Jonathan D. Wirtschafter

Continuous myofiber remodeling in uninjured extraocular myofibers: myonuclear turnover and evidence for apoptosis.

Unlike normal mature limb skeletal muscles, in which satellite cells are quiescent unless the muscle is injured, satellite cells in mammalian adult extraocular muscles (EOM) are chronically activated. This is evidenced by hepatocyte growth factor, the myogenic regulatory factor, Pax‐7, and the cell‐cycle marker, Ki‐67, localized to the satellite cell position using serial sections and the positional markers laminin and dystrophin. Bromodeoxyuridine (brdU) labeling combined with dystrophin immunostaining showed brdU‐positive myonuclei, presumably the result of fusion of activated satellite cells into existing myofibers. One new myonucleus was added to every 1000 myofibers in cross‐section using a 12‐hour brdU‐labeling paradigm. The EOM thus appear to retain a stable nuclear population by an opposing process of apoptosis that results in myonuclear removal as visualized by terminal deoxynucleotidyltransferase‐mediated nick end labeling (TUNEL). Activated caspase‐3 was present in localized cytoplasmic domains extending from 10 to 210 μm within individual myofibers, suggesting segmental cytoplasmic reorganization. Understanding the cellular mechanisms that maintain this process of continuous myonuclear addition and removal in normal adult EOM may suggest new hypotheses to explain the preferential involvement or sparing of these muscles in skeletal muscle disease.

Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles.

Because complex structural differences in adult extraocular muscles may have physiological and pathophysiological significance, the three-dimensional pattern of myosin heavy chain (MHC) isoform expression within the orbital and global layers of the muscle bellies compared with the distal tendon ends was quantitatively assessed. Three of the six extraocular muscles of adult rabbits were examined for immunohistologic expression of all fast, fast IIA/X, slow, neonatal and developmental MHC isoforms. The percentages of myofibers positive for each of these 5 myosin isoforms were determined in the orbital and global layers. There were relatively similar patterns of fast and slow MHC expression in the orbital and global layers of each of the three muscles examined. There were high levels of developmental MHC in the orbital layers, but significantly fewer developmental MHC positive myofibers in the global layer. The most variable expression was found with the neonatal MHC. There were significant differences between the longitudinal expression of the various isoforms in the middle of each muscle compared with the tendon end. In the orbital layer of all three muscles examined, the large numbers of fibers positive for fast MHC in the middle of the muscle dramatically decreased at the tendon end, with a concomitant increase in expression of slow myosin. There was a greater number of developmental MHC-positive myofibers at the tendon end than in the middle of the muscle in all three muscles examined. In the global layer, the IIA/X-positive myofibers comprised only half of the total number of fast-positive myofibers whereas in the orbital layer they comprised all or almost all of the fast positive myofibers. The configuration of the extraocular muscles is more complex than might be indicated by previous studies. The lateral rectus muscle had the most individual pattern of MHC expression when compared with the inferior rectus and inferior oblique muscles. Together with dramatic cross-sectional MHC fiber type differences between the orbital and global layers of the muscles, there are pronounced longitudinal differences in the proportions of myofibers expressing these five MHC isoforms in the middle region of the muscles and those in the distal tendon ends. This longitudinal progression appears to occur both within single myofibers, as well as within the series of myofibers that comprise the length of the muscle. We also confirm that the number of myofibers is reduced at the tendonous end while the cross-sectional area of each of the remaining myofibers is proportionally increased with regard to those in the muscle belly. Future studies may yet require two additional schemes for anatomic classification of the named extraocular muscles. One will be based on immunohistochemical features of their constituent myofibers as a supplement to classifications based on their electron microscopic appearance, innervation patterns or relative position with regard to the globe and orbit. Another will be based on the proportional length and longitudinal position of individual myofibers within an individual extraocular muscle.

Continuous myonuclear addition to single extraocular myofibers in uninjured adult rabbits

Extraocular muscles (EOM) are unique among mammalian skeletal muscles in that they normally express molecules associated with muscle development and regeneration. In this study we show that satellite cells of EOM, unlike those of other skeletal muscles, continually divide in the normal, uninjured adult. Adult EOM contained activated satellite cells positive for the myogenic regulatory factor MyoD. EOM satellite cells did not require a prolonged activation period prior to onset of cell division and differentiation in vitro. EOM satellite cells incorporated bromodeoxyuridine (brdU), a marker for cell division, and with longer postlabeling survival, brdU‐labeled nuclei populated EOM myofibers. This was not seen with leg muscle. These findings suggest the possibility that continual division of satellite cells and fusion of their daughter myocytes with existing adult EOM myofibers contribute to the unique sparing or susceptibility of EOM to certain muscle diseases.

Hemifacial Spasm Due to Intracranial Tumor: An International Survey of Botulinum Toxin Investigators

Hemifacial spasm (HFS) due to intracranial mass lesions is rare. Most cases are thought to be due to compression of the facial nerve by small vessels near the root of the facial nerve. A survey was undertaken of all botulinum toxin investigators to determine the incidence of imaged mass lesions causing HFS. Responders contributed information on 1676 patients with HFS. Of this group, nine tumors were reported for an incidence of 0.54% of patients. However, of this group only 52.5% underwent computed tomography (CT) or magnetic resonance (MR) scanning so the incidence of tumor causing HFS could be as high as 1.0%. No one tumor type was predominant, and most patients were women older than 50 years of age. The incidence compares with another large series of HFS patients in which one tumor was found in 367 patients. The authors also report as an illustrative case a 26-year-old man with HFS due to a presumed lipoma of the cerebellopontine angle. This diagnosis can be made with increased certainty with MR scanning. If the incidence of unsuspected diagnostically significant mass lesions is 1 in 200 patients with HFS referred for botulinum toxin injection, the cost of detecting one such lesion would be

Glaucoma visual field analysis by computed profile of nerve fiber function in optic disc sectors.

100,000 at an average imaging cost of

Abnormalities of the Central Visual Pathways in Prader–Willi Syndrome Associated with Hypopigmentation

500 per MR imaging or CT examination. Although mass lesions are uncommon, any patient with HFS whose general clinical course could justify intervention should be considered for imaging studies to rule out treatable conditions other than vascular compression.

Time course of the regenerative response in bupivacaine injured orbicularis oculi muscle

Automated perimetry has decreased the subjective aspects of data collection, but analysis has remained largely subjective. A microcomputer permits more objective analysis by regrouping and averaging data points in the threshold static visual field according to their retinotopic projection onto the optic disc rather than according to their eccentricity from the point of fixation. The applications of the technique include (1) following glaucomatous visual field loss, (2) differentiating glaucomatous from other forms of visual loss, and (3) studying the effects of aging on the visual field. Six formulas for data analysis are described, and their relative usefulness discussed. Percent loss or gain seemed to convey the most diagnostic information to the clinician. Percent of expected sensitivity was less than the decimal visual acuity when the diagnosis was glaucoma but greater when the diagnosis was cataract. In some cases this analytic method should provide information that could favorably affect patient management.

Homonucleotide expansion and contraction mutations of PAX2 and inclusion of Chiari 1 malformation as part of renal-coloboma syndrome.

Patients with oculocutaneous or ocular albinism have misrouting of optic fibers, with fibers from 20 degrees or more of the temporal retina crossing at the chiasm instead of projecting to the ipsilateral hemisphere. Misrouting can result in strabismus and nystagmus. Because patients with the Prader-Willi syndrome may also have hypopigmentation and strabismus, we wondered whether they too might have misrouting of optic fibers. We therefore studied six patients with Prader-Willi syndrome selected for a history of strabismus, using pattern-onset visually evoked potentials with binocular and monocular stimulation to look for evidence of misrouted retinal-ganglion fibers. Four had hypopigmentation, and three of these four had abnormal evoked potentials indistinguishable from those recorded in human albinos. The two with normal pigmentation had normal responses. These findings indicate that patients with Prader-Willi syndrome who have hypopigmentation have a brain abnormality characterized by misrouting of retinal-ganglion fibers at the optic chiasm--a finding previously reported only in forms of albinism.

Optic nerve axoplasm and papilledema

Abstract Local anesthetics, particularly bupivacaine, are known to be myotoxic to skeletal muscle. Injury is followed by satellite cell mediated regeneration. The eyelid is a common site for the injection of local anesthetics. Due to the complex anatomy of this region and the unique properties of facial musculature compared to limb skeletal muscle, the response of the orbicularis oculi to local injection of bupivacaine was examined to determine the time course of maximum satellite cell activation and division. The lower eyelids of rabbits were injected with two doses of a combination of bupivacaine and hyaluronidase, spaced 18 h apart. To assess the time course of satellite cell division, bromodeoxyuridine (BrdU) was injected immediately or, 1, 2, 3, 6 or 13 days after the second bupivacaine injury. The rabbits were sacrificed 24 h later. The eyelids were prepared for immunohistological examination and morphometric analysis of the presence of CD11-positive monocytes, neutrophils and macrophages, MyoD expression in satellite cells and/or myoblasts, and co-expression of BrdU and the developmental myosin heavy chain isoform. One day after bupivacaine injury of the orbicularis oculi, there was a large influx of CD11-positive cells which gradually decreased over time. Maximum activation of satellite cells, as defined by MyoD expression, occurred 2 and 3 days after the injury. Using double labeling techniques, the peak of BrdU incorporation occurred on day 3 and was identified in developmental myosin co-labeled cells 4 days after injury. The peak of satellite cell activation and division occurred 3 days after bupivacaine induced injury, as demonstrated by both MyoD expression and after pulse labeling with BrdU as identified in double labeled cells positive for BrdU and the developmental myosin heavy chain isoform. The process of regeneration in this muscle extended beyond the duration of this study. Muscle fibers remained small in cross-sectional area and positive for developmental myosin 2 weeks after injury, at a time when the fiber number had reached control, uninjured levels.

Clinical Doxorubicin Chemomyectomy: An Experimental Treatment for Benign Essential Blepharospasm and Hemifacial Spasm

Renal‐Coloboma syndrome, an autosomal dominant disorder characterized by colobomatous eye defects, vesicoureteral reflux, and abnormal kidneys, results from mutations in PAX2. The purpose of this study was to identify mutations in PAX2 and understand the associated patient phenotypes. We report a severely affected girl and a mildly affected mother and daughter, all of whom have PAX2 homoguanine tract (7 G) missense mutations. The mother and daughter have optic nerve colobomas and the daughter has vesicoureteral reflux. The severely affected girl developed renal failure and has bilateral colobomatous eye defects. Additionally, this girl developed hydrocephalus associated with platybasia and a Chiari 1 malformation. We examined genomic DNA from these individuals by SSCP and sequencing. The mother and daughter had a novel mutation: a contraction in a string of 7 Gs to 6 Gs in one allele of PAX2, leading to a premature stop codon two amino acids downstream. The severely affected girl had an expansion to 8 Gs, leading to a premature stop codon 27 amino acids downstream. The 8 G expansion has been found in other patients without brain anomalies and has occurred spontaneously in a mouse model, PAX21Neu. We expand the known phenotype associated with mutations in PAX2 to include brain malformations. The homoguanine tract in PAX2 is a hot spot for spontaneous expansion or contraction mutations and demonstrates the importance of homonucleotide tract mutations in human malformation syndromes. Hum Mutat 14:369–376, 1999.