Anne C. Belliveau

Myf5-/- :MyoD-/- amyogenic fetuses reveal the importance of early contraction and static loading by striated muscle in mouse skeletogenesis.

The mechanical loading of striated muscle is thought to play an important role in shaping bones and joints. Here, we examine skeletogenesis in late embryogenesis (embryonic day 18.5) in Myf5−/−:MyoD−/− fetuses completely lacking striated muscle. The phenotype includes enlarged and fused cervical vertebrae and postural anomalies, some viscerocranial anomalies, long bone truncation and fusion, absent deltoid tuberosity of the humerus, scapular and clavicular hypoplasia, cleft palate, and cleft sternum. In contrast, neurocranial bone development was essentially normal. While the magnitude of individual effects varied throughout the skeletal system, the results are consistent with skeletal development depending on functional muscles. Novel abnormalities in the amyogenic fetuses relative to less severely paralyzed phenotypes extend our understanding of skeletogenic dependence on embryonic muscle contraction and static loading.

Importance of Normal Aging in Estimating the Rate of Glaucomatous Neuroretinal Rim and Retinal Nerve Fiber Layer Loss

PURPOSE To describe longitudinal rates of change of neuroretinal parameters in patients with glaucoma and healthy controls, and to evaluate the influence of covariates. DESIGN Prospective longitudinal study. PARTICIPANTS Treated patients with glaucoma (n = 192) and healthy controls (n = 37). METHODS Global disc margin-based neuroretinal rim area (DMRA) was measured with confocal scanning laser tomography, while Bruchs membrane opening-minimum rim width (BMO-MRW), BMO area (BMOA), and peripapillary retinal nerve fiber layer thickness (RNFLT) were measured with optical coherence tomography at 6-month intervals. Individual rates of change were estimated with ordinary least-squares regression, and linear mixed effects modeling was used to estimate the average rate of change and differences between the groups, and to evaluate the effects of baseline measurement and baseline age on rates of change. MAIN OUTCOME MEASURES Rates of change for each parameter. RESULTS Subjects were followed for a median (range) of 4 (2-6) years. The proportion of controls who had significant reduction of neuroretinal parameters was 35% for BMO-MRW, 31% for RNFLT, and 11% for DMRA. The corresponding figures for patients with glaucoma were not statistically different (42%, P = 0.45; 31%, P = 0.99; 14%, P = 0.99, respectively). Controls had a significant reduction of BMO-MRW (mean: -1.92 μm/year, P < 0.01) and RNFLT (mean: -0.44 μm/year, P = 0.01), but not DMRA (mean: -0.22×10(-2) mm(2)/year, P = 0.41). After adjusting for covariates, patients with glaucoma had faster, but not statistically different, rates of deterioration compared with controls, by -1.26 μm/year (P = 0.07) for BMO-MRW, -0.40 μm/year (P = 0.11) for RNFLT, and -0.38×10(-2) mm(2)/year (P = 0.23) for DMRA. Baseline BMO-MRW and RNFLT significantly influenced the respective rates of change, with higher baseline values relating to faster reductions. Older age at baseline was associated with a slower reduction in rates of BMO-MRW. Reductions in intraocular pressure were related to increases in BMO-MRW and DMRA. There was a tendency for BMOA to decrease over time (-0.38×10(-2) mm(2)/year; P = 0.04). CONCLUSIONS Age-related loss of neuroretinal parameters may explain a large proportion of the deterioration observed in treated patients with glaucoma and should be carefully considered in estimating rates of change.

The role of neurotrophins in the maintenance of the spinal cord motor neurons and the dorsal root ganglia proprioceptive sensory neurons

The aim of this study was to approach the question of neuronal dependence on neurotrophins during embryonic development in mice in a way other than gene targeting. We employed amyogenic mouse embryos and fetuses that develop without any skeletal myoblasts or skeletal muscle and consequently lose motor and proprioceptive neurons. We hypothesized that if, in spite of the complete inability to maintain motor and proprioceptive neurons, the remaining spinal and dorsal root ganglia tissues of amyogenic fetuses still contain any of the neurotrophins, that particular neurotrophin alone is not sufficient for the maintenance of motor and proprioceptive neurons. Moreover, if the remaining spinal and dorsal root ganglia tissues still contain any of the neurotrophins, that particular neurotrophin alone may be sufficient for the maintenance of the remaining neurons (i.e., mostly non‐muscle‐ and a few muscle‐innervating neurons). To test the role of the spinal cord and dorsal root ganglia tissues in the maintenance of its neurons, we performed immunohistochemistry employing double‐mutant and control tissues and antibodies against neurotrophins and their receptors. Our data suggested that: (a) during the peak of motor neuron cell death, the spinal cord and dorsal root ganglia distribution of neurotrophins was not altered; (b) the distribution of BDNF, NT‐4/5, TrkB and TrkC, and not NT‐3, was necessary for the maintenance of the spinal cord motor neurons; (c) the distribution of BDNF, NT‐4/5 and TrkC, and not NT‐3 and Trk B, was necessary for the maintenance of the DRG proprioceptive neurons; (d) NT‐3 was responsible for the maintenance of the remaining neurons and glia in the spinal cord and dorsal root ganglia (possibly via TrkB).

A significant reduction of the diaphragm in mdx:MyoD−/−9th embryos suggests a role for MyoD in the diaphragm development

To further investigate the role of MyoD during skeletal myogenesis, we backcrossed mdx mutant mice (lacking dystrophin) with MyoD knock-out mice to obtain viable mice with MyoD allele on a pure mdx background. However, after nine generations of backcrossing, it was not possible to obtain a viable mdx:MyoD-/- phenotype (designated as: mdx:MyoD-/-(9th)). The compound-mutant embryos were examined just before birth. Essentially normal Myf5-dependent and most of the MyoD-dependent musculature was observed. By contrast, the skeletal muscle compartment of the diaphragm was significantly reduced. The mesenchymal compartment of the diaphragm was intact and no herniations were observed. Other examined organs (e.g., liver, kidney, brain, etc.) showed no histological abnormalities. Pulmonary hypoplasia was determined as the cause of neonatal death. Therefore, using a different approach, our new data supplement our previous findings and suggest an essential role for MyoD in development of skeletal muscle of the diaphragm. The failure of mdx:MyoD-/-(9th) diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program. Our data also suggest that functions of MyoD and Myf5 (and the respective muscle precursor cell sub-populations) are not entirely redundant by term, as previously suggested, since Myf5 is not capable of fully substituting for MyoD in the diaphragm development.

Beta and Gamma Peripapillary Atrophy in Myopic Eyes With and Without Glaucoma

PURPOSE To determine whether beta and gamma peripapillary atrophy (PPA) areas measured with optical coherence tomography (OCT) enhances glaucoma diagnosis in myopic subjects. METHODS We included 55 myopic glaucoma patients and 74 myopic nonglaucomatous controls. Beta-PPA comprised the area external to the clinical disc margin, with absence of retinal pigment epithelium and presence of Bruchs membrane. Gamma-PPA comprised the area external to the disc margin, with absence of both RPE and Bruchs membrane. OCT scans colocalized to fundus photographs were used to measure PPA, choroidal thickness, border tissue of Elschnig configuration, optic disc area, and optic disc ovality. RESULTS Beta-PPA area was larger in glaucoma patients compared with controls (median [interquartile range], 1.0 [0.66-1.53] mm2 and 0.74 [0.50-1.38] mm2, respectively), whereas gamma-PPA was smaller in glaucoma patients compared with controls (0.28 [0.14-0.50] mm2 and 0.42 [0.17-0.74] mm2, respectively). However, the distributions of both beta- and gamma-PPA in the two groups overlapped widely. The areas under the receiver operating characteristic curve of beta- and gamma-PPA areas were 0.60 and 0.59, respectively. Larger beta-PPA area was associated with larger disc area, thinner choroidal thickness, longer axial length, less oblique border tissue configuration, older age, and greater disc ovality. Larger gamma-PPA area was associated with greater disc ovality, more oblique border tissue configuration, and longer axial length. CONCLUSIONS Subclassifying PPA with OCT into beta and gamma zones reveals association with different covariates, but does not enhance the diagnostic performance for glaucoma in a population of predominantly Caucasians myopic subjects.

Presence of neurotrophic factors in skeletal muscle correlates with survival of spinal cord motor neurons

To determine which combination of skeletal muscle‐derived neurotrophic factors may be important for the survival of specific subpopulations of developing spinal cord motor neurons, we used Myf5 and MyoD (myogenic regulatory factors) knockouts, containing differentially committed myogenic precursor cells (MPCc) and immunohistochemistry against several muscle‐secreted neurotrophic factors. At the peak of motor neuron cell death, skeletal muscle development is delayed in the back and body wall muscles of Myf5−/− embryos and in the limb muscles of MyoD−/− embryos. We hypothesized that, if the skeletal muscle was indeed an important source of survival factors for motor neurons, the back, the abdominal wall, and the forelimb MPCs of Myf5−/− or MyoD−/− embryos should produce at least some neurotrophic factors necessary for the survival of motor neurons. In this report, we demonstrate that (1) different MPCs lacking Myf5, MyoD, or Myf5/MyoD have different capabilities in providing factors potentially required for the survival of motor neurons and intramuscular nerve branching, (2) MPCs in double‐mutant embryos do not contain neurotrophic factors in the absence of myogenic specification, and (3) different subpopulations of MPCs contain different combinations of neurotrophic factors potentially required for the survival of the specific subpopulations of innervating motor neurons. Developmental Dynamics 234:659–669, 2005.

Rates of Change in the Visual Field and Optic Disc in Patients with Distinct Patterns of Glaucomatous Optic Disc Damage

PURPOSE To investigate the rate of visual field and optic disc change in patients with distinct patterns of glaucomatous optic disc damage. DESIGN Prospective longitudinal study. PARTICIPANTS A total of 131 patients with open-angle glaucoma with focal (n = 45), diffuse (n = 42), and sclerotic (n = 44) optic disc damage. METHODS Patients were examined every 4 months with standard automated perimetry (SAP, SITA Standard, 24-2 test, Humphrey Field Analyzer, Carl Zeiss Meditec, Dublin, CA) and confocal scanning laser tomography (CSLT, Heidelberg Retina Tomograph, Heidelberg Engineering GmbH, Heidelberg, Germany) for a period of 4 years. During this time, patients were treated according to a predefined protocol to achieve a target intraocular pressure (IOP). Rates of change were estimated by robust linear regression of visual field mean deviation (MD) and global optic disc neuroretinal rim area with follow-up time. MAIN OUTCOME MEASURES Rates of change in MD and rim area. RESULTS Rates of visual field change in patients with focal optic disc damage (mean -0.34, standard deviation [SD] 0.69 dB/year) were faster than in patients with sclerotic (mean -0.14, SD 0.77 dB/year) and diffuse (mean +0.01, SD 0.37 dB/year) optic disc damage (P = 0.003, Kruskal-Wallis). Rates of optic disc change in patients with focal optic disc damage (mean -11.70, SD 25.5 ×10(-3) mm(2)/year) were faster than in patients with diffuse (mean -9.16, SD 14.9 ×10(-3) mm(2)/year) and sclerotic (mean -0.45, SD 20.6 ×10(-3) mm(2)/year) optic disc damage, although the differences were not statistically significant (P = 0.11). Absolute IOP reduction from untreated levels was similar among the groups (P = 0.59). CONCLUSIONS Patients with focal optic disc damage had faster rates of visual field change and a tendency toward faster rates of optic disc deterioration when compared with patients with diffuse and sclerotic optic disc damage, despite similar IOP reductions during follow-up.

Comparison between confocal scanning laser tomography, scanning laser polarimetry and optical coherence tomography on the ability to detect localised retinal nerve fibre layer defects in glaucoma patients

Background/aim: To compare the ability of confocal scanning laser tomography (CSLT), scanning laser polarimetry (SLP) and optical coherence tomography (OCT) in recognising localised retinal nerve fibre layer (RNFL) defects. Methods: 51 eyes from 43 patients with glaucoma were identified by two observers as having RNFL defects visible on optic disc photographs. 51 eyes of 32 normal subjects were used as controls. Three masked observers evaluated CSLT, SLP and OCT images to determine subjectively the presence of localised RNFL defects. Results: Interobserver agreement was highest with OCT, followed by SLP and CSLT (mean kappa: 0.83, 0.69 and 0.64, respectively). RNFL defects were identified in 58.8% of CSLT, 66.7% of SLP and 54.9% of OCT (p = 0.02 between SLP and OCT) by at least two observers. In the controls, 94.1% of CSLT, 84.3% of SLP and 94.1% of OCT scans, respectively, were rated as normal (p = 0.02 between CSLT and SLP, and SLP and OCT). Conclusion: Approximately 20–40% of localised RNFL defects identified by colour optic disc photographs are not detected by CSLT, SPL or OCT. SLP showed a higher number of false-positive results than the other techniques, but also had a higher proportion of correctly identified RNFL defects in the glaucoma population.