Linda K. McLoon - US grants

Embryonic retina, superior colliculus or occipital cortex transplanted to the midbrain region of a newborn rat differentiates and forms extensive connections with appropriate visual nuclei in the host brain. These same fetal regions, when transplanted to the midbrain region of an adult rat, also survive and differentiate. However, in marked contrast to the newborn transplants, axonal outgrowth into the adult midbrain is very limited, extending a maximum of 6 mm into the host tissue. The project proposed here will further examine transplants of fetal retina, superior colliculus and cortex into adult brain in order to gain insight into ways in which this outgrowth can be increased as well as to further assess the potential use of these transplants as a clinical technique. The project has three major parts. First will be the examination in vitro of whether the presence of various glial populations, i.e. optic nerve glia, Muller cells, astrocytes, and Schwann cells, can facilitate neuritic outgrowth from explant cultures of embryonic and adult rat retina, tectum or cortex. Second will be to evaluate the possible ability of the various glial populations to increase axonal outgrowth from the transplants in vivo. This will involve the preparation of reaggregates of the embryonic visual system tissue combined with enriched populations of the various glial types. Third will be to determine if transplanted optic nerve or sciatic nerve grafts might be able to elicit and sustain outgrowth between areas of newborn or adult host visual system. This project will provide a greater understanding of the possible mechanisms active in the adult visual system that limit its regenerative capacity and ascertain ways to increase this outgrowth into adult tissue to foster recovery after injury. With this understanding, we can begin to assess means whereby the adult visual system might be treated to restore functon after injury to the system.

Blepharospasm, hemifacial spasm and other diseases involving forceful, uncontrolled muscle spasms of the face and neck are extremely debilitating conditions that result in functional blindness, often causing affected persons to withdraw from social and economic activities. Indirect treatment with orally administered medications is often accompanied by unacceptable side effects. Surgical treatments are costly and often unsatisfactory. Current medical treatment involves the injection of botulinum toxin into the affected muscles. While usually effective, this treatment provides only temporary relief. We have developed a novel permanent, non-surgical treatment for these diseases, doxorubicin chemomyectomy. Doxorubicin (DXN), a potent anti-metabolic and anti-mitotic cancer drug, is injected directly into the eyelids. In laboratory studies injection of DXN results in a loss of up to 70% of the muscle fibers in the orbicularis oculi muscle. This muscle loss is permanent, with no further changes in muscle fiber number after one month. In the Phase I patient clinical trial, over half of the treated patients have now gone over a year with significant relief from their muscle spasms and without requiring further treatment. One problem with the current protocol is that the maximal relief of muscle spasms requires three sets of injections at the maximal safe dose. Each injection results in a substantial inflammatory reaction within the lid, and each injection increases the chance of skin ulceration at the injection site. Inflammation and local pain at the injection site is the major deterrent for patients in choosing this treatment alternative. This proposal focuses on the improvement of this treatment for these patients. First, the acute and chronic inflammatory reactions after doxorubicin administration will be assessed. Attempts will be made to ameliorate inflammation and skin injury with a variety of anti- inflammatory mediators. Issues of skin toxicity are a major concern for patients. The effect of doxorubicin on epithelial cell turnover in the treated eyelids will be assessed and the role of lipid peroxidation on doxorubicin induced skin toxicity will be determined. The generation of oxidants will be assessed in the treated eyelids. Further strategies will be tested that alter anti- and pro-oxidant generating molecules within the eyelid in an attempt to increase doxorubicin myotoxicity and/or decrease skin injury and inflammation. A variety of agents known to increase doxorubicin toxicity will be tested in an effort to improve the efficiency of muscle loss. These studies will allow us to develop the most clinically effective and cost effective treatment for these patients.

9407826 McLoon ABSTRACT The extraocular muscles, the muscles that move the eye in the orbit, are different from other mature skeletal muscles in mammals. They have a number of characteristics seen only in immature or regenerating muscle, such as the continued expression of immature or embryonic proteins and as well as immature patterns of innervation. This study proposes to examine the possibility that either these muscles fail to fully mature or that the extraocular muscles, unlike other skeletal muscles continue to divide in the mature mammal to produce new muscle fibers. This will hopefully shed light on possible mechanisms that control when and how muscle cells divide.

DESCRIPTION: (Applicant's Abstract) Extraocular muscles (EOM) are spared or preferentially involved in various skeletal muscle diseases. We propose a novel and controversial alternative mechanism that may form the basis for the differences between extraocular muscles and limb skeletal muscles. We have strong preliminary evidence to suggest that there is ongoing, continuous myofiber remodeling in the adult extraocular muscles. This would involve both myogenic and apoptotic components. These conclusions are based on 4 lines of evidence. In this proposal we are asking questions to confirm our original observations. We have shown that the EOM continue to express cells positive for myogenic regulatory factors, such as myoD. Activated satellite cells are always present in adult EOM. BrdU labeling experiments using both 2 week and 4 week continuous labeling protocols followed by various brdU-free periods, a protocol that labels dividing cells, demonstrated mature myofibers with brdU-positive nuclei within them. Our working hypothesis is that there are mechanisms present in adult EOM that allow continuous satellite cell activation and division, resulting in either continuous remodeling of existing myofibers by fusion of new myoblasts with existing adult myofibers or formation of entirely new myofibers by the fusion of myoblasts with each other. This proposal asks the following questions: What are the mechanisms of myofiber remodeling in adult EOM? What is the time course and extent of fiber remodeling? What role does apoptosis play in myofiber remodeling and by what mechanism? How do surgical and chemodenervation manipulations simulating strabismus treatments cause changes in myofiber remodeling that may be affecting long-term surgical outcomes? The ability of adult EOM to continuously remodel provides a wealth of testable hypotheses for some long-standing enigmas involving the EOM and their preferential sparing or involvement in various muscle diseases. Ultimately, we hope to use this information to develop new therapeutic strategies.for the treatment of strabismus and other ocular and non-ocular muscle diseases.

DESCRIPTION (provided by applicant): Our studies are directed at advancing treatments to cure infantile and acquired strabismus. At least 3% of the children born in the U.S. are diagnosed with strabismus each year. Early treatment of strabismus can prevent loss of visual function, but strabismus management remains challenging. This is partly because we lack a definitive understanding of its etiology. Our studies will fill this gap in knowledge and lead to improved therapies for strabismus. Eye misalignment in some cases is likely due to improper calibration of the tonic innervation of individual extraocular muscles (EOM) or specific muscle compartments. Acquired strabismus may follow injury of EOM or their innervation. Ultimately, eye alignment, gaze-holding and eye movements all depend on the quality of ocular motor innervation supplied to the EOM. Our studies will determine how current surgical methods and novel application of growth factors alter oculomotor neuronal properties associated with eye alignment. Our long term goal is to develop pharmacologic therapies to modulate force in an underacting or overacting EOM by changing intrinsic neuronal firing properties and innervational density and modulating perineuronal nets on the motor neurons that innervate the EOM to allow for synaptic plasticity and motor fusion. We will test these strategies in a non-human primate model of sensory-induced strabismus. We also hope to prevent maladaptations that occur after normal human strabismus surgery in order to reduce surgical failure rate. We have 5 specific aims: I. What are the molecular signals that influence the growth of axons and maintain the innervational pattern of the extraocular muscles? We will analyze patterns of growth factor and receptor expression in rabbit EOM, followed by use of exogenously added growth factors or antibodies to modulate the innervational pattern and density. II. Can EOM innervation be manipulated so that maladaptations at the muscle level that occur after surgical recession and/or resection are prevented? We will manipulate nerve growth in EOM by either promoting or preventing proliferation of satellite cells and/or nerve sprouting and neuromuscular junction formation. III. Can we modulate the perineuronal nets around mature motor neurons, and does this result in altered synaptic connections after sustained growth factor treatments? We will define perineuronal net structure and determine efficacy of sustained release BDNF, IGF1, and BMP4 to modulate perineuronal nets, and determine if it will restore motor neuron synaptic plasticity. IV. How do our proven growth factors alter oculomotor neuronal firing properties in the ocular motor system of normal non- human primates? We will analyze neuronal firing rates after treatments we pioneered, IGF-1 and BMP4, which increase or decrease force generation and muscle size. V. We hypothesize that growth factors can effectively treat strabismus. We will use our established methods to produce sensory-induced strabismus in infant monkeys, and then evaluate novel treatments employing growth factor treatments to correct eye misalignment.

DESCRIPTION (provided by applicant): The extraocular muscles (EOM) are spared in Duchenne muscular dystrophy patients (DMD) and continue to function after most skeletal muscles in the body have completely degenerated. The reason for this sparing in DMD is unknown. Unlike limb skeletal muscle, normal adult EOM retain a population of activated satellite cells, the regenerative cell in adult skeletal muscle. Satellite cells actively fuse into normal myofibers in the EOM throughout life, even in aging EOM, resulting in a continuous process of myofiber remodeling. The EOM and their satellite cells are extremely resilient to injury, denervation, disease, and aging, retaining normal morphology when limb muscle would normally atrophy. In addition, the early genes controlling EOM development are distinct from those that control somite development. We will test the hypothesis that this continuous remodeling is the process by which EOM are spared in DMD. We will examine 1) the rate of myogenic precursor cell turnover in two mouse models of DMD, the mdx and mdx/utrophin+/- (mdx/utrhet) mice, and 2) test whether inhibition of cell division in the EOM prevent their sparing in the two mouse models for DMD. The sparing of the EOM in DMD, aging, and injury and differences in the myogenic precursor cell populations in skeletal muscle suggest myogenic precursor cells (mpcs) within EOM may be significantly enriched or phenotypically distinct compared with adult limb muscle cells. There are 5-8 fold more myogenic precursor cells in adult EOM compared to limb. Additionally, mpcs from EOM are more resistant to apoptosis than similar cells from limb. We will test the hypothesis that continuous remodeling allows for EOM sparing in DMD by examining 1) the rate of myogenic precursor cell turnover in two mouse DMD models, the mdx and mdx/utrophin+/- heterozygote (mdx/utrhet) mice, and 2) whether inhibition of cell division by gamma irradiation of the EOM prevents sparing in the DMD models. Sparing of the EOM in DMD and differences in their mpcs suggest that the EOM mpcs may be significantly enriched or phenotypically distinct compared with adult limb mpcs. One population is increased;these cells are CD34+ and negative for Sca1, CD31, an endothelial lineage marker, CD45, an hematopoietic lineage marker, and negative for various satellite cell markers (EOMCD34). These EOMCD34 cells are present in the EOM and limb muscles of neonatal mice, but only maintained throughout adulthood in EOM. These cells are also present in the EOM of mdx and mdx/utrophin-/- mice. Our hypothesis is that this population of EOMCD34 cells enriched in adult EOM may be, at least in part, responsible for the sparing of EOM in DMD. We will test these hypotheses in two Specific Aims. Specific aim 1 asks: 1) is the rate of myofiber remodeling increased in the DMD mice models? 2) Does inhibition of cell division in adult EOM prevent sparing in the EOM of mdx/utrophin +/- heterozygote mice? We will isolate specific subpopulations of mononucleated cells from EOM and limb muscles and in specific aim 2 ask: 1) Are the mpcs more resistant to injury?2) Are the mpcs from EOM more multipotent than those from limb muscle? 3) Do the myogenic precursor cells from EOM have greater proliferative potential than those derived from limb skeletal muscle? The long term goal is to define and isolate a myogenic precursor cell type from EOM that has greater proliferative and survival potential compared with limb. These cells would be tested in a myoblast transfer model to determine their potential for use in the treatment of DMD. These may offer advantages over other cells, as autologous transplants would be possible. PUBLIC HEALTH RELEVANCE: The extraocular muscles (EOM) are spared in Duchenne muscular dystrophy (DMD), and the cause of this sparing is unknown. The unique ability of EOM to continuously remodel throughout life suggests that this ability may be responsible for this sparing. This may be due to an enriched myogenic precursor cell population within the EOM that has a greater ability to survive injury, aging and disease. While the eye muscles are spared in DMD, the rate of regeneration in the EOM is similar to that in the leg muscles of the mdx mouse model of DMD. We will attempt to demonstrate that muscle precursor cell division is responsible for sparing of the EOM by using irradiation to inhibit cell division. It is well known that the EOM survive injury better than limb muscle. This in turn suggests that muscle progenitors in EOM may be more robust and long-lived than those from limb muscles. If these hypotheses are true, ultimately we hope to exploit this by using identified myogenic precursor cells from EOM as a new source of donor cells in myoblast therapy in mice models of muscle injury and DMD.

? DESCRIPTION (provided by applicant): This is a revised application for a training program focused on translational vision science from an established group of vision scientists. We request funds for six predoctoral students with a training focus in translational vision science to reflect the current strengths of the vision faculty as well as current NEI research priorities. Thi is an exciting time to pursue a career in vision research, as molecular biology and imaging advances have allowed visual outcomes to be monitored in real time. Our vision community has become increasingly focused on translational research with well-known senior faculty who are highly respected investigators in vision research as well as younger investigators clearly on a trajectory for long term success. The training faculty consists of a core group of 6 NEI funded vision scientists and an additional 10 vision scientists who have actively funded research programs in translational vision research. With the arrival of our new chairperson in the Department of Ophthalmology and Visual Neurosciences, we have greatly increased the collaborations between vision scientists at the University of Minnesota and the clinical faculty in the medical school. Several key elements of our training program in Translational Vision Research include a course designed for trainees, Basic and Clinical Vision Sciences, co-taught by the training faculty in this proposal, and an annual Vision Symposium, where nationally known and in-house experts in vision science present their most recent research along with our trainees and faculty. Trainees will come from one of 4 programs: the Graduate Programs in Neuroscience; Molecular, Cellular, Developmental Biology and Genetics; Psychology; Biomedical Engineering, or Microbiology, Immunology and Cancer Biology. Trainees will be eligible for support once they have completed their first year of graduate school and have begun focused research under the mentorship of a grant preceptor, culminating in their thesis research. They will earn a Ph.D. or an M.D./Ph.D. and will be able to receive support from two to a maximum of four years. The diverse research interests of the vision scientists at the University of Minnesota, and the broad-based and collaborative nature in which they study the visual system - from molecular biology to visual perception - makes this an excellent environment for vision research with translatable components. Each trainer directs a successful research program and has demonstrated commitment to teaching and training. In addition, all trainees will be connected to a practicing Ophthalmologist in their research area, as well as expected to attend related grand rounds. An impressive array of scientific and institutional resources is available to the trainees, but their choice of vision research laboratories will be greatly facilitated by access to support from this training grant. As we move toward increasingly collaborative research between our basic and clinically trained faculty members, we believe this approach will maximize our ability to prepare vision scientists with a multi-disciplinary education needed to meet the audacious goals of the NEI - focused on preventing, treating, and rehabilitating blinding eye disease.

HISTOLOGY CORE ? SUMMARY Histology Module. The Histology module provides a fully equipped histology laboratory and the services of the module histologist to Core Grant investigators and staff. Services offered by our skilled histologist, Ms. Heidi Roehrich, include (1) embedding and sectioning of paraffin embedded and frozen tissue, (2) hematoxylin and eosin staining of sections and other histological stains as required (e.g. Oil Red O, cresyl violet, Masson's trichome), (3) immunohistochemistry using both DAB and fluorescence based procedures, (4) in situ cell death detection, (5) in situ hybridization, (6) Q-PCR analysis of RNA, (7) nuclease protection assays, (8) RNA extraction, (9) protein assays, (10) image acquisition and analysis using the Bioquant Nova Prime software, and (11) preparation of publication quality photomicrographs. The Histology module has proven to be a great asset and is in high demand by a wide range of Core Grant investigators. It is utilized to capacity and, for the past several years, we have had to hire a part-time student assistant to help our histologist, who is fully occupied with the more complex histological procedures required by our investigators.