mg/kg body weight) as a bolus 30 min prior to ischemia (Ph group). Sham-operated animals served as controls (CO group). Plasma malondialdehyde, potassium, and microvascular perfusion (monitored by laser flowmetry) were assessed. Histomorphometry and electron microscopy were performed from major adductor muscles.

Results: Two hours after reperfusion, potassium levels were significantly elevated in IR compared to Ph group (6.7 +/- 1.2 versus 4.9 +/- 0.9 mmol/l, P < 0.006). Enhanced lipid peroxidation, apparent by increased plasma malondialdehyde levels, was ameliorated in the Ph group (1.0 +/- 0.1 versus 0.7 +/- 0.1 nmol/ml, P < 0.0001). No-reflow (reduction of blood flow by 62% in IR group) was not observed in the Ph group (P < 0.004). Phlogenzym treatment prevented microvascular constriction (17.6 +/- 2.3 versus 12.6 +/- 1.1 microm(2), P < 0.0001) and mollified interstitial edema (21.5 +/- 2.0 versus 26.0 +/- 3.7%, P < 0.017), resulting in mild ultrastructural alterations in contrast to pronounced sarcolemmal and mitochondrial damage in untreated rabbits.

Conclusions: Phlogenzym had a protective effect on skeletal muscle during I/R injury expressed by prevention of no-reflow and preservation of muscle tissue.
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http://dx.doi.org/10.1016/j.jss.2005.12.005DOI Listing
June 2006

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

Invest Ophthalmol Vis Sci 2005 Dec;46(12):4548-54

Center of Anatomy and Cell Biology, Integrative Morphology Group, Medical University Vienna, Austria.

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.
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http://dx.doi.org/10.1167/iovs.05-0726DOI Listing
December 2005

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

Invest Ophthalmol Vis Sci 2005 Jan;46(1):155-65

Center of Anatomy and Cell Biology, Integrative Morphology Group, Vienna, Austria.

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.
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http://dx.doi.org/10.1167/iovs.04-1087DOI Listing
January 2005

Combined L-arginine and antioxidative vitamin treatment mollifies ischemia-reperfusion injury of skeletal muscle.

J Vasc Surg 2004 Apr;39(4):868-77

Department of Vascuar Surgery, Ludwig Blotzmann Research Institute for Vascular Medicine, University of Vienna, Austria.

Enhanced production of superoxide in L-arginine-depleted environments and concomitant reduction of nitric oxide (NO) concentration are involved in ischemia-reperfusion (I/R) injury. Treatment with L-arginine or antioxidative vitamins alone and in combination was used to mollify I/R injury in skeletal muscle. Untreated rabbits were compared with those treated with L-arginine/antioxidative vitamin cocktail Omnibionta only, or a combination of L-arginine/ antioxidative vitamins during hind limb I/R (2.5 hours/2 hours). NO was continuously measured in vivo. Plasma malondialdehyde (MDA) served as the measure of oxygen free radical formation. Interstitial edema formation, microvessel diameter alterations, microvessel plugging, and blood flow changes were used as indicators of I/R injury. The MDA level in untreated animals 2 hours after reperfusion was significantly higher than in control animals (0.81 micromol/L +/- 0.14 micromol/L vs 0.57 micromol/L +/- 0.11 micromol/L; P<.05), indicating enhanced production of oxygen free radicals. This sequela paralleled the decreasing concentration of NO, which dropped below the detection limit (1 nmol/L) after reperfusion. Microvascular changes during I/R injury were expressed as a 40% decrease in microvessel diameter and adhesion of neutrophils in 20% of microvessels, which led to a consequent 60% reduction in blood flow, demonstrating "no reflow" (reperfusion failure after restoration of blood flow). The increase in the fraction of muscle interfiber area by 85% indicated prominent edema formation. Treatment with antioxidative vitamins alone had a minimally positive effect on edema formation and microvascular plugging, possibly by suppression of oxygen free radical production, as expressed by the reduction in plasma MDA levels. However, this therapy failed to preserve basal NO production and to protect from microvascular constriction and no reflow. Treatment with L-arginine alone had a stronger protective effect, maintaining basal NO production, further reduction of neutrophil plugging, abolition of microvascular constriction, and no reflow. The combination of antioxidative vitamins and L-arginine was the best treatment against I/R injury, expressed not only by the protection of microvessel constriction, but also by abolition of microvascular plugging, increase in NO production (68 nmol/L +/- 5 nmol/L) over the basal level (52 nmol/L +/- 7 nmol/L), and higher blood flow, as compared with treatment with L-arginine or antioxidative vitamins alone.
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http://dx.doi.org/10.1016/j.jvs.2003.10.060DOI Listing
April 2004

Impaired VE-cadherin/beta-catenin expression mediates endothelial cell degeneration in dilated cardiomyopathy.

Circulation 2003 Sep 8;108(13):1585-91. Epub 2003 Sep 8.

Laboratory for Cardiovascular Research, Department of Anatomy, University of Vienna, Vienna, Austria.

Background: The cross-talk between vascular endothelial growth factor (VEGF)-A, angiopoietin (Ang), and VE-cadherin coregulates endothelial cell (EC) survival. Cardiac expression of VEGF-A but not its receptor KDR is blunted in dilated cardiomyopathy (DCM). Whether VE-cadherin/Ang function is affected in DCM is unknown.

Methods And Results: The myocardial expression of VE-cadherin/beta-catenin, Ang-1, Ang-2, and their receptor Tie-2 was examined in DCM, ischemic cardiomyopathy (ICM), and in control subjects through the use of real-time RT-PCR, Western blotting, and immunocytochemistry. EC degeneration was quantified by TEM. RNA interference against VE-cadherin and VEGF deprivation and stimulation were applied to cultured DCM myocardium and human microvascular ECs to examine the interplay between VEGF, VE-cadherin/beta-catenin, and Ang-2. Analysis of tissue sections with similar rates of EC degeneration in both patient groups showed that VE-cadherin/beta-catenin expression was downregulated in DCM only (P<0.05). Although Ang-1 was not changed, Ang-2 expression was downregulated and Tie-2 protein expression was upregulated both in DCM and ICM (P<0.05). The ratio of degenerated to normal ECs was significantly higher in DCM versus ICM (P<0.05). Targeted VE-cadherin gene silencing in cultured human ECs resulted in similar degenerative effects observed in myocardial ECs of DCM patients. In vitro experiments indicated that VE-cadherin/beta-catenin expression is independent of VEGF.

Conclusions: These results indicate for the first time that the EC survival is impaired in myocardium of patients with DCM involving VE-cadherin/beta-catenin, probably independent of VEGF. Targeting VE-cadherin might be of benefit to counteract the selective EC pathology in DCM.
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http://dx.doi.org/10.1161/01.CIR.0000091085.12422.19DOI Listing
September 2003

Muscle spindles and Golgi tendon organs in bovine calf extraocular muscle studied by means of double-fluorescent labeling, electron microscopy, and three-dimensional reconstruction.

Exp Eye Res 2003 Oct;77(4):447-62

Department of Anatomy, University of Vienna, Austria.

In the present study muscle spindles (MSps) and Golgi tendon organs (GTOs) in bovine extraocular muscles (EOMs) were analyzed in detail. The innervation pattern of these proprioceptors was investigated with transmission electron microscope and confocal laser scanning microscope after double-fluorescent labeling. Three-dimensional (3D) reconstructions were performed of GTOs. Muscle spindles. MSps are numerous, each containing two nuclear bag fibers and up to eight nuclear chain fibers. In the equatorial region and paraequatorial region thin axons enwrapping the intrafusal muscle fibers form numerous nerve contacts on the muscle fiber surface. Double staining of such nerve terminals with synaptophysin and alpha-bungarotoxin and their fine structural features confirm their sensory nature. In the encapsulated part of the polar region neuromuscular contacts have structural features of motor nerve terminals and stain positively with alpha-bungarotoxin. Golgi tendon organs. GTOs are numerous in bovine EOMs. Each GTO contains collagen bundles but frequently also intracapsular muscle fibers. Intracapsular muscle fibers either terminate inside the GTO in collagen bundles or pass through the proprioceptor. GTOs are richly supplied with sensory nerve terminals which intermingle with the collagen bundles. Nerve terminals on intracapsular muscle fibers exhibit fine structural characteristics of motor nerve terminals and are alpha-bungarotoxin positive. The 3D images of GTOs show the detailed spatial arrangement of the GTO tissue components. These new insights in the complex and specific morphology of MSps and GTOs in bovine EOMs indicate that we deal with highly developed proprioceptors. These are supposed to provide important information for EOM innervation.
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http://dx.doi.org/10.1016/s0014-4835(03)00157-xDOI Listing
October 2003

Ischemia/reperfusion injury of skeletal muscle: plasma taurine as a measure of tissue damage.

Surgery 2003 Jan;133(1):91-100

Department of Vascular Surgery, Ludwig Boltzmann Research Institute for Interdisciplinary Vascular Medicine, University of Vienna Medical School, Vienna, Austria.

Background: Cell membrane rupture by oxygen-derived free radicals is a systematic feature of ischemia/reperfusion (I/R) injury. High taurine concentration gradients in skeletal muscle prompted us to evaluate whether plasma taurine levels (pTau) are a useful marker of I/R injury after different periods of ischemia.

Methods: Rabbits were randomly assigned to either 1 or 2.5 hours of hind-limb ischemia followed by 2 hours of reperfusion (groups IR1 [n = 12] and IR2.5 [n = 13], respectively). Corresponding sham groups (SHAM1 [n = 8] and SHAM2.5 [n = 9]) were used as controls. Analyzed parameters included histomorphometry and electron microscopy of skeletal muscle biopsies, pTau, and plasma level of malondialdehyde. Skeletal muscle function was assessed 3 weeks after I/R injury.

Results: No significant morphologic changes were detectable at the end of ischemia. After reperfusion, mild interstitial edema with intact muscle cell membranes developed in IR1 group; pTau was not increased. IR2.5 group, by contrast, showed severe interstitial edema formation (interfiber area increased by 112%, P <.005), microvascular constriction (microvessel area decreased by 33%, P <.0005), and damage to the muscle cell membranes that was confirmed by the increased plasma malondialdehyde. pTau was higher than in the SHAM2.5 group (P <.0005). Pronounced cell damage in IR2.5 group resulted in impaired muscle function (maximal tetanic tension was reduced 2 times, P <.005) but not in IR1 group.

Conclusion: Skeletal muscle tolerates 1 h/2 h but not 2.5 h/2 h of I/R, the latter resulting in interstitial edema formation, microvascular constriction, and a late muscle dysfunction. Cell membrane rupture through stimulated lipid peroxidation promotes leakage of intracellular taurine, leading to increased pTau after reperfusion and may be considered as prognostically unfavorable in terms of organ function reversibility. In the rabbit model, pTau seems to be a sensitive marker of I/R injury to skeletal muscle.
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http://dx.doi.org/10.1067/msy.2003.65DOI Listing
January 2003
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