2013

Lacrimal gland inflammation during autoimmune Sjögren's syndrome (SS) leads to ocular surface inflammation - Keratoconjunctivitis sicca (KCS). This condition afflicts both the cornea and conjunctiva that form the ocular surface. Thrombospondin-1 (TSP-1) deficiency in mice results in lacrimal gland and corneal inflammation that resembles the human disease. In this study we report conjunctival pathology in this mouse model of SS. We found that TSP-1 null mice develop inflammation in the conjunctiva and associated loss of goblet cell function similar to that seen in patients with SS. Increased expression of Th1 (IFN-γ, TNF-α) and Th17 (IL-6, IL-17A) inflammatory cytokines and related transcription factors (Tbet and RORγt) were detected in TSP-1 null conjunctiva as well as their draining lymph nodes (LNs). The conjunctival inflammation was also accompanied by an increase in local lymphatic vessels. Interestingly, migration of antigen-bearing dendritic cells (DCs) from the ocular surface to the LNs was dependent on the TSP-1 available in the tissue. These results not only reveal potential immunopathogenic mechanisms underlying KCS in SS but also highlight the therapeutic potential of TSP-1.

Ocular surface inflammation associated with Sjögren's syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren's syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-α and IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.

PURPOSE: To compare the anatomy of the graft-host junction and anterior chamber angle after Boston Keratoprosthesis (KPro) placement using oversized (9.5-mm) and standard (8.5-mm) back plates. METHODS: Six patients with 9.5-mm titanium back plates and 10 patients with 8.5-mm titanium back plates were imaged by anterior segment optical coherence tomography 6 to 12 months after KPro placement. The location of the graft-host junction in relation to the back plate, the corneal thickness at the graft-host junction, and the anterior chamber angle were assessed. The clinical outcomes and incidence of retroprosthetic membrane (RPM) formation in this cohort were retrospectively evaluated. RESULTS: The oversized back plates completely covered the graft-host junction in all quadrants, allowing the complete apposition of the posterior surface of the carrier graft with the host cornea, with decreased graft-host junction wound thickness. The standard back plates covered the posterior aspect of the carrier graft but not the graft-host junction or the host cornea, resulting in a significantly thicker graft-host junction. None of the patients with larger back plates developed a significant RPM during a 12-month follow-up period. One patient with a larger back plate developed a corneal melt at the KPro stem as a result of chronic exposure. CONCLUSIONS: Oversized KPro back plates effectively cover the graft-host junction without any adverse effects on angle anatomy or wound healing. This may be a strategy to provide better wound apposition, reduce RPM formation, and reduce angle closure from iris synechiae to the wound.

The Boston keratoprosthesis (B-KPro), currently the most commonly used artificial cornea worldwide, can provide rapid visual rehabilitation for eyes with severe corneal opacities not suitable for standard corneal transplantation. However, the B-KPro presently needs a corneal graft as a tissue carrier. Although corneal allograft tissue is readily available in the United States and other developed countries with established eye banks, the worldwide need vastly exceeds supply. Therefore, a simple, safe, and inexpensive alternative to corneal allografts is desirable for the developing world. We are currently exploring reasonable alternative options such as corneal autografts, xenografts, noncorneal autologous tissues, and laboratory-made tissue constructs, as well as modifications to corneal allografts, such as deep-freezing, glycerol-dehydration, gamma irradiation, and cross-linking. These alternative tissue carriers for the B-KPro are discussed with special regard to safety, practicality, and cost for the developing world.

PURPOSE: To compare the operating room performance of ophthalmology residents trained by traditional wet-lab versus surgical simulation on the continuous curvilinear capsulorhexis (CCC) portion of cataract surgery. SETTING: Academic tertiary referral center. DESIGN: Prospective randomized study. METHODS: Residents who chose to participate and provided informed consent were randomized to preoperative CCC training in the wet lab or on a simulator. Residents completed pre-practice demographic questionnaires including habits of daily living. After completion of their preoperative training (wet lab versus simulator), residents performed their first CCC of the clinical rotation under the direct supervision of an attending physician as part of their standard training at the facility. Residents then completed satisfaction questionnaires regarding their preoperative training. Two attending surgeons reviewed and graded each video of operating room performance. The mean score between the 2 attending physicians was used as the individual performance score for each of the 12 performance criteria. The overall score was calculated as the sum of these 12 individual performance scores (standardized). RESULTS: Ten residents trained in the wet lab and 11 on the simulator. There was no significant difference in overall score between the 2 groups (P=.608). There was no significant difference in any individual score except time (wet-lab group faster than simulator group) (P=.038). CONCLUSIONS: Preoperative simulator training prepared residents for the operating room as effectively as the wet lab. The time to pass the simulator curriculum was predictive of the time and overall performance in the operating room. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

PURPOSE: Orbital osteotomy risks injury to the eyeball and orbit soft tissues. Used extensively in oral and maxillofacial surgery, piezoelectric technology offers a greater margin of safety than traditional bone cutting instruments. The authors describe the novel use of this system in a variety of orbital surgeries. METHODS: This interventional case series was performed in accordance with institutional review board regulations. The medical records of all patients who had undergone orbital surgery using the piezoelectric blade at 3 institutions were reviewed. Indication for surgery, gender, age, duration of follow up, intraoperative complications, surgical result, and postoperative course was recorded. RESULTS: Sixteen patients underwent surgery on 18 orbits using the piezoelectric system between August 2011 and June 2012. Surgeries performed included orbital decompression (8), lateral orbitotomy (5), cranio-orbitotomy (4), and external dacryocystorhinostomy (1). Eight were female and 8 were male patients. Mean age was 55 years old (standard deviation 15 years). Mean follow up was 82 days. The osteotomy created by the blade was narrow and smooth in every case. The surgeons uniformly appreciated the precision and safety of the instrument compared with traditional electric saw blades. There were no soft tissue lacerations or intraoperative complications and reconstructions were uniformly uneventful. Postoperative healing was rapid with no unexpected inflammation, and no palpable bony defects were appreciated in the reconstructed cases. CONCLUSIONS: Because it does not cut soft tissue and cuts a narrow trough, the self-irrigating piezoelectric saw blade appears safer and more precise than traditional electric saw blades in and around the orbit.

Growth hormone (GH) is a protein secreted by the anterior pituitary and circulates throughout the body to exert important actions on growth and metabolism. GH stimulates the secretion of insulin-like growth factor-I (IGF-I) that mediates some of the growth promoting actions of GH. The GH/IGF-I axis has recently been recognized as important in terms of longevity in organisms ranging from Caenorhabditis elegans to mice. For example, GH transgenic mice possess short lifespans while GH receptor null (GHR-/-) mice have extended longevity. Thus, the actions of GH (or IGF-I) or lack thereof impact the aging process. In this review, we summarize the proteomic analyses of plasma and white adipose tissue in these two mouse models of GH action, i.e. GH transgenic and GHR-/- mice. At the protein level, we wanted to establish novel plasma biomarkers of GH action as a function of age and to determine differences in adipose tissue depots. We have shown that these proteomic approaches have not only confirmed several known physiological actions of GH, but also resulted in novel protein biomarkers and targets that may be indicative of the aging process and/or new functions of GH. These results may generate new directions for GH and/or aging research.

PURPOSE: Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells. METHODS: To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated. RESULTS: We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9. CONCLUSIONS: Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA-related induction of MGD.

PURPOSE: Letter acuity, the predominant clinical assessment of vision, is relatively insensitive to slow vision loss caused by eye disease. While the contrast sensitivity function (CSF) has demonstrated the potential to monitor the slow progress of blinding eye diseases, current tests of CSF lack the reliability or ease-of-use to capture changes in vision timely. To improve the current state of home testing for vision, we have developed and validated a computerized adaptive test on a commercial tablet device (iPad) that provides an efficient and easy-to-use assessment of the CSF. METHODS: We evaluated the reliability, accuracy, and flexibility of tablet-based CSF assessment. Repeated tablet-based assessments of the spatial CSF, obtained from four normally-sighted observers, which each took 3 to 5 minutes, were compared to measures obtained on CRT-based laboratory equipment; additional tablet-based measures were obtained from six subjects under three different luminance conditions. RESULTS: A Bland-Altman analysis demonstrated that tablet-based assessment was reliable for estimating sensitivities at specific spatial frequencies (coefficient of repeatability 0.14-0.40 log units). The CRT- and tablet-based results demonstrated excellent agreement with absolute mean sensitivity differences <0.05 log units. The tablet-based test also reliably identified changes in contrast sensitivity due to different luminance conditions. CONCLUSIONS: We demonstrate that CSF assessment on a mobile device is indistinguishable from that obtained with specialized laboratory equipment. We also demonstrate better reliability than tests used currently for clinical trials of ophthalmic therapies, drugs, and devices.

The fundamental role of the visual system is to guide behavior in natural environments. To optimize information transmission, many animals have evolved a non-homogeneous retina and serially sample visual scenes by saccadic eye movements. Such eye movements, however, introduce high-speed retinal motion and decouple external and internal reference frames. Until now, these processes have only been studied with unnatural stimuli, eye movement behavior, and tasks. These experiments confound retinotopic and geotopic coordinate systems and may probe a non-representative functional range. Here we develop a real-time, gaze-contingent display with precise spatiotemporal control over high-definition natural movies. In an active condition, human observers freely watched nature documentaries and indicated the location of periodic narrow-band contrast increments relative to their gaze position. In a passive condition under central fixation, the same retinal input was replayed to each observer by updating the video's screen position. Comparison of visual sensitivity between conditions revealed three mechanisms that the visual system has adapted to compensate for peri-saccadic vision changes. Under natural conditions we show that reduced visual sensitivity during eye movements can be explained simply by the high retinal speed during a saccade without recourse to an extra-retinal mechanism of active suppression; we give evidence for enhanced sensitivity immediately after an eye movement indicative of visual receptive fields remapping in anticipation of forthcoming spatial structure; and we demonstrate that perceptual decisions can be made in world rather than retinal coordinates.

Mutations in the CRB1 gene cause severe retinal degenerations, which may present as Leber congenital amaurosis, early onset retinal dystrophy, retinitis pigmentosa, or cone-rod dystrophy. Some clinical features should alert the ophthalmologist to the possibility of CRB1 disease. These features are nummular pigmentation of the retina, atrophic macula, retinal degeneration associated with Coats disease, and a unique form of retinitis pigmentosa named para-arteriolar preservation of the retinal pigment epithelium (PPRPE). Retinal degenerations associated with nanophthalmos and hyperopia, or with keratoconus, can serve as further clinical cues to mutations in CRB1. Despite this, no clear genotype-phenotype relationship has been established in CRB1 disease. In CRB1-disease, as in other inherited retinal degenerations (IRDs), it is essential to diagnose the specific disease-causing gene for the disease as genetic therapy has progressed considerably in the last few years and might be applicable.

Cone photoreceptors carry out phototransduction in daylight conditions and provide the critical first step in color vision. Despite their importance, little is known about the developmental mechanisms involved in their generation, particularly how they are determined relative to rod photoreceptors, the cells that initiate vision in dim light. Here, we report the identification of a cis-regulatory module (CRM) for the thyroid hormone receptor beta (Thrb) gene, an early cone marker. We found that ThrbCRM1 is active in progenitor cells biased to the production of cones and an interneuronal cell type, the horizontal cell (HC). Molecular analysis of ThrbCRM1 revealed that it is combinatorially regulated by the Otx2 and Onecut1 transcription factors. Onecut1 is sufficient to induce cells with the earliest markers of cones and HCs. Conversely, interference with Onecut1 transcriptional activity leads to precocious rod development, suggesting that Onecut1 is critically important in defining cone versus rod fates.

AIM: To report the incidence rate of acute postoperative endophthalmitis secondary to therapeutic intravitreal injections. METHODS: A retrospective review of all consecutive eyes after intravitreal injections was performed at the Massachusetts Eye and Ear Infirmary, Boston, from 1 January 2007 to 31 December 2011. RESULTS: During the 5-year study interval, 10 208 intravitreal injections were performed. The overall incidence rate of endophthalmitis was 0.029% per injection (3 of 10 208 injections). In the three cases, in our series, the endophthalmitis occurred at an average of seven injections, which lies within the SD of the mean number of injections received by each eye in this study, suggesting approximately equal probability of infection for each eye after receiving multiple, sequential injections. Bacterial cultures and Gram stain revealed coagulase-negative Staphylococcus species (n=1), moderate bacteria with negative culture (n=1) and moderate Staphylococcus epidermidis (n=1). All cases were successfully treated using either intravitreal antibiotics and steroids or pars plana vitrectomy. Best-corrected visual acuity reduction was not clinically significant at the last visit (>7 months for all cases). CONCLUSIONS: Acute endophthalmitis is a rare potential complication after intravitreal injection. Further studies are required to elucidate the best prophylactic and aseptic techniques to prevent this rare complication.

BACKGROUND: The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease. RESULTS: We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3' and 5' alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products. CONCLUSIONS: To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.

PURPOSE: To investigate the necessity and usefulness of prophylactic postoperative antibiotics in patients undergoing enucleation or ocular evisceration. METHODS: A retrospective, multicenter, comparative case series was designed. After obtaining Institutional Review Board authorization, a medical records' review was conducted. Demographics, indication for surgery, surgical technique, postoperative antibiotic dosing, and postoperative course were evaluated. Records were grouped according to antibiotic protocols, and presence or absence of postoperative wound infection (orbital cellulitis) was recorded. Rates of postoperative infection were analyzed statistically. RESULTS: Between 1996 and 2011, 666 evisceration or enucleation surgeries were conducted at 4 institutions. Six hundred forty-eight records were available for analysis, of which 4 were excluded due to insufficient follow-up data. All the remaining 644 patients received a single, perioperative, intravenous dose of antibiotics. Five hundred seventy-eight patients (90%) received an orbital implant, while 66 (10%) did not. Three hundred eighty-one patients (59%) received postoperative antibiotics, and 263 patients (41%) did not. Two cases were identified with signs suggestive of infection, but no culture-positive infections were found, and no patient was admitted to the hospital for management. Of the 2 suspicious cases, 1 was found in the group that received postoperative antibiotics (group 1) and 1 in the group that did not receive postoperative antibiotics (group 2). No statistically significant difference in postoperative infection rate was noted between the 2 groups (p=0.52). While patients with infectious indications for surgery were more likely to receive postoperative antibiotics (p<0.001), there was no statistically significant difference in rates of infection among patients with infectious indications for surgery based on receiving or not receiving postoperative antibiotics (p=0.79), and no patients with infectious indications for surgery not receiving postoperative antibiotics developed a postoperative infection. CONCLUSIONS: This study demonstrates the clinical safety of withholding postoperative prophylactic antibiotics in orbital surgery even when implanting alloplastic material in a sterile field. Furthermore, Centers for Disease Control and Prevention guidelines mandate cessation of postoperative antibiotics within 24 hours of surgery. Surgeons are cautioned not to generalize these results to nonsterile surgery such as sinonasal or nasolacrimal surgery.

PURPOSE: To assess whether dorzolamide 2%-timolol 0.5% (D/T) and/or brimonidine 0.2%-timolol 0.5% (B/T) alters retinal vascular autoregulation (RVA) and seated ocular perfusion pressure (sOPP) in primary open angle glaucoma (POAG) patients who demonstrate retinal vascular dysregulation (RVD) on timolol 0.5% alone. METHODS: In this prospective, observer-masked, crossover study, 21 POAG patients with untreated intraocular pressure (IOP) >21 mmHg were treated for 6 weeks with timolol 0.5%. Subsequently, we measured inferior temporal retinal artery blood flow in the left eye with subjects seated and then while reclined for 30 min using the Canon Laser Blood Flowmeter. Subjects with a change in retinal blood flow in response to posture change outside of the range previously found in healthy subjects were designated as having RVD and randomized to either D/T or B/T for 6 weeks and re-tested. This was followed by treatment with the opposite medication. RESULTS: Seven of the 21 subjects demonstrated RVD in response to posture change following timolol 0.5%. Multiple linear regression analysis indicated that lower sOPP was the main determinant of RVD (P=0.033). After treatment with D/T, all 7 converted from RVD to normal RVA status (P=0.001). Four of 6 subjects showed a similar return to normal RVA following B/T (P=0.066). Mid-morning sOPP was 41.1±5.5 mmHg post-timolol, 46.3±6.5 mmHg post-D/T, and 38.6±6.0 mmHg post-B/T (D/T vs. B/T, P=0.026). CONCLUSIONS: D/T significantly improved RVA in POAG patients exhibiting RVD while on timolol 0.5% alone. D/T also increased sOPP compared to B/T. There was no significant difference (P=0.37) between D/T and B/T in improving RVA.

PURPOSE: To evaluate the regulatory cross-talk of the vascular and neural networks in the cornea. METHODS: b-FGF micropellets (80 ng) were implanted in the temporal side of the cornea of healthy C57Bl/6 mice. On day 7, blood vessels (hemangiogenesis) and nerves were observed by immunofluorescence staining of corneal flat mounts. The next group of mice underwent either trigeminal stereotactic electrolysis (TSE), or sham operation, to ablate the ophthalmic branch of the trigeminal nerve. Blood vessel growth was detected by immunohistochemistry for PECAM-1 (CD31) following surgery. In another set of mice following TSE or sham operation, corneas were harvested for ELISA (VEGFR3 and pigment epithelium-derived factor [PEDF]) and for quantitative RT-PCR (VEGFR3, PEDF, and CD45). PEDF, VEGFR3, beta-3 tubulin, CD45, CD11b, and F4/80 expression in the cornea were evaluated using immunostaining. RESULTS: No nerves were detected in the areas subject to corneal neovascularization, whereas they persisted in the areas that were neovessel-free. Conversely, 7 days after denervation, significant angiogenesis was detected in the cornea, and this was associated with a significant decrease in VEGFR3 (57.5% reduction, P = 0.001) and PEDF protein expression (64% reduction, P < 0.001). Immunostaining also showed reduced expression of VEGFR3 in the corneal epithelial layer. Finally, an inflammatory cell infiltrate, including macrophages, was observed. CONCLUSION: Our data suggest that sensory nerves and neovessels inhibit each other in the cornea. When vessel growth is stimulated, nerves disappear and, conversely, denervation induces angiogenesis. This phenomenon, here described in the eye, may have far-reaching implications in understanding angiogenesis.