Edward R. Chu

Natural History of Conversion of Leber's Hereditary Optic Neuropathy: A Prospective Case Series

PURPOSE To illustrate the natural history of Lebers hereditary optic neuropathy (LHON). DESIGN Prospective observational case series. PARTICIPANTS The Soave-Brazil pedigree of m.11778G>A/ND4 mitochondrial DNA LHON mutation. METHODS A prospectively acquired database of the Soave-Brazil pedigree was reviewed. Data from 285 individuals were included in the database over a 15-year period. The pedigree was reviewed for unaffected mutation carriers who converted to affected status, 6 patients with LHON were identified. The medical records were reviewed 1 year preconversion to 1 year postconversion for visual acuity (logarithm of the minimum angle of resolution [logMAR]), Humphrey Visual Field (HVF) mean deviation (MD), and retinal nerve fiber layer (RNFL) thickness, as measured by Cirrus (Carl Zeiss, Oberkochen, Germany) optic coherence tomography (OCT). The RNFL thickness values were normalized for age. Visual acuity, HVF, and processed RNFL data from each of the 12 eyes were then sorted into 2-month time periods relative to the date of conversion, within which they were averaged. MAIN OUTCOME MEASURES The main outcome measures were visual acuity, HVF MD, and RNFL thickness. RESULTS Decreased visual acuity preceded conversion by up to 2 months and then declined up to 8 months postconversion. Decrease in HVF MD occurred at least 4 months preceding conversion, after which values decreased until plateau at 6 to 8 months. Average RNFL thickness was above normal baseline thickness in all 4 quadrants as measured by OCT at the time of conversion. Increase in RNFL thickness preceded conversion as early as 4 to 6 months, peaked at conversion, and decreased until individual plateaus. The temporal quadrant was first to be involved, then the inferior and superior quadrants, and the nasal quadrant showed the latest and least changes. CONCLUSIONS Subclinical changes preceded the date of conversion and may reflect the complicated nature of identifying the date of conversion in LHON. Early increases in RNFL preceding conversion suggest that structural changes precede clinically significant vision loss. Asynchronous quadrant involvement supports a previously published mathematical model. The natural history of LHON is not a subacute process, as previously believed, but progresses more slowly, taking up to 8 months to plateau.

Tissue-Based Imaging Model of Human Trabecular Meshwork

We have developed a tissue-based model of the human trabecular meshwork (TM) using viable postmortem corneoscleral donor tissue. Two-photon microscopy is used to optically section and image deep in the tissue to analyze cells and extracellular matrix (ECM) within the original three-dimensional (3D) environment of the TM. Multimodal techniques, including autofluorescence (AF), second harmonic generation (SHG), intravital dye fluorescence, and epifluorescence, are combined to provide unique views of the tissue at the cellular and subcellular level. SHG and AF imaging are non-invasive tissue imaging techniques with potential for clinical application, which can be modeled in the system. We describe the following in the tissue-based model: analysis of live cellularity to determine tissue viability; characteristics of live cells based on intravital labeling; features and composition of the TMs structural ECM; localization of specific ECM proteins to regions such as basement membrane; in situ induction and expression of tissue markers characteristic of cultured TM cells relevant to glaucoma; analysis of TM actin and pharmacological effects; in situ visualization of TM, inner wall endothelium, and Schlemms canal; and application of 3D reconstruction, modeling, and quantitative analysis to the TM. The human model represents a cost-effective use of valuable and scarce yet available human tissue that allows unique cell biology, pharmacology, and translational studies of the TM.

Intraocular pressure measurement in acepromazine‐sedated mice

trexate because of adverse effects, and adalimumab and certolizumab failed to control her symptoms so she was started on mycophenolate mofetil (MM). Although MM resulted in quiescence of uveitis and improved her macular oedema, it did not control the psoriatic lesions, and therefore, GLM was added to her regimen. On the current regimen of GLM and MM, patient achieved complete resolution of macular oedema, and uveitis remained quiet with exception of one flare that resolved quickly with topical steroids for 1 month. Patient later developed rapidly progressive cataract OD despite the satisfactory control of uveitis, resulting in decreased VA. GLM was discontinued after 9 months because of insufficient control of arthritis. In this study, we present outcomes of GLM treatment in three patients with SpA-associated uveitis. There are reports of GLM therapy for juvenile idiopathic arthritis and uveitis syndrome (JIA) uveitis, Behçet uveitis, human leukocyte antigen -B27 (HLA-B27)-associated uveitis and idiopathic retinal vasculitis (summary presented in Table 2). To our best knowledge, this is the first report of GLM treatment in patients with ASand PsAassociated uveitis. In contrast with the prior report about GLM treatment in HLA-B27 associated uveitis, our case series details response of both systemic and eye symptoms to GLM and its related adverse effects. While on GLM, patients 1 and 2 achieved satisfactory control of their ocular inflammation, but only patient 1 also achieved systemic disease control. For patient 3, uveitis was well controlled on GLM and MM regimen with complete resolution of macular oedema. However we cannot attribute these effects only to GLM as the patient was receiving another immunosuppressant simultaneously. This patient’s decreased vision was secondary to cataract progression that could be due to longstanding inflammation and/or previous long-term steroid use. Patient 3 did not achieve systemic disease control with GLM. Two of the three patients tolerated GLM well despite having adverse reactions to other anti-TNF agents, and one patient developed side-effects with all TNF inhibitors including GLM. This report suggests that GLM can be effective treatment for controlling uveitis in SpA patients who may or may not have responded to other TNF-inhibitors. We acknowledge that this is a small case series and further studies with longer follow up are required to better evaluate the efficacy and safety of GLM in treatment of SpA-associated uveitis.

Correcting Finger Counting to Snellen Acuity

ABSTRACT In this paper, the authors describe an online tool with which to convert and thus quantify count finger measurements of visual acuity into Snellen equivalents. It is hoped that this tool allows for the re-interpretation of retrospectively collected data that provide visual acuity in terms of qualitative count finger measurements.

Total Outflow Facility in Live C57BL/6 Mice of Different Age

Purpose: To characterize total outflow facility across the live adult mouse lifespan as a reference for mouse glaucoma studies and the common C57BL/6 background strain. Methods: Microperfusion was performed by single-needle cannulation and feedback-controlled coupling of pressure and flow to maintain a constant pressure in the anterior chambers of live C57BL/6NCrl mice aged 3-4 months (n = 17), 6-9 months (n = 10), and 23-27 months (n = 12). This mouse age range represented an equivalent human age range of young adult to elderly. We characterized the following across age groups in vivo: (1) outflow facility based on constant pressure perfusion in a pressure range of 15-35 mm Hg, (2) perfusion flow rates, and (3) anterior segment tissue histology after perfusion. Thirty-nine live mice underwent perfusion. Results: Pressure-flow rate functions were consistently linear for all age groups (all R2 > 0.96). Total outflow facility in mice aged 3-4, 6-9, and 23-27 months was 0.0066, 0.0064, and 0.0077 μL/min/mm Hg, respectively. Facility was not significantly different between age groups (all p > 0.4). The groups had closely overlapping frequency distribution profiles with right-sided tails. Post hoc estimates indicated that group facility differences of at least 50% would have been detectable, with this limit set mainly by inherent variability in the strain. A trend toward higher perfusion flow rates was seen in older mice aged 23-27 months, but this was not significantly different from that of mice aged 3-4 months or 6-9 months (p > 0.2). No histological disruption or difference in iridocorneal angle or drainage tissue structure was seen following perfusion in the different age groups. Conclusion: We did not find a significant difference in total outflow facility between different age groups across the live C57BL/6 mouse adult lifespan, agreeing with some human studies. The possibility that more subtle differences might exist ought to be judged with respect to the heterogeneity in facility at different ages. Our findings provide reference data for live perfusion studies pertaining to glaucoma involving the C57BL/6 strain.

Smooth muscle features of mouse extraocular muscle

We have been intrigued to find smooth muscle markers within mouse extraocular muscle (EOM) while studying contractile features of the aqueous humour drainage tissues by immunohistochemistry. Smooth muscle is known to be present in connective tissue fascial pulleys ensheathing EOM1 but not in the EOM per se. The mouse has many available engineered strains, shares many biological similarities with primates, and is widely used to model human biology, including that of EOM.2 Herein, we show that classic smooth muscle proteins of alpha-smooth muscle actin (α-SMA), myosin heavy chain (MHC), caldesmon, and tropomyosin are intimately associated with EOM fibre bundles, a finding that may be relevant to better understanding EOM control. We studied C57BL/6 mice (2-3 months, Charles River, Wilmington, MA) that were housed in a temperature-controlled room with 12h light and dark cycles and fed ad libitum. Animal care and use complied with the Institutional Animal Care and Use Committee as well as the Association for Research in Vision and Ophthalmology guidelines. Anterior (insertion) and posterior (retro-equator) portions of four rectus EOM of C57BL/6 mice (n=5) were carefully isolated, quickly embedded in OCT compound and snap-frozen in liquid nitrogen. Eight μm-thick cryosections were fixed with 4% paraformaldehyde, permeabilised and blocked (0.3% Triton X-100+5% BSA), incubated with primary antibodies (Abcam) to α-SMA, MHC non-muscle, caldesmon, and tropomyosin overnight at 4°C, then secondary antibodies and Alexa 568-phalloidin. Vascular smooth muscle labeling of the same tissues represented positive controls, while normal IgG isotype labeling represented negative controls. Sections were analysed by Leica SP5 or Zeiss LSM 710 confocal microscopy. The same tissue slides were further processed for hematoxylin and eosin staining to confirm structure. Figure 1 shows representative longitudinal and cross sectional immunohistochemistry images of the mouse anterior EOM at their globe insertions. F-actin labeling localized to contractile regions that were mostly consistent with striated muscle fibre bundles. Positive labeling for α-SMA, MHC, and caldesmon labeling was present in the periphery of phalloidin-positive muscle fibre bundles. Positive tropomyosin labeling was seen centrally and peripherally in muscle bundles, corresponding to regions of striated and presumed smooth muscle elements. Smooth muscle marker labeling partially overlapped with phalloidin labeling in fibre bundles. Figure 1 Smooth muscle profile in the anterior part of 4 different rectus extraocular muscles (EOMs). Co-localisation of α-SMA, MHC, caldesmon, or tropomyosin with phalloidin in the anterior portion of EOM. All 4 different rectus EOMs demonstrated a similar ... Figure 2 shows the distribution of smooth muscle markers in representative longitudinal and cross sections of rectus muscles posteriorly. All recti showed this pattern. As in the anterior EOM, α-SMA, MHC, and caldesmon labeling localized to the periphery of phalloidin-labeled bundles, while tropomyosin was present peripherally and centrally. Positive immunolabeling using the same prior antibody panel was seen in vascular smooth muscle from the same tissues (not shown). Figure 2 Smooth muscle profile of the posterior part of 4 different rectus EOMs. Co-localisation of α-SMA, MHC, caldesmon, or tropomyosin with phalloidin in the posterior portion of EOM was shown. All 4 different rectus EOMs demonstrated a similar in situ ... Orbital smooth muscle is present in superior and inferior palpebral muscles, inferior orbital fissure and EOM fascial pulleys1 as part of a periorbital smooth muscle network.5 To our best knowledge smooth muscle has not been described in EOM, which is considered striated muscle. Here we describe classic smooth muscle markers within mouse EOM, mostly in the periphery of striated muscle fiber bundles. Smooth muscle was present in all rectus muscles at their insertions and retro-equatorially, indicating this organization is widely present in mouse EOM. The EOM and their supporting structures represent a complex that helps orchestrate eye movement. Striated EOM are organized as global and orbital layers, each with different structural, vascular, neural, mechanical and metabolic features.3-4 Fascial sheaths and pulleys influence muscle actions on the globe1 under possible smooth muscle modulation. As with autonomically-innervated palpebral smooth muscle that works with striated muscle to regulate eyelid position,5 we postulate that smooth muscle associated with striated EOM plays a role in determining eye position in mice, and possibly in humans.