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1.
Br J Ophthalmol ; 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345604

RESUMO

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.

2.
Exp Eye Res ; 190: 107831, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31606450

RESUMO

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.

3.
DNA Repair (Amst) ; 74: 26-37, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30665830

RESUMO

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.


Assuntos
Algoritmos , DNA/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Fatores de Tempo
4.
Nano Lett ; 18(11): 6981-6988, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30285455

RESUMO

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.

5.
Sci Rep ; 8(1): 3916, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29500375

RESUMO

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 ).


Assuntos
Aprendizado de Máquina , Oxigênio/toxicidade , Retina/patologia , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Animais , Animais Recém-Nascidos , Camundongos , Retina/efeitos dos fármacos , Retina/metabolismo , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/metabolismo , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/metabolismo
6.
Sci Rep ; 7(1): 2869, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28588217

RESUMO

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.


Assuntos
Bioensaio , Quimiotaxia de Leucócito , Neutrófilos/fisiologia , Fatores Quimiotáticos , Humanos , Microscopia de Fluorescência
7.
Exp Cell Res ; 357(1): 40-50, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28442266

RESUMO

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.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Linhagem Celular , Humanos , Lisossomos/metabolismo , Transporte Proteico , Solubilidade , Rede trans-Golgi/metabolismo
8.
Sci Rep ; 7: 42112, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28181546

RESUMO

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).


Assuntos
Automação/métodos , Doenças da Coroide/diagnóstico por imagem , Doenças da Coroide/patologia , Corioide/diagnóstico por imagem , Corioide/patologia , Processamento de Imagem Assistida por Computador/métodos , Tomografia de Coerência Óptica/métodos , Algoritmos , Humanos
9.
Invest Ophthalmol Vis Sci ; 58(1): 461-469, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28122088

RESUMO

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).


Assuntos
Calcinose/prevenção & controle , Córnea/efeitos dos fármacos , Doenças da Córnea/prevenção & controle , Tomografia de Coerência Óptica/métodos , Xilazina/toxicidade , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Agonistas de Receptores Adrenérgicos alfa 2/toxicidade , Animais , Calcinose/patologia , Cálcio/metabolismo , Córnea/metabolismo , Córnea/patologia , Doenças da Córnea/induzido quimicamente , Doenças da Córnea/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Xilazina/administração & dosagem
10.
Nat Commun ; 7: 11636, 2016 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-27198043

RESUMO

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.


Assuntos
Fotodegradação , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Animais , Cães , Genômica/métodos , Humanos , Lasers , Células Madin Darby de Rim Canino
11.
Bioinformatics ; 31(8): 1279-85, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25480371

RESUMO

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.


Assuntos
Algoritmos , Movimento Celular , Rastreamento de Células , Corantes Fluorescentes/química , Neutrófilos/citologia , Análise por Conglomerados , Humanos , Neutrófilos/metabolismo
12.
Methods Cell Biol ; 119: 125-40, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24439282

RESUMO

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.


Assuntos
Adsorção , Técnicas de Cultura de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Lasers , Luz , Microscopia Confocal/métodos , Óptica e Fotônica , Fotodegradação
13.
Biophys J ; 105(2): 328-37, 2013 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-23870254

RESUMO

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.


Assuntos
Aquaporina 4/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Membrana Celular/metabolismo , Polaridade Celular , Interpretação Estatística de Dados , Cães , Células Madin Darby de Rim Canino , Microtúbulos/metabolismo , Transporte Proteico , Processos Estocásticos
14.
J Vis ; 12(2)2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22303024

RESUMO

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.


Assuntos
Aberrometria , Lasers , Modelos Neurológicos , Campos Visuais/fisiologia , Percepção Visual/fisiologia , Aberrometria/instrumentação , Aberrometria/métodos , Aberrometria/normas , Adulto , Calibragem , Convergência Ocular/fisiologia , Desenho de Equipamento , Humanos , Pessoa de Meia-Idade , Estimulação Luminosa/instrumentação , Estimulação Luminosa/métodos , Pupila/fisiologia , Valores de Referência , Adulto Jovem
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