Linda K. McLoon

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.

Intranasal delivery of neurotrophic factors BDNF, CNTF, EPO, and NT-4 to the CNS

Injury to the central nervous system (CNS) generally results in significant neuronal death and functional loss. In vitro experiments have demonstrated that neurotrophic factors such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and neurotrophin-4/5 (NT-4/5) can promote neuronal survival. However, delivery to the injured CNS is difficult as these large protein molecules do not efficiently cross the blood–brain barrier. Intranasal delivery of 70 μg [125I]-radiolabeled BDNF, CNTF, NT-4, or erythropoietin (EPO) resulted in 0.1–1.0 nM neurotrophin concentrations within 25 min in brain parenchyma. In addition, not only did these neurotrophic factors reach the CNS, they were present in sufficient concentrations to activate the prosurvival PI3Kinase/Akt pathway, even where lower levels of neurotrophic factors were measured. Currently traumatic, ischemic and compressive injuries to the CNS have no effective treatment. There is potential clinical relevancy of this method for rescuing injured CNS tissues in order to maintain CNS function in affected patients. The intranasal delivery method has great clinical potential due to (1) simplicity of administration, (2) noninvasive drug administration, (3) relatively rapid CNS delivery, (4) ability to repeat dosing easily, (5) no requirement for drug modification, and (6) minimal systemic exposure.

Experimental and Clinical Evidence for Brimonidine as an Optic Nerve and Retinal Neuroprotective Agent: An Evidence-Based Review

OBJECTIVE To review the available evidence for the neuroprotective qualities of brimonidine tartrate in optic nerve and retinal injury. METHODS References for this study were obtained by running a search of the PubMed database using keywords brimonidine, neuroprotection, ischemic optic neuropathy, and alpha2-adrenergic agonists. References focusing on ocular hypertension were excluded. RESULTS Forty-eight articles addressing 1 of 4 criteria for neuroprotection were included. The literature confirms that brimonidine therapy meets the first 3 criteria for neuroprotection: receptors on its target tissues, adequate penetration into the vitreous and retina at pharmacologic levels, and induction of intracellular changes that enhance neuronal resistance to insults or interrupt apoptosis in animal models. Brimonidine did not meet the final neuroprotective criterion of success in humans. CONCLUSIONS Experimental evidence has demonstrated that brimonidine is a potential neuroprotective agent. However, to date, clinical trials have failed to translate into similar efficacy in humans.

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.

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

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.

Cultured embryonic retinae transplanted to rat brain: Differentiation and formation of projections to host superior colliculus

Retinae of rats on embryonic day 14 were placed in explant culture for 2-14 days prior to transplantation adjacent to the superior colliculus of newborn rats. In explant culture cell division and neuronal differentiation continued unabated. One month after transplantation host brains were examined for transplant survival, differentiation and formation of projections to the host brain. The cultured retinal transplants survived and developed a morphology typical of mature retina, with normal cell and fiber laminae present. HRP injections into the host superior colliculus labeled neurons in the ganglion cell layer of the transplant which closely resembled ganglion cells in vivo. A small number of transplants received lesions. Degeneration material was traced into the superior colliculus and pretectal nuclei confirming that the cultured transplants had projections appropriate for retina entering the host brain. These results correlate closely with those seen after transplantation of embryonic rat retinae that had not been cultured. Thus, a period of explant culture of up to two weeks does not affect the ability of embryonic retinal transplants to differentiate and form projections into the host brain.

Sparing of Extraocular Muscle in Aging and Muscular Dystrophies: A Myogenic Precursor Cell Hypothesis

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin(-/-) (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.

Myonuclear Addition to Uninjured Laryngeal Myofibers in Adult Rabbits

Objectives: In normal mature limb skeletal muscle, satellite cells are quiescent and myonuclei do not divide after formation of their associated myofibers in the absence of injury. The possibility of myonuclear addition in uninjured laryngeal myofibers of adult rabbits was investigated in an immunohistochemical pilot study. Methods: Bromodeoxyuridine (brdU), a marker for cell division, was administered by intraperitoneal injection over a 12-hour period in rabbits. The number of brdU-positive myonuclei per myofiber was determined on cross sections through the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles. Results: In the TA muscle, 0.13% ± 0.03% (mean ± SEM) of the myofibers counted had a brdU-positive nucleus. In the PCA muscle, 0.13% ± 0.01% of the myofibers counted had a brdU-positive nucleus. Approximately 0.2% and 0.3% of the myofibers of the TA and PCA muscles, respectively, had brdU-positive satellite cells associated with them. Tibialis anterior and pectoralis major muscle controls were negative for brdU-positive myonuclei. Conclusions: These data support the possibility of continuous remodeling in uninjured adult laryngeal myofibers and accentuate the distinct nature of laryngeal muscle relative to limb skeletal muscle in the rabbit model.

Regional differences in the orbicularis oculi muscle: conservation between species

The orbicularis oculi muscle is a complex facial muscle involved in eyelid closure. The central parts of pretarsal and preseptal regions of the palpebral part of the orbicularis oculi muscle in rabbit and cynomolgus monkey lower eyelids were examined histologically and were analyzed for muscle fiber number, muscle fiber cross-sectional area and fiber type composition. Distinct regional differences were seen in the muscle fiber composition in these two regions of the muscle. The pretarsal portion of the muscle, that closest to the eyelid margin, was quite homogeneous and almost completely composed of type 2 fibers. These fibers were the smallest in cross-sectional area. Type 2 fibers also predominated in the preseptal portion of the muscle, but this region contained between 10 and 20% type 1 fibers. They appeared to be a gradient in muscle fiber size, whereby the fiber size increased as a function of the distance from the eyelid margin. The same pattern of regional differences were found in both rabbit and monkey orbicularis oculi. Thus, there is a clear conservation of these regional differences in these two species. While the developmental significance is unknown, the identification of this pattern may facilitate the evaluation of chemomyectomy agents for treatment of eyelid spasms in humans and allow a more accurate analysis of biopsy material from this muscle.