Comparing 2-dimensional macular pigment optical density with objective and subjective perimetry and visual acuity in age-related macular degeneration.

PURPOSE: To compare diagnostic power for different severities of age-related macular degeneration (AMD) of two-dimensional macular pigment optical densities (2D-MPOD) and spatially matched objective perimetry, with standard perimetry and best-corrected visual acuity (BCVA). METHODS: The ObjectiveField Analyser (OFA) provided objective perimetry, and a Heidelberg Spectralis optical coherence tomography (OCT) measured 2D-MPOD in AMD patients, both completed twice over 0.99 ± 0.16 years. From each 2D-MPOD image, we extracted 20 regions/macula, matched to the 20 OFA stimuli/macula. For each region, we calculated 7 measures from the 2D-MPOD pixel values and correlated those with OFA sensitivities and delays. We quantified 2D-MPOD changes, the ability of 2D-MPOD and OFA to discriminate AMD stages, and the discriminatory power of Matrix perimetry and BCVA using percentage area under receiver operator characteristic plots (%AUROC). RESULTS: In 58 eyes of 29 subjects (71.6 ± 6.3 years, 22 females), we found significant correlations between 2D-MPOD and OFA sensitivities for Age-Related Eye Disease Studies (AREDS)-3 and AREDS-4 severities. Delays showed significant correlations with AREDS-2. For AREDS-4, correlations extended across all eccentricities. Regression associated with the Bland-Altman plots showed significant changes in 2D-MPOD over the study period, especially variability measures. MPOD per-region medians discriminated AREDS-1 from AREDS-3 eyes at a %AUROC of 80.0 ± 6.3%, outperforming OFA, Matrix perimetry, and BCVA. CONCLUSIONS: MPOD changes correlated with central functional changes and significant correlations extended peripherally in later-stage AMD. Good diagnostic power for earlier-stage AMD and significant change over the study suggest that 2D-MPOD and OFA may provide effective biomarkers.

Objective perimetry identifies regional functional progression and recovery in mild Diabetic Macular Oedema.

PURPOSE: Retinal function beyond foveal vision is not routinely examined in the clinical screening and management of diabetic retinopathy although growing evidence suggests it may precede structural changes. In this study we compare optical coherence tomography (OCT) based macular structure with function measured objectively with the ObjectiveFIELD Analyzer (OFA), and with Matrix perimetry. We did that longitudinally in Type 2 diabetes (T2D) patients with mild Diabetic Macular Oedema (DMO) with good vision and a similar number of T2D patients without DMO, to evaluate changes in retinal function more peripherally over the natural course of retinopathy. METHODS: Both eyes of 16 T2D patients (65.0 ± 10.1, 10 females), 10 with baseline DMO, were followed for up longitudinally for 27 months providing 94 data sets. Vasculopathy was assessed by fundus photography. Retinopathy was graded using to Early Treatment of Diabetic Retinopathy Study (ETDRS) guidelines. Posterior-pole OCT quantified a 64-region/eye thickness grid. Retinal function was measured with 10-2 Matrix perimetry, and the FDA-cleared OFA. Two multifocal pupillographic objective perimetry (mfPOP) variants presented 44 stimuli/eye within either the central 30° or 60° of the visual field, providing sensitivities and delays for each test-region. OCT, Matrix and 30° OFA data were mapped to a common 44 region/eye grid allowing change over time to be compared at the same retinal regions. RESULTS: In eyes that presented with DMO at baseline, mean retinal thickness reduced from 237 ± 25 µm to 234.2 ± 26.7 µm, while the initially non-DMO eyes significantly increased their mean thickness from 250.7 ± 24.4 µm to 255.7 ± 20.6 µm (both p<0.05). Eyes that reduced in retinal thickness over time recovered to more normal OFA sensitivities and delays (all p<0.021). Matrix perimetry quantified fewer regions that changed significantly over the 27 months, mostly presenting in the central 8 degrees. CONCLUSIONS: Changes in retinal function measured by OFA possibly offer greater power to monitor DMO over time than Matrix perimetry data.

Rapid Objective Testing of Visual Function Matched to the ETDRS Grid and Its Diagnostic Power in Age-Related Macular Degeneration.

Purpose: To study the power of an 80-second multifocal pupillographic objective perimetry (mfPOP) test tailored to the ETDRS grid to diagnose age-related macular degeneration (AMD) by Age-Related Eye Disease Study (AREDS) severity grade. Design: Evaluation of a diagnostic technology. Methods: We compared diagnostic power of acuity, ETDRS grid retinal thickness data, new 80-second M18 mfPOP test, and two wider-field 6-minute mfPOP tests (Macular-P131, Widefield-P129). The M18 stimuli match the size and shape of bifurcated ETDRS grid regions, allowing easy structure-function comparisons. M18, P129, and P131 stimuli test both eyes concurrently. We recruited 34 patients with early-stage AMD with a mean ± standard deviation (SD) age of 72.6 ± 7.06 years. The M18 and P129 plus P131 stimuli had 26 and 51 control participants, respectively with mean ± SD ages of 73.1 ± 8.17 years and 72.1 ± 5.83 years, respectively. Multifocal pupillographic objective perimetry testing used the Food and Drug Administration-cleared Objective FIELD Analyzer (OFA; Konan Medical USA). Main Outcome Measures: Percentage area under the receiver operator characteristic curve (AUC) and Hedge's g effect size. Results: Acuity and OCT ETDRS grid thickness and volume produced reasonable diagnostic power (percentage AUC) for AREDS grade 4 eyes at 83.9 ± 9.98% and 90.2 ± 6.32% (mean ± standard error), respectively, but not for eyes with less severe disease. By contrast, M18 stimuli produced percentage AUCs from 72.8 ± 6.65% (AREDS grade 2) to 92.9 ± 3.93% (AREDS grade 4), and 82.9 ± 3.71% for all eyes. Hedge's g effect sizes ranged from 0.84 to 2.32 (large to huge). Percentage AUC for P131 stimuli performed similarly and for P129 performed somewhat less well. Conclusions: The rapid and objective M18 test provided diagnostic power comparable with that of wider-field 6-minute mfPOP tests. Unlike acuity or OCT ETDRS grid data, OFA tests produced reasonable diagnostic power in AREDS grade 1 to 3 eyes.

Comparing Objective Perimetry, Matrix Perimetry, and Regional Retinal Thickness in Mild Diabetic Macular Edema.

Purpose: To compare per-region macular sensitivity and delay from objective perimetry with Matrix perimetry and retinal thickness in mild diabetic macular edema (DMO). Methods: Thirty-three patients with type 2 diabetes (T2D) aged 59.2 ± 10.5 years participated in a longitudinal study. Macular thickness, sensitivities and delays from the objectiveFIELD Analyzer (OFA), and Matrix perimeter sensitivities were mapped onto a common spatial layout to compute per-region correlations between structure/function measures. A generalized linear mixed-effects logistic regression model determined which variables contributed to clinical diagnosis of DMO. Results: For OFA, the mean sensitivity differences compared with normal in patients with T2D were negative and the mean delay differences positive, indicating lowered sensitivities and prolonged delays, both increasing with diabetes duration. Shorter diabetes duration could produce either localized peripheral hypersensitivities or shorter delays. Functional change could occur when retinal thickness was stable. Peripheral macular thickness correlated with central and peripheral OFA sensitivity and delay, all P < 0.0012 in DMO and a median of P = 0.001 without DMO; this was not true for Matrix sensitivities. The logistic model determined that peripheral thickness, OFA sensitivity (P = 0.043), and time in the study (P = 0.001) contribute independently to the odds of DMO versus no DMO. Conclusions: Mean sensitivities decreased and mean delays increased with duration of diabetes. Peripheral macular thickness correlated significantly with central and peripheral macular OFA sensitivity and delay. Peripheral macular thickness and functional measures may provide sensitive prognostic data. Translational Relevance: Functional loss can precede structural change in DMO, so including such functional assessment for deciding on treatment may be beneficial.

A Retrospective Analysis of Adverse Events in Hyperbaric Oxygen Therapy (2012-2015): Lessons Learned From 1.5 Million Treatments.

OBJECTIVE: To describe the distribution and occurrence of adverse events recorded during hyperbaric oxygen (HBO) therapy from 2012 to 2015. In this analysis, events are defined as otic/sinus barotrauma, confinement anxiety, hypoglycemia, oxygen toxicity, pneumothorax, seizure, and shortness of breath. DATA AND ANALYSIS: The data for the analysis were drawn from a proprietary electronic health data system that contained information on 1,529,859 hyperbaric treatments administered during 53,371 treatment courses from 2012 to 2015 in outpatient wound care centers across the United States managed by Healogics, Inc, Jacksonville, Florida. RESULTS: Of the 1.5 million treatments included in the analysis, 0.68% were associated with an adverse event. Barotrauma and confinement anxiety were the most frequently reported events. Medically severe events were extremely uncommon, with fewer than 0.05 instances of oxygen toxicity per 1000 treatments and only 1 confirmed case of pneumothorax. CONCLUSIONS: Results indicate that the occurrence of adverse events associated with HBO therapy is infrequent and typically not serious. The findings of this study suggest that when administered according to the appropriate therapeutic protocols HBO therapy is a safe and low-risk intervention.

Elevated maxi-K(+) ion channel current in glaucomatous lamina cribrosa cells.

The connective tissue plates of the lamina cribrosa (LC) region are continuously exposed to a mechanically dynamic environment. To study how the LC cells respond to these mechanical forces, we measured the mechano-sensitive calcium dependent maxi-K(+) ion channel current in the cell membrane of LC cells of glaucoma and normal subjects. Primary culture LC cells from 7 normal and 7 age matched glaucoma donors were studied. Perfusion of cells with hypotonic solution was used to stretch the cell membrane. Whole-cell patch-clamp technique was used to measure the basal (non stretched) and hypotonic stretch-induced changes in maxi-K(+) ion channel activity in normal and glaucoma LC cells. The role of membrane-type Ca(2+) entry channel inhibition (verapamil) and internal Ca(2+) store re-uptake blockade (2-APB) on maxi-K(+) activity was also examined. Basal and stretched-induced maxi-K(+) current were significantly elevated in the glaucoma LC cells compared to normal controls (p < 0.05). In normal LC cells hypotonic stretch elevated the mean maxi-K(+) current from 18.5 ± 5.7 pA/pF (at Vp = +100 mV) to 88.4 ± 12.4 pA/pF (P < 0.05), and from 39.5 ± 7.3 pA/pF to 133.1 ± 18.5 pA/pF in glaucoma LC cells (P < 0.02). Verapamil and 2-APB significantly reduced basal maxi-K(+) current in glaucoma LC cells (33.1 ± 8.2 pA/pF to 17.9 ± 5.6 pA/pF; and 32.2 ± 8.3 pA/pF to 17.3 ± 5.4 pA/pF, P < 0.05, respectively) but not in normal LC cells (P > 0.05). Following hypotonic stretch, verapamil and 2-APB significantly (P < 0.05) reduced the maxi-K(+) current in both normal and glaucoma LC cells. Baseline and hypotonic stretch induced Ca(2+)-dependent maxi-K(+) channel activity are elevated in LC cells of glaucoma patients, which may result from the abnormally high levels of intracellular calcium in glaucoma LC cells.

Interventional treatment of carotid cavernous fistula.

We reviewed retrospectively the outcomes of interventional endovascular treatment of direct or dural (indirect) carotid cavernous fistulas in 24 consecutive patients requiring endovascular treatment at Royal Prince Alfred Hospital between 1994 and 2009. Data was collected from each patient's neurological, ophthalmological and radiological reports. Of the 12 patients with direct fistulas all had signs of orbital and ocular venous congestion and ophthalmoplegia; nine also had reduced vision ranging from 6/9 to nil perception of light, two had normal vision and one was unconscious. Nine of the 12 direct fistulas were embolized transarterially, two transvenously, one by a combination of both approaches and all were successfully closed. After treatment, seven of the nine patients with reduced vision had complete or nearly complete restoration of vision,while two who presented with nil perception of light (one in both eyes) had no recovery of vision. In contrast, seven of the 12 patients with dural fistulas had ophthalmoplegia, three had reduced vision, down to 6/24 and one did not have any sign of venous congestion. Vision recovered in all three patients after embolization of the dural fistula. Dural fistulas were embolized transvenously in 11 and transarterially in one patient. Apart from ophthalmoplegia, all other ocular signs and symptoms rapidly resolved after closure of the fistula in each of the 24 patients. The diagnosis was delayed by being missed either during the first admission or at the first specialist consultation in three of the 12 patients with direct fistulas, and in seven of the 12 patients with dural fistulas. One patient with a direct and another with a dural fistula had limited cerebral infarctions during embolization. In this series, endovascular interventional treatment of carotid cavenous fistulas restored visual loss in 10 of 12 patients with visual loss. The two who did not recover had presented with nil perception of light, one after a delay in diagnosis of 6 weeks. Some degree of ophthalmoplegia tended to remain. This emphasizes the need for early diagnosis and treatment before visual loss or ophthalmoplegia becomes severe.

Nanotechnology-based electrochemical sensors for biomonitoring chemical exposures.

The coupling of dosimetry measurements and modeling represents a promising strategy for deciphering the relationship between chemical exposure and disease outcome. To support the development and implementation of biological monitoring programs, quantitative technologies for measuring xenobiotic exposure are needed. The development of portable nanotechnology-based electrochemical (EC) sensors has the potential to meet the needs for low cost, rapid, high-throughput, and ultrasensitive detectors for biomonitoring an array of chemical markers. Highly selective EC sensors capable of pM sensitivity, high-throughput and low sample requirements (<50 microl) are discussed. These portable analytical systems have many advantages over currently available technologies, thus potentially representing the next generation of biomonitoring analyzers. This paper highlights research focused on the development of field-deployable analytical instruments based on EC detection. Background information and a general overview of EC detection methods and integrated use of nanomaterials in the development of these sensors are provided. New developments in EC sensors using various types of screen-printed electrodes, integrated nanomaterials, and immunoassays are presented. Recent applications of EC sensors for assessing exposure to pesticides or detecting biomarkers of disease are highlighted to demonstrate the ability to monitor chemical metabolites, enzyme activity, or protein biomarkers of disease. In addition, future considerations and opportunities for advancing the use of EC platforms for dosimetric studies are discussed.

Activation of stretch-activated channels and maxi-K+ channels by membrane stress of human lamina cribrosa cells.

PURPOSE: The lamina cribrosa (LC) region of the optic nerve head is considered the primary site of damage in glaucomatous optic neuropathy. Resident LC cells have a profibrotic potential when exposed to cyclical stretch. However, the mechanosensitive mechanisms of these cells remain unknown. Here the authors investigated the effects of membrane stretch on cell volume change and ion channel activity and examined the associated changes in intracellular calcium ([Ca(2+)](i)). METHODS: The authors used primary LC cells obtained from normal human donor eyes. Confocal microscopy was used to investigate the effect of hypotonic cell membrane stretch on cell volume changes. Whole-cell patch-clamp and calcium imaging techniques were used to investigate the effect of hypotonicity on ion channel(s) activity and [Ca(2+)](i) changes, respectively. RT-PCR was used to examine for the maxi-K(+) signature in LC cells. RESULTS: In this study, LC cells showed significant volume changes in response to hypotonic cell swelling. The authors characterized a large conductance K(+) channel (maxi-K(+)) in LC cells and demonstrated its increased activity during cell membrane hypotonic stretch. RT-PCR revealed the presence of maxi-K(+) signature in LC cells. The authors showed the [Ca(2+)](i) and maxi-K(+) channels to be dependent on extracellular Ca(2+) and inhibited by gadolinium, which blocks stretch-activated channels (SACs). Pretreatment with thapsigargin, which blocks the release of Ca(2+) from endoplasmic reticulum stores, showed no significant difference in [Ca(2+)](i) concentration on hypotonic swelling. CONCLUSIONS: The results show that hypotonic stress of human LC cells activates SAC and Ca(2+)-dependent maxi-K(+) channels and that the increase in [Ca(2+)](i) during cell swelling was predominantly from extracellular sources (or intracellular stores other than the endoplasmic reticulum). These findings improve the understanding of how LC cells respond to cell membrane stretch. Further experiments in this area may reveal future targets for novel therapeutic intervention in the management of glaucoma.

Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2.

A proteomic map of Sulfolobus solfataricus P2, an archaeon that grows optimally at 80 degrees C and pH 3.2, was developed using high-resolution 2-DE and peptide mass fingerprinting. A total of 867 protein spots (659 aqueous Tris-soluble spots and 208 aqueous Tris-insoluble) were mapped over IPG 3-10, 4-7, and 6-11, with second-dimensional gels made of 8-18% polyacrylamide. Three hundred and twenty-four different gene products were represented by the 867 spots, with 274 gene products being identified in the Tris-soluble fractions and 100 gene products in the Tris-insoluble portion. Fifty gene products were found on gels from both fractions. Additionally, an average of 1.50 +/- 0.12 isoforms/protein was identified. This mapping study confirmed the expression of proteins involved in numerous metabolic, transport, energy production, nucleic acid replication, translation, and transcription pathways. Of particular interest, phosphoenolpyruvate carboxykinase (SSO2537) was detected even though the pathway for gluconeogenesis is unknown for this archaeon. Tris-soluble fractions contained many cytosolic proteins while Tris-insoluble fractions contained many membrane-associated proteins, including ABC transporters and an ATP synthase. This study provides an optimized 2-DE approach for investigating the biochemical pathways and post-translational modifications employed by Sulfolobus to survive in its extreme environment.

Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics.

Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias to some degree, assigned ranks among the techniques revealed that for most LC-FTICR-MS analyses linear regression normalization ranked either first or second. However, the lack of a definitive trend among the techniques suggested the need for additional investigation into adapting normalization approaches for label-free proteomics. Nevertheless, this study serves as an important step for evaluating approaches that address systematic biases related to relative quantification and label-free proteomics.

Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment.

We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 nonredundant proteins ( approximately 34% of the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models. The proteomic approach presented here may have broad applications for rapid proteomic analyses of various mouse models of human brain diseases.

Proteomic analyses using an accurate mass and time tag strategy.

An accurate mass and time (AMT) tag approach for proteomic analyses has been developed over the past several years to facilitate comprehensive high-throughput proteomic measurements. An AMT tag database for an organism, tissue, or cell line is established by initially performing standard shotgun proteomic analysis and, most importantly, by validating peptide identifications using the mass measurement accuracy of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) and liquid chromatography (LC) elution time constraint. Creation of an AMT tag database largely obviates the need for subsequent MS/MS analyses, and thus facilitates high-throughput analyses. The strength of this technology resides in the ability to achieve highly efficient and reproducible one-dimensional reversed-phased LC separations in conjunction with highly accurate mass measurements using FTICR MS. Recent improvements allow for the analysis of as little as picrogram amounts of proteome samples by minimizing sample handling and maximizing peptide recovery. The nanoproteomics platform has also demonstrated the ability to detect >10(6) differences in protein abundances and identify more abundant proteins from subpicogram amounts of samples. The AMT tag approach is poised to become a new standard technique for the in-depth and high-throughput analysis of complex organisms and clinical samples, with the potential to extend the analysis to a single mammalian cell.

Quantitative evaluation of sample application methods for semipreparative separations of basic proteins by two-dimensional gel electrophoresis.

The use of cup-loading for sample application has become widely used in two-dimensional electrophoresis (2-DE) for resolution of basic proteins, but no side-by-side quantitative study has been published which compares cup-loading with the alternative passive and active rehydration methods to fully promote one type of loading method over another. Replicate 2-D gels from each loading method were quantitatively evaluated for gel-to-gel reproducibility using IPG 6-11 strips and semipreparative protein loads (300 microg). Gels were stained with SYPRO Ruby and analyzed with PDQuest. An inexpensive home-made assembly for cup-loading was used with the Protean IEF Cell for separation of whole cell extracts from the archaeon, Sulfolobus solfataricus. Cup-loading was determined to be far superior for IPG 6-11 separations than active or passive rehydration methods. Cup-loading consistently produced the greatest number of detectable spots, the best spot matching efficiency (56%), lowest spot quantity variations (28% coefficient of variation, CV), and the best-looking gels qualitatively. The least satisfactory results were obtained with active rehydration, followed closely by passive rehydration in off-line tubes. Passive rehydration experiments, performed using an on-line isoelectric focusing (IEF) tray, produced comparable spot numbers to cup-loading (84%), with 55% of the spots having higher intensity but 10% more spot quantity variance than cup-loading.