Ocular rigidity and neuroretinal damage in patients with vasospasticity: a pilot study.

OBJECTIVE: Evidence suggests that ocular blood flow dysregulation in patients with vasospasticity could occur in response to biomechanical stimuli, contributing to optic nerve head susceptibility in glaucoma. We evaluate the role of vasospasticity in the association between ocular rigidity (OR) and neuroretinal damage, hypothesizing that low OR correlates with greater glaucoma damage in patients with vasospasticity. DESIGN: Cross-sectional study. PARTICIPANTS: Patients with open-angle glaucoma (OAG), suspect discs, or no glaucoma. METHODS: OR was measured using a noninvasive, validated method developed by our group. Retinal nerve fibre layer (RNFL) and ganglion cell complex thicknesses were acquired using spectral domain optical coherence tomography. Vasospasticity was assessed by a standardized questionnaire that was based on existing validated questionnaires and adapted to our requirements. Atherosclerosis was evaluated based on Broadway and Drance's (1998) cardiovascular disease score. Correlations between OR and structural parameters were assessed in patients with vasospasticity and those with atherosclerosis. RESULTS: Of 118 patients with either OAG (n = 67), suspect discs (n = 26), or no glaucoma (n = 25) who were recruited consecutively, 10 were classified as having vasospasticity, and 37 as having atherosclerosis. In the vasospastic group, significant correlations were found between OR and the minimum ganglion cell complex thickness (rs = 0.681, p = 0.030), the average RNFL thickness (rs = 0.745, p = 0.013), and the RNFL in the temporal quadrant (rs = 0.772, p = 0.009), indicating more damage with lower OR. Similar trends were maintained when applying multiple testing correction; however, only the eighth RNFL clock hour corresponding to the inferior-temporal peripapillary region remained significantly correlated with OR in the vasospastic group (p = 0.015). In contrast, no correlation was found in the atherosclerotic group (p > 0.05). CONCLUSIONS: The findings of the current pilot study indicate a trend for more neuronal structural damage in less-rigid eyes of patients with vasospasticity, meaning that OR may play a greater role in glaucoma in vasospastic patients than in patients with atherosclerosis. Although these results provide interesting insight into the pathophysiology of OAG, further investigation is needed to confirm our observations.

Correlation of ocular rigidity with intraocular pressure spike after intravitreal injection of bevacizumab in exudative retinal disease.

BACKGROUND/AIMS: To evaluate the non-invasive measurement of ocular rigidity (OR), an important biomechanical property of the eye, as a predictor of intraocular pressure (IOP) elevation after anti-vascular endothelial growth factor (anti-VEGF) intravitreal injection (IVI). METHODS: Subjects requiring IVI of anti-VEGF for a pre-existing retinal condition were enrolled in this prospective cross-sectional study. OR was assessed in 18 eyes of 18 participants by measurement of pulsatile choroidal volume change using video-rate optical coherence tomography, and pulsatile IOP change using dynamic contour tonometry. IOP was measured using Tono-Pen XL before and immediately following the injection and was correlated with OR. RESULTS: The average increase in IOP following IVI was 19±9 mm Hg, with a range of 7-33 mm Hg. The Spearman correlation coefficient between OR and IOP elevation following IVI was 0.796 (p<0.001), showing higher IOP elevation in more rigid eyes. A regression line was also calculated to predict the IOP spike based on the OR coefficient, such that IOP spike=664.17 mm Hg·µL×OR + 4.59 mm Hg. CONCLUSION: This study shows a strong positive correlation between OR and acute IOP elevation following IVI. These findings indicate that the non-invasive measurement of OR could be an effective tool in identifying patients at risk of IOP spikes following IVI.

The Association Between Ocular Rigidity and Neuroretinal Damage in Glaucoma.

Purpose: Ocular rigidity (OR) is an important biomechanical property, thought to be relevant in the pathophysiology of open-angle glaucoma (OAG). This study aims to evaluate the relationship between OR and neuroretinal damage caused by glaucoma. Methods: One hundred eight subjects (22 with healthy eyes, 23 with suspect discs, and 63 with OAG) were included in this study. OR was measured using a noninvasive optical coherence tomography (OCT)-based method developed by our group. We also measured central corneal thickness (CCT), corneal hysteresis (CH), and corneal resistance factor (CRF). Pearson and partial correlations were performed to evaluate the relationship between OR and glaucomatous damage represented by ganglion cell complex (GCC), retinal nerve fiber layer (RNFL) thicknesses, and neuroretinal rim area. Results: Significant positive correlations were found between OR and minimum GCC thickness (r = 0.325, P = 0.001), average GCC thickness (r = 0.320, P = 0.002), rim area (r = 0.344, P < 0.001), and RNFL thickness in the superior (r = 0.225, P = 0.023), and inferior (r = 0.281, P = 0.004) quadrants. These correlations were generally greater than those found for CCT, CH, and CRF. Furthermore, no correlation was found between OR and corneal biomechanical parameters. After adjusting for age, sex, and ethnicity, significant correlations were found between OR and minimum and average GCC thickness (r = 0.357, P = 0.001 and r = 0.344, P = 0.001, respectively), rim area (r = 0.327, P = 0.001), average RNFL thickness (r = 0.331, P = 0.001), and RNFL thickness in the superior (r = 0.296, P = 0.003) and inferior (r = 0.317, P = 0.001) quadrants. Conclusions: In this study, we found a positive correlation between structural OCT-based parameters and OR, indicating more neuroretinal damage in eyes with lower OR. These findings could provide insight into the pathophysiology of OAG.

Novel Anti-Interleukin-1ß Therapy Preserves Retinal Integrity: A Longitudinal Investigation Using OCT Imaging and Automated Retinal Segmentation in Small Rodents.

Retinopathy of prematurity (ROP) is the leading cause of blindness in neonates. Inflammation, in particular interleukin-1ß (IL-1ß), is increased in early stages of the disorder, and contributes to inner and outer retinal vasoobliteration in the oxygen-induced retinopathy (OIR) model of ROP. A small peptide antagonist of IL-1 receptor, composed of the amino acid sequence, rytvela, has been shown to exert beneficial anti-inflammatory effects without compromising immunovigilance-related NF-κB in reproductive tissues. We conducted a longitudinal study to determine the efficacy of "rytvela" in preserving the integrity of the retina in OIR model, using optical coherence tomography (OCT) which provides high-resolution cross-sectional imaging of ocular structures in vivo. Sprague-Dawley rats subjected to OIR and treated or not with "rytvela" were compared to IL-1 receptor antagonist (Kineret). OCT imaging and custom automated segmentation algorithm used to measure retinal thickness (RT) were obtained at P14 and P30; gold-standard immunohistochemistry (IHC) was used to confirm retinal anatomical changes. OCT revealed significant retinal thinning in untreated animals by P30, confirmed by IHC; these changes were coherently associated with increased apoptosis. Both rytvela and Kineret subsided apoptosis and preserved RT. As anticipated, Kineret diminished both SAPK/JNK and NF-κB axes, whereas rytvela selectively abated the former which resulted in preserved monocyte phagocytic function. Altogether, OCT imaging with automated segmentation is a reliable non-invasive approach to study longitudinally retinal pathology in small animal models of retinopathy.

Non-invasive in vivo measurement of ocular rigidity: Clinical validation, repeatability and method improvement.

Ocular rigidity (OR) is thought to play a role in the pathogenesis of glaucoma, but the lack of reliable non-invasive measurements has been a major technical challenge. We recently developed a clinical method using optical coherence tomography time-lapse imaging and automated choroidal segmentation to measure the pulsatile choroidal volume change (ΔV) and calculate OR using Friedenwald's equation. Here we assess the validity and repeatability of this non-invasive technique. We also propose an improved mathematical model of choroidal thickness to extrapolate ΔV from the pulsatile submacular choroidal thickness change more accurately. The new mathematical model uses anatomical data accounting for the choroid thickness near the equator. The validity of the technique was tested by comparing OR coefficients obtained using our non-invasive method (OROCT) and those obtained with an invasive procedure involving intravitreal injections of Bevacizumab (ORIVI) in 12 eyes. Intrasession and intersession repeatability was assessed for 72 and 8 eyes respectively with two consecutive measurements of OR. Using the new mathematical model, we obtained OR values which are closer to those obtained using the invasive procedure and previously reported techniques. A regression line was calculated to predict the ORIVI based on OROCT, such that ORIVI = 0.655 × OROCT. A strong correlation between OROCT and ORIVI was found, with a Spearman coefficient of 0.853 (p < 0.001). The intraclass correlation coefficient for intrasession and intersession repeatability was 0.925, 95% CI [0.881, 0.953] and 0.950, 95% CI [0.763, 0.990] respectively. This confirms the validity and good repeatability of OR measurements using our non-invasive clinical method.

An open-source algorithm for rapid unbiased determination of DNA fiber length.

DNA fiber fluorography is widely employed to study the kinetics of DNA replication, but the usefulness of this approach has been limited by the lack of freely-available automated analysis tools. Quantification of DNA fibers usually relies on manual examination of immunofluorescence microscopy images, which is laborious and prone to inter- and intra-operator variability. To address this, we developed an unbiased, fully automated algorithm that quantifies length and color of DNA fibers from fluorescence microscopy images. Our fiber quantification method, termed FiberQ, is an open-source image processing tool based on edge detection and a novel segment splicing approach. Here, we describe the algorithm in detail, validate our results experimentally, and benchmark the analysis against manual assessments. Our implementation is offered free of charge to the scientific community under the General Public License.

In Vivo Laser-Mediated Retinal Ganglion Cell Optoporation Using KV1.1 Conjugated Gold Nanoparticles.

Vision loss caused by retinal diseases affects hundreds of millions of individuals worldwide. The retina is a delicate central nervous system tissue stratified into layers of cells with distinct roles. Currently, there is a void in treatments that selectively target diseased retinal cells, and current therapeutic paradigms present complications associated with off-target effects. Herein, as a proof of concept, we introduce an in vivo method using a femtosecond laser to locally optoporate retinal ganglion cells (RGCs) targeted with functionalized gold nanoparticles (AuNPs). We provide evidence that AuNPs functionalized with an antibody toward the cell-surface voltage-gated K+ channel subunit KV1.1 can selectively deliver fluorescently tagged siRNAs or fluorescein isothiocyanate-dextran dye into retinal cells when irradiated with an 800 nm 100 fs laser. Importantly, neither AuNP administration nor irradiation resulted in RGC death. This system provides a novel, non-viral-based approach that has the potential to selectively target retinal cells in diseased regions while sparing healthy areas and may be harnessed in future cell-specific therapies for retinal degenerative diseases.

A machine learning approach for automated assessment of retinal vasculature in the oxygen induced retinopathy model.

Preclinical studies of vascular retinal diseases rely on the assessment of developmental dystrophies in the oxygen induced retinopathy rodent model. The quantification of vessel tufts and avascular regions is typically computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network. Such manual measurements are time-consuming and hampered by user variability and bias, thus a rapid and objective method is needed. Here, we introduce a machine learning approach to segment and characterize vascular tufts, delineate the whole vasculature network, and identify and analyze avascular regions. Our quantitative retinal vascular assessment (QuRVA) technique uses a simple machine learning method and morphological analysis to provide reliable computations of vascular density and pathological vascular tuft regions, devoid of user intervention within seconds. We demonstrate the high degree of error and variability of manual segmentations, and designed, coded, and implemented a set of algorithms to perform this task in a fully automated manner. We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools. The source code of our implementation is released under version 3 of the GNU General Public License ( https://www.mathworks.com/matlabcentral/fileexchange/65699-javimazzaf-qurva ).

A Haptotaxis Assay for Neutrophils using Optical Patterning and a High-content Approach.

Neutrophil recruitment guided by chemotactic cues is a central event in host defense against infection and tissue injury. While the mechanisms underlying neutrophil chemotaxis have been extensively studied, these are just recently being addressed by using high-content approaches or surface-bound chemotactic gradients (haptotaxis) in vitro. Here, we report a haptotaxis assay, based on the classic under-agarose assay, which combines an optical patterning technique to generate surface-bound formyl peptide gradients as well as an automated imaging and analysis of a large number of migration trajectories. We show that human neutrophils migrate on covalently-bound formyl-peptide gradients, which influence the speed and frequency of neutrophil penetration under the agarose. Analysis revealed that neutrophils migrating on surface-bound patterns accumulate in the region of the highest peptide concentration, thereby mimicking in vivo events. We propose the use of a chemotactic precision index, gyration tensors and neutrophil penetration rate for characterizing haptotaxis. This high-content assay provides a simple approach that can be applied for studying molecular mechanisms underlying haptotaxis on user-defined gradient shape.

CLN5 is cleaved by members of the SPP/SPPL family to produce a mature soluble protein.

The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children.

Open-source algorithm for automatic choroid segmentation of OCT volume reconstructions.

The use of optical coherence tomography (OCT) to study ocular diseases associated with choroidal physiology is sharply limited by the lack of available automated segmentation tools. Current research largely relies on hand-traced, single B-Scan segmentations because commercially available programs require high quality images, and the existing implementations are closed, scarce and not freely available. We developed and implemented a robust algorithm for segmenting and quantifying the choroidal layer from 3-dimensional OCT reconstructions. Here, we describe the algorithm, validate and benchmark the results, and provide an open-source implementation under the General Public License for any researcher to use (https://www.mathworks.com/matlabcentral/fileexchange/61275-choroidsegmentation).

Preventing Corneal Calcification Associated With Xylazine for Longitudinal Optical Coherence Tomography in Young Rodents.

Purpose: Spectral-domain optical coherence tomography (SD-OCT) is widely used in clinical ophthalmology and recently gained popularity in laboratory research involving small rodents. Its noninvasive nature allows repeated measurements, thereby decreasing the number of animals required. However, when used at a conventional dosage, xylazine (an α2-adrenoceptor) can cause irreversible corneal calcification, especially among young rodents. In the present study, we test whether corneal calcification associated with xylazine is mediated by the α2-adrenoceptor. Methods: Our study tested Sprague-Dawley rats, Long-Evans rats, and CD-1 mice (postnatal day [P]14). Retinal images were captured by SD-OCT. Quantitative PCR (qPCR) was used to study gene expression, whereas receptor localization was examined by immunofluorescent staining followed by confocal microscopy. Calcium deposits were detected via von Kossa staining. Results: When used at dosages appropriate for adult animals, ketamine-xylazine anesthetics led to a high rate of respiratory failure, increased apoptotic activity in the corneal epithelium, and irreversible corneal calcification in P14 rat pups. Meanwhile, OCT image quality decreased drastically as a result of corneal calcification among animals recovering from anesthesia. α2-Adrenoceptor subtypes were highly expressed on P14, in line with rodents' age-specific sensitivity to xylazine. Clonidine, a potent α2-adrenoceptor agonist, dose-dependently induced corneal calcification, which could be prevented by an α2-adrenoceptor antagonist. Conclusions: These data suggest that α2-adrenoceptors contribute to corneal calcification in young rodents. Therefore, we developed a suitable OCT imaging protocol for this cohort, including a carefully tailored ketamine-xylazine dosage (60 mg/kg and 2.5 kg/mg, respectively).

Live single-cell laser tag.

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types.

Adaptive settings for the nearest-neighbor particle tracking algorithm.

BACKGROUND: The performance of the single particle tracking (SPT) nearest-neighbor algorithm is determined by parameters that need to be set according to the characteristics of the time series under study. Inhomogeneous systems, where these characteristics fluctuate spatially, are poorly tracked when parameters are set globally. RESULTS: We present a novel SPT approach that adapts the well-known nearest-neighbor tracking algorithm to the local density of particles to overcome the problems of inhomogeneity. CONCLUSIONS: We demonstrate the performance improvement provided by the proposed method using numerical simulations and experimental data and compare its performance with state of the art SPT algorithms. AVAILABILITY AND IMPLEMENTATION: The algorithms proposed here, are released under the GNU General Public License and are freely available on the web at http://sourceforge.net/p/adaptivespt. CONTACT: javier.mazzaferri@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Laser-assisted adsorption by photobleaching.

This chapter presents a simple method to produce substrate-bound protein patterns of micrometer resolution. Our approach uses only low power visible lasers and commercially available reagents to obtain arbitrary patterns of wide concentration range. We provide useful and detailed information on how to assemble the experimental setup to create engineered cell culture substrates using laser scanning or widefield illumination modalities. A protocol that includes the biochemistry, the optics, and the computer programming needed to fabricate functional micropatterns of single and multiple components is explained for readers without experience in optical engineering. Finally, we introduce a novel widefield illumination scheme for fabricating large surface patterns as well as how to make simple patterns using a standard commercial confocal microscope.

Analysis of AQP4 trafficking vesicle dynamics using a high-content approach.

Aquaporin-4 (AQP4) is found on the basolateral plasma membrane of a variety of epithelial cells, and it is widely accepted that microtubules play an important role in protein trafficking to the plasma membrane. In the particular case of polarized trafficking, however, most evidence on the involvement of microtubules has been obtained via biochemistry experiments and single-shot microscopy. These approaches have provided essential information, even though they neglect the dynamical details of microtubule transport. In this work, we present a high-content framework in which time-lapse imaging, and single-particle-tracking algorithms were used to study a large number (∼10(4)) of GFP-AQP4-carrying vesicles on a large number of cells (∼170). By analyzing several descriptors in this large sample of trajectories, we were able to obtain highly statistically significant results. Our results support the hypothesis that AQP4 is transported along microtubules, but to our surprise, this transport is not directed straight to the basolateral plasma membrane. On the contrary, these vesicles move stochastically along microtubules, changing direction repeatedly. We propose that the role of microtubules in the basolateral trafficking of AQP4 is to increase the efficiency, rather than determine the specificity of the target.

Wide two-dimensional field laser ray-tracing aberrometer.

There is an increasing interest in measuring the peripheral optical quality of the eye. Optical aberrations have been studied extensively in the center of the visual field due to the development of Hartmann-Shack wavefront sensor. However, experimental data of the peripheral field of view are still scarce, partly due to the fact that this evaluation presents various challenges. Here, we propose a novel device based on the laser ray-tracing (LRT) aberrometer, which is well suited for measuring the off-axis aberrations. The proposed instrument is able to measure a wide (±40°) 2D visual field and is based on three main design principles: spiral-shaped sampling of the visual field, real-time detection of the eye's entrance pupil, and automatic shaping and delivering of the ray bundle that optimally samples the eye pupil. We present experimental data obtained on 11 healthy subjects and a novel analysis based on a 2D quadratic model of the aberrations as a function of visual field and azimuth. The obtained results are consistent with previous findings.