fhl2b mediates extraocular muscle protection in zebrafish models of muscular dystrophies and its ectopic expression ameliorates affected body muscles.

In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies.

Obscurin Maintains Myofiber Identity in Extraocular Muscles.

Purpose: The cytoskeleton of the extraocular muscles (EOMs) is significantly different from that of other muscles. We aimed to investigate the role of obscurin, a fundamental cytoskeletal protein, in the EOMs. Methods: The distribution of obscurin in human and zebrafish EOMs was compared using immunohistochemistry. The two obscurin genes in zebrafish, obscna and obscnb, were knocked out using CRISPR/Cas9, and the EOMs were investigated using immunohistochemistry, qPCR, and in situ hybridization. The optokinetic reflex (OKR) in five-day-old larvae and adult obscna-/-;obscnb-/- and sibling control zebrafish was analyzed. Swimming distance was recorded at the same age. Results: The obscurin distribution pattern was similar in human and zebrafish EOMs. The proportion of slow and fast myofibers was reduced in obscna-/-;obscnb-/- zebrafish EOMs but not in trunk muscle, whereas the number of myofibers containing cardiac myosin myh7 was significantly increased in EOMs of obscurin double mutants. Loss of obscurin resulted in less OKRs in zebrafish larvae but not in adult zebrafish. Conclusions: Obscurin expression is conserved in normal human and zebrafish EOMs. Loss of obscurin induces a myofiber type shift in the EOMs, with upregulation of cardiac myosin heavy chain, myh7, showing an adaptation strategy in EOMs. Our model will facilitate further studies in conditions related to obscurin.

Myofiber Type Shift in Extraocular Muscles in Amyotrophic Lateral Sclerosis.

Purpose: To investigate changes in myofiber composition in the global layer (GL) and orbital layer (OL) of extraocular muscles (EOMs) from terminal amyotrophic lateral sclerosis (ALS) donors. Methods: Medial recti muscles collected postmortem from spinal-onset ALS, bulbar-onset ALS, and healthy control donors were processed for immunofluorescence with antibodies against myosin heavy chain (MyHC) IIa, MyHCI, MyHCeom, laminin, neurofilaments, synaptophysin, acetylcholine receptor γ-subunit, and α-bungarotoxin. Results: The proportion of myofibers containing MyHCIIa was significantly smaller and MyHCeom was significantly larger in the GL of spinal-onset ALS and bulbar-onset ALS donors compared to control donors. Changes in the GL were more prominent in the bulbar-onset ALS donors, with a significantly larger proportion of myofibers containing MyHCeom being present compared to spinal-onset ALS donors. There were no significant differences in the myofiber composition in the OL. In the spinal-onset ALS donors, the proportions of myofibers containing MyHCIIa in the GL and MyHCeom in the OL were significantly correlated with the disease duration. Neurofilament and synaptophysin were present at motor endplates of myofibers containing MyHCeom in ALS donors. Conclusions: The EOMs of terminal ALS donors displayed changes in the fast-type myofiber composition in the GL, with a more pronounced alteration in bulbar-onset ALS donors. Our results align with the worse prognosis and subclinical changes in eye movement function previously observed in bulbar-onset ALS patients and suggest that the myofibers in the OL might be more resistant to the pathological process in ALS.

Aniridia-related keratopathy relevant cell signaling pathways in human fetal corneas.

We aimed to study aniridia-related keratopathy (ARK) relevant cell signaling pathways [Notch1, Wnt/ß-catenin, Sonic hedgehog (SHH) and mTOR] in normal human fetal corneas compared with normal human adult corneas and ARK corneas. We found that fetal corneas at 20 weeks of gestation (wg) and normal adult corneas showed similar staining patterns for Notch1; however 10-11 wg fetal corneas showed increased presence of Notch1. Numb and Dlk1 had an enhanced presence in the fetal corneas compared with the adult corneas. Fetal corneas showed stronger immunolabeling with antibodies against ß-catenin, Wnt5a, Wnt7a, Gli1, Hes1, p-rpS6, and mTOR when compared with the adult corneas. Gene expression of Notch1, Wnt5A, Wnt7A, ß-catenin, Hes1, mTOR, and rps6 was higher in the 9-12 wg fetal corneas compared with adult corneas. The cell signaling pathway differences found between human fetal and adult corneas were similar to those previously found in ARK corneas with the exception of Notch1. Analogous profiles of cell signaling pathway activation between human fetal corneas and ARK corneas suggests that there is a less differentiated host milieu in ARK.

Genetic compensation between Pax3 and Pax7 in zebrafish appendicular muscle formation.

BACKGROUND: Migrating muscle progenitors delaminate from the somite and subsequently form muscle tissue in distant anatomical regions such as the paired appendages, or limbs. In amniotes, this process requires a signaling cascade including the transcription factor paired box 3 (Pax3). RESULTS: In this study, we found that, unlike in mammals, pax3a/3b double mutant zebrafish develop near to normal appendicular muscle. By analyzing numerous mutant combinations of pax3a, pax3b and pax7a, and pax7b, we determined that there is a feedback system and a compensatory mechanism between Pax3 and Pax7 in this developmental process, even though Pax7 alone is not required for appendicular myogenesis. pax3a/3b/7a/7b quadruple mutant developed muscle-less pectoral fins. CONCLUSIONS: We found that Pax3 and Pax7 are redundantly required during appendicular myogenesis in zebrafish, where Pax7 is able to activate the same developmental programs as Pax3 in the premigratory progenitor cells.

Cytoskeletal Proteins in Myotendinous Junctions of Human Extraocular Muscles.

Purpose: The purpose of this study was to investigate the cytoskeletal composition of myotendinous junctions (MTJs) in the human extraocular muscles (EOMs). Desmin and other major cytoskeletal proteins are enriched at the MTJs of ordinary myofibers, where they are proposed to be of particular importance for force transmission and required to maintain myofiber integrity. Methods: EOM and limb muscle samples were analyzed with immunohistochemistry using antibodies against the intermediate filament proteins desmin, nestin, keratin 19, vimentin, and different myosin heavy chain (MyHC) isoforms. MTJs were identified by labeling with antibodies against laminin or tenascin. Results: In contrast to MTJs in lumbrical muscle where desmin, nestin, and keratin 19 were always present, approximately one-third of the MTJs in the EOMs lacked either desmin and/or nestin, and all MTJs lacked keratin 19. Approximately 6% of the MTJs in the EOMs lacked all of these key cytoskeletal proteins. Conclusions: The cytoskeletal protein composition of MTJs in human EOMs differed significantly from that of MTJs in limb muscles. These differences in cytoskeletal protein composition may indicate particular adaptation to meet the functional requirements of the EOMs.

Absence of Desmin in Myofibers of the Zebrafish Extraocular Muscles.

Purpose: To study the medial rectus (MR) muscle of zebrafish (Daniorerio) with respect to the pattern of distribution of desmin and its correlation to distinct types of myofibers and motor endplates. Methods: The MRs of zebrafish were examined using confocal microscopy in whole-mount longitudinal specimens and in cross sections processed for immunohistochemistry with antibodies against desmin, myosin heavy chain isoforms, and innervation markers. Desmin patterns were correlated to major myofiber type and type of innervation. A total of 1382 myofibers in nine MR muscles were analyzed. Results: Four distinct desmin immunolabeling patterns were found in the zebrafish MRs. Approximately a third of all slow myofibers lacked desmin, representing 8.5% of the total myofiber population. The adult zebrafish MR muscle displayed en grappe, en plaque, and multiterminal en plaque neuromuscular junctions (NMJs) with intricate patterns of desmin immunolabeling. Conclusions: The MRs of zebrafish showed important similarities with the human extraocular muscles with regard to the pattern of desmin distribution and presence of the major types of NMJs and can be regarded as an adequate model to further study the role of desmin and the implications of heterogeneity in cytoskeletal protein composition. Translational Relevance: The establishment of a zebrafish model to study the cytoskeleton in muscles that are particularly resistant to muscle disease opens new avenues to understand human myopathies and muscle dystrophies and may provide clues to new therapies.

Complex Correlations Between Desmin Content, Myofiber Types, and Innervation Patterns in the Human Extraocular Muscles.

Purpose: To investigate whether the distribution of intermediate filament protein desmin is related to the different patterns of innervation in the human extraocular muscles (EOMs). Methods: EOM samples were analyzed with immunohistochemistry using antibodies against desmin, vimentin, different myosin heavy chain (MyHC) isoforms, and fetal and adult acetylcholine receptor (AChR) subunits. Neuromuscular junctions (NMJs) were identified with α-bungarotoxin or with antibodies against neurofilament and synaptophysin. Results: Desmin was present in the vast majority of myofibers, but it was weakly present or absent in a limited area in the close vicinity of the single en plaque NMJs in less than half of these myofibers. Desmin was either present or lacking in MyHCsto/I myofibers displaying multiple en grappe endings but present in MyHCsto/I myofibers receiving spiral nerve endings. In MyHCeom myofibers displaying multiterminal en plaque endings, desmin was either present or absent irrespective of AChR subunits or EOM layer. Vimentin did not substitute for the lack of desmin. Conclusions: The results indicate that the human EOMs have a more complex cytoskeletal organization than other muscles and suggest additional signalling mechanisms from the NMJs to the myofibers.

Gene expression profile of extraocular muscles following resection strabismus surgery.

This paper aims to identify key biological processes triggered by resection surgery in the extraocular muscles (EOMs) of a rabbit model of strabismus surgery by studying changes in gene expression. Resection surgery was performed in the superior rectus of 16 rabbits and a group of non-operated rabbits served as control. Muscle samples were collected from groups of four animals 1, 2, 4 and 6 weeks after surgery and processed for RNA-sequencing and immunohistochemistry. We identified a total of 164; 136; 64 and 12 differentially expressed genes 1, 2, 4 and 6 weeks after surgery. Gene Ontology enrichment analysis revealed that differentially expressed genes were involved in biological pathways related to metabolism, response to stimulus mainly related with regulation of immune response, cell cycle and extracellular matrix. A complementary pathway analysis and network analysis performed with Ingenuity Pathway Analysis tool corroborated and completed these findings. Collagen I, fibronectin and versican, evaluated by immunofluorescence, showed that changes at the gene expression level resulted in variation at the protein level. Tenascin-C staining in resected muscles demonstrated the formation of new tendon and myotendinous junctions. These data provide new insights about the biological response of the EOMs to resection surgery and may form the basis for future strategies to improve the outcome of strabismus surgery.

Sulfated Glycosaminoglycans as Viral Decoy Receptors for Human Adenovirus Type 37.

Glycans on plasma membranes and in secretions play important roles in infection by many viruses. Species D human adenovirus type 37 (HAdV-D37) is a major cause of epidemic keratoconjunctivitis (EKC) and infects target cells by interacting with sialic acid (SA)-containing glycans via the fiber knob domain of the viral fiber protein. HAdV-D37 also interacts with sulfated glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, we investigated the molecular requirements of HAdV-D37 fiber knob:GAG interactions using a GAG microarray and demonstrated that fiber knob interacts with a broad range of sulfated GAGs. These interactions were corroborated in cell-based assays and by surface plasmon resonance analysis. Removal of heparan sulfate (HS) and sulfate groups from human corneal epithelial (HCE) cells by heparinase III and sodium chlorate treatments, respectively, reduced HAdV-D37 binding to cells. Remarkably, removal of HS by heparinase III enhanced the virus infection. Our results suggest that interaction of HAdV-D37 with sulfated GAGs in secretions and on plasma membranes prevents/delays the virus binding to SA-containing receptors and inhibits subsequent infection. We also found abundant HS in the basement membrane of the human corneal epithelium, which may act as a barrier to sub-epithelial infection. Collectively, our findings provide novel insights into the role of GAGs as viral decoy receptors and highlight the therapeutic potential of GAGs and/or GAG-mimetics in HAdV-D37 infection.

Altered Signaling Pathways in Aniridia-Related Keratopathy.

Purpose: To study the Notch1, Wnt/beta-catenin, sonic hedgehog (SHH), and mammalian target of rapamycin (mTOR) cell signaling pathways in naïve and surgically treated corneas of aniridia cases with advanced aniridia-related keratopathy (ARK). Methods: Two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation, and two adult healthy control corneas were processed for immunohistochemistry in this descriptive study. Antibodies specific against elements of the Notch1 (Notch1; Dlk1; Numb), Wnt/beta-catenin (Wnt5a; Wnt7a; beta-catenin), SHH (glioma-associated oncogene homolog [Gli1]; Hes1), and mTOR (mTOR1; ribosomal protein S6 [rpS6]) signaling pathways were used as well as antibodies against PAX6 and keratin 13 (Krt13). Results: All ARK corneas presented signs of conjunctivalization and analogous signaling pathway changes in the subepithelial pannus and epithelium, with decreased detection of the Notch1 signaling pathway and an increased presence of the Notch1 inhibitors Numb and Dlk1. Increased detections of Wnt/beta-catenin (enhanced presence of Wnt5a, Wnt7a, and beta-catenin), SHH (detection of Gli1 and Hes1), and mTOR (identification of mTOR and rpS6) signaling pathways were found in the subepithelial pannus and epithelium of all ARK corneas, when compared with normal controls. Conclusions: The similarity in pathway alterations found in all ARK corneas, irrespective of limbal stem cell transplantation, further supports the discussion on the role of host-specific factors and limbal stem cell deficiency in ARK.

The Cytoskeleton in the Extraocular Muscles of Desmin Knockout Mice.

Purpose: To investigate the effect of absence of desmin on the extraocular muscles (EOMs) with focus on the structure and composition of the cytoskeleton. Methods: The distribution of synemin, syncoilin, plectin, nestin, and dystrophin was evaluated on cross and longitudinal sections of EOMs and limb muscles from 1-year-old desmin knockout mice (desmin-/-) by immunofluorescence. General morphology was evaluated with hematoxylin and eosin while mitochondrial content and distribution were evaluated by succinate dehydrogenase (SDH) and modified Gomori trichrome stainings. Results: The muscle fibers of the EOMs in desmin-/- mice were remarkably well preserved in contrast to those in the severely affected soleus and the slightly affected gastrocnemius muscles. There were no signs of muscular pathology in the EOMs and all cytoskeletal proteins studied showed a correct location at sarcolemma and Z-discs. However, an increase of SDH staining and mitochondrial aggregates under the sarcolemma was detected. Conclusions: The structure of the EOMs was well preserved in the absence of desmin. We suggest that desmin is not necessary for correct synemin, syncoilin, plectin, and dystrophin location on the cytoskeleton of EOMs. However, it is needed to maintain an appropriate mitochondrial distribution in both EOMs and limb muscles.

Chromatic Pupillometry in Children.

Chromatic pupillometry is a technique that is increasingly used to assess retinal disorders. As age may be one of the various factors which can influence the pupillary light reaction, this study aimed to evaluate the pupil responses to colored light stimuli in the pediatric population. Fifty-three children with normal vision and without any history of ocular disorders were tested with a portable pupillometer. Four test sequences were used: five dim blue (470 nm) stimuli presented in half log steps ranging from -3.15 to -1.15 log cd/m2 after 3 min of dark adaptation, five red (622 nm) stimuli of -1.15, -0.7, -0.15, 0.3, and 0.85 log cd/m2 after 1 min light adaptation, one bright blue stimulus of 2.2 log cd/m2 and one bright red of 2 log cd/m2. The results were grouped by age: a younger group included 27 children aged from 3 to 10 years old and an older group included 26 from 10 and 1 month to 18 years old. The younger group had a smaller pupil diameter after dark adaptation compared with the older group. A linear regression defining the photopic threshold showed that younger subjects had a higher threshold, e.g., needed a brighter red stimulus to evoke a threshold pupil response comparable that of subjects. Age thus seems to influence outer retinal sensitivity at least as evaluated by the pupillary photopic threshold intensity. The post-illumination pupillary reaction was used as a marker of intrinsic melanopsin activity and did not show any difference between the two age groups.

Aniridia-related keratopathy: Structural changes in naïve and transplanted corneal buttons.

BACKGROUND: To study structural changes in naïve and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK). METHODS AND FINDINGS: Two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor α11 integrin and laminin α3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, α-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naïve ARK, KLAL ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous. CONCLUSIONS: Given the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run.

Composition, Architecture, and Functional Implications of the Connective Tissue Network of the Extraocular Muscles.

Purpose: We examined the pattern and extent of connective tissue distribution in the extraocular muscles (EOMs) and determined the ability of the interconnected connective tissues to disseminate force laterally. Methods: Human EOMs were examined for collagens I, III, IV, and VI; fibronectin; laminin; and elastin using immunohistochemistry. Connective tissue distribution was examined with scanning electron microscopy. Rabbit EOMs were examined for levels of force transmission longitudinally and transversely using in vitro force assessment. Results: Collagens I, III, and VI localized to the endomysium, perimysium, and epimysium. Collagen IV, fibronectin, and laminin localized to the basal lamina surrounding all myofibers. All collagens localized similarly in the orbital and global layers throughout the muscle length. Elastin had the most irregular pattern and ran longitudinally and circumferentially throughout the length of all EOMs. Scanning electron microscopy showed these elements to be extensively interconnected, from endomysium through the perimysium to the epimysium surrounding the whole muscle. In vitro physiology demonstrated force generation in the lateral dimension, presumably through myofascial transmission, which was always proportional to the force generated in the longitudinally oriented muscles. Conclusions: A striking connective tissue matrix interconnects all the myofibers and extends, via perimysial connections, to the epimysium. These interconnections are significant and allow measurable force transmission laterally as well as longitudinally, suggesting that they may contribute to the nonlinear force summation seen in motor unit recording studies. This provides strong evidence that separate compartmental movements are unlikely as no region is independent of the rest of the muscle.

Exophiala phaeomuriformis keratitis in a subarctic climate region: a case report.

PURPOSE: To report a case of Exophiala phaeomuriformis mycotic keratitis in a patient from a subarctic climate region. Dematiaceous fungi (black yeasts) have been gaining importance as corneal keratitis and ulcer causative agents in certain regions, but no cases have been described in Scandinavia. METHODS: Case report of a patient with a persistent corneal erosion that eventually presented a brown-pigmented infiltrate. The patient had a history of several months of topical therapy comprising medication for glaucoma, corticosteroids and antibiotics. A therapeutic contact lens was used, and amniotic membrane transplantation was performed before the development of the pigmented infiltrate. RESULTS: Exophiala phaeomuriformis was identified on the microbiological cultures from the surgically obtained infiltrate scrapes. The patient responded to topical amphotericin and fluconazole, the erosion was cured and a stromal scar subsided. During follow-up, sequential slit-lamp images and anterior segment optical coherence tomography (OCT) scans were obtained. CONCLUSION: This is the first described case of keratitis caused by E. phaeomuriformis in a subarctic region, the first in Europe and, to our knowledge, the second reported case in the literature. It is important to remember that superficial corneal brown-pigmented infiltrates should raise the suspicion of an unusual fungal infection even in this climate. This is particularly important in patients with ocular surface disease treated with steroids and antibiotics for a long time.

Impact of Amyotrophic Lateral Sclerosis on Slow Tonic Myofiber Composition in Human Extraocular Muscles.

Purpose: To analyze the proportion and cross-sectional area of myofibers containing myosin heavy chain slow-twitch (MyHCI) and myosin heavy chain slow tonic (MyHCsto) in extraocular muscles of autopsied amyotrophic lateral sclerosis (ALS) patients with either spinal or bulbar site of disease onset. Methods: Whole-muscle cross sections from the middle portion of the medial rectus were labeled with antibodies against MyHCI or MyHCsto and laminin. Myofibers labeled with the MyHC antibodies (MyHCI+sto) and the total number of myofibers were quantified in the orbital and global layer of 6 control individuals and 18 ALS patients. The cross-sectional area of myofibers labeled for either MyHC was quantified in 130 to 472 fibers/individual in the orbital and in 180 to 573 fibers/individual in the global layer of each specimen. Results: The proportion of MyHCI+sto myofibers was significantly smaller in the orbital and global layer of ALS compared to control individuals. MyHCI+sto myofibers were significantly smaller in the global layer than in the orbital layer of ALS, whereas they were of similar size in control subjects. The decreased proportion of MyHCI+sto fibers correlated significantly with the age of death, but not disease duration, in patients who had the bulbar-onset variant of ALS but not in patients with spinal variant. Conclusions: ALS, regardless of site of onset, involves a loss of myofibers containing MyHCI+sto. Only in bulbar-onset cases did aging seem to play a role in the pathophysiological processes underlying the loss of MyHCI+sto fibers.

Unchanged Neurotrophic Factors and Their Receptors Correlate With Sparing in Extraocular Muscles in Amyotrophic Lateral Sclerosis.

Purpose: To investigate the impact of amyotrophic lateral sclerosis (ALS) on the extraocular muscles (EOMs) by examining the distribution of neurotrophic factors (NTFs) and their receptors in EOMs and limb muscles from ALS transgenic mice. Methods: Muscle samples collected from transgenic mice overexpressing human superoxide dismutase type 1 mutations (SOD1G93A, the most widely used mouse model of ALS) at 50 and 150 days as well as age-matched controls were analyzed with immunohistochemistry using antibodies against brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4), glial cell line-derived neurotrophic factor (GDNF), and the neurotrophin receptors p75NTR, tyrosine kinase (Trk) receptor TrkB and TrkC, and GDNF family receptor alpha-1 (GFRα-1). Results: There was an intrinsic difference in NTF expression between EOMs and limb muscles in control mice: EOMs presented significantly lower number of neuromuscular junctions (NMJs) labeled for BDNF and NT-4 at 50 days, and for BDNF and GDNF at 150 days, compared with the control limb muscles of corresponding age. In ALS transgenic mice at 150 days, NTF expression in limb muscles was significantly changed but not in EOMs: the limb muscles presented a significant decline in the number of NMJs labeled for BDNF, NT-4, GDNF, p75NTR, TrkB, and TrkC, which was not observed in EOMs. Conclusions: The significant differences in expression of NTFs on NMJs between EOMs and limb muscles in both control and ALS transgenic mice suggest that NTF may be involved in the pathogenesis of ALS and the resistance of EOMs to the disease.

Pax7-Positive Cells/Satellite Cells in Human Extraocular Muscles.

PURPOSE: We quantified and investigated the distribution of Pax7-positive cells/satellite cells (SCs) in the human extraocular muscles (EOMs). METHODS: An immunofluorescence multiple-marker method simultaneously combining two SC markers (Pax7, NCAM), detection of the basement membrane (laminin) and cell nuclei (4',6-diamidino-2-phenylindole [DAPI]), was used on the anterior, middle, and posterior portions of EOMs from five healthy donors. Pax7-positive cell and SC content, myonuclear content, myofiber cross-sectional area, and myonuclear domain were analyzed in single cross-sections. Between 3915 and 13,536 myofibers per muscle cross-section and myofibers from the entire EOM cross-section were analyzed for quantification of Pax7-positive cells per myofiber (Pax7/F). RESULTS: The number of Pax7/F in the human EOMs varies along the length of the muscle with twice as high Pax7/F in the anterior part of the EOMs, but within the range of what has been previously reported for normal adult limb muscles. Furthermore, there are Pax7-positive cells in positions other than the classical SC position and the myonuclear domain size of adult EOMs is noticeably smaller than that previously reported for other adult skeletal muscles. CONCLUSIONS: Previous data on differences in Pax7-positive cell/SC abundance between EOMs and limb muscles must be reconsidered and the characteristics of different Pax7-positive cell populations further investigated. Higher numbers of Pax7-positive cells in the anterior portion of the EOMs may have a bearing for strabismus surgery involving sectioning of the muscle fibers.

Fibre typing of intrafusal fibres.

The first descriptions of muscle spindles with intrafusal fibres containing striated myofibrils and nervous elements were given approximately 150 years ago. It took, however, another 100 years to establish the presence of two types of intrafusal muscle fibres: nuclear bag and nuclear chain fibres. The present paper highlights primarily the contribution of Robert Banks in fibre typing of intrafusal fibres: the confirmation of the principle of two types of nuclear bag fibres in mammalian spindles and the variation in occurrence of a dense M-band along the fibres. Furthermore, this paper summarizes how studies from the Umeå University group (Laboratory of Muscle Biology in the Department of Integrative Medical Biology) on fibre typing and the structure and composition of M-bands have contributed to the current understanding of muscle spindle complexity in adult humans as well as to muscle spindle development and effects of ageing. The variable molecular composition of the intrafusal sarcomeres with respect to myosin heavy chains and M-band proteins gives new perspectives on the role of the intrafusal myofibrils as stretch-activated sensors influencing tension/stiffness and signalling to nuclei.