Exploring the retinal connectome.

PURPOSE: A connectome is a comprehensive description of synaptic connectivity for a neural domain. Our goal was to produce a connectome data set for the inner plexiform layer of the mammalian retina. This paper describes our first retinal connectome, validates the method, and provides key initial findings. METHODS: We acquired and assembled a 16.5 terabyte connectome data set RC1 for the rabbit retina at ≈ 2 nm resolution using automated transmission electron microscope imaging, automated mosaicking, and automated volume registration. RC1 represents a column of tissue 0.25 mm in diameter, spanning the inner nuclear, inner plexiform, and ganglion cell layers. To enhance ultrastructural tracing, we included molecular markers for 4-aminobutyrate (GABA), glutamate, glycine, taurine, glutamine, and the in vivo activity marker, 1-amino-4-guanidobutane. This enabled us to distinguish GABAergic and glycinergic amacrine cells; to identify ON bipolar cells coupled to glycinergic cells; and to discriminate different kinds of bipolar, amacrine, and ganglion cells based on their molecular signatures and activity. The data set was explored and annotated with Viking, our multiuser navigation tool. Annotations were exported to additional applications to render cells, visualize network graphs, and query the database. RESULTS: Exploration of RC1 showed that the 2 nm resolution readily recapitulated well known connections and revealed several new features of retinal organization: (1) The well known AII amacrine cell pathway displayed more complexity than previously reported, with no less than 17 distinct signaling modes, including ribbon synapse inputs from OFF bipolar cells, wide-field ON cone bipolar cells and rod bipolar cells, and extensive input from cone-pathway amacrine cells. (2) The axons of most cone bipolar cells formed a distinct signal integration compartment, with ON cone bipolar cell axonal synapses targeting diverse cell types. Both ON and OFF bipolar cells receive axonal veto synapses. (3) Chains of conventional synapses were very common, with intercalated glycinergic-GABAergic chains and very long chains associated with starburst amacrine cells. Glycinergic amacrine cells clearly play a major role in ON-OFF crossover inhibition. (4) Molecular and excitation mapping clearly segregates ultrastructurally defined bipolar cell groups into different response clusters. (5) Finally, low-resolution electron or optical imaging cannot reliably map synaptic connections by process geometry, as adjacency without synaptic contact is abundant in the retina. Only direct visualization of synapses and gap junctions suffices. CONCLUSIONS: Connectome assembly and analysis using conventional transmission electron microscopy is now practical for network discovery. Our surveys of volume RC1 demonstrate that previously studied systems such as the AII amacrine cell network involve more network motifs than previously known. The AII network, primarily considered a scotopic pathway, clearly derives ribbon synapse input from photopic ON and OFF cone bipolar cell networks and extensive photopic GABAergic amacrine cell inputs. Further, bipolar cells show extensive inputs and outputs along their axons, similar to multistratified nonmammalian bipolar cells. Physiologic evidence of significant ON-OFF channel crossover is strongly supported by our anatomic data, showing alternating glycine-to-GABA paths. Long chains of amacrine cell networks likely arise from homocellular GABAergic synapses between starburst amacrine cells. Deeper analysis of RC1 offers the opportunity for more complete descriptions of specific networks.

Preloaded Descemet Membrane Endothelial Keratoplasty Donor Tissue: Surgical Technique and Early Clinical Results.

PURPOSE: To describe the technique, advantages, and early complication rates of using Descemet membrane endothelial keratoplasty (DMEK) donor tissue that is prestained and preloaded into an injector at the eye bank and delivered in a storage medium to the surgeon for transplantation 1 to 2 days later. METHODS: A total of 111 eyes with endothelial failure underwent DMEK using donors that were prestripped, prestained, S-stamped, and preloaded into a Straiko modified Jones tube and delivered in an Optisol-filled viewing chamber 1 to 2 days later. Scroll tightness, time to unscroll and center the tissue, postoperative rebubble rate, and graft failure rate were recorded. Endothelial cell density was measured at 3 and 6 months. RESULTS: All tissues remained well stained with easy visualization at the time of surgery (n = 111). The mean scroll tightness was 2.2 (range: 1-4). The mean time to center and unscroll the tissue was 3.5 minutes (range: 0.5-11.25 min). There was no primary graft failure. There were 16 cases with the placement of another bubble postoperatively (with a 14.4% rebubble rate). Of those 16 cases, 2 required a second rebubble. Endothelial cell loss at 3 and 6 months postoperatively was 26.7% (n = 63 eyes) and 30.9% (n = 67 eyes), respectively. CONCLUSIONS: This is the first report of the clinical use of prestained, preloaded tissue for DMEK. The characteristics and handling of the tissue were not different from those of surgeon-loaded tissue. Because punching, staining, and loading the graft intraoperatively is not necessary, the surgery time and risk of damaging donor tissue are reduced when using preloaded tissue.

Quantitative Analysis of Endothelial Cell Loss in Preloaded Descemet Membrane Endothelial Keratoplasty Grafts.

PURPOSE: Availability of preloaded Descemet membrane endothelial keratoplasty (pDMEK) tissue may increase acceptance of DMEK in surgical management of endothelial disease. The goal of this study was to determine the safety of pDMEK grafts for 24 hours before surgery by analyzing endothelial cell loss (ECL) using 2 image analysis software programs. METHODS: A total of 18 cadaveric corneas were prepared for DMEK using a standardized technique and loaded in a modified Jones tube injector. Nine of the corneas were injected into Calcein AM vital dye after 1 minute (controls), and the remaining 9 corneas were left preloaded for 24 hours before injection into vital dye for staining. The stained corneas were imaged using an inverted confocal microscope. ECL was then analyzed and quantified by 2 different graders using 2 image analysis software programs. RESULTS: The control DMEK tissue resulted in 22.0% ± 4.0% ECL compared with pDMEK tissue, which resulted in 19.2% ± 7.2% ECL (P = 0.31). Interobserver agreement was 0.93 for MetaMorph and 0.92 for Fiji. The average time required to process images with MetaMorph was 2 ± 1 minutes and with Fiji was 20 ± 10 minutes. Intraobserver agreement was 0.97 for MetaMorph and 0.93 for Fiji. CONCLUSIONS: Preloading DMEK tissue is safe and may provide an alternative technique for tissue distribution and surgery for DMEK. The use of MetaMorph software for quantifying ECL is a novel and accurate imaging method with increased efficiency and reproducibility compared with the previously validated Fiji.

Prevalence of Antiretinal Antibodies in Acute Zonal Occult Outer Retinopathy: A Comprehensive Review of 25 Cases.

PURPOSE: To perform a comprehensive review and to investigate the presence and role of autoimmune antibodies in 25 cases of acute zonal occult outer retinopathy (AZOOR) identified using the classification originally proposed by J. Donald Gass. DESIGN: Observational case series. METHODS: Setting: Institutional. STUDY POPULATION: Twenty-five patients were identified by characteristic symptoms (abrupt onset of photopsias, followed by large scotomata at or connected to the blind spot), ocular findings (paucity of pigmentary changes with no sign of vitreous inflammation and abnormal electroretinogram in at least 1 eye), and a negative family history for retinitis pigmentosa. OBSERVATION PROCEDURES: Patients underwent a full comprehensive ophthalmologic examination, fundus retinography, Goldmann kinetic visual field (GVF), and full-field electroretinogram (ffERG). Blood samples were also obtained to verify for the presence of antiretinal antibodies by Western blot analysis. MainOutcome Measures: Clinical presentation, best-corrected visual acuity (BCVA), fundus abnormalities, visual field defects, ffERG changes, and presence of antiretinal antibodies. RESULTS: Sixteen patients (64%) presented with photopsias, 56% (14/25) with night blindness, and 56% (14/25) with loss of peripheral vision. Sixty-four percent (16/25) of cases were bilateral. All patients demonstrated retinal vascular attenuation, optic nerve head pallor, and mottling of retinal pigment epithelium. The most common visual field changes included enlargement and expansion of the blind spot extending into large pericentral or other types of scotomata (64%). Both scotopic and photopic ffERG values were abnormal and affected to a similar degree in our patients. Nine patients (36%) had a greater than 20% asymmetry in ERG values between the 2 eyes. All patients had antiretinal antibodies on Western blot with an average of 6.6 bands. CONCLUSION: Evidence suggests that AZOOR is a unique form of autoimmune retinopathy and retinal manifestation suggests possible antiretinal antibody leakage from the disc margin with spread of immune products under the retina, resulting in large scotomata that connect to the optic nerve head.

Ocular surface disease in patients with diabetic peripheral neuropathy.

PURPOSE: To analyse clinical signs and symptoms of ocular surface disease in patients with diabetes mellitus (DM), based on severity of diabetic peripheral neuropathy (DPN). METHODS: This cross-sectional study included participants who were carefully phenotyped by a multidisciplinary team and categorised into groups based on severity of DPN. All study participants underwent ophthalmic evaluation and completed the Ocular Surface Disease Index (OSDI) and Visual Function Questionnaire (VFQ-25). RESULTS: The 34 study participants were healthy controls (n=9), patients with DM and mild or no DPN (n=16) and patients with DM and severe DPN (n=9). Tear osmolarity was increased, and corneal nerve fibre length was decreased, with increasing severity of DPN. In addition, patients with DM were found to have decreased Schirmer's test values when compared with healthy controls. No statistically significant differences were found between groups in OSDI, tear breakup time or corneal sensitivity. No statistically significant correlations were noted between the OSDI or VFQ-25 scores and clinical signs of dry eyes. CONCLUSION: This study demonstrates some increased clinical signs of ocular surface disease but not an increase in subjective symptoms of dry eyes, with increasing severity of DPN. Furthermore, no significant correlation was found between OSDI scores and clinical signs of dry eye. A periodic evaluation of the ocular surface is important for patients with DM, in addition to retinopathy screening, as they may be asymptomatic but have severe dry eye disease, which can lead to further ocular surface complications such as corneal ulceration. TRIAL REGISTRATION NUMBER: NCT01695629.

Objective comparison of 4 nonlongitudinal ultrasound modalities regarding efficiency and chatter.

PURPOSE: To compare efficiency and chatter of Infiniti Ozil with and without Intelligent Phacoemulsification (IP) and the Signature Ellips with and without FX. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Brunescent 2.0 mm human lens cubes were created by an instrument devised for this study. Cubes were tested (10 per test) for time of particle removal (efficiency) and for the number of times the lens particle bounced off the tip (chatter) at 300 mm Hg and 550 mm Hg, 50% and 100% power, and 50% and 100% amplitudes (amplitude for Ozil only). RESULTS: Of the ultrasound settings, efficiency varied from a mean of 3.3 seconds ± 1.4 (SD) to 50.4 ± 11.7 seconds and chatter from 0.0 to 52.0 ± 16.7 bounces per run. The Ozil-IP was generally more efficient than the Ozil and the Ellips FX more efficient than the Ellips. At optimized values, the Ozil-IP and Ellips-FX were similar. In general, efficiency and chatter were better at 550 mm Hg and at 50% power. The amplitude effect was complex. Efficiency closely correlated with chatter (Pearson r(2) = .31, P<.0001). CONCLUSIONS: Objective comparison of phacoemulsification efficiency and chatter found that optimized Ozil-IP and Ellips-FX were similar in both parameters and in general, both performed better than preceding technology. The study parameters can significantly affect efficiency and chatter, which strongly correlate with each other.

Evaluation of the American Society of Cataract and Refractive Surgery intraocular lens calculator for eyes with prior radial keratotomy.

BACKGROUND: The purpose of this study was to evaluate the American Society of Cataract and Refractive Surgery (ASCRS) intraocular lens (IOL) calculator for eyes with prior radial keratotomy and assess the accuracy of its methods in predicting IOL power in patients with previous radial keratotomy. METHODS: This retrospective study included data from 15 eyes with previous radial keratotomy and subsequent cataract surgery. The average central power and Humphrey Atlas methods from the ASCRS IOL calculator, along with an average IOL power produced from an average of these two methods (ASCRS average), were compared. Primary outcome measures for each method were mean arithmetic and absolute IOL prediction error, variance in mean arithmetic IOL prediction error, and the percentage of refractive outcomes within ±0.50, ±1.00, ±1.50, and ±2.00 diopters (D). RESULTS: The average central power method and the ASCRS average were significantly more accurate than the Humphrey Atlas method in terms of mean absolute IOL prediction error (1.03 D and 1.02 D versus 1.53; P = 0.04 and P = 0.01, respectively). In addition, the average central power method and ASCRS average produced a higher percentage of refractive outcomes within ±0.50 D when compared with the Humphrey Atlas method (60% and 46.67% versus 0%, respectively). A comparison of the average central power method and the ASCRS average demonstrated a smaller variance and higher percentage of patients within ±1.00 D when using the ASCRS average. CONCLUSION: The ASCRS calculator for eyes with prior radial keratotomy is an easily accessible and valuable online tool for calculating IOL power in patients with previous radial keratotomy. We found that the ASCRS average produced by the calculator provided the best IOL prediction. We recommend using it with the addition of 1.00 to 1.50 D to its IOL power prediction.

A comparison of the American Society of Cataract and Refractive Surgery post-myopic LASI K/PRK intraocular lens (IOL) calculator and the Ocular MD IOL calculator.

BACKGROUND: To compare the average values of the American Society of Cataract and Refractive Surgery (ASCRS) and Ocular MD intraocular lens (IOL) calculators to assess their accuracy in predicting IOL power in patients with prior laser-in-situ keratomileusis (LASIK) or photorefractive keratectomy. METHODS: In this retrospective study, data from 21 eyes with previous LASIK or photorefractive keratectomy for myopia and subsequent cataract surgery was used in an IOL calculator comparison. The predicted IOL powers of the Ocular MD SRK/T, Ocular MD Haigis, and ASCRS averages were compared. The Ocular MD average (composed of an average of Ocular MD SRK/T and Ocular MD Haigis) and the all calculator average (composed of an average of Ocular MD SRK/T, Ocular MD Haigis, and ASCRS) were also compared. Primary outcome measures were mean arithmetic and absolute IOL prediction error, variance in mean arithmetic IOL prediction error, and the percentage of eyes within ±0.50 and ±1.00 D. RESULTS: The Ocular MD SRK/T and Ocular MD Haigis averages produced mean arithmetic IOL prediction errors of 0.57 and -0.61 diopters (D), respectively, which were significantly larger than errors from the ASCRS, Ocular MD, and all calculator averages (0.11, -0.02, and 0.02 D, respectively, all P < 0.05). There was no statistically significant difference between the methods in absolute IOL prediction error, variance, or the percentage of eyes with outcomes within ±0.50 and ±1.00 D. CONCLUSION: The ASCRS average was more accurate in predicting IOL power than the Ocular MD SRK/T and Ocular MD Haigis averages alone. Our methods using combinations of these averages which, when compared with the individual averages, showed a trend of decreased mean arithmetic IOL prediction error, mean absolute upper limit of IOL prediction error, and variance, while increasing the percentage of outcomes within ±0.50 D.